KR102048869B1 - Process for synthesizing ramalin not using Tin as a heavy metal - Google Patents

Abstract

The present invention relates to a method for synthesizing ramaline, and more particularly, by reacting a glutamic acid derivative prepared using alkylchloroformate with a hydrazine salt compound prepared using a hydroxyl group-protected hydroxy aniline. The present invention relates to a method for synthesizing ramalin.

Description

Process for synthesizing ramalin not using Tin as a heavy metal}

The present application relates to a method for synthesizing a ramalline exhibiting the same physical properties and efficacy as a ramalin derived from a natural product without using heavy metal tin (Sn).

Ramalin is derived from lichens called Ramalina terebrata, which grow inhabiting King George Island in Antarctica. According to a study by the Korea Institute of Maritime Science and Technology, Ramalina Terrebreta is a lichen that grows only 1cm in 100 years in a barren area of 2,500m above sea level and -100 ° C. It has a persistent life force. Ramalina protects itself by making antioxidants that remove oxides by attaching themselves to the rocks on the shore and receiving UV light directly. Ramalin is the ingredient that was created by itself to overcome the strong ultraviolet rays of Antarctica.

Since the ramallin has been reported to have excellent anti-inflammatory activity, the Korea Maritime Institute of Science and Technology has been the first to obtain a patent for the extraction of ramalin from a dry sample of Ramalina Terebrata (Korea Patent Publication No. 10-2010-0052130 ).

Lichens are less well studied than higher plants because it is difficult to collect sufficient natural samples, and mass cultivation techniques are not known. However, as the tissue culture method, mass production method, and biochemical analysis method of lichens have been improved, research on this is being actively conducted (Behera, BC et al., Lebensm. Wiss. Technol., 39: 805). , 2006). Fatty acids, depsides and depsidones, debenzofurans, diterpenes, anthraquinones, anthraquinones, which have a variety of biological activities such as cytotoxicity, mold, antimicrobial, antioxidants, etc. Compounds including naphtoqui nones, usninic acid, pμlvinic acids, xanthones and epidithiopiperazinediones have been isolated from lichens (Muller) , K., Appl.Microbiol.Biotechnol., 56: 9-16, 2001).

Ramalin has excellent anti-oxidant and anti-inflammatory activity and needs to be mass-produced.However, when the separation method using methanol is used in the natural lichen, Ramalina terebrata, the slow growth rate unique to polar lichens and the mass in nature Due to the difficulty in collecting and the very small amount extracted from Ramalina terebrata, there has been a problem of costly and time-consuming production.

Accordingly, there has been an industry demand for a new chemical synthesis method of ramalin which is simple in process and competitive in price, and one of the prior arts is a glutamic acid derivative prepared using alkylchloroformate and hydroxyaniline. The present invention relates to a synthetic method of reacting a hydrazine salt compound prepared using hydroxyaniline or a hydroxyl group protected hydroxyaniline (Korean Patent Publication No. 10-2013-0085306).

However, the synthesis method of the prior art includes a process of using tin, which is a heavy metal, in the production of ramalin, which is used in cosmetics and pharmaceuticals, which is the main use of ramalin, even though a small amount of tin is contained in the final product as it is. There is a problem. In particular, the problem of residual tin in ramalin is due to the residual tin in vivo to generate organotin compounds in vivo and cause problems in the nervous system of the human body.

Accordingly, it is an object of the present application to chemically synthesize ramalins that exhibit the same efficacy as ramalin derived from natural products in a novel process without using heavy metal tin.

The present inventors confirmed that the diazo compound, which is an intermediate, was reduced with a tin chloride reducing agent in the synthesis process of hydrazine in which the hydroxyl group of the following formula (3) was protected by the benzyl group in the synthesis process of ramalin. However, various reducing agents were examined to exclude tin, which is a heavy metal, but it was difficult to synthesize a desired target because the benzyl group protected by the benzyl group in the hydroxyl group was decomposed. Therefore, as a result of earnest examination, the present inventors examined various protecting groups except the benzyl group, and changed into the allyl group among them. The allyl group-protected diazo compound was variously studied by the following various reducing agents, and thus allylhydrazine intermediates could be synthesized with high efficiency as in the examples.

The most typical reduction method is the reduction method using tin chloride (SnCl2), but several methods that do not use tin chloride were examined. We used ascorbic acid (Tetrahedron, 67, (2011), 10296 ~ 10303), triphenylphosphine (EP 1981860B1), Benzenere among the recently reported methods to synthesize allylhydrazine derivatives. Various methods including benzeneselenol (J. Chem. Soc. Chem. Commun., 1977, 131-132) were used. The method with the highest selectivity and high reaction conversion rate was the method using sodium sulfite. As a result of various experiments, in order to apply the method using sodium sulfite, when the protecting group of the phenol in the ortho position is composed of a hindered protecting group such as benzyl, the reaction conversion rate is not high. The reason for this is that the sulfonic acid group is introduced in the diazonium reduction process to narrow the intramolecular space, resulting in the steric hindrance effect of the benzyl group, which does not facilitate the conversion of the reaction.

Therefore, while suppressing this steric hindrance effect, an allyl group was selected to easily induce a deprotection reaction later. The aryldiazonium salt protected with the ortho-allyloxy group could be readily reduced to sodium sulfite to easily secure the allylhydrazine intermediate.

An object of the present application is a method for synthesizing Ramalin of Formula 1,

Coupling a glutamic acid derivative of Formula 2 with a hydrazine salt compound of Formula 3 to produce a ramine precursor of Formula 4;

Generating a glutamic acid derivative of Formula 5 by performing a deallyl reaction on the compound of Formula 4; And

It can be achieved by a method comprising the step of removing the protecting group by performing a hydrogenation reaction to the compound of formula 5 produced above:

+

+

→

→

→

→

In the above formula,

R ¹ is lower alkyl;

R ² is a hydroxy protecting group;

X is acid.

R ^{1, which} is a 'lower alkyl group' in the above formula, represents a straight or branched chain alkyl group having 1 to 8 carbon atoms, preferably 1 to 6 carbon atoms, particularly preferably 1 to 4 carbon atoms. In an embodiment, R ¹ is methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, isopropyl, isobutyl, sec-butyl, tert-butyl, iso A pentyl group etc. are mentioned, Preferably it is ethyl.

R ² is allyl, and X is hydrochloric acid, bromic acid, iodic acid or p-Toluenesulfonic acid, and among them, hydrochloric acid is preferable.

In another embodiment of the present invention, the hydrazine salt compound of formula 3 is a 2-nitrophenol in which the hydroxy group is protected with allyl, the nitro group is reduced to amine, and the hydroxy-protected hydroxy aniline salts azo After it can be prepared by reducing using sodium bisulfite.

In particular, in the method for synthesizing allylhydrazine, a compound of Formula 3, from a diazonium salt, the reduction reaction using sodium bisulfite is used in the range of 2 equivalents to 10 equivalents of sodium bisulfite, preferably in the range of 2 equivalents to 2.5 equivalents. Into the reaction conditions, ammonia water is added to adjust the pH of the reaction solution to pH 5.5 to pH 6.5, preferably pH 6.0, and the reaction temperature is adjusted to 50 ° C to 70 ° C, preferably 60 ° C. It is important to adjust to 6.0. At this time, an aqueous diazonium salt solution is added for 40 minutes and stirred at 60 ° C to 90 ° C, preferably at 75 ° C for about 30 minutes to 2 hours, preferably about 1 hour.

In addition, as another embodiment of the present invention, the glutamic acid derivative of Formula 2 may be prepared by reacting a glutamic acid derivative of Formula 6 with alkylchloroformate (R ¹ -O-CO-Cl) below:

+

→

+

→

In addition, the alkyl chloroform used in the synthesis of glutamic acid derivatives of Chemical Formula 2 may be used as a reactant, ethylchloroformate (ECF), methylchloroformate (MCF), preferably ethylchloroformate (ECF). The purchase was purchased from TCI or Aldrich.

In addition, as another embodiment of the present invention, in order to obtain a compound of the formula (5) through the allyl reaction, ammonium formate may be used in the range of 4 to 8 equivalents, preferably 6 equivalents. Further, the Pd / C catalyst may be used in the range of 5 to 20%, preferably 10% of the reactant, and MeOH, the reaction solvent, may be used at a volume ratio of 100 times that of the reactants, preferably at a volume ratio of 50 times use. The reaction temperature is in the range of 0 to 50 ° C, preferably in the range of 20 to 30 ° C.

In addition, the deprotection reaction for the removal of the allyl group screened various reducing catalysts such as Pd (PPh3) 4, Pd, Pd / C, etc., resulting in the best condition of 10% Pd / C and ammonium formate. there was.

Through the invention of the present application it was possible to chemically synthesize a ramine having the same physical properties as that of a natural product derived from a natural synthesis without using heavy metal tin. Furthermore, the method for synthesizing ramalin according to the present invention can synthesize ramin excellent in antioxidant and anti-inflammatory effects in a stable yield without using tin, which is a heavy metal. Therefore, the RAMALIN synthesized according to the present invention is cost competitive in mass production and can provide RAMALIN in high yield, which can be widely used in cosmetics and pharmaceuticals.

1 is ¹ H-NMR analysis data of ramalin synthesized according to the present application;
Figure 2 is ¹ H-NMR analysis data of ramallin extracted from natural lichens.

The present application will be described in detail with reference to the following examples, but the following examples are provided by way of example, and those skilled in the art will fully understand that the present application is not limited thereto.

Example 1

Synthesis Example of Ramalin

1-1. Preparation of Allyl Hydrazine Salt

Scheme 1-1

Into a _three -necked round bottom flask, 72 g of the compound of Formula 9, 68.8 g of allyl bromide, 214.6 g of K ₂ CO _{3, and} 720 ml of acetone were added at room temperature (rt). After the temperature was raised to 56-57 ° C., the mixture was refluxed for 4 hours to terminate the reaction. Cool the temperature to rt and concentrate the solution after celite filtration. After dissolving methylene chloride (MC), washing is performed using H ₂ O. Water was removed with Na ₂ SO ₄ and concentrated under reduced pressure to obtain 92.3 g (yield: 99.5%) of the compound of formula 10.

[Scheme 1-2]

In a three-neck round bottom flask, 1.02 g of compound 9 of formula 10, 455 ml of EtOH, Sat. 455 ml of NH ₄ Cl are added sequentially at room temperature. After adding 149.5g of iron powder, increase the stirring speed. Add 2 drops of 35% HCl and proceed to reflux. After filtration of celite, the mixture was concentrated under reduced pressure to remove EtOH, and extracted using ethyl acetate (EA). The organic layer was washed with H ₂ O, and the organic layer was concentrated under reduced pressure to obtain 74.65 g of a compound of Formula 11 (yield: 98.5%).

Scheme 1-3

71.74 g of a compound of Formula 11 and 6.54 g of H ₂ O 8 were added to a three-neck round bottom flask, followed by stirring at rt. HCl diluted with 100 g of 35% HCl and 76 g of H ₂ O was added to the reactor for 15 minutes at room temperature, and then cooled to 0 ° C. NaNO ₂ was dissolved using 57.53 g of H ₂ O and then charged for 1 hour while maintaining the temperature below 5 ° C. to obtain a compound of Formula 12.

After confirming that Compound 11 disappeared by TLC, Compound 12 was not separated, and continuously, 112.53 g of NaHSO _3, 217 g of H ₂ O, 105.4 g of NaCl, and 252 g of H ₂ O were dissolved at rt. Aqueous ammonia was added to make pH 6.0, and then the temperature was raised to 60 ° C and adjusted to pH 6.0 again. The temperature is maintained at 60 ° C., and the diazonium salt solution prepared above is added for 40 minutes. The mixture was heated to 75 ° C, stirred for 1 hour, cooled to 10 ° C, and adjusted to pH 10-11 using a 50% aqueous NaOH solution. The mixture was extracted with toluene, dried over sodium sulfate, and then toluene was concentrated under a reduced pressure to obtain 67.3 g of a compound of formula 13 (yield: 87.8%) in 99.2% purity.

1-2. Preparation of Glutamic Acid Derivatives and Synthesis of Ramalin

[Scheme 1-4]

75.39 g of the compound of Formula 6 and MC 754 ml were added to a three-neck round bottom flask, and the mixture was cooled to 0 ° C., and triethylamine (TEA) 24.65 g was added for 30 minutes. The reaction solution was cooled to 15 ° C. and 26.44 g of ethylchloroformate (ECF) was added for 40 minutes to activate. The mixture was stirred for 1 hour while kept at 0 ° C. or less to confirm that the compound of Formula 6 disappeared using TLC. .

Subsequently, a solution obtained by dissolving 40 g of the compound of formula 13, which is the allyl hydrazine salt, obtained in the above, in 360 ml of MC was kept at 0 ° C. or lower for 1 hour and 40 minutes, and heated to rt, followed by stirring for 10 hours. After the reaction was completed, 200 ml of H ₂ O was added and washed, and then the organic layer was washed once with 200 ml of 1N HCl and once with 200 ml of Sat. NaHCO ₃ . The organic layer was dried over Na ₂ SO ₄ and concentrated under reduced pressure to remove the solvent, dissolved in MeOH, and treated with activated carbon. After filtration of celite, the methanol solvent was again removed by distillation under reduced pressure, and crystallized using MC / Hex (methylene chloride / hexane) to obtain 93.61 g (yield: 89.09%) of the compound of formula 8 as a ramine precursor.

The compound of formula 8, ammonium formate 6eq, Pd / C 10%, MeOH 50v was added to the reactor and stirred. The reaction is terminated after confirming the disappearance of the compound of formula 8 using TLC, HPLC. The Pd catalyst was removed using Celite filtration, the solvent was concentrated under reduced pressure, and EA was added thereto, followed by washing with water to remove residual ammonium formate. This gave 8.18 g (yield: 90.05%) of the compound of the formula (5) which is another ramine precursor.

Then, the compound of formula 5, Pd / C 10%, MeOH 50v is stirred at rt. H ₂ is injected into the reactor and stirred for 10 hours. Celite filtration removes the catalyst. The organic layer was concentrated and the concentration was stopped when the solid was produced, and the concentrated methanol solution was added to EA prepared by stirring beforehand to precipitate a solid to obtain a white solid ramine (Formula 1). (Yield: 92%)

1-3. HPLC of RAMALIN, ^One H-NMR Comparative Analysis

The obtained RAMALIN (Formula 1) showed purity of 98 area% or more as a result of HPLC analysis, and ¹ H-NMR results are shown in FIG. 1, and for comparison, ¹ H-NMR of RAMALIN extracted from natural lichens for comparison. The analysis results are shown in FIG.

1 and 2, ¹ H-NMR (D ₂ O, 400 MHz, δ / ppm) of the synthetic ramine of the present invention was 6.86-6.92 (m, 4H), 3.81 (t, 1H), 2.55 (m, 2H), 2.21 (m, 2H), and ¹ H-NMR (D ₂ O, 400 MHz, δ / ppm) of the natural product ramalin is 6.77-6.80 (m, 4H), 3.73 (t, 1H), 2.45 (m, 2H), 2.11 (m, 2H). From these peaks, it was found that the synthetic RAMALINE of the present invention is consistent with natural RAMALIN.

While the invention has been described in some detail by way of illustration and example for clarity to those skilled in the art, it will be understood by those skilled in the art that numerous and various modifications may be made without departing from the spirit of the disclosure. Accordingly, the forms disclosed herein are illustrative only and are not intended to limit the scope of the present disclosure, and the scope of the present invention should be defined by the following claims, and together with the true scope and spirit of the present invention. It should be clearly understood that it is intended to include variations and alternatives.

Claims (6)

As a method of synthesizing ramalin of the formula (1),
Coupling a glutamic acid derivative of Formula 2 with a hydrazine salt compound of Formula 3 to produce a ramine precursor of Formula 4;
Generating a glutamic acid derivative of Formula 5 by performing a deallyl reaction on the compound of Formula 4; And
Performing a hydrogenation reaction on the compound of Chemical Formula 5 produced above to remove the protecting group,
The hydrazine salt compound of Formula 3 protects the hydroxy group with allyl in 2-nitrophenol, reduces the nitro group to amine, and azoizes the salt of hydroxy aniline, which is protected with hydroxy, and then sodium bisulfite. Prepared by reduction using:

+

→

→

→

In the above formula,
R ¹ is straight or branched chain alkyl of 1 to 6 carbon atoms;
R ² is allyl;
X is acid.
delete The method of claim 1, wherein the glutamic acid derivative of Formula 2 is prepared by reacting a glutamic acid derivative of Formula 6 with alkylchloroformate (R ¹ -O-CO-Cl) below.

+

→

The method of claim 1 or 3, wherein R ¹ is ethyl and X is hydrochloric acid. The method according to claim 1 or 3, wherein in the step of producing the glutamic acid derivative of Formula 5 and the removing of the protecting group, Pd or Pd / C is used as a catalyst. The method according to claim 1 or 3, wherein ammonium formate is used in the range of 4 to 8 equivalents in the course of obtaining the compound of formula (5) glutamic acid by deyllation of the compound of formula (4).

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