Betulin, betulonic acid, 3-aminobetulinic acid. Improved extraction and preparative syntheses of derivatives

We optimized the process of extraction and purification of betulin from birch bark and modified preparative syntheses of the simplest derivatives of betulin, namely, betulonic acid, betulonic aldehyde, and 3-aminobetulinic acid, as a platform for subsequent modifications.

Plant pentacyclic triterpenoids have a variety of biological activities: antitumor, antimicrobial, antiinfl ammatory, antiviral, hepatoprotective, etc. 1-5 These compounds are widely distributed in nature, in particular, they are found in the bark of white birch. Among triterpenoids, betulin (1), its derivatives, such as betulonic (2) and betulinic acids, betulonic aldehyde (3), as well as their modifi ed analogs and so-called hybrid compounds, attract close attention. 6-34 Relatively recently, after spread of SARS in 2003-2005, triterpenoids has been found to possess anti-coronavirus activity, 6 which makes them very promising platforms for drug development, especially in connection with SARS-CoV-2 infection (see, for example, the review 17 ).
We proposed an improved method for the isolation of betulin from natural raw materials and its purifi cation, as well as modifi ed methods for the synthesis of its simplest key derivatives, betulonic acid, betulonic anhydride, and 3-aminobetulinic acid (Scheme 1). The main attention was paid to the improvement of synthetic approaches so that they become more effi cient and feasible for implementation in the scientifi c laboratory.

Results and Discussion
Betulin (3,28-dihydroxy-20(29)-lupene, 1) (see Scheme 1) is a pentacyclic triterpene alcohol with a lupane skeleton. Betulin 1 is contained mainly in the outer part of the birch bark (up to 30-40%), giving it a characteristic white color and protecting the wood from the penetration of various microorganisms and insects, as well as refl ecting sunlight. The main method for isolation of betulin is extraction from natural raw materials. 19,20 To intensify the process, it was proposed to use extraction under microwave irradiation, 21 thinfi lm vapor-phase extraction, 22 as well as steam treatment of the feedstock under conditions of "explosive autohydrolysis". 23 An essential problem of the known methods is the contamination by compounds of the lupane series (lupeol, betulinic and betulonic acids).
In the present work, we propose an improved method for isolation and purifi cation of betulin 1 from ground and preliminarily dried birch bark. For the isolation of betulin 1, we used the extraction of plant raw materials with boiling aqueous propan-2-ol for 3 h. The degree of birch bark grinding signifi cantly aff ects the yield of betulin isolated by extraction, 9 therefore, the birch bark was ground to a particle size of 3×3 mm 2 .
The fi rst stage of the proposed method for purifi cation of betulin 1 was the treatment of the extract with 20% aqueous NaOH, which allowed us to remove impurities of betulinic and betulonic acids as their soluble Na salts. Betulin 1 itself does not react with alkalis. Next, resinous, slightly colored impurities were removed using silica gel chromatography. At the last stage, the impurities of lupeol and other compounds soluble in hexane were removed by extraction with boiling hexane. Such a purifi cation gave chemically pure betulin 1. The 1 Н NMR spectrum of betulin 1 exhibits singlets for six methyl groups and multiplets for cycloalkyl CH and CH 2 groups in the region of  0.7-2.0. The signals for other protons are found in the region of  2.0-4.7 and fully correspond to the structure of betulin 1.
The proposed method for the isolation of betulin 1 does not require its purifi cation by recrystallization from alcohols and the use of highly toxic benzene, instead of which we used hexane. The proposed method is convenient for scaling and application under laboratory conditions.
Betulin 1 was oxidized to betulonic acid 2 with a mixture of potassium bichromate and sulfuric acid supported on alumina in acetone at 0 С for 2 h (see Scheme 1) according to a modifi ed known procedure. 11 The use of Al 2 O 3 as a substrate allowed us to selectively oxidize betulin 1 to betulonic acid 2. In contrast to the procedure described in the work, 11 we did not use ultrasonic dispersion of betulin in acetone and changed the order of mixing the components. The 1 H and 13 C NMR spectra, as well as the IR spectrum of betulonic acid 2 confi rm its structure. The oxidation of the primary hydroxy group at atom C(28) of betulin 1 to the carboxy group is evidenced by the absence of signals for the C(28)H 2 protons at  3.36 and 3.80 in the 1 H NMR spectrum of acid 2, which are observed in the 1 H NMR spectrum of betulin 1. The 13 C NMR spectrum of compound 2 exhibits two downfi eld signals at  218.35 (С(3)) and 182.61 (С(28)), confi rming the oxidation of secondary and primary hydroxy groups. In addition, the presence in the IR spectrum of compound 2 of a strong absorption band in the region of 1705 cm -1 related to the stretching vibrations of carbonyl groups and the absence of a broad absorption band of stretching vibrations of hydroxy groups in the region of 3390 cm -1 , which is present in the IR spectrum of betulin 1, also confi rm the formation of betulonic acid 2.
For the oxidation of the hydroxy groups of betulin 1 at atoms C(28) and C(3) to the aldehyde and carbonyl groups, respectively, we used a selective oxidizing agent, pyridinium chlorochromate (PCC). The reaction was carried out according to a modifi ed known procedure 12 in anhydrous dichloromethane at room temperature for 2 h (see Scheme 1). To increase the conversion of betulin, we used an excess of PCC, which made it possible to increase the yield of betulonic aldehyde 3 to 93% and eliminate purifi cation of the product by recrystallization from hexane. The appearance in the 1 H NMR spectrum of aldehyde 3 of the singlet at  9.69 reliably confi rms the presence of an aldehyde group. In the 13 С NMR spectrum of aldehyde 3, the signal for the С(28) atom is observed in a lower fi eld ( 206.53) than the corresponding signal for acid 2 ( 182.61). Very strong absorption corresponding to the stretching vibrations of the C=O bond in the IR spectrum of aldehyde 3, like in the case of betulonic acid 2, is observed at 1705 cm -1 . It should be noted that in the 1 H NMR spectra of betulin 1, betulonic acid 2, and betulonic aldehyde 3, the C(29)H 2 methylene protons of the isopropenyl group resonate as two singlets at  4.60-4.65 and 4.70-4.78. In addition, the presence of the signals for atoms C(29) ( 109) and C(20) ( 150) in the 13 C NMR spectra of compounds 1, 2, and 3 also indicates that the transformations we carried out do not aff ect the double bond.
3-Aminobetulinic acid 4 was obtained from betulonic acid 2 by treatment with a solution of ammonia in methanol in the presence of ammonium acetate, followed by reduction with sodium borohydride (see Scheme 1). To remove the unreacted betulonic acid 2 as its soluble sodium salt, the crude product was treated with 1 M NaOH. Note that 3-aminobetulinic acid 4 does not form Na salt under these conditions. This method makes it possible to obtain 3-aminobetulinic acid from commercially available reagents in good yield. In this version of the synthesis, the reaction proceeds via an intermediate formation of a Schiff base, while the literature sources 28, 34 show obtaining of compound 4 via an intermediate oxime.
The 1 Н NMR spectrum of compound 4 exhibits a signal for the C(3)H proton at  3.13, which is absent in the spectrum of the starting betulonic acid 2. In addition, two singlets for the protons of the C(29)H 2 group are present at  4.55 and 4.68 in the 1 H NMR spectrum of aminobetulinic acid 4, like in the spectrum of the starting betulonic acid 2. The signal for carbon atom C(3), which in the 13 С NMR spectrum of betulonic acid 2 is observed at  218.35, in the case of 3-aminobetulinic acid 4 is signifi cantly shifted to the upfi eld region to  77.90, confi rming the formation of the C-N bond. At the same time, the chemical shifts for carbon atom C(28) of betulonic acid 2 ( 182.61) and 3-aminobetulinic acid 4 ( 181.90) are very close, which confi rms the presence of the carboxy group.
In conclusion, in the present work we proposed an improved method for the isolation and purifi cation of betulin from birch bark, which does not require the use of special equipment and can be implemented under laboratory conditions. Betulin was converted to betulonic acid, betulonic aldehyde, and 3-aminobetulinic acid in good yields, which were characterized by spectral methods.
In the future, we plan to synthesize hybrid betulinferrocene structures and study their pharmacological activity. Experimental 1 Н and 13 С NMR spectra were recorded on a Bruker Avance 400 instrument (400.13 and 100.925 MHz, respectively). Chemical shifts are given relative to the residual signals of protons and carbon atoms of the solvent. IR spectra were recorded on a Bruker TEM37 IR Fourier-transform spectrophotometer. Specifi c rotation was determined on a P3000 polarimeter (A.KRÜSS Optronic). Melting points were measured on a Stuart SMP30 apparatus. Solvent were purifi ed using standard procedures. Reaction progress was monitored by TLC. Dichloromethane, methanol, diethyl ether, hexane, and propan-2-ol (Acros Organics) were dehydrated before use. Ammonia was used as a 7 N solution in methanol (Acros Organics). The birch bark was collected in the Bitsevsky forest park in Moscow.
Lup-20(29)-ene-3,28-diol (betulin, 1). A mixture of airdried and ground birch bark (50 g, particle size 3×3 mm 2 ) and an azeotropic mixture of propan-2-ol-water (9 : 1, 300 mL) was refl uxed for 3 h with stirring and fi ltered, the fi ltrate was kept for 24 h in a freezer (-18 С). The white fl occulent precipitate was collected by fi ltration and washed with propan-2-ol. Then, it was dissolved in chloroform, washed with 20% aqueous NaOH and brine. The resulting solution was passed through a short layer of silica gel, eluting with a 9 : 1 mixture of chloroform-methanol. The solvent was vacuum evaporated to dryness on a rotary evaporator, the residue was diluted with hexane and refl uxed for 1 h, the precipitate was collected by fi ltration to obtain compound 1 (5.6 g, 37% based on the 30% content of betulin in birch bark), a white crystalline substance. M.p. 251 С (cf. Ref Lup-20(29)-en-3-oxo-28-oic acid (betulonic acid, 2). A mixture of K 2 Cr 2 O 7 (3 g, 10 mmol), H 2 SO 4 (5 mL), and water (7 mL) was added to a solution of betulin 1 (1.50 g, 3.38 mmol) and Al 2 O 3 (9.2 g) in acetone (150 mL). The mixture was stirred for 2 h at 0 С, the alumina was fi ltered off . Water (1 L) was added to the fi ltrate, and the resulting white fl occulent precipitate was collected by fi ltration and dried. Then, the precipitate was dissolved in benzene and the undissolved, unreacted betulin 1 was fi ltered off . A 10% aqueous NaOH (30 mL) was added to the fi ltrate, the resulting precipitate of the sodium salt was collected by fi ltration and dried. After its dissolution in methanol, a 15% aqueous HCl was added dropwise to the resulting solution until pH 1 was reached. The precipitate formed was collected by fi ltration, washed with water, and dried to obtain acid 2 ( Lup-20(29)-en-3-on-28-al 12 (betulonic aldehyde, 3). A mixture of betulin 1 (0.40 g, 0.9 mmol), dichloromethane (7 mL), and pyridinium chlorochromate (0.645 g, 3.0 mmol) was stirred for 2 h at room temperature, followed by the addition of diethyl ether (7 mL). The product was isolated by column chromatography on alumina, eluent diethyl ether. No human or animal subjects were used in this research.
The authors declare no competing interests.