Nitrogen-containing heterocyclic compound as well as preparation method, fluorescent probe and application thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a nitrogen-containing heterocyclic compound, a preparation method thereof, a fluorescent probe and application. The nitrogen-containing heterocyclic compound provided by the invention has a structure as shown in the following general formula (I): # imgabs0 #, is simple in structure, has good chemical and optical stability, can be used for imaging fungi such as cryptococcus neoformans, has application potential for judging fungal infection conditions, and in addition, has good application prospects. The compound can be combined with intein of fungi through sulfydryl reaction, shows good specificity as a targeted fluorescent probe, and realizes rapid evaluation and screening of Prp8 intein inhibitor candidate compounds.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a nitrogen-containing heterocyclic compound, a preparation method thereof, and a fluorescent probe. Background Art

[0002] Inteins are a class of polypeptides that can self-splice without the need for cofactors or external energy. Their primary function is to link extein fragments. Some pathogens, such as Cryptococcus neoformans and Aspergillus fumigatus, contain inteins within the RNA spliceosome Prp8. Given their absence in human cells, inteins have become attractive targets for the development of antifungal drugs and fungal detection methods. Prp8 is a core protein of the RNA spliceosome, a large ribonucleoprotein complex that removes introns during pre-mRNA splicing and plays a crucial role in the splicing process. Prp8 forms the catalytic core of the spliceosome by cross-linking to the 5′ splice site, branch point, and 3′ splice site of pre-mRNA and binding to U5 and U6 snRNAs. Prp8 is an essential gene in all organisms, from fungi to humans. Small molecule compounds inhibiting the Prp8 intein of Cryptococcus neoformans lead to fungal cell death. Therefore, studying the characteristics of Prp8 intein will not only help in the detection and clinical diagnosis of fungal pathogens such as Cryptococcus, but also provide an important tool for the development of antifungal drugs.

[0003] Diseases caused by fungal infections have brought a significant burden to the global healthcare system. Many fungal pathogens, such as Cryptococcus neoformans, Aspergillus fumigatus, Candida albicans, and Candida auris, are listed by the World Health Organization as fungi that are of vital or high importance to human health. Among them, Cryptococcus neoformans (C. neoformans) is an opportunistic infectious fungus distributed throughout the world and widely present in the natural environment. Cryptococcus neoformans usually enters the human lungs through the respiratory tract in the form of spores, causing lung, brain, and even systemic infections, and has an extremely high mortality rate for patients with impaired immune system function. Cryptococcus neoformans infection is highly hidden in the early stages, and the clinical manifestations are complex and non-specific. Due to its unique "Trojan horse infection method", infections with this type of fungus are extremely prone to misdiagnosis and missed diagnosis. Therefore, the development of new methods for rapid, efficient, and specific detection of Cryptococcus provides effective information for studying the pathogenic mechanism of Cryptococcus, drug development, and early diagnosis of the disease, and has very important research and application value. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide a nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof, which can be used as a fluorescent probe molecule targeting intein, and used in the evaluation and screening of antifungal drugs.

[0005] The technical solutions of the present invention are as follows:

[0006] A nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof, having the structure shown in the following general formula (I):

[0007]

[0008] A is selected from one of the following groups:

[0009] wherein R1 is independently selected from hydrogen, amino, alkyl-substituted amino, and alkoxy; n represents the number of olefinic bonds, and n is any integer from 1 to 3; p represents the number of R1 substituents on the benzene ring, and p is any integer from 0 to 6;

[0010] L is selected from one of the following groups:

[0011] Wherein, m represents the number of alkylene groups, and m is any integer from 1 to 3;

[0012] Y is selected from hydrogen or halogen;

[0013] X is selected from one of the following groups:

[0014]

[0015] Indicates the attachment site.

[0016] The present invention also provides a method for preparing the above-mentioned nitrogen-containing heterocyclic compound, comprising the following steps:

[0017] Prepare an intermediate compound B1 by aminolysis of a dicarboxylic anhydride compound; carry out a condensation reaction of the intermediate compound B1 with a carboxylic acid compound; or

[0018] 4-methylquinoline is subjected to a nucleophilic substitution reaction and a catalytic hydrogenation reaction to prepare an intermediate compound B2; the intermediate compound B2 and a carboxylic acid compound are subjected to a condensation reaction to prepare an intermediate compound D; the compound D and compound K are subjected to a Knoevenagel condensation reaction;

[0019] The structural formula of the intermediate compound B1 includes

[0020] The structural formula of the intermediate compound B2 includes

[0021] The structural formula of the intermediate compound D includes

[0022] The structural formula of the compound K includes

[0023] Wherein, A, L, Y, X, m, n, p and R1 are defined the same as above.

[0024] The present invention also provides a fluorescent probe, comprising the nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof, or the nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof prepared according to the above preparation method.

[0025] The present invention also provides the use of the fluorescent probe in detecting fungi.

[0026] The present invention also provides a method for evaluating and screening antifungal drugs, comprising the following steps:

[0027] preparing an experimental group comprising the antifungal drug to be evaluated and screened, the fungus, and the fluorescent probe;

[0028] preparing a control group comprising the fungus and the fluorescent probe;

[0029] Prepare a blank group: including a buffer solution and the above-mentioned fluorescent probe;

[0030] The fluorescence intensities of the experimental group, control group and blank group were tested respectively, and the average fluorescence intensities of the experimental group, control group and blank group were used as the basis for drug screening.

[0031] The beneficial effects of the present invention are: the nitrogen-containing heterocyclic compound of the present invention has a simple structure, the compound has good chemical and optical stability, can image fungi such as Cryptococcus neoformans, and has application potential for judging fungal infection. In addition, the compound can bind to the intein of the fungus through a thiol reaction and exhibit good specificity as a targeted fluorescent probe.

[0032] Other advantages, objects, and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art upon examination of the following description or may be learned from practice of the present invention. The objects and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below with reference to the accompanying drawings, in which:

[0034] Figure 1: Responses of probes H1 and H2 to changes in solvent polarity, wherein Figure A-1 is the UV absorption spectrum of probe H1 (10 μM) in solvents of different polarities, Figure A-2 is the fluorescence emission spectrum of probe H1 (10 μM) in solvents of different polarities, Figure B-1 is the UV absorption spectrum of probe H2 (10 μM) in solvents of different polarities, and Figure B-2 is the fluorescence emission spectrum of probe H2 (10 μM) in solvents of different polarities.

[0035] Figure 2 : Responses of probes H3 and H4 to changes in solvent polarity, wherein Figure A-1 is the UV absorption spectrum of probe H3 (10 μM) in solvents of different polarities, Figure A-2 is the fluorescence emission spectrum of probe H3 (10 μM) in solvents of different polarities, Figure B-1 is the UV absorption spectrum of probe H4 (10 μM) in solvents of different polarities, and Figure B-2 is the fluorescence emission spectrum of probe H4 (10 μM) in solvents of different polarities.

[0036] Figure 3 : Response of probe H5 to changes in solvent polarity, wherein Figure A is the UV absorption spectrum of probe H5 (10 μM) in solvents of different polarities, and Figure B is the fluorescence emission spectrum of probe H5 (10 μM) in solvents of different polarities.

[0037] Figure 4 : Responses of probes V1 and V2 to changes in solvent polarity, wherein Figure A-1 is the UV absorption spectrum of probe V1 (10 μM) in solvents of different polarities, Figure A-2 is the fluorescence emission spectrum of probe V1 (10 μM) in solvents of different polarities, Figure B-1 is the UV absorption spectrum of probe V2 (10 μM) in solvents of different polarities, and Figure B-2 is the fluorescence emission spectrum of probe V2 (10 μM) in solvents of different polarities.

[0038] Figure 5 : Different fluorescent probes (40 μM) and Cryptococcus neoformans H99 (1.0×10 7 CFU / mL) after incubation.

[0039] Figure 6 : Fluorescence response curves of probe H2 in PBS buffer solution and different concentrations of Cryptococcus neoformans H99.

[0040] Figure 7 :V series probes (10 μM) were used to detect the activity of Cryptococcus neoformans H99 (1.0×10 7 CFU / mL) fluorescence response curve.

[0041] Figure 8 : Fluorescence imaging of probe V1 in Cryptococcus neoformans.

[0042] Figure 9: Fluorescence response graph and fluorescence intensity histogram of probe H2 after pretreatment of Cryptococcus neoformans with different concentrations of inhibitor 6G-318S, where Figure 9 A represents the fluorescence response curve of probe H2, Figure 9 B shows the fluorescence intensity histogram of probe H2.

[0043] Figure 10 : Fluorescence changes of probe H2 after pretreatment of Cryptococcus neoformans with two inhibitors of different MIC values at different concentrations. In the figure, I is the fluorescence intensity of the experimental group, I d is the fluorescence intensity of the probe in PBS solution, and I0 is the fluorescence intensity of the probe without drug pretreatment.

[0044] Figure 11 : Fluorescence imaging of probe H2 in untreated Cryptococcus neoformans and in C. neoformans treated with inhibitors with different MIC values. DETAILED DESCRIPTION

[0045] The following describes the embodiments of the present invention by means of specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0046] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual drawings, and are not to be construed as limiting the present invention. To better illustrate the embodiments of the present invention, some parts of the accompanying drawings may be omitted, enlarged, or reduced, and do not represent the dimensions of the actual product. For those skilled in the art, it is understandable that some well-known structures and their descriptions may be omitted in the accompanying drawings. The terms describing positional relationships (such as "upper," "lower," "left," "right," "front," "back," etc.) in the accompanying drawings of the embodiments of the present invention are for illustrative purposes only and are not to be construed as limiting the present invention. Those skilled in the art may understand the specific meanings of the above terms based on the specific circumstances.

[0047] In the present invention, “further”, “further”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of the present invention.

[0048] In the present invention, "A and B are independently selected from x, y or z" means that A and B are independent events, and event A does not affect the occurrence of event B. Therefore, when A is selected from x, B can be selected from any one of x, y or z; when A is selected from y, B can be selected from any one of x, y or z; when A is selected from z, B can be selected from any one of x, y or z.

[0049] In the present invention, "alkyl" can refer to a linear, branched, and / or cyclic alkyl group. The number of carbon atoms in the alkyl group can be 1 to 50, 1 to 30, 1 to 20, 1 to 10, or 1 to 6. Phrases containing this term, for example, "C1-C9 alkyl" refer to alkyl groups containing 1 to 9 carbon atoms, and each occurrence can independently be C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, or C9 alkyl. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, 2-ethylbutyl, 3,3-dimethylbutyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, cyclopentyl, 1-methylpentyl, 3-methylpentyl, 2-ethylpentyl, 4-methyl-2-pentyl, n-octyl, n-hexyl, 1-methylhexyl, 2-ethylhexyl, 2-butylhexyl, cyclohexyl, 1,1-dimethylbutyl, adamantane, and the like.

[0050] The term "alkoxy" refers to a group having an -O-alkyl group, i.e., an alkyl group as defined above connected to a parent core structure via an oxygen atom. Suitable examples of phrases containing this term include, but are not limited to, methoxy (-O-CH or -OMe), ethoxy (-O-CHCH or -OEt), and tert-butoxy (-OC(CH) or -OtBu).

[0051] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0052] In the present invention, the single bond connecting the substituent runs through the corresponding ring, indicating that the substituent can be connected to any position of the ring, for example R can be connected to any substitutable position of the benzene ring.

[0053] The present invention provides a nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof, having a structure represented by the following general formula (I):

[0054]

[0055] A is selected from one of the following groups: wherein R1 is independently selected from hydrogen, amino, alkyl-substituted amino, and alkoxy; n represents the number of olefinic bonds, and n is any integer from 1 to 3; p represents the number of R1 substituents on the benzene ring, and p is any integer from 0 to 6;

[0056] L is selected from one of the following groups: Wherein, m represents the number of alkylene groups, and m is any integer from 1 to 3;

[0057] Y is selected from hydrogen or halogen;

[0058] X is selected from one of the following groups: Indicates the attachment site.

[0059] The nitrogen-containing heterocyclic compound of the present invention has a simple structure and good chemical and optical stability. It can image fungi such as Cryptococcus neoformans and has application potential for judging fungal infection. In addition, the compound can bind to the fungal intein through a thiol reaction, exhibiting good specificity as a targeted fluorescent probe, and realizes rapid evaluation and screening of candidate Prp8 intein inhibitor compounds.

[0060] In some examples, A is selected from one of the following groups: wherein R1 is independently selected from hydrogen, amino, dimethylamino, and p is 0 or 1.

[0061] In some examples, L is selected from one of the following groups:

[0062] In some examples, Y is selected from hydrogen or fluorine.

[0063] In some of these examples, X is selected from

[0064] In some specific examples, the compound has any of the following structures:

[0065]

[0066]

[0067] The present invention also provides a method for preparing the above compound, comprising the following steps:

[0068] Prepare intermediate compound B1 by aminolysis reaction of dicarboxylic anhydride; carry out condensation reaction of the intermediate compound B1 and a carboxylic acid compound; or

[0069] 4-methylquinoline is subjected to a nucleophilic substitution reaction and a catalytic hydrogenation reaction to prepare an intermediate compound B2; the intermediate compound B2 and a carboxylic acid compound are subjected to a condensation reaction to prepare an intermediate compound D; the compound D and compound K are subjected to a Knoevenagel condensation reaction;

[0070] The structural formula of the intermediate compound B1 includes

[0071] The structural formula of the intermediate compound B2 includes

[0072] The structural formula of the intermediate compound D includes

[0073] The structural formula of the compound K includes

[0074] It can be understood that the above groups A, L, Y, and X have the same definitions as those in the general formula (I).

[0075] In some specific examples, a dicarboxylic anhydride compound and an amino compound are subjected to an aminolysis reaction to prepare an intermediate compound B1; and the intermediate compound B1 and a carboxylic acid compound are subjected to a condensation reaction to prepare compound H.

[0076] In some specific examples, the structural formula of the diformic anhydride compound includes The structural formula of the amino compound includes

[0077] In some specific examples, the method may further include a step of preparing a diformic anhydride compound by condensing diformic acid.

[0078] In some specific examples of preparing compound H, when L is selected from When the carboxylic acid compound is C1; the structural formula of C1 is More specifically, the C1 is prepared by a known method, for example, when X is selected from C1-1 was prepared according to the method disclosed in document CN118878531A. The structural formula of C1-1 is When X is selected from C1-2 was prepared according to the method disclosed in document CN118878531A. The structural formula of C1-2 is When X is selected from When, it can be prepared according to the method disclosed in document CN118878531A.

[0079] As an example of preparing compound H, when the diformic anhydride compound is L is When, the synthetic route 1 is as follows:

[0080]

[0081] As a more specific example, the synthetic route 1-1 for preparing compound H1 is shown below:

[0082]

[0083] As another more specific example, the synthetic route 1-2 for preparing compound H2 is shown below:

[0084]

[0085] As another more specific example, the synthetic route 1-3 for preparing compound H5 is shown below:

[0086]

[0087] In other specific examples of preparing compound H, when L is selected from When the carboxylic acid compound is C2; the structural formula of C2 is

[0088] In some more specific examples, the preparation of the carboxylic acid compound C2 includes the following steps: condensing an aniline carboxylic acid compound F and a carboxylic acid compound C1; the structural formula of the aniline carboxylic acid compound F is The synthetic route 2 for preparing compound C2 is as follows:

[0089]

[0090] As another example of preparing compound H, when the diformic anhydride compound is L is selected from When, synthetic route 3 is as follows:

[0091]

[0092] As a more specific example, the synthetic route 3-1 for preparing compound H3 is shown below:

[0093]

[0094]

[0095] As another more specific example, the synthetic route 3-2 for preparing compound H4 is shown below:

[0096]

[0097] In some examples, 4-methylquinoline is subjected to a nucleophilic substitution reaction and a catalytic hydrogenation reaction to prepare an intermediate compound B2; the intermediate compound B2 and a carboxylic acid compound are subjected to a condensation reaction to prepare an intermediate compound D; and the compound D and compound K are subjected to a Knoevenagel condensation reaction.

[0098] In some specific examples, 4-methylquinoline and a halogenated nitro compound G are reacted through a nucleophilic substitution reaction to prepare an intermediate compound I; the intermediate compound I is subjected to a catalytic hydrogenation reaction to prepare an intermediate compound B2;

[0099] The structural formula of the halogenated nitro compound G is: Z is selected from halogen;

[0100] The structural formula of the intermediate compound I is:

[0101] The synthetic route 4 for preparing the intermediate compound B2 is as follows:

[0102]

[0103] Furthermore, the intermediate compound B2 and the carboxylic acid compound C1 are subjected to a condensation reaction to prepare the intermediate compound D. Synthesis route 5 is shown below:

[0104]

[0105] Furthermore, based on the synthetic route 5, the compound D and the compound K are subjected to a Knoevenagel condensation reaction to prepare the compound V; the structural formula of the compound K includes The synthetic route 6 for preparing compound V is shown below:

[0106]

[0107] As a specific example, the compound K is (E)-3-(4-dimethylamino)phenylpropenal, and the structural formula is

[0108] The synthetic route 6-1 for preparing compound V1 is shown below:

[0109]

[0110]

[0111] As another more specific example, the compound K is (E)-3-(4-dimethylamino)phenyl acrolein, and the synthetic route 6-2 for preparing compound V2 is as follows:

[0112]

[0113] The present invention also provides a fluorescent probe, comprising the nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof of the nitrogen-containing heterocyclic compound prepared according to the above preparation method.

[0114] The present invention also provides the use of the fluorescent probe in detecting fungi.

[0115] In some specific examples, the fungus includes Cryptococcus.

[0116] In some embodiments, the fungus has an intein, and the intein includes a Prp8 intein.

[0117] The fluorescent probes provided by the present invention exhibit weak or no fluorescence in highly polar solvents or buffer solutions. However, these fluorescent probes bind to intein-bearing fungi (such as Cryptococcus), enhancing their fluorescence. Based on this principle, the fluorescent probes of the present invention have promising application prospects in detecting intein-bearing fungi.

[0118] In some specific examples, detecting fungi using the fluorescent probes includes the following steps:

[0119] The mixed solution of the fluorescent probe and the fungus to be detected was co-incubated as the experimental group; the buffer solution containing the fluorescent probe was used as the control group; and the fluorescence intensity of the experimental group and the control group was tested respectively.

[0120] The present invention also provides a method for evaluating and screening antifungal drugs, comprising the following steps:

[0121] preparing an experimental group comprising the antifungal drug to be evaluated and screened, the fungus, and the fluorescent probe;

[0122] preparing a control group comprising the fungus and the fluorescent probe;

[0123] Prepare a blank group: including a buffer solution and the above-mentioned fluorescent probe;

[0124] The fluorescence intensities of the experimental group, control group and blank group were tested respectively, and the average fluorescence intensities of the experimental group, control group and blank group were used as the basis for drug screening.

[0125] In some specific examples, the fluorescence intensity ratio (I) is calculated based on the average fluorescence intensity of the experimental group, the control group and the blank group. d ) / (I0-I d ). Comparison of the fluorescence intensity ratio is used as a basis for drug screening. More specifically, the fluorescence intensity ratio (II d ) / (I0-I d ) is smaller, indicating that the average fluorescence intensity of the experimental group is significantly reduced, and the antifungal drug to be evaluated and screened is a strong inhibitor drug, otherwise, it is a weak inhibitor drug. Wherein, I value is the fluorescence intensity of the experimental group, I dis the fluorescence intensity of the probe in buffer solution, and I0 is the fluorescence intensity of the probe without drug pretreatment.

[0126] In some specific examples, the fungus includes Cryptococcus.

[0127] In some embodiments, the fungus has an intein, and the intein includes a Prp8 intein.

[0128] In some specific examples, the buffer solution includes a PBS buffer solution.

[0129] The fluorescent probe of the present invention is a small molecule fluorescent probe, which can more easily penetrate biological membranes and enter cells, enabling detection and imaging of intracellular biomolecules. It has the advantages of short detection time, low cost, high sensitivity, and good specificity. The present invention specifically provides a fluorescent probe targeting the Prp8 intein. This type of probe can bind to the Prp8 intein and can quickly detect and image fungi containing inteins (such as Cryptococcus). It can also evaluate and screen intein inhibitors through optical signals, achieving rapid evaluation of the inhibitory activity of compounds against inteins in a short time. This provides a sensitive and efficient tool for the development of new fungal target inhibitors.

[0130] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention. Alternatively, the methods may be based on the experimental manuals or conventional conditions in the art, the conditions recommended by the manufacturer, or experimental methods known in the art. For reagents or instruments where the manufacturer is not specified, all are conventional products that can be purchased commercially.

[0131] EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, analytical grade;

[0132] HBTU: O-Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, analytical grade;

[0133] HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, analytical grade;

[0134] DIPEA: N,N-diisopropylethylamine;

[0135] Compound C1-1 and compound C1-2 can both be prepared according to the method disclosed in document CN118878531A.

[0136] Example 1 Preparation of Compound H1

[0137] (1) Preparation of Compound 1

[0138] 4-Aminophthalic acid (500 mg, 2.76 mmol) and 30 mL of methanol were placed in a 100 mm round-bottom flask and stirred at room temperature. 15 mL of formaldehyde solution and 200 mg of Pd / C were then added and reacted at room temperature under hydrogen for 6 h. After completion of the reaction, the reaction solution was filtered using a diatomaceous earth-lined funnel, and the filter cake was washed with methanol. The filtrate was then concentrated to give 500 mg of a yellow solid in an 87% yield.

[0139]

[0140] (2) Preparation of Compound 2

[0141] At room temperature, compound 1 (1.0 g, 4.78 mmol) and 10 mL of acetic anhydride were placed in a 50 mL round-bottom flask and stirred for 5 minutes. The oil bath was then heated to 140°C and reacted under argon for 3 hours. After the reaction, the reaction solution was naturally cooled to room temperature. During this process, yellow crystals precipitated. The filter cake was filtered and washed with acetic anhydride to obtain 680 mg of a yellow solid, with a yield of 74%.

[0142]

[0143] (3) Preparation of Compound B1-1

[0144] In a 50 mL round-bottom flask, compound 2 (1.0 g, 5.23 mmol) and 4-aminobenzylamine dihydrochloride (767 mg, 6.28 mmol) were dissolved in 15 mL of DMF. After stirring at room temperature for 5 minutes, the mixture was heated to 140°C and reacted under argon for 3 hours. After completion of the reaction, the DMF was spin-dried to obtain the crude product, which was further purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:1, v / v) to obtain 600 mg of a yellow solid, with a yield of 42%.

[0145] 1 H NMR(400MHz,DMSO-d6)δ7.60(d,J=8.4Hz,1H),7.02(d,J=2.0Hz,1H),6.95(s,1H),6 .94(d,J=3.6Hz,2H),6.47(d,J=8.4Hz,2H),5.01(s,2H),4.49(s,2H),3.08(s,6H).

[0146]

[0147] (4) Preparation of Compound H1

[0148] Compound B1-1 (400 mg, 1.35 mmol), compound C1-1 (400 mg, 1.76 mmol), EDC (400 mg, 1.35 mmol), and HBTU (140 mg, 0.7 mmol) were dissolved in 8 mL of anhydrous dichloromethane and stirred for 5 min. 1.4 mL of DIPEA solution was then slowly added dropwise to the reaction mixture. The reaction was allowed to proceed at 40°C under argon protection. As the reaction proceeded, a yellow solid gradually formed. The reaction was continued for 12 h. After the reaction was completed, the mixture was filtered, and the filter cake was washed with ethyl acetate. The filter cake was collected to obtain the crude product. The crude product was hot filtered using ethyl acetate to obtain 210 mg of a yellow solid with yellow fluorescence, with a yield of 27%.

[0149] Test data of compound H1: 1 H NMR (400MHz, DMSO-d6) δ11.13(s,1H),8.13(d,J=8.4Hz,2H),7.76(d,J=8.4Hz,2H),7.65-7.62(m,3H) ,7.31(d,J=8.4Hz,2H),7.06(d,J=2.4Hz,1H),6.95(dd,J=8.4,2.4Hz,1H),4.71(s,2H),3.10(s,6H); 13 C NMR(100MHz,DMSO-d6)δ168.6,168.1,158.2,156.5,154.8,141.1,137.7,134.7,134.1,131.2 ,130.3,128.4,128.0,126.7,125.1,121.1,116.7,115.4,105.9,40.5; ESI-HRMS(m / z):[M+H] + calcd.forC 26 H 20 ClN5O5S2,582.0667; found 582.0673.

[0150]

[0151] Example 2 Preparation of Compound H2

[0152] The steps for preparing compounds 1 and 2 were the same as those in Example 1.

[0153] (1) Preparation of Compound B1-2

[0154] In a 50 mL round-bottom flask, compound 2 (1.0 g, 5.23 mmol) and 2-(4-aminophenyl)ethylamine (800 mg, 5.20 mmol) were dissolved in 15 mL of DMF. After stirring at room temperature for 5 minutes, the mixture was heated to 110°C under argon and allowed to react for 3 hours. After completion of the reaction, the DMF was spin-dried to obtain the crude product, which was purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:1, v / v) to yield 622 mg of a yellow solid with yellow fluorescence, a yield of 40%.

[0155] 1 H NMR (400MHz, DMSO-d6) δ7.57(d,J=8.4Hz,1H),6.99(d,J=2.0Hz,1H),6.89(dd,J=8.4,2.0Hz,2H),6.81(d,J =8.4Hz,2H),6.44(d,J=8.4Hz,2H),4.87(s,2H),3.64(t,J=7.2Hz,2H),3.07(s,6H),2.69(t,J=7.2Hz,2H).

[0156]

[0157] (2) Preparation of Compound H2

[0158] Compound B1-2 (400 mg, 1.3 mmol), compound C1-1 (400 mg, 1.7 mmol), EDC (500 mg, 2.6 mmol), HBTU (160 mg, 0.7 mmol), and 8 mL of dichloromethane were placed in a 50 mL round-bottom flask and stirred for 5 minutes. Then, 1.4 mL of DIPEA solution was added. The reaction was allowed to proceed at 40°C. As the reaction proceeded, a yellow solid gradually formed, and the reaction was complete after 12 hours. The mixture was filtered, and the filter cake was washed with ethyl acetate. The crude product was collected and hot filtered with ethyl acetate to obtain 300 mg of a yellow solid with yellow fluorescence, for a yield of 39%.

[0159] Test data of compound H2: 1 H NMR (400MHz, DMSO-d6) δ11.00(s,1H),8.14(d,J=8.4Hz,2H),7.76(d,J=8.8Hz,2H),7.59(d,J=8.4Hz,3H),7.21(d,J=8. 4Hz,2H),7.00(d,J=2.0Hz,1H),6.90(dd,J=8.4,2.4Hz,1H),3.78(t,J=7.2Hz,2H),3.07(s,6H),2.90(t,J=6.8Hz,2H); 13C NMR (100MHz, DMSO-d6) δ168.1,167.7,157.8,155.8,154.5,154.2,140.6,137.2,136.1,134.9,134. 1,130.7,129.9,129.1,124.4,120.2,116.3,114.8,105.3,40.0,38.4,33.4;ESI-HRMS(m / z):[M+H] + calcd.for C 27 H 22 ClN5O5S2,596.0824; found 596.0832.

[0160]

[0161] Example 3 Preparation of Compound H3

[0162] The steps for preparing compounds 1 and 2 were the same as those in Example 1.

[0163] (1) Preparation of Compound B1-3

[0164] In a 50 mL round-bottom flask, compound 2 (320 mg, 1.67 mmol) was dissolved in 10 mL of DMF solution and stirred at room temperature for 5 minutes. Ethanolamine (120 mg, 2.0 mmol) was then added to the reaction flask. The oil bath was heated to 140°C and the reaction was carried out under argon for 4 hours. After the reaction, DMF was removed by distillation under reduced pressure. A large amount of pure water was added to the reaction solution, and the mixture was extracted three times with dichloromethane. The organic phase was washed with saturated sodium chloride solution and finally evaporated to dryness. The organic phase was further purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:1, v / v) to obtain 260 mg of a yellow fluorescent solid with a yield of 66%.

[0165] Test data of compound B1-3: 1 HNMR(400MHz,DMSO-d6)δ7.59(d,J=8.8Hz,1H),7.00(d,J=2.4Hz,1H),6.91( dd,J=8.4,2.4Hz,1H),4.83(t,J=5.6Hz,1H),3.56-3.53(m,4H),3.07(s,6H).

[0166]

[0167] (2) Preparation of Compound C2

[0168] In a 150 mL round-bottom flask, 2-fluoro-5-amino-phenylacetic acid (300 mg, 1.78 mmol), compound C1-1 (540 mg, 2.39 mmol), EDC (400 mg, 1.3 mmol), and HBTU (140 mg, 0.7 mmol) were dissolved in 8 mL of dichloromethane. After stirring for 5 minutes, 1.4 mL of DIPEA was added and the mixture was allowed to react at room temperature for 12 hours. After completion of the reaction, the organic solvent was rotary evaporated to obtain the crude product, which was purified by column chromatography (eluent: dichloromethane:methanol = 15:1, v / v) to afford 310 mg of a white solid, with a yield of 39%.

[0169] 1 H NMR (400MHz, CDCl3) δ9.23 (s, 1H), 8.21 (d, J = 8.8Hz, 2H), 7.67 (dd, J = 6.0, 2.4Hz, 1H), 7.58-7.56 (m, 3H), 7.11 (t, J = 8.8Hz, 1H), 3.77 (s, 2H).

[0170]

[0171] (3) Preparation of Compound H3

[0172] Compound B1-3 (120 mg, 263 μmol), compound C2 (70 mg, 263 μmol), EDC (100 mg, 220 μmol), HBTU (20 mg, 100 μmol), and 10 mL of dichloromethane were placed in a 50 mL round-bottom flask and stirred at room temperature for 5 minutes. 0.2 mL of DIPEA was then added to the reaction solution, and the reaction was allowed to proceed at room temperature for 4 hours. After the reaction, the organic phase was removed to obtain a crude product, which was dissolved in dichloromethane. The organic phase was washed with saturated sodium chloride solution, concentrated, and purified by column chromatography (eluent: petroleum ether:ethyl acetate = 10:1, v / v) to obtain 60 mg of a yellow solid, with a yield of 36%.

[0173] 1 H NMR (400MHz, DMSO-d6) δ11.08(s,1H),8.15(d,J=8.4Hz,2H),7.77(d,J=8.8Hz,2H),7.62-7.59(m,3H),7.15(t,J=9.6Hz,1H) ,7.01(d,J=2.0Hz,1H),6.89(dd,J=8.8,2.4Hz,1H),4.29(t,J=5.2Hz,2H),3.79(t,J=5.6Hz,2H),3.68(s,2H),3.05(s,6H); 13C NMR(100MHz,DMSO-d6)δ170.4,168.6,168.1,159.0,158.4,156.6,156.4,154.8,141.1,137.7,134.6,134.3,131.2,1 30.3,124.9,124.1,122.2,122.1,121.6,116.9,115.9,115.8,115.3,105.7,62.3,36.8,34.4; ESI-HRMS(m / z):[M+H] + calcd.for C 29 H 23 ClFN5O7S2,672.0784; found 672.0790.

[0174]

[0175] Example 4 Preparation of Compound H4

[0176] The steps for preparing compounds 1 and 2 were the same as those in Example 1.

[0177] The steps for preparing compound C2 are the same as those in Example 3.

[0178] (1) Preparation of Compound B1-4

[0179] Compound 2 (500 mg, 2.26 mmol) was dissolved in 10 mL of DMF solution in a 50 mL round-bottom flask and stirred at room temperature for 5 minutes. 3-Amino-1-propanol (500 mg, 2.26 mmol) was then added to the reaction flask. The oil bath was heated to 140°C and reacted under argon for 4 hours. After the reaction, the DMF solution was removed from the reaction solution by spin drying. A large amount of pure water was added, and the solution was extracted three times with dichloromethane. The organic phase was washed with saturated sodium chloride solution, concentrated, and purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:1, v / v) to obtain 259 mg of a yellow solid with yellow fluorescence, with a yield of 40%.

[0180] 1 H NMR (400MHz, DMSO-d6) δ7.59(d,J=8.4Hz,1H),7.07(d,J=2.0Hz,1H),6.91(dd,J=8.8,2.4Hz,1H),4 .50(t,J=4.8Hz,1H),3.57(t,J=7.2Hz,2H),3.43(t,J=6.0Hz,2H),3.08(s,6H),1.74-1.67(m,2H).

[0181]

[0182] (2) Preparation of Compound H4

[0183] Compound B1-4 (100 mg, 220 μmol), compound C2 (80 mg, 350 μmol), EDC (100 mg, 220 μmol), HBTU (20 mg, 100 μmol), and 10 mL of dichloromethane were placed in a 50 mL round-bottom flask and stirred at room temperature for 5 minutes. 0.2 mL of DIPEA was then added and the mixture was allowed to react at room temperature for 4 hours. After the reaction, the organic solvent was removed by rotary evaporation, and the crude product was dissolved in dichloromethane. The organic phase was washed with saturated sodium chloride solution, dried, and purified by column chromatography (eluent: petroleum ether:ethyl acetate = 10:1, v / v) to obtain 70 mg of a yellow solid in a 43% yield.

[0184] 1 H NMR (400MHz, DMSO-d6) δ11.13(s,1H),8.15(d,J=8.8Hz,2H),7.76(d,J=8.4Hz,2H),7.68(dd,J=6.0,1.6Hz,1H),7.65-7.59(m,2H),7.24(t,J=8.8Hz ,1H),7.02(d,J=1.6Hz,1H),6.91(dd,J=8.8,2.0Hz,1H),4.10(t,J=6.0Hz ,2H),3.68(s,2H),3.62(t,J=6.8Hz,2H),3.07(s,6H),1.94–1.91(m,2H); 13 C NMR(100MHz,DMSO-d6)δ170.4,168.8,168.4,158.4,156.5,154.8,141.1,137.7,134.7,134.3,131.2,130.3,124.9, 124.2,122.5,122.3,121.8,117.0,116.0,115.8,115.3,105.7,63.1,40.4,34.8,34.4,27.7; ESI-HRMS(m / z):[M+H] + calcd.for C 30 H 25 ClFN5O7S2,686.0941; found 686.0948.

[0185]

[0186] Example 5 Preparation of Compound H5

[0187] The steps for preparing compounds 1 and 2 were the same as those in Example 1.

[0188] The steps for preparing compound B1-2 are the same as those in Example 2.

[0189] Compound B1-2 (600 mg, 1.9 mmol), compound C1-2 (550 mg, 1.9 mmol), EDC (600 mg, 3.0 mmol), HBTU (200 mg, 0.9 mmol), and 15 mL of dichloromethane were placed in a 50 mL round-bottom flask and stirred for 5 minutes. 2.0 mL of DIPEA solution was then added. The reaction was allowed to proceed at room temperature. As the reaction proceeded, a yellow solid gradually formed. The reaction was completed after 8 hours. The mixture was filtered and the filter cake was washed with ethyl acetate. The filter cake was collected to obtain a crude product, which was then hot-filtered with ethyl acetate to obtain 510 mg of a yellow solid with yellow fluorescence, with a yield of 46%.

[0190] 1 H NMR (400MHz, DMSO-d6) δ11.09(s,1H),7.94(d,J=8.4Hz,2H),7.69(d,J=8.4Hz,2H),7.63(d,J=8.4Hz,2H),7.55(d,J=8.8Hz ,2H),7.17(d,J=8.4Hz,2H),6.90(s,1H),6.86(d,J=8.4Hz,1H),3.75(t,J=6.8Hz,2H),3.05(s,6H),2.87(t,J=6.8Hz,2H); 13 CNMR(100MHz,DMSO-d6)δ174.3,168.6,168.2,156.9,154.7,152.5,143.7,137.6,136 .4,134.5,130.2,129.4,128.1,124.9,121.4,116.7,115.3,105.6,40.5,38.9,33.8.

[0191]

[0192] Example 6 Preparation of Compound V1

[0193] (1) Preparation of Compound I-1

[0194] Lepidin (1.0 g, 1 mmol) and 4-nitrophenylethyl bromide (1.61 g, 1 mmol) were weighed and placed in a 100 mL round-bottom flask. 15 mL of anhydrous acetonitrile was added. The mixture was refluxed at 97°C under inert gas for 24 hours. After completion of the reaction, the mixture was filtered and washed with ethyl acetate to obtain 1.8 g of an off-white solid in an 88% yield.

[0195] 1H NMR (400MHz, DMSO-d6) δ9.34(d,J=6.0Hz,1H),8.73(d,J=8.8Hz,1H),8.57(d,J=8.4Hz,1H),8.31-8.26(m,1H),8.16( d,J=8.4Hz,2H),8.10-8.02(m,2H),7.59(d,J=8.4Hz,2H),5.37(t,J=7.2Hz,2H),3.48(t,J=7.2Hz,2H),3.01(s,3H).

[0196]

[0197] (2) Preparation of Compound B2-1

[0198] Compound I-1 (1.0 g, 1 mmol), Pd / C (381 mg, 5% palladium on carbon), and 13 mL of ethyl acetate were placed in a 100 mL round-bottom flask and reacted under hydrogen at room temperature for 18 hours. After the reaction, the reaction solution was filtered through a diatomaceous earth-lined funnel. The filter cake was washed with methanol, and the filtrate was concentrated to obtain 780 mg of a dark green solid in an 86% yield.

[0199] 1 H NMR (400MHz, DMSO-d6) δ9.08(d,J=6.0Hz,1H),8.65(d,J=9.2Hz,1H),8.54(d,J=8.4Hz,1H),8.29-8.26(m,1H),8.06(t,J=7.6Hz,1H),7. 96(d,J=6.0Hz,1H),6.74(d,J=8.4Hz,2H),6.43(d,J=8.4Hz,2H),5.18(t,J=7.2Hz,2H),4.99(s,2H),3.10(t,J=7.2Hz,2H),2.99(s,3H).

[0200]

[0201] (3) Preparation of Compound D1

[0202] Compound C1-1 (35 mg, 1.0 mmol) and HATU (112 mg, 2.0 mmol) were weighed and placed in a 25 mL round-bottom flask. 3 mL of anhydrous dichloromethane was added. Under inert gas, the mixture was stirred at room temperature for 30 minutes. DIPEA (40 μL, 2.0 mmol) and compound B2-1 (30 mg, 1.0 mmol) were then added, and the mixture was stirred at room temperature for 4 hours. After the reaction was complete, the crude product was concentrated and subjected to column chromatography (eluent: dichloromethane:methanol = 40:1, v / v) to afford 34 mg of a pink solid, a 54% yield.

[0203] 1 H NMR (400MHz, DMSO-d6) δ11.08(s,1H),9.15(d,J=6.0Hz,1H),8.69(d,J=8.8Hz,1 H),8.56(d,J=8.4Hz,1H),8.3(t,J=7.6Hz,1H),8.15(d,J=8.8Hz,2H),8.08(t,J= 9.6Hz,1H),7.99(d,J=6.0Hz,1H),7.78(d,J=8.8Hz,2H),7.62(d,J=8.0Hz,2H), 7.21(d,J=8.0Hz,2H),5.28(t,J=6.8Hz,2H),3.32(t,J=6.8Hz,2H),3.01(s,3H).

[0204]

[0205] (4) Preparation of Compound V1

[0206] Compound (E)-3-(4-dimethylamino)phenylpropenal (50 mg, 1.0 mmol) and compound D1 (156 mg, 1.0 mmol) were weighed and placed in a 25 mL round-bottom flask. 5 mL of anhydrous N,N-dimethylformamide was added, followed by 2 drops of methanesulfonic acid. The mixture was refluxed at 150°C under inert gas for 3 h. After completion of the reaction, the bulk of the DMF was removed by rotary evaporation, and the product was extracted with dichloromethane. The product was washed with deionized water, dried over anhydrous NaSO₄, and filtered and concentrated to obtain the crude product. Further column chromatography (eluent: dichloromethane:methanol = 80:1, V / V) afforded 18 mg of a purple-black solid (10% yield).

[0207] 1 H NMR (400MHz, DMSO-d6) δ11.09(s,1H),8.89(d,J=6.4Hz,1H),8.77(d,J=8.4Hz,1H),8.56(d,J=8 .4Hz,1H),8.25-8.21(m,2H),8.15(d,J=8.8Hz,2H),8.09-8.0(m,3H),7.77(d,J=8.8Hz,2H),7.7 1(d,J=15.2Hz,1H),7.63(d,J=8.0Hz,2H),7.49(d,J=8.4Hz,2H),7.23(d,J=8.4Hz,2H),7.15(t, J=15.2Hz,1H),6.77(d,J=8.8Hz,2H),5.15(t,J=5.6Hz,2H),3.28(t,J=5.6Hz,2H),3.02(s,6H); 13C NMR(100MHz,DMSO-d6)δ158.4,156.4,154.8,153.1,151.9,146.8,146.7,144.2,141.1,138.3,137.7,136.2,135.4,133.5,131 .3,131.2,130.3,130.0,129.9,129.3,126.4,124.3,123.9,120.8,120.3,119.6,114.7,112.6,57.4,34.9; ESI-HRMS(m / z):[M] + calcd.for C 38 H 33 ClN5O3S2 + ,706.1708;found,706.1704.

[0208]

[0209] Example 7 Preparation of Compound V2

[0210] (1) The steps for preparing compound I-1 were the same as those in Example 6.

[0211] (2) The steps for preparing compound B2-1 are the same as those in Example 6.

[0212] (3) Preparation of Compound D2

[0213] Compound C1-2 (100 mg, 1.0 mmol) and HATU (342 mg, 2.0 mmol) were weighed and placed in a 25 mL round-bottom flask. 5 mL of anhydrous dichloromethane was added. Under inert gas, the mixture was stirred at room temperature for 30 minutes. DIPEA (120 μL, 2.0 mmol) and compound B2-1 (91 mg, 1 mmol) were then added, and the mixture was stirred at room temperature for 4 hours. After the reaction was complete, the crude product was concentrated and subjected to column chromatography (eluent: dichloromethane:methanol = 80:1, v / v) to afford 205 mg of a pink oil (81% yield).

[0214] 1H NMR (400MHz, CDCl3) δ9.22 (s, 1H), 8.64 (d, J = 6.0Hz, 1H), 8.39 (t, J = 8.0Hz, 2H), 8.25 (t, J = 8.4Hz, 1H), 8.02-7.98 (m, 3H), 7.67 (d, J = 6. 0Hz, 1H), 7.58 (d, J = 8.0Hz, 2H), 7.49 (d, J = 8.4Hz, 2H), 7.08 (d, J = 8.0Hz, 1H), 5.2 (t, J = 6.4Hz, 2H), 3.39 (t, J = 6.4Hz, 2H), 2.06 (s, 3H).

[0215]

[0216] (4) Preparation of Compound V2

[0217] Compound (E)-3-(4-dimethylamino)phenylpropenal (50 mg, 1.0 mmol) and compound D2 (152 mg, 1.0 mmol) were weighed and placed in a 25 mL round-bottom flask. 5 mL of anhydrous N,N-dimethylformamide was added, followed by 2 drops of methanesulfonic acid. The mixture was refluxed at 150°C under inert gas for 3 h. After completion of the reaction, the excess DMF was removed by rotary evaporation, and the product was extracted with dichloromethane. The product was washed with deionized water, dried over anhydrous NaSO₄, and filtered and concentrated to yield the crude product. Further column chromatography (eluent: dichloromethane:methanol = 80:1, v / v) afforded 10 mg of a purple-black solid in a 14% yield.

[0218] 1 H NMR (400MHz, DMSO-d6) δ10.94(s,1H),8.91(d,J=6.4Hz,1H),8.77(d,J=8.8Hz,1H),8.57(d,J=8 .8Hz,1H),8.22(d,J=6.4Hz,2H),8.08-7.95(m,3H),7.89(d,J=8.4Hz,2H),7.80(d,J=8.4Hz,2H) ,7.76-7.73(m,1H),7.68(d,J=8.4Hz,2H),7.49(d,J=8.8Hz,2H),7.21(d,J=8.4Hz,2H),7.16(t, J=14.4Hz,1H),6.77(d,J=8.8Hz,2H),5.15(t,J=6.8Hz,2H),3.27(t,J=6.8Hz,2H),3.02(s,6H); 13C NMR (100MHz, DMSO-d6) δ166.6,158.7,153.1,151.9,148.2,146.8(d,J=68Hz),146.6,144.2,138.3,137.6,136.9,136.2,135.4,1 32.8,132.3,131.4,129.9,129.8,129.3,126.4,124.4,123.9,121.1,120.3,119.6,114.8,112.6,57.4,35.0; ESI-HRMS(m / z):[M] + calcd.for C 38 H 33 ClN5O2S2 + ,690.1759;found 690.1691.

[0219]

[0220] Test Example 1

[0221] Test Example 1 used the above compound as a fluorescent probe targeting intein peptides to test the response to changes in the polarity of the probe solvent. The specific steps are as follows:

[0222] Fluorescent probe stock solution preparation: Analytical grade dimethyl sulfoxide (DMSO) was used as the solvent for the preparation of fluorescent probe stock solutions. First, compounds H1, H2, H3, H4, H5, V1, and V2 were dissolved in DMSO to obtain a 1 mM fluorescent probe stock solution. The solution was then stored in a -4°C refrigerator in the dark.

[0223] The UV absorption spectrum and fluorescence emission spectrum of the detection probe in different polar solvents, including PBS buffer, dimethyl sulfoxide (DMSO), methanol (MeOH), ethyl acetate (EA), and dichloromethane (DCM). First, 3960 μL of each solvent was taken and placed in a centrifuge tube. Then, 40 μL of the probe mother solution was added to each centrifuge tube, and the final concentration of the fluorescent probe was 10 μM. Subsequently, the UV absorption spectrum and fluorescence emission spectrum were measured using a 10 mm quartz four-way cuvette at room temperature.

[0224] Figure 1 These are the response results of probes H1 and H2 to changes in solvent polarity. Figure A-1 is the UV absorption spectrum of probe H1 (10 μM) in solvents of different polarities, Figure A-2 is the fluorescence emission spectrum of probe H1 (10 μM) in solvents of different polarities, Figure B-1 is the UV absorption spectrum of probe H2 (10 μM) in solvents of different polarities, and Figure B-2 is the fluorescence emission spectrum of probe H2 (10 μM) in solvents of different polarities. Figure 2 The following are the responses of probes H3 and H4 to changes in solvent polarity. Figure A-1 is the UV absorption spectrum of probe H3 (10 μM) in solvents of different polarities, Figure A-2 is the fluorescence emission spectrum of probe H3 (10 μM) in solvents of different polarities, Figure B-1 is the UV absorption spectrum of probe H4 (10 μM) in solvents of different polarities, and Figure B-2 is the fluorescence emission spectrum of probe H4 (10 μM) in solvents of different polarities. Figure 1 and Figure 2 It can be seen that probes H1, H2, H3, and H4 have similar ultraviolet absorption characteristics, with relatively obvious absorption peaks at wavelengths of around 325nm and 400nm. In non-polar solvents, the ultraviolet absorption peaks of these probes are more pronounced, while in PBS buffer, the maximum absorption peak is slightly red-shifted. In subsequent fluorescence emission spectroscopy tests, 400nm was selected as the excitation wavelength. In terms of fluorescence emission spectra, the fluorescence intensity of the probes decreased significantly with increasing solvent polarity, and the maximum emission wavelength also underwent a significant red-shift. The fluorescence emission peaks of probes H1 and H2 in dichloromethane solution and PBS buffer were 500nm and 550nm, respectively, red-shifted by approximately 50nm. Among them, the fluorescence intensities of probes H1 and H2 at the maximum emission wavelength in the non-polar solvent dichloromethane were approximately 63 times and 76 times that of the fluorescence intensity in PBS buffer solution. The fluorescence intensities of probes H3 and H4 at the maximum emission wavelength in the non-polar solvent dichloromethane were approximately 78 times and 90 times that of the maximum emission wavelength in PBS buffer solution, respectively.

[0225] Figure 3 Figure 1 is the UV absorption spectrum and fluorescence emission spectrum of probe H5 (10 μM) in solutions of different polarities, where Figure A is the UV absorption spectrum of probe H5 (10 μM) in solvents of different polarities, and Figure B is the fluorescence emission spectrum of probe H5 (10 μM) in solvents of different polarities. Figure 3 As can be seen, the UV absorption spectrum of probe H5 is similar to that of the aforementioned probes, primarily exhibiting characteristic absorption peaks of the fluorophore, with distinct absorption peaks at wavelengths of 325 nm and 400 nm. In PBS buffer, the maximum absorption peak exhibits a slight red shift. Furthermore, with increasing solvent polarity, the fluorescence intensity of probe H5 decreases significantly, while the maximum emission wavelength also undergoes a significant red shift, with the maximum red shift being approximately 50 nm.

[0226] Figure 4Figure 1 is the response result of probe V1 and V2 to the change of solvent polarity. Figure A-1 is the UV absorption spectrum of probe V1 (10 μM) in solvents of different polarities. Figure A-2 is the fluorescence emission spectrum of probe V1 (10 μM) in solvents of different polarities. Figure B-1 is the UV absorption spectrum of probe V2 (10 μM) in solvents of different polarities. Figure B-2 is the fluorescence emission spectrum of probe V2 (10 μM) in solvents of different polarities. Figure 4 Probes V1 and V2 exhibit a maximum absorption peak around 550 nm. However, in dichloromethane, this peak exhibits a slight red-shift of approximately 80 nm. Fluorescence emission spectra show a significant increase in fluorescence intensity with decreasing solvent polarity, with a maximum emission peak around 725 nm. These results demonstrate the probes' sensitivity to solvent polarity.

[0227] Test Example 2

[0228] Test Example 2 is to use the above compound as a targeted intein fluorescent probe to detect Cryptococcus, and the specific steps are as follows:

[0229] (1) Activation and expansion of strains: The frozen Cryptococcus neoformans H99 was inoculated into YPD solid medium and air-dried under sterile conditions, and then inverted and cultured at 30°C for 48 hours. When experiments were required, a small amount of activated colonies was gently taken and inoculated into 4 mL YPD liquid medium and cultured at 30°C and 220 r / min for 16 hours. In order to avoid the interference of YPD medium on fluorescence detection, the fungal cells were transferred to a 5 mL centrifuge tube, centrifuged for 3 minutes (4000 r / min) to remove the medium, and washed 3 times with PBS buffer.

[0230] (2) Strain counting: First, add 980 μL of PBS buffer solution and 20 μL of the bacterial solution of the strain to be tested to a 2 mL centrifuge tube to achieve a 50-fold dilution of the strain, and then shake the mixture to fully mix it for subsequent use. Before counting bacteria, use alcohol to thoroughly clean the blood cell counting plate, then select one end of the counting plate and cover it with a coverslip to seal the counting chamber. Next, slowly pipette 200 μL of the diluted bacterial solution and add it to the counting chamber from the sample loading port, ensuring that the bacterial solution evenly fills the entire counting chamber. After the bacterial solution naturally settles, select a 20x objective lens and place the blood cell counting plate on the microscope stage for observation and counting. Count the number of strains in the five large squares of the upper left, lower left, upper right, lower right and center, and count the edge strains according to the principle of "record the upper part but not the lower part, and count the left but not the right". After completing the counting, calculate the concentration of the original solution of the strain according to the corresponding formula.

[0231] (3) Detection of Cryptococcus: Three tubes were set up in parallel for the experimental group and the control group. Experimental group: 900 μL of fungal solution was taken from the fungal mother solution and added to three 2 mL centrifuge tubes, and then 100 μL of the prepared fluorescent probe storage solution was added to make the probe reach the specified concentration. The final concentration of the strain to be tested was 1.0×10 5 CFU / mL, 5.0×10 5 CFU / mL, 1.0×10 6 CFU / mL, 5.0×10 6 CFU / mL, 1.0×10 7 CFU / mL, 5.0×10 7 CFU / mL; 900 μL of sterile buffer solution and 100 μL of probe stock solution were added to the three centrifuge tubes of the control group. All centrifuge tubes were incubated on a shaker for the specified time, and then their fluorescence intensity was measured using a fluorescence spectrophotometer. The appropriate ultraviolet absorption wavelength of the fluorescent probe was used as the excitation wavelength, and its fluorescence emission peak was collected. The final concentration of the H series probe was 40 μM, and the excitation and emission wavelengths were 400 nm and 510 nm, respectively. The final concentration of the V series probe was 10 μM, and the excitation and emission wavelengths were 520 nm and 620 nm, respectively.

[0232] Figure 5 Different fluorescent probes (40 μM) were used to detect the interaction between Cryptococcus neoformans H99 (1.0×10 7 CFU / mL) after incubation. Figure 5 The dotted line represents the autofluorescence of different fluorescent probes (40 μM) in PBS buffer, and the solid line represents the fluorescence of different fluorescent probes (40 μM) after incubation with Cryptococcus neoformans H99 (1.0×107 CFU / mL). Specifically, “H5+C.neoformans H99” represents the fluorescence intensity of the bacterial solution after co-incubation of probe H5 with Cryptococcus neoformans, “H2+C.neoformans H99” represents the fluorescence intensity of the bacterial solution after co-incubation of probe H2 with Cryptococcus neoformans, “H1+C.neoformans H99” represents the fluorescence intensity of the bacterial solution after co-incubation of probe H1 with Cryptococcus neoformans, “H3+C.neoformans H99” represents the fluorescence intensity of the bacterial solution after co-incubation of probe H3 with Cryptococcus neoformans, and “H4+C.neoformans H99” represents the fluorescence intensity of the bacterial solution after co-incubation of probe H3 with Cryptococcus neoformans. H99" represents the fluorescence intensity of the bacterial solution after the probe H4 was co-incubated with Cryptococcus neoformans; H5, H1, H2, H3, and H4 represent the fluorescence intensity of the probes H5, H1, H2, H3, and H4 in PBS buffer solution, respectively. Figure 5 As shown in the figure, in PBS buffer solution, the five probes showed no obvious fluorescence changes. 7After incubation with 500 μg / mL (100 μg / mL), all five probes exhibited enhanced fluorescence emission, with a slight blue shift in their maximum fluorescence emission peaks. These results indicate that upon binding to the intein protein, the probes enter the enzyme cavity, placing the fluorophore in a low-polarity microenvironment and generating an enhanced fluorescence signal.

[0233] Figure 6 The fluorescence response curves of probe H2 in PBS buffer solution and different concentrations of Cryptococcus neoformans. The concentrations of Cryptococcus neoformans were 1.0×10 5 CFU / mL, 5.0×10 5 CFU / mL, 1.0×10 6 CFU / mL, 5.0×10 6 CFU / mL, 1.0×10 7 CFU / mL, 5.0×10 7 CFU / mL. Figure 6 As shown, probe H2 showed good detection effect on Cryptococcus neoformans, and the fluorescence intensity increased with the increase of Cryptococcus neoformans concentration, thereby enabling accurate quantitative analysis of the pathogen, which further verified that the probe had good detection effect on Cryptococcus neoformans of different concentrations and high sensitivity.

[0234] Figure 7 Figure 5 represents the fluorescence response curves of the V series probes to Cryptococcus neoformans. V2+C. neoformans H99 (solid line) represents the fluorescence response curve of probe V2 to Cryptococcus neoformans, V1+C. neoformans H99 (dashed line) represents the fluorescence response curve of probe V1 to Cryptococcus neoformans, and C. neoformans H99 (dotted line) represents the fluorescence intensity of Cryptococcus neoformans itself. Figure 8 This figure shows the fluorescence imaging of Cryptococcus neoformans by the V1 probe. Figure 7 and Figure 8 It also demonstrated that the V series probes had a good detection effect on Cryptococcus neoformans, indicating that the above probes all had a rapid and sensitive response to Cryptococcus neoformans.

[0235] Test Example 3

[0236] Test Example 3 involves an evaluation and screening method for antifungal drugs based on intein fluorescent probes. Taking the V1 probe as an example, the specific steps are as follows:

[0237] (1) Prepare the drug library and probe stock solution

[0238] The drug to be screened and the V1 probe are prepared in DMSO solvent to the desired concentration. For each drug screening, a control group and a screening group are set up for comparison with the positive drug group.

[0239] (2) Screening process

[0240] The drug is pre-incubated with cryptococcus under conditions sufficient for binding to the intein, and then incubated with the fluorescent probe under conditions sufficient for binding to the intein; a blank group and a control group are set up at the same time.

[0241] Experimental Group: 900 μL of Cryptococcus bacterial suspension and various concentrations of the drug stock solution to be screened were added to a centrifuge tube and incubated for 30 minutes. After incubation, 100 μL of the fluorescent probe solution was added to the tube to achieve the specified probe concentration, and the incubation continued for 90 minutes.

[0242] Control group: 900 μL of Cryptococcus bacterial suspension and different volumes of DMSO were added to a centrifuge tube and incubated for 30 minutes. After the incubation, 100 μL of fluorescent probe solution was added to the control tube to achieve the specified probe concentration and incubated for another 90 minutes.

[0243] For blank control groups, 900 μL of buffer solution and various volumes of DMSO were added to a centrifuge tube and incubated for 30 minutes. After the incubation period, 100 μL of fluorescent probe solution was added to the blank control tube to achieve the specified probe concentration, and the tubes were incubated for another 90 minutes.

[0244] (3) Data Collection

[0245] Spectral data of the co-incubated product is collected using a spectrometer. Spectral data used in the present invention encompasses the optical signals generated by the probe and the analyte. This includes, but is not limited to, microscopy, fluorescence microscopy, UV / Vis spectrometry, and flow cytometry, using a "microplate reader" to collect optical signals, such as fluorescence, absorbance, and luminescence, from samples contained in a microplate.

[0246] Fluorescence signal reading using a fluorescence spectrophotometer: Add the test solutions from the experimental, control, and blank groups to microcuvettes and collect the fluorescence signal using a fluorescence spectrophotometer. The excitation and emission wavelengths for the H-series probes are 400 nm and 510 nm, respectively. The excitation and emission wavelengths for the V-series probes are 520 nm and 620 nm, respectively.

[0247] Fluorescence imaging: Before imaging, the test liquid was centrifuged at 4000 rpm for 3 minutes and the supernatant was removed. The stained fungal cells were dispersed in PBS buffer solution. 5 μL of bacterial solution was added to a glass slide using a micropipette and covered with a coverslip. The slide was then inverted and allowed to stand for 15 minutes before fluorescence imaging. Figure 8 As shown, Cryptococcus neoformans H99 emits strong fluorescence.

[0248] Fluorescence signal reading using a microplate: Cryptococcal cells were seeded in a 96-well plate, incubated with the drug for 30 minutes, and then with the probe for 90 minutes. After incubation, fluorescence data was collected using a microplate reader. The excitation and emission wavelengths for the H-series probes were 400 nm and 510 nm, respectively. The excitation and emission wavelengths for the V-series probes were 520 nm and 620 nm, respectively.

[0249] (4) Data processing.

[0250] The fluorescence intensity ratio (I d ) / (I0-I d The comparison of fluorescence intensity ratio is used as the basis for drug screening. d ) / (I0-I d ) is smaller, indicating that the average fluorescence intensity of the experimental group is significantly reduced, and the antifungal drug to be evaluated and screened is a strong inhibitor drug, otherwise, it is a weak inhibitor drug. Wherein, I value is the fluorescence intensity of the experimental group, I d is the fluorescence intensity of the probe in PBS solution, and I0 is the fluorescence intensity of the probe without drug pretreatment.

[0251] Taking the highly inhibitory drug 6G-318S as an example, different concentrations of the drug were selected and the above steps were followed to conduct a screening experiment for cryptococcal inhibitors. Figure 9 The fluorescence response graph and fluorescence intensity histogram of probe H2 after pretreatment of Cryptococcus neoformans with different concentrations of inhibitor 6G-318S. The concentrations of inhibitor 6G-318S are 0μM, 2μM, 4μM, 8μM, 16μM, and 32μM. Figure 9 As shown in the figure, as the concentration of the inhibitor 6G-318S decreases, the fluorescence intensity gradually increases. When 32μM 6G-318S is used to pre-treat Cryptococcus neoformans H99, the fluorescence intensity only increases by 2 times; however, when 2μM 6G-318S is used to pre-treat Cryptococcus neoformans H99, the fluorescence intensity increases by about 10 times. This result further verifies the significant competitive relationship between 6G-318S and probe H2. The correlation between fluorescence intensity and 6G-318S concentration can be used to judge the inhibitory activity of drugs.

[0252] Figure 10 Figure 2 shows fluorescence changes of probe H2 after pretreatment of Cryptococcus neoformans with the strong inhibitor 6G-318S and the weak inhibitor TY4 at different concentrations and specific concentrations. Data analysis shows that the strong inhibitory drug 6G-318S (MIC 0.62 μg / mL) exhibits a significant fluorescence reduction, while the weak inhibitory drug TY4 causes almost no fluorescence change. Figure 11The fluorescence imaging of probe H2 in untreated and treated Cryptococcus neoformans with inhibitors of different MIC values was performed. The fungal cells were pretreated with the inhibitor for 30 minutes and then incubated with probe H2 for 90 minutes. After the incubation was completed, the fluorescence imaging data was collected. Figure 11 As shown in the figure, Cryptococcus H99 without 6G-318S treatment showed obvious green fluorescence in the dark field, which clearly reflected the distribution of Cryptococcus neoformans H99. In contrast, Cryptococcus H99 treated with the inhibitor 6G-318S showed only weak green fluorescence, and its distribution was not very obvious, while Cryptococcus H99 treated with the weak inhibitor TY4 showed stronger green fluorescence.

[0253] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions, which should all be included in the scope of the claims of the present invention.

Claims

1. A nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof, characterized in that: It has the structure shown in the following general formula (I): A is selected from one of the following groups: wherein R1 is independently selected from hydrogen, amino, alkyl-substituted amino, and alkoxy; n represents the number of olefinic bonds, and n is any integer from 1 to 3; p represents the number of R1 substituents on the benzene ring, and p is any integer from 0 to 6; L is selected from one of the following groups: Wherein, m represents the number of alkylene groups, and m is any integer from 1 to 3; Y is selected from hydrogen or halogen; X is selected from one of the following groups: Indicates the attachment site.

2. The nitrogen-containing heterocyclic compound according to claim 1, wherein Satisfy one or more of the following (1) to (4): (1) A is selected from one of the following groups: wherein R1 is independently selected from hydrogen, amino, dimethylamino, and p is 0 or 1; (2) L is selected from one of the following groups: (3) Y is selected from hydrogen or fluorine; (4) X is selected from one of the following groups:

3. The nitrogen-containing heterocyclic compound according to any one of claims 1 or 2, wherein Has any of the following structures:

4. A method for preparing a nitrogen-containing heterocyclic compound according to any one of claims 1 to 3, characterized in that: The steps include: Prepare an intermediate compound B1 by aminolysis of a dicarboxylic anhydride compound; carry out a condensation reaction of the intermediate compound B1 with a carboxylic acid compound; or 4-Methylquinoline is subjected to a nucleophilic substitution reaction and a catalytic hydrogenation reaction to prepare an intermediate compound B2; The intermediate compound B2 and the carboxylic acid compound are subjected to a condensation reaction to prepare the intermediate compound D; subjecting the compound D and the compound K to a Knoevenagel condensation reaction; The structural formula of the intermediate compound B1 includes The structural formula of the intermediate compound B2 includes The structural formula of the intermediate compound D includes The structural formula of the compound K includes Wherein, the definitions of A, L, Y, X, m, n, p and R1 are the same as those in any one of claims 1 to 3.

5. The preparation method according to claim 4, characterized in that The preparation of the intermediate compound B2 by 4-methylquinoline through a nucleophilic substitution reaction and a catalytic hydrogenation reaction comprises the following steps: 4-Methylquinoline and a halogenated nitro compound G are reacted through a nucleophilic substitution reaction to prepare an intermediate compound I; subjecting the intermediate compound I to a catalytic hydrogenation reaction; The structural formula of the halogenated nitro compound G is: Z is selected from halogen; The structural formula of the intermediate compound I is:

6. The preparation method according to claim 4, characterized in that When L is selected from When the carboxylic acid compound is C1; and / or when L is selected from When the carboxylic acid compound is C2; The structural formula of C1 is The structural formula of C2 is 7. The preparation method according to claim 6, characterized in that The preparation of the carboxylic acid compound C2 comprises the following steps: subjecting the aniline carboxylic acid compound F and the carboxylic acid compound C1 to a condensation reaction; The structural formula of the aniline carboxylic acid compound F is 8. A fluorescent probe, characterized in that The invention relates to a nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, or a nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof prepared by the preparation method according to any one of claims 4 to 7.

9. Use of the fluorescent probe according to claim 8 in detecting fungi.

10. A method for evaluating and screening antifungal drugs, characterized in that: The steps include: preparing an experimental group comprising the antifungal drug to be evaluated and screened, the fungus, and the fluorescent probe according to claim 8; Prepare a control group comprising the fungus and the fluorescent probe according to claim 8; Prepare a blank group: comprising a buffer solution and the fluorescent probe according to claim 8; The fluorescence intensities of the experimental group, control group and blank group were tested respectively, and the average fluorescence intensities of the experimental group, control group and blank group were used as the basis for drug screening.

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