A near-infrared fluorescence-photoacoustic dual-mode probe for detecting nitric oxide and a preparation method and application thereof

Abstract

The application discloses a kind of preparation and its application of near-infrared fluorescence-photoacoustic bimodal probe for detecting nitric oxide.The pyrrolopyrrole nitrogen hetero fluorine boron fluorophore is used as parent body, and the classic photoinduced electron transfer system is constructed by introducing adjacent diamino phenyl strong electron-donating group.Because the structure has photoinduced electron transfer effect, the fluorophore has no fluorescence emission under normal physiological conditions.But in the disease of overexpression of nitric oxide, adjacent phenylenediamine group is converted into benzotriazole by reacting with nitric oxide, and the photoinduced electron transfer effect is inhibited, and the fluorophore can emit near-infrared fluorescence.In addition, the fluorophore has high molar extinction coefficient and ratio absorption change, can be used for photoacoustic imaging, and can efficiently and accurately detect the content of nitric oxide in disease.

Description

Technical Field

[0001] This invention relates to a nitric oxide-activated near-infrared fluorescent-photoacoustic compound, its preparation method, and its application as a nitric oxide probe, belonging to the field of fluorescence detection technology. Background Technology

[0002] As the core organ of human metabolism, the liver's health directly affects overall physiological function and quality of life. However, liver injury, a common pathological condition, has complex and diverse mechanisms, encompassing various types such as drug-induced liver injury, alcoholic liver disease, and viral hepatitis, posing significant challenges to clinical diagnosis and treatment. Currently, detecting liver function indicators in the blood (such as alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) is one of the important means of assessing liver injury. However, blood tests can only reflect the overall functional state of the liver and cannot accurately determine the specific location and extent of damage. Therefore, there is an urgent need to develop a technology that can achieve accurate and real-time monitoring of liver injury.

[0003] Nitric oxide (NO), as a key signaling molecule, exhibits significant concentration fluctuations during the progression of liver injury, making it an ideal biomarker for monitoring liver damage. However, its detection faces considerable challenges due to its extremely short half-life. In-situ detection technology can provide real-time and accurate feedback on changes in NO concentration, offering crucial evidence for the early diagnosis of liver injury.

[0004] Near-infrared fluorescence imaging offers several significant advantages: First, near-infrared light exhibits lower scattering characteristics when penetrating biological tissues, effectively reducing signal attenuation and providing higher resolution and image clarity. Second, interference from autofluorescence of pigments within the organism is significantly reduced, improving the imaging signal-to-noise ratio. Finally, near-infrared light has weak optical absorption in biological tissues, resulting in less tissue damage and excellent biocompatibility. Furthermore, photoacoustic imaging combines the high selectivity of optical imaging with the deep penetration of ultrasound imaging, enabling the acquisition of high-resolution and high-contrast tissue images. Its principle effectively avoids interference from light scattering, overcoming the depth limitation of traditional high-resolution optical imaging (approximately 1 mm), and achieving imaging of deep living tissues, with a maximum imaging depth of up to 50 mm. Therefore, developing fluorophores with both fluorescence and / or photoacoustic properties for the detection of liver injury is urgently needed. Summary of the Invention

[0005] The purpose of this invention is to provide a near-infrared fluorescence-photoacoustic dual-modal probe based on nitric oxide responsive to photoinduced electron transfer effect, its preparation method, and its application.

[0006] The technical solution of the present invention is as follows:

[0007] The present invention first provides a compound represented by the following formula IA:

[0008]

[0009] Where X is selected from O, N, S, Se, or Te;

[0010] R0 is selected from C 1-20 Alkyl, C 1-20 Alkoxy, C 3-12 Cycloalkyl or 3-12 membered heterocyclic groups;

[0011] R1 may be the same or different, and is independently selected from H, unsubstituted or optionally substituted with one, two or three substituents Ra, and the following groups: C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-14 Aryl or 5-14 heteroaryl groups;

[0012] R2 values ​​are the same or different, and are independently selected from H and C. 1-20 Alkyl, C 1-20 Alkoxy groups, unsubstituted or optionally substituted with one, two or three substituents Rc, and unsubstituted or optionally substituted with one, two or three substituents Rd, of the following groups: C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-14 Aryl or 5-14 heteroaryl groups;

[0013] Ra and Rc may be the same or different, and are independently selected from C. 1-20 Alkyl, C 1-20 Alkoxy, C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-14 Aryl, 5-14 heteroaryl;

[0014] Rb and Rd may be the same or different, and are independently selected from C. 1-20 Alkyl, C 1-20 Alkoxy, C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-14 Aryl, 5-14 heteroaryl, -N(C 6-14 aryl)2,-N(5-14 heteroaryl)2,-C 6-14 Aryl-N(C) 6-14 Aryl)2;

[0015] The condition is that at least one of R1 and R2 is selected from an ortho-diaminophenyl that is unsubstituted or optionally substituted by one, two or three substituents Ra or Rc;

[0016] m is 3; n is 3.

[0017] According to an embodiment of the present invention, X is selected from O, N, S, Se, or Te;

[0018] R0 is selected from C 1-16 Alkoxy;

[0019] R1 may be the same or different, and is independently selected from H, unsubstituted or optionally substituted with one, two or three substituents Ra, and the following groups: C 6-14 Aryl or 5-14 heteroaryl groups;

[0020] R2 may be the same or different, and independently selected from H, unsubstituted or optionally substituted with one, two or three substituents Rc, or unsubstituted or optionally substituted with one, two or three substituents Rd, of the following groups: C 6-14 Aryl or 5-14 heteroaryl groups;

[0021] Ra and Rc may be the same or different, and are independently selected from C. 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-14 Aryl, 5-14 heteroaryl;

[0022] Rb and Rd may be the same or different, and are independently selected from C. 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-14 Aryl, 5-14 membered heteroaryl, -N(phenyl)2, -phenyl-N(phenyl)2;

[0023] The condition is that at least one of R1 and R2 is selected from an ortho-diaminophenyl that is unsubstituted or optionally substituted by one, two or three substituents Ra or Rc;

[0024] m is 3; n is 3.

[0025] According to an embodiment of the present invention, X is selected from O, N, S, Se, or Te;

[0026] R0 is selected from C 1-16 Alkoxy;

[0027] R1 may be the same or different, and is independently selected from H, unsubstituted or optionally substituted with one, two or three substituents Ra, and the following groups: C 6-14 Aryl or 5-14 heteroaryl groups;

[0028] R2 may be the same or different, and independently selected from H, unsubstituted or optionally substituted with one, two or three substituents Rc, or unsubstituted or optionally substituted with one, two or three substituents Rd, of the following groups: C 6-14 Aryl or 5-14 heteroaryl groups;

[0029] Ra and Rc may be the same or different, and are independently selected from C. 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-14 Aryl, 5-14 heteroaryl;

[0030] Rb and Rd may be the same or different, and are independently selected from C. 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-14 Aryl, 5-14 membered heteroaryl, -N(phenyl)2, -phenyl-N(phenyl)2;

[0031] The condition is that at least one of R1 and R2 is selected from an ortho-diaminophenyl that is unsubstituted or optionally substituted by one, two or three substituents Ra or Rc;

[0032] m is 3; n is 3.

[0033] According to an embodiment of the present invention, X is selected from O, N, S, Se, or Te;

[0034] R0 is selected from C 1-16 Alkoxy;

[0035] R1 may be the same or different, and is independently selected from H, o-diaminophenyl (e.g., 2,3-diamino-1-phenyl, 3,4-diamino-1-phenyl), -phenyl-N(phenyl)2, or C-terminated by one or two Cs. 1-6 Alkyl-substituted fluorenyl, phenyl-substituted carbazole, and thiophene group substituted with -phenyl-N(phenyl)2;

[0036] R2 may be the same or different, and are independently selected from H, o-diaminophenyl (e.g., 2,3-diamino-1-phenyl, 3,4-diamino-1-phenyl), -phenyl-N(phenyl)2, or separated by one or two Cs. 1-6 Alkyl-substituted fluorenyl, phenyl-substituted carbazole, and thiophene group substituted with -phenyl-N(phenyl)2;

[0037] The condition is that at least one of R1 and R2 is selected from o-diaminophenyl;

[0038] m is 3; n is 3.

[0039] According to an embodiment of the present invention, the compound represented by formula IA is selected from the compounds represented by IA' below:

[0040]

[0041] Each group has the definition given above or below.

[0042] According to an embodiment of the present invention, R0 is OCH2CH(C6H) 13 (C8H) 13 );

[0043] R1 and R2 may be the same or different, and are independently selected from H.

[0044] One of them;

[0045] The condition is that at least one of R1 and R2 is selected from...

[0046] As an exemplary embodiment of the present invention, the compound represented by Formula IA is selected from the following compounds FNO1, FNO2 and FNO3:

[0047]

[0048] The present invention also provides a method for preparing the compound shown in Formula IA, comprising:

[0049] The compound shown in Formula I' reacts with compounds R1-L3 and / or R2-L3 to give the compound shown in Formula IA;

[0050]

[0051] L1 is selected from H, halogens, unsubstituted groups, or groups optionally substituted by one, two, or three substituents Rb, such as: C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-14 Aryl or 5-14 heteroaryl groups;

[0052] L2 is selected from H, halogens, unsubstituted groups, or optionally substituted by one, two, or three substituents Rd, as follows: C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-14 Aryl or 5-14 heteroaryl groups;

[0053] L3 and L4 are selected from

[0054] X, m, n, R0, R1, R2, Rb, and Rd are defined as described above.

[0055] As an exemplary embodiment of the present invention, the method for preparing compounds FNO1, FNO2, and FNO3 includes:

[0056] (1) Mix 2-hydroxy-3-amino-5-bromopyridine or 2-hydroxy-3-aminopyridine, 1-bromo-2-hexyldecane and potassium carbonate, add to the first solvent under the protection of an inert gas, and react at 70-100℃ for 1-24h to obtain an intermediate;

[0057] (2) The intermediate and 3,6-bis(2-thienyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione were mixed, and a second solvent was added under the protection of an inert gas. The mixture was stirred evenly and refluxed for 0.5-12 h. Then titanium tetrachloride was added and refluxed for 0.5-12 h. Then triethylamine was added and refluxed for 0.5-12 h. Finally, boron trifluoride ether was added and refluxed for 0.5-12 h to obtain compound (I) or compound (II).

[0058] (3) Compound (Ⅰ), 4-(diphenylamino)phenylboronic acid pinacol ester, potassium carbonate and tetra(triphenylphosphine)palladium are mixed and added to a third solvent under an inert gas atmosphere. The mixture is reacted at 80-110℃ for 0.5-12h to obtain compound (Ⅲ).

[0059] (4) Dissolve compound (II) or compound (III) and N-bromosuccinimide in a fourth solvent in an ice bath and react for 1-24 h to obtain compound (IV) or compound (V);

[0060] (5) Mix compound (Ⅰ) or compound (Ⅳ) or compound (Ⅴ), 3,4-diaminophenylboronic acid pinacol ester, potassium carbonate and tetra(triphenylphosphine)palladium, add to the fifth solvent under an inert gas, and react at 80-110℃ for 1-12h to obtain compounds FNO1, FNO2 and FNO3;

[0061]

[0062] The first solvent is N,N-dimethylformamide; the second solvent is anhydrous toluene; the third solvent is 1,4-dioxane and water; the fourth solvent is anhydrous dichloromethane; and the fifth solvent is 1,4-dioxane and water.

[0063] According to an embodiment of the present invention, the molar ratio of 2-hydroxy-3-amino-5-bromopyridine or 2-hydroxy-3-aminopyridine: 1-bromo-2-hexyldecane: potassium carbonate is 1:0.8-1.0:3.8-4.2.

[0064] According to an embodiment of the present invention, the molar ratio of 3,6-bis(2-thienyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione: intermediate: titanium tetrachloride: triethylamine: boron trifluoride ether is 1.0: 8.0-10.0: 4-4.5: 15-18: 8-9.

[0065] According to an embodiment of the present invention, the PPAB derivative (Ⅰ): 4-(diphenylamino)phenylboronic acid pinacol ester: potassium carbonate: tetra(triphenylphosphine)palladium = 1.0: 5.0-6.0: 40.0-45.0: 0.2-0.3 in molar ratio.

[0066] According to an embodiment of the present invention, the molar ratio of PPAB derivative (II) or PPAB derivative (III) to N-bromosuccinimide is 1.0: 2.0 to 3.0.

[0067] According to an embodiment of the present invention, the molar ratio of the PPAB derivative (Ⅰ) or PPAB derivative (Ⅳ) or PPAB derivative (Ⅴ) is: 3,4-diaminophenylboronic acid pinacol ester: potassium carbonate: tetrakis(triphenylphosphine)palladium = 1.0: 5.0-6.0: 40.0-45.0: 0.2-0.3.

[0068] The present invention also provides a method for nanoforming the compound shown in formula (IA), comprising: dissolving the compound shown in formula (IA) and an amphiphilic compound in an organic solvent, and dropping the organic phase into water under ultrasonication to obtain nanoparticles FNO NPs.

[0069] According to an embodiment of the present invention, the amphiphilic compound is selected from one of DSPE-PEG (e.g., DSPE-PEG 2000), F127, BSA, and HSA.

[0070] According to an embodiment of the present invention, the nanoforming is carried out in an ice-water bath.

[0071] According to an embodiment of the present invention, the organic solvent is dimethyl sulfoxide.

[0072] According to an embodiment of the present invention, the concentration of the solution prepared by dissolving the compound of formula (IA) in an organic solvent is 0.01-1000 μM, for example 0.1-100 μM, such as 1-10 μM, such as 1 μM.

[0073] According to an embodiment of the present invention, the molar ratio of the compound shown in formula (IA) to the amphiphilic compound is (0.1 to 5.0): 3.0, for example (1.0 to 3.0): 3.0, such as 2.0: 3.0.

[0074] According to an embodiment of the present invention, the ultrasound time is 1-50 min, for example 5-30 min, such as 15 min.

[0075] The present invention also provides FNO NPs nanoparticles prepared by the method described above.

[0076] The present invention also provides the use of compounds or nanoparticles FNO NPs as shown in formula (IA) above for qualitative or quantitative detection of nitric oxide.

[0077] According to embodiments of the present invention, the compound or nanoparticles FNO NPs are used for qualitative or quantitative detection of nitric oxide for non-disease treatment or diagnostic purposes to detect liver damage.

[0078] This invention also provides a method for the quantitative detection of nitric oxide using compounds or nanoparticles FNO NPs as shown in formula (IA) above, comprising the following steps:

[0079] Step 1: Add nitric oxide solutions of different concentrations to compounds or nanoparticles FNO NPs of the same concentration as shown in formula (IA) to prepare more than 5 standard solutions containing compounds or nanoparticles FNO NPs of the same formula (IA) with different nitric oxide contents.

[0080] The concentration of the compound or nanoparticle FNO NPs represented by formula (IA) in the standard solution is 0.1–100 μM, for example 1–10 μM, such as 10 μM;

[0081] The nitric oxide content in the standard solution ranges from 100 nM to 40 μM.

[0082] Step 2: Measure the UV absorption spectrum or fluorescence emission spectrum of the standard solution. The maximum absorption wavelengths of the compound or nanoparticles FNO NPs shown in formula (IA) before and after responding to nitric oxide are 821 nm and 754 nm, respectively, with an excitation wavelength of 680 nm. Plot the ratio of nitric oxide concentration to the concentration of the compound or nanoparticles FNO NPs shown in formula (IA) ([NO] / [probe]) on the x-axis and the ratio of Abs concentration on the y-axis. 754 / Abs 821 or I 794 Establish a standard curve with the vertical axis as the ordinate;

[0083] Abs 754 This indicates the absorbance of the standard solution at a wavelength of 754 nm; Abs 821 This indicates the absorbance of the standard solution at a wavelength of 821 nm; I 794 This indicates the fluorescence intensity value of the standard solution at 794 nm;

[0084] Step 3: Add the sample to be tested into a solution of the compound or nanoparticles FNO NPs of the standard concentration shown in formula (IA), measure its ultraviolet absorption spectrum or fluorescence emission spectrum under excitation light of 680 nm, and then calculate the content of nitric oxide in the sample according to the standard curve.

[0085] The present invention also provides the use of compounds or nanoparticles FNO NPs as shown in formula (IA) above as near-infrared fluorescent probes for nitric oxide imaging in liver injury.

[0086] According to an embodiment of the present invention, when used as a near-infrared fluorescent probe for nitric oxide imaging in liver injury, it is used for the detection of liver injury for non-disease diagnosis and treatment purposes.

[0087] The present invention also provides the use of compounds or nanoparticles FNO NPs as shown in formula (IA) above for the preparation of kits for the qualitative or quantitative detection of nitric oxide.

[0088] According to an embodiment of the present invention, the kit is used to detect nitric oxide, thereby detecting liver damage.

[0089] According to an embodiment of the present invention, when the kit is used to detect nitric oxide and thus detect liver damage, it is used for non-disease diagnosis and treatment purposes.

[0090] Beneficial effects

[0091] The near-infrared fluorescence-photoacoustic dual-mode probe based on pyrrolopyrrole fluorophores provided by this invention has a good response to nitric oxide, good selectivity, and high sensitivity, enabling qualitative and quantitative detection of nitric oxide. Furthermore, the probe's ultraviolet absorption and fluorescence emission are both in the near-infrared region, enabling photoacoustic imaging. It also has excellent tissue penetration, and the ratiometric absorption improves sensitivity and accuracy while reducing background interference.

[0092] Furthermore, the preparation method of this probe compound is simple and reproducible, and has certain application prospects.

[0093] Terminology Definitions and Explanations

[0094] Unless otherwise defined, all technical terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains.

[0095] Partial substituents The location indicates the connection point.

[0096] "More than three" means three or more.

[0097] The term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0098] Term "C" 1-20"alkyl" should be understood to represent a straight-chain or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms, preferably "C". 1-16 Alkyl or C 1-6 Alkyl group. "C" 1-6 "Alkyl" means a straight-chain or branched alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl and the like or their isomers.

[0099] Term "C" 1-20 "Alkoxy" should be understood as -OC 1-20 Alkyl groups, preferably "-OC" 1-16 "alkyl", where C 1-20 Alkyl groups have the above definition.

[0100] Term "C" 3-12 "Cycloalkyl" should be understood as representing a saturated monovalent monocyclic, bicyclic, or polycyclic hydrocarbon ring (also called a fused ring hydrocarbon ring) with 3-12 carbon atoms. Bicyclic or polycyclic cycloalkyl includes fused cycloalkyl, bridged cycloalkyl, and spirocyclic cycloalkyl; fused ring refers to a fused ring structure formed by two or more cyclic structures sharing two adjacent ring atoms (i.e., sharing a bond). Bridged ring refers to a fused ring structure formed by two or more cyclic structures sharing two non-adjacent ring atoms. Spirocyclic refers to a fused ring structure formed by two or more cyclic structures sharing a single ring atom. For example, the C 3-12 Cycloalkyl groups can be C 3-8 Monocyclic cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, or C 7- C 12 And cycloalkyl groups, such as decahydronaphthalene rings.

[0101] The term "3-12 membered heterocyclic group" refers to a saturated or partially saturated monovalent monocyclic or bicyclic hydrocarbon ring containing 1-5, preferably 1-3, heteroatoms selected from N, O, and S. The heterocyclic group can be connected to the rest of the molecule via any one of the carbon atoms or a nitrogen atom (if present). Specifically, the heterocyclic group can include, but is not limited to: 4-membered rings, such as azirmonobutyl or oxobutyl; 5-membered rings, such as tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, imidazoyl, pyrazolyl, or pyrrololinyl; or 6-membered rings, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazinyl, or trithiaalkyl; or 7-membered rings, such as diazacycloheptyl. Optionally, the heterocyclic group can be benzofused. The heterocyclic group may be bicyclic, such as, but not limited to, a 5,5-membered ring, like a hexahydrocyclopentano[c]pyrrolo-2(1H)-yl ring, or a 5,6-membered bicyclic ring, like a hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl ring. The nitrogen-containing ring may be partially unsaturated, i.e., it may contain one or more double bonds, such as, but not limited to, 2,5-dihydro-1H-pyrroloyl, 4H-[1,3,4]thiadiazinyl, 4,5-dihydrooxazolyl, or 4H-[1,4]thiazinyl, or it may be benzofused, such as, but not limited to, dihydroisoquinolinyl.

[0102] Term "C" 6-14 "Aryl" should be understood to preferably represent a monovalent aromatic or partially aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring ("C") having 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms. 6-14 Aryl), particularly a ring with 6 carbon atoms (“C6 aryl”), such as phenyl; or biphenyl, or a ring with 9 carbon atoms (“C9 aryl”), such as indenyl or indenyl, or a ring with 10 carbon atoms (“C9 aryl”). 10 Aryl groups, such as tetrahydronaphthyl, dihydronaphthyl, or naphthyl, or rings with 13 carbon atoms (“C”). 13 Aryl groups, such as fluorene groups, or rings with 14 carbon atoms (“C”). 14 Aryl), for example, anthracene. When the C 6-14 When the aryl group is substituted, it can be monosubstituted or polysubstituted. Furthermore, there are no restrictions on the substitution site; for example, it can be ortho, para, or meta substituted.

[0103] The term "5-14-membered heteroaryl" should be understood to include monovalent monocyclic, bicyclic, or tricyclic aromatic ring systems having 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ring atoms, particularly 5, 6, 9, or 10 carbon atoms, and containing 1 to 5, preferably 1 to 3, heteroatoms independently selected from N, O, and S. Furthermore, in each case, it may be benzofused. Specifically, the heteroaryl group is selected from thienyl, furanyl, pyrroleyl, oxazolyl, thiazolyl, imidazoleyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thia-4H-pyrazolyl, and their benzo[derivatives], such as benzofuranyl, benzothienyl, benzooxazolyl, benzoisooxazolyl, benzoimidazolyl, benzotriazolyl, indazole, indolyl, isindolyl, etc.; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and their benzo[derivatives], such as quinolinyl, quinazolinyl, isoquinolinyl, etc.; or acrylinyl, inazinyl, purinyl, and their benzo[derivatives]; or terpenolyl, phthalazinyl, quinazolinyl, quinoxolinyl, naphridinyl, pteridinyl, carbazolyl, acridineyl, phenazinyl, phenothiazinyl, phenothiazinyl, etc.

[0104] Similarly, C 6-14 Aryl and 5-14 heteroaryl groups have the same definition throughout the text. Attached Figure Description

[0105] Figure 1 This is a graph showing the changes in ultraviolet absorption of the near-infrared fluorescence-photoacoustic dual-mode probe of the present invention in response to nitric oxide.

[0106] Figure 2 This is a fluorescence change diagram of the near-infrared fluorescence-photoacoustic dual-mode probe of the present invention in response to nitric oxide;

[0107] Figure 3 This is a photoacoustic imaging change diagram of the near-infrared fluorescence-photoacoustic dual-mode probe of the present invention and the nitric oxide response;

[0108] Figure 4 This is a fluorescence signal diagram of the near-infrared fluorescence-photoacoustic dual-modal probe of the present invention in major mouse tissues. Detailed Implementation

[0109] To better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to the embodiments.

[0110] It should be understood that the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0111] The reaction flow charts for Examples 1-3 are shown below:

[0112]

[0113] Example 1

[0114] Preparation of the near-infrared fluorescent-photoacoustic dual-modal probe FNO1:

[0115] 3.9 g of 2-hydroxy-3-amino-5-bromopyridine (20.6 mmol), 6.3 g of 1-bromo-2-hexyldecane (20.6 mmol), and 11.4 g of potassium carbonate (82.5 mmol) were mixed, and 50 mL of N,N-dimethylformamide was added under N2 atmosphere. The mixture was reacted at 85 °C for 12 h. After the solution cooled to room temperature, the organic phases were extracted with ethyl acetate and combined. Excess water in the organic phase was removed with anhydrous sodium sulfate, and then ethyl acetate was removed by rotary evaporation to obtain the intermediate product. 50 mg of 3,6-bis(2-thienyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (0.17 mmol) was mixed with 560 mg of the intermediate (1.3 mmol). 40 mL of anhydrous toluene was added under N2 atmosphere, and the mixture was refluxed at 130 °C for 2 h. Then, 0.1 mL of titanium tetrachloride was added, and the reaction was carried out at 130 °C for 1 h. Finally, 0.35 mL of triethylamine was added, and the reaction was carried out at 130 °C for 3 h. Then, 0.35 mL of boron trifluoride diethyl ether was added, and the reaction was carried out at 130 °C for 2 h. Heating was then stopped, and the mixture was allowed to cool to room temperature. The organic phases were extracted with dichloromethane and combined. Excess water in the organic phase was removed with anhydrous sodium sulfate, and then dichloromethane was removed by rotary evaporation to obtain the crude product of compound I. The crude product was purified by column chromatography using dichloromethane / petroleum ether (v / v, 1:1) as the eluent to obtain the green solid compound I. 50 mg of compound I (0.042 mmol) was mixed with 50 mg of pinacol 3,4-diaminophenylboronic acid (0.21 mmol), 238 mg of potassium carbonate (1.68 mmol), and 9.7 mg of tetrakis(triphenylphosphine)palladium (0.0084 mmol). This mixture was then added to 5 mL of 1,4-dioxane and water (v / v, 4:1) under N2 conditions, and the reaction was carried out at 101 °C for 4 h. After cooling to room temperature, a large amount of methanol was added, and the mixture was filtered to obtain 40 mg of green solid FNO1.

[0116] 1H NMR (400MHz, deuterated chloroform) δ9.52(d,J=3.9Hz,2H),8.16(s,2H),7.73(d,J=4.9Hz,2H),7.44(d,J=1.8Hz,2H),7.31(d,J=4.9Hz,2H),7.01(d,J=6.5 Hz,4H),6.82(d,J=8.1Hz,2H),4.10(d,J=5.5Hz,4H),3.55(s,8H),2.12(p,J=6.1Hz,2H),1.49–1.06(m,48H),0.88(td,J=6.9,3.5Hz,12H).

[0117] Example 2

[0118] Preparation of near-infrared fluorescent-photoacoustic dual-modal probe FNO2:

[0119] 4.62 g of 2-hydroxy-3-aminopyridine, 12.82 g of 1-bromo-2-hexyldecane, and 22 g of potassium carbonate were mixed, and 60 mL of N,N-dimethylformamide was added under N2 atmosphere. The mixture was reacted at 85 °C for 12 h. After the solution cooled to room temperature, the organic phases were extracted with ethyl acetate and combined. Excess water in the organic phase was removed with anhydrous sodium sulfate, and then ethyl acetate was removed by rotary evaporation to obtain the intermediate product. 50 mg of 3,6-bis(2-thienyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione was mixed with the intermediate product, and 40 mL of anhydrous toluene was added under N2 atmosphere. The mixture was refluxed at 130 °C for 2 h, followed by the addition of 0.1 mL of titanium tetrachloride. The reaction was carried out at 130 °C for 1 h, then 0.35 mL of triethylamine was added, and the reaction was carried out at 130 °C for 3 h. Finally, 0.36 mL of boron trifluoride diethyl ether was added, and the reaction was carried out at 130 °C for 2 h. The heating was then turned off, and the mixture was allowed to cool to room temperature. The organic phases were extracted with dichloromethane and combined. Excess water in the organic phase was removed with anhydrous sodium sulfate, and then dichloromethane was removed by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography using dichloromethane / petroleum ether (v / v, 1:1) as the eluent to obtain the green solid compound II. 50 mg of compound II (0.048 mmol) was mixed with 19 mg of N-bromosuccinimide (0.107 mmol) in an ice bath, and 8 mL of dichloromethane was added. The mixture was reacted at room temperature for 12 h to give compound IV. 50 mg of compound IV (0.042 mmol) was mixed with 50 mg of pinacol 3,4-diaminophenylboronic acid (0.21 mmol), 238 mg of potassium carbonate (1.68 mmol), and 9.7 mg of tetrakis(triphenylphosphine)palladium (0.0084 mmol). This mixture was then added to 5 mL of 1,4-dioxane and water (v / v, 4:1) in a nitrogen atmosphere and reacted at 101 °C for 4 h. After cooling to room temperature, a large amount of methanol was added, and the mixture was filtered to give 43 mg of a dark red solid, FNO2.

[0120] 1 ¹H NMR (400MHz, deuterated dimethyl sulfoxide-d6) δ 10.01 (d, J = 4.1 Hz, 2H), 8.01 (d, J = 6.1 Hz, 2H), 7.69 (d, J = 8.1 Hz, 2H), 7.35 (d, J = 4.2 Hz, 2H), 7.29 (t, J = 7.1 Hz, 2H), 7.00–6.88 (m, 4H), 6.60 (d, J = 8.0 Hz, 2H), 5.17 (s, 4H), 4.83 (s, 4H), 4.13 (s, 4H), 2.04–1.92 (m, 2H), 1.33 (s, 10H), 1.24–1.01 (m, 38H), 0.75 (dt, J = 12.9, 6.4 Hz, 12H).

[0121] Example 3

[0122] Preparation of near-infrared fluorescent-photoacoustic dual-modal probe FNO3:

[0123] 50 mg of compound I (0.042 mmol) was mixed with 80 mg of 4-(diphenylamino)phenylboronic acid pinacol ester (0.21 mmol), 238 mg of potassium carbonate (1.68 mmol), and 9.7 mg of tetra(triphenylphosphine)palladium (0.0084 mmol). This mixture was then added to 5 mL of 1,4-dioxane and water (v / v, 4:1) under N2 atmosphere and reacted at 101 °C for 4 h. After cooling to room temperature, a large amount of methanol was added, and the mixture was filtered to obtain compound III. 50 mg of compound III (0.033 mmol) was mixed with 30 mg of N-bromosuccinimide (0.165 mmol) in an ice bath, and 8 mL of dichloromethane was added. The mixture was reacted at room temperature for 12 h to obtain compound V. 70 mg of compound V (0.042 mmol) was mixed with 50 mg of pinacol 3,4-diaminophenylboronic acid (0.21 mmol), 238 mg of potassium carbonate (1.68 mmol), and 9.7 mg of tetrakis(triphenylphosphine)palladium (0.0084 mmol). This mixture was then added to 5 mL of 1,4-dioxane and water (v / v, 4:1) under N2 conditions, and the reaction was carried out at 101 °C for 4 h. After cooling to room temperature, a large amount of methanol was added, and the mixture was filtered to obtain 49 mg of a dark red solid, FNO3.

[0124] 1¹H NMR (600 MHz, deuterated dichloromethane-d²) δ 9.93 (s, 2H), 8.16 (s, 2H), 7.57 (d, J = 8.4 Hz, 4H), 7.42 (d, J = 8.6 Hz, 12H), 7.18 (s, 8H), 7.13 (s, 2H), 7.01 (s, 10H), 6.75 (s, 2H), 4.15–4.12 (m, 4H), 3.68 (s, 4H), 3.51 (s, 4H), 2.22 (s, 2H), 1.25 (d, J = 26.1 Hz, 48H), 0.82 (s, 12H).

[0125] Example 4

[0126] Near-infrared fluorescence-photoacoustic dual-mode probe and in vitro measurement of nitric oxide response ultraviolet absorption and fluorescence emission:

[0127] The probe compound FNO1 obtained in Example 1 and the probe compound FNO2 obtained in Example 2 were weighed and dissolved in 2.9 mL of DMSO to prepare a 1 mM probe stock solution.

[0128] Different concentration gradients were set for the probe FNO1 or FNO2 (10 μM) and the NO donor (DEA NONOate) within the range of 0–30 μM or 0–40 μM. Specific concentrations are as follows: Figure 1 and 2 (As shown) After being stored at 37℃ for 2 minutes, the ultraviolet absorption spectrum was obtained using a UV-Vis spectrophotometer and the fluorescence spectrum was obtained using a fluorescence spectrophotometer (excitation wavelength 680 nm). The obtained data were processed using Origin software. Figure 1 and 2 .Depend on Figure 1 It can be seen that in the ultraviolet spectrum of the reaction between FNO1 solution and nitric oxide, the absorption peaks change proportionally; that is, after the addition of the nitric oxide donor, the absorption peak at 821 nm gradually decreases, while the absorption peak at 754 nm gradually increases. For example... Figure 2 As shown, the fluorescence value at 794 nm gradually increased with the addition of NO, indicating that the probe had a good response to nitric oxide in solution.

[0129] Example 5

[0130] Near-infrared fluorescence-photoacoustic dual-modal probe and in vitro photoacoustic signal measurement experiment of nitric oxide response:

[0131] Dissolve 30 mg DSPE-PEG 2000 in 1 mL of FNO2 mother liquor, add the organic phase dropwise to 10 mL of water under sonication, and concentrate by centrifugation to obtain FNO2 nanoparticles NPs.

[0132] The probe FNO2 NPs (10 μM) and NO donor (DEA NONOate, 0–40 μM) were stored at 37 °C for 10 minutes, and photoacoustic spectra were obtained using a photoacoustic imager. The obtained data were then processed using Origin software. Figure 3 In the photoacoustic spectrum of the reaction between FNO2 NPs solution and nitric oxide, the photoacoustic signal changes proportionally. That is, after the addition of the nitric oxide donor, the photoacoustic signal at 782 nm gradually decreases, while the photoacoustic signal at 745 nm gradually increases. The ratio of the photoacoustic signal at 745 nm to that at 782 nm (PA) is used to represent the change in photoacoustic signal intensity. 745 / PA 782 Plotting the nitric oxide concentration to probe concentration ratio ([NO] / [probe]) on the ordinate and the probe nanoparticles in solution on the abscissa shows that the probe nanoparticles have a good response to nitric oxide.

[0133] Example 6

[0134] Near-infrared fluorescence-photoacoustic dual-modal probe for in vivo fluorescence detection in mice:

[0135] The FNO2 NPs prepared in Example 5 were prepared into a 100 μM solution and injected into mice via the tail vein at a dose of 5 mg / kg (the mice were liver injury model mice, established by dissolving 30 mg of acetaminophen in 2 mL of water and heating to 60°C to ensure complete dissolution; then injecting 400 mg / kg intraperitoneally into the mice for 6 h). Two h after injection, the mice were sacrificed, the organs were removed, and images were acquired using a small animal in vivo imaging system. Figure 4 As can be seen, compared with the blank control group, the probe only showed fluorescence in the liver region, indicating that the probe can respond to liver injury in mice.

[0136] The technical solutions provided in this application have been described in detail above. The descriptions of the embodiments above are only for the purpose of helping to understand the methods and core ideas of this application; at the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. The compound represented by formula IA: in, X is selected from O, N, S, Se, or Te; R0 is selected from C 1-20 Alkyl, C 1-20 Alkoxy, C 3-12 Cycloalkyl or 3-12 membered heterocyclic groups; R1 may be the same or different, and is independently selected from H, unsubstituted or optionally substituted with one, two or three substituents Ra, and the following groups: C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-14 Aryl or 5-14 heteroaryl groups; R2 values ​​are the same or different, and are independently selected from H and C. 1-20 Alkyl, C 1-20 Alkoxy groups, unsubstituted or optionally substituted with one, two or three substituents Rc, and unsubstituted or optionally substituted with one, two or three substituents Rd, of the following groups: C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-14 Aryl or 5-14 heteroaryl groups; Ra and Rc may be the same or different, and are independently selected from C. 1-20 Alkyl, C 1-20 Alkoxy, C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-14 Aryl, 5-14 heteroaryl; Rb and Rd may be the same or different, and are independently selected from C. 1-20 Alkyl, C 1-20 Alkoxy, C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-14 Aryl, 5-14 heteroaryl, -N(C 6-14 aryl)2,-N(5-14 heteroaryl)2,-C 6-14 Aryl-N(C) 6-14 Aryl)2; The condition is that at least one of R1 and R2 is selected from an ortho-diaminophenyl that is unsubstituted or optionally substituted by one, two or three substituents Ra or Rc; m is 3; n is 3.

2. The compound according to claim 1, wherein, X is selected from O, N, S, Se, or Te; R0 is selected from C 1-16 Alkoxy; R1 may be the same or different, and is independently selected from H, unsubstituted or optionally substituted with one, two or three substituents Ra, and the following groups: C 6-14 Aryl or 5-14 heteroaryl groups; R2 may be the same or different, and independently selected from H, unsubstituted or optionally substituted with one, two or three substituents Rc, or unsubstituted or optionally substituted with one, two or three substituents Rd, of the following groups: C 6-14 Aryl or 5-14 heteroaryl groups; Ra and Rc may be the same or different, and are independently selected from C. 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-14 Aryl, 5-14 heteroaryl; Rb and Rd may be the same or different, and are independently selected from C. 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-14 Aryl, 5-14 membered heteroaryl, -N(phenyl)2, -phenyl-N(phenyl)2; The condition is that at least one of R1 and R2 is selected from an ortho-diaminophenyl that is unsubstituted or optionally substituted by one, two or three substituents Ra or Rc; m is 3; n is 3; Preferably, X is selected from O, N, S, Se, or Te; R0 is selected from C 1-16 Alkoxy; R1 may be the same or different, and is independently selected from H, unsubstituted or optionally substituted with one, two or three substituents Ra, and the following groups: C 6-14 Aryl or 5-14 heteroaryl groups; R2 may be the same or different, and independently selected from H, unsubstituted or optionally substituted with one, two or three substituents Rc, or unsubstituted or optionally substituted with one, two or three substituents Rd, of the following groups: C 6-14 Aryl or 5-14 heteroaryl groups; Ra and Rc may be the same or different, and are independently selected from C. 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-14 Aryl, 5-14 heteroaryl; Rb and Rd may be the same or different, and are independently selected from C. 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-14 Aryl, 5-14 membered heteroaryl, -N(phenyl)2, -phenyl-N(phenyl)2; The condition is that at least one of R1 and R2 is selected from an ortho-diaminophenyl that is unsubstituted or optionally substituted by one, two or three substituents Ra or Rc; m is 3; n is 3.

3. The compound according to claim 1 or 2, wherein, X is selected from O, N, S, Se, or Te; R0 is selected from C 1-16 Alkoxy; R1 may be the same or different, and is independently selected from H, o-diaminophenyl (e.g., 2,3-diamino-1-phenyl, 3,4-diamino-1-phenyl), -phenyl-N(phenyl)2, or C-terminated by one or two Cs. 1-6 Alkyl-substituted fluorenyl, phenyl-substituted carbazole, and thiophene group substituted with -phenyl-N(phenyl)2; R2 may be the same or different, and are independently selected from H, o-diaminophenyl (e.g., 2,3-diamino-1-phenyl, 3,4-diamino-1-phenyl), -phenyl-N(phenyl)2, or separated by one or two Cs. 1-6 Alkyl-substituted fluorenyl, phenyl-substituted carbazole, and thiophene group substituted with -phenyl-N(phenyl)2; The condition is that at least one of R1 and R2 is selected from o-diaminophenyl; m is 3; n is 3; Preferably, R0 is OCH2CH(C6H) 13 (C8H) 13 ); R1 and R2 may be the same or different, and are independently selected from H. One of them; The condition is that at least one of R1 and R2 is selected from... Preferably, the compound represented by formula IA is selected from the following compounds FNO1, FNO2 and FNO3:

4. A method for preparing the compound according to any one of claims 1-3, wherein, include: The compound shown in Formula I' reacts with compounds R1-L3 and / or R2-L3 to give the compound shown in Formula IA; L1 is selected from H, halogens, unsubstituted groups, or groups optionally substituted by one, two, or three substituents Rb, such as: C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-14 Aryl or 5-14 heteroaryl groups; L2 is selected from H, halogens, unsubstituted groups, or optionally substituted by one, two, or three substituents Rd, as follows: C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-14 Aryl or 5-14 heteroaryl groups; L3 and L4 are selected from X, m, n, R0, R1, R2, Rb, and Rd have the definitions described in any one of claims 1-3.

5. A method for nanoforming the compound according to any one of claims 1-3, wherein, include: The compound described in any one of claims 1-3 and the amphiphilic compound were dissolved in an organic solvent, and the organic phase was dropped into water under ultrasonication to obtain FNO nanoparticles NPs; Preferably, the amphiphilic compound is selected from one of DSPE-PEG, F127, BSA, and HSA.

6. The nanoparticles FNO NPs prepared by the method of claim 5.

7. Use of the compound according to any one of claims 1-3 or the nanoparticles FNO NPs according to claim 6 for qualitative or quantitative detection of nitric oxide; Preferably, the method for qualitative or quantitative detection of nitric oxide is used to detect liver damage for non-disease treatment or diagnostic purposes. A method for quantitative detection of nitric oxide using the compound of any one of claims 1-3 or the nanoparticles FNO NPs of claim 6, wherein... Includes the following steps: Step 1: Add nitric oxide solutions of different concentrations to compounds or nanoparticles FNO NPs of formula (IA) of the same concentration to prepare more than 5 standard solutions containing compounds or nanoparticles FNO NPs of formula (IA) with different nitric oxide contents. The concentration of the compound or nanoparticle FNO NPs represented by formula (IA) in the standard solution is 0.1–100 μM, for example 1–10 μM, such as 10 μM; The nitric oxide content in the standard solution ranges from 100 nM to 40 μM. Step 2: Measure the UV absorption spectrum or fluorescence emission spectrum of the standard solution. The maximum absorption wavelengths of the compound or nanoparticles FNO NPs shown in formula (IA) before and after responding to nitric oxide are 821 nm and 754 nm, respectively, with an excitation wavelength of 680 nm. Plot the ratio of nitric oxide concentration to the concentration of the compound or nanoparticles FNO NPs shown in formula (IA) ([NO] / [probe]) on the x-axis and the ratio of Abs concentration on the y-axis. 754 / Abs 821 or I 794 Establish a standard curve with the vertical axis as the ordinate; Abs 754 This indicates the absorbance of the standard solution at a wavelength of 754 nm; Abs 821 This indicates the absorbance of the standard solution at a wavelength of 821 nm; I 794 This indicates the fluorescence intensity value of the standard solution at 794 nm; Step 3: Add the sample to be tested into a solution of the compound or nanoparticles FNO NPs of the standard concentration shown in formula (IA), measure its ultraviolet absorption spectrum or fluorescence emission spectrum under excitation light of 680 nm, and then calculate the content of nitric oxide in the sample according to the standard curve.

8. Use of the compound according to any one of claims 1-3 or the nanoparticles FNO NPs according to claim 6 as a near-infrared fluorescent probe for nitric oxide imaging in liver injury; Preferably, when used as a near-infrared fluorescent probe for nitric oxide imaging in liver injury, it is used for detecting liver injury for non-disease diagnosis and treatment purposes.

9. Use of the compound according to any one of claims 1-3 or the nanoparticles FNO NPs according to claim 6 for the preparation of a kit for the qualitative or quantitative detection of nitric oxide; Preferably, the kit is used to detect nitric oxide, thereby detecting liver damage.

10. A kit for detecting nitric oxide to detect liver injury, comprising: The compound according to any one of claims 1-3 or the nanoparticles FNO NPs according to claim 6.

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