Cyanine compound and application thereof in fluorescence labeling of mycobacterium tuberculosis

Abstract

The invention relates to a cyanine compound and application thereof in fluorescence labeling of mycobacterium tuberculosis. The cyanine compound is a compound as shown in I, a tautomer or a pharmaceutically acceptable salt thereof. The cyanine compound provided by the invention can effectively mark Mtb, so that the cyanine compound has a prospect of being developed into an Mtb detection probe.

Description

Cyanine compounds and their application in fluorescent labeling of Mycobacterium tuberculosis

[0001] This application claims the benefit of Chinese Patent Application No. 2023111978040, filed September 15, 2023. This application incorporates the entirety of the aforementioned Chinese Patent Application. Technical Field

[0002] The present invention relates to the field of mycobacterium tuberculosis labeling, in particular to cyanine compounds and applications thereof in fluorescent labeling of mycobacterium tuberculosis. Background Art

[0003] Tuberculosis (TB) is a deadly infectious disease caused by a single pathogen, Mycobacterium tuberculosis (Mtb). It is primarily transmitted through airborne droplets and is the leading cause of death from a single infection source. According to WHO estimates, the 2022 World Health Organization (WHO) Global Tuberculosis Report indicated that approximately 10.6 million new cases of TB were diagnosed and 1.6 million deaths were reported worldwide in 2021, resulting in a TB mortality rate of 15%. Approximately one-quarter of the global population is already infected with Mtb, of whom approximately 5% to 10% will eventually progress from latent TB infection (LTBI) to active TB. Further studies have shown that 10% of LTBI cases will eventually develop active TB and may develop drug resistance. Currently, the global TB epidemic is extremely severe, and TB remains a major global public health challenge.

[0004] Rapid tuberculosis (TB) diagnosis is a critical first step in TB prevention and control. Currently, commonly used clinical diagnostic methods fall into five main categories: bacteriology, imaging, pathology, immunology, and molecular biology. In 2021, 5.3 million people were diagnosed with TB, 63% of whom were confirmed through bacteriological methods, meaning that bacteriology remains the most widely used diagnostic method worldwide. Bacteriological detection methods are primarily divided into two categories: bacterial culture and sputum smear microscopy. Bacterial culture is the gold standard for TB diagnosis, but it is time-consuming. For example, the classic Roche culture method requires approximately three months of incubation. The emergence of improved fully automated liquid culture systems (Bactec MGIT 960, BacT / Alert MB) has significantly shortened the diagnostic timeline, but results still require three to eight weeks. Compared to culture, sputum smear microscopy is faster and more suitable for rapid TB detection. Among them, the traditional Zinc-Auramine O staining method has low detection cost and low requirements for laboratories and equipment. It is widely used in many economically underdeveloped areas, but its sensitivity and positive detection rate are low. The auramine O fluorescence staining method has a higher positive detection rate than Zinc-Auramine O staining because the bacteria are colored as bright yellow fluorescence under the field of view, which contrasts sharply with the dark field background. However, both methods are prone to false positive results caused by dye residues, impure proteins, fibers, food residues, etc., and cannot distinguish between live and dead bacteria, and cannot guide adjustments to clinical medication plans.

[0005] Cyanine (Cy) fluorescent dyes are a class of synthetic fluorescent dyes characterized by a polymethylene bridge chemical structure. Classic Cy dyes contain two nitrogen-containing heterocycles, one of which is positively charged, forming an interionic compound with a delocalized positive charge effect. Therefore, the molar absorptivity of Cy dyes is very high. The length of the bridge chain and the chromophores at both ends directly control the absorption and emission peaks of the dye, allowing the Cy series of dyes to cover almost all commonly used fluorescence bands from ultraviolet to far infrared. Due to the unique photophysical properties and easy synthetic modification of Cy dyes, they are widely used in technical engineering, pharmacology, and medicine, and can be used to detect a variety of targets such as ions, enzymes, and cellular microenvironments.

[0006] Currently, there are no reports of Cy dyes directly fluorescently labeling Mtb. Cy dyes typically require specific enzyme-recognized structural fragments to achieve specific recognition of Mtb. For example, the cyanine compounds described in the paper (Imaging tuberculosis with endogenous β-lactamase reporter enzyme fluorescence in live mice, DOI: 10.1073 / pnas.1000643107) recognize Mtb via specific enzyme-recognized structural fragments. Other common Mtb-specific binding fragments include auramine O and Mtb cell wall components (long fatty chain structures). Therefore, exploring the application of Cy dyes in fluorescent labeling of Mtb will be of great value.

[0007] Summary of the Invention

[0008] The present invention aims to improve existing methods for labeling Mycobacterium tuberculosis. To this end, the present invention provides a cyanine compound and its use in fluorescently labeling Mycobacterium tuberculosis. The cyanine compound provided by the present invention can effectively label Mtb and further exhibits the advantages of high fluorescence intensity and low false positives.

[0009] The present invention provides a use of a compound as represented by formula I, a tautomer thereof, or a pharmaceutically acceptable salt thereof in the preparation of a Mycobacterium tuberculosis detection reagent or kit.

[0010] in,

[0011] R 1 and R 2 are independently hydrogen, halogen, hydroxyl, carboxyl, -SO3H, C 1-6 Alkyl, benzoyl, amino, phenyl, benzyl, 4-7 membered heterocyclic group, one or more R 3 Substituted amino, one or more R 4 Substituted phenyl, one or more R 5 substituted benzyl or one or more R 6 a substituted 4-7 membered heterocyclic group; the heteroatoms in each of the 4-7 membered heterocyclic groups are independently selected from one, two or three of N, O and S, and the number of heteroatoms is independently 1, 2 or 3; wherein S is optionally substituted with 1 or 2 oxo (=O) groups (i.e., S(=O) or S(=O)2);

[0012] n is 0, 1, or 2;

[0013] Each R 3 Independently C 1-6 alkyl;

[0014] Each R 4 , each R 5 and each R 6 Independently C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, amino, -CN, halogen or -(C=O)OC 1-6 alkyl;

[0015] Ring A and Ring A' are independently C 6-14 aryl;

[0016] Each W is independently -C(R 8 )2-, -O-, -S- or -Se-;

[0017] Each R 8 Independently C 1-6 alkyl;

[0018] X and L 1 are independently hydroxyl, C 1-14 Alkyl, C 1-6 Alkoxy, C 6-14 Aryl, 5-10 membered heteroaryl, The heteroatoms of the 5-10 membered heteroaryl group are selected from one, two or three of N, O and S, and the number of heteroatoms is 1, 2 or 3;

[0019] each p is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14;

[0020] Y – F – 、Cl – Br – , I – 、ClO4 – PF6 – 、CH3COO – or

[0021] Z is CH or

[0022] R 7 C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, amino, -CN or halogen;

[0023] m is 1, 2 or 3.

[0024] In a preferred embodiment, R 1 、R 2 , each R4 , each R 5 , each R 6 and R 7 wherein the halogen is independently fluorine, chlorine, bromine or iodine.

[0025] In a preferred embodiment, R 1 、R 2 , each R 3 , each R 4 , each R 5 , each R 6 、R 7 and each R 8 In the C 1-6 Alkyl is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl; for example, methyl.

[0026] In a preferred embodiment, R 1 and R 2 wherein the 4-7 membered heterocyclic group and the one or more R 6 The 4- to 7-membered heterocyclic group in the substituted 4- to 7-membered heterocyclic group is independently a monocyclic heterocycloalkyl group.

[0027] In a preferred embodiment, R 1 and R 2 wherein the 4-7 membered heterocyclic group and the one or more R 6 The 4- to 7-membered heterocyclic group in the substituted 4- to 7-membered heterocyclic group is independently a 5- to 6-membered heterocyclic group.

[0028] In a preferred embodiment, each R 4 , each R 5 and each R 6 In the -(C=O)OC 1-6 C in the alkyl group 1- 6. Alkyl is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.

[0029] In a preferred embodiment, each R 4 , each R 5 , each R 6 、R 7 , X and L 1 In the C 1-6 Alkoxy is independently methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy or tert-butoxy.

[0030] In a preferred embodiment, ring A, ring A', X and L 1 In the C 6-14 Aryl is independently phenyl or naphthyl, for example phenyl.

[0031] In a preferred embodiment, X and L 1 In the C 1-14 The alkyl groups are independently C 1-6 Alkyl, preferably, the C 1- 6 Alkyl is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl; for example, methyl.

[0032] In a preferred embodiment, X and L 1 wherein the 5-10 membered heteroaryl group is independently a 5-6 membered heteroaryl group.

[0033] In a preferred embodiment, each R 8 In the C 1-6 The alkyl group is methyl.

[0034] In a preferred embodiment, R 1 and R 2 is hydrogen, halogen or C 1-6 Alkyl; preferably, R 1 and R 2 For hydrogen.

[0035] In a preferred embodiment, n is 0.

[0036] In a preferred embodiment, Z is CH.

[0037] In a preferred embodiment, each W is independently -C(R 8 )2-; preferably, each W is -C(CH3)2-.

[0038] In a preferred embodiment, each p is independently 1, 2, 3, 4, 5 or 6.

[0039] In a preferred embodiment, wherein p is independently 1, 2 or 3, for example 1.

[0040] In a preferred embodiment, The p in is 4, 5, or 6, for example, 4.

[0041] In a preferred embodiment, X and L 1 Independently C 1-14 Alkyl, C 1-6 Alkoxy, Preferably, X and L 1 Independently C 1-14 alkyl,

[0042] In a preferred embodiment, X is C1-14 alkyl,

[0043] In a preferred embodiment, X is methyl,

[0044] In a preferred embodiment, L 1 C 1-14 Alkyl or

[0045] In a preferred embodiment, L 1 Methyl or

[0046] In a preferred embodiment, Y is F – 、Cl – Br – or I – , preferably, Y is Br – or I – .

[0047] In a preferred embodiment, R 1 and R 2 is hydrogen, halogen or C 1-6 alkyl;

[0048] n is 0, 1, or 2;

[0049] Ring A and Ring A' are independently C 6-14 aryl;

[0050] Each W is independently -C(R 8 )2-;

[0051] Each R 8 Independently C 1-6 alkyl;

[0052] X and L 1 Independently C 1-14 Alkyl, C 1-6 Alkoxy,

[0053] Each p is independently 1, 2, 3, 4, 5 or 6;

[0054] Z is CH;

[0055] Y is F – 、Cl – Br – or I – .

[0056] In a preferred embodiment, the tautomer of the compound represented by formula I is the compound represented by formula IA,

[0057] In a preferred embodiment, the compound represented by formula I is any one of the following compounds:

[0058] The present invention also provides a compound as shown in formula II, its tautomers or pharmaceutically acceptable salts thereof,

[0059] in,

[0060] R 1 and R 2 are independently hydrogen, halogen, hydroxyl, carboxyl, -SO3H, C 1-6 Alkyl, benzoyl, amino, phenyl, benzyl, 4-7 membered heterocyclic group, one or more R 3 Substituted amino, one or more R 4 Substituted phenyl, one or more R 5 substituted benzyl or one or more R 6 a substituted 4-7 membered heterocyclic group; the heteroatoms in each of the 4-7 membered heterocyclic groups are independently selected from one, two or three of N, O and S, and the number of heteroatoms is independently 1, 2 or 3; wherein S is optionally substituted with 1 or 2 oxo (=O) groups (i.e., S(=O) or S(=O)2);

[0061] Each R 3 Independently C 1-6 alkyl;

[0062] Each R 4 , each R 5 and each R 6 Independently C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, amino, -CN, halogen or -(C=O)OC 1-6 alkyl;

[0063] Ring A and Ring A' are independently C 6-14 aryl;

[0064] Each W is independently -C(R 8 )2-, -O-, -S- or -Se-;

[0065] Each R 8 Independently C 1-6 alkyl;

[0066] X a for

[0067] L 1a C1-14 Alkyl or

[0068] Each p is independently an integer from 0 to 14;

[0069] Y – F – 、Cl – Br – , I – 、ClO4 – PF6 – 、CH3COO – or OTs – .

[0070] In a preferred embodiment, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 8 , Ring A, Ring A', W, p and Y – The definition of can also be as described in any of the embodiments of the compound represented by formula I.

[0071] In a preferred embodiment, L 1a In the C 1-14 The alkyl groups are independently C 1-6 Alkyl, preferably, the C 1-6 Alkyl is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl; for example, methyl.

[0072] In a preferred embodiment, X a Methyl,

[0073] In a preferred embodiment, L 1a Methyl or

[0074] In a preferred embodiment, the tautomer of the compound shown in formula Ⅱ is the compound shown in formula II-A,

[0075] In a preferred embodiment, the compound shown in formula II is any one of the following compounds:

[0076] The present invention provides a detection reagent, which includes the compound represented by Formula II according to any one of the present invention, its tautomer or a pharmaceutically acceptable salt thereof.

[0077] The detection reagent can be used to detect Mycobacterium tuberculosis (Mycobacterium smegmatis).

[0078] The present invention provides a kit comprising a compound as shown in Formula II according to any one of the present invention, a tautomer thereof, or a pharmaceutically acceptable salt thereof.

[0079] The kit can be used to detect Mycobacterium tuberculosis (Mycobacterium smegmatis).

[0080] The present invention also provides a method for detecting Mycobacterium tuberculosis, comprising the following steps: incubating a sample to be tested with the compound of Formula I, its tautomers or pharmaceutically acceptable salts thereof in a culture medium, and then detecting the fluorescence intensity of the mixture.

[0081] In a preferred embodiment, the method for detecting Mycobacterium tuberculosis is an in vitro detection method, a diagnostic or non-diagnostic method.

[0082] In a preferred embodiment, the sample to be tested is a clinical sample, and the source of the sample is not particularly limited, and can be derived from humans or other animals, such as livestock.

[0083] In a preferred embodiment, the sample to be tested is a sample suspected of containing Mycobacterium tuberculosis and non-tuberculosis mycobacteria, including but not limited to sputum samples. Preferably, the sample to be tested is sputum, pleural effusion, ascites, urine, cerebrospinal fluid, gastric juice, pus (secretions, puncture fluid, etc.), pathological tissue or cheese mass, feces, throat cotton swab or bronchial lavage fluid.

[0084] In a preferred embodiment, the fluorescence intensity is detected using a fluorescence microscope, microfluidic chip technology, microwell array chip technology or flow cytometer technology.

[0085] In a preferred embodiment, the fluorescence intensity detection is performed using a flow cytometer; preferably, the excitation wavelength of the fluorescence intensity detection is 500-760 nm (eg, 545 nm); the emission wavelength of the fluorescence intensity detection may be 550-790 nm (eg, 565 nm).

[0086] In a preferred embodiment, the detection method includes the following scheme 1 or scheme 2:

[0087] Protocol 1 comprises the following steps: placing the sample to be tested in a centrifuge tube, adding NALC-NaOH (NALC: N-acetylcysteine) solution, then adding PBS, and centrifuging; discarding the supernatant, adding culture medium and the compound represented by Formula I as described above, incubating, smearing, and measuring the fluorescence intensity;

[0088] Scheme 2 includes the following steps: placing the sample to be tested in a centrifuge tube, adding NALC-NaOH solution, adding the compound represented by formula I as described above, incubating, then adding PBS, centrifuging, discarding the supernatant, smearing, and testing the fluorescence intensity.

[0089] In a preferred embodiment, the first embodiment comprises the following steps: placing the sample to be tested in a centrifuge tube, adding NALC-NaOH solution, then adding PBS, and centrifuging; discarding the supernatant, adding culture medium and the compound shown in Formula I as described above, incubating, centrifuging, discarding the supernatant, then adding PBS, smearing, and testing the fluorescence intensity.

[0090] In a preferred embodiment, the detection method includes the following scheme 1 or scheme 2:

[0091] Protocol 1: First, process the sample to be tested. Place the sample (2 mL) into a sterile plastic centrifuge tube (50 mL) and add an equal volume of NALC-NaOH solution, with a final NaOH concentration of 1%. Mix thoroughly (close the lid tightly and invert), and vortex (place on a vortexer) for 20 seconds. Let the solution stand at room temperature for 15 minutes. (After complete digestion, add PBS (pH 6.8) to 50 mL) and centrifuge horizontally for 15 minutes (3000 rpm). Discard the supernatant and add 7H9 medium (2 mL). Incubate the treated bacteria with the compound of Formula I described above for 5 minutes to 24 hours, with a final concentration of 2-50 μM (e.g., 10 μM). After 24 hours of incubation, smear the sample and evenly spread it into a 10×20 mm oval smear. Cover with 3% hydrochloric acid solution for 3 minutes for staining, and gently rinse with running water. Then, 0.5% KMnO4 aqueous solution was added for counterstaining for 1 minute, rinsed with running water, and counted under a fluorescence microscope after natural drying;

[0092] Protocol 2: First, process the sample to be tested. Place the sample to be tested (2 mL) into a sterile plastic centrifuge tube (50 mL). Add an equal volume of NALC-NaOH solution to a final NaOH concentration of 1%. Simultaneously add the compound of Formula I described above to a final concentration of 2-50 μM (e.g., 10 μM). Mix thoroughly (close the lid tightly and invert the tube), and vortex for 20 seconds (on a vortexer). Let the solution stand at room temperature for 15 minutes. (After complete digestion, add PBS (pH 6.8) to 50 mL) and centrifuge horizontally at 3000 rpm for 15 minutes. Discard the supernatant and smear the sample, evenly spreading it into a 10×20 mm oval sputum film. Cover with 3% hydrochloric acid solution for 3 minutes to stain, then gently rinse with running water. Then, counterstain with 0.5% KMnO₄ aqueous solution for 1 minute. Rinse with running water, allow to air dry, and count under a fluorescence microscope.

[0093] In a preferred embodiment, Scheme 1 comprises the following steps: first, the sample to be tested is processed by placing the sample to be tested (2 mL) into a sterile plastic centrifuge tube (50 mL), adding an equal volume of NALC-NaOH solution, with the final NaOH concentration in the centrifuge tube being 1%. The mixture is mixed (with the lid tightly closed and inverted), and vortexed (on a vortexer) for 20 seconds. The solution is allowed to stand at room temperature for 15 minutes. (After complete digestion, PBS (pH 6.8) is added (to 50 mL), and the mixture is centrifuged horizontally for 15 minutes (3000 rpm). The supernatant is discarded and added to 7H9 medium (2 mL). The compound of Formula I described above is co-incubated with the treated bacteria for 5 minutes to 24 hours, with the final concentration of the compound of Formula I described above being 2-50 μM (e.g., 10 μM). The incubation period is 24 hours. After 24 hours, the mixture is centrifuged horizontally for 15 minutes (3000 rpm). Discard the supernatant, add PBS (pH 6.8) (2 mL), and smear the sample, evenly spreading it into an oval sputum film measuring 10 x 20 mm. Cover with 3% hydrochloric acid and ethyl acetate solution for 3 minutes, then rinse gently with running water. Then, counterstain with 0.5% KMnO₄ aqueous solution for 1 minute, rinse with running water, and allow to air dry before counting under a fluorescence microscope.

[0094] The present invention also provides a method for diagnosing tuberculosis, comprising:

[0095] a) obtaining a test sample from a patient;

[0096] b) incubating the test sample with the compound of formula I according to any one of the present invention, its tautomer or a pharmaceutically acceptable salt thereof in a culture medium; and

[0097] c) Detecting the fluorescence intensity of the mixture.

[0098] Definition of terms

[0099] The term "plurality" refers to 2, 3, 4 or 5, preferably 2 or 3.

[0100] The terms “comprise” and “include” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0101] As used herein, numerical ranges defined in substituents, such as 0 to 4, 1-4, 1 to 3, etc., indicate integers within the range, such as 1-6, which represents 1, 2, 3, 4, 5, or 6.

[0102] When any variable (such as R 4 ) appears multiple times in the definition of a compound, the definition of each position of the variable is independent of the definition of the other positions, and their meanings are independent of each other and do not affect each other. Therefore, if a group is replaced by 1, 2 or 3 R 4 group substituted, that is, the group may be replaced by up to 3 R 4 Substitution, where a position R 4 Definition and other positions R 4 In addition, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0103] When a group is listed without specifying that it has a substituent, such group is only meant to be unsubstituted. For example, when "C 1-6 When there is no “substituted or unsubstituted” before “alkyl”, it only refers to “C 1-6 Alkyl" itself or "unsubstituted C 1-6 alkyl".

[0104] The term "alkyl" refers to a straight or branched chain alkyl group having the specified number of carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0105] The term "alkoxy" refers to the group -OR X , where R X is an alkyl group as defined above.

[0106] The term "heterocycle" refers to a stable 4- to 7-membered monocyclic ring, which may be saturated or unsaturated, aromatic or non-aromatic, and is composed of carbon atoms and 1 to 4 heteroatoms selected from N, O and S, wherein the nitrogen and sulfur heteroatoms may be selectively oxidized, preferably a 6-membered heterocycle, such as pyridine, piperidine, pyrazine, piperazine, morpholine or thiomorpholine.

[0107] The term "heterocycloalkyl" refers to a saturated cyclic group having heteroatoms, preferably a 4-7 membered saturated monocyclic ring containing 1, 2, 3 or 4 ring heteroatoms independently selected from N, O and S. Examples of heterocycloalkyl are preferably 6-membered heterocycles, such as pyridine, piperidine, morpholine or thiomorpholine.

[0108] The term "aryl" refers to an aromatic group comprising a conjugated hydrocarbon ring system consisting of carbon atoms that satisfies the 4n+2 rule, with each ring being aromatic. In one embodiment, "aryl" refers to an aromatic group having 6 to 18 (preferably 6 to 10) carbon atoms. Examples of aryl groups include, but are not limited to, phenyl and naphthyl.

[0109] The term "heteroaryl" means a conjugated ring system group having carbon atoms and 1 to 5 heteroatoms selected from nitrogen, oxygen and sulfur within the ring. Unless otherwise specified in this specification, the heteroaryl group may be a monocyclic, bicyclic, tricyclic or more ring system. When it is a bicyclic, tricyclic or more ring system (condensed ring), it may also include a cycloalkyl or heterocyclic group fused to a cycloalkyl or heterocyclic group as defined herein, provided that the heteroaryl group is connected to the rest of the molecule via a single bond via an atom on the aromatic ring. Preferably, the 5-10 membered heteroaryl group comprises 1, 2, 3 or 4 heteroatoms selected from N, O and S, more preferably the 5-6 membered heteroaryl group comprises 1, 2, 3 or 4 heteroatoms selected from N, O and S. Examples of heteroaryl groups include, but are not limited to, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, oxadiazolyl, isoxazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, benzimidazolyl, benzopyrazolyl, indolyl, furanyl, pyrrolyl, triazolyl, tetrazolyl, triazinyl, indolizinyl, isoxazolyl, thiadiazolyl, isoindolyl, indazolyl, isoindazolyl, purinyl, quinolinyl, isoquinolinyl, naphthazinyl, naphthyridinyl, quinoxalinyl, pteridinyl, carbazolyl, carbolinyl, phenanthridinyl, phenanthrolinyl, acridinyl, phenazinyl, isothiazolyl, benzothiazolyl, benzisothiazolyl, benzothienyl, oxatriazolyl, cinnolinyl, quinazolinyl, indolizinyl, o-phenanthroline, isoxazolyl, phenoxazinyl, phenothiazinyl, benzoxazolyl or benzisoxazolyl.

[0110] As used herein, "pharmaceutically acceptable salts" refer to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as described in SM Berge et al., describing pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19.

[0111] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0112] The reagents and raw materials used in the present invention are commercially available.

[0113] The present invention has at least the following beneficial effects:

[0114] The present invention discovers for the first time that cyanine (Cy) compounds can effectively label Mycobacterium tuberculosis. Experiments have shown that the cyanine compounds of the present invention can effectively label Mycobacterium tuberculosis. Furthermore, tuberculosis samples labeled with the compounds of the present invention have high fluorescence intensity, with obvious contrast against the dark field background. Under the microscope, the typical morphology of Mycobacterium tuberculosis (short, thick rod-shaped bacteria) can be clearly seen, with low false positive rates. This is of great significance for guiding the adjustment of clinical medication regimens, and therefore has the prospect of being developed into Mtb detection probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1 shows the fluorescence excitation and emission spectra of Cy3-B.

[0116] FIG2 is a graph showing the mean fluorescence intensity of DMSO-treated E. coli and M. smeg and compound-treated E. coli and M. smeg analyzed by flow cytometry.

[0117] FIG3 is a schematic diagram of the fluorescence change fold of Cy3-B labeled Escherichia coli and Mycobacterium smegmatis analyzed by flow cytometry.

[0118] FIG4 is a graph showing the results of microscopic conditions for Cy3-B-labeled Escherichia coli and Mycobacterium smegmatis.

[0119] FIG5 is a graph showing the mean fluorescence intensity of Mycobacterium smegmatis labeled with Cy3-A, Cy3-B, and Cy3-C by flow cytometry analysis.

[0120] FIG6 is a schematic diagram showing the fluorescence fold change of Cy3-A, Cy3-B and Cy3-C labeled Mycobacterium smegmatis analyzed by flow cytometry.

[0121] FIG7 is a diagram showing the results of microscopic analysis of Cy3-A staining of Mycobacterium tuberculosis in clinical sputum smear samples; wherein A, B, C and D represent the sputum smear staining results of four patients, respectively. DETAILED DESCRIPTION

[0122] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0123] Example 1: Cy3-A Preparation Method and Structural Characterization Data

[0124] 4-Nitrobenzyl bromide (30 mmol) and an indole compound (20 mmol) were refluxed in acetonitrile to produce indole intermediate 1, which was used directly in the next step without further purification. Indole intermediate 1 (15 mmol) was dissolved in 2N NaOH (40 mL) and an appropriate amount of ethanol was added for solubilization. The mixture was stirred at room temperature for 30 minutes, and the reaction system was observed to change from red to yellow liquid. Dichloromethane and water were then added for extraction. The organic phase was collected and concentrated to obtain the target intermediate 2, which was used directly in the next step without further purification. Intermediate 2 (10 mmol) was dissolved in 30 mL of dichloromethane, and DMF (860 μL, 12 mmol) was added. The mixture was then stirred in an ice bath, and phosphorus oxychloride (1.1 mL, 12 mmol) was slowly added dropwise and stirred for 3 hours. Upon completion, 2 mL of water was added to quench the reaction, and stirring was continued for 30 minutes. The reaction mixture was then adjusted to pH 10 by adding NaOH solution, and the temperature was raised to 70°C and stirred for 1 hour. After the product was completely hydrolyzed, dichloromethane and water were added for extraction. The organic phase was concentrated and purified by column chromatography to obtain intermediate 3. Intermediate 3 (5 mmol) and intermediate 4 (5 mmol) were dissolved in 30 mL of acetic anhydride and heated under reflux for 2 hours. After the reaction was complete, the acetic anhydride was evaporated and purified by column chromatography to obtain Cy3-A (yield 58%).

[0125] 1 H NMR (400MHz, CDCl3) δ8.41(t,J=13.3Hz,1H),8.22(d,J=8.6Hz,2H),7.48(d,J=8.7Hz,2H),7.44(d,J=7.4Hz,2H),7.39(dd,J=11.7,7.3Hz,2H),7.32(d d,J=13.9,7.3Hz,2H),7.20(d,J=7.9Hz,1H),7.05(d,J=7.9Hz,1H),6.53(d d,J=17.4,13.5Hz,2H),5.50(s,2H),3.68(s,3H),1.81(s,6H),1.74(s,6H).

[0126] 13C NMR (100MHz, CDCl3) δ175.83,173.55,150.86,147.75,142.32,142.19,141.50,140.67,139.97,129.14,129.07,127.4 7,126.26,125.48,124.38,122.39,122.10,111.39,110.54,105.29,103.55,49.47,49.04,46.77,31.62,28.39,27.83.

[0127] HR-MS (ESI): theoretical value C 31 H 32 N3O2 + ,[M+]m / z:478.2489, measured value:478.2487.

[0128] Example 2: Cy3-B preparation method and structural characterization data

[0129] 6-Bromohexanoic acid (15 mmol) and an indole compound (10 mmol) were refluxed in acetonitrile to generate indole intermediate 5, which was used directly in the next step without further purification. Intermediate 3 (5 mmol) and intermediate 5 (5 mmol) were dissolved in 30 mL of acetic anhydride and heated under reflux for 2 hours. After the reaction was completed, the acetic anhydride was evaporated and purified by column chromatography to obtain intermediate 6. Intermediate 6 (2.5 mmol) was dissolved in ethanol, 10 times the amount of iron powder was added, 10 equivalents of NH4Cl aqueous solution was added dropwise, the temperature was raised to 80°C, and the reaction was stirred vigorously for 1 hour. The reaction endpoint was monitored by LC-MS, and Cy3-B was purified by HPLC (yield 75%).

[0130] 1 H NMR (400MHz, CDCl3) δ11.25 (s, 1H), 8.39 (t, J = 13.5Hz, 1H), 7.83 (s, 2H), 7.47 (s, 1H), 7.4 3(d,J=7.5Hz,2H),7.39(dd,J=9.6,5.7Hz,2H),7.32(d,J=8.0Hz,1H),7.25(d,J=7.9Hz,1 H),7.15(dd,J=13.1,7.9Hz,2H),6.17(dd,J=24.8,13.3Hz,2H),5.26(s,1H),4.01(s,2H) ,2.37-2.24(m,2H),1.85-1.76(m,8H),1.72(s,6H),1.65-1.60(m,2H),1.45-1.37(m,2H).

[0131] 13 C NMR (200MHz, CDCl3) δ176.58,174.91,174.02,160.96,150.65,142.31,141.51,140.74,140.09,137.77,129.20,127.62,126.09,125.66, 123.19,122.25,116.88,115.43,111.37,110.88,103.89,103.54,49 .52,49.21,44.33,33.61,28.22,27.94,26.93,25.83,24.40,24.06.

[0132] HR-MS (ESI): theoretical value C 36 H 42 N3O2 + ,[M+]m / z:548.3272, measured value:548.3268.

[0133] Effect Example 1 Cy3-B excitation spectrum and emission spectrum experiment

[0134] Instrument: Multifunctional microplate reader ( Flash, Thermo Scientific).

[0135] Reagents and materials: Cy3-B, ddH2O, DMSO, 96-well ELISA plate (Corning, 3603).

[0136] Procedure: Prepare a 10mM Cy3-B DMSO stock solution. Add 5μL of freshly prepared Cy3-B DMSO stock solution to 45μL of DMSO to obtain 50μL of a 1mM Cy3-B DMSO working solution. Add 1μL of the 1mM Cy3-B DMSO working solution and 99μL of ddH2O to one well of a 96-well microtiter plate for a final Cy3-B concentration of 10μM. Perform two replicates per well. Mix thoroughly and analyze fluorescence excitation and emission spectra on a microtiter plate.

[0137] The results are shown in FIG1 . Cy3-B can emit fluorescence (565 nm) under excitation light of a specific wavelength (545 nm), that is, the maximum excitation wavelength of Cy3-B is 545 nm, and the maximum emission wavelength is 565 nm.

[0138] Effect Example 2 Fluorescence Response of Cy Dye in Bacteria

[0139] Instrument: Flow cytometer (BD, LSR Fortessa)

[0140] Reagents and materials: Cy3-B, Mycobacterium smegmatis (M. smeg, ATCC 700084) were purchased from Shanghai Jingnuo Biotechnology Co., Ltd., Escherichia coli (E. coli, ATCC 700926) was kindly provided by Zhang Jingren Laboratory, School of Medicine, Tsinghua University, 7H9 medium, LB medium, DMSO.

[0141] Procedure: Prepare a 10 mM Cy3-B DMSO stock solution. Add 1 μL of freshly prepared Cy3-B DMSO stock solution to 99 μL of DMSO to obtain 100 μL of a 0.1 mM Cy3-B DMSO working solution. M. smeg and E. coli were cultured in 7H9 and LB medium, respectively, until the logarithmic growth phase. The cells were centrifuged at 3000 rpm for 15 minutes, the supernatant discarded, and the suspension resuspended in PBS. Separately, 300 μL of the bacterial suspension was added to 3 μL of the 0.1 mM Cy3-B DMSO working solution to a final concentration of 1 μM. A negative control was treated with an equal amount of DMSO. After incubation at 37°C for 1 hour, the suspension was centrifuged at 3000 rpm for 15 minutes, the supernatant discarded, and the suspension resuspended in PBS. Fluorescence intensity was analyzed by flow cytometry and microscopy.

[0142] Flow cytometric analysis of bacterial fluorescence intensity is shown in Figures 2 and 3. While the fluorescence of the Cy3-B-labeled E. coli group did not show a significant increase compared to the control group without compound addition, the fluorescence of the Cy3-B-labeled M. smeg group was significantly enhanced compared to the control group without compound addition, demonstrating the selective labeling ability of Cy3-B for M. smeg. The fluorescence intensity of Cy3-B increased by 6.5-fold in the E. coli group and 216.6-fold in the M. smeg group, respectively.

[0143] In practice, Mycobacterium tuberculosis is often detected through direct microscopic observation. A 6-fold fluorescence change detected by flow cytometry is typically invisible under a microscope. However, Cy3-B-labeled M. smeg exhibits a 217-fold fluorescence change under the same conditions. This significant fluorescence difference results in only M. smeg fluorescence being observed under a microscope, while E. coli fluorescence is not.

[0144] The results of microscopic analysis of bacterial fluorescence intensity are shown in FIG4 . As can be seen from FIG4 , under the same conditions, the microscopic observation results show that the fluorescence emitted by Mycobacterium smegmatis labeled with Cy3-B can be seen, while the fluorescence emitted by Escherichia coli labeled with Cy3-B cannot be seen.

[0145] The change in fluorescence intensity refers to the condition in which the sample under test is in a non-fluorescent state or in a state of autofluorescence prior to contact with the Cy dye. After contact with the Cy dye, the sample can be excited by 500-760 nm excitation light and emits 550-790 nm fluorescence, with the emitted fluorescence exhibiting a change in intensity. Specifically, the fluorescence change factor in the present invention is calculated by dividing the fluorescence intensity measured in the compound-treated group by the fluorescence intensity measured in the DMSO-treated group (i.e., the autofluorescence of the bacteria).

[0146] Effect Example 3 Fluorescence Response of Cy Dye in Bacteria

[0147] Instrument: Flow cytometer (BD, LSR Fortessa)

[0148] Reagents and materials: Cy3-A, Cy3-B, Cy3-C (Cy3-C was purchased from Zhengzhou Huiju Chemical Co., Ltd.), Mycobacterium smegmatis (M. smeg), 7H9 culture medium, DMSO

[0149] Procedure: Prepare 10 mM DMSO stock solutions of Cy3-A, Cy3-B, and Cy3-C. Add 1 μL of each freshly prepared DMSO stock solution of Cy3-A, Cy3-B, and Cy3-C to 99 μL of DMSO to obtain 100 μL of 0.1 mM DMSO working solutions of Cy3-A, Cy3-B, and Cy3-C. Cultivate the bacteria to the logarithmic growth phase, centrifuge at 3000 rpm for 15 minutes, discard the supernatant, and resuspend in PBS. Separately, 300 μL of bacterial solution was added with 3 μL of 0.1 mM Cy3-A, Cy3-B and Cy3-C DMSO working solution to make the final concentration of the compound 1 μM. An equal amount of DMSO was added to the negative control group. After incubation at 37°C for 1 hour, the cells were horizontally centrifuged at 3000 rpm for 15 minutes, the supernatant was discarded, and the cells were resuspended in PBS. The bacterial fluorescence intensity was analyzed by flow cytometry.

[0150] The results are shown in Figures 5 and 6. The fluorescence of the Cy3-A, Cy3-B, and Cy3-C groups was significantly enhanced compared to the control group without compound addition. The fluorescence of Cy3-A, Cy3-B, and Cy3-C increased by 1497.1 times, 216.6 times, and 1851.4 times, respectively.

[0151] Effect Example 4 Detection Ability of Cy3-A for Mycobacterium tuberculosis in Clinical Sputum Smear Samples

[0152] Sputum samples were collected from four patients (source: patients diagnosed with tuberculosis with positive acid-fast staining at Beijing Chest Hospital). 2 mL of sputum was placed in sterile 50 mL plastic centrifuge tubes. An equal volume of NaOH solution was added to the tubes, resulting in a final NaOH concentration of 1%. The tubes were tightly capped, mixed by inversion, and vortexed for 20 seconds. The solution was allowed to stand at room temperature for 15 minutes. After complete digestion, PBS (pH 6.8) was added to 50 mL. The tubes were centrifuged horizontally at 3000 rpm for 15 minutes. The supernatant was discarded, and 2 mL of 7H9 medium containing Cy3-A was added to a final concentration of 10 μM. The samples were incubated at 37°C for 24 hours. The tubes were then centrifuged horizontally at 3000 rpm for 15 minutes, smeared, dried, and eluted with 3% hydrochloric acid-ethanol solution for 3 minutes. The tubes were then washed with water and counterstained with 0.5% potassium permanganate for 1 minute. After washing and drying, the tubes were observed under a fluorescence microscope.

[0153] The results are shown in FIG7 . It can be seen that the typical morphology of Mycobacterium tuberculosis (rod-shaped, short and thick bacteria) can be clearly seen under the microscope. The results indicate that Cy3-A can stain Mycobacterium tuberculosis in clinical samples.

[0154] The above examples illustrate that the Cy dye of the present invention can selectively label Mycobacterium tuberculosis and has a strong fluorescence intensity on Mycobacterium tuberculosis. It can detect Mycobacterium tuberculosis in sputum smear samples and has the application prospect of being developed into a Mycobacterium tuberculosis detection reagent.

[0155] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0156] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. Use of a compound as shown in formula I, its tautomer or a pharmaceutically acceptable salt thereof in the preparation of a Mycobacterium tuberculosis detection reagent or kit, in, R 1 and R 2 are independently hydrogen, halogen, hydroxyl, carboxyl, -SO3H, C 1-6 Alkyl, benzoyl, amino, phenyl, benzyl, 4-7 membered heterocyclic group, 3 Substituted amino, one or more R 4 Substituted phenyl, substituted by one or more R 5 substituted benzyl or one or more R 6 substituted 4-7 membered heterocyclic group; the heteroatoms in each 4-7 membered heterocyclic group are independently selected from one, two or three of N, O and S, and the number of heteroatoms is independently 1, 2 or 3; wherein S is optionally substituted by 1 or 2 oxo groups; n is 0, 1 or 2; Each R 3 Independently for C 1-6 alkyl; Each R 4 , each R 5 and each R 6 Independently for C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, amino, -CN, halogen or -(C=O)OC 1-6 alkyl; Ring A and Ring A' are independently C 6-14 Aryl; Each W is independently -C(R 8 )2-, -O-, -S- or -Se-; Each R 8 Independently for C 1-6 alkyl; X and L 1 are independently hydroxyl, C 1-14 Alkyl, C 1-6 Alkoxy, C 6-14 Aryl, 5-10 membered heteroaryl, 5. The heteroatoms of the 10-membered heteroaryl group are selected from one, two or three of N, O and S, and the number of heteroatoms is 1, 2 or 3; each p is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14; Y – F – , Cl – Br – ,I – 、ClO4 – PF6 – 、CH3COO – or OTs – Z is CH or R 7 C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, amino, -CN or halogen; m is 1, 2 or 3.

2. Use of the compound of formula I, its tautomer or its pharmaceutically acceptable salt as claimed in claim 1 in the preparation of a Mycobacterium tuberculosis detection reagent or kit, characterized in that: It meets one or more of the following conditions: (1)R 1 , R 2 , each R 4 , each R 5 , each R 6 and R 7 wherein the halogen is independently fluorine, chlorine, bromine or iodine; (2)R 1 , R 2 , each R 3 , each R 4 , each R 5 , each R 6 , R 7 and each R 8 In the C 1-6 Alkyl is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl; for example, methyl; (3)R 1 and R 2 wherein the 4-7 membered heterocyclic group and the one or more R 6 The 4-7 membered heterocyclic group in the substituted 4-7 membered heterocyclic group is independently a monocyclic heterocycloalkyl group; (4)R 1 and R 2 wherein the 4-7 membered heterocyclic group and the one or more R 6 The 4-7-membered heterocyclic group in the substituted 4-7-membered heterocyclic group is independently a 5-6-membered heterocyclic group; (5) Each R 4 , each R 5 and each R 6 In the above-(C=O)OC 1-6 C in the alkyl group 1-6 Alkyl is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl; (6) Each R 4 , each R 5 , each R 6 , R 7 , X and L 1 In the C 1-6 Alkoxy is independently methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy or tert-butoxy; (7) Ring A, Ring A', X and L 1 In the C 6-14 Aryl is independently phenyl or naphthyl, for example phenyl; (8)X and L 1 In the C 1-14 The alkyl groups are independently C 1-6 Alkyl, preferably, the C 1-6 Alkyl is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl; for example, methyl; and (9)X and L 1 In the above, the 5-10 membered heteroaryl group is independently a 5-6 membered heteroaryl group.

3. Use of the compound of formula I as claimed in claim 1 or 2, its tautomer or a pharmaceutically acceptable salt thereof in the preparation of a Mycobacterium tuberculosis detection reagent or kit, characterized in that: It meets one or more of the following conditions: (1)R 1 and R 2 is hydrogen, halogen or C 1-6 Alkyl; preferably, R 1 and R 2 is hydrogen; (2) n is 0; (3) Z is CH; (4) each W is independently -C(R 8 )2-; Preferably, each W is -C(CH3)2-; (5) each p is independently 1, 2, 3, 4, 5 or 6; Preferably, wherein p is independently 1, 2 or 3, for example 1; The p in can be 4, 5 or 6, for example 4; (6) X and L 1 Independently for C 1-14 Alkyl, C 1-6 Alkoxy, Preferably, X and L 1 Independently for C 1-14 alkyl, Preferably, X is C 1-14 alkyl, L 1 Can be C 1-14 Alkyl or (7)Y is F – , Cl – Br – or I – Preferably, Y is Br – or I – ; and (8) The tautomer of the compound represented by formula I is the compound represented by formula IA, 4. Use of the compound of formula I, its tautomer or its pharmaceutically acceptable salt as claimed in claim 1 in the preparation of a Mycobacterium tuberculosis detection reagent or kit, characterized in that: It meets one or both of the following conditions: (1) X is methyl, and (2)L 1 Methyl or 5. Use of the compound of formula I, its tautomer or its pharmaceutically acceptable salt as claimed in claim 1 in the preparation of a Mycobacterium tuberculosis detection reagent or kit, characterized in that: R 1 and R 2 is hydrogen, halogen or C 1-6 alkyl; n is 0, 1 or 2; Ring A and Ring A' are independently C 6-14 Aryl; Each W is independently -C(R 8 )2-; Each R 8 Independently for C 1-6 alkyl; X and L 1 Independently for C 1-14 Alkyl, C 1-6 Alkoxy, Each p is independently 1, 2, 3, 4, 5 or 6; Z is CH; Y – F – , Cl – Br – or I – .

6. Use of the compound of formula I, its tautomer or its pharmaceutically acceptable salt as claimed in claim 1 in the preparation of a Mycobacterium tuberculosis detection reagent or kit, characterized in that: The compound shown in formula I is any of the following compounds:

7. A compound of formula II, its tautomer or a pharmaceutically acceptable salt thereof, in, X a for L 1a C 1-14 Alkyl or R 1 , R 2 , Ring A, Ring A', W, p and Y – The definition is as described in any one of claims 1-6.

8. The compound of formula II according to claim 7, its tautomer or a pharmaceutically acceptable salt thereof, characterized in that: It meets one or more of the following conditions: (1)L 1a In the C 1-14 The alkyl groups are independently C 1-6 Alkyl, preferably, the C 1-6 Alkyl is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl; for example, methyl; (2)X a for (3)L 1a Methyl or and (4) The tautomer of the compound represented by formula Ⅱ is the compound represented by formula II-A, 9. The compound of formula II according to claim 7, its tautomer or a pharmaceutically acceptable salt thereof, characterized in that: The compound shown in formula II is any of the following compounds:

10. A detection reagent or kit, characterized in that: It includes a compound as shown in formula II as described in any one of claims 7 to 9, a tautomer thereof or a pharmaceutically acceptable salt thereof.

11. A method for detecting Mycobacterium tuberculosis, characterized in that: The detection method comprises the following steps: performing fluorescence intensity detection on a mixture of a sample to be tested and a compound as shown in formula I as claimed in any one of claims 1 to 6, a tautomer thereof or a pharmaceutically acceptable salt thereof, after incubation in a culture medium; Preferably, the detection method satisfies one or more of the following conditions: (1) The method for detecting Mycobacterium tuberculosis is an in vitro detection method; (2) The sample to be tested is a clinical sample, and the sample source can be human or animal, for example, the animal is livestock; (3) The sample to be tested is a sample suspected of containing Mycobacterium tuberculosis and non-tuberculous mycobacteria. Preferably, the sample to be tested is sputum, pleural effusion, ascites, urine, cerebrospinal fluid, gastric juice, pus, pathological tissue or cheese mass, feces, throat cotton swab or bronchial lavage fluid; and (4) The fluorescence intensity detection is performed using a fluorescence microscope, microfluidic chip technology, microwell array chip technology or flow cytometer technology; preferably, the fluorescence intensity detection is performed using a flow cytometer; more preferably, the excitation wavelength of the fluorescence intensity detection is 500-760nm; the emission wavelength of the fluorescence intensity detection can be 550-790nm.

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