WO2025130957A1 - Squaric acid compound, pharmaceutically acceptable salt or deuterated compound thereof, preparation method therefor and use thereof - Google Patents

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

Related Applications

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 uses thereof”, the entire text of which is hereby incorporated by reference.

Technical Field

The present application relates to the field of organic fluorescent probes, and in particular to a square acid compound or a pharmaceutically acceptable salt or deuterated compound thereof, and a preparation method and application thereof.

Background Art

Optical imaging, especially fluorescence imaging, which has developed rapidly and been widely used in recent years, uses specific fluorescent molecular probes to mark specific molecules or cells. Its spatial resolution can reach the mm level. It is well known to life science workers and has been widely used in in vitro imaging. It is very popular among scientists. It has many advantages such as high sensitivity, quick and simple, low cost, and relatively high throughput. The key issues of optical imaging include autofluorescence, quenching, photobleaching, and low tissue penetration depth.

Compared with fluorescence imaging in the visible light region (400-700nm), fluorescence imaging in the near-infrared (NIR) window (700-1700nm) has considerable advantages in reducing photon scattering, lowering absorption, and minimizing autofluorescence interference (Chem. Soc. Rev., 2018, 47, 4258.). Near-infrared imaging has high resolution, high signal-to-noise ratio, and great potential in molecular diagnosis and treatment applications. In the past decade, near-infrared fluorescence imaging in the first region (NIR-I, 700–900nm) has been widely used in basic research and preclinical and clinical diagnosis; such as FDA-approved indocyanine green (ICG) and methyl blue (MB). However, compared with NIR-I fluorescence imaging, NIR-II has a deeper penetration depth, more significant imaging effect, signal-to-noise ratio, and sensitivity due to reduced tissue autofluorescence, reduced photon scattering, and low photon absorption levels. At present, biological NIR-II fluorescence imaging agents mainly include carbon nanotubes, quantum dots, rare earth-doped nanoparticles, organic small molecules and conjugated polymers.

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 tried 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 enrich the NIR fluorescent probe library by rationally designing the main chain and the substituent groups of the dye structure. For example, cyanines and D-A-D types have shown good water solubility, quantum yield, and molar absorption coefficient 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. D-A-D dyes have the disadvantages of large molecular weight, small molar absorption coefficient, and slow metabolism in the body.

Summary of the invention

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 zone II contrast agent.

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

wherein 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 R ₁ is independently selected from at least one of H, C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ alkoxy, C ₁ -C ₈ alkylsilyl, hydroxy C ₁ -C ₈ alkyl, amino C ₁ -C ₈ alkyl, aldehyde C ₁ -C ₈ alkyl, mercapto C ₁ -C ₈ alkyl, halogenated C ₁ -C ₈ alkyl, acyloxy C ₁ -C ₈ alkyl, amino, halogen, nitro, carboxyl C ₁ -C ₆ alkyl, substituted or unsubstituted C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl, -(CH ₂ ) n ₁ -COOCH ₂ CH ₂ Si(CH ₃ ) ₃ , -(CH ₂ ) n ₂ -(CH ₂ CH ₂ O) n ₃ -R and -(CH ₂ ) n ₂ -(OCH ₂ CH ₂ ) n ₃ -R;

R ₂ is at least one selected from hydroxyl, C ₁ -C ₁₂ alkoxy, C ₁ -C ₈ alkylsilyl, hydroxy C ₁ -C ₈ alkyl, carboxyl, amino, -NR ₅ R ₆ , mercapto, -SR ₇ and malononitrile;

_R3 , _R4 , _R5 , _R6 and _R7 are independently selected from H, _C1 - _C8 alkyl, hydroxyl, amino, carboxyl, _C1 - _C12 alkyl, _C1 - _C12 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 - _C10 aryl or _5-10 membered heteroaryl, -( _CH2 ) _{n1 - COOCH2CH2Si} ( _CH3 ) ₃ , -( _{CH2 ) n2-} ( _CH2CH2O ) _n3 -R and -(CH ₂ )n ₂ -(OCH ₂ CH ₂ )n ₃ -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, C ₁ -C ₈ alkyl, hydroxy, amino, carboxyl, sulfonic acid, halogen, mercapto, and At least one of;

Indicates the connection site.

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

In one embodiment, the substituents in the C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl are selected from at least one of C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, C ₁ -C ₈ alkylsilyl, hydroxy C ₁ -C ₈ alkyl, amino C ₁ -C ₈ alkyl, mercapto C ₁ -C ₈ alkyl, halogenated C ₁ -C ₈ alkyl and carboxyl C1 -C6 alkyl.

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

In one embodiment, ^X- is independently selected _from ^I- , ^Br- , _BF4- or ^{ClO4- .}

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

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

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

Compound 1 undergoes a nucleophilic substitution reaction with compound a-1 to obtain compound 2-1, wherein compound a-1 is halogenated R ₃ ; compound 1 undergoes a nucleophilic substitution reaction with compound a-2 to obtain compound 2-2, wherein compound a-2 is halogenated R ₄ ;

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 compound 4 (i.e., compound IV), 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 R ₂ ;

wherein R ₁ , R ₂ , R ₃ and R ₄ are as defined above.

In one embodiment, the compound a-1 is Br-R ₃ .

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

In one embodiment, the compound a-2 is Br-R ₄ .

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

In one embodiment, the compound b is methylmagnesium chloride.

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

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

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

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

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

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

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

Compound 6 reacts with compound d-2 to obtain the compound shown in II, wherein the compound d-2 contains R ₂ ;

wherein R ₁ , R ₂ , R ₃ and R ₄ are as defined above.

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

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

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

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 (i.e., compound VI);

Compound 9 reacts with compound d-3 to obtain the compound shown in III, wherein the compound d-3 contains R ₂ ;

wherein R ₁ , R ₂ , R ₃ and R ₄ are as defined above.

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

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

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

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

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

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

This application has at least the following beneficial effects:

The squaric acid compounds provided in the present application use squaric acid as the central ring, and by changing the donor and the acceptor, a class of donor-π-acceptor (D-π-A) type near-infrared light-emitting fluorescent molecules are developed. The squaric acid compounds are stable to light, 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 be further used to prepare near-infrared zone II contrast agents for in vivo imaging application research such as in vivo metabolism research in small animals and lymphangiography.

The preparation process of the square acid compound of the present application is simple, the raw materials are easily available, the cost is low, it is very suitable for scale-up production, and has broad application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the conventional technology, the drawings required for use in the embodiments or the conventional technology descriptions are briefly introduced below. Obviously, the drawings described below are merely embodiments of the present application, and ordinary technicians in this field can obtain other drawings based on the disclosed drawings without paying any creative work.

Figure 1 is the UV absorption spectrum of compound Ⅰa.

Figure 2 is the fluorescence emission spectrum of compound Ⅰa.

FIG3 is a diagram showing the metabolism of compound Ia in normal mice.

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

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

FIG. 6 is a qPCR graph of primary hepatocytes extracted from carbon tetrachloride liver injury model mice.

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

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

DETAILED DESCRIPTION

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

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 belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

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 explicit limiting terms such as “only,” “consisting of,” etc. are used.

In the present application, “further”, “furthermore”, “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.

In the present application, "at least one" means more than one, such as one, two and more than two. "Multiple" or "several" means at least two, such as two, three, etc., and "multilayer" means at least two layers, such as two layers, three layers, etc., unless otherwise clearly and specifically defined. In the description of the present application, "several" means at least one, such as one, two, etc., unless otherwise clearly and specifically defined.

When a numerical range is disclosed herein, the above range is considered to be 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 features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

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 also 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.

Unless mentioned to the contrary, terms in the singular may include plural forms and should not be construed as being one in number.

In this application, "above" or "below" includes the number itself. For example, "1 below" includes 1.

The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

In this application, the number of atoms described by a 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.

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

In this application, Indicates the connection site.

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

In the present application, the term "alkyl" refers to a monovalent residue formed by the loss of a 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. A phrase containing the term, for example, "C1-C10 alkyl" refers to an alkyl containing 1 to 10 carbon atoms, and each occurrence may be independently 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, -CH ₃ ), ethyl (Et, -CH ₂ CH ₃ ), 1-propyl (n-Pr, n-propyl, -CH ₂ CH ₂ CH ₃ ), 2-propyl (i-Pr, i-propyl, -CH(CH ₃ ) ₂ ), 1-butyl (n-Bu, n-butyl, -CH ₂ CH ₂ CH ₂ CH ₃ ), 2-methyl-1-propyl (i-Bu, i-butyl, -CH ₂ CH(CH ₃ ) ₂ ), 2-butyl (s-Bu, s-butyl, -CH(CH ₃ )CH ₂ CH ₃ ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH ₃ ) ₃ ), 1-pentyl (n-pentyl, -CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 2-pentyl (-CH(CH _{3 ) 2} )CH ₂ CH ₂ CH ₃ ), 3-pentyl (-CH(CH ₂ CH ₃ ) ₂ ), 2-methyl-2-butyl (-C(CH ₃ ) ₂ CH ₂ CH ₃ ), 3-methyl-2-butyl (-CH(CH ₃ )CH(CH ₃ ) ₂ ), 3-methyl-1-butyl (-CH ₂ CH ₂ CH(CH ₃ ) ₂ ), 2-methyl-1-butyl (-CH ₂ CH(CH ₃ )CH ₂ CH ₃ ), 1-hexyl (-CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ) _, 2-hexyl (-CH(CH ₃ )CH ₂ CH ₂ CH ₂ CH ₃ ), 3-hexyl (-CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )), 2-methyl-2-pentyl (-C(CH ₃ ) ₂ CH _{2 3} ), 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 ) .}

In this application, "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" means that each hydrogen atom in the alkyl group is replaced by a halogen atom. A haloalkyl group that is "fully halogenated" with 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, etc.

In the present 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, and representative examples of cycloalkyl containing double bonds include cyclopentenyl, cyclohexenyl, cyclohexadienyl and cyclobutadienyl.

In the present application, "ring atoms" refers to the number of atoms in the atoms constituting 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 bonding to form a ring. When the ring is substituted by a substituent, the atoms contained in the substituent are not included in the ring atoms. The same is true for the "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 is 12.

In the present 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.

In the present application, the term "arylene" refers to an aromatic hydrocarbon group derived from an aromatic ring compound by removing two hydrogen atoms, which may 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 containing 6 to 10 carbon atoms, and each occurrence may be independently 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.

In the present application, the term "cycloalkylene" refers to a hydrocarbon group having two monovalent group centers derived from a cycloalkyl group by removing two hydrogen atoms, which may be a monocycloalkylene group, a spirocycloalkylene group, or a bridged cycloalkylene group. For example, "C3-C10 cycloalkylene" refers to a cycloalkylene group containing 3 to 9 carbon atoms, which may be independently C3 cycloalkylene, C4 cycloalkylene, C5 cycloalkylene, C6 cycloalkylene, C7 cycloalkylene, C8 cycloalkylene or C9 cycloalkylene each time it occurs. 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, and representative examples of cycloalkylene groups containing double bonds include cyclopentenylene, cyclohexenylene, cyclohexadienylene and cyclobutadienylene.

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.

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

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 by 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 by substituents acceptable in the art, and the selected substituents include but are 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, hydroxyl, trifluoromethyl, nitro or halogen; it is understandable that R and R' in -NRR' are each independently substituted by a group acceptable 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, 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 having 5-10 ring atoms, or a 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.

Inorganic nanomaterials have attracted great interest due to their excellent optical properties, but inorganic materials are slowly metabolized in the body and remain in the liver and spleen for a long time, and their long-term biosafety limits their further clinical transformation. 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 tried 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 enrich the NIR fluorescent probe library by rationally designing the main chain and the substituent groups of the dye structure. Many organic dyes, such as cyanines, D-A-D types, BODIPY types, and porphyrins, have shown good water solubility, quantum yield, and molar absorption coefficient in biological systems and tissues. However, currently only cyanines and D-A-D types have been widely developed for NIR-II fluorescent probes. Since cyanine dyes are photoinstable, have poor chemical stability, low photothermal conversion efficiency, small Stokes shift, and most of the developed dyes are located in NIR-I, D-A-D type dyes have disadvantages such as large molecular weight, small molar absorption coefficient, and slow metabolism in the body.

The liver has very complex and important physiological functions. As the main metabolic organ of the human body, it is closely related to a variety of complex biological processes such as drug metabolism, excretion, bile secretion, phagocytosis and immunity. The incidence and mortality of liver diseases are very high worldwide. In particular, acute and chronic liver damage, drug-induced and alcoholic liver damage seriously threaten people's life and health. And as new drugs continue to enter the market, drug-induced hepatotoxicity will be a major problem with clinical significance. However, the commonly used biomarkers, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), are not sensitive to early liver function damage, and the body's muscle damage and kidney damage may also lead to false positive diagnosis. In addition, with the secretion and accumulation of ALT and AST, when these two indicators rise to a level that can be identified and monitored, it indicates that the liver disease has reached a very serious level. At this time, the opportunity for timely treatment has been lost, which will cause the disease to further develop and threaten life. Even at the end of the disease, the ALT level will decrease. Therefore, ALT cannot be regarded as a true predictive indicator. Histopathological examination, i.e., liver biopsy, is currently considered the gold standard for liver disease diagnosis and disease staging and grading. However, this technique is invasive and patient compliance is poor. In addition, complications may occur at any time during the operation of liver tissue collection, and the partial liver tissue samples collected often do not represent the actual state of the entire liver, so there may be misjudgment of the course of liver injury. Early detection of liver function damage can allow timely liver protection intervention. Therefore, the development of a reliable liver injury monitoring method will greatly help ensure drug safety and improve treatment efficiency. Although the probes reported so far have the main advantages of high selectivity and real-time feedback, there are relatively few near-infrared zone II small molecule fluorescent probes for early monitoring of drug-induced liver injury. The FDA-approved fluorescent probe ICG for clinical use is mainly used for liver resection surgery navigation and preoperative and postoperative liver function assessment. There are few studies on its use for monitoring acute liver injury, and ICG is limited in its application in biological imaging due to its unstable photophysical properties.

At present, some squaric acid dyes have been reported. Squaric acid dyes have advantages such as high molar absorption coefficient (>10 ⁵ M ^-1 cm ^-1 ), excellent photostability, easy absorption and wavelength adjustment. 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.

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 thereof have the characteristics of emitting near-infrared light, being stable to light, having a small molecular weight, a large Stokes shift and a large molar absorption coefficient, and are water-soluble and have high bioavailability, so they can be used for fluorescent probes and the preparation of contrast agents, especially near-infrared zone II contrast agents.

The technical solution is as follows:

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

wherein X ^m- , X ^n- and X ^p- are independently anions;

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

R ₁ and R _1' are each independently selected from at least one of H, C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ alkoxy, C ₁ -C ₈ alkylsilyl, hydroxy C ₁ -C ₈ alkyl, amino C ₁ -C ₈ alkyl, aldehyde C ₁ -C ₈ alkyl, mercapto C ₁ -C ₈ alkyl, halogenated C ₁ -C ₈ alkyl, acyloxy C ₁ -C ₈ alkyl, amino, halogen, nitro, carboxyl C ₁ -C ₆ alkyl, substituted or unsubstituted C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl, -(CH ₂ ) n ₁ -COOCH ₂ CH ₂ Si(CH ₃ ) ₃ , -(CH ₂ ) n ₂ -(CH ₂ CH ₂ O) n ₃ -R and -(CH ₂ ) n ₂ -(OCH ₂ CH ₂ ) n ₃ -R;

R ₂ is at least one selected from hydroxyl, C ₁ -C ₁₂ alkoxy, C ₁ -C ₈ alkylsilyl, hydroxy C ₁ -C ₈ alkyl, carboxyl, amino, -NR ₅ R ₆ , mercapto, -SR ₇ and malononitrile;

_R3 , _R4 , _R5 , _R6 and _R7 are independently selected from H, _C1 - _C8 alkyl, hydroxyl, amino, carboxyl, _C1 - _C12 alkyl, _C1 - _C12 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 - _C10 aryl or 5-10 membered heteroaryl _, -( _CH2 ) _{n1 - COOCH2CH2Si} ( _CH3 ) ₃ , -( _{CH2 ) n2-} ( _CH2CH2O ) _n3 -R and -(CH ₂ )n ₂ -(OCH ₂ CH ₂ )n ₃ -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, C ₁ -C ₈ alkyl, hydroxy, amino, carboxyl, sulfonic acid, halogen, mercapto, and At least one of;

Indicates the connection site.

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

In one embodiment, each R ₁ is independently selected from hydrogen, nitro, methyl (Me, -CH ₃ ), ethyl (Et, -CH ₂ CH ₃ ), 1-propyl (n-Pr, n-propyl, -CH ₂ CH ₂ CH ₃ ), 2-propyl (i-Pr, i-propyl, -CH(CH ₃ ) ₂ ), 1-butyl (n-Bu, n-butyl, -CH ₂ CH ₂ CH ₂ CH ₃ ), 2-methyl-1-propyl (i-Bu, i-butyl, -CH ₂ CH(CH ₃ ) ₂ ), 2-butyl (s-Bu, s-butyl, -CH(CH ₃ )CH ₂ CH ₃ ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH ₃ ) ₃ ), 1-pentyl (n-pentyl, -CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 2-pentyl (-CH(CH ₃ )CH ₂ CH ₂ CH ₃ ), 3-pentyl (-CH(CH ₂ CH ₃ ) ₂ ), 2-methyl-2-butyl (-C(CH ₃ ) ₂ CH ₂ CH ₃ ), 3-methyl-2-butyl (-CH(CH ₃ )CH(CH ₃ ) ₂ ), 3-methyl-1-butyl (-CH ₂ CH ₂ CH(CH ₃ ) ₂ ), 2-methyl-1-butyl (-CH ₂ CH(CH ₃ )CH ₂ CH ₃ ), 1-hexyl (-CH ₂ CH ₂ CH ₂ CH ₂ CH _{3 )} , 2-hexyl (-CH(CH ₃ )CH ₂ CH ₂ CH ₂ CH ₃ ), 3-hexyl (-CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )), 2-methyl-2-pentyl (-C(CH ₃ ) ₂ CH ₂ CH ₂ CH ₃ ), 3-methyl-2-pentyl (-CH(CH ₃ )CH(CH ₃ )CH ₂ CH ₃ ), 4-methyl-2-pentyl (-CH(CH ₃ )CH ₂ CH(CH ₃ ) ₂ ), 3-methyl-3-pentyl (-C(CH ₃ )(CH ₂ CH ₃ ) ₂ ), 2-methyl-3-pentyl (-CH(CH ₂ CH ₃ )CH(CH ₃ ) ₂ ), 2,3-dimethyl-2-butyl (-C(CH ₃ ) ₂ CH(CH ₃ ) ₂ ), 3,3-dimethyl-2-butyl (-CH(CH ₃ )C(CH ₃ ) ₃ , and octyl (-(CH ₂ ) ₇ CH ₃ ).

In one embodiment, each R ₁ is independently selected from hydroxy C ₁ -C ₈ alkyl, and in one embodiment is hydroxy C ₁ -C ₄ alkyl.

In one embodiment, each R ₁ is independently selected from amino C ₁ -C ₈ alkyl, and in one embodiment is amino C ₁ -C ₄ alkyl.

In one embodiment, the substituent replacing the C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl in each R ₁ is at least one selected from C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, C ₁ -C ₈ alkylsilyl, hydroxy C ₁ -C ₈ alkyl, amino C ₁ -C ₈ alkyl, mercapto C ₁ -C ₈ alkyl, halogenated C ₁ -C ₈ alkyl and carboxyl C1 -C6 alkyl.

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(CH ₃ )CH ₂ CH ₂ CH ₃ ), 3-pentyl (-CH(CH ₂ CH ₃ ) ₂ ), 2-methyl-2-butyl (-C(CH ₃ ) ₂ CH ₂ CH ₃ ), 3-methyl-2-butyl (-CH(CH ₃ )CH(CH ₃ ) ₂ ), 3-methyl-1-butyl (-CH ₂ CH ₂ CH(CH ₃ ) ₂ ), 2-methyl-1-butyl (-CH ₂ CH(CH ₃ )CH ₂ CH ₃ ), 1-hexyl (-CH ₂ CH ₂ CH ₂ CH ₂ CH _{3 )} , 2-hexyl (-CH(CH ₃ )CH ₂ CH ₂ CH ₂ CH ₃ ), 3-hexyl (-CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )), 2-methyl-2-pentyl (-C(CH ₃ ) ₂ CH ₂ CH ₂ CH ₃ ), 3-methyl-2-pentyl (-CH(CH ₃ )CH(CH ₃ )CH ₂ CH ₃ ), 4-methyl-2-pentyl (-CH(CH ₃ )CH ₂ CH(CH ₃ ) ₂ ), 3-methyl-3-pentyl (-C(CH ₃ )(CH ₂ CH ₃ ) ₂ ), 2-methyl-3-pentyl (-CH(CH ₂ CH ₃ )CH(CH ₃ ) ₂ ), 2,3-dimethyl-2-butyl (-C(CH ₃ ) ₂ CH(CH ₃ ) ₂ ), 3,3-dimethyl-2-butyl (-CH(CH ₃ )C(CH ₃ ) ₃ , and octyl (-(CH ₂ ) ₇ CH ₃ ).

In one embodiment, R ₃ and R ₄ are independently selected from hydroxy C ₁ -C ₈ alkyl, and in one embodiment, hydroxy C ₁ -C ₄ alkyl.

In one embodiment, R ₃ and R ₄ are independently selected from amino C ₁ -C ₈ alkyl, and in one embodiment, amino C ₁ -C ₄ alkyl.

In one embodiment, the substituents replacing the C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl in R ₃ and R ₄ are selected from at least one of C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, C ₁ -C ₈ alkylsilyl, hydroxy C ₁ -C ₈ alkyl, amino C ₁ -C ₈ alkyl, mercapto C ₁ -C ₈ alkyl, halogenated C ₁ -C ₈ alkyl and carboxyl C ₁ -C ₆ alkyl.

In the present application, R ₂ is at least one selected from hydroxyl, O ⁻ , C ₁ -C ₁₂ alkoxy, C ₁ -C ₈ alkylsilyl, hydroxy C ₁ -C ₈ alkyl, carboxyl, amino, —NR ₆ R ₇ , mercapto, —SR ₈ and malononitrile.

In one embodiment, R ₅ , R ₆ and R ₇ are independently selected from hydrogen, methyl (Me, -CH ₃ ), ethyl (Et, -CH ₂ CH ₃ ), 1-propyl (n-Pr, n-propyl, -CH ₂ CH ₂ CH ₃ ), 2-propyl (i-Pr, i-propyl, -CH(CH ₃ ) ₂ ), 1-butyl (n-Bu, n-butyl, -CH ₂ CH ₂ CH ₂ CH ₃ ), 2-methyl-1-propyl (i-Bu, i-butyl, -CH ₂ CH(CH ₃ ) ₂ ), 2-butyl (s-Bu, s-butyl, -CH(CH ₃ )CH ₂ CH ₃ ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH ₃ ) ₃ ), 1-pentyl (n-pentyl, -CH ₂ CH ₂ CH ₂ CH _{2 3} ), 2-pentyl (-CH(CH ₃ )CH ₂ CH ₂ CH ₃ ), 3-pentyl (-CH(CH ₂ CH ₃ ) ₂ ), 2-methyl-2-butyl (-C(CH ₃ ) ₂ CH ₂ CH ₃ ), 3-methyl-2-butyl (-CH(CH ₃ )CH(CH ₃ ) ₂ ), 3-methyl-1-butyl (-CH ₂ CH ₂ CH(CH ₃ ) ₂ ), 2-methyl-1-butyl (-CH ₂ CH(CH ₃ )CH ₂ CH ₃ ), 1-hexyl (-CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 2-hexyl (-CH(CH ₃ )CH ₂ CH ₂ CH ₂ CH ₃ ) _, 3-hexyl (-CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃₎ )), 2-methyl-2-pentyl (-C(CH ₃ ) ₂ CH ₂ CH ₂ CH ₃ ), 3-methyl-2-pentyl (-CH(CH ₃ )CH(CH ₃ )CH ₂ CH ₃ ), 4-methyl-2-pentyl (-CH(CH ₃ )CH ₂ CH(CH ₃ ) ₂ ), 3-methyl-3-pentyl (-C(CH ₃ )(CH ₂ CH ₃ ) ₂ ), 2-methyl-3-pentyl (-CH(CH ₂ CH ₃ )CH(CH ₃ ) ₂ ), 2,3-dimethyl-2-butyl (-C(CH ₃ ) ₂ CH(CH ₃ ) ₂ ), 3,3-dimethyl-2-butyl (-CH(CH ₃ )C(CH ₃ ) ₃ , octyl (-(CH ₂ ) ₇ CH ₃ ), -(CH ₂ )n ₁ -COOCH ₂ CH ₂ Si(CH ₃ ) ₃ , -(CH ₂ )n ₂ -(CH ₂ CH ₂ O)n ₃ -R and -(CH ₂ )n ₂ -(OCH ₂ CH ₂ )n ₃ -R.

In one embodiment, R ₅ , R ₆ and R ₇ are independently selected from hydroxy C ₁ -C ₈ alkyl, and in one embodiment, hydroxy C ₁ -C ₄ alkyl.

In one embodiment, R ₅ , R ₆ and R ₇ are independently selected from amino C ₁ -C ₈ alkyl groups, and in one embodiment, amino C ₁ -C ₄ alkyl groups.

In one embodiment, the substituents replacing the C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl in R ₅ , R ₆ and R ₇ are selected from at least one of C ₁ -C ₈ alkyl, C _{1 -C 8 alkoxy, C 1 -C 8 alkylsilyl} , hydroxy C ₁ -C ₈ alkyl, amino C ₁ -C ₈ alkyl, mercapto C ₁ -C ₈ alkyl, halogenated C ₁ -C ₈ alkyl and carboxyl C1 -C6 alkyl.

It can be understood that in the present application, ^Xm- , ^Xn- and ^Xp- are all anions, and m, n and p are independently selected from any integer from 1 to 10, that is, 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, that is, 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, and X- _is independently selected from ^I- , ^Br- , _BF4- or ^{ClO4- .}

It can be 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.

It is understandable 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.

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.

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

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

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

Compound 1 undergoes a nucleophilic substitution reaction with compound a-1 to obtain compound 2-1, wherein compound a-1 is halogenated R ₃ ; compound 1 undergoes a nucleophilic substitution reaction with compound a-2 to obtain compound 2-2, wherein compound a-2 is halogenated R ₄ ;

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 compound 4 (i.e., compound IV), 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 R ₂ ;

wherein R ₁ , R ₂ , R ₃ and R ₄ are as defined above.

In one embodiment, the compound a-1 is Br-R ₃ .

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

In one embodiment, the compound a-2 is Br-R ₄ .

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

In one embodiment, the compound b is methylmagnesium chloride.

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

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

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

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

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

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

Compound 1 (5.91 mmol) was dissolved in DMF, sodium hydride (7.09 mmol) was added in batches at 0°C under ice bath conditions and stirred for 10 minutes, followed by addition of iodomethane (0.56 mL, 7.09 mmol) under ice bath conditions, followed by reaction at room temperature overnight under nitrogen protection. After the reaction was completed, it was extracted with saturated sodium chloride aqueous solution and ethyl acetate, the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was sampled with silica gel and passed through a column to obtain compound 2-1;

Take compound 2-1 (5.07mmol) in a two-necked bottle, dissolve it with 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 sodium fluoroborate aqueous solution and stir for 30 minutes, and solids will appear in the solution. The green solid is centrifuged by a centrifuge, and the solid is washed with a small amount of water and dried to obtain compound 3-1;

Squaric acid (0.87 mmol) and compound 3-1 (1.75 mmol) were added to the flask, and 10 mL of n-butanol and 10 mL of toluene were added as reaction solvents. Toluene was added to the water separator to the outlet, and nitrogen was replaced. The reaction solution was stirred at 130°C for 3-4 hours. After the reaction was completed, toluene was removed by vacuum rotary evaporation, the obtained solution was filtered, and the solid was washed with anhydrous ether until the ether was colorless, and passed through a silica gel column to obtain compound 4-1;

Compound 4-1 (0.3 mmol) was added to a two-necked bottle, nitrogen was replaced, anhydrous dichloromethane was added to dissolve, nitrogen was replaced again, methyl trifluoromethanesulfonate (0.36 mmol) was added with a syringe, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the reaction was quenched with a 5 wt% sodium bicarbonate aqueous solution, extracted with water and dichloromethane, the organic phase was dried with anhydrous sodium sulfate, the filtrate was sampled with silica gel, and passed through a column to obtain a compound I.

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

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

Compound 6 reacts with compound d-2 to obtain the compound shown in II, wherein the compound d-2 contains R ₂ ;

wherein R ₁ , R ₂ , R ₃ and R ₄ are as defined above.

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

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

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

Add squaric acid (0.87mmol) and compound 5-1 (1.75mmol) to the flask, add 10mL of n-butanol and 10mL of toluene as the reaction solvent, add toluene to the water separator to the outlet, replace nitrogen, and stir the reaction solution at 130°C for 3-4 hours. After the reaction is completed, remove toluene by vacuum rotary evaporation, filter the resulting solution, and wash the solid with anhydrous ether until the ether is colorless. Pass through a silica gel column to obtain compound 6-1;

Compound 6-1 (0.3 mmol) was added to a two-necked bottle, nitrogen was replaced, anhydrous dichloromethane was added to dissolve, nitrogen was replaced again, methyl trifluoromethanesulfonate (0.36 mmol) was added with a syringe, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the reaction was quenched with a 5 wt% sodium bicarbonate aqueous solution, extracted with water and dichloromethane, the organic phase was dried with anhydrous sodium sulfate, the filtrate was sampled with silica gel, and passed through a column to obtain a compound II.

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

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 (i.e., compound VI);

Compound 9 reacts with compound d-3 to obtain the compound shown in III, wherein compound d-3 contains R ₂ ;

wherein R ₁ , R ₂ , R ₃ and R ₄ are as defined above.

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

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

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

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

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 nitrogen was replaced and the mixture was heated to reflux at 90°C overnight to react. 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;

Compound 7-1 (1.20 mmol) was added to a bottle, ethanol was added to dissolve, 40% sodium hydroxide aqueous solution was added under reflux conditions to react for 2-3 hours, the reaction was completed, cooled to room temperature, 2M HCl solution was added to adjust the pH to neutral, the reaction solution was concentrated, 5 mL of ice ethanol solution was added to produce solids, and the compound 8-1 was obtained by filtration;

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

Compound 9-1 (0.3 mmol) was added to a two-necked bottle, nitrogen was replaced, anhydrous dichloromethane was added to dissolve, nitrogen was replaced again, methyl trifluoromethanesulfonate (0.36 mmol) was added with a syringe, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the reaction was quenched with a 5% sodium bicarbonate aqueous solution, extracted with water and dichloromethane, the organic phase was dried with anhydrous sodium sulfate, the filtrate was sampled with silica gel, and passed through a column to obtain a compound III.

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

A fluorescent probe comprises the above-mentioned square acid compound or a pharmaceutically acceptable salt or deuterated compound thereof.

A near-infrared zone II contrast agent comprises the above-mentioned squaric acid compound or a pharmaceutically acceptable salt or deuterated compound thereof.

A method for monitoring early liver damage, comprising imaging a subject in need thereof using a squaric acid compound as described above, or a pharmaceutically acceptable salt or deuterated compound thereof, a fluorescent probe as described above, or a near-infrared zone II contrast agent as described above. In this aspect, there is also provided the use of a squaric acid compound as described above, or a pharmaceutically acceptable salt or deuterated compound thereof, a fluorescent probe as described above, or a near-infrared zone II contrast agent as described above in monitoring early liver damage in a subject in need thereof.

Specific embodiments are listed below to illustrate the present application.

Table 1 Structures of the compounds synthesized in the examples

Example 1: Synthesis of Compound Ia

Weigh compound 1a (3 g, 17.73 mmol) and potassium tert-butoxide (1.99 g, 17.73 mmol) in a three-necked flask, add ultra-dry THF as a solvent to dissolve the two compounds. Replace nitrogen, reflux the reaction solution at 70 ° C for 10 minutes, then add 1b (2.41 g, 17.73 mmol, 1.81 mL) through a syringe and react for 2 hours. A white precipitate is produced during the reaction. The reaction progress is monitored by high performance liquid chromatography (HPLC). After the reaction of reactant 1a is complete, no post-treatment operation is required, and the next step can be carried out directly in the bottle.

After compound 1a is completely reacted to generate 1c, stop heating, add 18-crown ether-6 (4.69g, 17.73mmol) and stir for 10 minutes. After replacing nitrogen protection; slowly add methylmagnesium chloride (4.64g, 62.1mmol, 20.70mL) with a syringe. Continue the reaction for 2-3 hours, monitor the reaction progress by HPLC; after the reaction is complete, cool the reaction solution to room temperature. Pour the reaction solution into 3M HCl (35.5mL) under ice bath conditions and stir for 20 minutes, add 30mL of ethanol and continue stirring for 10 minutes. The resulting suspension solution is centrifuged, the solid is washed with acetonitrile, filtered and dried to obtain a green solid 1d. (4.1g, yield is 75.9%)

¹ H 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).

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.

Compound 1e (0.2 g, 1.75 mmol) and compound 1d (1.12 g, 3.51 mmol) were added to a flask, and 10 mL each of n-butanol and toluene were added as solvents. Toluene was added to the water separator to the outlet, and nitrogen was replaced. The reaction solution was stirred at 130 ° C for 3-4 hours. After the reaction was completed, toluene was removed by vacuum rotary evaporation, the obtained solution was filtered, and the solid was washed with anhydrous ether until the ether was colorless. The obtained solid was dissolved in water and passed through a reverse phase silica gel column to obtain a green solid of compound Ⅰa.

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).

^13C 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.

ESI-LR:

Ⅰa：expected M.W:about685.Found:about 685

Example 2: Synthesis of Compound Ib

At 0°C, sodium hydride (0.28 g) was added in batches to a DMF solution of 1a (1 g), stirred for 15 min, and iodoethane (567 μL) was added, and the mixture was reacted at room temperature for 12 h. The reaction was monitored by TLC spot plate, and the reaction solution was poured into saturated brine, extracted with ethyl acetate three times, and the organic layers were combined, dried, and passed through a silica gel column to obtain compound 2b (0.8 g, yield 68%).

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).

Nitric acid was added to an acetic acid solution of 2b (0.5 g) at 0°C, and the reaction solution was then reacted at 50°C for 12 h. The reaction was completed by monitoring by TLC. The reaction solution was extracted with water and ethyl acetate, the organic layer was dried, and compound 2c (0.46 g, yield 75%) was obtained by silica gel column.

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).

Compound 2c (0.5 g) was dissolved in THF, and nitrogen was replaced. Methylmagnesium chloride (2.5 mL) was then added and reacted at 70°C for 12 h. The reaction was completed by HPLC monitoring. The mixture was cooled to room temperature and poured into ice water containing HCl (2.5 mL). KI (1 g) was then added and filtered to obtain compound 2d (0.4 g, yield 76%).

¹ H 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).

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, and compound 2e (0.2 g, yield 40%) was obtained by column chromatography.

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).

Compound 2e (10 mg) was dissolved in THF, methyl trifluoromethanesulfonate (100 μL) was added, and the mixture 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 dried compound in ultra-dry DMSO, add NH ₂ -PEG5K (3 mg) and react at room temperature for 12 h. After the reaction was completed, the mixture was dialyzed with a dialysis bag and purified by a C18 reverse phase column. The final product was purified by MALDI-TOF-MS.

MALDI-TOF-MS:

Ⅰb：expected M.W:about5558.Found：about 5550

Example 3: Synthesis of Compound Ic

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, add 3-amino-N-tert-butyloxycarbonylalanine (3 mg) and react at room temperature for 12 h. After the reaction was completed, the dialysis bag was used for dialysis and purified by C18 reverse phase column. The final product was verified by MALDI-TOF-MS.

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).

MALDI-TOF-MS:

Ⅰc：expected M.W:about771.Found：about 771

Example 4: Synthesis of Compound Id

Compound 1e (0.1 g, 1.75 mmol) and compound 3a (0.675 g, 3.51 mmol) were added to a flask, and 10 mL each of n-butanol and toluene were added as solvents. Toluene was added to the water separator to the outlet, and nitrogen was replaced. The reaction solution was stirred at 130 ° C for 3-4 hours. After the reaction was completed, toluene was removed by vacuum rotary evaporation, the obtained solution was filtered, and the solid was washed with anhydrous ether until the ether was colorless. The obtained solid was dissolved in water and passed through a silica gel column to obtain compound Ⅰd (0.2 g, yield 38.75%).

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).

Example 5: Synthesis of Compound Ie

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 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, yield 52%).

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).

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

¹ H 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).

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 nitrogen atmosphere was replaced and the reaction was carried out at 130°C for 12 h. The solvent was removed and the mixture was passed through a column to obtain compound Ⅰe (90 mg, yield: 49%).

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).

Example 6 Synthesis of Compound IF

Compound Ⅰa (10 mg) was dissolved in THF, methyl trifluoromethanesulfonate (100 μL) was added, and the reaction was stirred at room temperature for 5 hours. 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. Peptide cRGDyk (15 mg) was added and reacted at room temperature for 12 hours. After the reaction was completed, dialyzed with a dialysis bag and purified with a C18 reverse phase column. The final product was verified by MALDI-TOF-MS. MALDI-TOF-MS.If:expected M.W:about 1286.46.Found:about 1286.5

Example 7: Synthesis of Compound Ig

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, and the solvent was removed by rotary evaporation. The sample was mixed and passed through a column to obtain compound 5c.

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

Compounds 5c and 1d were added to a bottle, followed by toluene and n-butanol (10 ml each), nitrogen was replaced, the reaction was carried out 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 bottle, TFA was added for deprotection, the reaction was continued for 5-6 hours, the solvent was removed, and the mixture was passed through a reverse phase column to obtain compound IG.

¹ H NMR (400MHz, DMSO-d ₆ )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＝8.1 Hz,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).

^13C 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.

Example 8: Synthesis of Compound Ih

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, and the solvent was removed by rotary evaporation. The sample was mixed and passed through a column to obtain compound 6c.

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).

Compounds 6c and 1d were added to the bottle, followed by toluene and n-butanol (10 ml each), and nitrogen was replaced. The mixture was reacted 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.

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).

Example 9: UV absorption spectrum of compound Ⅰa

Dissolve compound Ia in deionized water and methanol, take 1.5 mL of each solution and add it to a 1 cm thick quartz cuvette. Measure the absorption spectra of different probes on a UV2600 ultraviolet-visible spectrophotometer, subtract the solvent used to prepare the solution and adjust the background to zero, and record the wavelength range of 300-1400 nm. The absorption peaks of compound Ia in water and methanol are 738 nm and 858 nm, respectively. The experimental results show that compound Ia has strong near-infrared light absorption characteristics. In addition, the absorption spectra of compound Ia in different solvents are different, which may be related to the aggregation state of compound Ia in different solvents.

Example 10: Fluorescence emission spectrum of compound Ia

Dissolve compound Ia in deionized water and methanol, take 200 μL of each, add to a 2 cm thick quartz cuvette, excite with 808 nm laser, record the emission wavelength with fluorescence spectrometer IHR320, and the wavelength measurement range is 825-1400 nm. The emission peak of compound Ia is 925 nm, and its spectrum falls in the near infrared region II. The experimental results show that compound Ia can emit near infrared region II light, and it is suitable for use as a near infrared region II fluorescent contrast agent.

Example 11: Biodistribution of Compound Ia in Normal Mice

The mice used in the experiment were purchased with the approval of Shanghai Experimental Animal Center, and the animal experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee (IACUC) of 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 two-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.

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

C57BL/6J male mice aged 6-8 weeks with a body weight of 18-22 g were selected from the same batch. The mice in the liver injury group were intraperitoneally injected with 10% carbon tetrachloride (CCl ₄ ) olive oil solution at a dose of 2 mg/kg. The mice in the normal group were intraperitoneally injected with the same dose of 0.9% saline. 24 h after CCl ₄ treatment, blood samples were collected by retroorbital bleeding. The activity of alanine aminotransferase (ALT) in serum was determined, which is a common liver function indicator. The concentration of ALT in the mice in the liver injury group increased sharply, indicating that the liver injury model was successfully established. 18 h after the injection of carbon tetrachloride, compound Ia dissolved in phosphate buffered saline (PBS) was injected into the tail vein of each mouse in the liver injury group and the control group. In vivo imaging was studied using a near-infrared zone II camera, excited by an 808 nm laser, and the filter was a 1100 nm long-pass filter. The results are shown in Figures 4 and 5. Compared with normal mice, the metabolism of compound Ia in mice in the carbon tetrachloride liver injury model group was significantly slowed down, indicating that compound Ia can be used for faster monitoring of early liver damage.

Example 13: Mechanism experiment of carbon tetrachloride liver injury model mice

According to the steps in Example 12, carbon tetrachloride liver injury mouse modeling was performed, and then the mice were anesthetized. Mouse liver primary cells were extracted, and the genes of the relevant efflux transporters in the liver tissue were subsequently tested by qPCR experiments. As shown in Figure 6, it can be seen that the levels of slc47a1, slc47a2, abcc3, abcc6, and abcg2 gene expression were all reduced, and the level of slc47a1 gene expression 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 mouse primary hepatocytes, a damaged cell model was constructed at the cellular level, and slc47a1 was confirmed by qPCR to be a potential target of compound Ia.

Example 14: Experiment of Compound Ia in Slc47a1-related Mate 1 transporter inhibitor model mice 6-8 weeks old C57BL/6j male mice weighing 18-22g were selected from the same batch. For the imatinib group, the inhibitor imatinib was weighed on an electronic balance according to the dosage of 25mg/kg or 50mg/kg, and then imatinib was first dissolved with a small amount of DMSO and then a mixed solution of polyethylene glycol 300 and 1×PBS with a volume ratio of 1:1 was added to a final volume of 200μL. After waiting for 30min, 200μL of the pre-prepared solution of Compound Ia was injected, and the fluorescence change images of the abdomen of each mouse were collected at 1, 3, 5, 8, 10, 15, 25, 30, 45, 60, 90 and 120min after the injection of Compound Ia. The acquisition conditions were: 808 nm wavelength, 1100 nm long pass filter to filter the receiving emission wavelength, and 200 ms exposure time. The results are shown in Figure 7. Compared with the normal group, it can be clearly seen at any time point of 10 min, 15 min, 30 min and 45 min that the metabolism of compound Ia in the liver of mice in the imatinib group was significantly inhibited and the metabolism was significantly slowed down. The experimental results show that imatinib affects the metabolic process of compound Ia by inhibiting the Mate1 transporter associated with slc47a1.

Example 15: Photostability test of compound Ia

The photostability of compound Ia SQ 905 was determined using indocyanine green (ICG), a near-infrared dye widely used in clinical practice and approved by the FDA, as a control. ICG was dissolved in water, and compound Ia SQ 905 was dissolved in methanol. Over a period of 25 minutes, a 108mW/ ^cm2 808nm laser was continuously irradiated to the methanol solution of compound Ia and the aqueous solution of ICG, during which the fluorescence intensity was recorded using a near-infrared zone II camera (MARS, Artemis Intelligent Imaging, Shanghai, China). The fluorescence intensity was recorded every minute for the first 15 minutes, and then recorded every 5 minutes. The fluorescence intensity of the region of interest (ROI) in the captured image was calculated using the software ImageJ, and compared with the initial fluorescence intensity to obtain the ratio of the fluorescence intensity at each time point to the initial fluorescence intensity. The curve was drawn using the software GraphPad Prism 8.0.2, which reflects the photostability of compound Ia.

Photostability is an important indicator for evaluating probes. If the photostability of the probe is not good, 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 embodiment. 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 significantly decay after continuous irradiation of 808nm laser for 25 minutes; however, the fluorescence intensity of the aqueous solution of ICG as a control was decayed by 83% after continuous irradiation of 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.

Compound Ib, Compound Ic, Compound Id, Compound Ie, Compound If, Compound Ig and Compound Ih were tested according to the methods described in Examples 9 to 15. The experimental results show that these compounds also have strong near-infrared light absorption characteristics, can emit near-infrared zone II light, and are suitable for use as near-infrared zone II fluorescent contrast agents. These compounds also have fast metabolism in the body, no obvious toxicity, good biocompatibility, and good photostability, and are suitable for faster monitoring of early liver damage.

The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims.

Claims (11)

A squaric acid compound or a pharmaceutically acceptable salt or deuterated compound thereof, characterized in that the general structural formula of the squaric acid compound is as shown in Formula I, II, III, IV, V or VI:
wherein 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 R ₁ is independently selected from at least one of H, C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ alkoxy, C ₁ -C ₈ alkylsilyl, hydroxy C ₁ -C ₈ alkyl, amino C ₁ -C ₈ alkyl, aldehyde C ₁ -C ₈ alkyl, mercapto C ₁ -C ₈ alkyl, halogenated C ₁ -C ₈ alkyl, acyloxy C ₁ -C ₈ alkyl, amino, halogen, nitro, carboxyl C ₁ -C ₆ alkyl, substituted or unsubstituted C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl, -(CH ₂ ) n ₁ -COOCH ₂ CH ₂ Si(CH ₃ ) ₃ , -(CH ₂ ) n ₂ -(CH ₂ CH ₂ O) n ₃ -R and -(CH ₂ ) n ₂ -(OCH ₂ CH ₂ ) n ₃ -R; R ₂ is at least one selected from hydroxyl, O ^- , C ₁ -C ₁₂ alkoxy, C ₁ -C ₈ alkylsilyl, hydroxy C ₁ -C ₈ alkyl, carboxyl, amino, -NR ₅ R ₆ , mercapto, -SR ₇ and malononitrile; R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are independently selected from H, C ₁ -C ₈ alkyl, hydroxy, amino, carboxyl, C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ alkoxy, C ₁ -C ₈ alkylsilyl, hydroxy C ₁ -C ₈ alkyl, amino C ₁ -C ₈ alkyl, aldehyde C ₁ -C ₈ alkyl, mercapto C ₁ -C ₈ alkyl, halogenated C ₁ -C ₈ alkyl, acyloxy C ₁ -C ₈ alkyl, amino, halogen, sulfonic acid, carboxyl C ₁ -C ₆ alkyl, substituted or unsubstituted C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl, -(CH ₂ ) n ₁ -COOCH ₂ CH ₂ Si(CH ₃ ) ₃ , -(CH ₂ ) n ₂ -(CH ₂ CH ₂ O) n ₃ -R and -(CH ₂ )n ₂ -(OCH ₂ CH ₂ )n ₃ -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, C ₁ -C ₈ alkyl, hydroxy, amino, carboxyl, sulfonic acid, halogen, mercapto, and At least one of; Indicates the connection site. The squaric acid compound or a pharmaceutically acceptable salt or deuterated compound thereof according to claim 1, characterized in that the general structural formula of the squaric acid compound is as shown in Formula I-1, II-1, III-1, IV-1, V-1 or VI-1:
The squaric acid compound or the pharmaceutically acceptable salt or deuterated compound thereof according to claim 1 or 2, characterized in that the substituent replacing the C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl is selected from at least one of C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, C ₁ -C ₈ alkylsilyl, hydroxy C ₁ -C ₈ alkyl, amino C ₁ -C ₈ alkyl, mercapto C ₁ -C ₈ alkyl, halogenated C ₁ -C ₈ alkyl and carboxyl C1-C6 alkyl. The square 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. The squaric acid compound or the 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, and ^X- is independently selected from ^I- , ^{Br- ,} _BF4- or _ClO4- ^. The square 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 a structure shown in any of the following:

A method for preparing the squaric acid compound or a pharmaceutically acceptable salt or deuterated compound thereof according to any one of claims 1 to 6, characterized in that it comprises the following steps: (1) Compound 1 undergoes a nucleophilic substitution reaction with compound a-1 to obtain compound 2-1, wherein compound a-1 is halogenated R ₃ ; compound 1 undergoes a nucleophilic substitution reaction with compound a-2 to obtain compound 2-2, wherein compound a-2 is halogenated R ₄ ; 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 R ₂ ;
(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 the compound d-2 contains R ₂ ;
(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 compound d-3 contains R ₂ ;
wherein R ₁ , R ₂ , R ₃ and R ₄ are as defined in any one of claims 1 to 6. The method for preparing a squaric acid compound or a pharmaceutically acceptable salt or deuterated compound thereof according to claim 7, characterized in that at least one of the following (1) to (9) is satisfied: (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 (1-1.5):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). A fluorescent probe, characterized in that it comprises the square acid compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt or deuterated compound thereof. A near-infrared zone II contrast agent, characterized in that it comprises the squaric acid compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt or deuterated compound thereof. A method for monitoring early liver damage, comprising imaging a subject in need thereof using the squaric acid compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt or deuterated compound thereof, the fluorescent probe according to claim 9 or the near-infrared zone II contrast agent according to claim 10.