Synthesis of Two New Hemisynthetic Diterpenylhydroquinones from Natural Ent-Labdanes

The synthesis and structural determination of two new diterpenylhydroquinones: 2β-acetoxy-15-phenyl-(22,25-dihydroxy)-ent-labda-8(17),13(E)-diene (1) and 2β-hydroxy-15-phenyl-(22,25-dihydroxy)-ent-labda-8(17),13(E)-diene is reported (2). These compounds were obtained by coupling via Electrophilic Aromatic Substitution (EAS) of 1,4-hydroquinone with primary or tertiary allyl alcohol derivatives of the natural ent-labdanes 3 and 4. With this new method, the best results were observed when mixtures of the primary alcohol derivatives 5-6 (26% yield of compound 1) and diol derivatives 9-10 (28% yield of compound 2) were used.

The more recurrent synthetic strategies used for synthesizing terpenylquinones/hydroquinones, involve, as a first step, the separate preparation of the appropriate terpenyl and aromatic nucleus fragments. The crucial step is the attachment of the aromatic synthon to the terpenyl skeleton and the final step is the formation of oxygenated functions (generally by oxidation of hydroxyl or methoxyl groups) on the quinone moiety. The more used methods for the coupling reactions are the following: 1) Nucleophilic addition of aryllithium derivatives to carbonyls of the terpenyl unit (synthesis of (+)puupehenone [13]); 2) Diels-Alder and hetero Diels-Alder cycloaddition (synthesis of (-)cyclozonarone [14] and (+)-cyclozonarone [15]); 3) Enolate alkylation with benzyl bromide derivatives (synthesis of (-)-ilimaquinone [16]); 4; Conjugated 1,4-addition; Michael addition (synthesis of (-)-yahazunol [17]); 5) Coupling of Horner-Wadsworth-Emmons (HWE) (synthesis of metachromin A [18]). All these structures are shown in Figure 1. In this work we report the first synthesis and the structural determination of two new bicyclic diterpenyl-hydroquinones 1 and 2 (Schemes 1 and 2) with ent-labdane skeletons and a bridge between the terpenyl unit and hydroquinonic nuclei consisting of five carbon atoms and an (E)-trisubstituted double bond, as found in the structure of metachromin A.
For the coupling reaction (key step of the synthesis) between both fragments, a strategy involving Electrophilic Aromatic Substitution (EAS) of diterpenyl allylic alcohol derivatives 5-8 ( Figure 2) and diols 9-10 with 1,4-hydroquinone were used, as was described for the synthesis of a taondiol derivative [19][20], although it is important to emphasize that the use of an EAS reaction as the like key coupling reaction step has not been previously reported for the syntheses of terpenylquinones. The results of the biological activities against cancer cellular lines of these compounds will be reported in complete detail elsewhere at a later date.

Results and Discussion
In previous investigations we reported the isolation and structural determination of the mixture of ent-labdanes 3-4 from Calceolaria inamoena and the preparation of derivatives 5 and 6 ( Figure 2) [22]. Our next step was the preparation of the tertiary alcohol 7 and/or 8, from primary allyl alcohols 5 or 6. Nevertheless, when these compounds or a mixture of both, were reacted with SOCl 2 , in the three cases small amounts of the epimeric mixture 7-8 was obtained (see Scheme 1) along with a complex unidentified mixture of compounds which was obtained as the major component. The highest yield of 7-8 mixture (33.7%) was observed when compound 6 was treated with SOCl 2 . Unfortunately this mixture could not be separated by conventional chromatography and the proportion of epimers in the mixture were determined as 7:8 =0 THF, N 2 , T=20°C, 24 h, 11%; from 5: 10% and from 6: 20%. c. From 5-6: K 2 CO 3 /MeOH, r.t, 0.5 h, C.C., 9, 11.3%; 9-10, 20.4% and 10, 55.4%. The next step was the coupling reaction between the 7-8 mixture with 1,4-hydroquinone catalyzed by BF 3 . THF, according to the previously described protocol [19][20]. After the corresponding workup and purification of the crude product by column chromatography (CC), only two major products were isolated: 0.678 g of an unidentified complex mixture (non-polar fraction) and 0.127 g (11%) of the desired compound 1. The structural determination of compound 1 was mainly accomplished by 1 H-, 13 C-, DEPT-135, gs-2D HSQC and gs-2D HMBC NMR techniques. In its IR spectrum absorptions at 3,401 and 1,701 cm -1 were observed, whereas the 1 H-NMR spectrum showed the existence of three aromatic hydrogens at δ = 6.67 (d, J= 8.   Additionally we decided to try the coupling reaction with 1,4-hydroquinone separately, using the compounds 5, 6 and a mixture of both (see Scheme 1), following the previously protocol described for the 7-8 mixture. In the case of alcohol 5 (1.39 g), after usual workup followed of CC separation, three fractions were obtained: 1.33 g of a non-identified complex mixture (non-polar fraction), 0.175 g (10% yield) of compound 1 and 0.37 g of unreacted 1,4-hydroqinone. In the case of alcohol 6 (1.54 g) were obtained: 0.983 g of complex mixture, 0.390 g (20% yield) of compound 1 and 0.28 g of unreacted c) 1,4-hydroquinone. In the case of a 5-6 mixture (3.92 g, approx. ratio 5:6 = 0.25:0.75) 2.47 g of complex mixture, 1.29 g ( 26% yield) of compound 1 and 0.963 g of unreacted 1,4-hydroquinone were obtained. The characterization of compound 1 was accomplished by simple inspection and comparison of 1 H-and 13 C-NMR spectral data with that previously obtained.
We also performed the coupling reaction using the diols 9, 10 and a mixture of both (see Scheme 1) following the experimental procedure previously described. The diols were obtained by alkaline hydrolysis with K 2 CO 3 /MeOH, acidification and later separation and purification by CC. From 2.93 g of the acetate mixture 5-6, 0.292 g of diol 9 (11.3% yield), 0.527 g (yield 20.4%) of a 9-10 mixture and 1.43 g (55.4% yield) of diol 10 were obtained. The structures of compounds 9 and 10 were mainly established by 1 H-and 13 C-NMR spectroscopic data (see Experimental section) and compared with those reported for acetates 5 and 6, respectively [21]. The proportion of geometrical isomers in the mixture were determined as 9:10 =0.2:1 (ratio calculated based on the integrals of the H-17a and H-17b signals in the 1 H-NMR spectrum). When compound 9 (0.238 g) was reacted with 1,4hydroquinone, a non polar unidentified complex mixture (0.183 g) and 40 mg (13%) of compound 2 were obtained. From the compound 10 (0.897 g) 0.420 g of complex mixture and 0.246 g (21%) of compound 2 were obtained. On the other hand, from 9-10 mixture (0.258 g) 0.122 g of the complex mixture, 57 mg (17%) of diol 11 and 94 mg (28%) of compound 2 were isolated (see Scheme 2). The structural determination of compound 2 was established by comparison of the spectral data of compound 1 and using the same criteria. In the IR spectrum of compound 2 a strong absorption at 3,375 cm -1 was mainly observed, whereas the 1 H-NMR spectrum showed the existence of three aromatic hydrogens at δ = 6.67 (d, J= 8. 6  The structure of compound 11 was established by spectroscopic correlations of its 1 H-and 13 C-NMR data with the 7-8 mixture and the stereochemistry in C-13 position was tentatively assigned as "R" by comparison and correlations with δ 13 C data of epimers 7 and 8, which showed a chemical shift at δ C = 73.5 ppm for this carbon. As previously indicated in all the coupling reactions, the formation of a complex mixture of products was observed. Nevertheless by simple inspection of the 1 H-and 13 C-NMR spectra of this mixture, we observed the presence of hydrogen (δ H : 6.45-6.14 ppm) and carbon (δ C : 150.6-142.4 ppm) vinyl signals that were probably due to the formation of mixtures of alkenes (IV and V), presumably obtained via formation of a cationic intermediary III (formed by reaction of the allyl alcohols with BF 3 . THF, followed by rearrangement of intermediate I and/or II), later elimination E 1 products or in one case SN 1 (for example the formation of compound 11), that compete with the formation of Electrophilic Aromatic Substitution (EAS) products (see Figure 4)

Conclusions
In this work we have described the synthesis and structural determination of two new diterpenylhydroquinones from natural ent-labdanes. These compounds were obtained by coupling primary or tertiary allyl alcohol ent-labdane derivatives with 1,4-hydroquinone by Electrophilic Aromatic Substitution (EAS). This method of coupling between terpenyl fragments and aromatic nucleus (key step in all synthesis of terpenyl-(hydro)quinones) had not been previously described, and worked for all the allylic alcohols used here. Nevertheless, the best results were observed when the mixtures of primary alcohols 5-6 (26% yield of compound 1) and mixture of diols 9-10 (28% yield of compound 2) were used for coupling reaction. We think that the possible formation of very stable intermediary III (Figure 4) is mainly responsible for the formation of a complex mixture of alkenes and the low yields observed in the coupling reactions.

General
Unless otherwise stated, all chemical reagents purchased (Merck or Aldrich) were of the highest commercially available purity and were used without previous purification. Melting points were measured (in triplicate) on a Stuart-Scientific SMP3 apparatus and are uncorrected. IR spectra were recorded as thin films in a Nicolet Impact 420 spectrometer and frequencies are reported in cm -1 .  (7) and 2β-acetoxy-(R)-13-hydroxyent-labda-8(17), 14-diene (8) from 5: A solution of 5 (1.54 g, 0.44 mmol) in dry CH 2 Cl 2 (50 mL) and dry pyridine (1 mL), was prepared under a N 2 atmosphere and cooled to -10°C (ice/acetone/brine bath). Then SOCl 2 (0.6 mL, 8.26 mmol) was slowly added dropwise while maintaining slow agitation. After one hour, the completion of the reaction were verified by TLC. Then saturated aqueous solution of NaHCO 3 (50 mL) was added and the mixture was extracted with EtOAc (2 x 25 mL) and the combined organic layers were washed with water (2 x 20 mL), dried over Na 2 SO 4 , filtered and evaporated. The crude was redissolved in CH 2 Cl 2 (5 mL) and chromatographed on silica-gel with petroleum ether/EtOAc mixtures of increasing polarity (19.8:0.2→8.8:11.2). Two fractions were obtained: Fraction I: 1.08 g of a non-polar unidentified complex mixture (colorless viscous oil) Fraction II: colorless viscous oil, 0.34 g (22.1%) of a mixture of 7-8.  THF (0.5 mL, 3.98 mmol) in freshly distilled 1,4-dioxane (5 mL) was slowly added dropwise, with stirring at room temperature and under a N 2 atmosphere, a solution of 7-8 mixture (0.91 g, 2.61 mmol) in 1,4-dioxane (5 mL). After the addition was complete, stirring at room temperature and under N 2 atmosphere was continued overnight. When the completion of the reaction was verified by TLC, the mixture was poured onto crushed ice (app. 30 g) and the organic layer extracted with diethyl ether (3 x 30 mL), the ethereal layer was washed with 5% NaHCO 3 (30 mL), then with water (2 x 20 mL) and dried over Na 2 SO 4 , filtered and evaporated. The crude was redissolved in CH 2 Cl 2 (5 mL) and chromatographed on silicagel with petroleum ether/EtOAc mixtures of increasing polarity (19.8 THF (1.5 mL, 11.9 mmol) and 5-6 mixture (3.92 g, 11.25 mmol) were used. After workup and CC purification: 2.47 g of complex mixture, 1.29 g (26%) of 1 and 0.963 g of unreacted 1,4-hydroqinone, were obtained.  After the TLC analysis indicated the completion of the reaction, the solvent was removed until a volume of approximately 5 mL remained and water (30 mL) was added, then 5% HCl (15 mL) was added, the mixture was extracted with EtOAc (3 x 30 mL) and the combined organic layers were washed successively with 10% NaHCO 3 and water, dried over Na 2 SO 4 , filtered and evaporated. The crude (2.45 g) was redissolved in CH 2 Cl 2 (10 mL) and chromatographed eluting with mixtures of petroleum ether/EtOAc of increasing polarity ( THF (0.2 mL, 1.59 mmol) in freshly distilled 1,4-dioxane (5 mL) was slowly added dropwise with stirring at room temperature and under a N 2 atmosphere, a solution of 9 (0.238 g, 0.777 mmol) in 1,4-dioxane (5 mL). After the addition was complete, stirring at room temperature and under N 2 atmosphere was continued overnight. When the end of the reaction was verified by TLC, the mixture was poured onto crushed ice (app. 30 g) and the