Isolation of high-purity friedelin and 3-hydroxyfriedel-3-en-2-one from cork smoker wash solids

Abstract Cork smoker wash solids (black wax) that is obtained in the process of making corkboard by treating the ground cork with superheated steam is useless waste, generating environmental problems. On the other hand, black wax is a rich source of friedelin (1) and 3-hydroxyfriedel-3-en-2-one (3), pentacyclic triterpenoids belonging to the friedelane family. In the present work, a simple, fast, and efficient procedure for the extraction of highly purified friedelin and 3-hydroxyfriedel-3-en-2-one from black wax is presented. Extraction of black wax with hexane or acetone afforded a mixture of friedelin and 3-hydroxyfriedel-3-en-2-one. However, chromatographic separation of 1 and 3, due to their very similar Rf coefficients, is difficult, especially on a multigram scale. Therefore, the conversion of 3-hydroxyfriedel-3-en-2-one to an acetyl derivative 4 was proposed by acetylation of the above mixture. Appropriate selection of the eluent sequence allows for rapid and efficient separation of the resulting mixture of 1 and 4.


Introduction
Natural products and their derivatives play an important role in drug discovery and development. [1][2][3] They are also sustainable starting materials in other industrial sectors, including food, agriculture, chemistry, etc. Special attention should be paid to compounds that can be isolated from waste products obtained during the commercial processing of plant material. For example, triterpenoids, compounds widely distributed in plants, are very promising sources of new potentially highly active therapeutic agents. [4] During the past decade, our group has studied the chemistry and biological properties of betulina pentacyclic triterpenoid isolated with impressive yield (20-30% w/ w) from the outer part of birch barkand its derivatives. [5][6][7] Similarly, the bark of cork oak, Quercus suber L. (Fagaceae) is a rich source of another pentacyclic triterpenoid friedelin (1, Scheme 1). Friedelin and its derivatives appear to be of interest, as a broad spectrum of their biological activities have been demonstrated. [8][9][10] Cork oak is a native tree in the Mediterranean area, and its bark plays an important role in the wine, construction, and furniture industries. The main components of the triterpene fraction of cork extractives are friedelin (1) and cerin (2) isolated in approximately 1.5% of the total yield. [11,12] However, the very low density of ground cork makes large-scale extraction of these triterpenoids difficult. On the other hand, the cork processing industry generates large amounts (approximately 2500 tons/year) of cork smoker wash solids, also known as black wax, obtained during the corkboard manufacturing process by treating ground cork with the superheated steam. [13] It is removed from the steam exhaust ducts as useless waste, which poses environmental problems and is usually burnt to generate energy. Black wax is a convenient source of friedelin (1, up to 9.5%, w/w) and 3-hydroxyfriedel-3en-2-one (3, up to 6%, w/w). [10,14,15] The first isolation of friedelin from black wax was described in 1956 by Corey and Ursprung [16] , who obtained it by treating black wax with acetone, column chromatography of the acetone-insoluble residue, and crystallization of the crude product. "Material which was satisfactory for most purposes" was isolated in ca. 20% yield, suggesting rather low purity of the prepared friedelin. Further recrystallization of the crude product afforded pure friedelin, but the yield was not given. Kane and Stevenson isolated friedelin and friedelane-2,3-dione by extracting black wax with ethanol, both in low yields. [17] Stevenson proposed the purification of friedelin obtained from black wax by its oxime. [18] The pretreatment of black wax with aqueous sodium hydroxide, followed by extraction with organic solvents, and repeated crystallization, was also used to isolate friedelin of high purity. However, the overall yield of high-purity friedelin was low (usually below 3%). [19][20][21] While the removal of most impurities and compounds belonging to other chemical families is relatively easy by extracting black wax with suitable solvents, the separation of friedelin (1) and 3-hydroxyfriedel-3-en-2-one (3) is challenging. Both compounds crystallize from the same solvents and their solubilities are similar. Furthermore, their chromatographic mobility is virtually identical in most eluents, with minor differences in the R f coefficients in the dichloromethane-hexane system. This solvent mixture was used by Moiteiro et al. for the chromatographic separation of 1 and 3. [14] However, due to the marginal difference in the R f coefficients, this is an exhaustive procedure.
Herein, we present an alternative simple procedure for the isolation of high-purity friedelin from black wax, with virtually no loss of this valuable ingredient. In addition, high-purity 3-hydroxyfriedel-3-en-2-one, usually lost during the separation of friedelin, is also obtained.

Results and discussion
The first step of friedelin isolation consisted of extracting the required components from the cork smoker wash solids with hexane or acetone. TLC analysis showed that extraction with hexane in a Soxhlet apparatus gave a complex extract. However, most impurities (the b-sitosterol fraction, as well as other unidentified compounds) could be easily removed by washing with acetone, from which an insoluble mixture of 1 and 3 was isolated by filtration. Alternatively, precipitation of the insoluble mixture of 1 and 3 during extraction of black wax with acetone gave only slightly contaminated products. In both cases, the resulting crude product contained only traces of tarry impurities. TLC analysis of the residues indicated that virtually all friedelin and 3-hydroxyfriedel-3-en-2-one were extracted from black wax in both cases.
In the next step, the crude mixture of products was acetylated under standard conditions (Ac 2 O, Et 3 N) providing a mixture of friedelin (1) and 3-acetoxyfriedel-3-en-2-one (4). Chromatographic separation of both compounds was possible, although the careful selection of eluents was required. For example, chromatographic separation by eluting with a mixture of hexane and ethyl acetate was poor despite significant differences in the R f coefficients of 1 and 4. In comparison, pure friedelin was eluted as the first product with a mixture of dichloromethane and hexane, then compound 4 was eluted as the second product with a mixture of hexane, ethyl acetate, and methanol. Trace amounts of tar impurities still present in both fractions were removed by crystallization from ethyl acetate.
In this procedure, high-purity friedelin (1) was isolated in 6.5% yield (based on black wax) together with 3-acetoxyfriedel-3-en-2-one (4) which was obtained in 2.7% yield (based on black wax and recalculated to the deacetylated product 3). The amounts of separated products depend on the batch of cork smoker wash solids and can vary considerably. According to elemental analysis, the purity of both products was greater than 95%.
We then attempted to deacetylate compound 4 by treating it with potassium carbonate in methanol. However, two products: the expected compound 3 and the more polar product 5 were obtained in an approx. 2:1 ratio. In the mass spectra of 5, the most important fragments were found at m/z 509, corresponding to [M þ Na] þ , and at m/z 995, associated with [2M þ Na] þ . In the 1 H NMR spectra, two singlets were observed at d ¼ 3.12 ppm and d ¼ 3.31 ppm (three protons each) whose positions corresponded to protons of the -OCH 3 groups. Two characteristic signals were recorded in the 13 C NMR; the first at d ¼ 207.7 ppm for the carbonyl group and the second at d ¼ 100.6 ppm, which may be related to the acetal function. Therefore, we supposed that in this reaction, dimethylacetal 5 was formed as a byproduct (Scheme 1). This assumption was confirmed by the HMBC and NOE correlations (Figure 1). The observed reaction is an example of extremely rare transformation in which an acetal is formed from the a-ketoenol derivative upon treatment with methanol in the presence of base. [22] To avoid the formation of acetal 5, in the next attempt to compound 3, we carried out the reaction under mild conditions (30% H 2 O 2 , NaHCO 3 ), previously used for the deacetylation of fragile taxol derivatives. [23] In this case, the required 3-hydroxyfriedel-3en-2-one (3) was isolated as the only product in excellent yield (91%).
Compound 4 may be considered as the convenient starting material for the synthesis of derivatives of 3hydroxyfriedel-2-one. We have shown that hydrogenation of 4 selectively afforded compound 6 (Scheme 1), in which the 3-hydroxy and 23-methyl groups are in a cis relationship. The structure of isomer 6 was confirmed by the NOE correlations observed between protons at positions H-3 and H-4 and the apical 24methyl group, as shown in Figure 1. Steric interactions on the a side of the friedelan core are probably responsible for the high selectivity of the hydrogenation reaction (Figure 1). 3a-Hydroxyfriedel-2-one (7), having 3-hydroxy and 23-methyl groups in the trans relation (Figure 2), was isolated from plant material [24][25][26] ; its synthesis and biological properties were studied. [10,14,27] The synthesis of its unnatural (3a,4a)-isomer (obtained as acetate 6) opens the way for further studies of the biological activity of these triterpenoids.

Conclusions
Here, we describe a simple, fast, and efficient procedure for the extraction of highly purified friedelin (1) and 3-hydroxyfriedel-3-en-2-one (3) from cork smoker wash solids (black wax) on a multigram scale. The required compounds were isolated in 6.5% and 2. 7% yields (based on raw material), respectively. It should be remembered that the yield can vary considerably depending on the batch of raw material used. Due to the similar physicochemical properties of these compounds, their efficient separation into high-purity components is challenging. The procedures described in the literature are based on multiple crystallization, Scheme 1. Natural fridelane-type triterpenoids isolated from cork (1 and 2) and cork smoker wash solids (1 and 3 which significantly reduces the efficiency of the process, and are focused on separating only friedelin. The proposed method is based on the acetylation of a mixture of 1 and 3 leading to a mixture of friedelin (1) and acetate 4, and their chromatographic separation. A simple method was also developed for the deacetylation of sensitive a-ketoenol 4 to recover the unprotected compound 3 in high yield. To our knowledge, this is currently the most efficient process for the isolation of high-purity friedelin and the only procedure dedicated to isolating pure 3-hydroxyfriedel-3-en-2one (3) from by-products of the cork industry. Virtually all friedelin and 3-hydroxyfriedel-3-en-2-one are isolated from black wax. We also showed that acetate 4 can be easily transformed by stereoselective hydrogenation into triterpene 6, starting material for the synthesis of derivatives of the unnatural isomer of 3-hydroxyfriedel-2-one.

Materials and methods
General experimental procedures. Silica gel HF 254 and Silica gel 230-400 mesh (E. Merck) were used for TLC and column chromatography, respectively. 1 H and 13 Cf 1 Hg NMR spectra were recorded at 298 K with Varian NMR-vnmrs600 or vnmrs500 spectrometers, using standard experimental conditions and Varian software (ChemPack 4.1). Internal TMS was used as the 1 H and 13 C NMR chemical shift standard. High-resolution mass spectra (HRMS ESI) were acquired with Mariner and MaldiSYNAPT G2-S HDMS (Waters) mass spectrometers. Optical rotations were measured with a Jasco P-2000 automatic polarimeter, and IR spectra were measured with a Jasco FT/IR-6200 and Shimadzu IRTracer-100. Melting points were measured with the OptiMelt MPA100 automated melting point system and were not corrected. Most of the solvents were recovered and reused.

Isolation of friedelin (1) and 3-hydroxyfriedel-3-en-2-one (3) from black wax
Method A. The powdered cork smoker wash solids (505 g) were extracted in a Soxhlet apparatus with hexane for 5 h. The extract was evaporated to dryness in vacuum, the remaining solids were suspended in refluxing acetone (2.5 L) and kept at room temperature overnight. The solid was collected, washed with acetone, and dried under reduced pressure.
Method B. The powdered cork smoker wash solids (420 g) were slowly added to acetone (3.5 L) and stirred until the particles were completely dissolved. The precipitated solid was washed four times with acetone (2 L each) by decantation. Then it was filtered, washed with acetone until an almost colorless filtrate was obtained, and dried under reduced pressure.
In both cases, the light brown solid obtained was dissolved in dichloromethane and filtered to remove insoluble contaminants. The concentration of the filtrate under reduced pressure produced a crude mixture (55.40 g in Method A and 45.40 g in Method B) of friedelin (1) and 3-hydroxyfriedel-3-en-2-one (3).

Acetylation of friedelin (1) and 3-hydroxyfriedel-3en-2-one (3) mixture
A crude mixture (55.40 g) of friedelin (1) and 3hydroxyfriedel-3-en-2-one (3) was dissolved in dichloromethane (CH 2 Cl 2 , 500 mL) and treated with acetic anhydride (Ac 2 O, 15 mL) and triethylamine (Et 3 N, 35 mL) for 48 h. Then the organic layer was  washed twice with water (100 mL each), and the organic solvents were evaporated under reduced pressure. The remaining solid was suspended in refluxing ethyl acetate (300 mL) and kept overnight at room temperature. The crystals were collected, washed with ethyl acetate until an almost colorless filtrate was obtained, and dried under reduced pressure to afford a crude mixture of friedelin (1) and 3-acetoxyfriedel-3-en-2-one (4). The mixture was dissolved in a minimum volume of CH 2 Cl 2 and subjected to a chromatographic column using at least 180 g of silica gel for every 25 g of product. Friedelin (1) was eluted with a mixture of CH 2 Cl 2 hexane (5:2) as eluent. When all friedelin was removed from the silica gel, 3-acetoxyfriedel-3-en-2one (4) was eluted with hexane-ethyl acetate-methanol mixture (5:3:0.5). Both products were crystallized from ethyl acetate to obtain 31.20 g (6.2%) of 1 as white crystals and 12.52 g (2.3%, based on the deacetylated product) of 4 as pale beige crystals.
Additional yield could be obtained from the concentration of the mother liquors. All organic filtrates after acetylation and crystallization of the acetylated product were collected and evaporated to dryness. Tarry contaminants were removed by column chromatography (hexane-ethyl acetate, 5:1) leaving a mixture of 1 and 4, which was separated into individual components as described above to provide additional batches of 1 (1.86 g, 0.3%) and 4 (2.15 g, 0.4%). All yields were calculated in relation to the black wax.