Sarsquaric acid compound or pharmaceutically acceptable salt thereof, or deuterated compound, and preparation method and application thereof

Abstract

The invention relates to a squaric acid compound or a pharmaceutically acceptable salt thereof, or a deuterated compound, and a preparation method and application thereof. The structural general formula of the squaric acid compound is shown as formula I, II, III, IV, V or VI: # imgabs0 # imgabs1 #. The compound emits near-infrared light, is stable to light, small in molecular weight, large in Stokes shift and large in molar absorption coefficient, and can be used for fluorescent probes and preparation of contrast agents.

Description

Technical Field

[0001] The present invention relates to the field of organic fluorescent probes, and particularly to a squaric acid compound or a pharmaceutically acceptable salt thereof, or a deuterated compound, and a preparation method and application thereof. Background Art

[0002] Optical imaging, especially fluorescence imaging which has developed rapidly and been widely applied in recent years, uses specific fluorescent molecular probes to label specific molecules or cells. Its spatial resolution can reach the mm level, which is well-known to life science researchers and has been widely used in in vitro imaging, and is very popular among scientific personnel. It has many advantages such as high sensitivity, quickness, low cost, and relatively high throughput. The key problems of optical imaging include autofluorescence, quenching, photobleaching, and low tissue penetration depth.

[0003] Compared with fluorescence imaging in the visible light region (400 - 700 nm), fluorescence imaging in the near-infrared (NIR) window (700 - 1700 nm) has considerable advantages in reducing photon scattering, decreasing 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 therapeutic applications. In the past decade, near-infrared I fluorescence imaging (NIR-I, 700–900 nm) has been widely used in basic research, pre-clinical, and clinical diagnosis; such as indocyanine green (ICG) and methylene blue (MB) approved by the FDA. However, compared with NIR-I fluorescence imaging, NIR-II has deeper penetration depth, better imaging effect, signal-to-noise ratio, and sensitivity because it can reduce tissue autofluorescence, photon scattering, and photon absorption level. Currently, biological NIR-II fluorescence imaging reagents mainly include carbon nanotubes, quantum dots, rare-earth-doped nanoparticles, organic small molecules, and conjugated polymers, etc.

[0004] Compared with inorganic nanomaterials, organic small molecule dyes have gradually attracted the interest of researchers due to their advantages such as well-defined structures, small molecular weights, easy metabolism, and safety. Researchers have attempted to push the emission wavelength of small molecule dyes into the near-infrared region by optimizing the structural design and synthesis routes. These diverse NIR dye structures enrich the NIR fluorescence probe library by rationally designing the main chain and substituents of the dye structure. For example, cyanine dyes and D-A-D type dyes show good water solubility, quantum yield, and molar extinction coefficient in biological systems and tissues, and are widely developed for NIR-II fluorescence probes. However, cyanine dyes are light-unstable, have poor chemical stability, low photothermal conversion efficiency, small Stokes shift, and most of the developed dyes are in NIR-I. D-A-D type dyes have disadvantages such as large molecular weight, small molar extinction coefficient, and slow in vivo metabolism rate. There is an urgent need in the industry to develop an organic fluorescent dye with light stability, small molecular weight, large Stokes shift, and large molar extinction coefficient. Summary of the Invention

[0005] Based on this, the object of the present invention is to provide an aromatic acid compound or a pharmaceutically acceptable salt thereof, or a deuterated compound that emits near-infrared light, is light-stable, has a small molecular weight, a large Stokes shift, and a large molar extinction coefficient, which can be used for fluorescent probes and the preparation of contrast agents.

[0006] The technical solution is as follows:

[0007] A squaric acid compound or a pharmaceutically acceptable salt thereof, or a deuterated compound, wherein the squaric acid compound has the structural general formula shown in I, II, III, IV, V or VI:

[0008]

[0009]

[0010] Wherein, X m- 、X n- and X p- are each independently an anion;

[0011] m, n, and p are each independently selected from any integer from 1 to 10;

[0012] Each R1 is independently selected from H, C1-C 12 alkyl, C1-C 12At least one of alkoxy, C1-C8 alkylsilyl, hydroxy C1-C8 alkyl, amino C1-C8 alkyl, aldehyde C1-C8 alkyl, mercapto C1-C8 alkyl, halo C1-C8 alkyl, acyloxy C1-C8 alkyl, amino, halogen, nitro, carboxy 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 -(CH2)n2-(OCH2CH2)n3-R;

[0013] R2 is selected from at least one of hydroxy, C1-C 12 alkoxy, C1-C8 alkylsilyl, hydroxy C1-C8 alkyl, carboxy, amino, -NR5R6, mercapto, -SR7, and malononitrile;

[0014] R3, R4, R5, R6, and R7 are each independently selected from H, C1-C8 alkyl, hydroxy, amino, carboxy, 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, halo C1-C8 alkyl, acyloxy C1-C8 alkyl, amino, halogen, sulfo, carboxy 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 -(CH2)n2-(OCH2CH2)n3-R;

[0015] 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;

[0016] R is selected from at least one of H, C1-C8 alkyl, hydroxy, amino, carboxy, sulfo, halogen, mercapto, and at least one of;

[0017] represents the attachment site.

[0018] In one embodiment, the structural general formula of the squaric acid compound is as shown in Formula I-1, II-1, III-1, IV-1, VI-1, or VI-1:

[0019]

[0020] In one embodiment, the substituents replacing the C6-C10 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, halo C1-C8 alkyl and carboxy C1-C6 alkyl.

[0021] In one embodiment, m, n and p are each independently selected from any integer from 1 to 4.

[0022] In one embodiment, X - is independently selected from I - , Br - , BF4- or ClO4 - .

[0023] In one embodiment, the squaric acid compound has any one of the following structures:

[0024]

[0025]

[0026] The present invention also provides a preparation method of the squaric acid compound as described above, and the technical solution is as follows:

[0027] (1) A preparation method of the squaric acid compound as described in Formula I above, comprising the following steps:

[0028] Compound 1 undergoes a nucleophilic substitution reaction with compound a-1 to obtain compound 2-1, where compound a-1 is halo R3, and compound 1 undergoes a nucleophilic substitution reaction with compound a-2 to obtain compound 2-2, where compound a-2 is halo R4;

[0029] 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, where compound b is a Grignard reagent;

[0030] Compound 3-1, compound 3-2 and compound c undergo a condensation reaction to obtain compound 4 (i.e., compound IV), where compound c is squaric acid;

[0031] Compound 4 reacts with compound d-1 to obtain the compound shown in I, where compound d-1 contains R2;

[0032]

[0033] Among them, the definitions of R1, R2, R3 and R4 are as described above.

[0034] In one embodiment, the compound a-1 is Br-R3.

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

[0036] In one embodiment, the compound a-2 is Br-R4.

[0037] In one embodiment, the molar ratio of the compound 1 to the compound a-2 is 1:(1 to 1.5).

[0038] In one embodiment, the compound b is methylmagnesium chloride.

[0039] In one embodiment, the molar ratio of the compound 2-1 to the compound b is 1:(1 to 1.5).

[0040] In one embodiment, the molar ratio of the compound 2-2 to the compound b is 1:(1 to 1.5).

[0041] In one embodiment, the molar ratio of the compound 3-1 to the compound c is (1 to 3):1.

[0042] In one embodiment, the molar ratio of the compound 3-2 to the compound c is (1 to 3):1.

[0043] In one embodiment, the molar ratio of the compound 4 to the compound d-1 is 1:(1 to 1.5).

[0044] (2) A method for preparing a squaric acid compound as described in formula II above, comprising the following steps:

[0045] The compound 5 undergoes a condensation reaction with the compound c to obtain the compound 6 (i.e., the compound V), where the compound c is squaric acid; the compound 6 reacts with the compound d-2 to obtain the compound shown in II, and the compound d-2 contains R2;

[0046]

[0047] Among them, the definitions of R1, R2, R3, and R4 are as described above.

[0048] In one embodiment, the molar ratio of the compound 5 to the compound c is (2 to 3):1.

[0049] In one embodiment, the molar ratio of the compound 6 to the compound d-2 is 1:(1 to 1.5).

[0050] (3) A preparation method of the squaric acid compound as described in Formula III above, comprising the following steps:

[0051] Compound 3-1 reacts with compound e to form compound 7 by substitution, and the compound e is diethyl squarate;

[0052] Compound 7 reacts with compound f to form compound 8 by hydrolysis reaction, and the compound f is a base;

[0053] Compound 8 reacts with compound 5 to form compound 9 (i.e., compound VI) by condensation reaction;

[0054] Compound 9 reacts with compound d-3 to obtain the compound shown in III, and the compound d-3 contains R2;

[0055]

[0056] Among them, the definitions of R1, R2, R3 and R4 are as described above.

[0057] In one embodiment, the molar ratio of the compound 3-1 to the compound e is (1 to 1.5):1;

[0058] In one embodiment, the molar ratio of the compound 8 to the compound 5 is 1:(1 to 1.5);

[0059] In one embodiment, the molar ratio of the compound 9 to the compound d-3 is 1:(1 to 1.5).

[0060] The present invention also provides an application of the squaric acid compound as described above, and the technical solution is as follows:

[0061] A fluorescent probe, comprising the squaric acid compound as described above or its salt or deuterated form.

[0062] The present invention also provides an application of the fluorescent probe as described above in the preparation of a near-infrared second-region contrast agent.

[0063] The present invention has at least the following beneficial effects:

[0064] The squaric acid compound provided by the present invention uses squaric acid as the central ring, and by changing the donor and acceptor, a class of donor-π-acceptor (D-π-A) type near-infrared light-emitting fluorescent molecules is developed. It has stable light, small molecular weight, large Stokes shift and molar extinction coefficient, and has a certain photothermal effect. It is very suitable for use as a fluorescent probe, especially a near-infrared fluorescent probe, and can further be used to prepare a near-infrared second-region contrast agent for in vivo imaging application research such as in vivo metabolism research and lymphangiography of small animals.

[0065] The preparation process of the squaric acid compounds of the present invention is simple, the raw materials are easily available, and the cost is low. It is very suitable for large-scale production and has broad application prospects. Description of the Drawings

[0066] Figure 1 is the ultraviolet absorption spectrum of Compound Ia;

[0067] Figure 2 is the fluorescence emission spectrum of Compound Ia;

[0068] Figure 3 is the in vivo metabolism map of Compound Ia in normal mice;

[0069] Figure 4 is the metabolism map of Compound Ia in normal mice and mice with carbon tetrachloride-induced liver injury model;

[0070] Figure 5 is the qPCR map of primary hepatocytes extracted from mice with carbon tetrachloride-induced liver injury model.

[0071] Figure 6 is the metabolism map of Compound Ia in mice with Mate 1 transporter inhibitor model. Detailed Embodiments

[0072] The present invention will be further described below in conjunction with the embodiments and examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the protection scope of the appended claims of the present invention.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0074] When using the terms "including", "having", and "comprising" described herein, it is intended to cover non-exclusive inclusion. Unless a clear limiting term is used, such as "only", "consisting of", etc., another component can also be added.

[0075] In the present invention, terms such as "preferably", "more preferably", "more preferably", "even more preferably" refer to embodiments of the present invention that can provide certain beneficial effects in certain cases. However, in the same or other cases, other embodiments may also be preferred. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not available, nor is it intended to exclude other embodiments from the scope of the present invention. That is, in the present invention, "preferably", "more preferably", "more preferably", "even more preferably", etc. are only used to describe embodiments or examples with better effects, but do not constitute a limitation on the protection scope of the present invention.

[0076] In the present invention, terms such as "further", "even further", "particularly" are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the protection scope of the present invention.

[0077] In the present invention, the meaning of "at least one" is more than one, such as one, two or more. The meaning of "multiple" or "several" is at least two, such as two, three, etc., and the meaning of "multiple layers" is at least two layers, such as two layers, three layers, etc., unless otherwise specifically defined. In the description of the present invention, the meaning of "several" is at least one, such as one, two, etc., unless otherwise specifically defined.

[0078] When a numerical range is disclosed herein, the above range is considered continuous and includes the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0079] Unless otherwise mentioned, all steps of the present invention can be carried out sequentially or randomly. For example, the method includes steps (a) and (b), which means that the method can include steps (a) and (b) carried out sequentially, or can also include steps (b) and (a) carried out sequentially. For example, when it is mentioned that the method may further include step (c), it means that step (c) can be added to the method in any order. For example, the method can include steps (a), (b) and (c) in sequence, or can also include steps (a), (c) and (b), or can also include steps (c), (a) and (b), etc.

[0080] Unless otherwise mentioned, terms in the singular form can include the plural form and should not be understood as having a quantity of one.

[0081] In the present invention, "above" or "below" both include the number itself. For example, "below 1" includes 1.

[0082] The temperature parameter in the present invention, unless otherwise specified, allows both constant temperature treatment and variation 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. Fluctuation within a range such as ±5°C, ±4°C, ±3°C, ±2°C, or ±1°C is allowed.

[0083] In the present invention, the number of atoms described by a numerical range includes both integer endpoints of the numerical range and each integer within the two endpoints. For example, "C1-C10 alkyl" represents an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.

[0084] In the present invention, When R is selected from a single bond, it represents that the connection site of the unsubstituted substituent R to the benzene ring is not defined.

[0085] In the present invention, it represents the connection site.

[0086] In the present invention, "halogen" or "halo group" refers to -F, -Cl, -Br, or -I.

[0087] In the present invention, the term "alkyl" refers to a monovalent residue formed by removing one hydrogen atom from a saturated hydrocarbon containing primary (normal) carbon atoms, or secondary carbon atoms, or tertiary carbon atoms, or quaternary carbon atoms, or a combination thereof. A phrase containing this term, for example, "C1-C10 alkyl" refers to an alkyl group containing 1 to 10 carbon atoms, and each occurrence may independently be a 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(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).

[0088] In the present invention, "haloalkyl" refers to an alkyl group substituted by one or more halogen (chlorine, fluorine, bromine or iodine) atoms. Polyhaloalkyl has the same or mixed types of halogen atoms. "Perhaloalkyl" means that each hydrogen atom in the alkyl group is substituted by a halogen atom. A haloalkyl group in which a particular carbon atom is "fully halogenated" 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.

[0089] In the present invention, "cycloalkyl" refers to a non-aromatic hydrocarbon containing ring carbon atoms, which can be a monocyclic alkyl group, or a spirocyclic alkyl group, or a bridged cyclic alkyl group. A phrase containing this term, for example, "C3-C10 cycloalkyl" refers to a cycloalkyl group containing 3 to 10 carbon atoms, and each occurrence can independently be a C3 cycloalkyl group, a C4 cycloalkyl group, a C5 cycloalkyl group, a C6 cycloalkyl group, a C7 cycloalkyl group, a C8 cycloalkyl group, a C9 cycloalkyl group or a C10 cycloalkyl group. 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 groups containing double bonds include cyclopentenyl, cyclohexenyl, cyclohexadienyl and cyclobutadienyl.

[0090] In the present invention, "number of ring atoms" refers to the number of atoms in the ring itself of a structural compound formed by bonding atoms in a ring (for example, a monocyclic compound, a fused-ring compound, a crosslinked compound, a carbocyclic compound, a heterocyclic compound). When the ring is substituted by a substituent, the atoms contained in the substituent are not included in the ring-forming atoms. The same applies to the "number of ring atoms" described below under the condition of no special explanation. For example, the number of ring atoms in a benzene ring is 6, the number of ring atoms in a naphthalene ring is 10, and the number of ring atoms in a biphenyl is 12.

[0091] In the present invention, the term "aryl, aromatic group or aromatic moiety" refers to a hydrocarbon group containing at least one aromatic ring, such as: benzene, naphthalene, anthracene, fluoranthene, phenanthrene, benzo[a]phenanthrene, acenaphthene, fluorene, biphenyl, terphenyl and derivatives of the above aryl groups.

[0092] In the present invention, the term "arylene" refers to an aromatic hydrocarbon group derived by removing two hydrogen atoms from an aromatic ring compound, and can be a monocyclic arylene, or a fused-ring arylene, or a polycyclic arylene. For polycyclic ring species, 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 can independently be a C6 arylene, a C7 arylene, a C8 arylene, a C9 arylene or a C10 arylene. Suitable examples include but are not limited to: phenylene, biphenylene, naphthylene, anthrylene, phenanthrylene, pyrenylene, triphenylene and their derivatives.

[0093] In the present invention, the term "cycloalkylene" refers to a hydrocarbon group having two monovalent group centers derived by removing two hydrogen atoms from a cycloalkyl group, and can be a monocyclic cycloalkylene, or a spirocycloalkylene, or a bridged cycloalkylene. For example, "C3-C10 cycloalkylene" refers to a cycloalkylene containing 3 to 9 carbon atoms, and each occurrence can independently be a C3 cycloalkylene, a C4 cycloalkylene, a C5 cycloalkylene, a C6 cycloalkylene, a C7 cycloalkylene, a C8 cycloalkylene or a 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, and representative examples of cycloalkylene containing double bonds include cyclopentenylene, cyclohexenylene, cyclohexadienylene and cyclobutadienylene.

[0094] In the present invention, "A and B are each 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.

[0095] In the present invention, "substituted" means that a hydrogen atom in the substituent is replaced by a substituent.

[0096] In the present invention, "substituted or unsubstituted" means that the defined group may be substituted or may not be substituted. When the defined group is substituted, it should be understood that it is optionally substituted by a group acceptable in the art, including but not limited to: a straight-chain alkyl group having 1-20 carbon atoms, a branched or cycloalkyl group having 3-20 carbon atoms, a heterocyclic group having 3-20 ring atoms, an aryl group having 5-20 ring atoms, a heteroaryl group having 5-20 ring atoms, a silyl group, a carbonyl group, an alkoxycarbonyl group, an aryloxycarbonyl group, a carbamoyl group, a halocarbonyl group, a formyl group, -NRR', a cyano group, an isocyano group, an isocyanate group, a thiocyanate group, an isothiocyanate group, a hydroxyl group, a trifluoromethyl group, a nitro group or a halogen, and the above groups may also be further substituted by substituents acceptable in the art, and the selected substituents include but not limited to: a straight-chain alkyl group having 1-20 carbon atoms, a branched or cycloalkyl group having 3-20 carbon atoms, a heterocyclic group having 3-20 ring atoms, an aryl group having 5-10 ring atoms, a heteroaryl group having 5-10 ring atoms, -NRR', a cyano group, a hydroxyl group, a trifluoromethyl group, a nitro group or a halogen; it is understood 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 heterocyclic group having 3-8 ring atoms, an aryl group having 5-10 ring atoms or a heteroaryl group having 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 heterocyclic 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 (preferably phenyl or naphthyl) or a heteroaryl group having 5-10 ring atoms.

[0097] Inorganic nanomaterials have attracted great interest due to their excellent optical properties. However, inorganic materials have slow metabolism in the body and remain in the liver and spleen for a long time, and their long-term biosafety limits further clinical translation. 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 into the near-infrared region by optimizing the structure design and synthesis route. These diverse NIR dye structures enrich the NIR fluorescence probe library by rationally designing the main chain and the substituents of the dye structure. Many organic dyes, such as cyanine dyes, D-A-D type, BODIPY type, and porphyrin type, etc., show good water solubility, quantum yield, and molar extinction coefficient in biological systems and tissues. However, currently only cyanine dyes and D-A-D type are widely developed for NIR-II fluorescence probes. Due to the poor light stability, chemical stability, low photothermal conversion efficiency, small Stokes shift of cyanine dyes, and most of the developed dyes are in NIR-I, D-A-D type dyes have disadvantages such as large molecular weight, small molar extinction coefficient, and slow metabolism rate in the body.

[0098] The liver has very complex and important physiological functions. As the main metabolic organ of the human body, it is closely related to various complex biological processes such as drug metabolism, excretion, bile secretion, phagocytosis, and body immunity. Liver diseases have high morbidity and mortality rates globally. In particular, acute and chronic liver injuries, drug-induced and alcoholic liver injuries, etc. seriously threaten people's lives and health. Moreover, with the continuous entry of new drugs into the market, drug-induced hepatotoxicity will be a major problem of clinical significance. However, the commonly used biomarkers, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), are not sensitive to early liver function injury, and the manifestations of muscle injury and kidney injury in the body may also lead to false positives in diagnosis. In addition, with the secretion and accumulation of ALT and AST, when these two indicators rise to a recognizable level for monitoring, it indicates that the liver disease has reached a very serious stage. At this time, the opportunity for timely treatment has been lost, and the disease will further develop and threaten life. Even in the terminal stage of the disease, the ALT level will instead decline. Therefore, ALT cannot be regarded as a true predictive indicator. Histopathological examination, that is, liver biopsy, is currently considered the gold standard for the diagnosis, staging, and grading of liver diseases. However, this technique is invasive and patients have poor compliance. In addition, complications may occur at any time during the operation of taking liver tissue, and the collected partial liver tissue samples often do not represent the actual state of the entire liver. Therefore, misjudgment of the course of liver injury may occur. Early detection of liver function injury can enable timely liver protection intervention. Therefore, developing a reliable method for monitoring liver injury will greatly help ensure drug safety and improve treatment efficiency. Although the probes reported currently have main advantages such as high selectivity and real-time feedback, relatively few near-infrared region II small molecule fluorescent probes are used for the early monitoring of drug-induced liver injury. The clinically applied fluorescent probe ICG approved by the FDA is mainly used for liver resection surgery navigation and liver function evaluation before and after surgery. There are few studies on its use for the monitoring of acute liver injury, and due to the instability of its own photophysical properties, ICG is restricted in its application in biological imaging. Therefore, it is very necessary to develop water-soluble small molecule squaric acid dyes with near-infrared region II fluorescence and good diagnostic monitoring.

[0099] A part of squaric acid dyes have been reported currently. Squaric acid dyes have relatively high molar extinction coefficients (>10 5 M -1 cm -1 ), excellent photostability, easy modification, and adjustable wavelengths, etc. However, most of the reported squaric acid dyes have emission spectra located in the near-infrared region I, with limited penetration depth and high tissue background, which affect the reliability of experimental results; at the same time, the reported dyes are basically lipophilic, with poor water solubility and low bioavailability.

[0100] Based on this, the object of the present invention is to provide an aromatic acid compound or a pharmaceutically acceptable salt thereof, or a deuterated compound that emits near-infrared light, is stable to light, has a small molecular weight, a large Stokes shift, and a large molar extinction coefficient, and can be used as a fluorescent probe and for preparing a contrast agent.

[0101] The technical solution is as follows:

[0102] A squaric acid compound or a pharmaceutically acceptable salt thereof, or a deuterated compound, as shown in structural general formulas I, II, III, IV, V or VI:

[0103]

[0104] Wherein, X m- 、X n- and X p- are each independently an anion;

[0105] m, n and p are each independently selected from any integer from 1 to 10;

[0106] R1 and R 1’ are each independently selected from at least one of 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, halo C1-C8 alkyl, acyloxy C1-C8 alkyl, amino, halogen, nitro, carboxy 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 -(CH2)n2-(OCH2CH2)n3-R;

[0107] R2 is selected from at least one of hydroxy, C1-C 12 alkoxy, C1-C8 alkylsilyl, hydroxy C1-C8 alkyl, carboxy, amino, -NR5R6, mercapto, -SR7, and malononitrile;

[0108] R3, R4, R5, R6 and R7 are each independently selected from H, C1-C8 alkyl, hydroxy, amino, carboxy, C1-C 12 alkyl, C1-C 12At least one of an alkoxy group, a C1-C8 alkylsilyl group, a hydroxy C1-C8 alkyl group, an amino C1-C8 alkyl group, an aldehyde C1-C8 alkyl group, a mercapto C1-C8 alkyl group, a halogenated C1-C8 alkyl group, an acyloxy C1-C8 alkyl group, an amino group, a halogen, a sulfonic acid group, a carboxy C1-C6 alkyl group, a substituted or unsubstituted C6-C10 aryl group or a 5-10 membered heteroaryl group, -(CH2)n1-COOCH2CH2Si(CH3)3, -(CH2)n2-(CH2CH2O)n3-R, and -(CH2)n2-(OCH2CH2)n3-R;

[0109] 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;

[0110] R is selected from H, a C1-C8 alkyl group, a hydroxy group, an amino group, a carboxy group, a sulfonic acid group, a halogen, a mercapto group, and at least one of;

[0111] represents a connection site.

[0112] In one embodiment, the structural general formula of the squaric acid compound is as shown in Formula I-1, II-1, III-1, IV-1, VI-1 or VI-1:

[0113]

[0114] 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).

[0115] In one embodiment, each R1 is independently selected from hydroxy C1-C8 alkyl, more preferably hydroxy C1-C4 alkyl.

[0116] In one embodiment, each R1 is independently selected from amino C1-C8 alkyl, more preferably amino C1-C4 alkyl).

[0117] In one embodiment, the substituents in each R1 that substitute the C6-C10 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, halo C1-C8 alkyl, and carboxy C1-C6 alkyl.

[0118] In one embodiment, R3 and R4 are each 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).

[0119] In one embodiment, R3 and R4 are each independently selected from hydroxy C1-C8 alkyl, more preferably hydroxy C1-C4 alkyl.

[0120] In one embodiment, R3 and R4 are each independently selected from amino C1-C8 alkyl, more preferably amino C1-C4 alkyl).

[0121] In one embodiment, the substituents in R3 and R4 that substitute the C6-C10 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, halo C1-C8 alkyl, and carboxy C1-C6 alkyl.

[0122] In the present invention, R2 is selected from hydroxy, O - , C1-C 12 alkoxy, C1-C8 alkylsilyl, hydroxy C1-C8 alkyl, carboxy, amino, -NR6R7, mercapto, -SR8, and malononitrile.

[0123] In one embodiment, R5, R6, and R7 are each 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, octyl (-(CH2)7CH3), -(CH2)n1-COOCH2CH2Si(CH3)3, -(CH2)n2-(CH2CH2O)n3-R, and -(CH2)n2-(OCH2CH2)n3-R.

[0124] In one embodiment, R5, R6, and R7 are each independently selected from hydroxy C1-C8 alkyl, more preferably hydroxy C1-C4 alkyl.

[0125] In one embodiment, R5, R6, and R7 are each independently selected from amino C1-C8 alkyl, more preferably amino C1-C4 alkyl).

[0126] In one embodiment, the substituents on R5, R6 and R7 that substitute the C6-C10 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, halo C1-C8 alkyl and carboxy C1-C6 alkyl.

[0127] It is understood that in the present invention, X m- , X n- and X p- are all anions, and m, n and p are each independently selected from any integer from 1 to 10, that is, m, n and p are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Preferably, m, n and p are each independently selected from any integer from 1 to 4, that is, 1, 2, 3 or 4. Further preferably, m, n and p are each independently selected from 1 or 2. Particularly preferably, m, n and p are all 1, and X - are each independently selected from I - , Br - , BF4 - or ClO4 - .

[0128] It is understood that in the present invention, n1 is any integer from 0 to 10, that is, n1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Preferably, n1 is any integer from 0 to 6.

[0129] It is understood that in the present invention, n2 is any integer from 0 to 10, that is, n2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Preferably, n2 is any integer from 0 to 6.

[0130] It is understood that in the present invention, 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. Preferably, n3 is any integer from 1 to 300. Further preferably, n3 is any integer from 1 to 150.

[0131] In one embodiment, the squaric acid compound has any of the following structures:

[0132]

[0133]

[0134] The present invention also provides a preparation method of the squaric acid compound as described above, and the technical solution is as follows:

[0135] (1) A preparation method of a squaric acid compound as described in the above formula (I), comprising the following steps:

[0136] Compound 1 undergoes a nucleophilic substitution reaction with compound a-1 to obtain compound 2-1, where compound a-1 is halogenated R3, and compound 1 undergoes a nucleophilic substitution reaction with compound a-2 to obtain compound 2-2, where compound a-2 is halogenated R4;

[0137] 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, where compound b is a Grignard reagent;

[0138] Compound 3-1, compound 3-2 and compound c undergo a condensation reaction to obtain compound 4 (i.e., compound IV), where compound c is squaric acid;

[0139] Compound 4 reacts with compound d-1 to obtain the compound shown in I, where compound d-1 contains R2;

[0140]

[0141] Among them, the definitions of R1, R2, R3 and R4 are as described above.

[0142] In one embodiment, compound a-1 is Br-R3.

[0143] In one embodiment, the molar ratio of compound 1 to compound a-1 is 1:(1 - 1.5).

[0144] In one embodiment, compound a-2 is Br-R4.

[0145] In one embodiment, the molar ratio of compound 1 to compound a-2 is 1:(1 - 1.5).

[0146] In one embodiment, compound b is methylmagnesium chloride.

[0147] In one embodiment, the molar ratio of compound 2-1 to compound b is 1:(1 - 1.5).

[0148] In one embodiment, the molar ratio of compound 2-2 to compound b is 1:(1 - 1.5).

[0149] In one embodiment, the molar ratio of compound 3-1 to compound c is (1 - 3):1.

[0150] In one embodiment, the molar ratio of the compound 3-2 to the compound c is (1 to 3):1.

[0151] In one embodiment, the molar ratio of the compound 4 to the compound d-1 is 1:(1 to 1.5).

[0152] In one embodiment, a method for preparing a squaric acid compound as described in the above formula (I) comprises the following steps:

[0153]

[0154] Take the compound 1 (5.91 mmol) and dissolve it in DMF. Under the condition of an ice bath at 0 °C, add sodium hydride (7.09 mmol) in batches and stir for 10 minutes. Subsequently, add methyl iodide (0.56 mL, 7.09 mmol) under the ice bath condition, and then react at room temperature overnight under nitrogen protection. After the reaction is completed, extract with saturated sodium chloride aqueous solution and ethyl acetate. The organic phase is dried with anhydrous sodium sulfate, filtered, and the filtrate is sampled with silica gel and passed through a column to obtain the compound 2-1;

[0155] Take the compound 2-1 (5.07 mmol) in a two-necked flask, dissolve it with anhydrous tetrahydrofuran, and purge with nitrogen. Add methylmagnesium chloride (2.03 mL, 6.08 mmol) under heating at 60 °C and reflux overnight. After the reaction is completed, pour the reaction solution into a 2 M hydrochloric acid solution under an ice bath to quench the reaction. Subsequently, add a saturated aqueous solution of sodium fluoroborate and stir for 30 minutes. Solids are produced in the solution. Centrifuge the green solids, wash the solids with a small amount of water and dry to obtain the compound 3-1;

[0156] Add squaric acid (0.87 mmol) and the compound 3-1 (1.75 mmol) to a flask, add 10 mL of n-butanol and 10 mL of toluene as reaction solvents, add toluene to the split port in the water separator, purge with nitrogen, and stir the reaction solution at 130 °C for 3 - 4 hours. After the reaction is completed, rotary evaporate under reduced pressure to remove toluene. Filter the obtained solution, and wash the solid with anhydrous ether until the ether is colorless. Pass through a silica gel column to obtain the compound 4-1;

[0157] Add the compound 4-1 (0.3 mmol) to a two-necked flask, purge with nitrogen, dissolve it with anhydrous dichloromethane, purge with nitrogen again, add methyl trifluoromethanesulfonate (0.36 mmol) with a syringe, and react at room temperature overnight. After the reaction is completed, quench the reaction with a 5 wt% aqueous sodium bicarbonate solution, extract with water and dichloromethane. The organic phase is dried with anhydrous sodium sulfate, and the filtrate is sampled with silica gel and passed through a column to obtain a compound I.

[0158] (2) A method for preparing a squaric acid compound as described in the above formula (II) comprises the following steps:

[0159] Compound 5 undergoes a condensation reaction with compound c to obtain compound 6 (i.e., compound V), and the said compound c is squaric acid;

[0160] Compound 6 reacts with compound d-2 to obtain the compound shown in II, and the said compound d-2 contains R2;

[0161]

[0162] Among them, the definitions of R1, R2, R3 and R4 are as described above.

[0163] In one embodiment, the molar ratio of the said compound 5 to the said compound c is (2-3):1.

[0164] In one embodiment, the molar ratio of the said compound 6 to the said compound d-2 is 1:(1-1.5).

[0165] In one embodiment, a preparation method of a squaric acid compound as described in the above formula (II) includes the following steps:

[0166]

[0167] Squaric acid (0.87 mmol) and compound 5-1 (1.75 mmol) are added to a flask, 10 mL of n-butanol and 10 mL of toluene are added as reaction solvents, toluene is added to the water separator to the split port, nitrogen is evacuated and replaced, and the reaction solution is stirred at 130 °C for 3-4 hours. After the reaction is completed, toluene is removed by rotary evaporation under reduced pressure, the obtained solution is filtered, and the solid is washed with anhydrous ether until the ether is colorless. Compound 6-1 is obtained by passing through a silica gel column;

[0168] Compound 6-1 (0.3 mmol) is added to a two-necked flask, nitrogen is evacuated and replaced, anhydrous dichloromethane is added for dissolution, nitrogen is evacuated and replaced again, methyl trifluoromethanesulfonate (0.36 mmol) is added with a syringe, and the reaction is carried out overnight at room temperature. After the reaction is completed, the reaction is quenched with a 5 wt% aqueous sodium bicarbonate solution, extracted with water and dichloromethane, the organic phase is dried with anhydrous sodium sulfate, the filtrate is sampled with silica gel, and a compound II is obtained by passing through a column.

[0169] (3) A preparation method of a squaric acid compound as described in the above formula (III) includes the following steps:

[0170] Compound 3-1 undergoes substitution with compound e to generate compound 7, and the said compound e is diethyl squarate;

[0171] Compound 7 undergoes a hydrolysis reaction with compound f to generate compound 8, and the said compound f is a base;

[0172] Compound 8 undergoes a condensation reaction with Compound 5 to form Compound 9 (i.e., Compound VI);

[0173] Compound 9 reacts with Compound d-3 to obtain the compound shown in III, and R2 is contained in Compound d-3;

[0174]

[0175] Among them, the definitions of R1, R2, R3, and R4 are as described above.

[0176] In one embodiment, the molar ratio of Compound 3-1 to Compound e is (1 to 1.5):1;

[0177] In one embodiment, the molar ratio of Compound 8 to Compound 5 is 1:(1 to 1.5);

[0178] In one embodiment, the molar ratio of Compound 9 to Compound d-3 is 1:(1 to 1.5).

[0179] In one embodiment, a method for preparing a squaric acid compound as described in formula (III) above includes the following steps:

[0180]

[0181] Add Compound 3-1 (3.98 mmol), diethyl squarate (3.32 mmol), and triethylamine (8.96 mmol) into a flask, add ethanol, evacuate and replace with nitrogen, heat under reflux at 90 °C overnight, cool the reaction solution to room temperature, remove the solvent, mix the sample, and purify by column chromatography to obtain Compound 7;

[0182] Add Compound 7-1 (1.20 mmol) into a flask, dissolve it with ethanol, add 40% aqueous sodium hydroxide solution under reflux conditions and react for 2 - 3 hours. After the reaction is completed, cool to room temperature, add 2M HCl solution to adjust the pH to neutral, concentrate the reaction solution, add 5 mL of ice-cold ethanol solution to produce a solid, and filter to obtain Compound 8-1;

[0183] Add Compound 8-1 (1.2 mmol) and Compound 5 (1.75 mmol) into a flask, add 10 mL of n-butanol and 10 mL of toluene as reaction solvents, add toluene to the demarcation point in the water separator, evacuate and replace with nitrogen, and stir the reaction solution at 130 °C for 3 - 4 hours. After the reaction is completed, rotary evaporate under reduced pressure to remove toluene, filter the obtained solution, wash the solid with anhydrous ether until the ether is colorless, and purify by silica gel column to obtain Compound 9-1;

[0184] Compound 9-1 (0.3 mmol) was added to a two-necked flask. Nitrogen was evacuated and replaced, and anhydrous dichloromethane was added to dissolve it. Nitrogen was evacuated and replaced again, and methyl trifluoromethanesulfonate (0.36 mmol) was added using a syringe. The reaction was carried out overnight at room temperature. After the reaction was completed, the reaction was quenched with 5% aqueous sodium bicarbonate solution, and extraction was carried out with water and dichloromethane. The organic phase was dried over anhydrous sodium sulfate, and the filtrate was sampled on silica gel and purified by column chromatography to obtain a compound III.

[0185] The present invention also provides the application of the squaric acid compounds as described above, and the technical solution is as follows:

[0186] A fluorescent probe, comprising the squaric acid compound as described above or a pharmaceutically acceptable salt thereof, or a deuterated compound.

[0187] The present invention also provides the application of the fluorescent probe as described above in the preparation of a near-infrared second-region contrast agent.

[0188] Specific examples are listed below to illustrate the present invention.

[0189] Table 1 Structures of the Compounds Synthesized in the Examples

[0190]

[0191]

[0192] Example 1: Synthesis of Compound Ia

[0193]

[0194] 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 ultradry THF was added as a solvent to dissolve the two compounds. Nitrogen was evacuated and replaced, and the reaction solution was refluxed and stirred at 70 °C for 10 minutes. Subsequently, 1b (2.41 g, 17.73 mmol, 1.81 mL) was added using a syringe and reacted for 2 hours. A white precipitate was formed during the reaction, and the reaction progress was monitored by high-performance liquid chromatography (HPLC). After the reactant 1a was completely reacted, no post-treatment operation was required, and the next step could be directly carried out in the flask.

[0195] After the reaction of Compound 1a was complete to form 1c, heating was stopped. 18-Crown-6 (4.69 g, 17.73 mmol) was added and stirred for 10 minutes. After purging with nitrogen, methylmagnesium chloride (4.64 g, 62.1 mmol, 20.70 mL) was slowly added dropwise with a syringe. The reaction was continued for 2 - 3 hours, and the reaction progress was monitored by HPLC. After the reaction was complete, the reaction solution was cooled to room temperature. Under ice bath conditions, the reaction solution was poured into 3M HCl (35.5 mL) and stirred for 20 minutes. 30 mL of ethanol was added and stirred for another 10 minutes. The resulting suspension was centrifuged, and the solid was washed with acetonitrile, filtered, and dried to obtain a green solid 1d (4.1 g, yield 75.9%).

[0196] 1 H NMR (400 MHz, D2O) δ 8.48 (d, J = 7.3 Hz, 1H), 8.39 (d, J = 8.1 Hz, 1H), 8.07 (dd, J = 9.6, 7.9 Hz, 2H), 7.84 (t, J = 7.7 Hz, 1H), 7.73 (t, J = 7.8 Hz, 1H), 4.52 (t, J = 7.6 Hz, 2H), 2.89 (t, J = 7.5 Hz, 2H), 2.06 (p, J = 7.7 Hz, 2H), 1.83 (p, J = 7.6 Hz, 2H).

[0197] 13 C NMR (126 MHz, D2O) δ 171.2, 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.

[0198] 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 up to the separation port, nitrogen was purged, and the reaction solution was stirred at 130 °C for 3 - 4 hours. After the reaction was completed, 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 a green solid of Compound Ⅰa.

[0199] 11H NMR (400 MHz, DMSO-d6) δ 9.00 (d, J = 7.4 Hz, 2H), 8.11 (d, J = 8.0 Hz, 2H), 7.88 (t, J = 7.7 Hz, 2H), 7.68 (d, J = 8.2 Hz, 2H), 7.60 (t, J = 7.7 Hz, 2H), 7.49 (d, J = 7.3 Hz, 2H), 6.27 (s, 2H), 4.32 (t, J = 7.3 Hz, 4H), 2.57 (t, J = 7.5 Hz, 5H), 1.89 (p, J = 7.4 Hz, 5H), 1.75 (h, J = 7.2, 6.2 Hz, 5H).

[0200] 13 13C NMR (126 MHz, DMSO) δ 181.6, 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.

[0201] ESI-LR:

[0202] Ia: expected M.W: about 685. Found: about 685

[0203] Example 2: Synthesis of Compound Ib

[0204]

[0205] At 0 °C, sodium hydride (0.28 g) was added portionwise to a DMF solution of 1a (1 g), and the mixture was stirred for 15 min. Then iodoethane (567 μL) was added, and the reaction was carried out at room temperature for 12 h. The reaction was monitored by TLC plate. After completion of the reaction, the reaction mixture was poured into saturated brine and extracted with ethyl acetate three times. The organic layers were combined, dried, and purified by silica gel column chromatography to obtain compound 2b (0.8 g, yield 68%).

[0206] 1 1H NMR (500 MHz, Chloroform-d) δ 7.93–7.87 (m, 1H), 7.81 (dd, J = 7.5, 1.6 Hz, 1H), 7.67 (t, J = 7.5 Hz, 1H), 7.64–7.56 (m, 2H), 6.99 (dd, J = 6.5, 2.5 Hz, 1H), 4.11 (q, J = 8.0 Hz, 2H), 1.31–1.24 (m, 3H).

[0207] At 0 °C, nitric acid was added to the acetic acid solution of 2b (0.5 g). Subsequently, the reaction solution was reacted at 50 °C for 12 h, and the reaction was monitored by TLC until completion. The reaction solution 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, yield 75%).

[0208] 1 H NMR (500 MHz, Chloroform-d) δ 9.01 (dd, J = 7.6, 1.5 Hz, 1H), 8.21 (d, J = 7.5 Hz, 1H), 7.86 (dd, J = 7.5, 1.5 Hz, 1H), 7.75 (t, J = 7.4 Hz, 1H), 7.25 (d, J = 7.5 Hz, 1H), 4.12 (q, J = 8.0 Hz, 2H), 1.28 (t, J = 8.0 Hz, 3H).

[0209] Compound 2c (0.5 g) was dissolved in THF, and nitrogen was evacuated and replaced. Subsequently, methylmagnesium chloride (2.5 mL) was added, and the reaction was carried out at 70 °C for 12 h. The reaction was monitored by HPLC until completion, cooled to room temperature, and the reaction solution was poured into ice water containing HCl (2.5 mL). Subsequently, KI (1 g) was added, and compound 2d (0.4 g, yield 76%) was obtained by filtration.

[0210] 1 H NMR (500 MHz, Chloroform-d) δ 9.02 (dd, J = 7.6, 1.6 Hz, 1H), 8.37 (d, J = 7.5 Hz, 1H), 7.83 (t, J = 7.5 Hz, 1H), 7.67 (dd, J = 7.5, 1.6 Hz, 1H), 7.53 (d, J = 7.5 Hz, 1H), 4.80 (q, J = 8.0 Hz, 2H), 2.88 (s, 2H), 1.53 (t, J = 8.0 Hz, 3H).

[0211] Compound 1e (100 mg) and compound 2d (710 mg) were weighed, 10 mL of n-butanol and toluene were added respectively, nitrogen was evacuated and replaced, and the reaction was carried out at 130 °C for 12 h. The reaction was monitored by TLC until completion, and compound 2e (0.2 g, yield 40%) was obtained by column chromatography.

[0212] 11H NMR (500 MHz, Chloroform-d) δ 9.01 (dd, J = 7.6, 1.6 Hz, 1H), 8.43 (d, J = 7.5 Hz, 1H), 8.26–8.18 (m, 3H), 7.75–7.66 (m, 3H), 7.65–7.58 (m, 2H), 7.54 (t, J = 7.5 Hz, 1H), 7.33 (d, J = 7.5 Hz, 1H), 4.83 (q, J = 8.0 Hz, 2H), 4.09 (q, J = 8.0 Hz, 2H), 1.50 (t, J = 8.0 Hz, 3H), 1.35 (t, J = 8.0 Hz, 3H).

[0213] Compound 2e (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 process was monitored by HPLC. After the reaction was completed, the solvent was removed by rotary evaporation. The next step could be carried out. The dried compound was dissolved in ultradry DMSO, and NH2-PEG5K (3 mg) was added and reacted at room temperature for 12 h. After the reaction was completed, dialysis was carried out using a dialysis bag and purified by a C18 reverse-phase column. The final product was purified by MALDI-TOF-MS.

[0214] MALDI-TOF-MS:

[0215] Ⅰe: expected M.W: about 5558. Found: about 5550

[0216] Example 3: Synthesis of Compound Ⅰc

[0217]

[0218] 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 process was monitored by HPLC. After the reaction was completed, the solvent was removed by rotary evaporation. The next step could be carried out. The dried compound was dissolved in ultradry DMSO, and 3-amino-N-Boc-alanine (3 mg) was added and reacted at room temperature for 12 h. After the reaction was completed, dialysis was carried out using a dialysis bag and purified by a C18 reverse-phase column. The final product was verified by MALDI-TOF-MS.

[0219] 11H NMR (500 MHz, Chloroform-d) δ 8.49 (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, 2.3 Hz, 3H), 7.71 (dt, J = 7.5, 1.5 Hz, 1H), 7.69–7.62 (m, 3H), 7.55 (dt, J = 7.5, 1.8 Hz, 2H), 7.50 (td, J = 7.4, 5.0 Hz, 2H), 7.38 (t, J = 7.5 Hz, 1H), 7.15 (dd, J = 7.5, 1.7 Hz, 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).

[0220] MALDI-TOF-MS:

[0221] Id: expected M.W: about 771. Found: about 771

[0222] Example 4: Synthesis of Compound Id

[0223]

[0224] Compound 1e (0.1 g, 1.75 mmol) and Compound 1d (0.675 g, 3.51 mmol) were added to a flask, and 10 mL each of n-butanol and toluene were used as solvents. Toluene was added to the water separator up to the separation port, nitrogen was evacuated and replaced, and the reaction solution was stirred at 130 °C for 3 - 4 hours. After the reaction was completed, 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 silica gel column to obtain Compound Ie (0.2 g, yield 38.75%).

[0225] 11H NMR (500 MHz, Chloroform-d) δ 7.80 (dd, J = 7.5, 2.3 Hz, 1H), 7.60 (dt, J = 2.2, 0.9 Hz, 1H), 7.41 (d, J = 7.5 Hz, 1H), 7.26 (dd, J = 7.6, 1.6 Hz, 1H), 6.92–6.88 (m, 1H), 6.71 (dd, J = 11.0, 1.4 Hz, 2H), 6.51 (dd, J = 7.5, 1.5 Hz, 1H), 3.49 (dq, J = 10.1, 8.0 Hz, 8H), 3.17 (t, J = 7.1 Hz, 2H), 2.91–2.83 (m, 2H), 2.87–2.76 (m, 2H), 2.75 (t, J = 7.1 Hz, 2H), 2.01 (p, J = 7.1 Hz, 2H), 1.64 (p, J = 7.1 Hz, 2H), 1.17 (t, J = 8.0 Hz, 12H).

[0226] Example 5: Synthesis of Compound Ie

[0227]

[0228] Compound 4b (0.5 g) was dissolved in ethanol (3 mL) and heated under 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 carried out for 12 h. After the reaction was completed, the reaction solution was cooled to room temperature, and the solvent was removed under reduced pressure. The solid was passed through a silica gel column to obtain Compound 4c (0.35 g, yield 52%).

[0229] 1 1H NMR (500 MHz, Chloroform-d) δ 7.84–7.78 (m, 1H), 7.81 (s, 3H), 7.67 (dt, J = 7.9, 1.7 Hz, 2H), 7.51–7.41 (m, 6H), 6.94 (dd, J = 7.5, 1.5 Hz, 2H), 4.19 (q, J = 8.1 Hz, 4H), 4.09 (q, J = 8.0 Hz, 4H), 1.47 (t, J = 8.0 Hz, 6H), 1.32 (t, J = 8.0 Hz, 6H).

[0230] Compound 4c (0.3 g) was dissolved in ethanol and heated under reflux. 40% aqueous sodium hydroxide solution (0.2 mL) was added, and the reaction was carried out for 2 h. The solvent was removed, and the product was passed through a column to obtain Compound 4d (0.21 g, yield 49%).

[0231] 11H NMR (500 MHz, Chloroform-d) δ 7.84–7.75 (m, 2H), 7.65–7.60 (m, 2H), 7.51–7.41 (m, 3H), 6.90 (dd, J=7.5, 1.6 Hz, 1H), 4.10 (q, J=8.0 Hz, 2H), 1.32 (t, J=8.0 Hz, 3H).

[0232] Compound 4d (0.1 g) and compound 3a (0.15 g) were added to a flask, 10 mL each of n-butanol and toluene were added, nitrogen was evacuated and replaced, and the reaction was carried out at 130 °C for 12 h. The solvent was removed, and compound If (90 mg, yield 49%) was obtained by column chromatography.

[0233] 1 1H NMR (500 MHz, Chloroform-d) δ 7.79 (ddd, J=10.6, 7.5, 2.0 Hz, 2H), 7.68–7.62 (m, 3H), 7.60 (dt, J=2.4, 0.9 Hz, 1H), 7.49 (q, J=7.5 Hz, 2H), 6.97 (dd, J=7.5, 1.5 Hz, 1H), 6.80 (dd, J=7.6, 1.6 Hz, 1H), 6.65–6.61 (m, 1H), 4.09 (q, J=8.0 Hz, 2H), 3.48 (q, J=8.0 Hz, 4H), 2.91–2.76 (m, 2H), 2.72 (t, J=7.1 Hz, 2H), 2.01 (p, J=7.1 Hz, 2H), 1.32 (t, J=8.0 Hz, 3H), 1.17 (t, J=8.0 Hz, 6H).

[0234] Example 8: Synthesis of compound Ig

[0235]

[0236] Compound 4a (100 mg) was dissolved in ethanol, then 5b (77 mg) and triethylamine (76 mg) were added thereto, and the reaction was stirred at room temperature for 12 h. The solvent was removed by rotary evaporation, and the sample was stirred and purified by column chromatography to obtain compound 5c.

[0237] 1 1H NMR (600 MHz, Chloroform-d) δ 4.74 (q, J=7.1 Hz, 2H), 1.48 (s, 9H), 1.44 (t, J=7.1 Hz, 3H).

[0238] Compound 5c and 1d were added into a flask, followed by the addition of toluene and n-butanol (10 ml each). Nitrogen was evacuated and replaced, and the reaction was carried out at 130 °C for 7 - 8 h. The reaction was stopped, cooled to room temperature, and filtered to obtain compound 5d. Subsequently, compound 5d was placed into a flask, deprotected with TFA, reacted for 5 - 6 h, the solvent was removed, and the product was purified by reverse-phase column chromatography to obtain compound Ig.

[0239] 1 H NMR (400 MHz, DMSO-d6) δ 10.00 (t, J = 6.2 Hz, 1H), 8.83 (d, J = 7.5 Hz, 1H), 8.32 (d, J = 8.1 Hz, 1H), 8.25 (d, J = 8.1 Hz, 1H), 8.18 (dd, J = 7.6, 4.6 Hz, 1H), 8.00 (q, J = 7.8 Hz, 2H), 7.89 (d, J = 8.0 Hz, 1H), 7.84–7.61 (m, 5H), 6.79 (s, 1H), 6.43 (s, 1H), 5.75 (s, 1H), 4.71 (d, J = 6.1 Hz, 2H), 4.39 (dt, J = 26.7, 7.7 Hz, 4H), 2.62 (dt, J = 19.7, 7.2 Hz, 4H), 2.07–1.97 (m, 2H), 1.96–1.71 (m, 6H).

[0240] 13 C NMR (126 MHz, DMSO) δ 174.3, 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.

[0241] Example 9: Synthesis of compound Ih

[0242]

[0243] Compound 4a (100 mg) was dissolved in ethanol, then 6b (60 mg) and triethylamine (76 mg) were added thereto. The reaction was stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, and the sample was mixed and purified by column chromatography to obtain compound 6c.

[0244] 1¹H NMR (600 MHz, Chloroform-d) δ 4.75 (q, J = 7.1 Hz, 2H), 4.68 (s, 1H), 3.53 (q, J = 6.6 Hz, 2H), 3.39 (t, J = 6.4 Hz, 2H), 1.89 (q, J = 6.6 Hz, 2H), 1.44 (t, J = 7.1 Hz, 3H).

[0245] Compound 6c and 1d were added into a flask, then toluene and n-butanol (10 mL each) were added. The mixture was evacuated and backfilled with nitrogen, and reacted at 130 °C for 7 - 8 h. The reaction was stopped, cooled to room temperature, filtered, and purified by column chromatography to obtain compound Ih.

[0246] 1 ¹H NMR (500 MHz, Chloroform-d) δ 7.82 (t, J = 7.6 Hz, 1H), 7.74–7.65 (m, 2H), 7.60 (dt, J = 7.5, 1.6 Hz, 1H), 7.58–7.53 (m, 3H), 7.50 (dd, J = 7.5, 1.5 Hz, 1H), 7.50–7.45 (m, 2H), 7.47–7.37 (m, 1H), 7.16 (dd, J = 7.3, 1.6 Hz, 1H), 6.74 (s, 1H), 6.62 (s, 1H), 6.44 (s, 2H), 6.10 (t, J = 4.3 Hz, 1H), 4.63 (t, J = 7.0 Hz, 2H), 4.16–4.08 (m, 2H), 3.24 (td, J = 7.1, 4.3 Hz, 2H), 3.15 (t, J = 7.1 Hz, 2H), 2.84 (t, J = 7.1 Hz, 2H), 2.81–2.75 (m, 2H), 2.09 (pd, J = 6.9, 0.7 Hz, 2H), 2.03–1.86 (m, 4H), 1.89–1.77 (m, 4H).

[0247] Example 10: Ultraviolet Absorption Spectrum of Compound Ia

[0248] Ia was dissolved in deionized water and methanol. 1.5 mL of the solution was taken and added into a 1-cm cuvette. The absorption spectra of different probes were measured on a UV2600 UV-visible spectrophotometer. The solvent used to prepare the solution was used to deduct the background and zero the instrument. The wavelength range was recorded as 300 - 1400 nm. The absorption peaks of compound Ia in water and methanol were 738 nm and 858 nm, respectively.

[0249] Example 11: Fluorescence Emission Spectrum of Compound Ia

[0250] Dissolve Ⅰa in deionized water and methanol. Pipette 200 μL and add it into a 2-cm-thick quartz cuvette. Excite it with an 808-nm laser, and record the emission wavelength with a fluorescence spectrometer IHR320. The wavelength measurement range is 825 - 1400 nm. The emission peak of compound Ⅰa is 925 nm, and its spectrum falls in the second near-infrared region.

[0251] Example 12: Biodistribution map of compound Ⅰa in normal mice

[0252] The mice used in the experiment were purchased after being approved by the Shanghai Laboratory Animal Center. The animal experiment was carried out according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. The normal 6-week-old Balb / c female mice were used in the experiment. Ⅰa was dissolved in PBS and injected into the mice via the tail vein. In vivo imaging was studied with a second near-infrared camera, excited by an 808-nm laser, and the filter was a 1000-nm long-pass filter.

[0253] The results are as Figure 3 shown, the biodistribution of the probe in the mice. The fluorescence intensity was concentrated in the liver and intestine, indicating that the probe was metabolized through the liver and intestine. The probe had a fast metabolism rate in vivo, no obvious toxicity, and good biocompatibility.

[0254] Example 13: Experiment on mice with a carbon tetrachloride-induced liver injury model of compound Ⅰa

[0255] Select the same batch of 6 - 8-week-old C57BL / 6J male mice weighing 18 - 22 g. The liver injury group was intraperitoneally injected with a 10% CCl4 olive oil solution at a dose of 2 mg / kg. The normal group of mice was intraperitoneally injected with the same dose of 0.9% normal saline. 24 h after CCl4 treatment, blood samples were collected by retro-orbital bleeding. The activity of alanine aminotransferase (ALT) in the serum was measured. As a common liver function index, the concentration of ALT in the CCl4-treated group increased sharply, indicating that the liver injury model was successfully established. 18 h after injecting carbon tetrachloride, in vivo imaging was studied with a second near-infrared camera, excited by an 808-nm laser, and the filter was a 1100-nm long-pass filter. The results are as Figure 4 shown, compared with the normal mice, the metabolism of compound Ⅰa in the model group was significantly slower.

[0256] Example 14: Mechanism experiment of compound Ⅰa in carbon tetrachloride model mice

[0257] Carbon tetrachloride-induced liver injury was modeled in accordance with the steps in Example 9, and then the mice were anesthetized. Primary mouse liver cells were extracted, and then the genes of related efflux transporters in the liver tissue were tested by qPCR experiment. The results are as Figure 5As shown, it can be seen that the expression levels of the slc47a1, slc47a2, abbc6, abcg5, and abcg2 genes have all decreased, and among them, the expression level of the slc47a1 gene has decreased most significantly.

[0258] Example 15: Experiment of Compound Ⅰa on a Mouse Model of Mate 1 Transporter Inhibitor

[0259] Select a group of 6 - 8 - week - old male C57BL / 6j mice with the same body weight of 18 - 22 g. For the imatinib group, which is an efflux Mate1 transporter inhibitor, first weigh the inhibitor imatinib on an electronic balance according to the dosing doses of 25 mg / kg or 50 mg / kg. Then, dissolve imatinib with a small amount of DMSO first and then add it to a mixed solution of polyethylene glycol 300 and 1×PBS with a volume ratio of 1:1 to a final volume of 200 μL. After waiting for 30 min, inject 200 μL of the pre - prepared solution of the probe FS39. At the time points of 1, 3, 5, 8, 10, 15, 25, 30, 45, 60, 90, and 120 min after injecting the probe, collect the fluorescence change images of the abdomen of each mouse. The collection conditions are: a wavelength of 808 nm, the emission wavelength is filtered and received by a 1100 nm long - pass filter, and the exposure duration is 200 ms. The results are as Figure 6 shown. It can be seen that, compared with the normal group, at any time point of 10 min, 15 min, 30 min, and 45 min, it can be clearly seen that the metabolism in the liver of the imatinib group has been significantly inhibited.

[0260] The technical features of the above - mentioned embodiments can be combined arbitrarily. For the sake of concise description, not all possible combinations of the technical features in the above - mentioned embodiments are described. However, as long as these combinations of technical features do not conflict, they should be considered as falling within the scope described in this specification.

[0261] The above - mentioned embodiments only represent several implementation manners of the present invention, but should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning, or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims, and the specification and drawings can be used to explain the content of the claims.

Claims

1. A squaric acid compound or a pharmaceutically acceptable salt thereof, or a deuterated compound, characterized in that, The general structural formula of the squaric acid compounds is shown as Formula I, II, III, IV, V or VI: Wherein, X m- , X n- and X p- are each independently an anion; m, n and p are each independently selected from any integer from 1 to 10; Each R1 is independently selected from at least one of 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, halo C1-C8 alkyl, acyloxy C1-C8 alkyl, amino, halogen, nitro, carboxy 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 -(CH2)n2-(OCH2CH2)n3-R; R2 is selected from at least one of hydroxy, O - , C1-C 12 alkoxy, C1-C8 alkylsilyl, hydroxy C1-C8 alkyl, carboxyl, amino, -NR5R6, mercapto, -SR7 and malononitrile; R3, R4, R5, R6 and R7 are each independently selected from H, C1-C8 alkyl, hydroxy, amino, carboxy, 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, halo C1-C8 alkyl, acyloxy C1-C8 alkyl, amino, halogen, sulfo, carboxy 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 -(CH2)n2-(OCH2CH2)n3-R, at least one of them; 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 at least one of H, C1-C8 alkyl, hydroxyl, amino, carboxyl, sulfonic acid group, halogen, mercapto, and ; Indicates the connection site.

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

3. The squaric acid compound or a pharmaceutically acceptable salt thereof, or a deuterated compound according to claim 1 or 2, characterized in that, The substituents substituting the C6-C10 aryl group or 5-10 membered heteroaryl group are selected from at least one of C1-C8 alkyl group, C1-C8 alkoxy group, C1-C8 alkylsilyl group, hydroxy C1-C8 alkyl group, amino C1-C8 alkyl group, mercapto C1-C8 alkyl group, halogenated C1-C8 alkyl group and carboxy C1-C6 alkyl group.

4. The squaric acid compound or a pharmaceutically acceptable salt thereof, or a deuterated compound according to claim 1 or 2, characterized in that, m, n and p are each independently selected from any integer from 1 to 4.

5. The squaric acid compound or a pharmaceutically acceptable salt thereof, or a deuterated compound according to claim 4, characterized in that, m, n, and p are all 1, and X - is independently selected from - I - , Br - , BF4 - or ClO4 6. The squaric acid compound or a pharmaceutically acceptable salt thereof, or a deuterated compound according to claim 1, characterized in that, The squaric acid compounds have the structure shown in any of the following:

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

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

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

10. Use of the fluorescent probe according to claim 9 in the preparation of a contrast agent for the second near-infrared region.

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