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Title:
NOVEL LUPEOL DERIVATIVE AND THE METHOD OF OBTAINING THE NOVEL LUPEOL DERIVATIVE
Document Type and Number:
WIPO Patent Application WO/2018/063010
Kind Code:
A9
Abstract:
The invention relates to a novel lupeol derivative, which is the ester of 4-picolinic acid (isonicotinic acid) and 20(29)-lupen-3.beta.-ol (lupeol), having structural Formula (1). The novel lupeol derivative is a result of modification of the structure of natural lupeol and is produced therefrom by claimed esterification processes.

Inventors:
MALINOWSKA MAGDALENA (PL)
SIKORA ELŻBIETA (PL)
OGONOWSKI JAN (PL)
Application Number:
PL2017/000070
Publication Date:
May 24, 2018
Filing Date:
July 12, 2017
Export Citation:
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Assignee:
POLITECHNIKA KRAKOWSKA IM TADEUSZA KOSCIUSZKI (PL)
International Classes:
C07J63/00; A61K31/58; A61P17/00; A61P39/06
Attorney, Agent or Firm:
DOSKOCZYŃSKA-GROYECKA, Anna (PL)
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Claims:
PATENT CLAIMS

A new lupeol derivative which is ester of 4-picolinic acid and 20(29)-Κιρεη-3β- ol and which graphic formula is presented on Formula (1)

A method for obtaining a novel lupeol derivative which is ester of 4-picolinic acid and 20(29)-lupen-3p-oi and which graphic formula is presented on Formula (1 ),

characterized in that lupeol, which graphic formula is presented on Formula (2),

is esterified with isonicotinic acid chloride, which graphic formula is presented on Formula (3),

or with 4-picolinic acid, which graphic formula is presented on Formula (4),

in addition, the esterification process is carried out in an organic solvent in the presence of a catalyst and pH regulators from an amino group.

The method according to claim 2, characterized in, that 2.3 mmol of lupeol presented on Formula (2)

is dissolved in 5-7 cm3 of tetrahydrofuran, then N-methylmorpholine is added until the pH of the system is 9-12, preferably 10-11, and then 4.6-9.2 mmol of 4-picolinic acid, presented on Formula (4)

and 2.3-2.6 mmol of catalyst which is N,N-dicyclohexylcarbodiimide, then the reaction mixture is heated to 40-80°C, preferably 50-70°C, and the reaction is carried out until creamy precipitate separates from the reaction mixture and then said precipitate is separated from the reaction mixture, preferably by filtering, washed with demineralized water and dried at room temperature for 1 -8 hours.

The method according to claim 2, characterized in, that 2.3 mmoles of lupeol, presented on Formula (2)

is dissolved in 5 - 7 cm of tetrahydrofuran, and then N-methylmorpholine added until the pH of the system is 9 - 12, preferably 10 - 11, then 4.6 - 9 mmol of 4-picolinic acid chloride, presented on Formula (3)

is added and 2.3 - 2.6 mmol of catalyst which is N, N- dicyclohexylcarbodiimide is added, then the reaction mixture is heated to the temperature of 40 - 80°C, preferably 50 - 70°C, and the reaction is carried out until creamy sediment precipitates from the reaction mixture and then said sediment is separated from the reaction mixture, preferably by filtering, washed with demineralized water and dried at room temperature for 1 - 8 hours.

The method according to claim 3 or 4, characterized in, that the lupebl, presented on Formula (2)

subjected to the esterification process is a natural plant-metabolite.

Description:
Novel lupeol derivative and the method of obtaining the novel lupeol derivative

TECHNICAL FIELD:

The invention relates to a novel lupeol derivative and the method of obtaining the novel lupeol derivative. The novel lupeol derivative can be used in cosmetic or pharmaceutical formulations.

BACKGROUND ART:

Lupeol is triterpene alcohol which chemical name is 20(29)-lupen-3P-ol. It is present in birch bark, in particular common birch (Betula pendula Ehrh), pubescent birch (Betula pubescens Ehrh.) and white birch (Betula pendula Roth). It is known that extract of birch bark has been used for years as a cure for skin diseases such as rash, infections, inflammation. Nowadays in medicine is used raw material obtained from two kinds of birch: common birch Betula pendula Ehrh and pubescent birch Betula pubescens Ehrh. [B. Bednarczycz-Cwynar, L. Zaprutko, Polish J. Cosmetol. 2003, 4, 218; K.H.C. Baser, B. Demirci, Arkivoc, 2007, VII, 335; A. Ozarowski, W. Jaroniewski, Medicinal plants and their practical application, IWZZ, Warsaw, 1987].

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

Triterpenes contained in birch extract demonstrate anti-inflammatory, antioxidant and regenerative properties, as well as antimicrobial and anti-cancer properties.

The US patent application US2006/216249 "Use of a cosmetic of pharmaceutical composition, comprising of a lupeol-rich extract as an active ingredient for stimulating the synthesis of heat shock proteins" and its analogs WO2005/9331 and EP 1648482, describe the studies that confirm the effectiveness of lupeol in stimulating skin cells to form structural proteins (collagen and elastin).

Methods of esterification of hydroxyl groups in triterpene compounds are known in literature.

Classic methods of esterification of 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, caprynyl chloride, lauroyl chloride, palmitoyl chloride, benzoyl chloride, in the presence of pyridine or tertiary amine. As the catalyst, 4-N,N-dimethylaminopyridine (DMAP) is most commonly used. The method which is frequently used for esterification is melting solid acid anhydrides with a triterpene compound. Most commonly used anhydrides are maleic anhydride, terephthalic anhydride, fumaric anhydride [J. Achrem- Achremowicz, emi-synthetic derivative of betulin cytotoxicity, PhD Dissertation, Department of Pharmacognosy, Faculty of Pharmacy, Jagiellonian University, Krakow, 2007].

The publication of M. Kvasnica, J. Sarek, E. Klinotova, P. Dzubak, M. Haj duch, 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: 4-fold molar excess of anhydride and 4-fold molar excess of DMAP (4-N,N-dimethylaminopyridine) was used per 1 mmol of triterpene alcohol. The whole mixture was dissolved in 10 cm 3 of pyridine. The synthesis of the esters was performed in a reflux condenser for 30 hours. The reaction mixture was washed twice with hydrochloric acid and twice with water. The organic phase was dried over anhydrous magnesium sulfate and then concentrated under reduced pressure. The resulting product was purified in a chromatographic column filed with silica gel, and as the eluant hexane - ethyl acetate system 9:1 - 5:1 (gradient elution) was used. Modification of triterpen alcohol, betulin, may also be carried out as oxidation process.

In the publication of O. Ashavina, O. Flekhter, F. Galin, N. Kabalnov, L. Baltin, G. Tolstikov, Chem. Nat. Compd., 2003, 39 (2), 201] is shown "cold" oxidation process of betulin, where strong acids and their anhydrides, among other things - trifluoroacetic acid, were used. Methylene chloride was used as a solvent, triethylamine was used as an alkalizing agent, and obtained products were purified on a chromatographic column with benzene or methylene chloride as eluents.

The publication of K. Papi Reddy, AB Singh, A. Puri, AK Srivastava, T. Narender, Bioorg. Med. Chem. Lett., 2009, 19 (15), 4463 discloses that esterification of the hydroxyl group of lupeol may proceed involving carboxylic acid, for example acetic acid, however, it is much more difficult than in the case of using acid anhydride. In the presence of N-methylmorpholine, an excess of carboxylic acid to alcohol is required, the most often 2- 4-fold molar excess of acid in relation to triterpene alcohol. As it was indicated, the catalyst systems used in the esterification of lupeol with carboxylic acids, in the most cases are the composition of two catalysts DCC - DMAP (N, N- dicyclohexylcarbodiimide and 4-N,N-dimethylaminopyridine). As a solvent anhydrous CH 2 C1 2 (methylene chloride) was used, and the reaction time was 4 hours.

In prior art methods of esterification of hydroxyl groups in triterpene compounds, toxic reagents and solvents such as methylene chloride, chloroform and also carcinogenic catalysts such as 4-N,N-dimethylaminopyridine were used [J. Achrem-Achremowicz, semi-synthetic derivative of betulin cytotoxicity, PhD Dissertation, Department of Pharmacognosy, Faculty of Pharmacy, Jagiellonian University, Krakow, 2007]. AIM OF THE INVENTION:

The advantages of lupeol, known from prior art have, prompted the authors of the present invention to work on obtaining a new lupeol derivative, characterized by biological activity identical or better than the lupeol biological activity. The aim of the invention was also to provide a method of obtaining the new lupeol derivative without using toxic reagents and solvents such as methylene chloride or chloroform, and without carcinogenic catalysts such as 4-N,N-dimethylaminopyridine.

The aim of the invention was achieved by obtaining a novel lupeoil ester using the method of the present invention. NATURE OF THE INVENTION:

The subject-matter of the invention is, in the first aspect, a novel lupeol derivative which is ester of 4-picolinic acid and 20(29)-1υρεη-3β-ο1 presented on Formula (1 ).

The novel lupeol derivative is a result of modification of the structure of natural lupeol

(triterpene alcohol 20(29)-1υρεη-3β-ο1) that is contained in birch bark.

The ester of 4-picolinic acid and 20(29)-lupen-3 -ol which is the matter of the present invention, shown on Formula (1 ), is a novel compound. Neither that novel derivative nor the method for obtaining it has been described in literature yet.

Another aspect of the invention is a method of obtaining the novel lupeol derivative which is ester of 4-picolinic acid and 20(29)-Κιρβη-3β-ο1 presented on Formula (1 ). According to the invention, the method for obtaining the novel lupeol derivative which is ester of 4-picolinic acid and 20(29)-lupen-3p-ol presented on

Formula (1 )

consists in that lupeol, 20(29)-lupen-3P-ol, which is presented on Formula (2),

is esterificated with 4-picolinic acid chloride, presented on Formula (3),

or with 4-picolinic acid presented on Formula (4)

The esterification process is carried out in an organic solvent in the presence of a catalyst and pH regulators from amino groups.

Preferably, the esterification process of lupeol is carried out in such a manner that lg (2.3 mmol) of lupeol, presented on Formula (2) is dissolved in 5 - 7 cm 3 of THF (tetrahydrofuran). To the resulting solution NMM (N- methylmorpholine) is added in such a volume that guarantees pH of 9 - 12, preferably 10 - Π . Then 0.57 - 1 .15g (4.6 - 9.2 mmol) of 4-picolinic acid, presented on Formula

(4)

or 0.66g - 1.33g (4.6 - 9.2 mmol) of 4-picolinic acid chloride, presented on Formula (3)

and 0.53 - 0.55g (2.3 - 2.6 mmol) of DCC (N, N-dicyclohexylcarbodiimide), as catalyst, are added to the solution.. The reaction mixture is heated under a reflux condenser at the temperature of 40 - 80°C, preferably 50 -70°C. The reaction is carried out until creamy sediment precipitates from the reaction mixture. 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. The dried product is a raw material for cosmetic and pharmaceutical industry. Preferably, the lupeol, presented on Formula (2)

subjected to the esterification process is a natural plant-metabolite.

DETAILED DESCRIPTION OF THE INVENTION:

The new lupeol derivative, obtained with the method according to the invention— ester of 4-picolinic acid and 20(29)-lupen-3p-ol, which also may be named lupeol izonicotinate or lupeol 4-picolinate, and which graphic formula is Formula (1 ) and molecular formula is C36H53NO2, has a solid form (white powder) and it is hardly soluble in water, easily soluble in methyl alcohol, ethyl alcohol and organic solvents such as chloroform, methylene chloride, acetone, ethyl acetate, tetrahydrofuran. Its melting point is in the range 257 - 259°C.

The new lupeol derivative is characterized by experimentally confirmed biological activity. Its ability to proliferate (regenerate) epidermal cells and its antioxidant activity is higher than the activity of lupeol, that was demonstrated by comparative studies described below. The studies included dermatological estimation (EpiDerm™ SIT test) and estimation of the antioxidant activity.

It is known that contact irritation of skin, as a consequence of chemical action, is reversible and occurs shortly after exposure to irritation. In contact irritation characteristic, reversible inflammatory reaction occurs.

In order to compare the irritant potential of lupeol and its new derivative, which is the subject of the invention, the method of EpiDerm™ SIT (EPI-200) was used, in accordance with guidelines of the Organization for Economic Cooperation and Development (OECD 439) [OECD Guideline For The Testing Of Chemicals. Test No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method.2013].

The applied method is an in vitro method, based on a model of the reconstructed human epidermis (RhE), whose structure allows the reproduction of the biochemical and physiological properties of the outer layers of human skin, i.e. epidermis. In the study the epidermis model EpiDerm provided by American Company MatTek was used. The epidermis was cultured on a collagen matrix.

The test consisted on applying the solution of the examined compound onto the surface of the epidermis model. The solutions of lupeol and its new derivative were prepared in Crodamol GTCC (Croda, UK) oil base, and the concentration of each solution was 0.5% (m/m). The whole procedure of measuring the irritant potential was performed according to SOP (Standard Operation Protocol) ECVAM [I Vitro Skin Irritatio Test: Human Skin Model, EpiDerm-200, Version: 7.0, 30.10.2007]. After 42 hours of incubation, the cell viability was assessed with MTT colorimetric test. Simultaneously with the tests of the compounds (lupeol and its new derivative), a positive test was carried - PC (5% sodium dodecyl sulfate (SDS) solution in Crodamol GTCC oil base) and a negative test - NC, which was pure Crodamol GTCC oil base.

The tests of irritant potential showed that the new lupeol derivative according to the invention, similarly to lupeol, does not cause inflammation of the epidermis cells. Moreover, the new lupeol derivative showed the ability to induce cell proliferation at the level of 134.1%, which could not have been predicted earlier. In contrast, for pure lupeol this value was 89.2%.

The results of evaluation of the irritant potential of lupeol, the new derivative of lupeol (ester of 4-picolinic acid and 20(29)-lupen-3p-ol ), the negative test (NC) and the positive test (PC), which were performed with the EpiDerm method, are presented in Table 1. Table 1

The results of the study clearly indicate that the new lupeol derivative has a direct affect on stimulating skin cell division, and that its activity is of over 50% higher than the activity of the original compound, i.e. lupeol. Thanks to these properties, the new lupeol derivative is suitable for use in formulas which support wound healing and skin regeneration.

The antioxidant properties of lupeol and its new derivative were tested with the method of chemical reduction of l,l-diphenyl-2-picrylhydrazyl (DPPH) radical. The effectiveness of antioxidant activity according to this method is expressed by the ability to deactivate the DPPH radical. This method enables to determine the total antioxidant activity of the compound, expressed as a percentage of inhibition.

The tests have shown that the new lupeol derivative according to the invention demonstrated the activity of 27.7% in the process of deactivation DPPH radical, while the activity of lupeol was only 1.4%. The results of the total antioxidant activity of both compounds are presented in Table 2.

Table 2

Name Inhibition of the oxidation process [%]

Lupeol 1.40 ± 0.08

New lupeol derivative 27.66 ± 0.12 DESCRIPTION OF THE FIGURES IN THE DRAWINGS:

Fig.1 shows the UV/VIS spectrum in the range of 200 - 280nm of the ester obtained in example 1;

Fig.2 shows the complete 'HNMR spectrum of ester of 4-picolinic acid and 20(29)- 1υρεη-3β-ο1, obtained in the example 1 ;

Fig.3 shows the nuclear magnetic resonance spectrum "CNMR of the ester obtained in example 1 ;

Fig.4 shows the complete chromatogram of ester of 4-picolinic acid and 20(29)-lupen- 3β-ο1, [High Performance Liquid Chromatography (HPLC-MS)]— example 1 ;

Fig.5 shows mass chromatogram [High Performance Liquid Chromatography (HPLC- MS)]— example 1;

Fig.6 mass and intensity of the fragment ions [High Performance Liquid Chromatography with Mass Detector (HPLC-MS)]— example 1 ;

The detailed description of the process is illustrated by the following examples.

EXAMPLES: Example 1

lg (2.3 mmol) of lupeol was dissolved in 5.5 cm 3 of THF (tetrahydrofuran). Then 1 cm 3 of NMM (N-methylmorpholine) was added and resulting mixture had pH of 10. Next 1.1 g (9.2 mmol) of 4-picolinic acid, which is also known as isonicotinic acid, and 0.54 g (2.3 mmol) of DCC (Ν,Ν-dicyclohexylcarbodiimide) were added. The reaction mixture was heated under a reflux condenser at the temperature of 50°C. The degree of conversion was controlled with TLC method. The reaction was carried out until creamy sediment precipitated from the reaction mixture and then said sediment was filtered on a funnel with sintered disc and then washed with 45 cm 3 of distilled water. The resulting compound was dried at room temperature for 6 hours. The reaction yield was calculated to be 57.9%. For the obtained compound the melting point was determined and thin layer chromatography analysis (TLC) was performed.

The structure of the obtained ester of 4-picolinic acid and 20(29)-Κιρεη-3β-ο1, was characterized with the following methods: UV VIS spectrophotometry; nuclear magnetic resonance ( MR), and elemental analysis (C, H and N content). The purity of the compound was determined with high performance liquid chromatography with mass spectroscopy method (HPLG - MS).

The melting point of the obtained crystals of ester of 4-picolinic acid and 20(2 )-lupen-3P-ol, was determined with a Stuart SMP10 apparatus.

The retention factor of the compound was determined with the thin layer chromatography method (TLC) using Macherey Nagel's Polygram SIL G UV254 plates. As an eluent the mixture of ethyl acetate and chloroform in the volume ratio of 1 :9 was used.

The first technique used to determine the identity of the compound was UV/VIS spectrophotometry. The UV/VlS spectra were made on Macherey Nagel's Nanpcolor UV/VIS spectrophotometer in ethyl alcohol solution. The wavelength range was 200 - 280 nm, the optical path length was 10 mm.

Another applied spectroscopic technique was nuclear magnetic resonance (NMR). The spectra 1HNMR and 13 CNMR were performed with an apparatus Mercury- VX, Varian, frequency 300 MHz, magnetic field induction 7.02 T, in CDC1 3 solution. Values of chemical shifts are given in ppm.

Elemental analysis of C, H and N content was made with an apparatus Perkin Elmer Type 2400.

The purity of ester of 4-picolinic acid and 20(29)-lupen-3p-ol was determined with a liquid chromatograph equipped with a mass analyzer (HPLC-MS). The HPLC- MS analysis was performed with Agilent Technologies 1 100 series apparatus, using Supelco column, Ascentis Express ® RP- Amide, 7,5cm x 2, 1mm x 2,7μιη. The parameters used for HPLC analysis were: injection volume 2 μΐ, analysis , time 25 minutes, mobile phase, acetonitrile (ACN) water mixture, gradient elution: 80-98-80% ACN (v/v), column temperature 40°C, mobile phase flow 0.5 cmVmin, DAD and MSD detectors, wavelength: 280.8 205.5 220.5 240.8 nm. Mass analyzer (MS) operated at the following parameters: APCI ion source, positive polarity, mass range 300 - 450, fragmentor 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, current density 5.0μΑ(+), 15μΑ(-).

Examination of purity and determination of the structure and properties of the new lupeol derivative obtained according to the procedure described in Example 1

Thin-layer chromatography (TLO

The TLC image allowed to determine the overall purity of the obtained compound, and also to determine its retention factor Rf (Retardation factor). The coefficient is one of the values that characterize organic compounds in a given eluent system.

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

Melting point

The melting point of the obtained compound was determined.

T , = 257 - 259°C - The narrow range of melting point indicates the high purity of the resulting compound. UV/VIS spectrophotometry:

The spectrophotometric measurement was performed with UV/VIS method for ethanol solution of lupeol isonicotinate with a concentration of 0,001 mmol/dm 3 . The attached drawing (Figure 1) shows the UV/VIS spectrum of the tested ester in-the range of 200 - 280nm.

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

The spectrum clearly shows a maximum absorbance at a wavelength of λ ηιαχ . = 209.8 nm

1H-NMR Spectrum

Spectroscopic studies performed with nuclear magnetic resonance (NMR) method have also confirmed the structure of the obtained ester of 4-picolinic acid and 20(29)-lupen- 3β-ο1.

The accompanying drawing (Figure 2) shows the complete l HNMR spectrum of ester of 4-picplinic acid and 20(29)-1υρεη-3β-ο1 obtained in the example 1.

Ή-NMR spectrum description (Figure 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, n-9), 1.46 (d, 1H, H-l l ), 1.40 (m, 4H, H-10, 4), 1.32(m, 4H, H-8,13), l . I6(m, 4H, Η- 6, 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) at 9.00 ppm and 8.37 ppm.

There was also observed a slight increase of chemical shift in relation to the spectrum of lupeol, due to a neighbouring aromatic ring and a nitrogen atom.

1 CNMR spectrum

Also the 1 CNMR spectra were made.

The accompanying drawing (Figure 3) shows the nuclear magnetic resonance spectrum

B C MR of the obtained ester.

The description of n CNM spectrum (Figure 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-23 ) 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(C-7) 17.97(C-28) 16.15(C-22) ] 5.96(C-26) 15.38(C-23) 14.50(C-24).

The 13 CNMR spectrum also confirms the structure of ester of 4-picolinic acid and 20(29)-lupen-3p-ol. The amount of the signals from the nuclei of carbon n C corresponds to the molecular structure (36 carbon atoms in a molecule). In the accompanying spectrum (Fig.3) singlets can be seen at 144.85 ppm, 143 59 ppm, 126.02 ppm, 85 07 ppm and 38.21 ppm coming from the 13 C nuclei of the aromatic ring. Such signals are not observed in the spectrum of lupeol. In addition, the signal at 161.83 ppm corresponds to the carbon atom of the ester group (-0-C = O).

Elemental Analysis (CHN)

In order to determine the contents of individual elements, a qualitative analysis determining the contents of C, H and N was performed,. The mass content of each element is shown below:

Theoretical values: C = 81.30%; H = 10.04%; N = 2.63% (m/m). Experimental values. C = 80.72 +/- 0.02%; H = 10.08 +/- 0.04%; N = 2.69 +/- 0.06% (m/m).

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

Molecular formula of the compound is C 36 H 53 N0 2 .

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 complete chromatogram of ester of 4-picolinic acid and 20(29)-1υρβη-3β-ο1 is shown on Figure 4, the mass chromatogram is shown on Figure 5 and the mass and intensity of the fragment ions on Figure 6.

Example 2

lg (2.3 mmol) of lupeol was dissolved in 6 ml of THF (tetrahydrofuran). Then 1 cm 3 of MM (N-methylmorpholine) was added and the resulting mixture had pH of 10.5. Then, 1.3g (9.2 mmol) of 4-picolinic acid chloride, which is also known as isonicotinic acid chloride, and 0.54 g (2.3 mmol) of DCC (Ν,Ν-dicyclohexylcarbodiimide) were added. The reaction mixture was heated under a reflux condenser at the temperature of 50°C. The degree of conversion was controlled with TLC method. The reaction was carried out until creamy sediment precipietated from the reaction mixture and then said sediment was filtered on funnel with sintered disc and then washed with 50 cm 3 of distilled water. The resulting compound was dried at room temperature for 8 hours. The reaction yield was calculated to be 57.6%. The results of analyzes confirming the structure of the obtained compound were, within the measurement error range, the same as in Example 1.

The performed studies made it possible to confirm that the above - mentioned method of esterification of lupeol, which is the matter of the present invention, leads to obtaining the novel compound, that is not described in literature yet - ester of4- picolinic acid and 20(29)-lupen-3p-ol, according to Formula (1).

The results of the analysis for the new lupeol derivative (ester of 4-picolinic acid and 20(29)-1υρεη-3β-ο1) obtained with isonicotinic acid chloride or with isonicotinic acid do not differ from each other and describe the same structure. The kind of the reagent does not affect the quality and purity of the obtained ester.

FAVORABLE EFFECTS OF THE INVENTION: The new lupeol derivative has proliferative affects on human skin cells with no toxic or irritating effects. In addition, it acts as an antioxidant and thanks to these properties it may be a component of skin care preparations and medicinal preparations for damaged skin or for a skin which requires quick regeneration.

The invention enables to obtain the active ingredient (ester of 4-picolinic acid and 20(29)-Κφβη-3β-ο1) which is a semi-synthetic, new derivative of lupeol - a natural plant-metabolite, which may be used in cosmetic and pharmaceutical formulations: Moreover, a particularly advantageous feature of the solution according to the invention is that the new lupeol derivative is obtained in ecologically safe conditions, i.e without using toxic solvents and catalysts. The DCC (N, N-dicyclohexylcarbodiimide) used in the process of the present invention, which has a catalytic function, is significantly less toxic than known from literature DMAP (4-N,N-dimethylaminopyridine), as far as the acting on skin and oral administration are concerned.

Toxic compounds such as methylene chloride, chloroform, pyridine, tributylamine, DMAP, hexane or ethyl acetate, which are present in esterification of triterpene alcohols described in literature [J. Achrem-Achremowicz, semi-synthetic derivative of betulin cytotoxicity, PhD Dissertation, Department of Pharmacognosy, Faculty of Pharmacy, Jagiellonian University, Krakow, 2007; M. Kvasnica, J . Sarek, E. Klinotova, P. Dzubak, M. Hajduch, Bioorg ά Med Chem. 2005, 13 (10), 3447], are eliminated from the esterification process, because in the method of the present invention the esterification of the lupeopl is carried out directly with 4-picolinic (isonicotinic acid) acid or with 4-picolinic acid chloride (isonicotinic acid chloride) in an organic solvent such as THF and using NMM (N-methylmorpholine) as the pH regulator.

INDUSTRIAL APPLICABILITY:

The above examples proved that both the novel lupeol derivative, as well as the method of obtaining the novel lupeol derivative according to this invention create the possibility of their industrial application.




 
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