KR20110033454A - Novel cyanine compound for labeling hydrophobic nanoparticle and preparation method thereof - Google Patents

Abstract

PURPOSE: A method for preparing a novel cyanin compound for labeling lipophilic nanoparticle is provided to improve fluorescent characteristics. CONSTITUTION: A cyanine compound is denoted by chemical formula 1. In chemical formula 1, R1 is hydrogen or a sulfonic acid group; A is hydrogen, or an acetyl group; and R4 is hydrogen or an alkyl group of 1-6 carbon atoms. A method for preparing the compound of chemical formula 1 comprises: a step of reacting a compound of chemical formula 6 with a compound of chemical formula 7 to obtain a compound of chemical formula 8; a step of reacting the compound of chemical formula 8 with the compound of chemical formula 6 to obtain the compound of chemical formula 1.

Description

Novel cyanine compound for lipophilic nanoparticle labeling and its preparation method {NOVEL CYANINE COMPOUND FOR LABELING HYDROPHOBIC NANOPARTICLE AND PREPARATION METHOD THEREOF}

The present invention relates to a novel cyanine compound capable of fluorescently labeling lipophilic nanoparticles used in biomolecular imaging, diagnosis, and treatment, and a method for producing the same.

Cyanine dyes generally have excellent optical and pH stability, have a narrow absorption and emission wavelength range, and have a fluorescence region of 500 to 800 nm, so that they do not overlap with the self fluorescence region of the biomolecule and thus the analysis is Although it is easy, and somewhat different depending on the solvent and solubility characteristics, there are many advantages such as high molar extinction coefficient. The following chemical formulas are the general structures of the cyanine dyes in the literature and the basic structures of hetero compounds known as derivatives.

The most commercially utilized cyanine dyes include indole structures as hetero rings and succinimidyl ester groups as reactors. The following chemical formulas are typical structures that are commercially available, and are those sold under the trade names Cy3, Cy5, and Cy7 by GE Healthcare.

An object of the present invention is to provide a novel cyanine compound that can be used for labeling lipophilic nanoparticles widely used in molecular imaging, diagnosis, treatment, and the like, and a method of preparing the same.

The object of the present invention as described above is achieved by providing a novel cyanine compound represented by the following formula (1) and a method for producing the same.

In the formula,

R ₁ is hydrogen, sulfonic acid group or sulfonate group,

Two R ₂ and R ₃ are each independently hydrogen or an alkyl group having 1 to 6 carbon atoms,

R ₄ is hydrogen, an alkyl group having 1 to 6 carbon atoms,

l and m are each independently an integer of 1-5.

In the case of cyanine dyes having commercially available succinimide ester groups, they bind to amine groups and the like in biomolecules, and have limited binding performance when they are combined with lipophilic nanoparticles. It is designed to have a hydrophobic property so as to be suitable for labeling lipophilic nanoparticles and improved fluorescence properties are expected.

The cyanine dye compounds according to the present invention are designed to target organic solvents, which are mediums for application to most lipophilic nanoparticles, and to be stable against heat.

The present invention also relates to a method for producing a novel cyanine compound represented by the formula (1).

Hereinafter, a method for preparing the compound of Formula 1 according to the present invention.

In the method for preparing the compound of Formula 1 according to the present invention, the compound of Formula 2 is reacted with the compound of Formula 3 as illustrated in Scheme 1a to obtain a compound of Formula 4a, which is used as a starting material.

In Formulas 2 to 4 and Scheme 1a, R ₁ , R _2, and R ₃ are the same as defined for Formula 1, respectively.

When R ₁ of Formula 4a is a sulfonic acid group, the compound of Formula 4a is treated with an inorganic base represented by the general formula MOH, preferably potassium hydroxide or sodium hydroxide, most preferably potassium hydroxide, as illustrated in Scheme 1b. To obtain a compound of formula 4b wherein R ₁ is a sulfonate group, which may be used as a starting material.

In Scheme 1b, M is potassium or sodium, R ₂ and R ₃ are each as defined for Formula 1, R ₁ in Formula 4a is a sulfonic acid group, and R ₁ in Formula 4b is a sulfonate group.

In preparing the compound of Formula 1 according to the present invention, the compound of Formula 4a or 4b is first reacted with the compound of Formula 5, respectively, as illustrated in Scheme 2 to obtain a compound of Formula 6.

In Formulas 5 and 6 and Scheme 2, R ₁ , R ₂ , R ₃ , R ₄ and m are as defined for Formula 1, respectively, X is selected from the group consisting of fluorine, chlorine, bromine and iodine Is a halogen atom.

Scheme 2 illustrates a method of preparing a compound of Formula 6 wherein R ₄ is hydrogen or an alkyl group having 1 to 6 carbon atoms.

Next, as illustrated in Scheme 3, the compound of Formula 6 is reacted with the compound of Formula 7 to obtain a compound of Formula 8.

In Formulas 7 and 8 and Scheme 3, R ₁ , R ₂ , R ₃ , R ₄ , l and m are as defined for Formula 1, respectively, and A is hydrogen or an acetyl group.

The compound of Formula 7 is N, N-diphenylformamidine (DPF) when l is 1, malonaldehyde dianile hydrochloride (MDH) when l is 2, and glutaconaldehyde when l is 3. Dianyl hydrochloride (GDH).

Next, as illustrated in Scheme 4, the compound of Formula 8 is reacted with the compound of Formula 6 to obtain a compound represented by Formula 1.

[Formula 1]

In Formula 10 and Scheme 5, R ₁ , R ₂ , R ₃ , R ₄ and m are as defined for Formula 1, respectively.

Example

Hereinafter, the present invention will be described in more detail through examples and comparative experiments. However, the examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

First, experimental apparatus, analytical equipment, and reagents used in Examples and Comparative Experiments will be described.

For the FT-NMR spectroscopic analysis, Bruker's Avance 300 and 400 were used, and for LC / MS, Varian's 1200L Quadrupole was used and measured by electrospray ionization (ESI).

Absorption values at the maximum and maximum wavelengths of the synthesized dyes were measured with a Hewlett-Packard company's HP 8452 diode array spectrophotometer, and the emission values at the maximum and maximum emission wavelengths were determined using LS-55 from Perkin Elmer. Obtained using.

Column chromatography for the separation and purification of organic compounds used Merck's kieselgel 60 (230-400 mesh) as a silica gel in the normal phase, silica gel 60 GF254 for thin layer chromatography (TLC) (0.25 mm, Merck) was used, and the compound identification on TLC was performed using ultraviolet rays of 254 nm and 365 nm, or 20-30% ethanol solution of phosphomolybdic acid (PMA) as a colorant or KMnO ₄ was used. In the reverse phase, TLC used a glass plate coated with silica gel 60 RP-18 F _254S (0.25 mm, Merck), and for column chromatography, Fraction, a medium pressure liquid chromatography (MPLC) instrument from Buchi A reverse phase column Lichroprep RP-18 (40-63 μm, available from Merck) was connected to Collector R-660. HPLC was used with Agilent's 1100 series equipped with Waterap's Bondapak C18 10 μm 125A.

Reagents were mainly used by Aldrich and TCI. Solvents requiring purification were used in accordance with known methods and all reactions were carried out under a nitrogen stream unless otherwise indicated. NMR solvents were DMSO- d ₆ or D ₂ O from Aldrich and Cambridge Isotope Laboratories Inc. The relative positions of the signals were based on tetramethylsilane (TMS) in the solvent or on NMR solvents. Chemical shifts are expressed in ppm from the standard material, and data indicate chemical shift multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), intergration, coupling constant ( Hz).

Example 1: Preparation of Compound 1-1

(1) Synthesis of Compound 8-1

1,2,3,3-tetramethyl-3H-indoliumiodide (6.626 g, 22 mmol, 1 eq) and glutaconaldehyde dianil hydrochloride (6.265 g, 22 mmol, 1 eq, TCI) were added to a 16 ml solution of acetic anhydride for 1 hour. Reaction at 100 degrees. After naturally cooling to room temperature, distilled water was added to obtain particles, followed by filtration, washed with distilled water several times, and dried under reduced pressure. (5.88 g, 54%)

R _f = 0.7 (normal phase, methylene chloride / methanol 5: 1 v / v)

¹ H NMR (400 MHz, CDCl ₃ ): δ 8.19-8.15 (d, J = 13.7 Hz, 1H), 7.84-6.89 (m, 13H), 5.45-5.39 (dt, J = 13.4 Hz, 11.7 Hz, 1H ), 4.2 (s, 3H), 1.92 (s, 3H), 1.72 (s, 6H), 1.64 (s, 3H)

(2) Synthesis of Compound 1-1

Compound 8-1 (3.97 g, 7.97 mmol, 1 eq) and 1,2,3,3, -Tetramethyl-3H-indolium iodide (2.39 g, 7.97 mmol, 1 eq, Aldrich) were added to a 30 ml Pyridine solution. Stir on nitrogen. The reaction is carried out at 60 degrees for 1 hour. After confirming the completion of the reaction, it is naturally cooled to room temperature and the solvent is recovered using a rotary evaporator and dried under reduced pressure. Pure dye material is separated by normal chromatography with a 5: 1 mixture of methylene chloride and methanol. (1.86 g, 43.6%)

R _f = 0.85 (normal phase, methylene chloride / methanol 5: 1 v / v)

¹ H NMR (400 MHz, DMSO): δ 7.89-7.80 (m, 2H), 7.76-7.70 (m, 1H), 7.65-7.54 (m, 3H), 7.39-7.36 (m, 5H), 6.54-6.51 (t, J = 12.6 Hz, 2H), 6.32-6.29 (d, J = 13.8 Hz, 2H), 3.57 (s, 6H), 1.70-1.62 (m, 12H)

LC / MS, calculated for C ₂₉ H ₃₃ N ₂ ⁺ 409.59, found 409.21

λ _abs (methanol): 737 nm, λ _fl (methanol): 774 nm

Example 2: Preparation of Compound 1-2

(1) Synthesis of Compound 6-1

2,3,3-trimethylindolenine (3.18 g, 20 mmol, 1 eq, Aldrich) and Ethyl iodide (20 ml, 250 mmol, 12.5 eq, TCI) were added and stirred under nitrogen. Heat to reflux for 24 hours. After cooling, check the particle state and filter. The obtained solid particles were washed 2-3 times with Hexane and dried under reduced pressure. (5.08 g, 80.6%)

R _f = 0.5 (normal phase, methylene chloride / methanol 5: 1 v / v)

(2) Synthesis of Compound 8-2

Add Chemical Formula 6-1 (3.15 g, 10 mmol, 1 eq) and Glutavonaldehyde dianil hydrochloride (2.85 g, 10 mmol, 1 eq, TCI) to 60 ml Acetic anhydride and stir. The reaction proceeds at 100 ° C. for 1 hour and then naturally cooled to room temperature. After removing the solution except particles, Diethyl ether is added to form solid particles. The resulting particles are filtered and dried under reduced pressure. (2.17 g, 42.4%)

R _f = 0.75 (normal phase, methylene chloride / methanol 5: 1 v / v)

LC / MS, calculated for C ₂₆ H ₂₉ N ₂ O ⁺ 385.52, found 385.16

(3) Synthesis of Compound 1-2

Add Formula 8-2 (5.11 g, 10 mmol, 1 eq) and Formula 6-1 (3.15 g, 10 mmol, 1 eq) to 20 ml pyridine solution and stir on nitrogen. After reacting at 40 ° C. for 1 hour, the mixture is cooled to room temperature. The reaction solution was recovered using a rotary evaporator and then dried under reduced pressure.

Pure dye material is separated by normal chromatography with a 5: 1: 1 mixture of methylene chloride, methanol, and hexane. (2.13 g, 37.8%)

R _f = 0.8 (normal phase, methylene chloride / methanol 5: 1 v / v)

¹ H NMR (400 MHz, DMSO): δ 7.91-7.84 (m, 2H), 7.79-7.72 (m, 1H), 7.58-7.54 (m, 3H), 7.41-7.35 (m, 5H), 6.56-6.50 (t, J = 12.6 Hz, 2H), 6.38-6.35 (d, J = 13.7 Hz, 2H), 4.12-4.09 (m, 4H), 1.68-1.62 (m, 12H), 1.32-1.24 (m, 6H )

LC / MS, calculated for C ₃₁ H ₃₇ N ₂ ⁺ 437.64, found 437.20

λ _abs (methanol): 739 nm, λ _fl (methanol): 774 nm

Example 3: Preparation of Compound 1-3

(1) Synthesis of Compound 6-2

2,3,3-trimethylindolenine (11 g, 70 mmol, 1 eq, Aldrich) and 1-iodoprapnae (70 ml, 840 mmol, 12 eq, TCI) were added and stirred over nitrogen. Heat to reflux for 24 hours. After cooling, check the particle state and filter. The obtained solid particles were washed 2-3 times with Hexane and dried under reduced pressure. (22.86 g, 99.2%)

R _f = 0.7 (normal phase, methylene chloride / methanol 5: 1 v / v)

(2) Synthesis of Compound 8-3

Add Chemical Formula 6-2 (10.5 g, 32 mmol, 1 eq) and Glutavonaldehyde dianil hydrochloride (9.1 g, 32 mmol, 1 eq, TCI) to 30 ml Acetic anhydride and stir. The reaction proceeds at 100 ° C. for 1 hour and then naturally cooled to room temperature. After removing the solution except particles, Diethyl ether is added to form solid particles. The resulting particles are filtered and dried under reduced pressure. (11.5 g, 68.3%)

R _f = 0.9 (normal phase, methylene chloride / methanol 5: 1 v / v)

LC / MS, calculated for C ₂₇ H ₃₁ N ₂ O ⁺ 399.55, found 399.20

(3) Synthesis of Compound 1-3

Formula 4c (5.26 g, 10 mmol, 1 eq) and formula 2b (3.29 g, 10 mmol, 1 eq) are added to a 72 ml pyridine solution and stirred on nitrogen. After reacting at 40 ° C. for 1 hour, the mixture is cooled to room temperature. The reaction solution was recovered using a rotary evaporator and then dried under reduced pressure.

Pure dye material is separated by normal chromatography with a 5: 1: 1 mixture of methylene chloride, methanol, and hexane. (1.96 g, 33.1%)

R _f = 0.75 (normal phase, methylene chloride / methanol 5: 1 v / v)

¹ H NMR (400 MHz, DMSO): δ 7.90-7.84 (m, 2H), 7.79-7.72 (m, 1H), 7.58-7.55 (m, 3H), 7.38-7.36 (m, 5H), 6.57-6.51 (t, J = 12.5 Hz, 2H), 6.40-6.37 (d, J = 13.6 Hz, 2H), 4.06-4.02 (m, 4H), 1.79-1.63 (m, 16H), 0.97-0.90 (m, 6H )

Example 4: Preparation of Compound 1-4

(1) Compound 8-4

Mix 1,2,3,3-tetramethyl-3H-indoliumiodide (1 g, 3.865 mmol, 1 eq) and malonaldehyde dianil hydrochloride (1.106 g, 3.672 mmol, 0.95 eq, TCI) with 5 ml acetic acid and 5 ml acetic anhydride Place in solution and heat to reflux for 4 hours. After naturally cooling to room temperature, the reaction solution was poured out, solid particles were produced using ethyl acetate, filtered, washed several times with n-butanol, and dried under reduced pressure. (1.56 g, 90%)

R _f = 0.60 (normal phase, methylene chloride / methanol 5: 1 v / v)

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 8.87-8.83 (d, J = 13.2 Hz, 1H), 8.47-8.44 (t, J = 14.9, 1H), 7.75-7.49 (m, 9H), 6.83-6.79 (d, J = 14.9, 1H), 5.62-5.59 (dt, J = 12.9 Hz, 11.5 Hz, 1H), 3.76 (s, 3H), 2.15 (s, 3H), 1.97 (s, 6H) , 1.66 (s, 3H)

LC / MS, calculated for C ₂₃ H ₂₅ N ₂ O ⁺ 345.46, found 345.14

(2) Compound 1-4

30 ml Acetic anhydride with 30 ml of compound 8-4 (5.5 g, 11.64 mmol, 1 eq) and 1,2,3,3, -Tetramethyl-3H-indolium iodide (3.51 g, 11.64 mmol, 1 eq, Aldrich) Pyridine mixed solution is added and stirred over nitrogen. The reaction is carried out at 100 degrees for 4 hours. After confirming the completion of the reaction, it is naturally cooled to room temperature and the solvent is recovered using a rotary evaporator and dried under reduced pressure. Pure dye material is separated by normal chromatography with a 5: 1 mixture of methylene chloride and methanol. (2.76 g, 46.5%)

R _f = 0.53 (normal phase, methylene chloride / methanol 5: 1 v / v)

LC / MS, calculated for C ₂₇ H ₃₁ N ₂ ⁺ 383.55, found 383.22

Example 5: Preparation of Compound 1-5

(1) Compound 8-5

Formula 6-1 (3.15 g, 10 mmol, 1 eq) and malonaldehyde dianil hydrochloride (2.59 g, 10 mmol, 1 eq, TCI) were added to a 20 ml acetic acid and 20 ml acetic anhydride solution and heated to reflux for 4 hours. . After naturally cooling to room temperature and removing the reaction solution, solid particles were produced using diethyl ether and filtered. The filtered particles were dried under reduced pressure. (2.12 g, 43.5%)

R _f = 0.65 (normal phase, methylene chloride / methanol 5: 1 v / v)

LC / MS, calculated for C ₂₂ H ₂₅ N ₂ ⁺ 317.45, found 317.14

(2) Compound 1-5

Compound 8-5 (2.32 g, 4.76 mmol, 1 eq) and compound 6-1 (1.5 g, 4.76 mmol, 1 eq, Aldrich) were added to a mixed solution of 10 ml Acetic anhydride and 10 ml Pyridine and stirred over nitrogen. The reaction is carried out at 100 degrees for 4 hours. After confirming the completion of the reaction, it is naturally cooled to room temperature and the solvent is recovered using a rotary evaporator and dried under reduced pressure. Pure dye material is separated by normal chromatography with a 5: 1: 1 mixture of methylene chloride, methanol, and hexane. (1.05 g, 40.8%)

R _f = 0.5 (normal phase, methylene chloride / methanol 5: 1 v / v)

¹ H NMR (400 MHz, DMSO): δ 8.37-8.30 (t, J = 12.9 Hz, 2H), 7.63-7.61 (m, 2H), 7.48-7.39 (m, 4H), 7.28-7.22 (m, 2H ), 6.59-6.53 (t, J = 12.4 Hz, 1H), 6.31-6.28 (d, J = 13.8 Hz, 2H), 4.14-4.04 (m, 4H), 1.75-1.60 (m, 12H), 1.27- 1.24 (m, 6H)

LC / MS, calculated for C ₂₉ H ₃₅ N ₂ ⁺ 411.6, found 411.23

λ _abs (methanol): 639 nm, λ _fl (methanol): 664 nm

Example 6: Preparation of Compound 1-6

(1) Compound 8-6

Formula 2b (10.54 g, 32 mmol, 1 eq) and malonaldehyde dianil hydrochloride (8.26 g, 32 mmol, 1 eq, TCI) were added to a 40 ml acetic acid and 40 ml acetic anhydride solution and heated to reflux for 4 hours. After naturally cooling to room temperature and removing the reaction solution, solid particles were produced using diethyl ether and filtered. The filtered particles were dried under reduced pressure. (9.29 g, 58.1%)

R _f = 0.75 (normal phase, methylene chloride / methanol 5: 1 v / v)

LC / MS, calculated for C ₂₅ H ₂₉ N ₂ ⁺ 373.51, found 373.17

(2) Compound 1-6

Compound 8-6 (6.01 g, 12 mmol, 1 eq) and compound 6-2 (3.95 g, 12 mmol, 1 eq, Aldrich) were added to a 60 ml Pyridine solution and stirred over nitrogen. The reaction is carried out at 60 degrees for 4 hours. After confirming the completion of the reaction, it is naturally cooled to room temperature and the solvent is recovered using a rotary evaporator and dried under reduced pressure. The pure dye material is separated by normal chromatography with a 5: 1: 1 mixture of methylene chloride, methanol, and hexane. (2.39 g, 35.2%)

R _f = 0.75 (normal phase, methylene chloride / methanol 5: 1 v / v)

¹ H NMR (400 MHz, DMSO): δ 8.36-8.30 (t, J = 12.9 Hz, 2H), 7.62-7.54 (m, 2H), 7.41-7.36 (m, 4H), 7.29-7.22 (m, 2H ), 6.60-6.55 (m, 1H), 6.33-6.29 (d, J = 13.8 Hz, 2H), 4.08-4.47 (m, 4H), 1.75-1.61 (m, 16H), 0.96-0.92 (m, 6H) )

LC / MS, calculated for C ₃₁ H ₃₉ N ₂ ⁺ 439.65, found 439.24

λ _abs (methanol): 641 nm, λ _fl (methanol): 667 nm

Examples 7-10

In the same manner as described in Examples 1 to 6 above, the compounds of Examples 7 to 10 (compounds 1-7 to 1-10) below were prepared. The structural confirmation data of these compounds is as follows.

Example 7: Preparation of Compound 1-7

R _f = 0.3 (RP-C18, acetonitrile / water 3: 7 v / v)

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 7.90-7.84 (m, 2H), 7.79-7.73 (m, 3H), 7.63-7.61 (m, 2H), 7.31-7.28 (m, 2H), 6.57-6.50 (t, J = 12.6 Hz, 2H), 6.38-6.30 (m, 2H), 4.11-4.09 (d, J = 7.4 Hz, 4H), 1.62 (m, 12H), 1.27-1.23 (m, 6H)

_{LC / MS, calculated for C 31} H 35 N 2 O 6 S 2 - 595.75, found 595.40

λ _abs (water): 744 nm, λ _fl (water): 780 nm

Example 8: Preparation of Compound 1-8

R _f = 0.22 (RP-C18, acetonitrile / water 3: 7 v / v)

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 7.91-7.84 (m, 2H), 7.79-7.73 (m, 3H), 7.62-7.60 (m, 2H), 7.32-7.30 (m, 2H), 6.57-6.50 (m, 2H), 6.40-6.36 (d, J = 13.6 Hz, 2H), 4.05-4.02 (m, 4H), 1.78-1.63 (m, 17H), 0.96-0.89 (m, 6H)

_{LC / MS, calculated for C 33} H 39 N 2 O 6 S 2 - 623.8, found 623.47

λ _abs (water): 747 nm, λ _fl (water): 783 nm

Example 9: Preparation of Compound 1-9

(1) Compound 9-9

R _f = 0.3 (RP-C18, acetonitrile / water 3: 7 v / v)

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 8.39-8.32 (t, J = 13 Hz, 2H), 7.89-7.81 (m, 2H), 7.64-7.63 (m, 2H), 7.38-7.31 ( m, 2H), 6.57 (m, 1H), 6.32-6.29 (d, J = 13.6 Hz, 2H), 4.14-4.12 (m, 4H), 1.67 (m, 12H), 1.26-1.23 (m, 6H)

_{LC / MS, calculated for C 29} H 33 N 2 O 6 S 2 - 569.71, found 569.19

λ _abs (water): 645 nm, λ _fl (water): 668 nm

Example 10: Preparation of Compound 1-10

R _f = 0.25 (RP-C18, acetonitrile / water 3: 7 v / v)

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 8.39-8.36 (m, 2H), 7.80 (s, 2H), 7.80- 7.61 (m, 2H), 7.34-7.32 (m, 2H), 6.61- 6.55 (t, J = 12.3 Hz, 1H), 6.27-6.22 (m, 2H), 4.08-4.04 (m, 4H), 1.74-1.68 (m, 16H), 0.95-0.91 (m, 6H)

_{LC / MS, calculated for C 31} H 37 N 2 O 6 S 2 - 597.77, found 597.33

λ _abs (water): 649 nm, λ _fl (water): 669 nm

Claims (4)

The cyanine compound represented by the following formula (1): [Formula 1]

In the formula, R ₁ is hydrogen, sulfonic acid group or sulfonate group, Two R ₂ and R ₃ are each independently hydrogen or an alkyl group having 1 to 6 carbon atoms, R ₄ is hydrogen, an alkyl group having 1 to 6 carbon atoms, l and m are each independently an integer of 1-5. The compound of claim 1, wherein the compound is selected from the group consisting of compounds represented by the formula:

The compound of claim 1, wherein the compound exhibits fluorescence at a wavelength of 600 to 800 nm. (1) reacting a compound of formula 6 with a compound of formula 7 to obtain a compound of formula 8, [Formula 6]

[Formula 7]

[Formula 8]

(2) A method of preparing a compound represented by Formula 1, comprising the step of reacting a compound of Formula 8 with a compound of Formula 6 to obtain a compound of Formula 1: [Formula 6]

[Formula 8]

[Formula 1]

In the formula, R ₁ is hydrogen, sulfonic acid group or sulfonate group, Two R ₂ and R ₃ are each independently hydrogen or an alkyl group having 1 to 6 carbon atoms, A is hydrogen or an acetyl group, R ₄ is hydrogen, an alkyl group having 1 to 6 carbon atoms, l and m are each independently an integer of 1-5.