CN112694431B

CN112694431B - Nitroreductase in hypersensitive fluorescent probe detection bacteria and application in bacterial infection

Info

Publication number: CN112694431B
Application number: CN201911008897.1A
Authority: CN
Inventors: 胡海宇; 张青扬; 武岭岭; 许胜男; 王庆华; 张蕾磊
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2019-10-23
Filing date: 2019-10-23
Publication date: 2022-09-16
Anticipated expiration: 2039-10-23
Also published as: CN112694431A

Abstract

The invention relates to a nitroreductase activated Cy fluorescent probe molecule I based on Cy dye and a preparation method and application of a Cy-aminoglycoside fluorescent probe II of a specific target bacterium, which is obtained by connecting the fluorescent probe and an aminoglycoside compound, and belongs to the field of biological fluorescence imaging. The two probe molecules can specifically detect nitroreductase, and the fluorescence is enhanced by the activation of the nitroreductase, so that the background noise of fluorescence imaging is reduced, and the imaging signal-to-noise ratio is improved. Compared with the reported nitroreductase detection probe molecules, the synthesized Cy-aminoglycoside fluorescent probe molecules have good water solubility, extremely high nitroreductase reaction rate and very low nitroreductase detection limit. In particular, Cy-aminoglycoside fluorescent probes have been reported as the first fluorescent probe molecules that can distinguish between bacterial infection and hypoxic tumor cells or tissues.

Description

Nitroreductase in hypersensitive fluorescent probe detection bacteria and application in bacterial infection

The technical field is as follows:

the invention belongs to the technical field of medicines. Relates to an aminoglycoside fluorescent probe used for detecting bacterial infection and having no influence on tumors, a preparation method and application thereof.

Background content:

medical imaging is a technology for visually presenting internal structures of a human body and providing accurate position information and morphological information for clinical diagnosis. Abnormal lesions were confirmed by comparison with normal anatomical physiology. ^[1-4] In contrast to conventional diagnostic imaging for visualization of pathological tissues, molecular imaging allows diagnosis at an early cellular and molecular level, observed before tissue damage occurs or obvious disease symptoms appear. ^[5,6] Molecular imaging techniques mainly used in clinical diagnosis mainly include positron diffraction imaging, single photon diffraction, nuclear magnetic resonance and medical optical imaging. Each method has its own advantages and disadvantages in view of spatial resolution, temporal resolution, sensitivity and cost. For example, positron emission tomography (pet) uses a radioactive isotope as a tracer to achieve high sensitivity, but requires an expensive cyclotron to acquire the radionuclide. Compared with positron diffraction imaging, the cost of single photon diffraction is obviously reduced, but the spatial resolution is reduced, so that the focus positioning is difficult. The nuclear magnetic resonance has the characteristics of high resolution and low sensitivity. Optical imaging has gained widespread attention due to its high sensitivity and specificity. The advent of rapidly evolving optical imaging microscopes and a series of readily available probe molecules and markers have facilitated the visual monitoring of physiological and pathological processes at the cellular, subcellular and molecular levels. Common probe molecules and labels include organic molecules, transition metal complexes, lanthanide complexes, quantum dots, nanoparticles, fluorescent proteins, and the like.

In optical imaging, the light intensity can intuitively reflect the concentration of the probe and the correspondingly positioned information. In multicolor imaging, different emission wavelengths of different probes have different emission colors. In order to avoid photobleaching of the luminescent dye and to improve the tissue penetration, it is necessary to enhance the photostability of the luminescent dye and to adjust the emitted light into the near infrared region to achieve a reduction in the light absorption of the biological sample itself, thereby enhancing the tissue penetration. Normally, the fluorescence intensity of the probe is proportional to the solution concentration of the sample, but in complex organismsIn a body environment, the accuracy and precision of detection can be reduced. This is because the fluorescence intensity is also affected by the uncertainty of the probe concentration in the cell or organism, the fluctuation of the excitation light source, and the fluorescence of the endogenous fluorescent substance. For example, flavin and flavoprotein in mitochondria can be excited at 450-500nm with an emission wavelength at 500-600 nm. The absorption wavelength of the different complexes of lipids and proteins in brain tissue is 400-550nm, and the emission wavelength covers the yellow region and extends into the near infrared region. The absorption and emission wavelengths of endogenous fluorescent substances cover the entire visible region, which results in interference with the collection of fluorescent signals from the probe, resulting in fluctuating changes. ^[7]

In vivo luminescence imaging, as one of optical imaging, includes bioluminescence imaging and fluorescence imaging. Bioluminescence imaging is the phenomenon that luciferase, or bacteria, fungi, parasites and mice genetically engineered to be luciferase-tagged, can produce luminescence in the presence of a luciferase substrate or in the absence of a luciferase substrate due to the presence of the lux operon, and the phenomenon that green fluorescent protein is directly activated by light to produce luminescence. All luciferases are oxidases, requiring large amounts of oxygen to function optimally; beetle luciferases also require intracellular adenosine triphosphate to function; for systems requiring exogenous luciferase substrates, the pharmacokinetics and in vivo distribution of the substrate must also be considered. These all limit their further applications. ^[8] The fluorescence imaging can realize the visual imaging of the specific organelles of the living cells and the whole animal, is a powerful biological research tool, and particularly has the value of clinical application in the emerging field of fluorescence guidance operation. ^[9] Fluorescence imaging has the advantages of high sensitivity, nondestructive rapid analysis and real-time monitoring. The small molecular probe has structure modifiability, so that the monitoring of various biological targets can be realized, and the real-time monitoring and imaging of various enzymes in cells under the original environment can be realized. The probe molecules with different structures are designed, so that the spectral properties of the probe molecules can be obviously changed when the probe molecules interact with enzyme, and the signal-to-noise ratio and the sensitivity of the molecular probe are further improved. ^[9]

Major problems with fluorescence imaging include autofluorescence, fluorescence quenching, photobleaching and low tissue penetration. Compared with fluorescence imaging in a visible light region (400-700nm), fluorescence imaging in a near-infrared light region (700-1700nm) has obvious advantages in the aspects of reducing light scattering, reducing light absorption of biological samples and interference of spontaneous fluorescence. ^[10] The near-infrared optical window can be artificially divided into a first near-infrared optical window (700-. However, the lack of a second near-infrared optical window bioprobe with both high quantum efficiency and biocompatibility has limited its widespread acceptance in clinical use. ^[11] Fluorescence imaging of the first near-infrared optical window has been widely performed in basic research, preclinical and clinical applications over the past decade. This is mainly due to the presence of a range of readily available fluorescent molecules such as indocyanine green (excitation wavelength 808nm, emission wavelength 822nm), methylene blue (excitation wavelength 665nm, emission wavelength 686nm), both of which have been approved by the U.S. food and drug administration for clinical use. ^[10]

Aminoglycoside antibiotics are a very important class of antibiotics that have been used clinically for decades. Such as neomycin, gentamicin, kanamycin, tobramycin, amikacin, and the like. The medicine has wide antibacterial spectrum and strong effect on aerobic gram-negative bacilli, such as escherichia coli, tubercle bacillus, brucella, salmonella and the like. Has weak effect on gram-positive bacteria except staphylococcus aureus and drug-resistant bacteria thereof, and is ineffective on anaerobic bacteria. The aminoglycoside medicine can enter the bacterial cell through the outer membrane of the bacterial cell, mainly acts on ribosome in the bacterial body, and inhibits the synthesis of bacterial protein through multiple links, thereby playing the role of antibiosis. The mechanism of drug resistance of the drugs is mainly through the change of permeability of cell outer membrane or the change of specific cell outer membrane transport system, so that the drug molecules cannot enter cells, and the drug molecules cannot reach enough concentration in the cells to reduce the efficacy of the drugs. In recent years, with the emergence of the problem of resistance, more and more scientists have studied new aminoglycoside antibioticsCan be used for the treatment of diseases. Scientists have reported a class of methods by linking positively charged liposomes (cationic lipids) to aminoglycoside antibiotics ^[12] The conjugate is formed, the transfer of the bacterial cell membrane to the aminoglycoside compound is increased, the uptake of the aminoglycoside compound is increased, the antibacterial activity and the drug-resistant bacteria resistance are improved, the activity to gram-positive bacteria is enhanced, and the antibacterial range is enlarged ^[13] 。

Various fluorescent dyes have been used to date for studies in vitro and in vivo animal experiments. Indocyanine green (ICG) was the first representative organic dye approved by the U.S. food and drug administration for use in humans. Thereafter, various groups synthesized ICG derivatives, Cy series cyanine dyes, IRDye series cyanine dyes for diagnostic detection. However, most dyes lack specificity for the target. Therefore, it is desirable to label them with specific targeting molecules, such as antibiotics.

Nitroreductases are present in bacteria, yeast, trypanosomes and hypoxic tumors. The nitroreductase can be applied to monitoring hypoxic tumors. Meanwhile, since nitroreductase enzymes are widely present in gram-positive and gram-negative bacteria, imaging for nitroreductase enzymes can be used for determination of bacterial infection. ^[14-17] The aromatic nitro compound has relatively high nitroreductase reaction activity, mainly due to pi-pi accumulation, hydrogen bond and other interactions between the aromatic nitro compound and the nitroreductase. Nitroreductases can reduce the corresponding aromatic nitro compound to an aromatic amine or an aromatic hydroxylamine by one of two mechanisms. In the first mechanism, a mitochondrial nitroreductase type I is oxygen tolerant, providing two electrons initially to reduce an aromatic nitro compound to an aromatic nitroso compound, followed by complete conversion to an aromatic amino compound. In the second mechanism, in II type mitochondrial nitroreductase, an electron is initially provided to reduce the aromatic nitro compound into aromatic nitro radical negative ions, the aromatic nitro radical negative ions can be completely converted into aromatic amino compounds under the low oxygen condition, and if the oxygen concentration is too high, the aromatic nitro radical negative ions are oxidized into the initial raw material aromatic nitro compound. ^[18] The nitroreductase activity in human cells is only byMitochondrial components isolated from mammalian hepatocytes, which are reported to be present in hypoxic tumors, also have nitroreductase activity, which may be present in type I mitochondrial nitroreductase. ^[19-21] The nitroreductase activity in hypoxic tumor tissues was detected by fluorescence, presumably type II mitochondrial nitroreductase. The reduction mechanism of nitroreductase can be divided into two types according to the difference of substrate structures. The first type is the direct reduction of aromatic nitro compounds to aromatic amino compounds. And the second type is that after the aromatic amino compound is obtained on the basis of the first type, electrons are rearranged and relevant carbon-oxygen bonds are induced to be broken to obtain a new compound.

According to the strategy, the novel fluorescent molecular probe is designed and synthesized, and then the aminoglycoside fluorescent probe is obtained by connecting a fluorescent molecule on the aminoglycoside drug. The two probes are near-infrared probe molecules which can be specifically activated by nitroreductase, have good water solubility, and avoid the aggregation-induced quenching problem of cyanine dye molecules in aqueous solution to a great extent. Meanwhile, the probe molecule shows good stability under different pH values. In the monitoring process of nitroreductase, the method shows good specificity, high sensitivity and ultra-fast reaction rate. At the same time, it can target the site of bacterial infection, but has no effect on hypoxic tumor cells that also contain nitroreductase.

The fluorescence imaging technology has the advantages of no wound, visualization, targeting property, high sensitivity and the like, has wide clinical application value in the aspects of accurately positioning bacterial infection, identifying bacterial species, distinguishing bacterial infection and aseptic inflammation and monitoring curative effect, and lays a solid foundation for dealing with the problems of bacterial infection and drug resistance thereof. The research of fluorescent molecular probes based on antibiotics will become the key of the technology. With the development of fluorescence imaging technology and the application of corresponding imaging equipment to clinic, the application of the antibiotic-based fluorescent molecular probe to the detection of bacterial infection has great clinical application value.

The invention content is as follows:

the invention solves the technical problem of providing a Cy fluorescent probe compound or acceptable salt thereof and an aminoglycoside fluorescent probe or acceptable salt thereof, a preparation method thereof and application thereof.

In order to solve the technical problem, the invention provides the following technical scheme:

the first aspect of the technical scheme of the invention provides a Cy fluorescent probe compound shown as a structure I or acceptable salt thereof:

wherein: r ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ 、R ₇ 、R ₈ 、R ₉ 、R ₁₀ 、R ₁₁ 、R ₁₂ 、R ₁₃ 、R ₁₄ 、R ₁₅ 、 R ₁₆ 、R ₁₇ 、R ₁₈ 、R ₁₉ Independently selected from hydrogen atoms, sulfonic groups, sulfamide, hydroxyl, amino, F, Cl, Br, I, nitro, C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, C1-6 alkoxy, C1-6 alkylamino, C1-6 carboxylic acid, C1-6 carboxylic acid methyl ester and C1-6 alkylamide; or R ₁ And R ₂ ，R ₂ And R ₃ ，R ₃ And R ₄ ，R ₆ And R ₇ ，R ₁₄ And R ₁₅ ，R ₁₆ And R ₁₇ ，R ₁₇ And R ₁₈ ，R ₁₈ And R ₁₉ The adjacent positions are connected in the form of benzene ring, naphthalene ring, anthracene ring and phenanthrene ring; the C1-6 is selected from C1, C2, C3, C4, C5 and C6;

R ₂₀ 、R ₂₁ 、R ₂₂ 、R ₂₃ independently selected from hydrogen atom, sulfonic group, sulfonamide, hydroxyl, amino, F, Cl, Br, I, nitro or C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl; the C1-6 is selected from C1, C2, C3, C4, C5 and C6;

R ₄ ，R ₆ and R ₇ ，R ₁₄ And R ₁₅ ，R ₁₆ And R ₁₇ ，R ₁₇ And R ₁₈ ，R ₁₈ And R ₁₉ The adjacent positions are connected in the form of benzene ring, naphthalene ring, anthracene ring and phenanthrene ring; the C1-6 is selected from C1, C2, C3, C4, C5 and C6;

R ₂₀ 、R ₂₁ 、R ₂₂ 、R ₂₃ independently selected from hydrogen atom, sulfonic group, sulfamide, hydroxyl, amino, F, Cl, Br, I, nitro or C1-6 alkyl, C1-6 alkenyl and C1-6 alkynyl; the C1-6 is selected from C1, C2, C3, C4, C5 and C6;

R ₂₄ is selected from

Vinyl, ethynyl, phenyl;

R ₂₅ selected from nitro-substituted benzene, nitro-substituted phenol, nitro-substituted halogeno-benzene, nitro-substituted toluene, nitro-substituted naphthalene, nitro-substituted anthracene and nitro-substituted anthracene

Nitro-substituted perylene, nitro-substituted benzopyrene, nitro-substituted furan, nitro-substituted thiophene, nitro-substituted pyrrole, nitro-substituted imidazole, nitro-substituted pyrazole, nitro-substituted thiazole, nitro-substituted oxazole, nitro-substituted pyridine, nitro-substituted pyrimidine, nitro-substituted pyridazine, nitro-substituted pyrazine, nitro-substituted purine, nitro-substituted quinoline, nitro-substituted isoquinoline, sulfonyl of nitro-substituted indole, sulfinyl, carbonyl or phosphorus oxide;

m represents 1,2,3, 4;

n represents 1,2,3, 4;

p represents 0,1,2,3,4,5,6,7,8, 9;

x represents F, Cl, Br, I;

L ₁ c1-9 alkyl, C1-9 alkenyl and C1-9 alkynyl, wherein C1-9 is selected from C1, C2, C3, C4, C5, C6, C7, C8 and C9;

L ₂ is selected from C1-20 alkyl, C1-20 alkenyl and C1-20 alkynyl, wherein the C1-20 is selected from C1, C2, C3, C4, C5, C6, C7, C8, C9, C10 and C811,C12,C13,C14,C15,C16,C17,C18,C19,C20；

R ₂₆ Is selected from

R ₂₇ Selected from methyl, hydroxyl, amino, formaldehyde, formic acid,

connecting a Cy fluorescent probe compound shown as a structure II with a targeted group aminoglycoside compound to obtain a Cy-aminoglycoside fluorescent probe compound or an acceptable salt thereof:

wherein: the aminoglycoside compound in the targeting group is selected from neomycin, streptomycin, gentamycin, kanamycin, tobramycin and amikacin.

R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ 、R ₇ 、R ₈ 、R ₉ 、R ₁₀ 、R ₁₁ 、R ₁₂ 、R ₁₃ 、R ₁₄ 、R ₁₅ 、R ₁₆ 、 R ₁₇ 、R ₁₈ 、R ₁₉ Independently selected from hydrogen atoms, sulfonic groups, sulfamide, hydroxyl, amino, F, Cl, Br, I, nitro, C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, C1-6 alkoxy, C1-6 alkylamino, C1-6 carboxylic acid, C1-6 carboxylic acid methyl ester and C1-6 alkylamide; or R ₁ And R ₂ ，R ₂ And R ₃ ，R ₃ And R ₄ ，R ₆ And R ₇ ，R ₁₄ And R ₁₅ ，R ₁₆ And R ₁₇ ，R ₁₇ And R ₁₈ ，R ₁₈ And R ₁₉ Adjacent position toBenzene ring, naphthalene ring, anthracene ring, phenanthrene ring form connected; the C1-6 is selected from C1, C2, C3, C4, C5 and C6;

R ₂₄ is selected from

Vinyl, ethynyl, phenyl;

R ₂₅ selected from nitro-substituted benzene, nitro-substituted phenol, nitro-substituted halogenated benzene, nitro-substituted toluene, nitro-substituted naphthalene, nitro-substituted anthracene and nitro-substituted anthracene

m represents 1,2,3, 4;

n represents 1,2,3, 4;

p represents 0,1,2,3,4,5,6,7,8, 9;

x represents F, Cl, Br, I;

L ₂ c1-20 alkyl, C1-20 alkenyl and C1-20 alkynyl, wherein the C1-20 alkynyl is selected from C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19 and C20;

R ₂₆ ，R ₂₈ is independently selected from

The preferable Cy fluorescent probe compound and the aminoglycoside-removed part of the Cy-aminoglycoside fluorescent probe compound are selected from Cy3, Cy3.3, Cy5, Cy5.5, Cy7 and Cy7.5, and have the following structures:

cy fluorescent probe compounds or acceptable salts thereof and Cy-aminoglycoside fluorescent probe compounds or acceptable salts thereof, also include the following compounds:

the fluorescent probe compound is characterized in that the acceptable salt is organic acid salt or inorganic acid salt of the compound, and the organic acid is trifluoroacetic acid, oxalic acid, succinic acid, acetic acid, succinic acid, maleic acid, fumaric acid or tartaric acid; the inorganic acid is hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid or phosphoric acid.

The second aspect of the technical scheme of the invention provides a preparation method of a Cy7 fluorescent probe or acceptable salt thereof and a Cy 7-aminoglycoside fluorescent probe or acceptable salt thereof, which comprises the following steps:

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ 、R ₇ 、R ₈ 、R ₉ 、R ₁₀ 、R ₁₁ 、R ₁₂ 、R ₁₃ 、R ₁₄ 、R ₁₅ 、 R ₁₆ 、R ₁₇ 、R ₁₈ 、R ₁₉ 、R ₂₀ 、R ₂₁ 、R ₂₂ 、R ₂₃ 、R ₂₄ 、R ₂₅ 、R ₂₆ 、R ₂₇ 、R ₂₈ 、L ₁ 、L ₂ As defined above for structures I and II;

R _a is selected from

R _b Selected from amino substituted benzene, amino substituted phenol, amino substituted halogenated benzene, amino substituted toluene, amino substituted naphthalene, amino substituted anthracene

Amino-substituted perylene, amino-substituted benzopyrene, amino-substituted furan, amino-substituted thiophene, amino-substituted pyrrole, amino-substituted imidazole, amino-substituted pyrazole, amino-substituted thiazole, amino-substituted oxazole, amino-substituted pyridine, amino-substituted pyrimidine, amino-substituted pyridazine, amino-substituted pyrazine, amino-substituted purine, amino-substituted quinoline, amino-substituted isoquinoline, sulfonyl of amino-substituted indole, sulfinyl, carbonyl or phosphorus oxide;

a, step a: adding into

Toluene is used as a solvent, and the mixture is refluxed and reacted for 24 hours;

step b: adding a compound 4 and acetic anhydride as a solvent, and reacting for 3 hours at 70 ℃; adding a compound 4 and absolute ethyl alcohol as a solvent, and reacting for 6 hours at 80 ℃;

step c: adding hydroxide anion salt, sodium amide and sodium mercaptan, and N, N-dimethylformamide as solvent, and reacting at 80 deg.C for 2 hr;

step d: adding nitro-substituted aromatic sulfonyl halide, aromatic sulfinyl halide, aromatic carbonyl halide or aromatic phosphorus oxide halide and dichloromethane serving as a solvent, and reacting at room temperature for 1 hour;

step e: adding a condensing agent or a coupling agent of amide, ester, sulfenamide, sulfenylidene, sulfonamide, sulfonyl ester and oxidized phospholipid, alkali and dichloromethane serving as solvents, refluxing, and reacting at room temperature for 6 hours;

step f: adding nitro-substituted aromatic sulfonyl halide, aromatic sulfinyl halide, aromatic carbonyl halide or aromatic phosphorus oxide halide and dichloromethane as a solvent, and reacting at room temperature for 1 hour; adding trifluoroacetic acid, reacting for 10 minutes at 0 ℃;

step g: adding tert-butyloxycarbonyl protected amino substituted aromatic sulfonyl halide, aromatic sulfinyl halide, aromatic carbonyl halide or aromatic phosphorus oxide halide and dichloromethane as solvent, reacting at room temperature for 1 hour; trifluoroacetic acid was added thereto, and the reaction was carried out at 0 ℃ for 10 minutes.

The third aspect of the technical scheme of the invention is to provide the application of the Cy7 fluorescent probe or the acceptable salt thereof and the Cy 7-aminoglycoside fluorescent probe or the acceptable salt thereof in the specific detection of nitroreductase at the molecular level.

The fourth aspect of the technical scheme of the invention provides an application of the Cy 7-aminoglycoside fluorescent probe or the acceptable salt thereof in targeted detection of bacterial infection at a cell or living body level, wherein the fluorescent probe comprises gram-positive bacteria and gram-negative bacteria.

The fifth aspect of the technical scheme of the invention is to provide the Cy 7-aminoglycoside fluorescent probe or the acceptable salt thereof in the first aspect, which can specifically detect bacteria without influencing hypoxic tumor cells and tumor tissues.

The sixth aspect of the technical scheme of the invention also provides application of the Cy7 fluorescent probe or the acceptable salt thereof and the Cy 7-aminoglycoside fluorescent probe or the acceptable salt thereof in preparation of diagnostic reagents.

Advantageous technical effects

The Cy7 fluorescent probe or acceptable salt thereof can detect nitroreductase and can be activated by nitroreductase so that the fluorescence intensity is obviously enhanced.

The Cy 7-aminoglycoside fluorescent probe or the acceptable salt thereof has water solubility, can detect nitroreductase and has the detection limit of 0.65 ng/mL. The fluorescence intensity is obviously enhanced by the activation of nitroreductase and can reach 8 times. And can stably exist in solutions with different pH values for a long time.

The Cy 7-aminoglycoside fluorescent probe or the acceptable salt thereof has a specific recognition function on nitroreductase, and simultaneously has a very fast reaction rate with nitroreductase, 0.2 mu g/mL nitroreductase can completely react 10 mu M of Cy 7-aminoglycoside fluorescent probe and the acceptable salt thereof in 1 minute in the presence of 500 mu M beta-nicotinamide adenine dinucleotide disodium salt hydrate, and is suitable for timely and fast diagnosis of bacteria.

The Cy 7-aminoglycoside fluorescent probe or its acceptable salt can specifically act on the bacterial infection site, but has no effect on the hypoxic tumor cells containing nitroreductase, and can distinguish the bacterial infection site from the hypoxic tumor tissue.

The Cy 7-aminoglycoside fluorescent probe or acceptable salt thereof is an intelligent near-infrared probe molecule which can be activated by nitroreductase and can obviously reduce background signals. In small animal in vivo experiments, the fluorescence signal reached a maximum at 30 minutes and then decreased. The method has good time resolution and spatial resolution in small animal living body imaging experiments.

The detection of the growth inhibition of the cells proves that the Cy 7-aminoglycoside fluorescent probe or the acceptable salt thereof is synthesized, and the Cy 7-aminoglycoside fluorescent probe has no inhibition effect on the cells at the concentration of 50 mu M within 24 hours, namely the toxicity is low.

Drawings

FIG. 1 shows absorption and emission spectra of

probes

1 and 2 and probe 3, a nitroreductase reduction product of probe 2.

FIG. 2 shows that probe 2 (10. mu. mol/L) is immediately reduced by nitroreductase (0.25. mu.g/L) in the presence of beta-nicotinamide adenine dinucleotide disodium salt hydrate, and the fluorescence intensity is significantly enhanced, up to 8-fold.

FIG. 3 shows the selective reduction of probe molecule probe 2 by nitroreductase.

FIG. 4 shows that probes 2 and 3 are not toxic to HepG2 cells at a concentration of 50. mu.M over 24 hours.

FIG. 5 shows that probe 2 has very good enzyme kinetic properties, with a Michaelis constant of 1.88. mu.M for nitroreductase and an initial maximum reaction rate of 0.178. mu.M/s.

FIG. 6 shows that the present probe 2 has a very low detection limit of nitroreductase, 0.65 ng/mL.

FIG. 7 shows that probe 2 was not monitored in hypoxic tumor cells in co-localized fluorescence imaging in HepG2 cells. In staphylococcus aureus, the imaging effect was good.

FIG. 8 shows that the test probe 2 of the present invention can selectively target bacteria imaging in small animal imaging simultaneously inoculated with CT26 tumor cells and Staphylococcus aureus, without affecting the tumor. And the fluorescence intensity reached a maximum at 30 minutes and then decreased. It is shown that probe 2 has good temporal and spatial resolution for imaging bacteria.

The specific implementation mode is as follows:

synthesis of

probe molecules

1,2, 3:

preparation example 1 preparation of Compound 2

Substrate 1(10g, 62.8mmol) was uniformly dispersed in toluene solvent (16mL), and after 6-bromohexanoic acid (12.2g,62.8mmol) was added, the reaction was refluxed for 24 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was washed with dehydrated ether (3X 32mL) and dichloromethane (3X 32mL) in this order, and the product was dried and used directly in the next step. Compound 2 was a white solid, 9.7g, 43.5% yield. ¹ H NMR(500 MHz,DMSO-d ₆ )δ12.00(s,1H),δ8.07–7.95(m,1H),7.91–7.80(m,1H),7.67– 7.56(m,2H),4.47(t,J＝7.7Hz,2H),2.87(s,3H),2.22(t,J＝7.3Hz,2H),1.84(p,J ＝7.8Hz,2H),1.59–1.51(m,8H),1.42(p,J＝8.0,7.4Hz,2H)； ¹³ C NMR(125MHz, DMSO-d ₆ ) δ 196.99,174.80,142.33,141.52,129.84,129.40,124.00,115.99,54.63, 47.93,33.83,27.42,25.86,24.49,22.47, 14.59; HRMS (ESI) with a theoretical value of m/z of C ₁₇ H ₂₄ NO ₂ ⁺ :274.1802[M] ⁺ (ii) a Found 274.1802.

Preparation example 2 preparation of Compound 5

Substrate 4(3.1g, 11.6mmol) in acetic anhydride solvent (16.5mL) was added dropwise at room temperatureTo a solvent (16.5mL) of acetic anhydride to substrate 3(2.0g, 11.6 mmol). After the reaction system was reacted at 70 ℃ for 3 hours, the reaction system was cooled to room temperature, and the solvent was distilled off under reduced pressure. The reaction mixture was diluted with anhydrous ethanol (33mL), and compound 2(4.9g, 13.9mmol) and sodium acetate (2.85g, 34.8mmol) were added sequentially. After the reaction system was reacted at 80 ℃ for 6 hours, the solvent was distilled off under reduced pressure, and the residue was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The residue was purified by medium pressure column chromatography (6% MeOH/CH) ₂ Cl ₂ ) Product 5 was obtained as a green solid, 2.9g, 38% yield. ¹ H NMR(500MHz,CDCl ₃ )δ8.37(d,J＝14.1Hz,1H),8.32(d,J＝ 13.9Hz,1H),7.44–7.33(m,4H),7.25–7.17(m,2H),7.13(d,J＝8.0Hz,1H),6.26 (d,J＝14.1Hz,1H),6.13(d,J＝14.0Hz,1H),4.20–4.12(m,4H),2.74(t,J＝6.1 Hz,2H),2.70(t,J＝6.0Hz,2H),2.60(t,J＝7.2Hz,2H),2.00(p,J＝6.2Hz,2H), 1.87(p,J＝7.8Hz,2H),1.79(p,J＝7.3Hz,2H),1.71(s,12H),1.57(q,J＝7.5,7.0 Hz,2H),1.44(t,J＝7.2Hz,3H)； ¹³ C NMR(125MHz,CDCl ₃ ) δ 176.01,172.97, 171.33,150.88,145.20,144.10,142.09,141.95,141.21,141.12,129.10,128.92, 128.06,127.62,125.69,125.15,122.39,122.33,111.27,110.47,101.93,100.42, 49.59,49.28,44.75,39.62,34.73,28.25,28.22,27.01,26.71,26.65,26.31,24.64, 20.79, 12.43; HRMS (ESI) with a theoretical value of m/z of C ₃₈ H ₄₆ ClN ₂ O ₂ ⁺ :597.3242[M] ⁺ (ii) a Found 597.3242.

Preparation example 3 preparation of Compound 6

Substrate 5(200mg, 0.295mmol) was dissolved in N, N-dimethylformamide (7.9mL) under argon blanket, and sodium acetate (73mg, 0.885mmol) was added slowly. The reaction system was reacted at 80 ℃ for 2 hours. After completion of the reaction, the reaction system was diluted with methylene chloride (40mL), washed successively with water (2X 14mL) and saturated brine (3X 14mL), dried over anhydrous sodium sulfate, suction filtered, concentrated under reduced pressure, and the residue was purified by medium pressure column chromatography (2% MeOH/CH) ₂ Cl ₂ ) Product 6 was obtained as a red solid, 100mg, 59% yield. ¹ H NMR(500MHz,CDCl ₃ )δ8.20(dd,J＝13.4,5.6Hz, 2H),7.18(p,J＝6.6Hz,4H),6.91(q,J＝7.1Hz,2H),6.68(t,J＝7.0Hz,2H),5.48 (dd,J＝13.3,6.8Hz,2H),3.74(q,J＝7.2Hz,2H),3.68(t,J＝7.5Hz,2H),2.61(q,J ＝5.3Hz,4H),2.40(t,J＝7.4Hz,2H),1.87(p,J＝6.3Hz,2H),1.74(t,J＝7.5Hz, 4H),1.67(d,J＝3.5Hz,12H),1.54–1.43(m,2H),1.27(t,J＝7.1Hz,3H)； ¹³ C NMR(125MHz,CDCl ₃ ) δ 186.58,177.69,162.79,162.45,144.08,143.62,139.87, 139.72,133.87,133.72,127.71,126.43,126.30,121.83,120.68,106.78,106.59, 92.69,92.36,53.50,46.66,42.41,37.11,34.15,30.59,28.72,28.66,26.71,26.03, 25.81,24.62,22.49, 11.18. HRMS (ESI) with a theoretical value of m/z of C ₃₈ H ₄₇ N ₂ O ₃ ⁺ :579.3581 [M+H] ⁺ (ii) a Found 579.3581.

Preparation example 4 preparation of Probe 1

Substrate 6(40mg, 0.069mmol) was dissolved in dichloromethane (1.7mL) under argon and p-nitrobenzenesulfonyl chloride (38mg, 0.173mmol) was added slowly. The reaction system was reacted at room temperature for 1 hour. After the reaction was completed, the reaction was quenched by adding anhydrous methanol (2 uL). The solvent was removed by concentration under reduced pressure and the residue was purified by medium pressure column chromatography (4% MeOH/CH) ₂ Cl ₂ ) The product was obtained as a green solid probe 1,26 mg, with a yield of 49%. ¹ H NMR(500MHz,CDCl ₃ )δ8.46(d,J＝8.4Hz,2H),8.23(d,J ＝8.4Hz,2H),7.79(dd,J＝14.1,6.8Hz,2H),7.36(t,J＝7.8Hz,2H),7.32(t,J＝6.6 Hz,2H),7.22(t,J＝7.5Hz,2H),7.13(t,J＝8.3Hz,2H),6.06(dd,J＝20.6,14.0Hz, 2H),4.12(q,J＝7.3Hz,2H),4.07(d,J＝7.7Hz,2H),2.57(q,J＝6.9Hz,3H),2.41 (t,J＝7.1Hz,2H),1.82(q,J＝6.9Hz,4H),1.70(h,J＝9.2Hz,3H),1.53(s,6H), 1.51(s,8H),1.40(t,J＝7.4Hz,3H)； ¹³ C NMR(125MHz,CDCl ₃ ) δ 172.53,171.73, 157.13,152.98,151.46,148.03,141.93,141.65,141.24,141.16,141.07,140.83, 140.54,130.10,129.04,127.67,125.82,125.64,124.87,123.73,123.29,122.38, 111.24,110.78,101.64,100.86,49.40,49.33,44.56,39.67,34.30,30.65,27.66,27.62, 27.00,26.31,25.48,24.53,20.36,19.26, 12.37. HRMS (ESI) with a theoretical value of m/z of C ₄₄ H ₅₀ N ₃ O ₇ S ⁺ :764.3364[M] ⁺ (ii) a Found 764.3368.

Preparation example 5 preparation of

probes

2,3

After dissolving the substrate 5(68mg, 0.098mmol) and the substrate 7(100mg, 0.082mmol) in a dichloromethane solution (1.0mL), 2- (7-benzotriazole oxide) -N, N '-tetramethyluronium hexafluorophosphate (62mg, 0.164mmol) and N, N' -diisopropylethylamine (32mg, 0.246mmol) were sequentially added, and the reaction was stirred at room temperature. After completion of the reaction as checked by thin layer chromatography, the reaction system was diluted with dichloromethane (3.0 mL), washed with water (4.0mL) and saturated brine (4.0mL), dried over anhydrous sodium sulfate, filtered under vacuum, concentrated under reduced pressure, and the residue was purified by medium pressure column chromatography (3% MeOH/CH) ₂ Cl ₂ ) After that, it was used directly in the next step.

The product of the previous step, 8(60mg, 0.034mmol), was dissolved in dichloromethane (680uL) under argon and p-nitrobenzenesulfonyl chloride (18.7mg, 0.085mmol) was added slowly. The reaction system was reacted at room temperature for 1 hour. After the reaction was completed, the reaction was quenched by adding anhydrous methanol (2 uL). The solvent was removed by concentration under reduced pressure, and the residue was purified by medium pressure column chromatography and used directly in the next step.

Trifluoroacetic acid (680uL) is added dropwise to the product obtained in the previous step under the condition of 0 ℃ under the protection of argon. The reaction system was allowed to continue at 0 ℃ for 10 minutes. After the reaction is finished, the reaction solution is directly concentrated under reduced pressure, and the residue is directly purified by a high-pressure chromatographic column to obtain green solid product probes 2 and 24mg, wherein the yield in the three steps is 12%. ¹ H NMR(500 MHz,D ₂ O)δ8.34–8.17(m,2H),7.95–7.78(m,2H),7.57–7.36(m,3H),7.23(d, J＝24.2Hz,2H),7.10(s,3H),6.89(s,3H),6.75(s,1H),5.90(s,1H),5.38–5.30(m, 1H),5.22(s,1H),4.31(s,1H),4.29–4.19(m,3H),4.17(s,3H),4.11–4.01(m,3H), 4.00–3.94(m,2H),3.95–3.83(m,5H),3.76(s,2H),3.70(t,J＝9.5Hz,2H),3.54 (s,2H),3.53–3.41(m,6H),3.41–3.28(m,2H),3.27–3.16(m,2H),2.45(s,1H), 2.14(s,6H),1.89(q,J＝13.1Hz,2H),1.61–1.51(m,2H),1.48–1.36(m,7H),1.24 (s,5H),1.22–1.13(m,13H),1.13–0.99(m,9H)； ¹³ C NMR(125MHz,D ₂ O)δ 176.67,171.36,167.68,167.62,166.76,164.94,162.92,162.64,162.35,162.07, 155.91,151.19,141.69,141.36,140.77,140.54,139.35,139.22,132.37,130.96, 130.61,129.71,129.60,129.52,128.96,128.56,124.70,122.82,121.38,119.87, 117.54,115.22,112.89,109.02,100.94,98.46,95.78,94.91,91.33,84.93,82.73, 80.88,77.44,75.04,73.42,72.25,70.42,70.24,70.12,69.85,68.12,67.53,67.42, 66.58,64.82,53.24,50.84,49.52,48.65,48.53,41.08,40.45,40.05,38.72,35.58, 30.43,27.87,26.91,25.94,25.04,18.93,17.95,16.65,13.31, 11.50; HRMS (ESI) m/z theoretical value of C ₆₇ H ₉₅ N ₁₀ O ₁₈ S ⁺ :1359.6541[M] ⁺ (ii) a Found 1359.6548.

Product 8(60mg, 0.034mmol) was dissolved in dichloromethane (680uL) under argon and t-butyloxycarbonyl-protected sulfanylsulphonyl chloride (24.7mg, 0.085mmol) was added slowly. The reaction system was reacted at room temperature for 1 hour. After the reaction was completed, the reaction was quenched by adding anhydrous methanol (2 uL). The solvent was removed by concentration under reduced pressure, and the residue was purified by medium pressure column chromatography and used directly in the next step.

Trifluoroacetic acid (680uL) is added dropwise to the product obtained in the previous step under the condition of 0 ℃ under the protection of argon. The reaction system was allowed to continue at 0 ℃ for 10 minutes. After the reaction is finished, the reaction solution is directly concentrated under reduced pressure, and the residue is directly purified by a high-pressure chromatographic column to obtain a green solid product, namely the probe 3, 21mg, wherein the yield of the three steps is 11.5%. ¹ H NMR (500MHz,D ₂ O)δ8.34–8.17(m,2H),7.95–7.78(m,2H),7.57–7.36(m,3H), 7.23(d,J＝24.2Hz,2H),7.10(s,3H),6.89(s,3H),6.75(s,1H),5.90(s,1H),5.38– 5.30(m,1H),5.22(s,1H),4.31(s,1H),4.29–4.19(m,3H),4.17(s,3H),4.11–4.01 (m,3H),4.00–3.94(m,2H),3.95–3.83(m,5H),3.76(s,2H),3.70(t,J＝9.5Hz, 2H),3.54(s,2H),3.53–3.41(m,6H),3.41–3.28(m,2H),3.27–3.16(m,2H),2.45 (s,1H),2.14(s,6H),1.89(q,J＝13.1Hz,2H),1.61–1.51(m,2H),1.48–1.36(m, 7H),1.24(s,5H),1.22–1.13(m,13H),1.13–0.99(m,9H)； ¹³ C NMR(125MHz, D ₂ O)δ176.67,171.36,167.68,167.62,166.76,164.94,162.92,162.64,162.35, 162.07,155.91,151.19,141.69,141.36,140.77,140.54,139.35,139.22,132.37, 130.96,130.61,129.71,129.60,129.52,128.96,128.56,124.70,122.82,121.38, 119.87,117.54,115.22,112.89,109.02,100.94,98.46,95.78,94.91,91.33,84.93, 82.73,80.88,77.44,75.04,73.42,72.25,70.42,70.24,70.12,69.85,68.12,67.53, 67.42,66.58,64.82,53.24,50.84,49.52,48.65,48.53,41.08,40.45,40.05,38.72, 35.58,30.43,27.87,26.91,25.94,25.04,18.93,17.95,16.65,13.31, 11.50; HRMS (ESI) m/z theoretical value of C ₆₇ H ₉₅ N ₁₀ O ₁₈ S ⁺ :1359.6541[M] ⁺ (ii) a Found 1359.6548.

Pharmacological experiments

Experimental example 1: absorption emission Spectroscopy of

probes

1,2 and 3

Probe

molecular probes

1,2 and 3 were dissolved in 0.05M tris solution (pH 7.4, 1.5% dmso) and the absorbance and normal emission spectra of the compounds were measured using a microplate reader. See fig. 1.

Experimental example 2: reduction of Probe 2 by Nitroreductase

Probe 2 was dissolved in 0.05M tris solution (pH 7.4, containing 1.5% dimethylsulfoxide), and β -nicotinamide adenine dinucleotide disodium salt hydrate (500 μ M) and nitroreductase (0.25 μ g/L) were added in this order, and the emission spectrum (excitation wavelength 750nm) of the compound was immediately monitored. The probe 2(10 mu mol/L) is immediately reduced by nitroreductase (0.25 mu g/L), and the fluorescence intensity is obviously enhanced and can reach 8 times. See fig. 2.

Experimental example 3: probe 2 can be selectively subjected to nitroreductase reduction experiments.

Probe 2 was dissolved in 0.05M tris solution (pH 7.4 containing 1.5% dimethylsulfoxide) and in the presence of β -nicotinamide adenine dinucleotide disodium salt hydrate (500 μ M), sodium chloride (50mM), potassium chloride (50mM), calcium chloride (50mM), magnesium chloride (50mM), vitamin C (1mM), cysteine (1mM), glutathione (1mM), sodium hypochlorite (1mM), DL-homocysteine (1mM), dithiothreitol (1mM), glucose (10mM), hydrogen peroxide (1mM), arginine (1mM), nitroreductase (0.25 μ g/L) was added to determine the emission spectrum (excitation wavelength 750nm) of the compound after 1 hour of reaction under 37 degree water bath conditions. Indicating that probe 2 can be specifically reduced by nitroreductase. See fig. 3.

Experimental example 4: probe 2 was not toxic to HepG2 cells at a concentration of 50. mu. mol/L.

Cell viability was determined using the MTS method. One experimental group and nine control groups were set. Adherent cells in a 96-well plate were treated sequentially with probe 2 at concentrations of 0.2. mu.M, 0.4. mu.M, 0.8. mu.M, 1.6. mu.M, 3.2. mu.M, 6.25. mu.M, 12.5. mu.M, 25. mu.M and 50. mu.M, at 37 ℃ with 5% CO ₂ After 24 hours of incubation under the conditions, 20. mu.L of MTS was added to each well, followed by incubation at 37 ℃ with 5% CO ₂ Culturing for 4h, and measuring the absorption OD value of each group on a microplate reader, wherein the wavelength is 490 nm. Cell viability was calculated by the formula based on the OD of each well. The formula is as follows: cell survival (%) × (experimental OD/control OD) × 100%. Probe 2 showed no cytotoxicity to HepG2 at a concentration of 50 μ M, indicating that probe 2 had low cytotoxicity. See fig. 4.

Experimental example 5: probe 2 enzyme kinetic property experiments.

Probe 2(3 μ M,4 μ M,5 μ M,6 μ M,7 μ M and 8 μ M) was dissolved in 0.05M tris solution (pH 7.4 containing 1.5% dimethylsulfoxide), and the change in the emission spectrum of the compound with time (excitation wavelength 750nm, emission wavelength 800nm) was measured immediately after the addition of nitroreductase (0.25 μ g/L) in the presence of β -nicotinamide adenine dinucleotide disodium salt hydrate (500 μ M). The apparent mie constant and the initial maximum reaction rate were calculated to be 11.88 μ M and 0.178 μ M/s, respectively, according to the mie equation V ═ Vmax [ probe ]/(Km + [ probe ]) (V represents the maximum reaction rate, [ probe ] represents the probe concentration, and Km represents the mie constant). See fig. 5.

Experimental example 6: experiment for representing lowest detection line of 2 enzymes in probe

Probe 2 was dissolved in 0.05M tris solution (pH 7.4 containing 1.5% dmso), and the emission spectrum of the compound (excitation wavelength 750nm, maximum emission wavelength 800nm) was measured immediately after nitroreductase (0.06, 0.05,0.04,0.03,0.02,0.01, and 0.005 μ g/L) was added in the presence of β -nicotinamide adenine dinucleotide disodium salt hydrate (500 μ M). 64795C (μ g/mL) + 1875.1 (R) according to linear equation y ² 0.9945) the limit of enzyme detection (3 δ/k, δ representing the standard deviation of 11 control samples, k representing the slope of the linear equation) was calculated to be 0.65 ng/mL. Thus, the detection limit of the nitroreductase of the probe 2 is very low.See fig. 6.

Experimental example 7: representing the Co-aggregate fluorescence imaging of Probe 2 in Staphylococcus aureus (ATCC29213) and HepG2 cells

Bacterial confocal imaging: probe 2 (10. mu.M) was incubated with Staphylococcus aureus at 37 ℃ for 1 hour and then stained with Hoechst 33342 at 37 ℃ for 0.5 hour. After 3 phosphate buffer washes, cells were imaged. The scale bar is 8 μm. Hypoxic tumor cell co-localization imaging: after 12 hours of incubation of HepG2 cells at 1% oxygen concentration, probe 2 (10. mu.M) was incubated for 30 minutes under normal oxygen conditions and then stained with Hoechst 33342 at 37 ℃ for 0.5 hour. After 3 washes with phosphate buffer, cells were imaged. The scale bar is 25 μm. In imaging staphylococcus aureus and HepG2 cells, Hoechst 33342 was used for nuclear staining with an excitation wavelength of 405nm and an emission wavelength collection range of 460 ± 30 nm. 670nm is selected as the excitation wavelength of the eight-hole plate containing the probe, and the collection range of the emission wavelength is 770 +/-30 nm. It is demonstrated that probe 2 has good imaging effect when applied to bacterial cells, but no effect in hypoxic tumor cells. See fig. 7.

Experimental example 8: the probe 2 is shown in application to small animal in vivo imaging.

BALB/c mice weighing 20 g on average for 6-8 weeks were injected subcutaneously with CT26 cell suspension (200. mu.L, approx. 2X 10) at the end of the left hind limb ⁶ Individual cells). After one week of culture, staphylococcus aureus (50 μ L, optical density (measurement wavelength 600nm) ═ 1) was injected subcutaneously into each right hind limb. Probe 2 (100. mu.L, 20. mu.M) was then injected immediately into the tail vein. Small animal in vivo imaging monitoring was performed at 0h, 0.5h, 1h, 2h, 4h and 6h, respectively (excitation wavelength 745nm, emission wavelength 820 nm). The results indicate that probe 2 can be used to specifically target bacteria without affecting tumor cells and that fluorescence intensity reaches a maximum at 30 minutes and then decreases. It is shown that probe 2 has good temporal and spatial resolution for imaging bacteria. See fig. 8.

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Claims

1. A compound Cy fluorescent probe 1 shown in the following structure or an acceptable salt thereof and a Cy-aminoglycoside fluorescent probe 2 or an acceptable salt thereof,

2. the compound of claim 1, which is Cy-based fluorescent probe 1 or an acceptable salt thereof, and Cy-aminoglycoside-based fluorescent probe 2 or an acceptable salt thereof, wherein the acceptable salt is an organic acid salt or an inorganic acid salt of the compound, and the organic acid is trifluoroacetic acid, oxalic acid, succinic acid, acetic acid, succinic acid, maleic acid, fumaric acid, or tartaric acid; the inorganic acid is hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid or phosphoric acid.

3. The use of the compound of claim 1, Cy-based fluorescent probe 1 or an acceptable salt thereof, and Cy-aminoglycoside-based fluorescent probe 2 or an acceptable salt thereof, for preparing a detection reagent specific for nitroreductase at a molecular level.

4. The use of the compounds Cy-aminoglycoside fluorescent probe 1 or an acceptable salt thereof and Cy-aminoglycoside fluorescent probe 2 or an acceptable salt thereof as claimed in claim 1 for the preparation of a reagent for the targeted detection of bacterial infections at the cellular or living level.

5. Use according to claim 4, wherein said bacteria are selected from the group consisting of gram-negative and gram-positive bacteria.

6. The use of the compound of claim 1, Cy-based fluorescent probe 1 or an acceptable salt thereof, and Cy-aminoglycoside-based fluorescent probe 2 or an acceptable salt thereof, for the preparation of a diagnostic reagent.