CYANINE-DERIVED COMPOUND, PREPARATION METHOD THEREFOR, AND APPLICATION THEREOF

Abstract

Disclosed are a cyanine-derived compound, a preparation method therefor, and application thereof. The compound has the structure represented by formula (1), and the preparation method for the compound is further disclosed. The series of compounds of the present invention can be used as fluorescent markers for live-cell imaging analysis or flow cytometric analysis, solving problems of high toxicity, high cost and poor imaging effect of current.

##STR00001##

Claims

1. A cyanine derivative, having a structure represented by formula (1): ##STR00015## wherein X is selected from —O— or —NH—; Z is selected from —C(O)— or —(CH.sub.2).sub.m—; Y.sup.− is a biocompatible anion; R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are selected from hydrogen, halogen, substituted or unsubstituted C.sub.1-20 alkyl, substituted or unsubstituted C.sub.1-20 alkoxy, substituted or unsubstituted C.sub.6-30 aryl, C.sub.1-20 ester or sulfonate, or any two adjacent groups of R.sub.1, R.sub.2, R.sub.3, and R.sub.4, together with a carbon atom bonded thereto, form an unsubstituted or substituted C.sub.3-10 aliphatic ring, C.sub.6-30 aromatic ring or C.sub.1-30 heteroaromatic ring, wherein a substituent is selected from halogen, alkyl, and alkoxy; n is an integer not less than 1; k is an integer of 1-3; and m is an integer of 1-6.

2. The cyanine derivative according to claim 1, wherein Z is selected from —C(O)—.

3. The cyanine derivative according to claim 1 or 2, wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are each selected from hydrogen; n=2; X is —O—; and Y is Br.

4. The cyanine derivative according to claim 1 or 2, wherein R.sub.1 and R.sub.2 are each selected from hydrogen; R.sub.3 and R.sub.4, together with the carbon atom bonded thereto, form phenyl; and Y is Br.

5. A preparation method for the cyanine derivative according to claim 1, comprising the following steps: step 1, performing an alkylation reaction on a compound of formula (2) as a raw material and a compound of formula (3) to obtain a compound of formula (4): ##STR00016## step 2, performing a condensation reaction on the compound of formula (4) and a compound of formula (5) to obtain a compound of formula (6): ##STR00017## wherein when k=1, the compound represented by formula (5) is triethyl orthoformate, and when k=2 or 3, the compound of formula (5) is: ##STR00018## and step 3, deacetylating the compound of formula (6) to obtain a product, and then performing an esterification reaction on the product and cyclooctetetraenoic formic acid to obtain the cyanine derivative of formula (1): ##STR00019##

6. The preparation method for the cyanine derivative according to claim 5, wherein a solvent used in the alkylation reaction of step 1 is toluene or acetonitrile; a solvent used in the condensation reaction of step 2 is acetic anhydride, with sodium acetate as a catalyst; and catalysts for the esterification reaction of step 3 are 2-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate HATU and triethylamine, with DMF as a solvent.

7. The preparation method for the cyanine derivative according to claim 5, wherein a molar ratio of the compound of formula (2) to the compound of formula (3) in the alkylation reaction is 1:1.5; a molar ratio of the compound of formula (4) to the compound of formula (5) in the condensation reaction is 2:1; and a molar ratio of the compound of formula (6) to the cyclooctatetraene formic acid in the esterification reaction is 1:2.5.

8. Application of the cyanine derivative according to any one of claims 1 to 4 as a mitochondrial fluorescent marker for live-cell imaging analysis or flow cytometric analysis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG. 1 shows normalized absorption and emission spectra, in methanol, of compounds of formula (1-4) and formula (1-5) synthesized in Embodiment 1 of the present invention;

[0058] FIG. 2 shows a comparative data diagram of phototoxicity on HeLa cells between the compound of formula (1-4) and a commercial dye MitoTracker Red CMXRos (MTR CMXRos);

[0059] FIG. 3 shows a comparative diagram of phototoxicity on HeLa cells between the compound of formula (1-5) and a commercial dye MitoTracker Deep Red FN(MTDR);

[0060] FIG. 4 shows a co-localization experiment of labeling mitochondria of HeLa cells with the compounds of formula (1-4) and formula (1-5) and the commercial dye MitoTracker green FM;

[0061] FIG. 5 shows a comparison diagram of phototoxicity on rat cardiomyocytes between the compounds of formula (1-4) and formula (1-5) and commercial dyes;

[0062] FIG. 6 shows experimental comparison data of 3D-laser confocal time-series imaging of rat cardiomyocytes labeled with the compound of formula (1-4) and the commercial dye MTR CMXRos; and

[0063] FIG. 7 shows a comparative data diagram of Hessian-SIM super-resolution mitochondrial imaging of COS7 cells labeled with the compound of formula (1-5) and the commercial dye MTDR.

DETAILED DESCRIPTION

Embodiment 1 Synthesis of Fluorescent Probe

[0064] All water-sensitive and air-sensitive reactions were carried out in a nitrogen atmosphere under an anhydrous condition. The reactions were monitored by thin-layer chromatography (TLC, GF254) under UV light by using a solution of phosphomolybdic acid and cerium sulfate in ethanol, as a visualizer. Compounds were isolated by silica gel flash column chromatography, unless otherwise specified. The nuclear magnetic resonance (NMR) spectra of the compounds were measured by a Bruker Advance 400 (.sup.1H 400 MHz) NMR spectrometer and calibrated with a residual undeuterated solvent (in .sup.1H NMR, 7.26 ppm deuterated chloroform, and 3.31 ppm methanol-d.sub.4). The following abbreviations were used to explain multiplicity: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad peak. Mass spectrometry data were acquired by using Acquity I class UPLC synapt G2-SI and electrospray ionization (ESI).

[0065] Embodiment 1-1. In the general formula (1) of the present invention, when n=2, k=1, X is O, and R.sub.1-R.sub.4 are hydrogen, the compound of this embodiment has the structure represented by formula (1-4), namely a probe 1-4, the preparation method of which was as follows.

[0066] 7.1 g of 2-bromoethanol and 3 g of 2,3,3-Trimethylindolenine were mixed and dissolved in 50 ml of DMF, and heated and stirred at 110° C. for 12 hours to obtain a reaction mixture. After the reaction mixture was cooled to room temperature, a white solid was precipitated. The white solid was filtered by suction and washed with ether to obtain 2.5 g of a compound of formula (7) with the purity of 46%.

[0067] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.90-7.82 (m, 1H), 7.82-7.74 (m, 1H), 7.70-7.59 (m, 2H), 4.70-4.63 (t, J=5.1 Hz, 2H), 4.08-4.01 (t, J=5.1 Hz, 2H), 1.62 (s, 6H).

##STR00008##

[0068] 200 mg of the compound of formula (7) and 133 mg of triethyl orthoformate were heated to 110° C. in 5 ml of acetic anhydride, stirred and reacted for 2 hours to obtain a reaction mixture. The reaction mixture was spin-dried and purified by HPLC to obtain 180 mg of a compound of formula (8).

[0069] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.60 (t, J=13.4 Hz, 1H), 7.58-7.51 (m, 2H), 7.45 (ddd, J=8.3, 7.1, 1.2 Hz, 2H), 7.44-7.37 (m, 2H), 7.32 (td, J=7.3, 1.2 Hz, 2H), 6.60 (d, J=13.4 Hz, 2H), 4.60-4.54 (m, 4H), 4.54-4.46 (m, 4H), 1.83 (s, 6H), 1.77 (s, 12H).

[0070] .sup.13C NMR (101 MHz, Methanol-d.sub.4) δ 175.63, 170.84, 151.24, 142.20, 140.66, 128.46, 125.49, 122.14, 111.21, 102.84, 60.23, 49.39, 43.22, 26.81, 19.16.

[0071] HRMS (ESI) calcd for C.sub.31H.sub.37N.sub.2O.sub.4.sup.+[M.sup.+] 501.2748, found 501.2753.

##STR00009##

[0072] 30 mg of the compound of formula (8) was dissolved in 2 ml of methanol, then, 15 mg of sodium hydroxide was added, and the reaction was carried out for 2 hours while stirring to obtain a mixture; and the mixture was evaporated under rotation to obtain a solid. The solid was mixed with 50 mg of 2-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), 10 mg of cyclooctetetraenoic formic acid (COTCOOH) and 20 μl of triethylamine in DMSO at room temperature to react for 12 hours. The reaction mixture was diluted with water, extracted three times with dichloromethane, and purified by HPLC to obtain 12 mg of a compound of formula (1-4).

[0073] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.59 (t, J=13.4 Hz, 1H), 7.56 (d, J=7.5 Hz, 2H), 7.51-7.38 (m, 4H), 7.35 (t, J=7.4 Hz, 2H), 6.88 (s, 1H), 6.53 (d, J=13.4 Hz, 2H), 5.95-5.66 (m, 12H), 4.65 (t, J=5.0 Hz, 4H), 4.54 (t, J=5.2 Hz, 4H), 1.78 (s, 12H).

[0074] .sup.13C NMR (101 MHz, Chloroform-d) δ 174.60, 165.25, 151.16, 143.54, 142.44, 140.28, 133.94, 132.94, 132.62, 132.02, 131.35, 130.04, 129.30, 128.88, 125.45, 122.02, 111.30, 104.52, 60.96, 49.15, 43.37, 28.09.

[0075] HRMS (ESI) calcd for C.sub.45H.sub.45N.sub.2O.sub.4.sup.+[M.sup.+] 677.3374, found 677.3377.

##STR00010##

[0076] Embodiment 1-2. In the general formula (1) of the present invention, when n=2, k=2, and X is O, the compound of this embodiment has the structure represented by formula (1-5), namely a probe 1-5, the preparation method of which was as follows:

[0077] The compound of formula (7) was prepared with the same method as described above. 100 mg of the compound of formula (7) and 50 mg of N-(3-(phenylamino)allylidene)aniline hydrochloride were heated to 110° C. in 5 ml of acetic anhydride, then reaction was carried out for 2 hours while stirring to obtain a mixture; and the mixture was spin-dried and purified by HPLC to obtain 71 mg of the compound of formula (9).

[0078] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.32 (t, J=13.0 Hz, 2H), 7.49 (dd, J=7.5, 1.2 Hz, 2H), 7.42 (td, J=7.7, 1.2 Hz, 2H), 7.34 (d, J=7.9 Hz, 2H), 7.27 (td, J=7.4, 1.0 Hz, 2H), 6.65 (t, J=12.4 Hz, 1H), 6.39 (d, J=13.7 Hz, 2H), 4.52 (t, J=5.1 Hz, 4H), 4.42 (t, J=5.1 Hz, 4H), 1.85 (s, 6H), 1.73 (s, 12H).

[0079] .sup.13C NMR (101 MHz, Methanol-d.sub.4) δ 174.30, 170.85, 154.67, 144.93, 142.28, 141.11, 128.23, 124.97, 122.03, 110.70, 103.40, 60.26, 49.30, 42.84, 26.43, 19.16.

[0080] HRMS (ESI) calcd for C.sub.33H.sub.39N.sub.2O.sub.4.sup.+ [M.sup.+] 527.2904, found 527.2915.

##STR00011##

[0081] 91 mg of the compound of formula (9) was dissolved in 5 ml of methanol, 45 mg of sodium hydroxide was added, and reaction was carried out for 2 hours while stirring to obtain a mixture; and the mixture was evaporated under rotation to obtain a solid. The solid was mixed with 50 mg of 2-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), 13 mg of cyclooctetetraenoic formic acid (COTCOOH) and 30 μl of triethylamine in DMSO at room temperature to react for 12 hours to obtain a reaction mixture. The reaction mixture was diluted with water, extracted three times with dichloromethane, and purified by HPLC to obtain 17 mg of a compound of formula (1-5).

[0082] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.30 (t, J=13.1 Hz, 2H), 7.50 (d, J=7.5 Hz, 2H), 7.43 (t, J=7.7 Hz, 2H), 7.35 (d, J=8.0 Hz, 2H), 7.29 (t, J=7.4 Hz, 2H), 6.89 (s, 2H), 6.61 (t, J=12.4 Hz, 1H), 6.39 (d, J=13.7 Hz, 2H), 5.92-5.70 (m, 12H), 4.62 (t, J=5.0 Hz, 4H), 4.49 (t, J=5.1 Hz, 4H), 1.74 (s, 12H).

[0083] .sup.13C NMR (101 MHz, Chloroform-d) δ 173.97, 165.30, 153.95, 143.97, 142.01, 141.09, 134.29, 133.38, 132.46, 132.03, 131.44, 129.94, 129.02, 128.59, 126.57, 125.37, 122.32, 60.61, 49.61, 43.01, 27.87.

[0084] HRMS (ESI) calcd for C.sub.47H.sub.47N.sub.2O.sub.4+[M.sup.+] 703.3530, found 703.3520.

##STR00012##

[0085] FIG. 1 shows the normalized absorption and emission spectra, in methanol, of the compounds of formula (1-4) and formula (1-5).

Embodiments 1-3 Preparation

[0086] The compound of formula (7) in the preparation method of Embodiment 1-1 was replaced with the compound of formula (7-1) to prepare the compound of formula (1-6), namely, a probe 1-6.

##STR00013##

[0087] .sup.1H NMR (400 MHz, Chloroform-d) δ 8.68 (t, J=13.1 Hz, 1H), 8.12 (d, J=8.4 Hz, H), 7.94 (dd, J=17.4, 8.5 Hz, 4H), 7.64 (ddd, J=8.3, 6.8, 1.3 Hz, 2H), 7.56 (d, J=13.2 Hz, 2H), 7.51 (ddd, J=8.0, 6.9, 1.0 Hz, 2H), 7.46 (d, J=8.8 Hz, 2H), 6.83 (s, 2H), 5.78 (s, 1H), 5.69-5.55 (m, 12H), 5.32 ((d, J=13.2 Hz, 4H), 4.84 (d, J=13.2 Hz, 4H), 2.06 (s, 12H).

[0088] HRMS (ESI C.sub.53H.sub.49N.sub.2O.sub.4.sup.+ found 777.5654.

[0089] Meanwhile, the compound of formula (1-6-1) was synthesized as the control of phototoxicity experiment:

##STR00014##

[0090] HRMS (ESI C.sub.49H.sub.45N.sub.2O.sub.4.sup.+ found 725.4713.

Embodiment 2 Colocalization Experiment with Existing Commercial Probe

[0091] HeLa cells were stained for 15 minutes with 250 nM probe 1-4 or probe 1-5 and a commercial dye MitoTracker Green FM; the stains were washed off, and then a colocalization assay was carried out. As shown in FIG. 4, the probe provided by the present invention exhibited excellent co-localization with the commercial dye and can label the mitochondria effectively.

Embodiment 3 Phototoxicity Assay of Probes Provided by the Present Invention and Existing Commercial Probes

[0092] HeLa cells were irradiated with corresponding LED light (same light intensity for the same channel) for different periods of time under a widefield fluorescence microscope. The irradiated cells were incubated for 2 hours in a cell incubator at 37° C. Then, the cells were stained with propidium iodide (PI) for viability counting. As shown in FIG. 2 and FIG. 3, two types of probes, i.e., the probes 1-4 and 1-5, in Embodiment 1 were about five times less phototoxic than the commercial dyes in the HeLa cells.

[0093] Rat cardiomyocytes were stained for 15 minutes with 250 nM probe provided by the present invention or commercial dye, and the dye was then washed off. The cells were re-covered with the culture medium, and were continuously irradiated and imaged with corresponding laser light in a high-throughput imaging system. Imaging results were analyzed, and the time of irreversible contraction of the cardiomyocytes was counted. As shown in FIG. 5, the probes 1-4 and 1-5 of the present invention were about five times less phototoxic than the commercial dye in the rat cardiomyocytes.

[0094] The probe 1-6 of the present invention and its reference compound of formula (1-6-1) were also subjected to phototoxicity comparison and determination according to the above steps. Results showed that the probe 1-6 of the present invention had an effect similar to those of the probes 1-4 and 1-5, but with lower phototoxicity. The phototoxicity data are listed in the table below.

TABLE-US-00001 Cell viability under Cell viability under Cell viability under 1-minute irradiation 5-minute irradiation 15-minute irradiation Probe 1-4 99.0%  94.0%.sup.  48.9%.sup.  MitoTracker Red CMXRos 86.9%   0%  0% Probe 1-5 100% 86% 69% MitoTracker Deep Red FM 100%  0%  0% Probe 1-6 100% 99% 95% 1-6-1 100% 95% 25%

Embodiment 4 Imaging Experiment on Cells with Probe Provided by the Present Invention

[0095] Target cells were stained with 250 nM probe provided by the present invention and dye for 10-15 minutes, the dye was then washed off, and a corresponding culture medium was added. Then, different microscopic imaging experiments were carried out. FIG. 6 shows an experimental comparison of 3D-laser confocal time-series imaging of rat cardiomyocytes labeled with the probe 1-5 and the commercial dye MTDR. The cells labeled with the commercial dye violently contracted to death after 8 time sequences, whereas the same process occurred to the cells labeled with the present invention after 23 time sequences.

[0096] FIG. 7 shows the comparison of Hessian-SIM super-resolution mitochondrial imaging of COS7 cells labeled with probe 1-4 versus the commercial dye MTDR. The MTDR-labeled mitochondria were already severely deformed and rounded at the 200th frame of imaging, whereas the similar process occurred to the probe 1-4 was still less severe than that of MTDR after 2000 frames of imaging.