Patent Publication Number: US-2022214351-A1

Title: Fluorescent dye, preparation method and uses thereof

Description:
RELATED APPLICATIONS 
     The present application is a U.S. National Phase of International Application Number PCT/CN2020/087311 filed Apr. 27, 2020 and claims priority to Chinese Application Number CN 201910352348.X filed Apr. 28, 2019. 
    
    
     INCORPORATION BY REFERENCE 
     The sequence listing provided in the file entitled 072_2110093US_SEQUENCE_LISTING_revised_27_10_2021.txt, which is an ASCII text file that was created on Oct. 27, 2021, and which comprises 2,896 bytes, is hereby incorporated by reference in its entirety. 
     TECHNICAL FIELD 
     The present invention relates to the technical field of fluorescent dye, and particularly relates to a fluorescent dye with viscosity responsiveness and low background fluorescence, as well as a preparation method and uses thereof. 
     BACKGROUND 
     Molecular rotors are a kind of dyes the fluorescence intensity of which changes with microenvironment viscosity. After excitation of molecular rotors, conformation of molecules is twisted and TICT (twisted intramolecular charge transfer) is formed, wherein the excited energy are mainly released in a non-radiative form; when the molecules are in a microenvironment of comparatively large viscosity or rigidity, the twisted molecular conformation will be restricted for this kind of molecules, and the excited energy of dye will be mainly released in the form of radioluminescence, namely, the fluorescence property of molecules is activated. It is important that the fluorescence intensity of this kind of molecules changes with the microenvironment viscosity, so that the viscosity change of the microenvironment is displayed in real time, in situ and in a sensitive and visual manner. 
     At present, besides the field of viscosity detection, the twisted conformation based on restrictions of the molecular rotors is also widely used for constructing a fluorescent activated probe, for example, after the combination of molecular rotors with BSA, the conformation of molecules is restricted by protein, and the fluorescence is lit up, but the excited energy of the dye that is not combined with protein is still dissipated in a non-radiative form, thereby detecting and quantifying the protein in real time. For another example, Thiazole Orange is in a state of fluorescence quenching before it is combined with DNA or RNA, and the molecular conformation is restricted after it is combined with DNA or RNA, as a result of which the fluorescence is activated, so Thiazole Orange is widely used for the detection and tracing of DNA and RNA; molecular rotors such as Malachite Green are coated with antibodies so as to limit the conformation changes of the molecules and are used for protein-activated fluorescence imaging; DHBI is combined with an adapter so as to construct fluorescent protein simulators for RNA tracing; for another example, the combination with amyloid protein can restrict the conformation changes of molecules, and can be used for the detection, research and so on of Alzheimer&#39;s disease. 
     However, current molecular rotors generally have the disadvantage of high fluorescence background, namely, the fluorescent intensity of molecular rotors in a free state is comparatively high, and thus can hardly be used for the sample detection and labeling with a small sample size, complicated components and low abundance of objects to be measured, such as endogenous proteins, nucleic acid, metabolites and so on in biological samples, so the development of a kind of molecular rotors with low background fluorescence can further expand the use of current molecular rotors. 
     SUMMARY OF THE INVENTION 
     The object of the present invention is to provide a fluorescent dye with viscosity responsiveness and low background fluorescence. 
     For one aspect, the present invention provides a fluorescent dye, wherein the fluorescent dye is shown as Formula (I), 
     
       
         
         
             
             
         
       
     
     wherein: 
     D- is HO— or N(X 1 )(X 2 )—, X 1  and X 2  are respectively and independently selected from hydrogen, alkyl and modified alkyl; and X 1  and X 2  are optionally interconnected, and form a lipid heterocyclic ring with N atoms; 
     R is selected from cyano group, carboxy, amide group, ester group, sulfoxide group, sulphone group, sulfonic ester group or sulfonamido group; Ar 1  and Ar 2  are respectively and independently selected from arylene and sub-heteroaryle; wherein hydrogen atoms in Ar 1  and Ar 2  being optionally, respectively and independently substituted by halogen atoms, hydroxyl group, aldehyde group, carboxyl group, ester group, amide group, cyano group, sulfonic acid group, phosphoric acid group, amino group, primary amino group, secondary amino group, alkyl or modified alkyl; 
     X 1  and X 2  optionally and independently form a lipid heterocyclic ring with Ar 1 ; 
     wherein: the “alkyl” is respectively and independently C 1 -C 10  straight or branched alkyl; optionally, the “alkyl group” is C 1 -C 7  straight or branched alkyl; optionally, the “alkyl group” is C 1 -C 5  straight or branched alkyl; optionally, the “alkyl group” is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tertiary butyl, sec-butyl, n-amyl, 1-methyl butyl, 2-methyl butyl, 3-methyl butyl, isoamyl, 1-ethyl propyl, neoamyl, n-hexyl, 1-methyl amyl, 2-methyl amyl, 3-methyl amyl, isohesyl, 1,1-dimethyl butyl, 2,2-dimethyl butyl, 3,3-dimethyl butyl, 1,2-dimethyl butyl, 1,3-dimethyl butyl, 2,3-dimethyl butyl, 2-ethyl butyl, n-heptyl, 2-methyl hexyl, 3-methyl hexyl, 2,2-dimethyl amyl, 3,3 dimethyl amyl, 2,3-dimethyl amyl, 2,4-dimethyl amyl, 3-ethyl amyl or 2,2,3-methyl butyl; 
     the “modified alkyl” is respectively and independently a group obtained by replacing any carbon atom in alkyl with one or more groups of halogen atom, —OH, —CO—, —O—, —CN, —S—, —SO 2 —, —(S═O)—, azido, primary amino group, secondary amino group, tertiary amino group, and quaternary ammonium base, and the modified alkyl has 1-10 carbon atoms, wherein the carbon-carbon single bond is optionally and independently replaced by a carbon-carbon double bond or a carbon-carbon triple bond; 
     the replacement of carbon atoms refers to that carbon atoms or the carbon atoms and hydrogen atoms thereon together are replaced by a corresponding group; 
     the “halogen atom” is respectively and independently F, Cl, Br or I; 
     the “lipid heterocyclic ring” is a saturated or unsaturated 4- to 15-membered monocyclic or polycyclic lipid heterocyclic ring containing one or more heteroatoms of N, O, S or Si on the ring, and the lipid heterocyclic ring is —S—, —SO— or —SO 2 — when there are S atoms on the ring; the lipid heterocyclic ring is optionally substituted by a halogen atom, an alkyl, an aryl or a modified alkyl; 
     the “arylene” is a 5- to 13-membered monocyclic or dicyclic or fused dicyclic or fused polycyclic subaromatic group; 
     the “sub-heteroaryle” is a 5- to 13-membered monocyclic or dicyclic or fused dicyclic or fused polycyclic sub-heteroaromatic group containing one or more heteroatoms of N, O, S or Si on the ring; 
     the “ester group” is R′(C═O)OR″ group; 
     the “amide group” is R′CONR″R′″ group; 
     the “sulfonic acid group” is R′SO 3 H group; 
     the “sulfonic ester group” is R′SO 2 OR″ group; 
     the “sulfonamido group” is R′SO 2 NR″R′″ group; 
     the “phosphoric acid group” is R′OP(═O)(OH) 2  group; 
     the “sulphone group” is R′SO 2 R″ group; 
     the “sulfoxide group” is R′SOR″ group; 
     the “primary amino group” is R′NH 2  group; 
     the “secondary amino group” is R′NHR″ group; 
     the “tertiary amino group” is R′NR″R′″ group; 
     the “quaternary ammonium base” is R′R″R′″ R″″N +  group; 
     each R′, R″, R′″, R″″ respectively and independently being single bond, hydrogen, alkyl, alkylene, modified alkyl or modified alkylene; 
     the “alkylene” is C 1 -C 10  straight or branched alkylene; optionally, it is C 1 -C 7  straight or branched alkylene; optionally, it is C 1 -C 5  straight or branched alkylene; 
     the “modified alkylene” is a group obtained by replacing any carbon atom in C 1 -C 10  (preferably C 1 -C 6 ) alkylene with a group selected from —O—, —OH, —CO—, —CS—, and —(S═O)—; 
     optionally, the “modified alkylene” is a group containing one or more groups selected from —OH, —O—, ethylene glycol unit (—(CH 2 CH 2 O) n —), monosaccharide unit, —O—CO—, —NH—CO—, —SO 2 —O—, —SO—, Me 2 N—, Et 2 N—, —S—S—, —CH═CH—, F, Cl, Br, I, cyano group; and 
     optionally, Ar 1  and Ar 2  respectively and independently are structures selected from the following Formulae (II-1) to (II-22). 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Optionally, the compound represented by Formula (I) is selected from the compounds below: 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     A second aspect of the present invention is to provide a method of preparing the afore-mentioned fluorescent dye, including a step of aldol condensation reaction between a compound of Formula (a) and a compound of Formula (b). 
     
       
         
         
             
             
         
       
     
     A third aspect of the present invention is to provide uses of the afore-mentioned fluorescent dye in viscosity testing, protein fluorescent labeling, nucleic acid fluorescent labeling, protein quantification or detection, or nucleic acid quantification or detection, wherein the uses are those other than for diagnostic methods of diseases. 
     A fourth aspect of the present invention is to provide uses of the afore-mentioned fluorescent dye in preparing reagents for viscosity testing, protein fluorescent labeling, nucleic acid fluorescent labeling, protein quantification or detection, or nucleic acid quantification or detection. 
     A fifth aspect of the present invention is to provide a fluorescent activated and lighted probe, comprising the afore-mentioned fluorescent dye. 
     A sixth aspect of the present invention is to provide uses of the afore-mentioned fluorescent activated and lighted probe in protein fluorescent labeling, nucleic acid fluorescent labeling, protein quantification or detection, or nucleic acid quantification or detection, wherein the uses are those other than for diagnostic methods of diseases. 
     A seventh aspect of the present invention is to provide uses of the afore-mentioned fluorescent activated and lighted probe in preparing reagents for protein fluorescent labeling, nucleic acid fluorescent labeling, protein quantification or detection, or nucleic acid quantification or detection. 
     The fluorescent dye of the present invention can be used for measuring viscosity of samples, such as for the tests of micro-viscosity. According to the embodiments of another aspect, the obtained fluorescent dye can be specifically combined with corresponding antibody, aptamer or amyloid, or bound to the protein tag or enzyme via a ligand or inhibitor, thereby obtaining a series of fluorescent activated and lighted probes used for fluorescent labeling, quantification or monitoring of protein, enzymes or nucleic acids. 
    
    
     
       DESCRIPTION OF DRAWINGS 
         FIG. 1  is a diagram showing the fluorescence emission intensity at different viscosity conditions of the molecular rotor III-3 (1×10 −5  M); 
         FIG. 2  is a diagram showing the linear relationship between viscosity conditions and fluorescence intensity of the molecular rotor III-3 (1×10 −5  M); 
         FIG. 3  is a diagram showing the fluorescence emission intensity at different viscosity conditions of the molecular rotor III-4 (1×10 −5  M); 
         FIG. 4  is a diagram showing the linear relationship between viscosity conditions and fluorescence intensity of the molecular rotor III-4 (1×10 −5  M); 
         FIG. 5  is a diagram showing the fluorescence emission intensity at different viscosity conditions of the molecular rotor III-28 (1×10 −5  M); 
         FIG. 6  is a diagram showing the linear relationship between viscosity conditions and fluorescence intensity of the molecular rotor III-28 (1×10 −5  M); 
         FIG. 7  is a diagram showing the fluorescence emission intensity at different viscosity conditions of the molecular rotor III-34 (1×10 5  M); 
         FIG. 8  is a diagram showing the linear relationship between viscosity conditions and fluorescence intensity of the molecular rotor III-34 (1×10 −5  M); 
         FIG. 9  is a diagram showing the fluorescence background contrast of molecular rotors III-11 and III-36 (1×10 −6  M) in PBS; 
         FIG. 10  is a diagram showing the fluorescence background contrast of molecular rotors III-34 and III-37 (1×10 −6  M) in PBS; 
         FIG. 11  is a diagram showing the fluorescence background contrast of molecular rotors III-31, III-32, III-33 and III-38 (1×10 −6  M) in PBS; 
         FIG. 12  is a diagram showing the fluorescence background contrast of molecular rotors III-3 and III-39 (1×10 −6  M) in PBS; 
         FIG. 13  is a diagram showing the fluorescence background contrast of molecular rotors II-21 and III-40 (1×10 −6  M) in PBS; 
         FIG. 14  is a diagram showing the fluorescence background contrast of molecular rotors III-28, III-29, I1-30 and I1-41 (1×10 −6  M) in PBS; 
         FIG. 15  is a diagram showing the fluorescence background contrast of molecular rotors I1-3 and I1-42 (1×10 −6  M) in PBS; 
         FIG. 16  is a diagram showing the fluorescence background contrast of molecular rotors I1-3 and I1-43 (1×10 −6  M) in PBS; 
         FIGS. 17A and 17B  are the application of molecular rotors III-3, III-4, III-6, III-7, III-8, III-18, III-21 in labeling intracellular RNA aptamers, wherein A are cells expressing the target RNA aptamers, and B are cells not expressing the target RNA aptamers; 
         FIGS. 18A and 18B  are the application of molecular rotors III-3, I1-43 in labeling intracellular mRNA. 
     
    
    
     SPECIFIC IMPLEMENTATION 
     Compound III-1 
     
       
         
         
             
             
         
       
     
     To a stirring solution of p-dimethylaminobenzaldehyde (0.35 g, 2.3 mmol) and 4-cyano-benzeneacetonitrile (0.4 g, 2.8 mmol) in 20 mL methanol, 2 drops of piperidine were added. After stirring at ambient temperature for 2 h, the mixture was cool to room temperature. A large amount of precipitate was appeared. Then the precipitate was obtained by filtration and washed with cold EtOH three times. The orange solid was obtained after dried under vacuum (0.60 g, yield 95%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=3.05 (s, 6H), 6.83 (d, J=9.2 Hz, 2H), 7.84-7.94 (m, 6H), 8.02 ppm (s, 1H). HRMS (ESI-TOF): Calcd. For C 18 H 16 O 3  [M+H] + : 274.1344. Found: 274.1345. 
     Example 2 
     Compound III-2 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1 (0.34, yield 89%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=1.23 (t, J=7.60 Hz, 6H), 3.05 (t, J=7.60 Hz, 4H), 6.84 (d, J=9.2 Hz, 2H), 7.84-7.95 (m, 6H), 8.09 ppm (s, 1H). HRMS (ESI-TOF): Calcd. For C 20 H 20 O 3  [M+H] + : 302.1657. Found: 302.1658. 
     Example 3 
     Compound III-3 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1 (0.33 g, yield 95%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=7.96 (s, 1H), 7.85 (d, J=16.0 Hz, 6H), 6.81 (d, J=8.0 Hz, 2H), 4.77 (s, 1H), 3.55 (d, J=28.0 Hz, 4H), 3.04 (s, 1H). HRMS (ESI-TOF): Calcd. For C 19 H 18 N 3 O [M+H] + : 304.1450. Found: 304.1451. 
     Example 4 
     Compound III-4 
     
       
         
         
             
             
         
       
     
     To stirring solution of compound III-3 (0.61 g, 2.0 mmol) and TEA (0.25 g, 2.2 mmol) in 40 mL dried DCM, 4-tosyl chloride (0.38 g, 2.0 mmol) in 10 mL DCM was added slowly under 0° C. The resulting mixture was stirred under Ar 1  atomo and was permitted to warm to room temperature. After complete the reaction, the mixture was quenched by 2 mL of water. The reaction mixture was extracted three times and the organic phase was dried with anhydrous Na 2 SO 4  and evaporation under reduced pressure, the residue was used in the next step without purified. 
     To a stirring solution of the residue in 20 mL CH 3 CN, 1 ml MeNH2 was added under Ar atmosphere. The mixture was heated to refluxed overnight. Upon completing the reaction, the reaction mixture was cooled to room temperature and the organic liquid was removed under reduce pressure. Then the residue was dissolved in 50 mL DCM and the organic phase was washed with water and brine (2×100 ml). Upon drying over anhydrous Na 2 SO 4  and evaporation under reduced pressure, the residue was purified by column chromatography on silica gel to afford orangered solid. (0.54 g, 82%).  1 H NMR (400 MHz, CDCl 3 ): δ=7.88 (d, J=9.0 Hz, 2H), 7.74-7.65 (m, 4H), 7.48 (s, 1H), 6.73 (d, J=9.1 Hz, 2H), 3.60-3.55 (m, 2H), 3.08 (s, 3H), 2.57-2.52 (m, 2H), 2.34 (s, 6H). HRMS (ESI-TOF): Calcd. For C 21 H 23 N 4  [M+H] + : 331.1923. Found: 331.1925. 
     Example 5 
     Compound III-5 
     
       
         
         
             
             
         
       
     
     To a stirring solution of 3,5-difluoro-4-hydroxybenzaldehyde (0.32 g, 2.0 mmol) and 4-cyano-benzeneacetonitrile (0.35 g, 2.4 mmol) in 40 mL anhydrous EtOH, 2 drops of piperidine were added. After stirring at ambient temperature for 2 h, the mixture was cool to room temperature. A large amount of precipitate was appeared. Then the precipitate was obtained by filtration and washed with cold EtOH three times. The orange solid was obtained after dried under vacuum.  1 H NMR (400 MHz, CDCl 3 ): δ=7.80 (d, J=9.0 Hz, 2H), 7.74-7.66 (m, 4H), 7.48 (s, 1H). HRMS (ESI-TOF): Calcd. For C 16 H 9 F 2 N 2 O [M+H] + : 283.0683. Found: 283.0684. 
     Example 6 
     Compound 5-(N-methyl-N-(2-hydroxyethyl)amino) pyrazine-2-carbaldehyde 
     
       
         
         
             
             
         
       
     
     To a stirring solution of N-methyl-N-(2-hydroxyethyl)amino (2.6 g, 35 mmol) and 5-chloro-pyrazine-2-carbaldehyde (0.50 g, 3.5 mmol) in 20 mL dry CH 3 CN, K 2 CO 3  (0.71 g, 5.3 mmol) was added in one portion. The mixture was heated to reflux under Ar atmosphere. The mixture was heated to refluxed for 24 h. Upon completing the reaction, the reaction mixture was cooled to room temperature and the organic liquid was removed under reduce pressure. Then the residue was dissolved in 100 mL DCM and the organic phase was washed with water and brine (2×100 ml). Upon drying over anhydrous Na 2 SO 4  and evaporation under reduced pressure, the residue was purified by column chromatography on silica gel to afford target compound. (0.48 g, 76%).  1 H NMR (400 MHz, CDCl 3 ): δ 9.88 (s, 1H), 8.62 (d, J=1.2 Hz, 1H), 8.14 (d, J=1.1 Hz, 1H), 3.92 (m, 2H), 3.88-3.83 (m, 2H), 3.28 (s, 3H). HRMS (ESI-TOF): Calcd. For C 8 H 12 N 3 O 2  [M+H] + : 182.1. Found: 182.1. 
     Compound III-6 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1 (0.36 g, 96%).  1 H NMR (400 MHz, CDCl 3 ): δ 8.39 (s, 1H), 8.30 (s, 1H), 7.80 (d, J=8.5 Hz, 2H), 7.72 (d, J=8.4 Hz, 2H), 7.51 (s, 1H), 3.93 (t, J=4.9 Hz, 2H), 3.88-3.83 (m, 2H), 3.29 (s, 3H). HRMS (ESI-TOF): Calcd. For C 17 H 16 N 5 O [M+H] + : 306.1355. Found: 306.1357. 
     Example 7 
     Compound III-7 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-4, (0.21 g, 67%) o  1 H NMR (400 MHz, DMSO-d 6 ): δ 8.37 (d, J=5.2 Hz, 2H), 8.06 (s, 1H), 8.00-7.85 (m, 4H), 3.77 (t, J=6.5 Hz, 2H), 3.20 (s, 3H), 2.56 (m, 2H), 2.23 (s, 6H). HRMS (ESI-TOF): Calcd. For C 19 H 21 N 6  [M+H] + : 333.1828. Found: 333.1829. 
     Example 8 
     Compound 6-(N-methyl-N-(2-hydroxyethyl)amino) pyridine-2-carbaldehyde 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of Compound 5-(N-methyl-N-(2-hydroxyethyl)amino) pyrazine-2-carbaldehyde: (0.45 g, 68%).  1 H NMR (400 MHz, CDCl 3 ): δ=9.69 (s, 1H), 8.43 (d, J=2.1 Hz, 1H), 7.86 (dd, J=9.0, 2.3 Hz, 1H), 6.56 (d, J=9.1 Hz, 1H), 3.86-3.79 (m, 4H), 3.15 (s, 3H). HRMS (ESI-TOF): Calcd. For C 9 H 13 O 2 N 2  [M+H] + : 181.1. Found: 181.1. 
     Compound III-8 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.39 g, 89%) o  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.54 (d, J=4.0 Hz, 1H), 8.30 (dd, J=9.3, 2.5 Hz, 1H), 8.03 (s, 1H), 7.92 (d, J=8.0 Hz, 2H), 7.85 (d, J=8.0 Hz, 2H), 6.84 (d, J=8.0 Hz, 1H), 4.77 (t, J=5.4 Hz, 1H), 3.67 (t, J=5.3 Hz, 2H), 3.60 (q, J=5.4 Hz, 2H), 3.15 (s, 3H). HRMS (ESI-TOF): Calcd. For C 18 H 27 N 4 O [M+H] + : 305.1402. Found: 305.1401. 
     Example 9 
     Compound III-9 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-4, (0.31 g, 92%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.55 (d, J=4.0 Hz, 1H), 8.31 (dd, J=9.3, 2.5 Hz, 1H), 8.05 (s, 1H), 7.93 (d, J=8.0 Hz, 2H), 7.84 (d, J=8.0 Hz, 2H), 6.85 (d, J=8.0 Hz, 1H), 4.78 (t, J=5.4 Hz, 1H), 3.67 (t, J=5.3 Hz, 2H), 3.60 (q, J=5.4 Hz, 2H), 3.17 (t, J=8.0 Hz, 4H), 1.17 (t, J=8.0 Hz, 6H). HRMS (ESI-TOF): Calcd. For C 22 H 26 N 5  [M+H]+: 360.2188. Found: 360.2187. 
     Example 10 
     4-(N,N-dimethylamino)-pyrazine-6-carbaldehyde 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-4, (0.31 g, 49%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=9.86 (d, J=0.6 Hz, 1H), 8.17 (d, J=2.9 Hz, 1H), 7.83 (d, J=8.9 Hz, 1H), 6.94 (dd, J=8.8, 2.9 Hz, 1H), 3.10 (s, 6H). HRMS (ESI-TOF): Calcd. For C 8 H 11 N 2 O [M+H] + : 151.1. Found: 151.1. 
     Compound III-10 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.36 g, 96%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=9.86 (d, J=0.6 Hz, 1H), 8.26 (s, 1H), 8.17 (d, J=2.9 Hz, 1H), 7.83 (d, J=8.9 Hz, 1H), 7.46 (m, 4H), 6.94 (dd, J=8.8, 2.9 Hz, 1H), 3.10 (s, 6H). HRMS (ESI-TOF): Calcd. For C 17 H 15 N 4  [M+H] + : 275.1297. Found: 275.1298. 
     Example 11 
     Compound 2-(N-methyl-N-(2-hydroxyethyl)amino) pyrimidine-5-carbaldehyde 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-4, (0.42 g, 72%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=9.89 (s, 1H), 8.73 (s, 2H), 3.64 (t, J=8.9 Hz, 2H), 3.45 (t, J=8.8 Hz, 2H), 3.10 (s, 3H). HRMS (ESI-TOF): Calcd. For C 8 H 12 N 3 O [M+H] + : 182.1. Found: 182.1. 
     Compound III-11 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.36 g, 96%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.26 (s, 1H), 8.73 (s, 2H), 7.64 (m, 4H), 3.64 (t, J=8.9 Hz, 2H), 3.44 (t, J=8.8 Hz, 2H), 3.11 (s, 3H). HRMS (ESI-TOF): Calcd. For C 17 H 16 N 5 O [M+H] + : 306.1355. Found: 306.1356. 
     Example 12 
     Compound 5-(N-methyl-N-(2-hydroxyethyl)amino) pyrimidine-2-carbaldehyde 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-4, (0.42 g, 72%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=9.98 (s, 1H), 8.21 (s, 2H), 3.64 (t, J=8.9 Hz, 2H), 3.44 (t, J=8.8 Hz, 2H), 3.12 (s, 3H). HRMS (ESI-TOF): Calcd. For C 8 H 12 N 3 O 2  [M+H] + : 182.1. Found: 182.1. 
     4-(1-cyano-2-(5-((2-hydroxyethyl)(methyl)amino)pyrimidin-2-yl)vinyl)benzonitrile1 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.56 g, 89%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.21 (s, 2H), 7.99 (s, 1H), 7.64 (s, 4H), 3.64 (t, J=8.9 Hz, 2H), 3.44 (t, J=8.8 Hz, 2H), 3.12 (s, 3H). HRMS (ESI-TOF): Calcd. For C 17 H 16 N 5 O [M+H] + : 306.1. Found: 306.1. 
     Compound III-12 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-4, (0.36 g, 96%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.21 (s, 2H), 7.99 (s, 1H), 7.64 (s, 4H), 3.77 (t, J=6.5 Hz, 2H), 3.20 (s, 3H), 2.56 (m, 2H), 2.23 (s, 6H). HRMS (ESI-TOF): Calcd. For C 19 H 21 N 6  [M+H] + : 333.1828. Found: 333.1829. 
     Example 13 
     5-cyano-2-acetonitrile-pyridine 
     
       
         
         
             
             
         
       
     
     To a stirring solution of 2-(bromomethyl)-benzonitrile (0.50 g, 2.5 mmol) in 50 mL THF, 10 ml NaCN aqueous solution (2 M) was added. The mixture was reflexed for 12 h under Ar atmosphere. Upon cooling to room temperature, the reaction mixture was extracted with DCM (3×100 ml). The organic phase was washed with water and brine (2×100 ml). Upon drying over anhydrous Na 2 SO 4  and evaporation under reduced pressure, the residue was purified by column chromatography on silica gel to afford target compound. (0.19 g, 56%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.78 (s, 1H), 7.95 (m, 1H), 7.56 (m, 1H), 4.01 (s, 2H). HRMS (ESI-TOF): Calcd. For C 8 H 6 N 3  [M+H] + : 144.1. Found: 144.1. 
     Compound III-13 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.45 g, 95%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.78 (s, 1H), 8.21 (s, 1H), 7.94 (m, 1H), 7.86 (d, J=8.0 Hz, 2H), 7.57 (m, 1H), 6.80 (d, J=8.0 Hz, 2H), 3.64 (t, J=8.9 Hz, 2H), 3.44 (t, J=8.8 Hz, 2H), 3.12 (s, 3H). HRMS (ESI-TOF): Calcd. For C 18 H 17  N 4 O [M+H] + : 305.1402. Found: 305.1403. 
     Example 14 
     5-cyano-2-acetonitrile-pyrazine 
     
       
         
         
             
             
         
       
     
     To a stirring solution of 2-(5-chloropyrazin-2-yl)acetonitrile (0.32 g, 2.0 mmol) in dry 30 mL DMSO, CuCN (0.93 g, 10.0 mmol) was added in one portation. The mixture was heated for 12 h under Ar atmosphere. Upon cooling to room temperature, the reaction mixture was poured into 100 mL water, then extracted with DCM (4×50 ml). The organic phase was washed with water and brine (2×100 ml). Upon drying over anhydrous Na 2 SO 4  and evaporation under reduced pressure, the residue was purified by column chromatography on silica gel to afford target compound (0.20 g, 69%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.60 (s, 1H), 8.48 (s, 1H), 3.92 (s, 2H). HRMS (ESI-TOF): Calcd. For C 7 H 5 N 4  [M+H] + : 145.1. Found: 145.1. 
     Compound III-14 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.25 g, 91%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.60 (s, 1H), 8.48 (s, 1H), 8.11 (s, 1H), 7.81 (d, J=8.2 Hz, 2H), 6.84 (d, J=8.2 Hz, 2H), 3.60 (t, J=9.2 Hz, 2H), 3.46 (t, J=9.2 Hz, 2H), 3.12 (s, 3H). HRMS (ESI-TOF): Calcd. For C 17 H 16 N 5 O [M+H] + : 306.1355. Found: 306.1354. 
     Example 15 
     Compound III-15 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.25 g, 91%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.22 (s, 1H), 8.00 (d, J=9.1 Hz, 1H), 7.77-7.69 (m, 1H), 7.43-7.34 (m, 1H), 6.88 (d, J=9.1 Hz, 1H), 4.81 (t, J=5.2 Hz, 1H), 3.31-3.25 (m, 4H), 2.66-2.63 (m, 4H), 1.89-1.81 (m, 4H). HRMS (ESI-TOF): Calcd. For C 22 H 20 N 3  [M+H] + : 326.1657. Found: 326.1658. 
     Example 16 
     Compound III-16 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.29 g, 94%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.11 (2H, d, J=10.4 Hz), 7.99 (3H, dd, J=8.6, 3.0 Hz), 7.54 (1H, dd, J=8.0, 8.0 Hz), 7.44 (1H, dd, J=8.0, 8.0 Hz), 6.88 (2H, d, J=9.2 Hz), 4.82 (1H, bt, t, J=5.2 Hz), 3.01-3.08 (m, 2H), 3.53-3.60 (m, 2H), 2.89 (s, 3H). HRMS (ESI-TOF): Calcd. For C 19 H 16 N 3  [M+H] + : 286.1344. Found: 286.1345. 
     Compound 17 
     6-(methylamino)benzo[b]thiophene-2-carbaldehyde 
     
       
         
         
             
             
         
       
     
     6-(methylamino)benzo[b]thiophene-2-carbaldehyde (0.42 g, 1.7 mmol), 40% aqueous N,N-Dimethylethylamin solution (1 g, 8.9 mmol), CuI (13.9 mg, 0.073 mmol), K 3 PO 4 .H 2 O (155.4 mg, 0.73 mmol), 1 mL 33% aqueous methylamine solution and stirring bar was sealed in a screwed tube and stirred at 60° C. for 12 h. upon cooling to room temperature, the mixture was poured into 50 mL water. The organic layer was separated and the aqueous layer was extracted with DCM (3×100 ml). Combined the organic phase and dried over anhydrous Na 2 SO 4  and evaporation under reduced pressure, the residue was purified by column chromatography on silica gel to afford target compound (0.23 g, 68%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=9.92 (1H, s), 8.14 (1H, s), 7.82 (1H, d, J=9.1 Hz), 7.18 (1H, d, J=2.1 Hz), 7.01 (1H, dd, J=9.1, 2.3 Hz), 3.05 (3H, s). HRMS (ESI-TOF): Calcd. For C 10 H 10 NOS [M+H] + : 192.0. Found: 192.0. 
     Compound III-17 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.29 g, 94%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.45 (s, 1H), 7.92 (d, J=8.6 Hz, 2H), 7.85 (d, J=8.3 Hz, 3H), 7.73 (dd, J=8.6, 3.9 Hz, 1H), 7.21 (d, J=1.9 Hz, 1H), 7.21 (d, J=1.9 Hz, 1H), 6.96 (dd, J=9.1, 2.3 Hz, 1H), 3.05 (s, 3H). HRMS (ESI-TOF): Calcd. For C 19 H 14 N 3 S [M+H] + : 360.1171. Found: 360.1173. 
     Example 18 
     6-((2-hydroxyethyl)(methyl)amino)benzo[b]thiophene-2-carbaldehyde 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound 6-(methylamino)benzo[b]thiophene-2-carbaldehyde, (0.54 g, 79%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=9.91 (s, 1H), 8.14 (s, 1H), 7.81 (d, J=5.2 Hz, 1H), 7.17 (d, J=2.0 Hz, 1H), 7.01 (dd, J=2.0, 8.8 Hz, 1H), 4.76 (t, J=5.6 Hz, 1H), 3.58 (t, J=4.2 Hz, 2H), 3.52 (t, J=4.2 Hz, 2H), 3.04 (s, 3H). HRMS (ESI-TOF): m/z Calcd. For C 12 H 14 NO 2 S, [M+H] + : 235.1. Found 236.1. 
     Compound III-18 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.21 g, 95%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.45 (s, 1H), 7.92 (d, J=8.6 Hz, 2H), 7.85 (d, J=8.3 Hz, 3H), 7.73 (dd, J=8.6, 3.9 Hz, 1H), 7.21 (d, J=1.9 Hz, 1H), 7.21 (d, J=1.9 Hz, 1H), 6.96 (dd, J=9.1, 2.3 Hz, 1H), 3.63-3.57 (m, 2H), 3.52 (t, J=5.7 Hz, 2H), 3.05 (s, 3H). HRMS (ESI-TOF): Calcd. For C 21 H 19 N 3 OS [M+H] + : 360.1171. Found: 360.1173. 
     Example 19 
     5-(N,N-dimethylamino)-thieno[3,2-b]thiophene-2-carbaldehyde 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound 6-((2-hydroxyethyl)(methyl)amino)benzo[b]thiophene-2-carbaldehyde, (0.54 g, 79%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=9.66 (s, 1H), 8.05 (s, 1H), 6.30 (s, 1H), 4.88 (bt, 1H), 3.07 (s, 6H). HRMS (ESI-TOF): m/z Calcd. For C 9 H 12 NOS 2  [M+H] + : 214.0; found 214.0. 
     Compound III-19 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.31 g, 90%) o  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.34 (s, 1H), 7.86 (d, J=8.0 Hz, 2H), 7.81 (s, 1H), 7.77 (d, J=8.0 Hz, 2H), 6.32 (s, 1H), 4.88 (t, J=4.0 Hz, 1H), 3.08 (s, 6H). HRMS (ESI-TOF): Calcd. For C 18 H 14 N 3 S 2  [M+H] + : 336.0629. Found: 336.0630. 
     Example 20 
     5-(N,N-diethylamino)-thieno[3,2-b]thiophene-2-carbaldehyde 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound 5-(N,N-dimethylamino)-thieno[3,2-b]thiophene-2-carbaldehyde, (0.44 g, 75%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=9.78 (s, 1H), 8.09 (s, 1H), 6.30 (s, 1H), 4.87 (bt, 1H), 3.27 (t, J=8.4 Hz, 4H), 1.26 (t, J=8.4 Hz, 4H). HRMS (ESI-TOF): m/z Calcd. For C 9 H 12 NOS 2  [M+H] + : 214.0; found 214.0. 
     Compound III-20 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.31 g, 90%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.34 (s, 1H), 7.86 (d, J=8.0 Hz, 2H), 7.81 (s, 1H), 7.77 (d, J=8.0 Hz, 2H), 6.32 (s, 1H), 4.88 (t, J=4.0 Hz, 1H), 3.27 (t, J=8.4 Hz, 4H), 1.26 (t, J=8.4 Hz, 4H). HRMS (ESI-TOF): Calcd. For C 20 H 18 N 3 S 2  [M+H] + : 364.0942. Found: 364.0943. 
     Example 21 
     5-((2-hydroxyethyl)(methyl)amino)-thieno[3,2-b]thiophene-2-carbaldehyde 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound 6-((2-hydroxyethyl)(methyl)amino)benzo[b]thiophene-2-carbaldehyde, (0.44 g, 75%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=9.66 (s, 1H), 8.05 (s, 1H), 6.30 (s, 1H), 4.88 (bt, 1H), 3.64 (t, J=5.6 Hz, 2H), 3.44 (t, J=5.6 Hz, 2H), 3.07 (s, 3H). HRMS (ESI-TOF): m/z Calcd. For C 10 H 12 NO 2 S 2  [M+H] + : 241.0; found 242.0. 
     Compound III-21 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.31 g, 90%)  1 H NMR (400 MHz, DMSO-d 6 ): δ 8.34 (s, 1H), 7.86 (d, J=8.0 Hz, 2H), 7.81 (s, 1H), 7.77 (d, J=8.0 Hz, 2H), 6.32 (s, 1H), 4.88 (t, J=4.0 Hz, 1H), 3.65 (q, J=5.5 Hz, 2H), 3.44 (t, J=5.5 Hz, 2H), 3.34 (s, 1H), 3.08 (s, 3H). HRMS (ESI-TOF): Calcd. For C 19 H 16 N 3 OS 2  [M+H] + : 366.0735. Found: 366.0736. 
     Example 22 
     Compound III-22 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.31 g, 90%) 1 H NMR (400 MHz, DMSO-d 6 ): δ=3.04 (s, 6H), 6.82 (d, J=9.2 Hz, 2H), 7.59 (d, J=9.1 Hz, 2H), 7.84-7.94 (m, 6H), 8.02 ppm (s, 1H). HRMS (ESI-TOF): Calcd. For C 24 H 19 O 3  [M+H] + : 350.1657. Found: 350.1656. 
     Example 23 
     Compound III-23 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1:  1 H NMR (400 MHz, DMSO-d 6 ): δ=3.02 (s, 6H), 6.72 (d, J=8.0 Hz, 2H), 7.24 (d, J=4.0 Hz, 1H), 7.49 (d, J=8.8 Hz, 2H), 7.55 (d, J=8.0 Hz, 1H), 7.69 (d, J=8.8 Hz, 2H), 8.02 ppm (s, 1H). HRMS (ESI-TOF): Calcd. For C 22 H 18 N 3 S [M+H] + : 356.1221. Found: 356.1220. 
     Example 24 
     Compound III-24 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, and compound 1 (With reference to the synthetic method of Chem. Commun. 2011, 47, 985-987):  1 H NMR (400 MHz, DMSO-d 6 ): δ=3.63 (m, 16H), 3.77 (m, 4H), 6.76 (d, J=8.8 Hz, 2H), 7.38 (d, J=4.0 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H), 7.59 (d, J=8.8 Hz, 2H), 7.72 (m, 4H), 8.28 (s, 1H). HRMS (ESI-TOF): Calcd. For C 30 H 32 O 3 N 4 S [M+H] + : 530.2114. Found: 530.2115. 
     Example 25 
     Compound III-25 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, and compound 2 (With reference to the synthetic method of J. Org. Chem. 2008, 73, 6587-6594):  1 H NMR (400 MHz, DMSO-d 6 ): δ=1.23 (t, J=7.2 Hz, 6H), 3.35 (m, J=7.2 Hz, 4H), 5.78 (d, J=4.0 Hz, 1H), 6.92 (d, J=4.0 Hz, 1H), 7.12 (d, J=4.0 Hz, 1H), 7.49 (d, J=8.8 Hz, 2H), 7.56 (d, J=4.0 Hz, 1H), 7.69 (d, J=8.8 Hz, 2H), 8.28 (s, 1H). HRMS (ESI-TOF): Calcd. For C 30 H 32 O 3 N 4 S [M+H] + : 390.1099. Found: 390.1097. 
     Example 26 
     Compound III-26 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1,  1 H NMR (400 MHz, DMSO-d 6 ): δ=3.30 (s, 6H), 5.71 (d, J=4.0 Hz, 1H), 6.93 (d, J=4.0 Hz, 1H), 7.15 (d, J=4.0 Hz, 1H), 7.47 (d, J=8.8 Hz, 2H), 7.56 (d, J=4.0 Hz, 1H), 7.64 (d, J=8.8 Hz, 2H), 8.28 (s, 1H). HRMS (ESI-TOF): Calcd. For C 20 H 17 O 2 N 2 S 2  [M+H] + : 381.0731. Found: 381.0730. 
     Example 27 
     Compound III-27 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, and compound 4 (With reference to the synthetic method of Heterocycles, 1997, 46, 489-501.)  1 H NMR (400 MHz, CDCl 3 ): δ 2.07 (m, 4H), 3.33 (t, J=6.6 Hz, 4H), 4.2 (s, 3H), 5.70 (d, J=4.4 Hz, 1H), 6.92 (d, J=4.0 Hz, 1H), 7.15 (d, J=4.0 Hz, 1H), 7.43 (d, J=8.2 Hz, 2H), 7.51 (d, J=8.2 Hz, 2H), 7.57 (d, J=4.0 Hz, 1H), 8.10 (s, 1H). HRMS (ESI-TOF): Calcd. For C 23 H 21 O 2 N 2 S 2  [M+H] + : 421.1044. Found: 521.1042. 
     Example 28 
     Compound III-28 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, and compound 5 (With reference to the synthetic method of WO2018014821).  1 H-NMR (400 MHz, DMSO-d 6 ): δ=7.84 (s, 1H), 7.59 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H), 7.24 (s, 1H), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H) o  HRMS (ESI-TOF): Calcd. For C 21 H 16 ON 3 S 3 . [M+H] + : 422.0455. Found: 422.0456. 
     Example 29 
     Compound III-29 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, and compound 6 (With reference to the synthetic method of WO2018014821) 1 H-NMR (400 MHz, DMSO-d 6 ): δ=7.84 (s, 1H) 7.59 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H), 7.24 (s, 1H), 3.56 (q, J=4.0 Hz, 2H), 3.01 (s, 6H), 1.21 (t, J=4.0 Hz, 3H). HRMS (ESI-TOF): Calcd. For C 22 H 19 O 2 N 2 S 3 . [M+H] + : 439.0609. Found: 439.0610. 
     Example 30 
     Compound III-30 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, and compound 7 (With reference to the synthetic method of WO 2014048547).  1 H-NMR (400 MHz, DMSO-d 6 ): δ=7.84 (s, 1H), 7.59 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H), 7.24 (s, 1H), 3.10 (s, 3H), 3.01 (s, 6H). HRMS (ESI-TOF): Calcd. For C 21 H 17 O 1 N 2 S 4 . [M+H] + : 429.0024. Found: 429.0026. 
     Example 31 
     Compound III-31 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, and compound 9 (With reference to the synthetic method of J. Chem. Pharm. Res., 2012, 4, 1661-1669).  1 H-NMR (400 MHz, DMSO-d 6 ): δ=7.84 (s, 1H), 7.59 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H), 7.24 (s, 1H), 3.14 (s, 3H), 3.01 (s, 6H). HRMS (ESI-TOF): Calcd. For C 22 H 23 O 2 N 2 S 3 Si. [M+H] + : 471.0691. Found: 471.0690. 
     Example 32 
     Compound III-32 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1.  1 H-NMR (400 MHz, DMSO-d 6 ): δ=7.84 (s, 1H), 7.59 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H), 7.24 (s, 1H), 3.77 (t, 2H, J=4.80 Hz), 3.41 (t, 2H, J=4.80 Hz), 3.00 (s, 3H). HRMS (ESI-TOF): Calcd. For C 22 H 24 O 3 N 3 S 3 Si. [M+H] + : 502.0749. Found: 502.0752. 
     Example 33 
     Compound III-33 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1.  1 H-NMR (400 MHz, CDCl 3 ): δ=7.89 (s, 1H), 7.59 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H), 7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz), 3.85 (t, 2H, J=4.80 Hz), 3.46 (t, 2H, J=4.80 Hz), 3.06 (s, 3H), 0.46 (s, 6H). Calcd. For C 23 H 22 ON 3 S 2 Si. [M+H] + : 448.0974. Found: 448.0972. 
     Example 34 
     Compound III-34 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1. H-NMR (400 MHz, CDCl 3 ): δ=7.83 (s, 1H), 7.59 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H), 7.11 (s, 1H), 3.85 (t, 2H, J=4.80 Hz), 3.46 (t, 2H, J=4.80 Hz), 3.06 (s, 3H), 1.46 (s, 6H). HRMS (ESI-TOF): Calcd. For C 24 H 24 O 2 N 3 S 2  [M+H] + :450.1310. Found: 450.1311. 
     Example 35 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of (K. T. Arun et. al. J. Phys. Chem. A. 2005, 109, 5571-5578.)  1 H-NMR (400 MHz, CDCl 3 ): δ=10.01 (s, 1H), 7.89 (s, 1H), 7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz), 3.52-3.65 (m, 20H), 3.37 (s, 3H), 2.97 (s, 3H). HRMS (ESI-TOF): Calcd. For C 24 H 22 ON 3 S 2 Si. [M+H] + :432.1204. Found: 432.1203. Calcd. For C 24 H 36 O 6 N 1 S 2 . [M+H] + : 497.3. Found: 497.3. 
     Compound III-35 
     With reference to the synthetic method of compound III-1.  1 H-NMR (400 MHz, CDCl 3 ): δ=7.89 (s, 1H), 7.59 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H), 7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz), 3.52-3.65 (m, 20H), 3.37 (s, 3H), 2.97 (s, 3H). HRMS (ESI-TOF): Calcd. For C 33 H 39 O 5 N 3 S 2 . [M+H] + : 622.2409. Found: 622.2409. 
     Control Example 1 
     Compound III-36 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.25 g, 91%) o. H NMR (400 MHz, DMSO-d 6 ): δ=8.21 (s, 2H), 7.99 (s, 1H), 7.64 (s, 4H), 3.64 (t, J=8.9 Hz, 2H), 3.44 (t, J=8.8 Hz, 2H), 3.12 (s, 3H). HRMS (ESI-TOF): Calcd. For C 16 H 17 N 4 O 4 S [M+H] + : 361.0971. Found: 361.0970 
     Control Example 2 
     Compound III-37 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.39 g, 910%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=7.83 (s, 1H), 7.59 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H), 7.11 (s, 1H), 3.85 (t, 2H, J=4.80 Hz), 3.46 (t, 2H, J=4.80 Hz), 3.05 (s, 3H), 1.46 (s, 6H). HRMS (ESI-TOF): Calcd. For C 23 H 23 N 2 O 4 S 3  [M+H] + : 487.0820. Found: 487.0821. 
     Control Example 3 
     Compound III-38 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, and compound 11 (With reference to the synthetic method of CN 106349105).  1 H-NMR (400 MHz, DMSO-d 6 ): δ=7.84 (s, 1H), 7.59 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H), 7.24 (s, 1H), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.01 (s, 3H). HRMS (ESI-TOF): Calcd. For C 22 H 23 O 4 N 2 S 3 Si. [M+H] + : 503.0589. Found: 203.0588. 
     Control Example 4 
     Compound III-39 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1.  1 H-NMR (400 MHz, DMSO-d 6 ): δ=7.84 (s, 1H), 7.59 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.01 (s, 3H). HRMS (ESI-TOF): Calcd. For C 18 H 19 O 4 N 2 S.[M+H] + : 359.1066. Found: 359.1065. 
     Control Example 5 
     Compound III-40 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1.  1 H-NMR (400 MHz, DMSO-d 6 ): δ=8.34 (s, 1H), 7.59 (d, J=8.0 Hz, 2H), 7.81 (s, 1H), 7.49 (d, J=8.0 Hz, 2H), 6.32 (s, 1H), 4.88 (t, J=4.0 Hz, 1H), 3.65 (q, J=5.5 Hz, 2H), 3.44 (t, J=5.5 Hz, 2H), 3.34 (s, 1H), 3.08 (s, 3H). HRMS (ESI-TOF): Calcd. For C 18 H 17 O 4 N 2 S 3 . [M+H] + : 421.0350. Found: 421.0351. 
     Control Example 6 
     Compound III-41 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1.  1 H-NMR (400 MHz, DMSO-d 6 ): δ=7.85 (s, 1H), 7.59 (d, J=8.8 Hz, 2H), 7.47 (d, J=8.8 Hz, 2H), 7.24 (s, 1H), 3.79 (t, 2H, J=4.80 Hz), 3.43 (t, 2H, J=4.80 Hz), 3.01 (s, 3H). HRMS (ESI-TOF): Calcd. For C 20 H 17 O 4 N 2 S 4 . [M+H] + : 477.0071. Found: 477.0070. 
     Control Example 7 
     Compound III-42 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.25 g, 91%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=8.22 (s, 1H), 8.00 (d, J=9.1 Hz, 1H), 7.77-7.69 (m, 1H), 7.43-7.34 (m, 1H), 6.88 (d, J=9.1 Hz, 1H), 4.81 (t, J=5.2 Hz, 1H), 3.64-3.52 (m, 3H), 3.09 (s, 1H). LR-HRMS (ESI-TOF): Calcd. For C 19 H 18 N 3 O 2  [M+H] + : 320.1399. Found: 320.1397. 
     Control Example 8 
     Compound III-43 
     
       
         
         
             
             
         
       
     
     With reference to the synthetic method of compound III-1, (0.29 g, 94%) 1 H NMR (400 MHz, DMSO-d 6 ): δ=8.11 (2H, d, J=10.4 Hz), 7.99 (3H, dd, J=8.6, 3.0 Hz), 7.54 (1H, dd, J=8.0, 8.0 Hz), 7.44 (1H, dd, J=8.0, 8.0 Hz), 6.88 (2H, d, J=9.2 Hz), 4.82 (1H, bt, t, J=5.2 Hz), 3.60 (2H, t, J=5.2 Hz), 3.56 (2H, t, J=5.2 Hz), 3.09 (3H, s). LR-HRMS (ESI-TOF): Calcd. For C 19 H 18 N 3 OS [M+H] + : 336.1171. Found: 336.1170. 
     Test Example 1 
     The fluorescent dyes (molecular rotors) prepared in Examples 1-35 were dissolved in DMSO with a concentration of 1×10 −2  M each, and each master batch was added to glycerol and methanol respectively, mixed well, and a solution with a final concentration of 1×10 −5  M each was prepared. According to the different fluorescent dyes, the fluorescence emission pattern of each fluorescent dye was detected under the same conditions using the maximum excitation wavelength of each fluorescent dye in turn, and the results are shown in Table 1, indicating that the fluorescent dyes of the present invention are sensitive to changes in viscosity. 
     
       
         
           
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                 Glycerol/methanol 
               
               
                   
                 Emission  
                 fluorescence 
               
               
                 Compound 
                 (nm) 
                 intensity ratio 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 III-1  
                 530 
                 990 
               
               
                 III-2  
                 530 
                 870 
               
               
                 III-3  
                 530 
                 1025 
               
               
                 III-4  
                 521 
                 892 
               
               
                 III-5  
                 525 
                 1028 
               
               
                 III-6  
                 490 
                 1148 
               
               
                 III-7  
                 485 
                 977 
               
               
                 III-8  
                 495 
                 1168 
               
               
                 III-9  
                 490 
                 920 
               
               
                 III-10 
                 520 
                 1620 
               
               
                 III-11 
                 470 
                 869 
               
               
                 III-12 
                 542 
                 855 
               
               
                 III-13 
                 545 
                 752 
               
               
                 III-14 
                 550 
                 785 
               
               
                 III-15 
                 561 
                 1011 
               
               
                 III-16 
                 555 
                 491 
               
               
                 III-17 
                 587 
                 828 
               
               
                 III-18 
                 595 
                 978 
               
               
                 III-19 
                 620 
                 991 
               
               
                 III-20 
                 620 
                 836 
               
               
                 III-21 
                 620 
                 544 
               
               
                 III-22 
                 650 
                 989 
               
               
                 III-23 
                 661 
                 687 
               
               
                 III-24 
                 662 
                 596 
               
               
                 III-25 
                 678 
                 783 
               
               
                 III-26 
                 676 
                 368 
               
               
                 III-27 
                 678 
                 486 
               
               
                 III-28 
                 662 
                 559 
               
               
                 III-29 
                 665 
                 684 
               
               
                 III-30 
                 660 
                 756 
               
               
                 III-31 
                 687 
                 624 
               
               
                 III-32 
                 690 
                 817 
               
               
                 III-33 
                 705 
                 691 
               
               
                 III-34 
                 689 
                 489 
               
               
                 III-35 
                 690 
                 710 
               
               
                   
               
            
           
         
       
     
     Test Example 2 
     Add molecular rotors III-3, III-4, III-28 and III-34 to a diethanol-glycerol mixed solution to prepare a solution with a final concentration of 1×10 −5  M, conduct excitation at 480 nm, and the fluorescence emission spectra at different viscosity conditions are shown as  FIGS. 1, 3, 5 and 7 , wherein molecular rotors of the same concentration have gradually increasing fluorescence intensity at different viscosity conditions, which indicates that the fluorescence intensity of molecular rotors increases following the increasing fluorescence of environmental viscosity, and that the relationship between the fluorescence intensity log and the solvent intensity log satisfies the Huffman equation and has a fine linear relation as shown in  FIGS. 2, 4, 6, 8 , proving that that molecular rotors are sensitive to viscosity and can be used for viscosity tests of unknown samples. 
     Test Example 3 
     Add molecular rotors III-11 and III-36; III-34 and III-37; III-31, III-32, III-33 and III-38; 11-3 and III-39; III-21 and III-40; III-28, III-29, III-30 and III-41; III-3 and III-42; III-3 and III-43 to a PBS solution to prepare a solution with a final concentration of 1×10 −6  M, conduct excitation respectively at the maximum excitation of each compound so as to detect their fluorescence intensities in PBS, and normalize each sample with the strongest fluorescence in each group as 100, as shown respectively in  FIG. 9 ,  FIG. 10 ,  FIG. 11 ,  FIG. 12 ,  FIG. 13 ,  FIG. 14 ,  FIG. 15  and  FIG. 16 . According to the results, compared with the molecular rotors with sulfonic acid group substitution and the rotors without substitution on the aromatic ring of the electron withdrawing group, the molecular rotors with cyano group, ester group, sulfoxide, sulphone, sulfonamido substitutions on the aromatic ring of the electron withdrawing group in the present application have lower background fluorescence. 
     Test Example 4 
     Compounds III-3, III-4, III-6, III-7, III-8, III-18, III-21 and RNA aptamer (Sequence 10: F30-8Pepper-5 RNA aptamer sequence UUGCCAUGUGUAUGUGGGUUCGCCCACAUACUCUGAUGAUCCCCAAUC GUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCG UGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGU GGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUG GCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCUUCGGAGAGG CACUGGCGCCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGAGA GGCACUGGCGCCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGA GAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGGAUCAUUCAUGGCAA) are specifically bound, and the compound fluorescence after binding is noticeably activated and emits bright fluorescence when being excited by excitation light with an appropriate wavelength, see Table 2 for the optical properties after binding; the compounds can also bind to this aptamer in cells, and cells transcribing the RNA aptamer have bright fluorescence, as shown in  FIG. 17A , and cells not expressing the RNA aptamer has no fluorescence, as shown in  FIG. 17B , indicating that dyes of this series can be used for nucleic acid labeling. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                   
                 ε (M −1   
                 QY  
                 Activation 
                 K d    
               
               
                 Name 
                 Ex/nm 
                 Em/nm 
                 cm −1 ) 
                 (−) 
                 Multiple 
                 (nM) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 III-7 
                 443 
                 485 
                 49100 
                 0.42 
                 691 
                 8.0 
               
               
                 III-6 
                 435 
                 497 
                 54700 
                 0.57 
                 16601 
                 6.7 
               
               
                 III-8 
                 458 
                 508 
                 42500 
                 0.30 
                 9091 
                 27.0 
               
               
                 III-4 
                 458 
                 514 
                 44100 
                 0.45 
                 4748 
                 12.0 
               
               
                 III-3 
                 485 
                 530 
                 65300 
                 0.66 
                 3595 
                 3.5 
               
               
                 III-18 
                 515 
                 599 
                 54400 
                 0.43 
                 708 
                 18.0 
               
               
                 III-21 
                 577 
                 620 
                 10000 
                 0.58 
                 12600 
                 6.1 
               
               
                   
               
               
                 Note: 
               
               
                 the fluorescence quantum yield was measured by the relative method with Rhodamine 6G as the standard (QY = 0.94). 
               
            
           
         
       
     
     Test Example 5 
     A stable cell line (293T/17 cell line) was constructed by fusing the skeleton protein mRNA with the aptamer (ACTB-4Pepper RNA aptamer sequence AUGGAUGAUGAUAUCGCCGCGCUCGUCGUCGACAACGGCUCCGGCAUG UGCAAGGCCGGCUUCGCGGGCGACGAUGCCCCCCGGGCCGUCUUCCCCU CCAUCGUGGGGCGCCCCAGGCACCAGGGCGUGAUGGUGGGCAUGGGUC AGAAGGAUUCCUAUGUGGGCGACGAGGCCCAGAGCAAGAGAGGCAUCC UCACCCUGAAGUACCCCAUCGAGCACGGCAUCGUCACCAACUGGGACGA CAUGGAGAAAAUCUGGCACCACACCUUCUACAAUGAGCUGCGUGUGGC UCCCGAGGAGCACCCCGUGCUGCUGACCGAGGCCCCCCUGAACCCCAAG GCCAACCGCGAGAAGAUGACCCAGAUCAUGUUUGAGACCUUCAACACCC CAGCCAUGUACGUUGCUAUCCAGGCUGUGCUAUCCCUGUACGCCUCUGG CCGUACCACUGGCAUCGUGAUGGACUCCGGUGACGGGGUCACCCACACU GUGCCCAUCUACGAGGGGUAUGCCCUCCCCCAUGCCAUCCUGCGUCUGG ACCUGGCUGGCCGGGACCUGACUGACUACCUCAUGAAGAUCCUCACCGA GCGCGGCUACAGCUUCACCACCACGGCCGAGCGGGAAAUCGUGCGUGAC AUUAAGGAGAAGCUGUGCUACGUCGCCCUGGACUUCGAGCAAGAGAUG GCCACGGCUGCUUCCAGCUCCUCCCUGGAGAAGAGCUACGAGCUGCCUG ACGGCCAGGUCAUCACCAUUGGCAAUGAGCGGUUCCGCUGCCCUGAGGC ACUCUUCCAGCCUUCCUUCCUGGGCAUGGAGUCCUGUGGCAUCCACGAA ACUACCUUCAACUCCAUCAUGAAGUGUGACGUGGACAUCCGCAAAGACC UGUACGCCAACACAGUGCUGUCUGGCGGCACCACCAUGUACCCUGGCAU UGCCGACAGGAUGCAGAAGGAGAUCACUGCCCUGGCACCCAGCACAAUG AAGAUCAAGAUCAUUGCUCCUCCUGAGCGCAAGUACUCCGUGUGGAUC GGCGGCUCCAUCCUGGCCUCGCUGUCCACCUUCCAGCAGAUGUGGAUCA GCAAGCAGGAGUAUGACGAGUCCGGCCCCUCCAUCGUCCACCGCAAAUG CUUCUAGCACUCGCUAGAGCAUGGUUAAGCUUCCCACGGAGGAUCCCCA AUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAA UCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAU CGUGGCGUGUCGGCCUCUCUUCGGAGAGGCACUGGCGCCGGAGAGGCAC UGGCGCCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGGAUCCU CCGUGGG), and, under the conditions of conventional mammalian cell culture (37° C., 5% carbon dioxide, 100% relative humidity), the cells were digested after the cell line and control cells (293T/17) grew to a cell confluence of 90%, and were centrifuged at 800 rpm, and then the cells were re-suspended with PBS containing 0.2 μM of III-3 and 0.2 μM of III-43 molecules, and were incubated for 5 minutes before flow detection, see  FIGS. 18A and 18B  for the detection results; the III-3 molecular rotors could specifically mark the mRNA of skeleton protein in cell lines expressing target RNA, and there was no obvious background fluorescence (as shown in  FIG. 18A ), while the background fluorescence of III-43 molecules was higher than III-3, and it was unclear whether ACTB was expressed (see  FIG. 18B ).