Selective transformations of friedelanes isolated from cork smoker wash solids

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Introduction
Natural products and their derivatives, which are often utilized in traditional medicine, play an important role in drug discovery and development [1,2].Due to the usually complex structure of natural compounds and the low-cost, sustainable sources of such compounds are highly desirable.In an ideal scenario, these compounds should be isolated from waste products generated during commercial processing of plant material.Triterpenoids, which are widely distributed in plants, represent highly promising starting materials for the synthesis of active therapeutic agents [3].An extremely rich source of triterpenoids is birch bark, a by-product of the paper industry.Birch bark contains large quantities of pentacyclic triterpenoids belonging to the lupane family, which may comprise up to 20-30% of its weight [4].Both, natural lupanes and their derivatives, have interesting biological properties [5][6][7][8][9][10].
An equally interesting source of natural triterpenoids is the bark of cork oak (cork), Quercus suber, a tree native to the Mediterranean area.Cork is obtained by environmentally friendly and sustainable methods and plays an important role in the wine, construction and furniture industries.The extraction of cork yields two pentacyclic triterpenoids of the friedelane-typefriedelin (1) and cerin (2) -which account for approximately 1.5% of the total bark weight (Fig. 1) [11,12].With this procedure, high-purity friedelin is available, but the very low density of ground cork makes large-scale extraction difficult.However, low quality cork or the fine pieces produced during industrial processing can be agglomerated by superheated steam treatment, resulting in a material known as corkboard, which is highly valued in ecological construction and insulation.This process generates large amounts of cork smoker wash solids, also known as black wax.Approximately 2500 tons of this byproduct is produced annually and is removed from the steam exhaust ducts as waste [13].Black wax has been identified as a promising source of friedelin (1, up to 9.5%, w/w) and 3-hydroxyfriedel-3-en-2-one (3, up to 6%, w/w) [14][15][16].Although isolation of both triterpenoids in the crude state by extraction of black wax with organic solvents is extremely simple, considerable losses are incurred during their separation and purification.Recently, we have shown that these triterpenoids can be efficiently obtained in their pure form without wasting raw materials.Acetylation of the crude mixture of 1 and 3 gives a mixture of unmodified friedelin and acetate 4.This mixture can be effectively separated on a multi-gram scale [17].
Owing to the wide spectrum of biological activities, friedelin and its derivatives are of significant interest [13,[18][19][20][21].They can provide a pathway for therapeutic development while utilizing industrial waste products.Despite the availability of friedelin, its chemical transformations remain relatively poorly understood compared to other pentacyclic triterpenoids, and studies investigating the structure-activity relationships are scarce.In our search for new facile transformations of the friedelin skeleton, herein, we are focusing on the selective functionalisations of friedelane-type triterpenoids 1 and 4, primarily within the A-ring.The presented methodology offers the simplest and shortest way to friedelanes containing diol or α-hydroxyketone functionalities bound to the A ring.

Experimental section
General methods.Silica gel HF 254 and Silica gel 230-400 mesh (E.Merck) were used for TLC and column chromatography, respectively.All eluent and solvent system compositions are given in volume percent (v/ v%). 1 H and 13 C{ 1 H} NMR spectra were recorded at 298 K with Varian NMR-vnmrs600 or vnmrs500 spectrometers, using standard experimental conditions and Varian software (ChemPack 4.1).Configurational assignments were based on the NMR measurements generated using two-dimensional techniques like COSY and 1 H- 13 C gradient selected HSQC (g-HSQC), as well as 1 H-13 C gradient selected HMBC (g-HMBC).Internal TMS was used as the 1 H and 13 C NMR chemical shift standard.J values are given in Hertz.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 melting points with OptiMelt MPA 100.Reaction conditions and yields were not optimised.
The crystals of 9 were obtained by slow diffusion of methanol to the dichloromethane solution at room temperature.The crystals of 15 were prepared in a mixture of hexane-ethyl acetate (5:1) by slow evaporation at room temperature.Single crystals of 9 (deposition number CCDC 2247865) and epi-cerin (15, deposition number CCDC 2247862) were selected and mounted on a Rigaku SuperNova diffractometer.The crystals were kept at 100.00 (10) K during data collection.Using Olex2 [22], the structures were solved with the SHELXS [23] structure solution program using Direct Methods and refined with the olex2.refinerefinement package using Gauss-Newton or Levenberg-Marquardt minimization [24].CCDC 2247862 and CCDC 2247865 contain the supplementary crystallographic data for this paper.These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via https://www.ccdc.cam.ac.uk/data_request/cif.

3β-Hydroxyfriedelane (epi-friedelinol, 5)
General method A. A mixture of friedelin (1, 1.000 g, 2.343 mmol), PtO 2 (150 mg), THF (5 mL), and ethyl alcohol (99%, 15 mL) was hydrogenated at 60 bar hydrogen pressure at 50 • C for 48 h.The insoluble material was dissolved by the addition of an appropriate volume of THF, the whole mixture was then filtered through a syringe filter, the catalyst was washed with THF, and the collected filtrates were evaporated to dryness.The title compound (1.000 g, quantitatively) sufficiently pure for further synthesis was obtained as a white powder.Crystallisation from dichloromethane gave pure 5 (850 mg, 85%) as white crystals.
M.p. 282-283 and 13 C NMR spectral data were in agreement with those published in the literature [26].

3α-Hydroxyfriedelane (friedelinol, 7)
General method C. Friedelin (1, 214 mg, 0.500 mmol) was dissolved in refluxing isopropyl alcohol (50 mL).Then, large pieces of sodium (~150-200 mg each) were slowly added.The reaction was controlled by TLC; usually approx.30-40 eq. of sodium was necessary to complete the reaction.The solvents were evaporated to dryness, the residue was mixed with a small amount of silica gel and purified by column chromatography (hexane-ethyl acetate, 20:1 for the elution of low polar contaminants, and hexane-ethyl acetate -methanol, 5:3:1 for the product) to afford 7 (170 mg, 79%) as an amorphous solid.
Acetylation of the crude product of the previous reaction using General method B also afforded acetate 8 (75% after two steps).

2β-Hydroxyfriedelan-3-one (epi-cerin) (15)
General method D. To a cooled in ice bath solution of compound 4 (966 mg (2.00 mmol) in dry THF (50 mL) was added LiAlH 4 (380 mg, 10.00 mmol) and stirred for 5 min.The cooling bath was removed and the mixture was stirred at room temperature for 45 min.Then ethyl acetate (5 mL) was slowly added and the entire mixture was filtered through a short silica pad using the mixture of hexane-ethyl acetate (1:1) as eluent.The solvents were evaporated to dryness to afford a crude product (840 mg, 95%) sufficiently pure for further synthesis.It can be purified by column chromatography (hexane-ethyl acetate, 5:1) to obtain pure 15 (810 mg, 91%) as white crystals.
M.p. 225-229 NMR spectral data were in agreement with those published in the literature [28,29].
Similarly, starting from 9 (200 mg, 0.413 mmol) and using General method D followed by acetylation as described in General method B diacetate 13 (145 mg, 67% after two steps) was obtained.
Compound 15 (150 mg, 0.452 mmol) was hydrogenated at 7 bar hydrogen pressure at room temperature for 24 h analogously to General method A to afford diol 21 (150 mg, quant.)sufficiently pure for further synthesis.Crystallisation from hexane-ethyl acetate gave pure 21 (133 mg, 89%) as white crystals.

2β,3β-Diacetoxyfriedelane (14)
Compound 21 (87 mg, 0.196 mmol) was acetylated according to General method B. The product was purified by column chromatography (hexane-ethyl acetate, 20:1) to afford the title compound (89 mg, 86%) as white crystals.The 13 C NMR spectral data were in agreement with those published in the literature [28].

Results and discussion
In the initial approach, we investigated the methods of friedelin (1) reduction.This reaction can be performed stereoselectively to yield epifriedelinol (5) or friedelinol (7) as illustrated in Scheme 1.In most cases, epi-friedelinol was prepared as preferred product.Typically, LiAlH 4 and NaBH 4 were employed as reducing reagents, providing product 5 selectively [14,25,38].Catalytic hydrogenation of friedelin in the presence of platinum in acetic acid has also been used to synthesise epifriedelinol [27].We have found that acetic acid can be replaced with a mixture of ethyl alcohol and THF.The product, sufficiently pure for further transformations, was obtained quantitatively by filtration of the reaction mixture and evaporation of the solvents.Crystallisation afforded pure alcohol 5 in excellent yield (85%).Alternatively, the crude product could be purified through acetylation to compound 6 (96%) and column chromatography.The proposed method is particularly suitable for multi-gram scale synthesis of 5, as it circumvents the need to handle large quantities of LiAlH 4 and the exhaustive isolation of the poorly soluble product.
The synthesis of friedelinol ( 7) is much more difficult.Currently, the reduction of the carbonyl group of friedelin with sodium, usually in boiling pentan-1-ol [25,38], or with lithium-ethylenediamine mixture [39] are the only fully selective methods known in the literature.We achieved comparable results by employing isopropyl alcohol as a solvent.The reactivity of isopropyl alcohol toward sodium remains marginal, while its low boiling point significantly simplifies the reaction workup.As compound 7 has low solubility in organic solvents, the most convenient method for the purification of friedelinol is its acetylation to acetate 8, followed by column chromatography (75% after two steps).
The configuration at the C3 carbon atom was deduced from the shape of the H-3 signal in the 1 H NMR spectra of acetates 6 and 8.The H-3 proton of 6 appeared at δ 4.89 as a ddd (J 2.9, 2.9, and 2.9 Hz) which indicated an equatorial orientation.The same proton for 8 was observed at δ 4.62 as a ddd (J 11.1, 11.1, and 5.0 Hz), with the coupling values being characteristic of an axial orientation.The 1 H and 13 C NMR spectral data for 6 were in agreement with those published in the literature [28,29].The configurational assignment of compound 8 was further confirmed by NMR measurements using two-dimensional techniques (see Supplementary data).
3-Acetoxyfriedel-3-en-2-one ( 4) is the second friedelane-type triterpenoid easily available from black wax.We recently discussed its catalytic hydrogenation in the presence of palladium, however, the structure of the product was incorrectly assigned as 3α,4α-isomer [17].This conclusion needs to be corrected due to the fact that the reanalysis of the NMR spectra, as well as the X-ray crystallographic analysis, unequivocally confirmed the 3α,4β configuration of 9 (Scheme 2, Supplementary data).Additionally, the NMR spectral data were consistent with those previously published [28].Apparently, the reaction's initial step involved the hydrogenation of the double bond, which resulted in the 3β,4β-isomer 10 (not detected in the reaction mixture).This product was rapidly isomerised to 3α,4β-epimer 9 in the presence of palladium.A similar epimerization of 10 to 9 on Al 2 O 3 had been reported earlier [40].This assumption was corroborated by the hydrogenation of 4 at room temperature in the presence of platinum.This reaction yielded an inseparable mixture of 9 and 10 in an approximate ratio of 1.4:1.0(26%, Scheme 2).The structure of the main component was confirmed by comparing the NMR spectra of the mixture with the original sample 9.The presence of 10 was confirmed by comparing the NMR spectra of the mixture with literature data.The signal of the axially oriented proton H-3 for 9 was observed at δ 4.95 ppm (d, J 12.4 Hz), while the equatorially oriented proton H-3 for 10 resonated at δ 4.98 ppm (d, J 4.2 Hz).These values were in line with the published data [17,40].
During the hydrogenation of 4 in the presence of platinum at 50 • C, a reduction of the carbonyl group occurred, leading to a complex mixture of products.This mixture was then acetylated and separated chromatographically to afford 2β-acetoxyfriedelane (11, 4% after two steps), 2α,3β-diacetate 12 (17% after two steps, pachysandiol-A diacetate) [28,29], and an inseparated mixture of diacetates 13 (2β,3α) and 14 (2β,3β).The latter appeared as the main products in an approximately 1:1 ratio (40% after two steps).Both epimers 13 and 14 can be easily distinguished by comparing the 1 H and 13 C NMR spectra of the mixture with literature data and the pure diacetates spectra obtained later in this study [28,29].The configurational assignments of 11 were based on the NMR measurements using two-dimensional techniques (see Supplementary data).
The reduction of 4 with sodium proceeded smoothly with complete conversion to 2β,3α-diol 18.The same diol 18 was prepared by reduction of 9 with LiAlH 4 or sodium.It was also obtained selectively by treating epi-cerin (15) or its silylated derivative 17 with sodium.It was purified and analysed as diacetate 13 (54-67%).
The reduction of 4 with NaBH 4 was less selective, providing an inseparable mixture of monoacetates 19 (2β,3β) and 20 (2α,3β) in a ratio of approximately 10:1 (74%).During the reaction, an acetyl migration from the O-3 to O-2 was observed (Scheme 4).The structure of the main product 19 was confirmed by comparing the NMR spectra of the mixture with the NMR spectra of pure sample 19, which was obtained later in this study.The 1 H NMR spectrum of 19 showed a signal of the axially oriented H-2 at δ 4.78 (ddd, J 3.3, 6.3, and 10.5 Hz).A broad singlet (half-height width 7.6 Hz) at δ 3.80 was assigned to the equatorially oriented H-3.Similarly, the H-2 proton of the minor compound 20 was observed at δ 4.94 (ddd, J 2.8, 2.8, and 2.8 Hz).This signal displayed the typical coupling constants of an equatorially oriented proton.Concurrently, a broad singlet at δ 3.55 (half-height width 8.5 Hz) was identified as equatorially oriented H-3.The structure of the minor compound 20 was confirmed by comparing the 1 H NMR spectra of the mixture with available literature data.The coupling constants and positions of the signals for H-2, H-3, and the methyl groups were in accordance with published data [40].
The treatment of epi-cerin (15) and its silylated derivative 17 with LiAlH 4 gave 2β,3β-diol 21 in an excellent yield (87% and 85%, respectively, Scheme 5).Catalytic hydrogenation of 15 gave the same diol quantitatively, whereas compound 17 remained intact, and the starting material was almost quantitatively recovered.Due to the extremely low solubility of 21, its analytical sample was acetylated to 14 (86%) to facilitate further analysis.When epi-cerin 16 was acetylated and subsequently reduced in the presence of platinum, monoacetate 19 (86%) was formed.The configurational assignments of compounds 19 and 21 were based on the NMR measurements using two-dimensional techniques (see Supplementary data).It is worth of noting, that ketone 9 remained inert during the platinum catalysed hydrogenation, while cerin acetate [44] decomposed rapidly under reaction conditions.

Conclusions
Methods for the synthesis of the number of A-ring functionalised friedelane triterpenoids have been presented.Friedelin (1) and 3-acetoxyfriedel-3-en-2-one (4), both readily available from cork smoker wash solids (black wax) on a multi-gram scale, were used as starting materials.
The key modification routes include reduction with LiAlH 4 or sodium and hydrogenolysis in the presence of platinum.Reduction of 4 with LiAlH 4 gave epi-cerin 15, which is also a convenient and valuable starting material in further transformations.Usually, reduction with LiAlH 4 and hydrogenation in the presence of platinum gave epimeric products compared to sodium reduction.Selection of the appropriate reaction conditions allows the synthesis of most of the products in high yield and excellent selectivity.
The methodology presented in this article offers the simplest and shortest way to obtain friedelanes containing diol and α-hydroxyketone  presence of functional groups and the ability to control the configuration on the stereogenic centres also allow us to plan further functionalisations of the structure, primarily within the A ring of the friedelin.Syntheses of seco-type compounds using diol cleavage or the Baeyer-Villiger oxidation reactions also become possible [44].We plan to use the friedelane-type triterpenoids in further biological studies.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.