PL230997B1 - Method for obtaining new derivative of lupeol - Google Patents

Abstract

The subject of the application is a method of preparing a new derivative of lupeol with the formula 1 and the chemical name ester of 3-picolinic acid and 20(29)-lupen-3ß-ol. This method involves esterifying lupeol of formula 2 with isonicotinic acid chloride of formula 3 or isonicotinic acid of formula 4, and the esterification is carried out in an organic solvent such as tetrahydrofuran, in the presence of a catalyst, which is N,N-dicyclohexylcarbodiimide and pH regulator from the group of amine compounds.

Description

Description of the invention

The subject of the invention is a method for the preparation of a new lupeol derivative, not described so far in the literature, with the chemical name 4-picolinic acid ester and 20 (29) -lupen-33-ol, with the formula 1, which is an ester obtained by modifying the structure of natural lupeol ( triterpene alcohol: 20 (29) -lupen-33-ol) found in the bark of birch, including especially warty birch (Betula pendula Ehrh.), mossy birch (Betula pubescens Ehrh.) and white birch (Betula Pendula Roth.), chloride 4-picolinic acid (isonicotinic acid chloride) or 4-picolinic acid (isonicotinic acid).

It is known that birch bark extract has been used for many years as a treatment for skin diseases, e.g. rashes, infections, inflammations. Currently, the medicine uses the raw material obtained from two species: birch Betula pendula Ehrh. and mossy birch Betula pubescens Ehrh. [B. Bednarczycz-Cwynar, L. Zaprutko, Polish J. Cosmetol., 2003, 4, 218; K.H.C. Ba§er, B. Demirci, Arkivoc, 2007, VII, 335; A. Ożarowski, W. Jaroniewski, Medicinal plants and their practical application, IWZZ, Warsaw, 1987].

Birch bark is characterized by a particularly high content of triterpenes, mainly betulin and lupeol. Their content in dry birch bark extract reaches 80% [A. Z. Abyshev, E. M. Agaev, A. B. Guseinov, Pharm. Chem. J., 2007, 41 (8) 22].

The triterpenes included in the birch extract have anti-inflammatory, antioxidant and regenerating properties, as well as antimicrobial and anticancer properties.

In patent application US2006 / 216249 "Use of a cosmetic of pharmaceutical composition, comprising a lupeol-rich extract as an active ingredient for stimulating the synthesis of heat shock proteins, and its analogues WO2005 / 9331 and EP1648482, studies are described that confirm the effectiveness of lupeol in the stimulation of skin cells to create structural proteins (collagen and elastin).

Classic esterification methods for the hydroxyl groups of triterpene compounds such as betulin are carried out with acid anhydrides such as acetic, propionic, phthalic, succinic or acid chlorides such as caproic acid chloride, capryloyl chloride, caprinoyl chloride, lauroyl chloride, palmitoyl chloride, benzoyl chloride. in the presence of pyridine or a tertiary amine. The most commonly used catalyst is 4-dimethylaminopyridine (DMAP). A common method of esterification is also to fuse solid acid anhydrides with the triterpene compound. The most commonly used anhydrides are maleic, terephthalic and fumaric [J. AchremAchremowicz, Cytotoxicity of semisynthetic betulin derivatives, Doctoral dissertation, Department of Pharmacognosy, Faculty of Pharmacy, Jagiellonian University Medical College, Krakow, 2007].

The publication by M. Kvasnica, J. Sarek, E. Klinotova, P. Dzubak, M. Hajduch, Bioorg & Med Chem., 2005, 13 (10), 3447 describes the esterification of triterpene alcohols with phthalic anhydride. The process was carried out under the following conditions: a 4-fold molar excess of anhydride and a 4-fold molar excess of DMAP (4-dimethylaminopyridine) were used for 1 mmol of triterpene alcohol. It was dissolved in 10 ml of pyridine. The ester synthesis was carried out at reflux for 30 hours. The reaction mixture was washed twice with hydrochloric acid solution and twice with water. The organic phase was dried over anhydrous magnesium sulfate and then concentrated under reduced pressure. The product obtained was purified by silica gel column chromatography using hexane-ethyl acetate 9: 1-5: 1 (gradient elution) as eluent.

In contrast, in the publication of O. Ashavin, O. Flekhter, F. Galin, N. Kabalnov, L. Baltina, G. Tolstikov, Chem. Nat. Compd., 2003, 39 (2), 201] shows the process of esterification of betulin, "cold", with the use of strong acids and their anhydrides, incl. trifluoroacetic acid.

In the publication of K. Papi Reddy, A.B. Singh, A. Puri, A.K. Srivastava, T. Narender, Bioorg. Med. Chem. Lett., 2009, 19 (15), 4463 discloses that esterification of the hydroxyl group of lupeol can be carried out with carboxylic acids such as acetic acid, but is much more difficult than when using an acid anhydride. In the presence of N-methylmorpholine, an excess of carboxylic acid over the alcohol is necessary. The most commonly used were 2-4 times the molar excess of acid in relation to triterpene alcohol. It has been shown that the catalytic systems used in the esterification of lupeol with carboxyls are most often a composition of two DCC-DMAP catalysts (N, N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine). Anhydrous CH2Cl2 (methylene chloride) was used as the solvent, and the reaction time was 4 hours.

The aim of the invention is to develop a preparation process from lupeol of the formula 2 and isonicotinic acid chloride of the formula 3 or isonicotinic acid of the formula 4 which has not been described in the literature so far.

The production of a new derivative of lupeol, with the chemical name 4-picolinic acid ester and 20 (29) -lupen-33-ol, of the formula 1, with the elimination of toxic reagents and solvents such as methylene chloride or chloroform from the process, as well as carcinogenic catalysts such as 4-dimethylaminopyridine.

The method according to the invention will make it possible to obtain a new semi-synthetic derivative of a natural plant metabolite (lupeol), which is a biologically active ester of 4-picolinic acid (isonicotinic) and pentacyclic triterpene alcohol (lupeol), which can be used as an active substance in the cosmetics or pharmaceutical industry.

According to the invention, the method for the preparation of a new lupeol derivative of the formula I and the chemical name 4-picolinic acid ester 20 (29) -lupen-33-ol consists in that lupeol (20 (29) -lupen-33-ol) The formula II is esterified with isonicotinic acid chloride (4-picolinic acid chloride) of the formula III or with isonicotinic acid (4-picolinic acid) of the formula IV, the esterification being carried out in an organic solvent in the presence of a catalyst and pH regulators from the group of amine compounds.

Preferably, the esterification of lupeol is carried out in such a way that 1 g (2.3 mmol) of lupeol is taken up in 5-7 ml of THF (tetrahydrofuran). NMM (N-methylmorpholine) is added to the resulting solution until the pH of the system is 9-12, preferably 10-11, and 0.57-1.15 g (4.6-9.2 mmol) of isonicotinic acid or 0.66 g -1.33 g (4.6-9.2 mmol) of isonicotinic acid chloride and 0.5-0.55 g (2.3-2.6 mmol) of DCC (N, N-dicyclohexylcarbodiimide) as a catalyst. The reaction mixture is then heated under reflux to 40-80 ° C, preferably 50-70 ° C. The reaction is continued until a cream colored solid precipitates from the reaction mixture. The precipitate, which is the reaction product (new lupeol derivative), is separated from the reaction mixture, preferably by filtration, and then washed with demineralized water and dried at room temperature for 1-8 hours. After drying, the product is a cosmetic and pharmaceutical raw material.

The new lupeol derivative prepared by the method according to the invention (ester of 4-picolinic acid and 20 (29) -lupen-33-ol), with the formula C36H53NO2, is in the form of a solid (white powder), it is very slightly soluble in water, it dissolves well. in methyl and ethyl alcohol and organic solvents such as chloroform, methylene chloride, acetone, ethyl acetate, tetrahydrofuran. Its melting point is in the range of 257-259 ° C. It has a proliferative effect in relation to human skin cells, without showing any toxic or irritating effects. In addition, it has an antioxidant effect, thanks to which it can be part of skin care and therapeutic preparations intended for damaged skin or skin that requires quick regeneration.

The following examples illustrate the detailed process flow.

Example 1 g lupeol (2.3 mmol) is taken up in 5.5 ml of THF (tetrahydrofuran). 1 ml of NMM (N-methylmorpholine) was added to obtain the pH of the system 10. Then 1.1 g (9.2 mmol) of isonicotinic acid and 0.54 g (2.3 mmol) of DCC (N, N-dicyclohexylcarbodiimide) were added. the reaction mixture was heated to reflux at 50 ° C. The conversion was monitored by TLC. The reaction was carried out until a cream-colored precipitate was formed from the reaction mixture, which was filtered on a fritted funnel, and then washed with 45 ml of distilled water. The obtained compound was dried at room temperature for 6 hours. The reaction yield was calculated to be 57.9%.

The melting point was determined for the compound thus obtained, and analysis by thin layer chromatography (TLC) was performed.

The structure of the obtained lupeol isonicotinate was determined by the following spectroscopic methods: UV / VIS spectroscopy, nuclear magnetic resonance (NMR) and also by elemental analysis (C, H and N content). Compound purity was determined by high performance liquid chromatography with mass spectroscopy (HPLC-MS).

The melting point of the obtained lupeol isonicotinate crystals was determined on a Stuart SMP10 apparatus.

Compound retention rate was determined by thin layer chromatography (TLC) using Polygram SIL G UV254 plates from Macherey Nagel. A 1: 9 v / v mixture of ethyl acetate and chloroform was used as eluent.

The first technique used to determine the identity of a compound was UV / VIS spectroscopy. The UV / VIS spectra were made on the Macherey Nagel Nanocolor UV / VIS apparatus in an ethyl alcohol solution. The wavelength range used was 200-280 nm, the optical path length was 10 mm.

PL 230 997 B1

Another spectrophotometric technique used was nuclear magnetic resonance (NMR). ¹ H NMR and ¹³ C NMR spectra were made on a Mercury-VX, Varian 300 MHz frequency, magnetic field induction 7.02 T, in CDCl3 solution. Chemical shift values are given in ppm.

Elemental analysis of C, H and N content was performed using a Perkin Elmer Type apparatus

2400.

The purity of lupeol isonicotinate was determined using a liquid chromatograph equipped with a mass analyzer (HPLC-MS). HPLC-MS analysis was performed on an Agilent Technologies 1100 series instrument using a Supelco column, Ascentis® Express RP-Amide, 7.5 cm x 2.1 1 mm x 2.7 µm. The HPLC analysis parameters used were: injection volume 2 μ (analysis time 25 minutes, mobile phase: mixture of acetonitrile (ACN) with water, gradient elution: 80-98-80% ACN (v / v), column temperature 40 ° C, mobile phase 0.5 ml / min, DAD and MSD detectors, wavelengths: 280.8, 205.5, 220.5, 240.8 nm The mass analyzer (MS) worked with the following parameters: APCI ion source, positive polarity , mass range 300-450, fragmenter 70, cycle time: 0.95 sec / cycle, positive polarity, gas flow 6-13 dm ³ / min, gas temperature 350 ° C, nebulizer pressure 60 psig, vaporizer temperature 500 ° C, capillary voltage 5000V, corona current 5.0 μA (+), 15 μA (-).

Testing the purity and determining the structure and properties of the new lupeol derivative obtained according to the procedure described in example 1

Thin layer chromatography (TLC)

A TLC image was made to determine the overall purity of the obtained compound and to determine its retardation factor (Rf). The factor is one of the quantities characterizing organic compounds in a given eluent system.

Rf = 0.81 (eluent system chloroform: ethyl acetate - 9: 1 v / v)

Melting temperature

The melting point of the obtained compound was determined.

Mp = 257-259 ° C

The narrow range of melting point proves the high purity of the compound obtained.

UV / VIS spectroscopy:

The spectrophotometric measurement was performed with the UV / VIS method for an ethanolic solution of lupeol isonicotinate with a concentration of 0.001 mmol / dm ³ . The attached drawing (Fig. 1) shows the UV / VIS spectrum of the tested ester in the range 200-280 nm.

Description of the UV / VIS spectrum (Fig. 1):

The spectrum clearly shows the maximum absorbance at Xmax = 209.8 nm.

¹ H NMR spectrum

Nuclear magnetic resonance (NMR) spectroscopic studies were also performed to confirm the structure of the obtained lupeol isonicotinate.

In the accompanying drawing (Fig. 2) is shown the full spectrum of ¹ H NMR isonicotinate lupeol obtained in the example.

Description ¹ H NMR spectrum (Fig. 2):

δ [ppm]: 9.00 (d, 2H, H-36.38), 8.37 (d, 2H, H-35.37), 4.81 (m, 1H, H-6), 4.57 (m, 2H, H- 5), 2.37 (3td, 1H, H-2), 1.93 (m, 2H, H-3), 1.78 (s, 3H, H-23), 1.65 (m, 6H, H-27.28), 1.50 (d, 1H, H-9), 1.46 (d, 1H, H-11), 1.40 (m, 4H, H-10.14), 1.32 (m, 4H, H-8.13), 1.16 (m , 4H, H-16.18), 1.04 (s, 3H, H-24), 1.00 (s, 3H, H-26), 0.91 (t, 8H, H-22.30.31), 0.86 (d , 1H, H-21), 0.76 (m, 4H, H-19.20), 0.63 (m, 1H, H-17).

The spectrum (Fig. 2) confirms the presence of an aromatic ring as evidenced by doublets (2 protons each) at 9.00 ppm and 8.37 ppm. There was also a slight increase in the chemical shift in relation to the lupeol spectrum caused by the vicinity of the aromatic ring and the nitrogen atom.

¹³ C NMR spectrum

Also made of the spectrum ¹³ C NMR. In the accompanying drawing (Fig. 3) is a nuclear magnetic resonance spectrum ^{13 C-NMR} of the ester.

Description ¹³ C NMR spectrum (Fig. 3):

δ [ppm]: 161.83 (C-32), 150.93 (C-25), 144.85 (C-36), 143.59 (C-38), 126.02 (C-35), 109.22 (C-30), 85.07 (C -37), 78.93 (C-6), 55.26 (C-21), 50.36 (C-31), 48.24 (C-4), 47.95 (C-5), 42.96 (C-1), 42.81 (C- 7), 40.81 (C-12), 39.95 (C-15), 38.83 (C-17), 38.68 (C-11), 38.21 (C-34), 37.98 (C-9), 37.08 (C-2 ), 35.51 (C-3), 34.11 (C-20), 29.80 (C-19), 28.18 (C-10), 27.98 (C-13), 27.41 (C-14), 25.03 (C-8) , 20.96 (C-16), 19.28 (C-18), 18.15 (0-7), 17.97 (C-28), 16.15 (C-22), 15.96 (C-26), 15.38 (C-23), 14.50 (C-24).

PL 230 997 B1

Also the ¹³ C NMR spectrum confirms the structure of lupeol isonicotinate. The number of signals from the nuclei of the ¹³ C carbon atoms corresponds to the structure of the molecule (36 carbon atoms per molecule). The accompanying spectrum (Fig. 3) shows singlets at 144.85 ppm, 143.59 ppm, 126.02 ppm, 85.07 ppm and 38.21 ppm from ¹³ C nuclei in the aromatic system. These signals, however, are not observed on the lupeol spectrum. Moreover, the signal at 161.83 ppm corresponds to the carbon atom next to the ester group (-OC = O).

Elemental analysis (CHN)

In order to determine the content of individual elements, a qualitative analysis was performed, the content of C, H and N was determined. The mass content of individual elements was presented below:

Theoretical values: C = 81.30%; H = 10.04%; N = 2.63% (m / m).

Experimental values: C = 080.72 +/- 0.02%; H = 10.08 +/- 0.04%; N = 2.69 +/- 0.06% (m / m).

The data obtained as a result of the experiment correspond to the theoretical values, which will confirm the qualitative and quantitative composition of the compound obtained. Small deviations from the measurement results are within the statistical error and may result from the measurement accuracy and possible contamination of the sample.

The formula for the compound is C36H53NO2.

High performance liquid chromatography (HPLC-MS)

MS (m / z,%): 425.5 (25.7), 424.4 (26.9), 423.3 (100), 410.3 (23.9), 409.4 (51.2), 407.2 (25.2), 311.3 (14.8).

The purity of the compound was 96.1%.

The full image of the lupeol isonicotinate chromatogram is shown in Fig. 4, the mass chromatogram is shown in Fig. 5 and the masses and intensities of the fragment ions are shown in Fig. 6.

Example 2 2 g of lupeol (2.3 mmol) are taken up in 6 ml of THF (tetrahydrofuran). 1.1 ml NMM (N-methylmorpholine) was added to give the system a pH of 10.5. Then 1.3 g (9.2 mmol) of isonicotinic acid chloride and 0.54 g (2.3 mmol) of DCC (N, N-dicyclohexylcarbodiimide) were added. The reaction mixture was then heated to reflux at 50 ° C. The conversion was monitored by TLC. The reaction was carried out until a cream-colored precipitate formed from the reaction mixture, which was filtered on a fritted funnel and then washed with 50 ml of distilled water. The obtained compound was dried at room temperature for 8 hours. The reaction yield was calculated to be 57.6%. The results of the analyzes confirming the structure of the obtained compound were, within the measurement error, the same as in example 1.

The performed tests allow for unequivocal confirmation that using the method of lupeol esterification described above, which is the subject of the invention, a new compound, not described so far in the literature, was obtained, i.e. lupeol isonicotinate of formula 1.

The presented results of analyzes for the new derivative of lupeol (lupeol isonicotinate) obtained with the use of isonicotinic acid chloride and isonicotinic acid do not differ from each other and describe the same structure. The choice of this reagent does not affect the quality and purity of the ester obtained.

The invention enables the preparation of an active ingredient (lupeol isonicotinate) which is a semi-synthetic, new derivative of lupeol - a natural plant metabolite, with potential application in cosmetic and pharmaceutical preparations.

Moreover, a particularly advantageous feature of the solution according to the invention is that it allows to obtain a new lupeol derivative in ecologically safe conditions, i.e. with the elimination of the use of toxic solvents and catalysts. The DCC (N, N-dicyclohexylcarbodiimide) catalyst used in the process according to the invention, has a much lower toxicity than DMAP (4-dimethylaminopyridine) known from the literature in terms of its action on the skin and after oral administration.

Due to the direct esterification of lupeol with isonicotinic acid or isonicotinic acid chloride in an organic solvent such as THF in the process according to the invention, using NMM (N-methylmorpholine) as the pH regulator, it was possible to eliminate toxic compounds such as methylene chloride from the process. , chloroform, pyridine and tributylamine, DMAP, hexane and ethyl acetate.

Claims (4)

Patent claims 1. Method for the preparation of a new lupeol derivative of formula 1 and chemical name 4'-picolinic acid ester and 20 (29) -lupen-33-ol, characterized in that lupeol of formula 2 is esterified with isonicotinic acid chloride of formula 3 or isonicotinic acid 4, wherein the esterification is carried out in an organic solvent in the presence of a catalyst and pH regulators from the group of amine compounds. 2. The method according to p. A process according to claim 1, characterized in that 2.3 mmol of lupeol is taken up in 5-7 ml of tetrahydrofuran, N-methylmorpholine is added until the pH of the system is 9-12, preferably 10-11, and then 4.6-9.2 mmoles of isonicotinic acid and the catalyst, which is DCC, in an amount of 2.3 to 2.6 mmol, then the reaction mixture is heated to 40-80 ° C, preferably 50-70 ° C, and reacted until precipitation from the cream precipitate which is separated from the reaction mixture, preferably by filtration, washed with demineralized water and dried at ambient temperature for 1-8 hours. 3. The method according to p. A process according to claim 1, characterized in that 2.3 mmol of lupeol is taken up in 5-7 ml of tetrahydrofuran, N-methylmorpholine is added until the pH of the system is 9-12, preferably 10-11, and then 4.6-9.2 mmoles of isonicotinic acid chloride and the catalyst, which is DCC, in the amount of 2.3 to 2.6 mmoles, then the reaction mixture is heated to 40-80 ° C, preferably 50-70 ° C, and the reaction is allowed to precipitate the cream precipitate which is separated from the reaction mixture, preferably by filtration, washed with demineralized water and dried at ambient temperature for 1-8 hours. 4. The method according to p. A method according to claim 2 or 3, characterized in that lupeol, which is a natural plant metabolite, is esterified.