Patent Publication Number: US-2023141569-A1

Title: Fluorescent dye, and preparation method and uses thereof

Description:
RELATED APPLICATIONS 
     The present application is a U.S. National Phase of International Application Number PCT/CN2021/080926, filed Mar. 16, 2021, and claims priority to Chinese Application Number 202010207772.8, filed Mar. 23, 2020. 
    
    
     INCORPORATION BY REFERENCE 
     The sequence listing provided in the file entitled 0722207066 PCT_US_seqn.txt, which is an ASCII text file that was created on Sep. 21, 2022, and which comprises 2,529 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 
     Chromophores of fluorescent proteins are mainly imidazolinone dye, which has good biocompatibility, and is also advantageous in photostability and adjustable fluorescence properties. According to research findings, this kind of dye is a molecular rotor, and its fluorescence property changes with viscosity variation, namely, when excited by light, free-state molecular rotors release excited-state energy in a non-radiation form by means of excited-state intra-molecular twist; and, when the molecules are in a relatively viscous or rigid environment, the excited-state intra-molecular twist is limited, and the excited-state energy is mainly released in a form of radio-luminescence and shows the property of fluorescence enhancement. Therefore, molecular rotors are often used for detecting changes in micro-environments, etc. 
     At present, intra-molecular twist and luminescence based on restricted molecular rotors are not only used for viscosity detection, but also are widely used in the construction of fluorescent sensors, protein labeling, nucleic acid labeling, etc.; for instance: after DCVJ binds to BSA and other proteins, the excited-state intra-molecular twist of the molecular rotors is restricted, and the excited-state energy is released in a form of radio-luminescence and is manifested as fluorescence activation, and thus can be used for the detection and quantification of target proteins; after imidazolinone and other dyes bind to DNA and RNA, molecular conformation is restricted so that fluorescence is illuminated, and thus can be used for the no-clean labeling and detection of DNA and RNA; with DHBI as the target molecule, aptamer that can specifically bind to DHBI can be obtained through SELEX screening, thereby constructing the fluorescent aptamer, overcoming the difficulty of lacking natural fluorescent RNA and making it possible to construct fluorescent RNA; with peacock green as the target molecule, a single-chain antibody that can specifically bind to peacock green derivatives and activate fluorescence is obtained by means of phage display, and can be used for labeling of cell membrane proteins; BODIPY and other molecular rotors can bind to amyloid and tua proteins and light up fluorescence, and thus can be used for researches of diseases such as Alzheimers and Parkinson&#39;s. 
     The types of imidazolinone molecular rotors completely derived from fluorescent protein chromophores are quite limited. Thanks to modification by means of organic synthesis, the spectral range of imidazolinone is significantly expanded, and other properties, such as light stability, oil-water distribution coefficient, etc. can be effective adjusted. However, molecular rotors obtained by this method generally have the disadvantage of high fluorescence background, which causes low detection signal-to-noise ratio and makes it difficult to detect and label samples with small sample sizes, complex components and low substrate abundance, so it is necessary to develop a kind of imidazolinone molecular rotors with low background fluorescence, so as to further expand uses of such 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, wherein the viscosity responsiveness of this kind of molecular rotors is that a ratio of fluorescence intensity in glycerol to that in methanol is greater than 10 at a condition of 10 −5  mol. 
     For one aspect, the present invention provides a fluorescent dye, which is shown as Formula (I), 
     
       
         
         
             
             
         
       
     
     wherein: 
     Ar is arylene or heteroarylene, and optionally, hydrogen atoms in Ar are independently substituted by halogen atoms; D- is HO— or N(X 1 )(X 2 )—, and X 1  and X 2  are independently selected from hydrogen, alkyl or modified alkyl; X 1  and X 2  are optionally interconnected, and form an alicyclic heterocycle with N atoms; and X 1  and X 2  are optionally and independently form an alicyclic heterocycle with Ar; 
     Y is 0 or S; 
     R 1  is hydrogen or alkyl; and 
     R 2  is a halogen atom, —OH or —CN; 
     wherein: 
     the “alkyl” is C 1 -C 10  straight or branched alkyl; optionally, the “alkyl” is C 1 -C 6  straight or branched alkyl; optionally, the “alkyl” is C 1 -C 4  straight or branched alkyl; optionally, the “alkyl” 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 independently a group obtained by replacing any carbon atom in C 1 -C 16  straight or branched alkyl with one or more groups selected from halogen atom, —OH, —CO—, —O—, —CN, —SO 3 H—, primary amino group, secondary amino group and tertiary amino group, or 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 independently F, Cl, Br or I; 
     the “alicyclic heterocycle” is a saturated or unsaturated 4- to 15-membered monocyclic or polycyclic alicyclic heterocycle containing one or more heteroatoms of N, O, S or Si on the ring, and the alicyclic heterocycle containing S heteroatom(s) on the ring include the ones that contain —S—, —SO— or —SO 2 — on the ring; the alicyclic heterocycle is optionally substituted by a halogen atom, an alkyl, an aryl or a modified alkyl; 
     the “arylene” is independently a 5- to 13-membered (optionally 6- or 10-membered) monocyclic or dicyclic or fused dicyclic or fused polycyclic subaromatic group; 
     the “heteroarylene” is independently a 5- to 13-membered (optionally 6- or 10-membered) monocyclic or dicyclic or fused dicyclic or fused polycyclic sub-heteroaromatic group containing one or more heteroatoms selected from N, O, S or Si on the ring; 
     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; 
     each R′, R″, R′″ is independently a single bond, hydrogen, alkyl, or 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; 
     optionally, the “modified alkylene” is a group containing one or more groups selected from —OH, —O—, —NH 2 , ethylene glycol unit (—(CH 2 CH 2 O) n —), —CN —O—CO—, —NH—CO—, —SO 2 —O—, —SO—, Me 2 N—, Et 2 N—, —CH═CH—, —C≡CH, F, Cl, Br, I, and cyano group; and optionally, Ar is a structure selected from the following Formulae (II-1) to (II-7): 
     
       
         
         
             
             
         
       
     
     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 for 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-up 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-up 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-up 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 specifically bind to corresponding antibody, aptamer or amyloid, or bind to the protein tag or enzyme via a ligand or inhibitor, thereby obtaining a series of fluorescent activated and lighted-up probes used for fluorescent labeling, quantification or monitoring of proteins, 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 IV-1 (1×10 −5  M); 
         FIG.  2    is a diagram showing the fluorescence emission intensity at different viscosity conditions of the molecular rotor IV-2 (1×10 −5  M); 
         FIG.  3    is a diagram showing the fluorescence emission intensity at different viscosity conditions of the molecular rotor IV-3 (1×10 −5  M); 
         FIG.  4    is a diagram showing the fluorescence emission intensity at different viscosity conditions of the molecular rotor IV-4 (1×10 −5  M); 
         FIG.  5    is a diagram showing the fluorescence emission intensity at different viscosity conditions of the molecular rotor IV-5 (1×10 −5  M); 
         FIG.  6    is a diagram showing the fluorescence emission intensity at different viscosity conditions of the molecular rotor IV-6 (1×10 −5  M); 
         FIG.  7    is a diagram showing the fluorescence emission intensity at different viscosity conditions of the molecular rotor IV-17 (1×10 −5  M); 
         FIG.  8    is a diagram showing the fluorescence emission intensity at different viscosity conditions of the molecular rotor IV-18 (1×10 −5  M); 
         FIG.  9    is a diagram showing the fluorescence emission intensity at different viscosity conditions of the molecular rotor IV-19 (1×10 −5  M); 
         FIG.  10    is a diagram showing the fluorescence emission intensity at different viscosity conditions of the molecular rotor IV-20 (1×10 −5  M); 
         FIG.  11    is a diagram showing the fluorescence emission intensity at different viscosity conditions of the molecular rotor IV-21 (1×10 −5  M); 
         FIG.  12    is a diagram showing the fluorescence emission intensity at different viscosity conditions of the molecular rotor IV-22 (1×10 −5  M); 
         FIG.  13    is a diagram showing the fluorescence background contrast of molecular rotors IV-39, IV-40, IV-44 and IV-1, IV-2, IV-3, IV-4, IV-5, IV-6, IV-7, IV-8 (1×10 −6  M) in PBS; 
         FIG.  14    is a diagram showing the fluorescence background contrast of molecular rotors IV-41, IV-43 and IV-17, IV-18, IV-19, IV-20, IV-21, IV-22 (1×10 −6  M) in PBS; 
         FIG.  15    is a diagram showing the fluorescence background contrast of molecular rotors IV-42 and IV-36 (1×10 −6  M) in PBS; 
         FIG.  16    is a diagram showing the fluorescence background contrast of molecular rotors IV-45, IV-46 and IV-3 (1×10 −6  M) in PBS; 
         FIG.  17    is a diagram showing the fluorescence background contrast of molecular rotors IV-47, IV-48  IV-20 (1×10 −6  M) in PBS; 
         FIG.  18    is a diagram showing the fluorescence background contrast of molecular rotors IV-49, IV-50  IV-5 (1×10 −6  M) in PBS; 
         FIG.  19    is a diagram showing the fluorescence background contrast of molecular rotors IV-51  IV-1 (1×10 −6  M) in PBS; 
         FIGS.  20 A and  20 B  are the application of molecular rotors IV-1, IV-2, IV-3, IV-4, IV-5, IV-6, IV-17, IV-18, IV-19, IV-20, IV-21, IV-22 in labeling intracellular RNA aptamer, wherein  FIG.  20 A  are cells expressing the target RNA aptamer, and  FIG.  20 B  are cells not expressing the target RNA aptamer; 
         FIGS.  21 A and  21 B  are a flow detection diagram for molecular rotors IV-21 and IV-41 for labeling mRNA. 
     
    
    
     EMBODIMENTS 
     Examples 
     Example 1 
     
       
         
         
             
             
         
       
     
     To a stirred solution of Compound 1 (0.504 g, 2 mmol), anhydrous zinc chloride (0.545 g, 4 mmol) in 100 mL THF, 4-Cyanobenzaidehyde (0.626 g, 5 mmol) was added. The complete solution were stirred at 80° C. under Ar atomophere. The progress of reaction was monitored on silica gel TLC. After completion of reaction, the solution allowing the reaction to cool to room temperature. The solvent is evaporated to dryness, to give a crude product, then purified by silica gel column chromatography to afford a target compound (0.292 g, 40%).  1 H NMR (400 MHz, DMSO-d 6 ) δ11.07 (s, 1H), 8.10 (d, J=8.5 Hz, 2H), 8.06 (dd, J=7.8, 2.1 Hz, 2H), 8.03 (s, 1H), 7.94 (d, J=8.3 Hz, 2H), 7.44 (d, J=15.9 Hz, 1H), 7.03 (s, 1H), 3.29 (s, 3H).MS(ESI): m/z Calcd. For C 20 H 13 F 2 N 3 O 2  365.0976; found 364.0902, [M−H] − . 
     Example 2 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.475 g, 65%)○ 1 H NMR (400 MHz, DMSO-d 6 ) δ11.04 (s, 1H), 8.49 (d, J=1.9 Hz, 1H), 8.21 (dt, J=8.0, 1.4 Hz, 1H), 8.10-8.02 (m, 2H), 8.00 (s, 1H), 7.88 (dt, J=7.7, 1.4 Hz, 1H), 7.67 (t, J=7.8 Hz, 1H), 7.43 (d, J=16.0 Hz, 1H), 7.01 (s, 1H), 3.29 (s, 3H).MS(ESI): m/z Calcd. For C 20 H 13 F 2 N 3 O 2  365.0976; found 364.0903, [M−H] − . 
     Example 3 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.221 g, 31%)○ 1 H NMR (400 MHz, DMSO-d 6 ) δ10.94 (s, 1H), 10.11 (s, 1H), 8.07-8.01 (m, 2H), 7.95 (d, J=15.7 Hz, 1H), 7.74 (d, J=8.7 Hz, 2H), 7.01 (d, J=15.7 Hz, 1H), 6.90 (s, 1H), 6.87-6.83 (m, 2H), 3.26 (s, 3H).MS(ESI): m/z Calcd. For C 19 H 14 F 2 N 2 O 3  356.0972; found 355.0901, [M−H] − . 
     Example 4 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.207 g, 29%)○ 1 H NMR (400 MHz, DMSO-d 6 ) δ9.70 (s, 1H), 8.05 (d, J=8.9 Hz, 2H), 7.98-7.89 (m, 1H), 7.30 (t, J=6.2 Hz, 1H), 7.26 (d, J=8.9 Hz, 1H), 7.15 (d, J=15.8 Hz, 1H), 6.97 (s, 1H), 6.87 (d, J=7.5 Hz, 1H), 3.27 (s, 3H).MS(ESI): m/z Calcd. For C 19 H 14 F 2 N 2 O 3  356.0972; found 355.0900, [M−H] − . 
     Example 5 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.186 g, 26%)○ 1 H NMR (400 MHz, DMSO-d 6 ) δ8.16 (d, J=8.5 Hz, 2H), 7.99-7.86 (m, 3H), 7.31 (t, J=8.9 Hz, 2H), 7.18 (d, J=15.9 Hz, 1H), 6.95 (s, 1H), 6.83-6.72 (m, 2H), 3.59 (t, J=5.9 Hz, 2H), 3.51 (t, J=5.9 Hz, 2H), 3.27 (s, 3H), 3.05 (s, 3H).MS(ESI): m/z Calcd. For C 19 H 13 F 3 N 2 O 2  358.0929; found 357.0856, [M−H] − . 
     Example 6 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.222 g, 31%)○ 1 H NMR (400 MHz, DMSO-d 6 ) δ11.02 (s, 1H), 8.09-8.03 (m, 2H), 8.01 (d, J=15.8 Hz, 1H), 7.85 (dt, =10.6, 2.1 Hz, 1H), 7.72 (d, =7.8 Hz, 1H), 7.51 (td, =8.0, 6.1 Hz, 1H), 7.34 (d, J=15.9 Hz, 1H), 7.28 (td, J=8.7, 2.7 Hz, 1H), 7.00 (s, 1H), 3.28 (s, 3H).MS(ESI): m/z Calcd. For C 19 H 13 F 3 N 2 O 2  358.0929; found 357.0857, [M−H] − . 
     Example 7 
     
       
         
         
             
             
         
       
     
     To a stirred solution of compound IV-5 (0.716 g, 2.0 mmol), TBSCI (0.450 g, 3.0 mmol), in 50 mL dry DMF, and imidazole (0.204 g, 3.0 mmol) was added. The complet solution was stirred for 3 hours at room temperature under Ar atmosphere. The mixture was poured into 150 mL water and extracted with DCM. The organic layer was dried over anhydrous MgSO 4 , filtered, and concentrated under reduced pressure to give the crude product, then purified by silica gel column chromatography to afford the compound 2 (0.927 g, 98%).  1 H NMR (400 MHz, DMSO-d 6 ) δ8.16 (d, J=8.5 Hz, 2H), 7.99-7.86 (m, 3H), 7.31 (t, J=8.9 Hz, 2H), 7.18 (d, J=15.9 Hz, 1H), 6.95 (s, 1H), 6.83-6.72 (m, 2H), 3.59 (t, J=5.9 Hz, 2H), 3.51 (t, J=5.9 Hz, 2H), 3.27 (s, 3H), 3.05 (s, 3H), 1.50 (s, 9H), 0.2 (s, 6H).MS(ESI): m/z Calcd. For C 25 H 28 F 3 N 2 O 2 Si 473.2; found 473.2, [M+H] + . 
     
       
         
         
             
             
         
       
     
     Compound 2 (0.473 g, 1 mmol), Lawesson&#39;s reagent (0.808 g, 2 mmol) was in 250 mL three neck boles and dissolved in 100 mi toluene. Two drops of aniline was added. The reaction mixture was reflexed until the TLC showed the complete the reaction. The solvent was removed under reduce pressure to give the crude product which was redissolved in 50 mL DCM, then TBAF (0.313 g, 1.2 mmol) was added. The mixture was stirred at rt under Ar atomophere. After complete the reaction, the solvent was removed under reduce pressure to give the crude product, then purified by silica gel column chromatography to afford a target compound IV-7 (0.209 g, 56%).  1 H NMR (400 MHz, DMSO-d 6 ) δ8.17 (d, J=8.5 Hz, 2H), 7.98-7.86 (m, 3H), 7.31 (t, J=8.9 Hz, 2H), 7.18 (d, J=15.9 Hz, 1H), 6.95 (s, 1H), 6.83-6.72 (m, 2H), 3.59 (t, J=5.9 Hz, 2H), 3.51 (t, J=5.9 Hz, 2H), 3.27 (s, 3H), 3.05 (s, 3H).MS(ESI): m/z Calcd. For C 18 H 13 F 3 N 2 NaOS 397.0598; found 397.0597, [M+Na] + . 
     Example 8 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for Compound IV-1 Compound 2, (0.932 g, 99%)○ 1 H NMR (400 MHz, DMSO-d 6 ) δ8.10 (d, J=8.5 Hz, 2H), 8.06 (dd, J=7.8, 2.1 Hz, 2H), 8.03 (s, 1H), 7.94 (d, J=8.3 Hz, 2H), 7.44 (d, J=15.9 Hz, 1H), 7.03 (s, 1H), 3.29 (s, 3H), 1.51 (s, 9H), 0.29 (s, 9H).MS(ESI): m/z Calcd. For C 26 H 28 F 2 N 3 O 2 Si 480.2; found 480.2, [M+H] + . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for Compound IV-7, (0.332 g, 49%)○ 1 H NMR (400 MHz, DMSO-d 6 ) δ11.00 (s, 1H), 8.11 (d, J=8.5 Hz, 2H), 8.07 (dd, J=7.8, 2.1 Hz, 2H), 8.04 (s, 1H), 7.94 (d, J=8.3 Hz, 2H), 7.44 (d, J=15.9 Hz, 1H), 7.03 (s, 1H), 3.29 (s, 3H).HMS(ESI): m/z Calcd. For C 20 H 13 F 2 N 3 NaOS 404.0645; found 404.0646, [M+Na] + . 
     Example 9 
     
       
         
         
             
             
         
       
     
     To a stirred solution of 3-Fluoro-4-hydroxybenzaldehyde (0.560 g, 4.0 mmol) and 5 mL 33% methylamine aqueous solution in 40 mL anhydrous ethanol, 10 g Na 2 SO 4  was added in one portion. The obtained mixture was stirred and kept at room temperature for 24 hr, then filtered and dried with additional Na 2 SO 4 . The solvent was removed under reduce pressure to give the intermediate which was used directly without any further purification. After re-dissolved in 10 mL anhydrous methanol, compound 4 (0.790 g, 5.0 mmol) was added. The complex was stirred and kept at room temperature for 12 hr, the precipitated product was filtered and washed with cooled methanol for three times to give the yellow compound 5. (0.796 g, 85%).  1 H NMR (400 MHz, DMSO-d 6 ) δ10.52 (s, 1H), 8.19 (m, 1H), 7.76 (m, 1H), 6.99 (t, J=8.8 Hz, 1H), 6.89 (s, 1H), 3.09 (s, 3H), 2.34 (s, 3H). MS(ESI): m/z Calcd. For C 12 H 10 FN 2 O 2 234.2; found 234.2, [M−H] − . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.239 g, 21%)○ 1 H NMR (400 MHz, DMSO-d 6 ) δ10.52 (s, 1H), 8.11 (d, J=8.5 Hz, 2H), 8.07 (d, J=7.8 Hz, 2H), 7.84 (d, J=8.0 Hz, 1H), 7.67 (d, J=8.4 Hz, 1H), 7.32 (m, 1H), 6.99 (t, J=8.8 Hz, 1H), 6.89 (s, 1H), 6.78 (m, 1H), 2.34 (s, 3H). HR-MS(ESI): m/z Calcd. For C 20 H 13 FN 3 O 2 346.0997; found 346.0998, [M−H] − . 
     Example 10 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for Compound 5, (0.812 g, 91%)○ 1 H NMR (400 MHz, CD 3 OD) δ7.28 (s, 1H), 7.19 (d, J=8.0 Hz, 2H), 3.59 (t, J=5.6 Hz, 3H), 3.12 (s, 3H), 1.23 (q, J=5.6 Hz, 3H). MS(ESI): m/z Calcd. For C 13 H 11 ClFN 2 O 2 281.0; found 281.0, [M−H] − . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.239 g, 21%)○ 1 H NMR (400 MHz, CD 3 OD) δ7.84 (s, 2H), 7.28 (s, 1H), 7.19 (d, J=8.0 Hz, 2H), 3.12 (s, 3H), 1.23 (q, J=5.6 Hz, 3H). HR-MS(ESI): m/z Calcd. For C 21 H 14 ClFN 3 O 2 395.0837; found 394.0764, [M−H] − . 
     Example 11 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for Compound 5, (0.812 g, 91%)○ 1 H NMR (400 MHz, CD 3 OD) δ7.28 (s, 1H), 7.19 (d, J=8.0 Hz, 2H), 3.81 (s, 3H), 3.12 (s, 3H), 3.12 (s, 3H), 1.58 (m, 1H), 1.11 (d, 6H). MS(ESI): m/z Calcd. For C 15 H 15 BrFN 2 O 2 353.0; found 3.0, [M−H] − . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.209 g, 22%)○ 1 H NMR (400 MHz, CD 3 OD) δ8.15 (d, J=8.8 Hz, 2H), 7.95 (d, J=15.7 Hz, 1H), 7.74 (d, J=8.7 Hz, 2H), 7.28 (s, 1H), 7.19 (d, J=8.0 Hz, 2H), 7.01 (d, J=15.7 Hz, 1H), 6.87-6.83 (m, 2H), 3.12 (s, 3H), 1.58 (m, 1H), 1.11 (d, 6H). HR-MS(ESI): m/z Calcd. For C 23 H 19 BrFN 3 O 2 467.0645; found 466.0572, [M−H] − . 
     Example 12 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for Compound 5, (0.812 g, 91%)○ 1 H NMR (400 MHz, CD3OD) δ10.52 (s, 1H), 7.76 (d, J=8.5 Hz, 2H), 6.95 (s, 1H), 3.79 (t, J=5.2 Hz, 2H), 3.35 (s, 3H), 2.39 (t, J=4.8 Hz, 2H), 1.40 (m, 2H), 1.20 (t, J=4.8 Hz, 3H). MS(ESI): m/z Calcd. For C 14 H 214 FIN 2 O 2 388.0; found 388.0, [M−H] − . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.156 g, 18%)○ 1 H NMR (400 MHz, CD 3 OD) δ10.52 (s, 1H), 7.98-7.86 (m, 3H), 7.76 (d, J=8.5 Hz, 2H), 7.31 (t, J=8.9 Hz, 2H), 7.18 (d, J=15.9 Hz, 1H), 6.95 (s, 1H), 3.79 (t, J=5.2 Hz, 2H), 3.35 (s, 3H), 2.39 (t, J=4.8 Hz, 2H), 1.40 (m, 2H), 1.20 (t, J=4.8 Hz, 3H). HR-MS(ESI): m/z Calcd. For C 21 H 17 ClFIN 2 O 2 510.0007; found 408.9936, [M−H] − . 
     Example 13 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for Compound 5, (0.732 g, 93%)○ 1 H NMR (400 MHz, CD 3 OD) δ10.52 (s, 1H), 7.76 (d, J=8.5 Hz, 2H), 6.95 (s, 1H), 3.10 (s, 3H), 2.39 (s, 3H). MS(ESI): m/z Calcd. For C 12 H 10 Cl 2 N 2 O 3 284.0; found 283.0, [M−H] − . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.156 g, 18%)○ 1 H NMR (400 MHz, CD 3 OD) δ10.52 (s, 1H), 8.07-8.01 (m, 2H), 7.95 (d, J=15.7 Hz, 1H), 7.76 (d, J=8.5 Hz, 2H), 7.01 (d, J=15.7 Hz, 1H), 6.95 (s, 1H), 6.87-6.83 (m, 2H), 3.10 (s, 3H), 2.39 (s, 3H).HR-MS(ESI): m/z Calcd. For C 19 H 13 BrCl 2 N 2 O 2 449.9537; found 448.9455, [M−H] − . 
     Example 14 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for Compound 5, (0.732 g, 93%)○ 1 H NMR (400 MHz, DMSO-d 6 ) δ10.52 (s, 1H), 8.19 (m, 1H), 7.76 (m, 1H), 6.99 (t, J=8.8 Hz, 1H), 6.89 (s, 1H), 3.01 (q, J=4.8 Hz, 2H), 2.34 (s, 3H), 1.21 (t, J=4.8 Hz, 3H). MS(ESI): m/z Calcd. For C 13 H 13 ClN 2 O 2 264.1; found 263.1, [M−H] − . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.156 g, 18%)○ 1 H NMR (400 MHz, DMSO-d 6 ) δ10.52 (s, 1H), 8.19 (m, 1H), 8.07-8.01 (m, 2H), 7.95 (d, J=15.7 Hz, 1H), 7.76 (m, 1H), 7.01 (d, J=15.7 Hz, 1H), 6.99 (t, J=8.8 Hz, 1H), 6.89 (s, 1H), 6.87-6.83 (m, 2H), 3.01 (q, J=4.8 Hz, 2H), 2.34 (s, 3H), 1.21 (t, J=4.8 Hz, 3H). HR-MS(ESI): m/z Calcd. For C 21 H 13 ClIN 2 O 2 486.9716; found 486.9715, [M−H] − . 
     Example 15 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for Compound 5, (0.732 g, 93%)○ 1 H NMR (400 MHz, CD 3 OD) δ10.52 (s, 1H), 8.19 (m, 1H), 7.76 (m, 1H), 6.99 (t, J=8.8 Hz, 1H), 6.89 (s, 1H), 3.05 (s, 3H), 2.34 (s, 3H). MS(ESI): m/z Calcd. For C 12 H 11 BrN 2 O 2 294.0; found 293.0, [M−H] − . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.156 g, 18%)○ 1 H NMR (400 MHz, CD 3 OD) δ10.52 (s, 1H), 8.19 (m, 1H), 7.95 (d, J=15.7 Hz, 1H), 7.76 (m, 1H), 7.49 (m, 1H), 7.40-7.22 (m, 3H), 7.01 (d, J=15.7 Hz, 1H), 6.99 (t, J=8.8 Hz, 1H), 6.89 (s, 1H), 3.05 (s, 2H), 2.34 (s, 3H). HR-MS(ESI): m/z Calcd. For C 19 H 14 BrClN 2 O 2 415.9927; found 414.9854, [M−H] − . 
     Example 16 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for Compound 5, (0.732 g, 93%)○ 1 H NMR (400 MHz, CD3 OD) δ10.52 (s, 1H), 7.76 (d, J=8.5 Hz, 2H), 6.95 (s, 1H), 2.39 (s, 3H). MS(ESI): m/z Calcd. For C 11 H 8 Cl 2 N 2 O 2 270.0; found 271.0, [M+H] − . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.156 g, 18%)○ 1 H NMR (400 MHz, CD 3 OD) δ10.52 (s, 1H), 8.07-8.01 (m, 2H), 7.95 (d, J=15.7 Hz, 1H), 7.76 (d, J=8.5 Hz, 2H), 7.01 (d, J=15.7 Hz, 1H), 6.95 (s, 1H), 6.87-6.83 (m, 2H), 2.39 (s, 3H).HR-MS(ESI): m/z Calcd. For C 18 H 11 BrCl 2 N 2 O 2 435.9381; found 436.9459, [M+H] − . 
     Example 17 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.286 g, 28%)○ 1 H NMR (400 MHz, DMSO-d 6 ): δ8.15 (d, J=8.6 Hz, 2H), 7.82 (d, J=15.8 Hz, 1H), 7.31-7.22 (m, 2H), 7.21-7.17 (m, 1H), 7.10 (d, J=15.8 Hz, 1H), 6.94 (s, 1H), 6.88-6.73 (m, 3H), 3.59 (t, J=5.8 Hz, 2H), 3.51 (t, J=6.0 Hz, 2H), 3.26 (s, 3H), 3.05 (s, 3H). HR-MS (ESI): m/z Calcd. For C 22 H 24 N 3 O 3  378.1818; found 378.1819, [M+H] + . 
     Example 18 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.486 g, 60%)○ 1 H NMR (400 MHz, DMSO-d 6 ): δ8.43 (d, J=1.8 Hz, 1H), 8.16 (t, J=8.5 Hz, 3H), 7.93 (d, J=15.9 Hz, 1H), 7.85 (dt, J=7.8, 1.4 Hz, 1H), 7.66 (t, J=7.8 Hz, 1H), 7.40 (d, J=15.9 Hz, 1H), 6.98 (s, 1H), 6.86-6.71 (m, 2H), 3.60 (t, J=5.9 Hz, 2H), 3.52 (t, J=5.9 Hz, 2H), 3.28 (s, 3H), 3.06 (s, 3H). HR-MS(ESI): m/z Calcd. For C 23 H 23 N 4 O 2  387.1821; found 387.1822, [M+H] + . 
     Example 19 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.286 g, 38%)○ 1 H NMR (400 MHz, DMSO-d 6 ): δ8.17 (d, J=8.6 Hz, 2H), 7.91 (d, J=15.8 Hz, 1H), 7.79 (dt, J=10.6, 2.1 Hz, 1H), 7.66 (d, J=7.8 Hz, 1H), 7.50 (td, J=8.0, 6.2 Hz, 1H), 7.34-7.20 (m, 2H), 6.97 (s, 1H), 6.84-6.73 (m, 2H), 3.59 (t, J=5.9 Hz, 2H), 3.52 (t, J=5.9 Hz, 2H), 3.27 (s, 3H), 3.06 (s, 3H). MS(ESI): m/z Calcd. For C 22 H 23 N 3 O 3 F 380.1774; found 380.1775, [M+H] 30 . 
     Example 20 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.222 g, 30%).  1 H NMR (400 MHz, DMSO-d 6 ): 8.14 (d, J=8.5 Hz, 2H), 7.84 (d, J=15.7 Hz, 1H), 7.68 (d, J=8.4 Hz, 2H), 6.97 (d, J=15.8 Hz, 1H), 6.88 (s, 1H), 6.84 (d, J=8.5 Hz, 2H), 6.79 (d, J=8.8 Hz, 2H), 3.59 (q, J=5.4 Hz, 2H), 3.51 (t, J=5.9 Hz, 2H), 3.24 (s, 3H), 3.05 (s, 3H). MS(ESI): m/z Calcd. For C 22 H 24 N 3 O 3  378.1818; found 378.1819, [M+H] + . 
     Example 21 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.352 g, 56%)○ 1 H NMR (400 MHz, DMSO-d 6 ): δ8.18 (d, J=8.5 Hz, 2H), 8.08-8.02 (m, 2H), 7.98-7.90 (m, 3H), 7.40 (d, J=15.9 Hz, 1H), 7.00 (s, 1H), 6.86-6.72 (m, 2H), 3.60 (t, J=5.9 Hz, 2H), 3.52 (t, J=5.6 Hz, 2H), 3.28 (s, 3H), 3.06 (s, 3H). MS(ESI): m/z Calcd. For C 23 H 23 N 4 O 2  387.1821; found 387.1820, [M+H] + . 
     Example 22 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.252 g, 32%)○ 1 H NMR (400 MHz, DMSO-d 6 ): δ8.16 (d, J=8.5 Hz, 2H), 7.93 (t, J=4.4 Hz, 2H), 7.90 (d, J=6.1 Hz, 1H), 7.31 (t, J=8.9 Hz, 2H), 7.18 (d, J=15.9 Hz, 1H), 6.95 (s, 1H), 6.83-6.72 (m, 2H), 3.59 (t, J=5.9 Hz, 2H), 3.51 (t, J=5.9 Hz, 2H), 3.27 (s, 3H), 3.05 (s, 3H). MS(ESI): m/z Calcd. For C 22 H 23 FN 3 O 2  380.1774; found 380.1775, [M+H] + . 
     Example 23 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.252 g, 32%)○ 1 H NMR (400 MHz, DMSO-d 6 ): δ8.43 (d, J=1.8 Hz, 1H), 8.16 (t, J=8.5 Hz, 3H), 7.93 (d, J=15.9 Hz, 1H), 7.85 (dt, J=7.8, 1.4 Hz, 1H), 7.66 (t, J=7.8 Hz, 1H), 7.40 (d, J=15.9 Hz, 1H), 6.98 (s, 1H), 6.86-6.71 (m, 2H), 3.60 (t, J=5.9 Hz, 2H), 3.52 (t, J=5.9 Hz, 2H), 3.28 (s, 3H), 3.06 (s, 3H). MS(ESI): m/z Calcd. For C 23 H 23 N 4 O 2  387.2; found 387.2, [M+H] + . 
     
       
         
         
             
             
         
       
     
     2 ml Allyl bromide was added to the mixture of compound 13 (0.774 g, 2.0 mmol), K 2 CO 3  (0.276 g, 2.0 mmol) in acetonitrile (100 ml) with constant stirring. This reaction mixture was heated to reflux. The progress of reaction was monitored on silica gel TLC. After completion of reaction, the reaction mixture was filtered then the solvent was removed under reduce pressure to Ove the crude product, then purified by silica gel column chromatography to afford a target compound 14.  1 H NMR (400 MHz, DMSO-d 6 ): δ8.43 (d, J=1.8 Hz, 1H), 8.16 (t, J=8.5 Hz, 3H), 7.93 (d, J=15.9 Hz, 1H), 7.85 (dt, J=7.8, 1.4 Hz, 1H), 7.66 (t, J=7.8 Hz, 1H), 7.40 (d, J=15.9 Hz, 1H), 6.98 (s, 1H), 6.86-6.71 (m, 2H), 3.81 (s, 2H), 3.60 (t, J=5.9 Hz, 2H), 3.52 (t, J=5.9 Hz, 2H), 3.41 (s, 3H), 3.06 (s, 3H). MS(ESI): m/z Calcd. For C 26 H 27 N 4 O 2  427.2; found 427.2, [M+H] + . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-7, (0.152 g, 72%)○ 1 H NMR (400 MHz, DMSO-d 6 ): δ8.43 (d, J=1.8 Hz, 1H), 8.16 (t, J=8.5 Hz, 3H), 7.93 (d, J=15.9 Hz, 1H), 7.85 (dt, J=7.8, 1.4 Hz, 1H), 7.66 (t, J=7.8 Hz, 1H), 7.40 (d, J=15.9 Hz, 1H), 6.98 (s, 1H), 6.86-6.71 (m, 2H), 3.81 (s, 2H), 3.60 (t, J=5.9 Hz, 2H), 3.52 (t, J=5.9 Hz, 2H), 3.41 (s, 3H), 3.06 (s, 3H). MS(ESI): m/z Calcd. For C 26 H 27 N 4 OS 443.1906; found 443.1905, [M+H] + . 
     Example 24 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-5, (0.692 g, 82%)○ 1 H NMR (400 MHz, CD 3 OD): δ=8.02 (d, J=2.4 Hz, 1H), 7.44 (dd, J=8.7 Hz, J=2.4 Hz, 1H), 7.09 (s, 1H), 6.51 (d, J=8.7 Hz, 1H), 3.56 (t, J HH=7.6 Hz, 2H), 3.08 (s, 6H), 1.66 (m, 2H), 2.38 (s, 3H), 0.95 (t, J=7.6 Hz, 3H). MS(ESI): m/z Calcd. For C 15 H 21 N 4 O 273.2; found 273.2, [M+H] + . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.452 g, 34%)○ 1 H NMR (400 MHz, CD 3 OD): δ=8.02 (d, J=2.4 Hz, 1H), 7.95 (m, 2H), 7.68-7.50 (m, 1H), 7.44 (dd, J=8.7 Hz, J=2.4 Hz, 1H), 7.34-7.06 (m, 2H), 7.09 (s, 1H), 7.00 (d, J=15.7 Hz, 1H), 6.51 (d, J=8.7 Hz, 1H), 3.56 (t, J HH=7.6 Hz, 2H), 3.08 (s, 6H), 1.66 (m, 2H), 2.38 (s, 3H), 0.95 (t, J=7.6 Hz, 3H). MS(ESI): m/z Calcd. For C 22 H 24 FN 4 O 379.2; found 379.2, [M+H] + . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-7, (0.152 g, 72%)○ 1 H NMR (400 MHz, CD 3 OD): δ=8.02 (d, J=2.4 Hz, 1H), 7.95 (m, 2H), 7.68-7.50 (m, 1H), 7.44 (dd, J=8.7 Hz, J=2.4 Hz, 1H), 7.34-7.06 (m, 2H), 7.09 (s, 1H), 7.00 (d, J=15.7 Hz, 1H), 6.51 (d, J=8.7 Hz, 1H), 3.56 (t, J HH=7.6 Hz, 2H), 3.08 (s, 6H), 1.66 (m, 2H), 2.38 (s, 3H), 0.95 (t, J=7.6 Hz, 3H). HR-MS(ESI): m/z Calcd. For C 22 H 24 FN 4 S 395.1706; found 395.1705, [M+H] + . 
     Example 25 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-5, (0.892 g, 80%)○ 1 H NMR (400 MHz, CD 3 OD) δ8.41 (d, J=1.5 Hz, 1H), 7.97 (d, J=1.5 Hz, 1H), 7.31 (s, 1H), 5.86 (s, 1H), 3.46 (t, J=6.6 Hz, 4H), 3.15 (s, 3H), 2.32 (s, 3H), 1.61 (m, 4H), 1.32 (m, 12H), 0.89 (t, 6H). MS(ESI): m/z Calcd. For C 22 H 36 N 5 O 386.3; found 386.3, [M+H] + . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.452 g, 34%)○ 1 H NMR (400 MHz, CD 3 OD) δ8.41 (d, J=1.5 Hz, 1H), 7.97 (d, J=1.5 Hz, 1H), 7.85 (d, J=15.7 Hz, 1H), 7.49 (m, 1H), 7.40-7.22 (m, 3H), 7.31 (s, 1H), 7.01 (d, J=15.7 Hz, 1H), 5.86 (s, 1H), 3.46 (t, J=6.6 Hz, 4H), 3.15 (s, 3H), 2.32 (s, 3H), 1.61 (m, 4H), 1.32 (m, 12H), 0.89 (t, 6H). MS(ESI): m/z Calcd. For C 29 H 39 ClN 5 O 508.2843; found 508.2842, [M+H] + . 
     Example 26 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-5, (0.812 g, 81%)○ 1 H NMR (400 MHz, CDCl 3 ) δ7.93 (s, 2H), 7.31 (s, 1H), 4.24 (t, J=6.8 Hz, 2H), 3.44 (s, 3H), 2.82 (s, 2H), 2.43 (s, 3H). MS(ESI): m/z Calcd. For Cl 4 H 12 N 6 O 285.1; found 285.1, [M+H] + . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.312 g, 26%)○ 1 H NMR (400 MHz, CDCl 3 ) δ=8.02 (d, J=15.7 Hz, 1H), 7.93 (m, 3H), 7.68-7.50 (n, 1H), 7.31 (s, 1H), 7.24-7.06 (m, 2H), 7.01 (d, J=15.7 Hz, 1H), 4.24 (t, J=6.8 Hz, 2H), 3.44 (s, 3H), 2.82 (s, 2H), 2.43 (s, 3H). MS(ESI): m/z Calcd. For C 21 H 20 IN 6 O 499.0743; found 499.0742, [M+H] + . 
     Example 27 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-5, (0.932 g, 85%)○ 1 H NMR (400 MHz, CDCl 3 ) δ=7.59 (s, 2H), 6.71 (s, 1H), 3.24 (t, J=5.6 Hz, 4H), 3.06 (t, J=8.4 Hz, 2H), 2.67 (t, J=6.2 Hz, 4H), 2.29 (s, 3H), 1.86-1.82 (m, 4H) 1.46 (m, 2H), 1.21 (t, J=8.4 Hz, 3H). MS(ESI): m/z Calcd. For C 20 H 25 N 3 O 323.2; found 324.2, [M+H] + . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-5, (0.932 g, 85%) ○ 1 H NMR (400 MHz, CDCl 3 ) δ=8.31 (dd, J=8.3 Hz, J=2.1 Hz, 1H), 8.16 (s, 1H), 7.94 (d, J=8.3 Hz, 1H), 7.95 (d, J=15.7 Hz, 1H), 7.59 (s, 2H), 7.01 (d, J=15.7 Hz, 1H), 6.71 (s, 1H), 3.24 (t, J=5.6 Hz, 4H), 3.06 (t, J=8.4 Hz, 2H), 2.67 (t, J=6.2 Hz, 4H), 2.29 (s, 3 II), 1.86-1.82 (m, 4H), 1.46 (m, 2H), 1.21 (s, J=8.4 Hz, 3H). MS(ESI): m/z Calcd. For C 28 H 28 N 4 O 2  436.2263; found 437.2340, [M+H] + . 
     Example 28 
     
       
         
         
             
             
         
       
     
     To a stirred solution of compound IV-21 (0.792 g, 2.0 mmol), p-toluenesulfonyl chloride (0.476 g, 2.5 mmol) in 100 mL dry DCM, TEA (0.303 g, 3.0 mmol) was added at rt under Ar atomophere. The progress of reaction was monitored on silica gel TLC. After completion of reaction, the reaction was poured into 200 mL water, and extracted with DCM three times. The organic layer was dried over anhydrous MgSO 4 , filtered, and concentrated under reduced pressure to give the crude product, then purified by silica gel column chromatography to afford a target compound 27 (0.822 g, 76%). 1 H-NMR (400 MHz, DMSO-d 6 ): δ8.18 (d, J=8.5 Hz, 2H), 8.08-8.02 (m, 2H), 7.98-7.90 (m, 3H), 7.40 (d, J=15.9 Hz, 1H), 7.18 (d, J=8.2 Hz, 2H), 7.00 (s, 1H), 6.86-6.72 (m, 2H), 6.47 (d, J=8.2 Hz, 2H), 4.06 (t, J=6.1 Hz, 2H), 3.49 (t, J=6.1 Hz, 2H), 3.28 (s, 3H), 2.77 (s, 3H), 2.31 (s, 3H). MS(ESI): m/z Calcd. For C 30 H 29 N 4 O 4 S 541.2; found 541.2, [M+H] + . 
     
       
         
         
             
             
         
       
     
     To a stirred solution of compound 20 (0.541 g, 1.0 mmol) in 20 mL dry DMF, sodium sulfite (0.630 g, 5.0 mmol) was added, the mixture solution was heated to 50° C. and stirred for 24 hrs under Ar atomophere. The progress of reaction was monitored on silica gel TLC. After completion of reaction, the solvent was removed under reduced pressure to give the crude product, then purified by silica gel column chromatography to afford a target compound 27 (0.301 g, 60%).  1 H-NMR(400 MHz, DMSO-d 6 ): 58.18 (d, J=8.5 Hz, 2H), 8.08-8.02 (m, 2H), 7.98-7.90 (m, 3H), 7.40 (d, J=15.9 Hz, 1H), 7.00 (s, 1H), 6.86-6.72 (m, 2H), 3.85 (m, 4H), 3.60 (t, J=5.9 Hz, 2H), 3.52 (t, J=5.6 Hz, 2H), 3.28 (s, 3H), 3.16 (m, 4H), 2.77 (s, 3H). MS(ESI): m/z Calcd. For C 23 H 21 N 4 O 4 S 499.1289; found 499.1288, [M−H] − . 
     Example 29 
     
       
         
         
             
             
         
       
     
     To a stirred solution of compound IV-21 (0.386 g, 1.0 mmol), compound 31 (0.265 g, 1.2 mmol), EDCl (0.382 g, 2.0 mmol) in 30 mL dry DMF, DMAP (0.183 g, 1.5 mmol) was added. The mixture solution was stirred at rt under Ar atomophere. The progress of reaction was monitored on silica gel TLC. After completion of reaction, the solvent was removed under reduced pressure to give the crude product, then purified by silica gel column chromatography to afford a target compound IV-29 (0.490 g, 83%).  1 H-NMR(400 MHz, DMSO-d 6 ): δ8.18 (d, J=8.5 Hz, 2H), 8.08-8.02 (m, 2H), 7.98-7.90 (m, 3H), 7.40 (d, J=15.9 Hz, 1H), 7.00 (s, 1H), 6.86-6.72 (m, 2H), 4.17 (s, 2H), 3.75 (s, 3H), 3.6-3.7 (m, 10H), 3.57 (m, 2H), 3.52 (t, J=5.6 Hz, 2H), 3.38 (s, 3H), 3.28 (s, 3H), 3.06 (s, 3H). MS(ESI): m/z Calcd. For C 32 H 39 N 4 O 7 S 591.2819; found 591.2820, [M+H] + . 
     Example 30 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-5, (0.612 g, 87%)○ 1 H NMR (400 MHz, CDCl 3 ) δ8.15 (d, J=9.0 Hz, 2H), 8.14 (d, J=9.0 Hz, 2H), 7.21 (s, 1H), 4.23 (s, 2H), 4.11 (s, 3H), 3.38 (t, J=6.4 Hz, 2H), 3.01 (s, 3H), 2.92 (t, J=6.4 Hz, 2H), 2.41 (s, 3H). MS(ESI): m/z Calcd. For C 18 H 25 N 4 O 3  345.2; found 345.2, [M+H] + . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.422 g, 36%)○ 1 H NMR (400 MHz, CDCl 3 ) δ8.15 (d, J=9.0 Hz, 2H), 8.14 (d, J=9.0 Hz, 2H), 7.95 (d, J=16.0 Hz, 1H), 7.82 (d, J=8.4 Hz, 2H), 7.32 (d, J=8.4 Hz, 2H), 7.21 (s, 1H), 7.01 (d, J=16.0 Hz, 1H), 4.23 (s, 2H), 4.11 (s, 3H), 3.38 (t, J=6.4 Hz, 2H), 3.01 (s, 3H), 2.92 (t, J=6.4 Hz, 2H).MS(ESI): m/z Calcd. For C 25 H 28 BrN 4 O 3  511.1345; found 511.1344, [M+H] + . 
     Example 31 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-23, (0.912 g, 89%)○ 1 H NMR (400 MHz, DMSO-d 6 ): δ8.17 (d, J=8.6 Hz, 2H), 7.91 (d, J=15.8 Hz, 1H), 7.79 (dt, J=10.6, 2.1 Hz, 1H), 7.66 (d, J=7.8 Hz, 1H), 7.50 (td, J=8.0, 6.2 Hz, 1H), 7.34-7.20 (m, 2H), 7.18 (d, J=8.2 Hz, 2H), 6.97 (s, 1H), 6.84-6.73 (m, 2H), 6.47 (d, J=8.2 Hz, 2H), 4.06 (t, J=6.1 Hz, 2H), 3.49 (t, J=6.1 Hz, 2H), 3.27 (s, 3H), 3.06 (s, 3H), 2.77 (s, 3H), 2.31 (s, 3H). MS(ESI): m/z Calcd. For C 29 H 29 FN 3 O 4 S 534.2; found 534.2, [M+H] + . 
     
       
         
         
             
             
         
       
     
     To a stirred solution of compound 23 (0.534 g, 1.0 mmol) in 35 mL dry DMF, NaN 3  (0.195 g, 3.0 mmol) was added carefully. The mixture solution heated to 50° C. over night under Ar atomophere. The solution was cooled down to rt and poured into 100 ml water and extracted with DCM for three times. The organic layer was dried over anhydrous MgSO 4 , filtered, and concentrated under reduced pressure to give the crude product, which was used for next step without further purification. 
     To a stirred solution of crud production and Ph 3 P (0.524 g, 2.0 mmol) in 30 mL THF, 2 mL water was added. The mixture solution was stirred at rt under Ar atomophere over night. After completion of reaction, the solvent was removed under reduced pressure to give the crude product, then purified by silica gel column chromatography to afford a target compound (0.301 g, 79%).  1 H-NMR(400 MHz, DMSO-d 6 ): δ8.17 (d, J=8.6 Hz, 2H), 7.91 (d, J=15.8 Hz, 1H), 7.79 (dt, J=10.6, 2.1 Hz, 1H), 7.66 (d, J=7.8 Hz, 1H), 7.50 (td, J=8.0, 6.2 Hz, 1H), 7.34-7.20 (m, 2H), 6.97 (s, 1H), 6.84-6.73 (m, 2H), 3.38 (t, J=6.4 Hz, 2H), 2.92 (t, J=6.4 Hz, 2H), 3.27 (s, 3H), 3.06 (s, 3H). MS(ESI): m/z Calcd. For C 22 H 24 FN 4 O 379.1934; found 379.1935, [M+H] + . 
     Example 32 
     
       
         
         
             
             
         
       
     
     To a stirred solution of compound 24 (0.534 g, 1.0 mmol) in 50 mL ethanol, Dimethylamine aqueous solution, the complet mixture was heated to reflux under Ar atomophere. The progress of reaction was monitored on silica gel TLC. After completion of reaction, the reaction cooled down to rt and the solvent was removed under reduced pressure to give the crude product, then purified by silica gel column chromatography to afford a target compound (0.276 g, 68%).  1 H-NMR(400 MHz, DMSO-d 6 ): δ8.17 (d, J=8.6 Hz, 2H), 7.91 (d, J=15.8 Hz, 1H), 7.79 (dt, J=10.6, 2.1 Hz, 1H), 7.66 (d, J=7.8 Hz, 1H), 7.50 (td, J=8.0, 6.2 Hz, 1H), 7.34-7.20 (m, 2H), 6.97 (s, 1H), 6.84-6.73 (m, 2H), 3.47 (t, J=7.6 Hz, 2H), 2.96 (s, 3H), 2.49 (t, J=7.6 Hz, 2H), 2.31 (s, 6H). MS(ESI): m/z Calcd. For C 24 H 28 FN 4 O 407.2247; found 407.2246, [M+H] + . 
     Example 33 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-5, (0.842 g, 89%)○ 1 H-NMR (400 MHz, CDCl 3 ) δ=7.97 (d, J=8.6 Hz, 1H), 7.88 (s, 1H), 6.85 (s, 1H), 6.56 (d, J=8.6 Hz, 1H), 5.40 (s, 1H), 3.07 (s, 3H), 2.84 (s, 3H), 2.31 (s, 3H), 1.94 (s, 3H), 1.32 (s, 6H). MS(ESI): m/z Calcd. For C 19 H 24 N 3 O 310.2; found 310.2, [M+H] + . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.222 g, 21%)○ 1 H NMR (400 MHz, CDCl 3 ) δ=7.97 (d, J=8.6 Hz, 1H), 7.90 (d, J=16.0 Hz, 1H), 7.88 (s, 1H), 7.87 (d, J=4.0 Hz, 2H), 7.43 (d, J=4.0 Hz, 2H), 7.01 (d, J=16.0 Hz, 1H), 6.85 (s, 1H), 6.56 (d, J=8.6 Hz, 1H), 5.40 (s, 1H), 3.07 (s, 3H), 2.84 (s, 3H), 1.94 (s, 3H), 1.32 (s, 6H).MS(ESI): m/z Calcd. For C 27 H 26 N 4 O 422.2107; found 423.2186, [M+H] + . 
     Example 34 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-5, (0.732 g, 81%)○ 1 H NMR (400 MHz, CDCl 3 ) δ=7.40 (dd, J=8.32, 1.93 Hz, 1H), 7.29 (d, J=1.89 Hz, 1H), 7.15 (s, 1H), 6.68 (d, J=8.35 Hz, 1H), 4.23-4.31 (m, 2H), 3.40-3.49 (m, 2H), 3.21 (s, 3H), 3.03 (s, 3H), 2.42 (s, 3H). MS(ESI): m/z Calcd. For C 15 H 18 N 3 O 2  227.1; found 227.1, [M+H] + . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.222 g, 21%)○ 1 H NMR (400 MHz, CDCl 3 ) δ=7.95 (d, J=16.0 Hz, 1H), 7.87 (d, J=4.0 Hz, 2H), 7.43 (d, J=4.0 Hz, 2H), 7.40 (dd, J=8.32, 1.93 Hz, 1H), 7.29 (d, J=1.89 Hz, 1H), 7.15 (s, 1H), 7.01 (d, J=16.0 Hz, 1H), 6.68 (d, J=8.35 Hz, 1H), 4.23-4.31 (m, 2H), 3.40-3.49 (m, 2H), 3.03 (s, 3H), 2.42 (s, 3H).MS(ESI): m/z Calcd. For C 22 H 20 FN 3 O 2  377.1540; found 378.1681, [M+H] + . 
     Example 35 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.732 g, 39%)○ 1 H NMR (400 MHz, CDCl 3 ) δ8.19 (d, J=8.5 Hz, 2H), 8.07 (m, 4H), 8.04 (s, 1H), 7.94 (d, J=8.3 Hz, 2H), 7.44 (d, J=15.9 Hz, 1H), 7.03 (s, 1H), 3.29 (s, 3H). MS(ESI): m/z Calcd. For C 20 H 17 N 4 O 3 359.1; found 359.1, [M+H] + . 
     
       
         
         
             
             
         
       
     
     To a stirred solution of compound 28 (0.718 g, 2.0 mmol) in 100 mL ethyl acetate, Anhydrous stannous chloride (0.758 g, 4.0 mmol) was added. The complet mixture was heated to reflux under Ar atomophere. The progress of reaction was monitored on silica gel TLC. After completion of reaction, the reaction was poured into 150 mL water, and extracted with ethyl acetate for three times. The ° manic layer was dried over anhydrous MgSO 4 , filtered, and concentrated under reduced pressure to give the crude product, then purified by silica gel column chromatography to afford a target compound (0.586 g, 89%).  1 H NMR (400 MHz, CDCl 3 ) 8.19 (d, J=8.5 Hz, 2H), 7.97 (m, 4H), 8.04 (s, 1H), 7.94 (d, J=8.3 Hz, 2H), 7.44 (d, J=15.9 Hz, 1H), 7.03 (s, 1H), 3.29 (s, 3H). MS(ESI): m/z Calcd. For C 20 H 17 N 4 O 329.1402; found 329.1403, [M+H] + . 
     Example 36 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-5, (0.756 g, 74%)○ 1 H NMR (400 MHz, CDCl 3 ) δ8.15 (1H, s), 7.81 (2H, m), 7.65 (1H, d, J=9.0 Hz), 7.16 (1H, dd, J=9.0, J=3.0 Hz), 6.95 (s, 1H), 6.88 (1H, d, J=3.0 Hz), 3.60 (t, J=5.9 Hz, 2H), 3.52 (t, J=5.9 Hz, 2H), 3.28 (s, 3H), 3.06 (s, 3H). MS(ESI): m/z Calcd. For C 19 H 21 N 3 O 2  323.2; found 324.2, [M+H] + . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.272 g, 24%)○ 1 H NMR (400 MHz, CDCl 3 ) δ8.15 (1H, s), 7.81 (2H, m), 7.65 (1H, d, J=9.0 Hz), 7.49 (m, 1H), 7.40-7.22 (m, 3H), 7.16 (1H, dd, J=9.0, J=3.0 Hz), 6.95 (s, 1H), 6.88 (1H, d, J=3.0 Hz), 3.60 (t, J=5.9 Hz, 2H), 3.52 (t, J=5.9 Hz, 2H), 3.28 (s, 3H), 3.06 (s, 3H). MS(ESI): m/z Calcd. For C 26 H 25 N 3 O 3  427.1896; found 428.1974, [M+H] + . 
     Example 37 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-13, (0.322 g, 79%)○ 1 H NMR(400 MHz, DMSO-d 6 ): δ8.16 (d, J=8.5 Hz, 2H), 7.93 (t, J=4.4 Hz, 2H), 7.90 (d, J=6.1 Hz, 1H), 7.31 (t, J=8.9 Hz, 2H), 7.18 (d, J=15.9 Hz, 1H), 6.95 (s, 1H), 6.83-6.72 (m, 2H), 4.21 (s, 2H), 3.59 (t, J=5.9 Hz, 2H), 3.51 (t, J=5.9 Hz, 2H), 3.27 (s, 3H), 3.05 (s, 3H). MS(ESI): m/z Calcd. For C 25 H 25 FN 3 O 2  418.1931; found 418.1932, [M+H] + . 
     Example 38 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-5, (0.816 g, 89%) ○ 1 H NMR (400 MHz, CDCl 3 ) δ9.02 (d, 1H, J=2.1 Hz), 8.59 (d, 1H, J=2.1 Hz), 7.93 (d, 1H, J=9.1 Hz), 7.34 (dd, 1H, J=9.1, 2.5 Hz), 7.21 (s, 1H), 7.00 (d, 1H, J=2.5 Hz), 3.11 (s, 6H), 3.06 (s, 3H). MS(ESI): m/z Calcd. For C 17 H 18 N 4 O 294.1; found 295.1, [M+H] + . 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.272 g, 24%)○ 1 H NMR (400 MHz, CDCl 3 ) δ=9.02 (d, 1H, J=2.1 Hz), 8.59 (d, 1H, J=2.1 Hz), 7.95 (d, J=16.0 Hz, 1H), 7.93 (d, 1H, J=9.1 Hz), 7.49 (m, 1H), 7.40-7.22 (m, 3H), 7.34 (dd, 1H, J=9.1, 2.5 Hz), 7.21 (s, 1H), 7.15 (d, J=16.0 Hz, 1H), 7.00 (d, 1H, J=2.5 Hz), 3.11 (s, 6H), 3.06 (s, 3H). MS(ESI): m/z Calcd. For C 25 H 21 N 5 O 407.1821; found 408.1825, [M+H] + . 
     Comparative Example 1 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.275 g, 75%)○ 1 H NMR (400 MHz, DMSO-d 6 ) δ11.00 (s, 1H), 8.08-8.04 (m, 2H), 8.02 (d, J=15.9 Hz, 1H), 7.92-7.87 (m, 2H), 7.49-7.45 (m, 2H), 7.26 (d, J=15.9 Hz, 1H), 6.98 (s, 1H), 3.29 (s, 3H). MS(ESI): m/z Calcd. For C 19 H 13 F 2 N 3 O 2  339.0951; found 339.0950, [M−H] − . 
     Comparative Example 2 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.327 g, 55%)○ 1 H NMR (400 MHz, DMSO-d 6 ) δ9.70 (s, 1H), 8.05 (d, J=8.9 Hz, 2H), 7.98-7.89 (m, 1H), 7.30 (t, J=6.2 Hz, 1H), 7.26 (d, J=8.9 Hz, 1H), 7.15 (d, J=15.8 Hz, 1H), 6.97 (s, 1H), 6.87 (d, J=7.5 Hz, 1H), 3.27 (s, 3H). MS(ESI): m/z Calcd. For C 18 H 12 F 2 N 3 O 2  340.0903; found 340.090, [M−H] − . 
     Comparative Example 3 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.342 g, 46%) ○ 1 H NMR(400 MHz, DMSO-d 6 ): δ=8.21 (d, 2H, J=8.8 Hz), 8.00 (d, 1H, J=16 Hz), 7.85 (d, 2H, J=8.0 Hz), 7.50-7.43 (m, 2H), 7.42 (d, J=2.6 Hz, 1H), 7.24 (s, 1H), 7.01 (s, 1H), 6.92 (d, 2H, J=8.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H). MS(ESI): m/z Calcd. For C 22 H 24 N 3 O 2  362.1869; found 362.1868, [M+H] + . 
     Comparative Example 4 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.312 g, 43%)○ 1 H NMR (400 MHz, CDCl 3 ) δ=8.15 (1H, s), 8.00 (d, 1H, J=16 Hz), 7.85 (d, 2H, J=8.0 Hz), 7.81 (2H, m), 7.65 (1H, d, J=9.0 Hz), 7.20 (s, 1H), 7.50-7.43 (m, 3H), 7.42 (d, 1H, J=16 Hz), 7.16 (1H, dd, J=9.0, J=3.0 Hz), 6.88 (1H, d, J=3.0 Hz), 3.65 (t, J=7.2 Hz, 2H), 3.45 (t, J=7.2 Hz, 2H), 3.35 (s, 3H) 3.03 (s, 3H).MS(ESI): m/z Calcd. For C 26 H 26 N 3 O 2  412.2025; found 412.2026, [M+H] + . 
     Comparative Example 5 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.412 g, 46%) ○ 1 H NMR(400 MHz, DMSO-d 6 ): δ=8,72 (s, 2H), 8.00 (d, 1H, J=16 Hz), 7.50-7.43 (m, 2H), 7.42 (d, J=2.6 Hz, 1H), 7.24 (s, 1H), 7.01 (s, 1H), 6.92 (d, 2H, J=8.8 Hz), 3.75 (t, J=6.8 Hz, 2H), 3.60 (t, 2H, J=6.8 Hz), 3.05 (s, 3H). MS(ESI): m/z Calcd. For C 21 H 21 IN 6 O 373.1777; found 373.1778, [M+H] + . 
     Comparative Example 6 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.192 g, 33%) ○ 1 H NMR (400 MHz, DMSO-d 6 ) δ7.96-7.99 (m, 4H), 7.33-7.37 (m, 3H), 6.98 (s, 1H), 3.24 (s, 3H). MS (ESI): m/z Calcd. For C 19 H 12 F 4 N 2 O 2  376.0835; found 376.0830, [M−H] − . 
     Comparative Example 7 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.192 g, 33%) ○ 1 H NMR (400 MHz, DMSO-d 6 ) δ7.96-7.99 (m, 4H), 7.33-7.37 (m, 3H), 6.98 (s, 1H), 3.24 (s, 3H). MS (ESI): m/z Calcd. For C 19 H 12 F 4 N 2 O 2  376.0835; found 376.0830, [M−H] − . 
     Comparative Example 8 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1,  1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 10.70 (s, 1H), 8.35 (s, 2H), 8.15 (s, 2H), 7.82 (d, J=15.8 Hz, 1H), 7.22 (d, J=15.9 Hz, 1H), 6.95 (s, 1H), 3.27 (s, 3H); HRMS (ESI) m/z: 546.8647 found (calcd for C 19 H 12 Br 2 C 12 N 2 O 3 , [M+H] +  546.8644). 
     Comparative Example 9 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1,  1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 10.38 (s, 1H), 8.18 (d, J=2.0 Hz, 2H), 8.12 (s, 2H), 7.81 (d, J=4.8 Hz, 1H), 7.18 (d, J=4.8 Hz, 1H), 6.93 (s, 1H), 6.78 (d, J=2.0 Hz, 2H), 3.25 (s, 3H), 3.04 (s, 6H); HRMS (ESI) m/z: 505.9855 found (calcd for C 21 H 19 Br 2 N 3 O 2 , [M+H] +  505.9896). 
     Comparative Example 10 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1,  1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 10.38 (s, 1H), 8.16 (d, J=2.0 Hz, 2H), 8.11 (s, 2H), 7.80 (d, J=4.8 Hz, 1H), 7.18 (d, J=4.8 Hz, 1H), 6.91 (s, 1H), 6.74 (d, J=2.0 Hz, 2H), 3.44 (d, J=4.8 Hz, 2H), 3.25 (s, 3H), 1.14 (t, J=4.8 Hz, 6H); HRMS (ESI) m/z: 534.0154 found (calcd for C 23 H 23 Br 2 N 3 O 2 , [M+H] + 534.0209). 
     Comparative Example 11 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the general procedure for CompoundIV-1, (0.156 g, 18%) ○ 1 H NMR (400 MHz, CD 3 OD) δ10.52 (s, 1H), 8.07-8.01 (m, 2H), 7.95 (d, J=15.7 Hz, 1H), 7.76 (d, J=8.5 Hz, 2H), 7.01 (d, J=15.7 Hz, 1H), 6.95 (s, 1H), 6.87-6.83 (m, 2H), 4.12 (s, 3H), 3.62 (s, 3H), 1.50 (s, 9H).HR-MS(ESI): m/z Calcd. For C 24 H 21 F 3 N 2 O 4  457.5381; found 457.5380, [M−H] − . 
     Comparative Example 12 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the published procedure (JACS, 2018, 140, 7381-7384.)○ 1 H NMR (400 MHz, CD 3 OD) δ8.17 (d, J=8.3 Hz, 2H), 7.79 (d, J=15.6 Hz, 1H), 7.55 (d, J=8.8 Hz, 2H), 6.84 (s, 1H), 6.80 (dd, J=9.9, 2.8 Hz, 2H), 6.75 (dd, J=8.8, 5.6 Hz, 2H), 6.66 (d, J=15.6 Hz, 1H), 4.82 (s, 2H), 4.38 (s, 2H), 3.59-3.43 (m 8H), 3.41-3.35 (m, 2H), 3.20-3.13 (m, 2H), 3.04 (s, 6H), 2.12-1.17 (m, 18H). HR-MS(ESI): m/z Calcd. For C 37 H 44 ClF 3 N 4 O 6  732.2901; found 733.2980, [M−H] − . 
     Comparative Example 13 
     
       
         
         
             
             
         
       
     
     This compound was obtained by following the published procedure (JACS, 2018, 140, 7381-7384.)○ 1 H NMR (400 MHz, CD 3 OD) δ8.17 (d, J=8.3 Hz, 2H), 7.79 (d, J=15.6 Hz, 1H), 7.55 (d, J=8.8 Hz, 2H), 6.84 (s, 1H), 6.80 (dd, J=9.9, 2.8 Hz, 2H), 6.75 (dd, J=8.8, 5.6 Hz, 2H), 6.66 (d, J=15.6 Hz, 1H), 4.82 (s, 2H), 4.38 (s, 2H), 3.59-3.43 (m 8H), 3.41-3.35 (m, 2H), 3.20-3.13 (m, 2H), 3.04 (s, 6H), 2.12-1.17 (m, 18H). HR-MS(ESI): m/z Calcd. For C 38 H 44 ClF 3 N 5 O 6  739.2948; found 740.3026, [M+H] − . 
     Test Example 1 
     Fluorescent dyes IV-1 to IV-38 (molecular rotors) prepared in Embodiments 1 to 38 were respectively dissolved in dimethyl sulfoxide to respectively prepare mother liquor with a concentration of 1×10 −2  M, wherein each mother liquor was respectively added to glycerin and methanol and blended, and thus respectively preparing a solution with a final concentration of 1×10 −5  M; according to different fluorescent dyes, fluorescence emission spectra thereof were successively detected by means of the maximum excitation wavelength of each fluorescent dye under the same condition, and the results are shown in Table 1, indicating that the fluorescent dye of the present disclosure sensitively responds to viscosity variations. 
     
       
         
           
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                 Maximum Excitation 
                 Glycerin/Methanol 
               
               
                 Compounds 
                 Wavelength (nm) 
                 Fluorescence Intensity Ratio 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 IV-1 
                 618 
                 987 
               
               
                 IV-2 
                 595 
                 687 
               
               
                 IV-3 
                 565 
                 951 
               
               
                 IV-4 
                 574 
                 687 
               
               
                 IV-5 
                 570 
                 861 
               
               
                 IV-6 
                 578 
                 921 
               
               
                 IV-7 
                 570 
                 873 
               
               
                 IV-8 
                 638 
                 861 
               
               
                 IV-9 
                 615 
                 697 
               
               
                 IV-10 
                 620 
                 779 
               
               
                 IV-11 
                 595 
                 698 
               
               
                 IV-12 
                 573 
                 898 
               
               
                 IV-13 
                 590 
                 711 
               
               
                 IV-14 
                 585 
                 699 
               
               
                 IV-15 
                 586 
                 730 
               
               
                 IV-16 
                 595 
                 689 
               
               
                 IV-17 
                 575 
                 655 
               
               
                 IV-18 
                 595 
                 890 
               
               
                 IV-19 
                 577 
                 821 
               
               
                 IV-20 
                 577 
                 781 
               
               
                 IV-21 
                 624 
                 655 
               
               
                 IV-22 
                 580 
                 689 
               
               
                 IV-23 
                 617 
                 915 
               
               
                 IV-24 
                 551 
                 934 
               
               
                 IV-25 
                 591 
                 918 
               
               
                 IV-26 
                 555 
                 937 
               
               
                 IV-27 
                 600 
                 890 
               
               
                 IV-28 
                 620 
                 942 
               
               
                 IV-29 
                 618 
                 829 
               
               
                 IV-30 
                 582 
                 921 
               
               
                 IV-31 
                 581 
                 902 
               
               
                 IV-32 
                 580 
                 999 
               
               
                 IV-33 
                 605 
                 729 
               
               
                 IV-34 
                 603 
                 810 
               
               
                 IV-35 
                 561 
                 776 
               
               
                 IV-36 
                 690 
                 792 
               
               
                 IV-37 
                 580 
                 801 
               
               
                 IV-38 
                 705 
                 529 
               
               
                   
               
            
           
         
       
     
     Test Example 2 
     Molecular rotors IV-1, IV-2, IV-3, IV-4, IV-5, IV-6, IV-17, IV-18, IV-19, IV-20, IV-21, IV-22 were added to a diethanol-glycerol mixed solution to prepare a solution with a final concentration of 1×10 −5  M, wherein the solution was excited at 480 nm, fluorescence emission spectra at different viscosity conditions are shown as  FIGS.  1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12   , and molecular rotors of the same concentration have gradually increasing fluorescence intensity at different viscosity conditions, indicating that the fluorescence intensity of molecular rotors increases following the increasing fluorescence of environmental viscosity, and proving that molecular rotors are sensitive to viscosity and are a kinds of molecular rotors. 
     Test Example 3 
     Molecular rotors (IV-39, IV-40, IV-44 and IV-1, IV-2, IV-3, IV-4, IV-5, IV-6, IV-7, IV-8; IV-41, IV-43 and IV-17, IV-18, IV-19, IV-20, IV-21, IV-22; IV-42 and IV-36; IV-45, IV-46 and IV-3; IV-47, IV-48 and IV-20; IV-49, IV-50 and IV-5; IV-51 and IV-1) were dissolved in DMSO to prepare mother liquor of 1×10 −3  M, wherein the afore-mentioned mother liquor was respectively taken and added to a PBS solution to prepare a solution with a final concentration of 1×10 −6  M, and was respectively excited by the maximum excitation wavelength of each compound, so that fluorescent intensities thereof in PBS were detected and each sample was normalized with the strongest fluorescence in each group as 100, as respectively shown in  FIG.  13   ,  FIG.  14   ,  FIG.  15   ,  FIG.  16   ,  FIG.  17   ,  FIG.  18    and  FIG.  19   . According to the results shown in  FIG.  13   ,  FIG.  14    and  FIG.  15   , relative to the molecular rotors with no substitution on the aromatic ring of the electron-withdrawing group, molecular rotors with hydroxyl radical, cyano group, fluorine atom, chlorine atom, bromine atom, iodine atom substitutions on the aromatic ring of the electron-withdrawing group in the present disclosure have lower background fluorescence; according to the results shown in  FIG.  16    and  FIG.  17   , relative to the molecular rotors with multiple substitutions on the aromatic ring of the electron-withdrawing group, molecular rotors with single substitution have relatively lower background fluorescence, and it may be that the electron cloud distribution of the aromatic structure on the electron-withdrawing group is changed by the multiple substitutions, which causes an increase in the fluorescence background; according to the results shown in  FIG.  18    and  FIG.  19   , the background fluorescence is even lower when simple alkyl substitution occurs at R 1 , and it may be caused by relatively large volume of R 1  or weak interaction between heteroatoms (e.g. oxygen atoms, nitrogen atoms) on modified alkyl group and the aromatic ring on the electron-withdrawing group, as a result, non-radioactive energy dissipation motion such as double bond rotation is inhibited and the fluorescence background of the compound is thus increased. 
     Test Example 4 
     Compounds IV-1, IV-2, IV-3, IV-4, IV-5, IV-6, IV-17, IV-18, IV-19, IV-20, IV-21, IV-22 specifically bound to RNA aptamer (UUGCCAUGUGUAUGUGGGAGGAAGAUUGUAAACACGCCGGAAGAUUGUAAACACGCCGGAAGAUU GUAAACACGCCGGAAGAUUGUAAACACGCCGAAAGGCGGACACUUCCGGCGGACACUUCCGGCGGAC ACUUCCGGCGGACACUUCCUCCCACAUACUCUGAUGAUCCUUCGGGAUCAUUCAUGGCAA), and the compound fluorescence after binding was noticeably activated and emitted bright fluorescence when being excited by excitation light at a suitable wavelength, see Table 2 for the optical properties after binding; the compounds could also bind to this aptamer in cells, and cells expressing the RNA aptamer had bright fluorescence, as shown in  FIG.  20 A ; and cells not expressing the RNA aptamer had no fluorescence, as shown in  FIG.  20 B , indicating that dyes of this series can be used for nucleic acid labeling. 
     
       
         
           
               
               
               
               
               
               
             
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 Compounds 
                 Ex/nm 
                 Em/nm 
                 ε (M −1  cm −1 ) 
                 QY (−) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 IV-1 
                 510 
                 618 
                 20667 
                 0.17 
               
               
                   
                 IV-2 
                 492 
                 595 
                 25000 
                 0.36 
               
               
                   
                 IV-3 
                 490 
                 565 
                 26000 
                 0.27 
               
               
                   
                 IV-4 
                 490 
                 574 
                 23333 
                 0.37 
               
               
                   
                 IV-5 
                 492 
                 570 
                 23000 
                 0.33 
               
               
                   
                 IV-6 
                 490 
                 578 
                 24000 
                 0.37 
               
               
                   
                 IV-17 
                 524 
                 582 
                 32000 
                 0.47 
               
               
                   
                 IV-18 
                 536 
                 600 
                 25000 
                 0.43 
               
               
                   
                 IV-19 
                 522 
                 590 
                 30000 
                 0.43 
               
               
                   
                 IV-20 
                 512 
                 577 
                 25500 
                 0.36 
               
               
                   
                 IV-21 
                 534 
                 624 
                 26222 
                 0.28 
               
               
                   
                 IV-22 
                 522 
                 580 
                 32000 
                 0.46 
               
               
                   
                   
               
               
                   
                 Note: 
               
               
                   
                 the fluorescence quantum yield was measured by the relative method with Rhodamine 6G as the standard (QY = 0.94). 
               
            
           
         
       
     
     Test Example 5 
     Stable cell line (293 T/17) of mRNA cytoskeletal protein, which is labeled by continuously-expressing RNA aptamer (AUGGAUGAUGAUAUCGCCGCGCUCGUCGUCGACAACGGCUCCGGCAUGUGCAAGGCCGGCUUCGC GGGCGACGAUGCCCCCCGGGCCGUCUUCCCCUCCAUCGUGGGGCGCCCCAGGCACCAGGGCGUGAU GGUGGGCAUGGGUCAGAAGGAUUCCUAUGUGGGCGACGAGGCCCAGAGCAAGAGAGGCAUCCUCA CCCUGAAGUACCCCAUCGAGCACGGCAUCGUCACCAACUGGGACGACAUGGAGAAAAUCUGGCACCA CACCUUCUACAAUGAGCUGCGUGUGGCUCCCGAGGAGCACCCCGUGCUGCUGACCGAGGCCCCCCUG AACCCCAAGGCCAACCGCGAGAAGAUGACCCAGAUCAUGUUUGAGACCUUCAACACCCCAGCCAUGU ACGUUGCUAUCCAGGCUGUGCUAUCCCUGUACGCCUCUGGCCGUACCACUGGCAUCGUGAUGGACU CCGGUGACGGGGUCACCCACACUGUGCCCAUCUACGAGGGGUAUGCCCUCCCCCAUGCCAUCCUGCG UCUGGACCUGGCUGGCCGGGACCUGACUGACUACCUCAUGAAGAUCCUCACCGAGCGCGGCUACAG CUUCACCACCACGGCCGAGCGGGAAAUCGUGCGUGACAUUAAGGAGAAGCUGUGCUACGUCGCCCU GGACUUCGAGCAAGAGAUGGCCACGGCUGCUUCCAGCUCCUCCCUGGAGAAGAGCUACGAGCUGCC UGACGGCCAGGUCAUCACCAUUGGCAAUGAGCGGUUCCGCUGCCCUGAGGCACUCUUCCAGCCUUC CUUCCUGGGCAUGGAGUCCUGUGGCAUCCACGAAACUACCUUCAACUCCAUCAUGAAGUGUGACGU GGACAUCCGCAAAGACCUGUACGCCAACACAGUGCUGUCUGGCGGCACCACCAUGUACCCUGGCAUU GCCGACAGGAUGCAGAAGGAGAUCACUGCCCUGGCACCCAGCACAAUGAAGAUCAAGAUCAUUGCU CCUCCUGAGCGCAAGUACUCCGUGUGGAUCGGCGGCUCCAUCCUGGCCUCGCUGUCCACCUUCCAG CAGAUGUGGAUCAGCAAGCAGGAGUAUGACGAGUCCGGCCCCUCCAUCGUCCACCGCAAAUGCUUC UAGCACUCGCUAGAGCAUGGUUAAGCUUGGAAGAUUGUAAACACGCCGGAAGAUUGUAAACACGCC GGAAGAUUGUAAACACGCCGGAAGAUUGUAAACACGCCGAAAGGCGGACACUUCCGGCGGACACUU CCGGCGGACACUUCCGGCGGACACUUCC) and control cells (293T/17) grew under a conventional mammalian cell culture condition (37° C., 5% carbon dioxide, 100% relative humidity), and the cells were digested after a cell confluence of 90% and were centrifuged at 800 rpm; then the cells were re-suspended with PBS containing 0.2 μM of IV-21 and IV-41 molecules, and were incubated for 5 minutes before flow detection, see  FIGS.  21 A and  21 B  for the detection results; IV-21 molecules could specifically label mRNA of ACTB in cell lines expressing target RNA, and there was no obvious background fluorescence (as shown in  FIG.  21 A ), while the background fluorescence of IV-41 molecules was higher than that of IV-21, and it was unclear whether ACTB was expressed (see  FIG.  21 B ).