CN118184667A - Rhodamine-cyanine compound containing hydrazide, tautomer thereof, preparation method and application thereof - Google Patents
Rhodamine-cyanine compound containing hydrazide, tautomer thereof, preparation method and application thereof Download PDFInfo
- Publication number
- CN118184667A CN118184667A CN202410306946.4A CN202410306946A CN118184667A CN 118184667 A CN118184667 A CN 118184667A CN 202410306946 A CN202410306946 A CN 202410306946A CN 118184667 A CN118184667 A CN 118184667A
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- China
- Prior art keywords
- reaction
- rhodamine
- hydrazide
- formula
- tautomer
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/10—Spiro-condensed systems
- C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention belongs to the technical field of biological detection, and provides a rhodamine-cyanine compound containing hydrazide, a tautomer thereof, a preparation method and application thereof. The rhodamine-cyanine compound containing the hydrazide and the tautomer thereof provided by the invention have the following structures. According to the invention, the electron cloud density of amide at the spiro ring is regulated and controlled through R, wherein fatty amine is introduced into 1 series, benzenesulfonamide is introduced into 2 series, and aniline is introduced into 3 series. In addition, the effect of hydrophobicity on probe imaging contrast was investigated by designing different chain lengths. The probe has novel design mechanism, can release fluorescence after being combined with oxidized collagen, is a targeted allysine small-molecule fluorescent probe capable of detecting fibrosis at animal and tissue level, has high imaging contrast and has great potential in the application of fibrosis drug screening.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a rhodamine-cyanine compound containing hydrazide, a tautomer thereof, a preparation method and application thereof.
Background
Fibrosis is a common feature of many chronic diseases, with a significant impact on morbidity and mortality worldwide, estimated to account for 45% of all deaths in the industrialized world. Improving the efficiency of fibrosis detection methods is critical to help solve the problem of lack of treatment for fibrosis, as these methods play a dual role in advancing early disease detection and accelerating drug discovery.
To date, many molecular imaging targets for fibrosis and fibrogenesis have been identified. Notably, oxidized collagen, in which lysine residues are oxidized to Lai Anquan (allysine) catalyzed by Lysyl Oxidase (LOX) in the extracellular matrix (ECM), is a common feature of active fibrogenesis. Under normal conditions, the levels of LOX and allysine are low in healthy, mature mammalian body tissues, however, allysine concentrations can reach hundreds of micromolar in pathological processes such as pulmonary and hepatic fibrosis.
Several molecular imaging probes for allysine have now been developed for detecting and analyzing fibrosis progression in animal models, as well as monitoring therapeutic response. However, reported probes targeting allysine are primarily associated with radiological imaging modalities (such as MRI, PET, and SPECT), and few fluorescent probes are designed for imaging allysine, a difference that is of interest. Unlike the radioactive mode, fluorescence imaging is non-radioactive, providing a safer alternative. It has high sensitivity and specificity, multiple functions and high spatial resolution, and makes it a multifunctional technology widely used in biomedical research and clinical diagnosis. The general fluorescent normally bright probe cannot distinguish allysine from background signals, and the challenge in designing a fluorescent probe for allysine imaging is how to have a higher signal-to-noise ratio and imaging contrast for the probe.
Disclosure of Invention
In view of the above, the present invention aims to provide a rhodamine-cyanine compound containing hydrazide, a tautomer thereof, a preparation method and an application thereof. The rhodamine-cyanine compound containing the hydrazide and the tautomer thereof can be specifically combined with allysine targets, and have higher signal-to-noise ratio and imaging contrast.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides a rhodamine-cyanine compound containing hydrazide and a tautomer thereof, wherein the rhodamine-cyanine compound containing hydrazide has the following structure:
In the formula I-1, the formula I-2 or the formula I-3, the value of n is independently 3, 5 or 7.
Preferably, the tautomer of the hydrazide-containing rhodamine-cyanine compound has the following structure:
in the formula II-1, the formula II-2 or the formula II-3, the value of n is independently 3, 5 or 7.
The invention also provides a preparation method of the rhodamine-cyanine compound containing the hydrazide and the tautomer thereof, which comprises the following steps:
4-diethylamino keto acid and cyclohexanone are subjected to ring-adding reaction under the conditions of concentrated sulfuric acid and perchloric acid to obtain a first intermediate with a structure shown in a formula 1;
the first intermediate performs Vilsmeier-Haack reaction under the condition of phosphorus oxychloride to obtain a second intermediate with a structure shown in a formula 2;
the second intermediate and the 1,2, 3-tetramethyl-3H-indolium iodide undergo nucleophilic substitution reaction under the condition of piperidine to obtain a fluorophore NIR-COOH with a structure shown in a formula 3;
One of methyl 4-sulfonamide benzoate and methyl 4-aminobenzoate and the fluorophore NIR-COOH are subjected to a first condensation reaction and a methyl ester hydrolysis reaction under the conditions of 4-dimethylaminopyridine and EDCI & HCl to obtain an intermediate compound 1 and an intermediate compound 2:
The intermediate compound 1 has a structure shown in a formula 4;
the intermediate compound 2 has a structure represented by formula 5:
Fmoc-NH- (CH 2) n-COOH and tert-butyl hydrazinoformate are subjected to a second condensation reaction and 9-fluorenylmethoxycarbonyl removal under EDCI & HCl conditions to obtain a side chain intermediate compound shown in formula 6;
And (3) performing a third condensation reaction and removing tert-butoxycarbonyl groups on one of the fluorophore NIR-COOH, the intermediate compound 1 and the intermediate compound 2 and the side chain intermediate compound under EDCI & HCl conditions to obtain the rhodamine-cyanine compound containing hydrazide and a tautomer thereof.
Preferably, the nucleophilic substitution reaction is carried out at normal temperature for 10-12 hours.
Preferably, the temperature of the first condensation reaction is 50-70 ℃ and the time is 12-14 h;
The reaction solvent of the methyl ester hydrolysis reaction is tetrahydrofuran, the alkali of the methyl ester hydrolysis reaction is lithium hydroxide solution, the concentration of the lithium hydroxide solution is 1mol/L, and the volume ratio of the lithium hydroxide solution to the tetrahydrofuran is 1:1, the temperature of the methyl ester hydrolysis reaction is normal temperature, and the time is 10 hours.
Preferably, the reaction solvent of the second condensation reaction is anhydrous dichloromethane, the temperature of the second condensation reaction is normal temperature, and the time is 6-10 h;
The solvent for removing the 9-fluorenylmethoxycarbonyl is methylene dichloride, and the base for removing the 9-fluorenylmethoxycarbonyl is diethylamine; the volume fraction of the diethylamine in the system for removing 9-fluorenylmethoxycarbonyl is 20-30%, the temperature for removing 9-fluorenylmethoxycarbonyl is normal temperature, and the time is 8 hours.
Preferably, the temperature of the third condensation reaction is normal temperature and the time is 8-10 h;
The solvent for removing the tert-butoxycarbonyl is methylene dichloride, the acid for removing the tert-butoxycarbonyl is hydrochloric acid, and the concentration of the hydrochloric acid is 2mol/L; the volume ratio of the hydrochloric acid to the dichloromethane is 2:1; the temperature for removing the tert-butoxycarbonyl group is normal temperature and the time is 8 hours.
The invention also provides the rhodamine-cyanine compound containing the hydrazide and a tautomer thereof or the application of the rhodamine-cyanine compound containing the hydrazide and the tautomer thereof prepared by the preparation method of the technical scheme in protein detection, wherein the protein contains aldehyde groups and/or ketone carbonyl groups.
The invention also provides application of the rhodamine-cyanine compound containing the hydrazide and the tautomer thereof or the rhodamine-cyanine compound containing the hydrazide and the tautomer thereof prepared by the preparation method of the technical scheme in diagnosis of non-diseases and detection of fibrosis collagen for treatment purposes.
The invention provides a rhodamine-cyanine compound containing hydrazide and a tautomer thereof, wherein the rhodamine-cyanine compound containing hydrazide has the following structure:
In the formula I-1, the formula I-2 or the formula I-3, the value of n is independently 3, 5 or 7.
According to the invention, the electron cloud density of amide at the spiro ring is regulated by R, fatty amine is introduced in the formula I-1 series, benzenesulfonamide is introduced in the formula I-2 series, and aniline is introduced in the formula I-3 series. In addition, the effect of hydrophobicity on probe imaging contrast was investigated by designing different chain lengths. The probe has novel design mechanism, can release fluorescence after being combined with oxidized collagen, is a targeted allysine small-molecule fluorescent probe capable of detecting fibrosis at animal and tissue level, has high imaging contrast and has great potential in the application of fibrosis drug screening.
The data of the examples show that: the rhodamine-cyanine compound containing hydrazide and the tautomer thereof provided by the invention not only can obviously distinguish normal liver/lung and fibrotic liver/lung, but also can effectively distinguish a model group of a mouse model induced by NASH high-fat high-cholesterol and carbon tetrachloride from an OCA drug treatment group. From the results, the fluorescence intensity of the model group is obviously higher than that of the OCA treatment group, and the results show that the rhodamine-cyanine compound containing the hydrazide has wide prospect in application of screening the drug effect of the fibrosis treatment drug.
Drawings
FIG. 1 is a quantitative image of fluorescence imaging of probes An/Bn/Cn (n=1, 2, 3) as indicators in liver tissue of normal mice and carbon tetrachloride-induced liver fibrosis mice;
FIG. 2 is a comparison of liver fluorescence imaging of probe B2 in normal mice and carbon tetrachloride-induced liver fibrosis mice;
FIG. 3 is a comparison of pulmonary fluorescence imaging of probe B2 in normal mice and bleomycin-induced pulmonary fibrosis mice;
Fig. 4 is a comparison of liver fluorescence imaging of probe B2 in untreated liver fibrosis mice and OCA treated liver fibrosis mice.
Detailed Description
The invention provides a rhodamine-cyanine compound containing hydrazide and a tautomer thereof, wherein the rhodamine-cyanine compound containing hydrazide has the following structure:
In the formula I-1, the formula I-2 or the formula I-3, the value of n is independently 3, 5 or 7.
In the present invention, the tautomer of the rhodamine-cyanine compound containing hydrazide has the following structure:
in the formula II-1, the formula II-2 or the formula II-3, the value of n is independently 3, 5 or 7.
The invention also provides a preparation method of the rhodamine-cyanine compound containing the hydrazide and the tautomer thereof, which comprises the following steps:
4-diethylamino keto acid and cyclohexanone are subjected to ring-adding reaction under the conditions of concentrated sulfuric acid and perchloric acid to obtain a first intermediate with a structure shown in a formula 1;
the first intermediate performs Vilsmeier-Haack reaction under the condition of phosphorus oxychloride to obtain a second intermediate with a structure shown in a formula 2;
the second intermediate and the 1,2, 3-tetramethyl-3H-indolium iodide undergo nucleophilic substitution reaction under the condition of piperidine to obtain a fluorophore NIR-COOH with a structure shown in a formula 3;
One of methyl 4-sulfonamide benzoate and methyl 4-aminobenzoate and the fluorophore NIR-COOH are subjected to a first condensation reaction and a methyl ester hydrolysis reaction under the conditions of 4-dimethylaminopyridine and EDCI & HCl to obtain an intermediate compound 1 and an intermediate compound 2:
The intermediate compound 1 has a structure shown in a formula 4;
the intermediate compound 2 has a structure represented by formula 5:
Fmoc-NH- (CH 2) n-COOH and tert-butyl hydrazinoformate are subjected to a second condensation reaction and 9-fluorenylmethoxycarbonyl removal under EDCI & HCl conditions to obtain a side chain intermediate compound shown in formula 6;
And (3) performing a third condensation reaction and removing tert-butoxycarbonyl groups on one of the fluorophore NIR-COOH, the intermediate compound 1 and the intermediate compound 2 and the side chain intermediate compound under EDCI & HCl conditions to obtain the rhodamine-cyanine compound containing hydrazide and a tautomer thereof.
In the present invention, the raw materials used in the present invention are preferably commercially available products unless otherwise specified.
The 4-diethylamino keto acid and cyclohexanone undergo a cyclization reaction under the conditions of concentrated sulfuric acid and perchloric acid to obtain a first intermediate with a structure shown in a formula 1;
in the present invention, the ratio of the amounts of the substances of the 4-diethylaminoketo acid and cyclohexanone is preferably 1:1 to 2. In the present invention, the mass concentration of the concentrated sulfuric acid is preferably 98%; the mass concentration of the perchloric acid is preferably 50%, and the volume ratio of the perchloric acid to the concentrated sulfuric acid is preferably 1:7. in the invention, the dosage ratio of the concentrated sulfuric acid to the cyclohexanone is preferably 14mL:10 to 15mmol, more preferably 14mL:12.74mmol.
In the invention, the ring-increasing reaction preferably comprises a first ring-increasing reaction and a second ring-increasing reaction which are sequentially carried out, wherein the temperature of the first ring-increasing reaction is preferably 80-100 ℃, more preferably 90 ℃, and the reaction time is preferably 2-3 h; the temperature of the second ring-increasing reaction is preferably ice water bath, and the time of the second ring-increasing reaction is not particularly limited in the invention, so long as the product is not precipitated any more.
In the present invention, the ring-increasing reaction of the 4-diethylamino-keto acid and cyclohexanone under the condition of concentrated sulfuric acid and perchloric acid specifically preferably comprises the following steps: adding cyclohexanone dropwise into concentrated sulfuric acid under ice-water bath, then adding 4-diethylamino keto acid in batches under intense stirring, and carrying out first cyclization reaction at a certain temperature; after the first cyclization reaction is finished, cooling to room temperature, slowly pouring the obtained first cyclization reaction feed liquid into ice cubes, and then slowly dripping perchloric acid into a reaction system under ice bath to perform a second cyclization reaction.
After the cyclization reaction, the invention preferably further comprises a post-treatment, wherein the post-treatment preferably comprises the following steps: and carrying out suction filtration on the obtained cyclization reaction feed liquid, and sequentially carrying out ice water washing and vacuum drying on the obtained filter residues.
After a first intermediate is obtained, the first intermediate is subjected to Vilsmeier-Haack reaction under the condition of phosphorus oxychloride to obtain a second intermediate with a structure shown in a formula 2;
In the present invention, the ratio of the first intermediate to phosphorus oxychloride is preferably 1.76 mmol/5 mL. In the present invention, the reaction solvent of the Vilsmeier-Haack reaction is preferably N, N-Dimethylformamide (DMF), and more preferably anhydrous N, N-dimethylformamide.
In the present invention, the Vilsmeier-Haack reaction preferably includes sequentially performing a first Vilsmeier-Haack reaction and a second Vilsmeier-Haack reaction; the temperature of the first Vilsmeier-Haack reaction is preferably ice water bath, and the time is preferably 0.5-2 h; the temperature of the second Vilsmeier-Haack reaction is preferably room temperature, and the time is preferably 1-2 h.
In the present invention, the first intermediate is subjected to the Vilsmeier-Haack reaction under the condition of phosphorus oxychloride, and particularly preferably comprises the following steps: adding a reaction solvent into a reaction bottle, carrying out nitrogen replacement on the reaction solvent, slowly dropwise adding phosphorus oxychloride into the reaction bottle under the conditions of ice bath and stirring, and carrying out a first Vilsmeier-Haack reaction in the ice bath to obtain a Vilsmeier reagent; dissolving the first intermediate by using a reaction solvent to obtain a first intermediate solution; and dropwise adding the first intermediate solution into the Vilsmeier reagent in an ice bath, recovering to room temperature, and performing a second Vilsmeier reaction.
After the Vilsmeier-Haack reaction, the present invention preferably further comprises a post-treatment, which preferably comprises: pouring the obtained Vilsmeier-Haack reaction system into crushed ice, and regulating the pH value to 5-6 by using NaOH solution with the mass concentration of 10% to obtain an alkaline system; extracting the alkaline system with dichloromethane, and collecting an organic layer; the organic layer was dried over anhydrous sodium sulfate and filtered, the obtained filtrate was distilled under reduced pressure to remove the solvent, and the obtained crude product was purified by silica gel column chromatography to obtain a pink solid. In the invention, the reagent of the silica gel column chromatography is Dichloromethane (DCM) and methanol (MeOH) with the volume ratio of 5: 1.
After a second intermediate is obtained, the second intermediate and 1,2, 3-tetramethyl-3H-indolium iodide undergo nucleophilic substitution reaction under the condition of piperidine to obtain a fluorophore NIR-COOH with a structure shown in a formula 3;
In the present invention, the ratio of the amounts of the second intermediate and the substance of 1,2, 3-tetramethyl-3H-indolium iodide is preferably 1:1.1 to 1.2. In the present invention, the ratio of the amounts of the second intermediate and the piperidine substance is preferably 1:1.
In the present invention, the reaction solvent for the nucleophilic substitution reaction is preferably ethanol.
In the invention, the temperature of the nucleophilic substitution reaction is preferably normal temperature, namely, no additional heating or no additional cooling is required; the time is preferably 10 to 12 hours. In the present invention, the nucleophilic substitution reaction is preferably monitored by TLC.
In the present invention, the nucleophilic substitution reaction of the second intermediate and 1,2, 3-tetramethyl-3H-indolium iodide under the condition of piperidine specifically preferably comprises the steps of: dissolving a second intermediate in a reaction solvent to obtain a second intermediate solution; adding the 1,2, 3-tetramethyl-3H-indolium iodide into the second intermediate solution, then dripping the piperidine into the solution under the condition of ice-water bath, and then carrying out nucleophilic substitution reaction after nitrogen gas is placed.
After the nucleophilic substitution reaction, the present invention preferably further includes a post-treatment, which preferably includes: and sequentially diluting and washing the nucleophilic substitution reaction system, drying and filtering the obtained organic layer, distilling the obtained filtrate under reduced pressure to remove the solvent, and purifying the obtained crude product by silica gel column chromatography to obtain a green solid compound NIR-COOH. In the present invention, the diluted reagent is preferably methylene chloride. In the present invention, the washing preferably includes sequentially performing dilute hydrochloric acid washing and saturated sodium chloride washing, the number of times of which is independently 3. In the present invention, the drying is preferably anhydrous sodium sulfate drying. The parameters and operations of the reduced pressure distillation are not particularly limited in the present invention. In the present invention, the reagent for silica gel column chromatography is preferably a mixed solution of Dichloromethane (DCM) and methanol (MeOH) in a volume ratio of 10:1.
In the invention, 4-diethylamino keto acid and cyclohexanone are used as raw materials, and the preparation formula is shown as follows through ring-adding reaction, vilsmeier-Haack reaction and nucleophilic substitution reaction:
After obtaining a fluorophore NIR-COOH, one of 4-sulfonamide methyl benzoate and 4-aminobenzoate and the fluorophore NIR-COOH are subjected to a first condensation reaction and a methyl ester hydrolysis reaction under the conditions of 4-dimethylaminopyridine and EDCI & HCl to obtain an intermediate compound 1 and an intermediate compound 2:
The intermediate compound 1 has a structure shown in a formula 4;
the intermediate compound 2 has a structure represented by formula 5:
In the present invention, the ratio of the amount of one of the methyl 4-sulfonamide benzoate and methyl 4-aminobenzoate to the substance of the fluorophore NIR-COOH is preferably 4.5 to 5.5:1. in the present invention, the ratio of the amounts of the substances of the fluorophores NIR-COOH and 4-dimethylaminopyridine is preferably 1:4.5 to 5.5. In the present invention, the ratio of the amounts of the substances of 4-dimethylaminopyridine and edci·hcl is preferably 1:1.
In the present invention, the reaction solvent of the first condensation reaction is preferably anhydrous methylene chloride.
In the present invention, the temperature of the first condensation reaction is preferably 50 to 70 ℃, and more preferably 60 ℃; the time is preferably 12 to 14 hours. In the present invention, the first condensation reaction is performed under heating reflux conditions.
After the first condensation reaction, the present invention preferably further comprises a post-treatment, preferably comprising: and (3) sequentially diluting, washing, drying, decompressing, evaporating and drying the obtained first condensation reaction feed liquid and performing silica gel column chromatography. In the present invention, the diluted reagent is preferably methylene chloride. In the invention, the washing preferably comprises the steps of sequentially carrying out acid washing and saturated saline water washing, wherein the reagent for the acid washing is preferably HCl with the concentration of 1 mol/L; the number of times of the acid washing and the saturated brine washing is preferably 3. In the present invention, the drying is preferably anhydrous sodium sulfate drying. The parameters and the operation of the reduced pressure distillation are not particularly limited, and the operation well known to the person skilled in the art can be adopted. In the invention, when the reaction raw material is 4-sulfonamide methyl benzoate, the reagent of the silica gel column chromatography is preferably a mixed solvent of petroleum ether and ethyl acetate with the volume ratio of 1:1; when the reaction raw material is methyl 4-aminobenzoate, the reagent for silica gel column chromatography is preferably a mixed solvent of petroleum ether and ethyl acetate with the volume ratio of 2:1.
In the present invention, the reaction solvent for the methyl ester hydrolysis reaction is preferably tetrahydrofuran. In the invention, the alkali for the methyl ester hydrolysis reaction is preferably lithium hydroxide solution, the concentration of the lithium hydroxide solution is preferably 1mol/L, and the volume ratio of the lithium hydroxide solution to tetrahydrofuran is preferably 1:1.
In the invention, the temperature of the methyl ester hydrolysis reaction is preferably normal temperature, namely, no additional heating or no additional cooling is needed; the time is preferably 10 hours.
After the methyl ester hydrolysis reaction, the present invention preferably further includes a post-treatment, which preferably includes: and (3) regulating the pH value of the obtained methyl ester hydrolysis reaction feed liquid to 5-6, and then sequentially carrying out dilution, washing, drying and reduced pressure evaporation to dryness. In the present invention, the reagent for adjusting the pH of the resulting methyl ester hydrolysis reaction feed solution to 5 to 6 is preferably HCl having a concentration of 1 mol/L. In the present invention, the diluted reagent is preferably methylene chloride. In the present invention, the washing reagent is saturated saline, and the number of times of washing is preferably 3. In the present invention, the drying is preferably anhydrous sodium sulfate drying. The parameters and the operation of the reduced pressure distillation are not particularly limited, and the operation well known to the person skilled in the art can be adopted.
The Fmoc-NH- (CH 2)n -COOH and tert-butyl hydrazinoformate of the invention undergo a second condensation reaction and removal of 9-fluorenylmethoxycarbonyl group under EDCI & HCl conditions to obtain a side chain intermediate compound shown in formula 6;
In the present invention, the ratio of the amounts of the Fmoc-NH- (CH 2)n -COOH and t-butyl hydrazinoformate is preferably 1:2. In the present invention, the ratio of the amounts of Fmoc-NH- (CH 2)n -COOH and EDCI. HCl) is preferably 2:3.
In the present invention, the reaction solvent of the second condensation reaction is preferably anhydrous methylene chloride.
In the present invention, the temperature of the second condensation reaction is preferably normal temperature, that is, no additional heating or no additional cooling is required; the time is preferably 6 to 10 hours, and the second condensation reaction is performed under stirring.
In the invention, the Fmoc-NH- (CH 2)n -COOH and tert-butyl hydrazinoformate are subjected to a second condensation reaction under EDCI. HCl conditions, wherein the Fmoc-NH- (CH 2)n -COOH and tert-butyl hydrazinoformate are dissolved in a reaction solvent, and then EDCI. HCl is added to perform the second condensation reaction.
After the second condensation reaction, the present invention preferably further comprises a post-treatment, preferably comprising: and the obtained second condensation reaction feed liquid is subjected to dilution, washing, drying, reduced pressure evaporation and silica gel column chromatography in sequence. In the present invention, the diluted reagent is preferably methylene chloride. In the invention, the washing preferably comprises the steps of sequentially carrying out acid washing and saturated saline water washing, wherein the reagent for the acid washing is preferably HCl with the concentration of 1 mol/L; the number of times of the acid washing and the saturated brine washing is preferably 3. In the present invention, the drying is preferably anhydrous sodium sulfate drying. The parameters and the operation of the reduced pressure distillation are not particularly limited, and the operation well known to the person skilled in the art can be adopted. In the invention, the reagent for silica gel column chromatography is preferably a mixed solvent of dichloromethane and ethyl acetate in a volume ratio of 20:1.
In the present invention, the solvent for removing 9-fluorenylmethoxycarbonyl is preferably methylene chloride. In the present invention, the base for removing 9-fluorenylmethoxycarbonyl is preferably diethylamine; the volume fraction of the diethylamine in the system for removing 9-fluorenylmethoxycarbonyl is 20-30%.
In the invention, the temperature for removing the 9-fluorenylmethoxycarbonyl group is preferably normal temperature, and the time is preferably 8 hours. In the present invention, the removal of 9-fluorenylmethoxycarbonyl is preferably performed under stirring.
In the present invention, the removal of 9-fluorenylmethoxycarbonyl group specifically preferably includes: and dissolving the second condensation reaction product in a solvent for removing 9-fluorenylmethoxycarbonyl, and adding diethylamine under the condition of ice water bath to remove the 9-fluorenylmethoxycarbonyl.
After the removal of the 9-fluorenylmethoxycarbonyl group, the present invention preferably further comprises a post-treatment, which preferably comprises: and (3) sequentially carrying out reduced pressure evaporation and silica gel column chromatography on the obtained removed 9-fluorenylmethoxycarbonyl. The parameters and the operation of the reduced pressure distillation are not particularly limited, and the operation well known to the person skilled in the art can be adopted. In the invention, the reagent for silica gel column chromatography is preferably a mixed solvent of dichloromethane and ethyl acetate in a volume ratio of 20:1.
After obtaining a fluorophore NIR-COOH, an intermediate compound 1, an intermediate compound 2 and a side chain intermediate compound, one of the fluorophore NIR-COOH, the intermediate compound 1 and the intermediate compound 2 and the side chain intermediate compound are subjected to a third condensation reaction and tert-butoxycarbonyl removal under EDCI & HCl conditions to obtain the rhodamine-cyanine compound containing hydrazide and a tautomer thereof.
In the present invention, the ratio of the amounts of the substances of the fluorophore NIR-COOH, one of the intermediate compound 1 and the intermediate compound 2 and the side chain intermediate compound is preferably 1:1 to 2.5. In the present invention, the ratio of the amounts of the substances of the fluorophore NIR-COOH, one of the intermediate compound 1 and the intermediate compound 2 and EDCI. HCl is preferably 1:1.5 to 2.
In the present invention, the reaction solvent of the third condensation reaction is preferably anhydrous methylene chloride.
In the present invention, the temperature of the third condensation reaction is preferably normal temperature, that is, no additional heating or no additional cooling is required; the time is preferably 8 to 10 hours.
After the third condensation reaction, the present invention preferably further comprises a post-treatment, preferably comprising: and adding water to quench the third condensation reaction, and sequentially diluting, washing, drying, evaporating to dryness under reduced pressure and performing silica gel column chromatography on the obtained third condensation reaction liquid. In the present invention, the diluted reagent is preferably methylene chloride. In the present invention, the washing includes sequentially performing water washing and saturated brine washing, and the number of times of the water washing and saturated brine washing is preferably 3. In the present invention, the drying is preferably anhydrous sodium sulfate drying. The parameters and the operation of the reduced pressure distillation are not particularly limited, and the operation well known to the person skilled in the art can be adopted. In the invention, when the raw material is NIR-COOH, the reagent for silica gel column chromatography is preferably a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 2:1; when the raw material is the intermediate compound 1, the reagent of the silica gel column chromatography is preferably a mixed solvent with the volume ratio of dichloromethane to methanol being 10:1; when the raw material is the intermediate compound 2, the reagent for silica gel column chromatography is preferably a mixed solvent of petroleum ether and ethyl acetate with the volume ratio of 1:4.
In the present invention, the solvent for removing t-butoxycarbonyl group is preferably methylene chloride. In the invention, the acid for removing the tert-butoxycarbonyl is preferably hydrochloric acid, and the concentration of the hydrochloric acid is preferably 2mol/L; the volume ratio of the hydrochloric acid to the dichloromethane is preferably 2:1; the hydrochloric acid is added dropwise under the condition of ice-water bath.
In the invention, the temperature for removing the tert-butoxycarbonyl group is preferably normal temperature; the time is preferably 8 hours. In the present invention, the removal of t-butoxycarbonyl group is preferably performed under stirring.
After the removal of the tert-butoxycarbonyl group, the present invention preferably further comprises a post-treatment, which preferably comprises: and (3) sequentially diluting, washing, drying and decompressing and evaporating the obtained material liquid with the tert-butoxycarbonyl removed. In the present invention, the diluted reagent is preferably methylene chloride. In the present invention, the washing preferably includes sequentially performing water washing and saturated brine washing, and the number of times of the water washing and saturated brine washing is preferably 3. In the present invention, the drying is preferably anhydrous sodium sulfate drying. The parameters and the operation of the reduced pressure distillation are not particularly limited, and the operation well known to the person skilled in the art can be adopted.
The invention also provides the rhodamine-cyanine compound containing the hydrazide and a tautomer thereof or the application of the rhodamine-cyanine compound containing the hydrazide and the tautomer thereof prepared by the preparation method of the technical scheme in protein detection. In the present invention, the protein contains aldehyde groups and/or ketocarbonyl groups.
The invention also provides application of the rhodamine-cyanine compound containing the hydrazide and the tautomer thereof or the rhodamine-cyanine compound containing the hydrazide and the tautomer thereof prepared by the preparation method of the technical scheme in diagnosis of non-diseases and detection of fibrosis collagen for treatment purposes.
The invention also provides application of the rhodamine-cyanine compound containing the hydrazide and the tautomer thereof or the rhodamine-cyanine compound containing the hydrazide and the tautomer thereof prepared by the preparation method of the technical scheme in fibrosis imaging for diagnosis and treatment of non-diseases.
In the present invention, the fibrosis imaging preferably includes a fibrosis detection of liver fibrosis tissue for non-disease diagnosis and treatment purposes, a fibrosis detection of lung fibrosis tissue for non-disease diagnosis and treatment purposes, or a fibrosis degree comparison detection of fibrosis tissue of a fibrosis model group and a drug intervention group for non-disease diagnosis and treatment purposes.
The application mode of the rhodamine-cyanine compound containing the hydrazide and the tautomer thereof is not particularly limited, and the rhodamine-cyanine compound containing the hydrazide and the tautomer thereof can be operated according to actual needs.
The preparation and application of the hydrazide-containing rhodamine-cyanine compound and the tautomer thereof provided in the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1: preparation of fluorophore NIR-COOH
Reaction conditions ：a)Cyclohexanone,Concentrated H2SO4,90℃;HClO4,0℃;b)POCl3,anhydrous DMF;c)1,2,3,3-tetramethyl-3H-indol-1-ium,Piperdine,EtOH.
The cyclization reaction prepares a first intermediate N1: cyclohexanone (1.32 mL,12.74 mmol) was added dropwise to concentrated sulfuric acid (14 mL) under ice-bath, followed by the addition of compound 4-diethylaminoketo acid (2 g,6.4 mmol) in portions with vigorous stirring. The reaction mixture was heated at 90℃for 2 hours, cooled to room temperature and then slowly poured into ice cubes (60 g). Finally, perchloric acid (2 mL) with a mass concentration of 50% was slowly added dropwise to the reaction system under ice bath, and a red-orange solid was observed to precipitate until no more red-orange solid precipitated, the obtained precipitate was suction filtered and washed with ice water (50 mL), and the obtained red solid N1 was vacuum-dried for direct use in the next reaction.
Preparation of second intermediate N2 by Vilsmeier-Haack reaction: 15mL of anhydrous N, N-dimethylformamide was added to a 100mL reaction flask, and the flask was replaced with nitrogen, phosphorus oxychloride (5 mL) was slowly added dropwise to the flask under ice-bath stirring, and reacted for half an hour to prepare a Vilsmeier reagent. First intermediate N1 (0.84 g,1.76 mmol) was dissolved in anhydrous N, N-dimethylformamide (10 mL), and first intermediate N1 was added dropwise to Vilsmeier reagent in ice bath, and the reaction was continued at room temperature for two hours. After completion of the TLC monitoring, the reaction system was poured into crushed ice (1000 g), pH was adjusted to 5-6 with 10% by mass NaOH solution, and the mixture was extracted with methylene chloride. The organic layer was dried over anhydrous sodium sulfate, filtered to obtain a filtrate, and the solvent was distilled off under reduced pressure. The crude product was further purified by silica gel column chromatography (DCM: meoh=5:1) to give second intermediate N2 as a pink solid (0.68 g, yield 54.79%).
Nucleophilic substitution reaction to prepare fluorophore NIR-COOH: the second intermediate N2 (0.1 g,0.23 mmol) was dissolved in ethanol (2 mL), 1,2, 3-tetramethyl-3H-indolium iodide (0.77 g,0.26 mmol) was added, piperidine (23.92. Mu.L, 0.23 mmol) was added dropwise under ice bath, the reaction solution was pink, nitrogen was replaced three times, and the reaction was carried out overnight at room temperature. After overnight reaction, the reaction mixture was green. After completion of the TLC monitoring reaction, methylene chloride (150 mL) was added for dilution, diluted hydrochloric acid (1M, 50 mL. Times.3) for washing, and saturated sodium chloride for washing (50 mL. Times.3). The organic layer was dried over anhydrous sodium sulfate, filtered to obtain a filtrate, and the solvent was distilled off under reduced pressure. The crude product was further purified by silica gel column chromatography (DCM: meoh=10:1) to give the green solid compound NIR-COOH (58 mg, 44.66% yield).
Example 2: preparation of intermediate compound 1 and intermediate compound 2
Reaction conditions: a) EDCI & HCl, DMAP, dry DCM; b) 1mol/L LiOH, THF.
Preparation of intermediate compound 1: the fluorophore NIR-COOH (0.35 g,0.63 mmol) was dissolved in anhydrous dichloromethane (15 mL) and placed in a 50mL vial, followed by addition of methyl 4-sulfonamide benzoate (0.67 g,3.13 mmol), EDCI. HCl (0.48 g,2.50 mmol) and DMAP (0.31 g,2.50 mmol). The reaction was heated to reflux at 60 ℃ overnight. The reaction solution gradually changed from dark green to light green. After the completion of the reaction, the crude product was diluted with methylene chloride, washed three times with 1mol/L HCl and saturated brine, dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and subjected to silica gel column chromatography (V petroleum ether/V ethyl acetate=1/1) to obtain green solid 1-COOEt (0.16 g, 28.8%). ESI-MS: m/z= [ m+h ] +calculated 756.31,found 756.31.
1-COOEt (0.22 mmol) was dissolved in tetrahydrofuran (3 mL), and 1mol/L LiOH (3 mL) was added to the solution in an ice bath and reacted overnight at room temperature. After the reaction is completed, the pH is adjusted to 5-6 by using 1mol/L HCl. The crude product was diluted with dichloromethane, washed three times with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to give a green solid.
Preparation of intermediate compound 2: referring to the preparation method of the intermediate compound 1, the 4-sulfonamide methyl benzoate is changed into 4-aminobenzoate, and the reaction solution gradually changes from dark green to light yellow. Silica gel column chromatography (V petroleum ether/V ethyl acetate=2/1) gave 2-COOEt as a pale yellow solid in 36.1% yield. ESI-MS: m/z= [ m+h ] +calculated 692.34,found 692.35. Hydrolysis of methyl ester referring to the preparation of intermediate compound 1, a pale green solid was obtained.
Example 3: preparation of side chain intermediate Compound 1a/1b/1c
The synthetic route is as follows:
Preparation of side chain intermediate compound 1 a: the compound Fmoc-NH- (CH 2)3 -COOH (5 g,15.40 mmol) and t-butyl hydrazinoformate (4.07 g,30.80 mmol) were dissolved in anhydrous dichloromethane (35 mL), EDCI. HCl (4.43 g,23.10 mmol) was added under dry tube protection, stirring overnight at room temperature, the reaction solution was gradually clarified and transparent from cloudiness, after the reaction was completed, the crude product was diluted with dichloromethane, washed three times with 1mol/l HCl and saturated saline each in turn, dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and chromatographed on silica gel column (V dichloromethane/V ethyl acetate=20/1) to give Fmoc-1a (6.3 g, 89.3%) as a white solid.
Fmoc-1a (0.50 g,1.14 mmol) was dissolved in dichloromethane (3 mL), diethylamine (0.8 mL) was added dropwise under ice and stirred overnight at ambient temperature. After the completion of the reaction, the reaction mixture was evaporated under reduced pressure and subjected to silica gel column chromatography (V dichloromethane/V methanol=10/1) to give 1a (0.13 g, 52.89%) as a pale yellow oil. ESI-MS: m/z= [ m+h ] +calculated 218.15,found 218.15.
Preparation of side chain intermediate compound 1 b: the preparation method of the reference compound 1a is used for obtaining white solid Fmoc-1b with the yield of 89.94 percent; light yellow oil 1b was obtained in 75.02% yield. ESI-MS: m/z= [ m+h ] +calculated 246.18,found 246.18.
Preparation of side chain intermediate compound 1 c: the preparation method of the reference compound 1a is used for obtaining white solid Fmoc-1c with the yield of 52.75%; a pale yellow oil 1c was obtained in 62.33% yield. ESI-MS: m/z= [ m+h ] +calculated 274.21,found 274.21.
Example 4: preparation of target Compounds
Reaction conditions: a) Side chain intermediate compound 1a/1b/1c, EDCI, dry DCM; b) EA.HCl, DCM.
Preparation of Compounds A1-Boc: after side chain intermediate compound 1a (46.67 mg,0.22 mmol) was dissolved in anhydrous dichloromethane (2 mL), fluorophore NIR-COOH (100 mg,0.18 mmol) and EDCI. HCl (69.01 mg,0.36 mmol) were added and reacted overnight at room temperature under dry tube protection. The reaction system is gradually changed from dark green to light green, and water is added to quench the reaction after the reaction is completed. The crude product was diluted with dichloromethane, washed three times with water and saturated brine, dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and chromatographed on silica gel (V petroleum ether/V ethyl acetate=2/1) to give a yellow solid A1-Boc (62 mg, 45.73%). ESI-MS: M/z= [ M+H ] +calculated 758.42,found 758.42.
Preparation of Compound A2-Boc: referring to the preparation of compound A1-Boc, the side chain intermediate compound 1a was replaced with side chain intermediate compound 1b to give A2-Boc as a yellow solid in 42.92% yield. ESI-MS: m/z= [ m+h ] +calculated 786.45,found 786.45.
Preparation of Compound A3-Boc: referring to the preparation of compound A1-Boc, the side chain intermediate compound 1a was replaced with side chain intermediate compound 1c to give A3-Boc as a yellow solid in 24.10% yield. ESI-MS: m/z= [ m+h ] +calculated 814.48,found 814.48.
Preparation of Compounds B1-Boc: after side chain intermediate compound 1a (0.58 g,0.27 mmol) was dissolved in anhydrous dichloromethane (2 mL), intermediate compound 1 (0.20 g,0.27 mmol) and EDCI. HCl (101.60 mg,0.53 mmol) were added and reacted overnight at room temperature under dry tube protection. The reaction system is gradually changed from dark green to light green, and water is added to quench the reaction after the reaction is completed. The crude product was diluted with dichloromethane, washed three times with water and saturated brine, dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and chromatographed on silica gel (V dichloromethane/V methanol=10/1) to give green solid B1-Boc (113 mg, 45.43%). ESI-MS: m/z= [ m+h ] +calculated 941.42,found 941.42.
Preparation of Compound B2-Boc: referring to the preparation of compound B1-Boc, the side chain intermediate compound was replaced with side chain intermediate compound 1B to give B2-Boc as a green solid in 22.29% yield. ESI-MS: m/z= [ m+h ] +calculated 969.45,found 969.45.
Preparation of Compound B3-Boc: referring to the preparation of compound B1-Boc, the side chain intermediate compound 1a was replaced with side chain intermediate compound 1c to give a green solid B3-Boc in a yield of 61.95%. ESI-MS: m/z= [ m+h ] +calculated 997.48,found 997.48.
Preparation of Compound C1-Boc: after side chain intermediate compound 1a (0.11 g,0.49 mmol) was dissolved in anhydrous dichloromethane (2 mL), intermediate compound 2 (0.17 g,0.22 mmol) and EDCI. HCl (83.19 mg,0.43 mmol) were added and reacted overnight at room temperature under dry tube protection. The reaction system is gradually changed from dark green to light green, and water is added to quench the reaction after the reaction is completed. The crude product was diluted with dichloromethane, washed three times with water and saturated brine, dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and chromatographed on silica gel (V petroleum ether/V ethyl acetate=1/4) to give C1-Boc (198mg, 91.84%) as a pale green solid. ESI-MS: m/z= [ m+h ] +calculated 877.46,found 877.46.
Preparation of Compound C2-Boc: referring to the preparation of compound C1-Boc, the side chain intermediate compound 1a was replaced with side chain intermediate compound 1b to give a pale green solid C2-Boc in 59.76% yield. ESI-MS: m/z= [ m+h ] +calculated 905.49,found 905.49.
Preparation of Compound C3-Boc: referring to the preparation of compound C1-Boc, the side chain intermediate compound 1a was replaced with side chain intermediate compound 1C to give a pale green solid C3-Boc in 45.24% yield. ESI-MS: m/z= [ m+h ] +calculated 933.52,found 933.52.
Preparation of compound An/Bn/Cn (n=1, 2, 3): after the compound An-Boc/Bn-Boc/Cn-Boc (n=1, 2, 3) was dissolved in methylene chloride, 2mol/L HCl (Methanol) was added dropwise to the solution in An ice bath in An amount of 2 times the volume of methylene chloride, and the solution was stirred at room temperature overnight. After the reaction is completed, the crude product is diluted by methylene dichloride, washed by water and saturated saline water for three times in sequence, dried by anhydrous sodium sulfate and evaporated to dryness under reduced pressure to obtain the target product. The nuclear magnetic hydrogen spectrum data of the target compound are shown in table 1.
TABLE 1 Nuclear magnetic Hydrogen Spectrometry data for target Compounds
Test example 1: evaluation of the ability of 9 probes An/Bn/Cn (n=1, 2, 3) to distinguish normal liver tissue from liver fibrosis 4 mice were housed, with a 12h:12h light/dark schedule, free to provide food and water. After pretreatment for 7d, male mice were intraperitoneally injected with 10% cc1 4 (olive oil dilution, 2mL/kg body weight, 3 times per week) to induce liver fibrosis for 8 weeks. The experiment will set up a control group and a liver fibrosis group. After tissue is collected, frozen liver tissue slices of a control group and a liver fibrosis group are obtained through the steps of fixing, freezing, slicing, airing and the like, 9 probes (5 mu M) are respectively incubated with frozen stop slices overnight, and tissue imaging shooting is carried out by adopting a small animal living body fluorescence image analysis system caliper (perkinelmer). The imaging results were quantitatively analyzed for fluorescence using Image J software, and the quantitative results are shown in fig. 1, wherein gray columns in fig. 1 are Oil groups, which are control groups injected with only olive Oil; the red bars are set CCl 4, which is the hepatic fibrosis model group injected with 10% CC1 4, and the ordinate Area is the fluorescent quantification. From FIG. 1, it can be seen that the fluorescence intensity of the A-series and C-series probes is lower, and the fluorescence intensity is presumed to be the reason for the weak ring opening capability of the probes, so that the balance of the transition from the spiro ring to the ring opening is far weaker than that of the transition from the ring opening to the spiro ring. Notably, all three probes of the B-series showed a strong ring opening ability, and the fluorescence imaging contrast was much higher for the fibrotic group and the control group than for the a-series and the C-series. Among them, probe B2 showed the highest imaging contrast, up to 6.89 times, and based on this conclusion, probe B2 was considered to have better potential for application to fibrosis imaging than the other 8 probes.
Test example 2: evaluation of Probe B2 ability to differentiate normal liver from fibrotic liver
The C57BL/J6 mice were all housed in groups (4 mice per cage in different experiments) under standard laboratory conditions (22.+ -. 1 ℃, 55.+ -. 5% humidity) using a 12h:12h light/dark schedule, with free supply of food and water. After pretreatment for 7d, male mice were intraperitoneally injected with 10% cc1 4 (olive oil dilution, 2mL/kg body weight, 3 times per week) to induce liver fibrosis for 8 weeks. The experiment will set up a control group and a liver fibrosis group. 160nm probe B2 was administered to each mouse (25 g) by tail vein injection. After 1 hour of tail vein injection, the mice were dissected and imaged using a small animal in vivo fluorescence image analysis system caliper (perkinelmer). The results are shown in FIG. 2, and in which normal mice are liver fibrosis mice in order from left to right in FIG. 2. As can be seen from fig. 2, the fluorescence intensity of liver sites of liver fibrosis mice is significantly higher than that of normal mice, indicating that probe B2 has a certain potential in applications for distinguishing normal liver from fibrotic liver.
Test example 3: evaluation Probe B2 for its ability to differentiate normal from fibrotic lungs
Healthy male 25-30 g C57BL/6 mice are selected, 10% chloral hydrate (0.3 mL-100 g -1) is used for intraperitoneal injection anesthesia, the rats are fixed on the experiment table in a supine mode, an mouth gag is used for fixing the oral cavity, the tongue is pulled out, a tongue depressor is used for pressing the tongue abdomen, under the direct view of a frontal mirror, a tracheal cannula (phi 2mm, 4-5 cm) is rapidly moved when the animals inhale, 5 mg-kg -1 BLM 0.2-0.3 mL is slowly injected, and the animals are immediately rotated, so that liquid medicine is uniformly distributed in the lungs and can drink water freely. In the molding process, part of mice begin to die and lose weight after about 5-6 days, and the mice are taken for experiments on the 11 th day of molding. 160nm probe B2 was administered to each mouse (25 g) by tail vein injection. Mice were sacrificed 1 hour after tail vein injection, their hearts, lungs, livers, spleens and kidneys were taken for in vitro tissue imaging, and were photographed using a small animal in vivo fluorescence image analysis system caliper (perkinelmer), and the results are shown in fig. 3, in which normal mice are lung fibrosis mice in order from left to right in fig. 3. As can be seen from fig. 3, the fluorescence intensity of the lung of the pulmonary fibrosis mice is significantly higher than that of the normal mice, indicating that probe B2 has a certain potential in applications for distinguishing normal and fibrotic lungs.
Test example 4: potential of evaluation probe B2 for evaluation of efficacy of fibrotic therapeutic drug
Mice were fed with rodent diet containing 40kcal% fat (palm oil), 20kcal% fructose and 2% cholesterol for 4 weeks, and NASH models were established. After 1 injection of 2.5% cc1 4 at 5 weeks, mice were randomized: (1) Model group (n=8) continued to inject 5% cc1 4 weeks; (2) OCA treatment group (n=8) was exposed to the same level of CC1 4 and 30mg/kg OCA orally for 4 weeks. Probe B2 was administered at 4. Mu. Mol/kg by tail vein injection. After 1 hour of tail vein injection, the mice were dissected and the fibrosis ex vivo tissue imaging was performed using a small animal in vivo fluorescence image analysis system caliper (perkinelmer), the results are shown in fig. 4, in which OCA treated mice and model mice are shown in fig. 4 in order from left to right. As can be seen from fig. 4, the fluorescence intensity of the liver of the mice in the model group is obviously higher than that of the liver of the mice in the OCA treatment group, which indicates that the probe B2 has a certain potential in the application of the drug efficacy evaluation of the fibrosis treatment drugs.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
1. The rhodamine-cyanine compound containing the hydrazide and the tautomer thereof are characterized in that the rhodamine-cyanine compound containing the hydrazide has the following structure:
In the formula I-1, the formula I-2 or the formula I-3, the value of n is independently 3, 5 or 7.
2. The hydrazide-containing rhodamine-cyanine compound according to claim 1, wherein the hydrazide-containing rhodamine-cyanine compound tautomer has the structure:
in the formula II-1, the formula II-2 or the formula II-3, the value of n is independently 3, 5 or 7.
3. The method for preparing a hydrazide-containing rhodamine-cyanine compound and a tautomer thereof according to any one of claims 1 to 2, characterized by comprising the steps of:
4-diethylamino keto acid and cyclohexanone are subjected to ring-adding reaction under the conditions of concentrated sulfuric acid and perchloric acid to obtain a first intermediate with a structure shown in a formula 1;
the first intermediate performs Vilsmeier-Haack reaction under the condition of phosphorus oxychloride to obtain a second intermediate with a structure shown in a formula 2;
the second intermediate and the 1,2, 3-tetramethyl-3H-indolium iodide undergo nucleophilic substitution reaction under the condition of piperidine to obtain a fluorophore NIR-COOH with a structure shown in a formula 3;
One of methyl 4-sulfonamide benzoate and methyl 4-aminobenzoate and the fluorophore NIR-COOH are subjected to a first condensation reaction and a methyl ester hydrolysis reaction under the conditions of 4-dimethylaminopyridine and EDCI & HCl to obtain an intermediate compound 1 and an intermediate compound 2:
The intermediate compound 1 has a structure shown in a formula 4;
the intermediate compound 2 has a structure represented by formula 5:
Fmoc-NH- (CH 2) n-COOH and tert-butyl hydrazinoformate are subjected to a second condensation reaction and 9-fluorenylmethoxycarbonyl removal under EDCI & HCl conditions to obtain a side chain intermediate compound shown in formula 6;
And (3) performing a third condensation reaction and removing tert-butoxycarbonyl groups on one of the fluorophore NIR-COOH, the intermediate compound 1 and the intermediate compound 2 and the side chain intermediate compound under EDCI & HCl conditions to obtain the rhodamine-cyanine compound containing hydrazide and a tautomer thereof.
4. The method according to claim 3, wherein the nucleophilic substitution reaction is carried out at room temperature for 10 to 12 hours.
5. The method according to claim 3, wherein the temperature of the first condensation reaction is 50 to 70 ℃ for 12 to 14 hours;
The reaction solvent of the methyl ester hydrolysis reaction is tetrahydrofuran, the alkali of the methyl ester hydrolysis reaction is lithium hydroxide solution, the concentration of the lithium hydroxide solution is 1mol/L, and the volume ratio of the lithium hydroxide solution to the tetrahydrofuran is 1:1, the temperature of the methyl ester hydrolysis reaction is normal temperature, and the time is 10 hours.
6. The method according to claim 3, wherein the reaction solvent of the second condensation reaction is anhydrous dichloromethane, and the temperature of the second condensation reaction is normal temperature for 6-10 hours;
the solvent for removing the 9-fluorenylmethoxycarbonyl group is dichloromethane, and the alkali is diethylamine; the volume fraction of the diethylamine in the system for removing 9-fluorenylmethoxycarbonyl is 20-30%, the temperature for removing 9-fluorenylmethoxycarbonyl is normal temperature, and the time is 8 hours.
7. The method according to claim 3, wherein the temperature of the third condensation reaction is room temperature for 8 to 10 hours;
The solvent for removing the tert-butoxycarbonyl is methylene dichloride, the acid for removing the tert-butoxycarbonyl is hydrochloric acid, and the concentration of the hydrochloric acid is 2mol/L; the volume ratio of the hydrochloric acid to the dichloromethane is 2:1; the temperature for removing the tert-butoxycarbonyl group is normal temperature and the time is 8 hours.
8. Use of the hydrazide-containing rhodamine-cyanine compound according to any one of claims 1 to 2 and its tautomer or the hydrazide-containing rhodamine-cyanine compound according to any one of claims 3 to 7 and its tautomer in the detection of proteins containing aldehyde groups and/or ketocarbonyl groups.
9. Use of the rhodamine-cyanine compound containing hydrazide and the tautomer thereof according to any one of claims 1 to 2 or the rhodamine-cyanine compound containing hydrazide and the tautomer thereof prepared by the preparation method according to any one of claims 3 to 7 in detection of fibrosis collagen for diagnosis and treatment of non-diseases.
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