Antiproliferative Activity of Natural Taiwaniaquinoids and Related

Article pubs.acs.org/jnp

Antiproliferative Activity of Natural Taiwaniaquinoids and Related Compounds Juan J. Guardia,† Rubén Tapia,† Soumicha Mahdjour,‡ Fernando Rodriguez-Serrano,§ Nuria Mut-Salud,§ Rachid Chahboun,*,† and Enrique Alvarez-Manzaneda*,† †

Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biotecnología, and §Instituto de Biopatología y Medicina Regenerativa (IBIMER), Universidad de Granada, 18071 Granada, Spain ‡ Laboratory Productions, Plant and Microbial Valuations (LP2VM), Department of Biotechnology, University of Sciences and Technology of Oran Mohamed Boudiaf, BP 1525, El M’Naouer, Oran, Algeria S Supporting Information *

ABSTRACT: The in vitro antiproliferative activities of some taiwaniaquinoids and related compounds with functionalized A, B, or C rings against human breast (MCF-7), colon (T-84), and lung (A-549) tumor cell lines were assayed. The most potent compounds, 16, 27, and 36, were more effective than the naturally occurring taiwaniaquinones A (4) and F (5) in all three cell lines. The structure−activity relationship study of these new taiwaniaquinoids highlighted the correlation between the bromo substituent and the antiproliferative activity, especially in MCF-7 cells. These findings indicate that some of the taiwaniaquinoids might be useful as cytostatic agents against breast, colon, and lung cancer cell lines.

F

also improves the effectiveness of a standard drug, additional studies will be merited.14,15 Many of the current anticancer drugs are natural products and their derivatives.16 A major advantage of drugs derived from natural products with respect to fully synthesized compounds is their greater structural complexity. This has been related to the fact that natural product derivatives tend to be more selective toward a wide range of targets.3,17,18 Taiwaniaquinoids belong to a group of terpenoids with an unusual rearranged 5(6→7) or 6-nor-5(6→7) abeo-abietane skeleton and have been isolated from certain species of East Asian conifers during the last 20 years.19 Among these, compounds bearing a 4a-methyltetrahydrofluorene skeleton, such as taiwaniaquinone D (1)20 or H (2),21 isolated from Taiwania cryptomerioides (Taxodiaceae), or dichroanone (3), obtained from Salvia dichroantha (Lamiaceae),22 are known. Taiwaniaquinoids with a 4a-methylhexahydrofluorene skeleton have also been isolated from T. cryptomerioides. Thus, compounds with a trans A/B junction, such as taiwaniaquinones A (4),23 F (5),24 and G (6),21 have been described, as well as others with a cis A/B junction, such as taiwaniaquinol B (7).23 Only a few studies, including a superficial description of the biological activity of this type of compounds, have been reported. Preliminary results show that some of these

or thousands of years, organisms in nature have served as a source for new drugs.1 In fact, most current chemotherapeutics are derived from natural products. In the last two decades, however, other agents have entered the therapeutic landscape, such as antibodies or small synthesized molecules, and these have been beneficial in the treatment of cancer. However, the benefit for patients with advanced solid and hematologic tumors remains insufficient, and in many cases the therapeutic options are only palliative. This shortcoming has revitalized interest in identifying new natural molecules with antitumor properties.2,3 In addition, advances in the understanding of the biology of cancer, together with new developments in chemistry and computer technology, have led to the synthesis of natural product derivatives with promising properties.4−8 Cancer is still one of the leading causes of mortality worldwide. Nowadays, breast, colon, and lung cancers have high prevalence and mortality in developed countries.9,10 Although treatments have advanced, many drugs produce systemic toxicity, and some patients acquire drug resistance, which may lead to disease recurrence and metastasis.11 Therefore, it is necessary to improve the treatment of cancer, finding more effective compounds against tumor cells that are less toxic toward normal cells.12,13 In this regard, drug discovery and development play an important role, including the synthesis of new compounds and the assessment of their biological properties.14 On the basis of its cytotoxic or antiproliferative activity, a compound is considered promising if it is able to obliterate cancer cells at low concentrations. If it © 2017 American Chemical Society and American Society of Pharmacognosy

Received: July 28, 2016 Published: January 25, 2017 308

DOI: 10.1021/acs.jnatprod.6b00700 J. Nat. Prod. 2017, 80, 308−318

Journal of Natural Products

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compounds exhibit cytotoxic21,25,26 and antiparasitic activities.27 It is known that taiwaniaquinones A, D, and F and taiwaniaquinol A exert some cytotoxic activity against KB87 human oral epidermoid carcinoma cells,21,25a while standishinal, isolated from the bark of the Japanese conifer Thuja standishii (Cupressaceae),25a shows an interesting activity as an inhibitor of the aromatase enzyme.19,28Aromatase inhibitors decrease plasma estrogen levels and, therefore, are useful therapeutic agents for estrogen-dependent cancers, such as luminal breast cancers.25c,26 In fact, it has been demonstrated that aromatase inhibitors reduce the rate of disease recurrence in luminal breast cancer patients.29 The promising biological activity and the unusual structure of these terpenoids have stimulated research into the synthesis of this type of compound, including total30 and stereoselective syntheses.31 The synthesis starting with natural terpenoids facilitates the formation of enantiopure taiwaniaquinoids.32 Thus, the first synthesis of a taiwaniaquinoid with a trans A/B junction, such as taiwaniaquinone G (6), was achieved by our group starting from (+)-sclareolide.32a,b Taiwaniaquinoids bearing a carbon function on the cyclopentane B ring, such as taiwaniaquinones A (4) and F (5), were first synthesized by us starting from (−)-abietic acid.32d,e This paper reports the synthesis of new taiwaniaquinoids starting from natural terpenoids and the evaluation of their in vitro antiproliferative activities, as well as that of other synthesized taiwaniaquinoids, against human breast, colon, and lung tumor cells.

Scheme 1. Synthesis of Taiwaniaquinoid Derivatives from α,β-Enone 8

The treatment of enone 8 with KOH in MeOH, in the presence of atmospheric oxygen, at −30 °C gave quinone 9.32d When the reaction was carried out at room temperature, epoxide 11 was formed. This compound was formed under similar reaction conditions starting from quinone 12.32e The successive treatment of acetoxyquinone 9 with NaBH4 and KOH afforded hydroxiquinone 10. When α,β-enone 8 was refluxed with trifluoroacetic acid (TFA) in tetrahydrofuran (THF), hydroquinone 13 was obtained. Subsequently, brominated taiwaniaquinoid-related compounds were synthesized. Thus, bromoquinones 15−17 were prepared from quinone 1432e (Scheme 2). Scheme 2. Synthesis of Bromoquinone Derivatives from Compound 14

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RESULTS AND DISCUSSION As indicated above, only a few studies of the biological activity of taiwaniaquinoids have been reported, probably due to their limited presence in natural sources. The development of synthetic sequences toward enantiopure taiwaniaquinoids from natural terpenoids prompted us to prepare some natural compounds and derivatives in order to study their cytotoxic activities. In addition, by obtaining this type of compound from A-ring-functionalized terpenoids, such as (−)-abietic acid, it becomes feasible to synthesize functionalized taiwaniaquinoids, which could be of interest in studies of the structure−activity relationship. The first step was to prepare taiwaniaquinoid-related compounds with a gem-dimethyl group in the A ring and functionalized in the B ring. Scheme 1 shows the transformation of α,β-enone 8, a precursor of taiwaniaquinoids previously synthesized,32e into compounds 9−11 and 13.

The treatment of acetoxybromoquinone 14 with 1,8diazabicyclo[5.4.0]undec-7-ene (DBU) in benzene at room temperature afforded alkene 15, which after treatment with SeO2 in dioxane under reflux gave allyl alcohol 16. Alcohol 17 was obtained when the acetoxy derivative 14 was successively treated with NaBH4 and KOH. The next aim involved the synthesis of taiwaniaquinoids functionalized on the A, B, or C ring, starting from commercial (−)-abietic acid (18). Scheme 3 shows the synthesis sequence from (−)-abietic acid (18) to functionalized taiwaniaquinoids. Aldol condensation of ketoaldehyde 20, obtained via ozonolysis of compound 19,32e afforded hydroxyaldehyde 21, which after successive reduction of the formyl group, dehydration, and allylic oxidation afforded α,β-enone 22, a suitable precursor of functionalized taiwaniaquinoids. 309



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The reduction of compound 21 with NaBH4 (2 equiv) in EtOH at room temperature for 90 min gave triol 23 in high yield as the sole product. A mixture of triol 23 and diol 24 was obtained when the reaction was carried out in the same solvent at 0 °C and in THF at −10 °C. When the reduction was performed using 1 equiv of NaBH4 in dimethoxyethane (DME), DMSO, CH2Cl2, or Et2O, the selective reduction of the formyl group afforded diol 24 as the sole product. Diol 24 was transformed into α,β-enone 22, a precursor of functionalized taiwaniaquinoids (Scheme 5). Acetylation of 24 afforded 25, which was dehydrated to give 26. Subsequent oxidation of 26 afforded ketone 22.

Scheme 3. Synthesis of Functionalized Taiwaniaquinoids from (−)-Abietic Acid (18)

Scheme 5. Synthesis of α,β-Enone 22 from Diol 24

The process to obtain hydroxyaldehyde 21 from (−)-abietic acid (18) is depicted in Scheme 4. Compound 21 was obtained in 82% yield after refluxing ketoaldehyde 20 and DBU in benzene for 3 h. Scheme 4. Synthesis of Hydroxyaldehyde 21 from (−)-Abietic Acid (18)

Next, the α,β-enone 22 was converted into different functionalized taiwaniaquinoids (Scheme 6). Treatment of enone 22 with Br2 in CH2Cl2 at room temperature gave bromoquinone 27 in 84% yield. When 27 was treated with NaOMe in MeOH at room temperature for 14 h, a mixture of exocyclic alkene 28 and alcohol 29, in a 2:3 proportion, was obtained. Oxidation of the latter with pyridinium dichromate (PDC) afforded aldehyde 30; this compound is similar to the natural taiwaniaquinone F (5), but with a 4-COOMe group instead of a 4-Me group. In an alternative approach, the α-acetoxy ketone 31 was obtained when enone 22 was treated with Pb(OAc)4 in benzene under reflux; further treatment of ketone 31 with HCl and O2 in MeOH led to hydroxyquinone 32. Finally, triol 23 was converted into bromoquinone 36 (Scheme 7). The dehydration of diacetoxy alcohol 33 gave alkene 34, which after treatment with pyridinium chlorochromate (PCC) led to α,β-enone 35. Bromination of the latter afforded bromoquinone 36. Following the synthesis of these taiwaniaquinoid-related compounds, the cytotoxicity of these and other synthesized taiwaniaquinoids was assayed. Biological Assays. In Vitro Cytotoxicity Assay. To evaluate the antiproliferative activity of the synthesized taiwaniaquinoids, their IC50 values against human breast (MCF-7), colon (T-84), and lung (A-549) tumor cell lines were assessed. The results are shown in Table 2, in which the compounds are ranked in order of increasing functionalization. Compounds possessing bromoquinone functionalities showed greater activity. Quinone 36 was the most effective in cell lines T-84 and A-549, with IC50 values of 1.6 and 1.1 μM,

The next stage in our study was the chemoselective reduction of the aldehyde group of compound 21. The most significant results of this process are presented in Table 1. Table 1. Reduction of Compound 21

1 2 3 4 5 6 7

NaBH4

solvent

T (°C)

t (min)

2 2 1 1 1 1 1

EtOH EtOH THF DME DMSO CH2Cl2 Et2O

21−25 0 −10 0−25 21−25 0−25 0

90 30 60 100 240 90 120

equiv equiv equiv equiv equiv equiv equiv

products (%) 23 23 23 24 24 24 24

(85) (23), 24 (64) (15), 24 (75) (91) (97) (98) (92) 310



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Scheme 6. Synthesis of Functionalized Taiwaniaquinoids from α,β-Enone 22

Scheme 7. Synthesis of Bromoquinone 36 from Triol 23

Fu. Regarding the cell line A-549, the IC50 of 5-Fu (4.2 μM) was higher than 36 (1.1 μM), 16 (1.5 μM), and 27 (2.4 μM) (entry 21). In T-84 cells, the IC50 value (4.1 μM) was higher than the IC50 values of compounds 36 (1.6 μM) and 27 (2.9 μM). Even in MCF-7, the IC50 of 5-Fu (1.6 μM) was a little higher than that of compounds 16 (1 μM) and 15 (1.3 μM). Other compounds with a significant degree of antiproliferative activity were 27 (IC50 < 3 μM in all three cell lines), 4132b (IC50 = 2.2 μM in MCF-7 and 3.2 μM in T-84) (entry 5), 3832b (IC50 = 3.2 μM in MCF-7 and 6.2 μM in T-84) (entry 2), 17 (IC50 2.1 μM in MCF-7 and 6.7 μM in T-84) (entry 12), 15 (IC50 = 1.3 μM in MCF-7 and 9.6 μM in T-84) (entry 14), and 4432e (IC50 = 4 μM in MCF-7) (entry 13). All of these compounds, as well as 36 and 16, are characterized by a 12bromo group, which appears to be the most important structural element enhancing the antiproliferative activity of these compounds. On average, the presence of the bromo group increased the activity of the new taiwaniaquinoids in

respectively. This compound was also highly active in the MCF-7 line, requiring an IC50 value of 1.8 μM to eliminate 50% of the cell population (entry 22). The most active compound against the MCF-7 line was 16, with an IC50 value of 1 μM, and this compound also showed significant activity against the A549 and T-84 cell lines, with IC50 values of 1.5 and 5.2 μM, respectively (entry 16). A notable finding is that these compounds are more effective than the natural taiwaniaquinoids 4 (taiwaniaquinone A) (entry 10) and 5 (taiwaniaquinone F) (entry 11). Thus, in the MCF-7 cell line, compound 16 was 28 and 15 times more effective than 4 and 5, respectively. In T-84 cells, compound 36 was 19 and 7 times more active than 4 and 5, respectively, while in A-549, 36 was 22 times more active than 4 and almost 9 times more active than 5. 5-Fluorouracil (5-Fu) was used as a positive control. This drug is frequently used for the treatment of breast and colon tumors, among others.33 Some of the new taiwaniaquinoids showed higher antiproliferative activities in vitro than 5311



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Table 2. Antiproliferative Activities of Taiwaniaquinoidsa

a

IC50 values expressed as means ± SEM of four independent determinations in the μM range.

compounds such as 3732b (IC50 = 15.0 μM) (entry 1) and 3832b (entry 2), which have the same structure as 3732b and the 12-bromo group (IC50 22.1 μM). Other examples are compounds 10 (without Br and IC50 = 12.5 μM) (entry 6), 17 (with Br and IC50 = 12.2 μM) (entry 12), 9 (without Br and

MCF-7, T-84, and A-549 cell lines 8.5, 2.4, and 1.9 times, respectively. However, in the A-549 cell line the 12-bromo group does not seem as crucial, since the presence of such a group does not contribute to enhanced activity. This fact is evidenced in 312



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IC50 = 21.1 μM) (entry 7), and 4432e (with Br and IC50 = 29.7 μM) (entry 13). In fact, although the correlation between the presence of a bromo group in the compounds and their IC50 values was statistically significant for the three cell lines, the analysis revealed a high correlation for MCF-7 (Spearman’s coefficient = 0.81, p < 0.01), a moderate one for T-84 (Spearman’s coefficient = 0.55, p < 0.01), and a low one for A549 (Spearman’s coefficient = 0.48, p < 0.05). Numerous articles have reported that halogen derivatives, and concretely bromo derivatives, are more active than the corresponding natural compounds. For example, bromo benzofuran-based derivates have shown significant activity against human liver carcinoma cells (HEPG2), exceeding that of the natural precursor (visnagin), doxorubicin, and 5-Fu.34 Moreover, in vivo assays have shown that bromo derivatives of brassisin can inhibit the growth of aggressive melanoma tumors, via a mechanism that may involve the inhibition of indoleamine 2,3-dioxygenase.35 Apart from the 12-bromo group, other substituents may be related to the activity of compounds. The presence of 5-OH in 16 (entry 16) increases the activity of 15 (entry 14), especially in T-84 and A-549 cells. Other substituents that might be relevant are 12-OMe as in 28, 29, and 30 and 18-COOMe as in 27 (entry 21), 28 (entry 20), 29 (entry 18), and 30 (entry 19). In fact, the presence of COOMe in certain positions in thiazolidinone derivatives increases the cytotoxicity in the A549 cell line, exceeding the activity of compounds with a bromo substituent in the same positions.36 Lipophilicity (log P) is a key parameter in the biological effect of many compounds, because it can determine their capacity to penetrate biological membranes and therefore influences intracellular bioavailability.33 The log P values of the compounds were determined using MarvinSketch 16.6.20 software (ChemAxon Ltd., 2016) (Table 3). A significant

cancer cells represented by the three cell lines used in different ways. Accordingly, the compounds may differentially affect the cellular processes and signaling pathways that influence the biology of each tumor cell type. Therefore, it would be interesting to conduct studies to clarify the mechanism of action of these compounds and to identify the cellular and molecular elements involved in the antiproliferative effects of the taiwaniaquinoids.

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General Experimental Procedures. Optical rotations were measured on a PerkinElmer 341 polarimeter, utilizing a 1 dm length cell and CHCl3 as a solvent. IR spectra were recorded as thin films on a Mattson Satellite FTIR spectrophotometer with samples between NaCl plates. NMR spectra were recorded at 500 MHz for 1H and at 125 MHz for 13C on a Varian Direct Drive spectrometer. Chemical shifts are expressed in parts per million (δ scale) downfield from tetramethylsilane. The 13C NMR signals were assigned utilizing DEPT experiments and on the basis of heteronuclear correlations. HRMS data were acquired on a Q-TOF Waters Synap G2 spectrometer, utilizing the ESI technique. TLC was performed using silica gel 60 F254 precoated plates (0.25 mm) and visualized by UV fluorescence quenching and phosphomolybdic acid solution staining. Separations were carried out by flash chromatography on silica gel 60 (230−400 mesh), utilizing MTBE/hexanes mixtures as eluent. tert-Butyl methyl ether (MTBE) was also utilized for extraction. CH2Cl2 was dried over CaH2. Benzene and THF were dried over Na-benzophenone. Solvents were removed on a rotary evaporator at temperatures below 45 °C. Syntheses of Compounds 10, 11, 13, 16, 17, and 20−36. (5S,7R,10S)-6-Hydroxy-5(6→7)abeo-abieta-8,12-diene-11,14-dione (10). NaBH4 (109 mg, 2.88 mmol) was added to a stirred solution of 9 (260 mg, 0.72 mmol) in THF (5 mL), and the mixture was stirred at 0 °C for 5 min, at which time TLC showed the complete consumption of 9. KOH (2 N) in MeOH (2 mL) was added, and the mixture was stirred at room temperature for 20 min. The solvent was removed under vacuum, and MTBE (50 mL)/H2O (20 mL) was added. The phases were shaken and separated, and the organic phase was washed with H2O (3 × 20 mL) and brine (3 × 20 mL) and dried over anhydrous Na2SO4. Removal of the solvent under vacuum afforded a crude product, which was purified by flash chromatography (20% MTBE/hexanes) to yield 167 mg of 10 (73%) as a yellow syrup: 1H NMR (CDCl3, 500 MHz) δ 1.05 (s, 3H), 1.14 (s, 3H), 1.17 (s, 3H), 1.22 (d, J = 7.1 Hz, 6H), 1.26 (m, 1H), 1.48−1.82 (m, 4H), 1.67 (d, J = 11.3 Hz, 1H), 2.20 (m, 1H), 3.26 (m, 1H), 3.39 (h, J = 7.1 Hz, 1H), 3.68 (dd, J = 11.7, 11.7 Hz, 1H), 4.65 (dd, J = 11.7, 1.8 Hz, 1H), 6.47 (s, 1H); HRESIMS m/z 339.1942 [M + Na]+ (calcd for C20H28O3Na, 339.1936). (5S,7S,8S,9R,10S)-6,12-Dihydroxy-8,9-epoxy-5(6→7)abeo-abiet12-ene-11,14-dione (11). KOH (2 N) in MeOH (1 mL) was added to a solution of 9 (78 mg, 0.225 mmol) in MeOH (5 mL), and the mixture was stirred at room temperature for 13 h, at which time TLC showed no remaining starting material. The solvent was removed under vacuum, MTBE (30 mL)/H2O (10 mL) was added, and the phases were shaken and separated. HCl (2 N, 2 mL) was added slowly to the aqueous phase, and the mixture was diluted with ether (30 mL). The organic phase was washed with H2O (3 × 20 mL) and brine (3 × 20 mL), dried over Na2SO4, and concentrated to afford a crude product, which was purified by flash chromatography on silica gel (35% MTBE/hexanes) to give 11 (54 mg, 73%) as a yellow syrup: [α]25D −32 (c 0.2, CHCl3); IR (film) 3398, 1696, 1638, 1463, 1382, 1286, 1258, 1094, 1041, 969, 904, 798 cm−1; 1H NMR (CDCl3, 500 MHz) δ 1.00 (s, 3H), 1.01 (s, 3H), 1.07 (s, 3H), 1.19 (d, J = 7.1 Hz, 3H), 1.23 (d, J = 7.1 Hz, 3H), 1.45 (dt, J = 13.4, 4.6 Hz, 1H), 1.52− 1.71 (m, 5H), 2.16 (dt, J = 12.8, 3.8, 1H), 2.54 (ddd, 11.5, 8.2, 2.7 Hz, 1H), 3.13 (h, J = 7.1 Hz, 1H), 3.65 (dd, J = 11.5, 8.2 Hz, 1H), 4.22 (dd, J = 11.5, 8.2 Hz, 1H), 6.97 (s, 1H); 13C NMR (CDCl3, 125 MHz) δ 32.1 (C-1), 19.0 (C-2), 41.6 (C-3), 33.4 (C-4), 48.3 (C-5), 61.9 (C6), 42.0 (C-7), 66.1 (C-8), 67.8 (C-9), 42.0 (C-10), 188.0 (C-11), 152.7 (C-12), 127.6 (C-13), 194.3 (C-14), 25.4 (C-15), 19.3 (C-16)*,

Table 3. Log P of Taiwaniaquinoids Determined Using MarvinSketch 16.6.20 Software compound

log P

compound

log P

32 29 30 11 4632e 4232e 432e 4532e 532e 28 10 36 27

2.6 2.7 2.8 3.0 3.1 3.2 3.4 3.5 3.5 3.7 3.7 3.9 4.1

9 4332e 17 4032b 3732b 16 4432e 3932b 13 3832b 15 4132b

4.2 4.3 4.3 4.4 4.4 4.5 4.7 4.8 4.9 5.0 5.3 5.4

EXPERIMENTAL SECTION

negative correlation (p < 0.05) between IC50 and log P values in MCF-7 and T-84 cells was found. However, the correlation is weak (Pearson’s correlation coefficient −0.48 for MCF-7 and −0.4 for T-84). Therefore, lipophilicity may affect the activity of the compounds in both cell lines, but does not seem to be the most important factor. The results in this paper indicate that the taiwaniaquinoids might be useful as cytostatic agents, in breast, colon, and lung cancer cell lines. The structure−activity relationship analysis suggests that taiwaniaquinoids could affect the different types of 313



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19.8 (C-17)*, 34.8 (C-18), 23.1 (C-19), 19.3 (C-20); HRESIMS m/z 371.1844 [M + Na]+ (calcd for C20H28O5Na, 371.1834); *interchangeable signals. (5S,7R,10S)-6-Acetyloxy-5(6→7)abeo-abieta-8,11,13-triene11,14-diol (13). TFA (182 mg, 1.6 mmol) was added to a solution of 9 (135 mg, 0.32 mmol) and H2O (0.1 mL) in THF (5 mL) at room temperature. The reaction mixture was stirred at this temperature for 1 h. The mixture was poured into MTBE (50 mL)/H2O (20 mL), and the phases were shaken and separated. The organic phase was washed with H2O (3 × 20 mL) and brine (3 × 20 mL) and dried over Na2SO4. Removal of the solvent under vacuum afforded a crude product, which was purified by flash chromatography on silica gel (25% MTBE/hexanes) to yield 95 mg of 13 (82%) as a colorless syrup: 1H NMR (CDCl3, 500 MHz) δ 1.07 (s, 6H), 1.15 (s, 3H), 1.16 (d, J = 7.0 Hz, 3H), 1.19 (d, J = 7.0 Hz, 3H), 1.20−1.78 (m, 5H), 1.75 (d, J = 11.0 Hz, 1H), 2.17 (s, 3H), 2.28 (m, 1H), 3.17 (m, 1H), 3.26 (h, J = 7.0 Hz, 1H), 4.56 (dd, J = 11.6, 7.1 Hz, 1H), 4.68 (dd, J = 11.6, 7.1 Hz, 1H), 6.47 (s, 1H), 7.36 (brs, 1H); HRESIMS m/z 383.2205 [M + Na]+ (calcd for C22H32O4Na, 383.2198). (10R)-12-Bromo-5-hydroxy-5(6→7)abeo-abieta-7(6),8,12-triene11,14-dione (16). SeO2 (143 mg, 1.29 mmol) was added to a solution of 15 (164 mg, 0.43 mmol) in dioxane (10 mL), and the mixture was refluxed for 7 h. After evaporation of the solvent, MTBE (30 mL) was added, and the resulting solution was washed with H2O (3 × 10 mL) and brine (2 × 10 mL), dried over anhydrous Na2SO4, and evaporated to give 16 (129 mg, 87%) as a yellow syrup: 1H NMR (CDCl3, 500 MHz) δ 0.75 (s, 3H), 1.00 (s, 3H), 1.12 (s, 3H), 1.23 (d, J = 6.9 Hz, 3H), 1.24 (d, J = 6.9 Hz, 3H), 1.20−1−70 (m, 6H), 2.66 (m, 1H), 3.45 (h, J = 6.9 Hz, 1H), 5.48 (s, 1H), 6.26 (s, 1H); 13C NMR (CDCl3, 125 MHz) δ 19.2 (CH2), 20.0 (CH3), 20.2 (CH3), 24.6 (CH), 26.0 (CH3) 28.1 (CH3), 29.9 (CH2), 34.2 (CH3), 35.2(CH2), 38.1 (C), 50.6 (C), 86.2 (C), 117.6 (CH2), 136.6 (C), 141.3 (C), 148.8 (C), 150.9 (C), 152.6 (C), 178.0 (C), 182.1 (C); HRESIMS m/ z 415.0890 [M + Na]+ (calcd for C20H25O3BrNa, 415.0885). (5S,7R,10S)-12-Bromo-6-hydroxy-5(6→7)abeo-abieta-8,12diene-11,14-dione (17). NaBH4 (62 mg, 1.64 mmol) was added to a stirred solution of 14 (182 mg, 0.41 mmol) in THF (4 mL), and the reaction mixture was further stirred at 0 °C for 5 min, at which time TLC showed the complete consumption of 14. Then, 2 N KOH in MeOH (1 mL) was added, and the mixture was stirred at room temperature for 20 min. The solvent was removed under vacuum, and MTBE (40 mL)/H2O (15 mL) was added. The phases were shaken and separated, and the organic phase was washed with H2O (3 × 20 mL) and brine (3 × 10 mL) and dried over anhydrous Na2SO4. Removal of the solvent under vacuum afforded a crude product, which was purified by flash chromatography (20% MTBE/hexanes) to yield 128 mg of 17 (78%) as a yellow syrup: 1H NMR (CDCl3, 500 MHz) δ 1.07 (s, 6H), 1.11 (s, 3H), 1.27 (d, J = 7.1 Hz, 3H), 1.30 (d, J = 7.1 Hz, 3H), 1.24 (m, 1H), 1.41−1.59 (m, 2H), 1.55−1.75 (m, 2H), 1.67 (d, J = 11.4 Hz, 1H), 2.32 (dt, J = 12.9, 4.1, 4.1 Hz, 1H), 3.14 (ddd, J = 11.4, 7.2, 1.8, Hz, 1H), 3.39 (h, J = 7.1 Hz, 1H), 3.68 (dd, J = 11.7, 7.2 Hz, 1H), 4.39 (dd, J = 11.7, 1.8 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ 19.4 (CH2), 19.8 (CH3), 20.1 (CH3), 20.4 (CH3), 20.4 (CH3) 22.6 (CH), 34.4 (CH2), 34.5 (CH3), 42.0 (CH2), 47.7 (CH), 48.0 (C), 57.1 (CH), 61.3 (C), 61.8 (CH2), 135.6 (C), 148.8 (C), 152.8 (C), 156.1 (C), 177.6 (C), 185.3 (C); HRESIMS m/z 417.1037 [M + Na]+ (calcd for C20H27O3BrNa, 417.1041). Methyl (4R,5R,9R,10S,13S,14R) 13,14-di-O-isopropyliden-7,8dioxo-7,8-seco-abietan-18-oate (20). An O3/O2 mixture at −78 °C was slowly bubbled through a stirred solution of 19 (4.76 g, 12.21 mmol) in CH2Cl2 (120 mL)/MeOH (40 mL), and the course of the reaction was monitored by TLC. When the starting material was consumed (50 min), the solution was flushed with Ar, and methyl sulfide (15 mL) was added. The mixture was further stirred at room temperature under an Ar atmosphere for 4 h, and the solvent was removed. Flash chromatography on silica gel (30% MTBE/hexanes) gave ketoaldehyde 20 (4.58 g, 89%) as colorless syrup: [α]25D +29 (c 0.1, CHCl3): IR (film) νmax 1720, 1370, 1239, 1209, 1089, 1055, 1006, 891, 755 cm−1; 1H NMR (CDCl3, 500 MHz) δ 0.91 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.8 Hz, 3H), 1.14 (s, 3H), 1.28 (s, 3H), 1.43 (s, 3H),

1.48 (s, 3H), 1.52−1.67 (m, 5H), 1.76−1.91 (m, 5H), 1.94−2.10 (m, 2H), 2.43 (dd, J = 5.1, 1.8 Hz, 2H), 2.94 (h, J = 6.8 Hz, 1H), 3.64 (s, 3H), 4.04 (s, 1H), 9.70 (s, 1H); 13C NMR (CDCl3, 125 MHz) δ 35.3 (C-1), 17.7 (C-2), 42.6 (C-3), 47.1 (C-4), 39.7 (C-5), 201.9 (C-6), 33.4 (C-7), 209.5 (C-8), 56.2 (C-9), 31.5 (C-10), 26.7 (C-11), 19.9 (C-12), 89.6 (C-13), 84.5 (C-14), 38.5 (C-15), 18.3 (C-16)*, 20.3 (C17)*, 178.5 (C-18), 22.6 (C-19), 16.4 (C-20), 109.8 (O−C−O), 27.4 (CH3−C-O), 27.6 (CH3−C−O), 52.1 (COOCH3); HRESIMS m/z 445.2575 [M + Na]+ (calcd for C24H38O6Na, 445.2566); *interchangeable signals. Methyl (4R,5R,7R,8S,9R,10S,13S,14S)-8-hydroxy-13,14-di-O-isopropyliden-6-oxo-5(6→7)abeo-abietan-18-oate (21). DBU (1.8 g, 11.84 mmol) was added to a stirred solution of ketoaldehyde 20 (2.5 g, 5.92 mmol) in benzene (30 mL), and the mixture was stirred under reflux for 3 h, at which time TLC showed the consumption of 20. Solvent was evaporated under vacuum, and the residue was purified by flash chromatography on silica gel (30% MTBE/hexanes) to afford aldehyde 21 (2.05 g, 82%) as colorless syrup: [α]25D +14 (c 0.2, CHCl3); IR (film) νmax 3464, 1725, 1456, 1378, 1244, 1208, 1145, 1108, 1050, 995, 772 cm−1; 1H NMR (CDCl3, 500 MHz) δ 0.97 (s, 3H), 0.98 (d, J = 6.8 Hz, 3H), 1.03 (d, J = 6.8 Hz, 3H), 1.32 (s, 3H), 1.41 (s, 3H), 1.42 (s, 3H), 1.45−1.57 (m, 4H), 1.60−1.80 (m, 8H), 1.91 (h, J = 6.8 Hz, 8H), 2.65 (d, J = 12.8 Hz, 1H), 2.96 (dd, J = 12.8, 1.4 Hz, 1H), 3.62 (s, 3H), 4.02 (s, 1H), 9.57 (d, J = 1.5 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ 37.0 (C-1), 15.9 (C-2), 39.2 (C-3), 44.6 (C-4), 50.5 (C-5), 206.2 (C-6), 55.3 (C-7), 78.7 (C-8), 60.9 (C-9), 41.5 (C-10), 27.9 (C-11), 18.8 (C-12), 85.9 (C-13), 80.3 (C-14), 37.6 (C-15), 18.1 (C-16)*, 18.2 (C-17)*, 178.8 (C-18), 17.3 (C-19), 16.8 (C-20), 106.6 (O−C−O), 26.5 (CH3−C−O), 27.1 (CH3−C−O), 52.0 (COOCH3); HRESIMS m/z 445.2559 [M + Na]+ (calcd for C24H38O6Na, 445.2566); *interchangeable signals. Reduction of Aldehyde 21. NaBH4 was added to a stirred solution of aldehyde 21 in the specified solvent (15 mL), and the reaction mixture was stirred at room temperature until TLC showed the complete consumption of 21 (see Table 1). The reaction mixture was quenched with H2O (5 mL), diluted with MTBE (70 mL), and washed with H2O (3 × 15 mL) and brine (3 × 20 mL). The organic phase was dried over Na2SO4 and concentrated to give a crude, which was purified by chromatography on silica gel (35% MTBE/hexanes). (4R,5R,7S,8S,9R,10S,13S,14S)-13,14-Di-O-isopropyliden-5(6→7) abeo-abietane-6,8,18-triol (23): white solid; mp 169 °C; [α]25D −14.5 (c 0.1, CHCl3); IR (KBr) νmax 3313, 1377, 1248, 1208, 1181, 1054, 995, 772 cm−1; 1H NMR (CDCl3, 500 MHz) δ: 0.81 (s, 3H), 0.92 (s, 3H), 1.00 (d, J = 6.9 Hz, 3H), 1.06 (d, J = 6.9 Hz, 3H), 1.10− 1.20 (m, 2H), 1.15 (s, 3H), 1.25 (s, 1H), 1.35−1.39 (m, 2H), 1.42 (s, 3H), 1.52−1.62 (m, 2H), 1.69−1.75 (m, 4H), 1.88 (d, J = 13.5 Hz, 1H), 1.91 (h, J = 6.9 Hz, 1H), 2.12 (dd, J = 13.5, 2.7 Hz, 1H), 2.73 (brs, 3H), 2.98 (d, J = 12.0 Hz, 1H), 3.63 (d, J = 12.0 Hz, 1H), 3.91 (dd, J = 12.9, 3.5 Hz, 1H), 4.18 (d, J = 12.9 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ 36.4 (C-1), 16.1 (C-2), 40.1 (C-3), 38.1 (C-4), 63.4 (C-5), 62.3 (C-6), 44.8 (C-7), 81.1 (C-8), 44.4 (C-9), 41.5 (C10), 28.9 (C-11), 19.3 (C-12), 85.5 (C-13), 78.5 (C-14), 37.7 (C-15), 18.1 (C-16)*, 18.3 (C-17)*, 72.3 (C-18), 18.5 (C-19), 16.9 (C-20), 106.5 (O−C−O), 26.6 (CH3-acetonide), 27.1 (CH3-acetonide); HRESIMS m/z 419.2780 [M + Na]+ (calcd for C23H40O5Na, 419.2773; *interchangeable signals. Methyl (4R,5R,7S,8S,9R,10S,13S,14S)-6,8-dihydroxy-13,14-di-Oisopropyliden-5(6→7)abeo-abietan-18-oate (24): colorless syrup; 1 H NMR (CDCl3, 500 MHz) δ 0.92 (s, 3H), 1.00 (d, J = 6.8 Hz, 3H), 1.04 (d, J = 6.8 Hz, 3H), 1.29 (s, 3H), 1.34 (s, 3H), 1.42 (s, 3H), 1.45−1.84 (m, 10H), 1.94 (h, J = 6.08 Hz, 1H), 1.99−2.09 (m, 2H), 2.33 (d, J = 13.5 Hz, 1H), 3.51 (d, J = 12.0 Hz, 1H), 3.58 (d, J = 8.9 Hz, 1H), 3.71 (s, 3H), 3.96 (dd, J = 21.4, 11.6 Hz, 1H), 4.10 (s, 1H), 5.05 (s, 1H); 13C NMR (CDCl3, 125 MHz) δ 38.5 (C-1), 16.1 (C-2), 39.4 (C-3), 45.0 (C-4), 52.7 (C-5), 59.9 (C-6), 46.7 (C-7), 79.2 (C-8), 43.6 (C-9), 40.8 (C-10), 28.7 (C-11), 19.1 (C-12), 86.9 (C-13), 79.9 (C-14), 37.5 (C-15), 18.16 (C-16)*, 18.19 (C-17)*, 181.9 (C-18), 17.1 (C-19), 16.7 (C-20), 106.3 (C-acetonide), 26.1 (CH3-acetonide), 27.1 (CH3-acetonide), 61.5 (COOCH3); HRESIMS m/z 447.2735 [M + Na]+ (calcd for C24H40O6Na, 447.2723); *interchangeable signals. 314



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Methyl (4R,5R,7S,8S,9R,10S,13S,14S)-6-acetyloxy-8-hydroxy13,14-di-O-isopropyliden-5(6→7)abeo-abietan-18-oate (25). To a solution of 24 (1.38 g, 3.26 mmol) in pyridine (12 mL) at 0 °C was added Ac2O (6 mL), and the reaction mixture was stirred at room temperature for 1 h, at which time TLC showed no remaining starting material. The reaction mixture was cooled to 0 °C, H2O (6 mL) was added to quench the reaction, and the mixture was stirred for an additional 10 min. Then, it was diluted with ether (100 mL) and washed with H2O (1 × 20 mL), 2 N HCl (5 × 20 mL), H2O (1 × 20 mL) again, saturated aqueous NaHCO3 (5 × 20 mL), and brine (1 × 20 mL), and the organic phase was dried over Na2SO4. Removal of the solvent under vacuum afforded a crude product, which was purified by flash chromatography on silica gel (15% MTBE/hexanes) to give 1.43 g of 25 (94%) as a colorless syrup: 1H NMR (CDCl3, 500 MHz) δ 0.95 (s, 3H), 0.96 (d, J = 6.9 Hz, 3H), 1.02 (d, J = 6.9 Hz, 3H), 1.33 (s, 3H), 1.34 (s, 3H), 1.44 (s, 3H), 1.51−1.79 (m, 12H), 1.87 (h, J = 6.9 Hz, 1H), 2.02 (s, 3H), 2.18 (d, J = 13.3 Hz, 1H), 2.37−2.49 (m, 1H), 3.63 (s, 3H), 3.91 (s, 1H), 3.95 (dd, J = 11.2, 6.2 Hz, 1H), 4.17 (dd, J = 11.2, 8.4 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ 38.1 (C1), 16.0 (C-2), 39.6 (C-3), 45.0 (C-4), 51.9 (C-5), 63.2 (C-6), 49.2 (C-7), 78.0 (C-8), 43.2 (C-9), 41.1 (C-10), 28.6 (C-11), 18.9 (C-12), 85.6 (C-13), 79.3 (C-14), 37.6 (C-15), 18.1 (C-16)*, 18.2 (C-17)*, 179.2 (C-18), 16.8 (C-19), 16.4 (C-20), 106.5 (C-acetonide), 26.5 (CH3-acetonide), 27.1 (CH3-acetonide), 171.4 (CH3COO), 21.0 (CH3COO), 61.9 (COOCH3); HRESIMS m/z 489.2814 [M + Na]+ (calcd for C26H42O7Na, 489.2828); *interchangeable signals. Methyl (4R,5R,7R,10S,13S,14S)-6-acetyloxy-13,14-di-O-isopropyliden-5(6→7)abeo-abiet-8-en-18-oate (26). SOCl2 (0.35 mL, 5.78 mmol) was added slowly to a solution of 25 (1.35 g, 2.89 mmol) and pyridine (2 mL) in dry CH2Cl2 (40 mL) at −60 °C. The reaction mixture was stirred at this temperature under an Ar atmosphere for 10 min, at which time TLC showed that the starting material had been consumed. The reaction was quenched with saturated aqueous NaHCO3 (1 mL), and the cooling bath was removed. The mixture was poured into MTBE (120 mL)/H2O (30 mL), and the phases were shaken and separated. The organic phase was washed with 2 N HCl (3 × 10 mL) and brine and dried over Na2SO4. Removal of the solvent under vacuum afforded a crude product, which was purified by flash chromatography on silica gel (10% MTBE/hexanes) to yield 1.12 g of 26 (86%) as a colorless syrup: [α]25D +13 (c 0.1, CHCl3); IR (film) νmax 1739, 1376, 1365, 1239, 1140, 1064, 1035, 861 cm−1; 1H NMR (CDCl3, 500 MHz) δ 0.88 (d, J = 6.9 Hz, 3H), 0.96 (s, 3H), 1.00 (d, J = 6.9 Hz, 3H), 1.30 (s, 3H), 1.35 (s, 3H), 1.39 (s, 3H), 1.74−1.91 (m, 3H), 1.57−1.73 (m, 6H) 1.97 (h, J = 6.9 Hz, 1H), 2.02 (s, 3H), 2.27 (d, J = 11.0 Hz, 1H), 2.83 (brs, 2H), 3.59 (s, 3H), 4.05 (dd, J = 11.4, 6.2 Hz, 1H), 4.14 (dd, J = 11.4, 6.2 Hz, 1H), 4.31 (s, 1H); 13C NMR (CDCl3, 125 MHz) δ 33.38 (C-1), 17.2 (C-2), 37.0 (C-3), 44.7 (C-4), 54.6 (C-5), 63.2 (C-6), 41.2 (C-7), 131.8 (C-8), 150.3 (C-9), 45.8 (C10), 24.5 (C-11), 18.6 (C-12), 85.0 (C-13), 73.5 (C-14), 33.39 (C15), 17.2 (C-16)*, 18.1 (C-17)*, 178.6 (C-18), 18.9 (C-19), 16.3 (C20), 108.5 (O−C−O), 28.81 (CH3−C−O), 28.85 (CH3−C−O), 171.1 (CH3COO), 20.9 (CH3COO), 51.8 (COOCH3); HRESIMS m/z 471.2731 [M + Na]+ (calcd for C26H40O6Na, 471.2723); *interchangeable signals. Methyl (4R,5R,7R,10S,13S,14S)-6-acetyloxy-13,14-di-O-isopropyliden-11-oxo-5(6→7)abeo-abiet-8-en-18-oate (22). PCC (1.17 g, 5.44 mmol), pyridine (0.75 mL, 12.65 mmol), and Celite (2 g) were added to a stirred solution of 26 (0.575 g, 1.28 mmol) in dry benzene (25 mL), and the mixture was kept stirring at reflux under an Ar atmosphere for 3 days, at which time TLC showed no remaining starting material. The reaction was worked up by the addition of MTBE (50 mL), and the resulting mixture was filtered through a silica gel pad and washed with MTBE (30 mL). The solvent was evaporated to yield a crude product, which was chromatographed on silica gel (25% MTBE/hexanes) to yield 22 (451 mg, 76%): [α]25D +3 (c 0.2, CHCl3); IR (film) νmax 1741, 1676, 1376, 1235, 1161, 1139, 1054, 1023 cm−1; 1H NMR (CDCl3, 500 MHz) δ 0.90 (d, J = 6.8 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H), 1.12 (s, 3H), 1.27 (s, 3H), 1.35 (s, 3H), 1.40 (s, 3H), 1.46−1.53 (m, 1H), 1.60−1.85 (m, 5H), 2.05 (s, 3H), 2.07 (h, J = 6.8 Hz, 1H), 2.27 (dt, J = 12.9, 4.1 Hz, 1H), 2.42 (d, J = 11.7 Hz,

1H), 2.53 (d, J = 16.5 Hz, 1H), 2.63 (d, J = 16.5 Hz, 1H), 3.00 (dt, J = 8.8, 4.1 Hz, 1H), 3.60 (s, 3H), 4.19 (d, J = 12.1 Hz, 1H), 4.21 (d, J = 12.1 Hz, 1H), 4.57 (s, 1H).; 13C NMR (CDCl3, 125 MHz) δ 36.6 (C1), 18.4 (C-2), 41.2 (C-3), 44.5 (C-4), 53.1 (C-5), 61.8 (C-6), 42.7 (C-7), 146.9 (C-8), 156.7 (C-9), 45.2 (C-10), 194.3 (C-11), 33.7 (C12), 87.9 (C-13), 72.8 (C-14), 34.7 (C-15), 18.8 (C-16)*, 19.4 (C17)*, 178.2 (C-18), 16.9 (C-19), 15.9 (C-20), 109.6 (O−C−O), 28.7 (CH 3 −C−O), 28.9 (CH 3 −C−O), 170.9 (CH 3 COO), 20.8 (CH3COO), 51.9 (COOCH3); HRESIMS m/z 485.2516 [M + Na]+ (calcd for C26H38O7Na, 485.2515); *interchangeable signals. Methyl (4R,5R,7R,10S)-6-acetyloxy-12-bromo-11,14-dioxo-5(6→ 7)abeo-abieta-8,12-dien-18-oate (27). Bromine (0.18 mL, 7.22 mmol) was added to a solution of 22 (398 mg, 0.86 mmol) in CH2Cl2 (15 mL), and the mixture was stirred at room temperature for 40 min, at which time TLC showed the absence of 22. A 5% NaHSO3 solution (2 mL) was added, and the mixture was diluted with MTBE (60 mL). The organic phase was washed with H2O (2 × 15 mL) and brine (2 × 15 mL) and dried over anhydrous Na2SO4. Removal of the solvent under vacuum afforded the resulting crude residue, which was purified by flash chromatography on silica gel (30% MTBE/hexanes), giving the bromoquinone derivative 27 (348 mg, 84%) as a yellow syrup: [α]25D −49 (c 0.1, CHCl3); IR (film) νmax 1741, 1664, 1383, 1244, 1135, 1082, 1034 cm−1; 1H NMR (CDCl3, 500 MHz) δ 1.15 (s, 3H), 1.25 (d, J = 7.0 Hz, 3H), 1.31 (d, J = 7.0 Hz, 3H), 1.37 (s, 3H), 1.61−1.86 (m, 5H), 1.99 (s, 3H), 2.31−2.36 (m, 1H), 2.58 (d, J = 11.5 Hz, 1H), 3.17 (ddd, J = 11.5, 5.6, 2.2 Hz, 1H), 3.38 (h, J = 7.0 Hz, 1H), 3.60 (s, 3H), 4.35 (dd, J = 11.5, 2.2 Hz, 1H), 4.42 (dd, J = 11.5, 5.6 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ 33.5 (C-1), 18.4 (C-2), 36.5 (C-3), 41.5 (C-4), 52.6 (C-5), 62.2 (C-6), 42.3 (C-7), 146.0 (C8), 154.5 (C-9), 46.7 (C-10), 183.2 (C-11), 152.7 (C-12), 134.8 (C13), 177.7 (C-14), 34.1 (C-15), 20.10 (C-16)*, 20.03 (C-17)*, 177.3 (C-18), 17.1 (C-19), 19.6 (C-20), 170.9 (CH3 COO), 20.7 (CH3COO), 52.1 (COOCH3); HRESIMS m/z 503.1050 [M + Na]+ (calcd for C23H29O6BrNa, 503.1045); *interchangeable signals. Treatment of 27 with NaOMe in MeOH. To a solution of 27 (185 mg, 0.38 mmol) in dry MeOH (10 mL) was added NaOMe (104 mg, 1.92 mmol), and the solution was stirred at room temperature for 14 h, at which time TLC showed the complete consumption of 27. The solvent was removed, and MTBE (60 mL)/H2O (10 mL) was added. The organic phase was washed with H2O (2 × 15 mL) and brine (2 × 15 mL) and dried over anhydrous Na2SO4. Removal of the solvent under vacuum afforded a crude product, which was purified by flash chromatography on silica gel (10% MTBE/hexanes), affording 54 mg of 28 (38%) as a yellow syrup (30% MTBE/hexanes) and 84 mg of 29 (56%) as a yellow syrup. Methyl (4R,5R,10S)-12-methoxy-11,14-dioxo-5(6→7)abeo-abieta-7(6),8,12-trien-18-oate (28): [α]25D −83 (c 0.2 CHCl3); IR (film) νmax 1724, 1656, 1572, 1459, 1255, 1215, 1131, 749, 667 cm−1; 1H NMR (CDCl3, 500 MHz) δ 1.14 (s, 3H), 1.21 (d, J = 7.1 Hz, 3H), 1.23 (d, J = 7.1 Hz, 3H), 1.43 (s, 3H), 1.51−1.91 (m, 5H), 2.38 (d, J = 12.8 Hz, 1H), 3.07 (s, 1H), 3.23 (h, J = 7.1 Hz, 1H), 3.69 (s, 3H), 3.95 (s, 3H), 5.06 (d, J = 2.8 Hz, 1H), 6.15 (d, J = 2.8 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ 33.6 (C-1), 18.7 (C-2), 37.3 (C3), 44.0 (C-4), 57.5 (C-5), 115.2 (C-6), 140.6 (C-7), 143.4 (C-8), 156.1 (C-9), 46.7 (C-10), 182.4 (C-11), 152.6 (C-12), 137.7 (C-13), 186.6 (C-14), 24.5 (C-15), 20.54 (C-16)*, 20.59 (C-17)*, 177.8 (C18), 15.6 (C-19), 21.2 (C-20), 52.3 (COOCH3), 61.1 (OCH3); HRESIMS m/z 395.1846 [M + Na]+ (calcd for C22H28O5Na, 395.1834); *interchangeable signals. Methyl (4R,5R, 7R,10S)-6-hydroxy-12-methoxy-11,14-dioxo5(6→7)abeo-abieta-8,12-dien-18-oate (29): [α]D25 −19 (c 0.1, CHCl3); IR (film) νmax 3565, 1723, 1658, 1556, 1429, 1245, 1218, 1129 cm−1; 1H NMR (CDCl3, 500 MHz) δ 1.17 (s, 3H), 1.19 (d, J = 7.0 Hz, 3H), 1.22 (d, J = 7.0 Hz, 3H), 1.65−1.84 (m, 5H), 2.49 (d, J = 11.8 Hz, 1H), 2.98 (ddd, J = 11.8, 5.5, 2.7 Hz, 1H), 3.21 (h, J =7.0 Hz, 1H), 3.33−3.42 (m, 1H), 3.71 (s, 3H), 3.95 (s, 3H), 4.02 (dd, J =12.3, 2.7 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ: 33.5 (C-1), 18.4 (C-2), 36.8 (C-3), 44.3 (C-4), 50.7 (C-5), 59.7 (C-6), 46.6 (C-7), 147.5 (C8), 155.9 (C-9), 45.9 (C-10), 181.8 (C-11), 154.1 (C-12), 137.5 (C13), 188.2 (C-14), 24.7 (C-15), 20.37 (C-16)*, 20.47 (C-17)*, 179.3 315



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(CDCl3, 125 MHz) δ 33.4 (C-1), 18.4 (C-2), 36.7 (C-3), 44.2 (C-4), 50.1 (C-5), 59.8 (C-6), 47.2 (C-7), 150.4 (C-8), 151.8 (C-9), 45.4 (C10), 181.1 (C-11), 150.7 (C-12), 125.2 (C-13), 187.6 (C-14), 24.1 (C15), 19.83 (C-16)*, 19.84 (C-17)*, 179.1 (C-18), 20.4 (C-19), 16.9 (C-20), 52.1 (COOCH3); HRESIMS m/z 399.1869 [M + Na]+ (calcd for C21H28O6Na, 399.1784); *interchangeable signals. (4R,5R,7S,8S,9R,10S,13S,14S)-6,18-Bis(acetyloxy)-13,14-di-O-isopropyliden-5(6→7)abeo-abietan-8-ol (33). To a solution of 23 (489 mg, 1.23 mmol) in pyridine (6 mL) at 0 °C was added Ac2O (36 mL), and the reaction mixture was stirred at room temperature for 1 h, at which time TLC showed the disappearance of starting material. The mixture was cooled to 0 °C, H2O (3 mL) was added to quench the reaction, and the mixture was stirred for an additional 10 min. The mixture was diluted with MTBE (60 mL) and washed with 2 N HCl (5 × 20 mL), H2O (1 × 20 mL), saturated aqueous NaHCO3 (2 × 20 mL), and brine (2 × 15 mL), and the organic phase was dried over Na2SO4. Removal of the solvent under vacuum afforded a crude product, which was purified by flash chromatography on silica gel (15% MTBE/hexanes) to yield 557 mg of 33 (94%) as a colorless syrup: [α]25D −12 (c 0.1, CHCl3); IR (film) νmax 1723, 1289, 1242, 1192, 1127, 1046, 1033, 772 cm−1; 1H NMR (CDCl3, 500 MHz) δ 0.96 (s, 3H), 0.97 (d, J = 6.8 Hz, 3H), 1.00 (s, 3H), 1.03 (d, J = 6.8 Hz, 3H), 1.23 (s, 1H), 1.32−1.39 (m, 3H), 1.34 (s, 3H), 1.44 (s, 3H), 1.45 (d, J = 12.7 Hz, 2H), 1.54 (d, J = 13.1 Hz, 2H), 1.61−1.71 (m, 4H), 1.87 (h, J = 6.8 Hz, 1H), 2.05 (s, 3H), 2.07 (s, 3H), 2.49 (ddd, J = 13.3, 8.4, 5.1 Hz, 1H), 3.74 (d, J = 11.3 Hz, 1H), 3.77 (d, J = 11.3 Hz, 1H), 3.97 (s, 1H), 4.20 (dd, J = 11.4, 8.5 Hz, 1H), 4.40 (dd, J = 11.4, 5.1 Hz, 1H). 13C NMR (CDCl3, 125 MHz) δ: 36.5 (C-1), 15.9 (C-2), 39.9 (C-3), 36.3 (C-4), 61.3 (C-5), 63.6 (C-6), 49.4 (C-7), 78.6 (C-8), 43.8 (C-9), 41.9 (C-10), 28.5 (C-11), 18.9 (C-12), 85.6 (C13), 80.1 (C-14), 37.6 (C-15), 18.02 (C-16)*, 18.08 (C-17)*, 73.4 (C18), 18.2 (C-19), 17.3 (C-20), 106.5 (C-acetonide), 26.5 (CH3acetonide), 28.9 (CH 3 -acetonide), 170.9 (CH 3 COO), 171.1 (CH3COO), 20.9 (CH3COO), 21.1 (CH3COO); HRESIMS m/z 503.2991 [M + Na]+ (calcd for C27H44O7Na, 503.2985); *interchangeable signals. (4R,5R,7R,10S,13S,14S)-6,18-Bis(acetyloxy)-13,14-di-O-isopropyliden-5(6→7)abeo-abiet-8-ene (34). SOCl2 (0.2 mL, 2.5 mmol) was added slowly to a solution of 33 (512 mg, 1.06 mmol) and pyridine (1 mL) in dry CH2Cl2 (10 mL) at −60 °C. The mixture was stirred at this temperature under an Ar atmosphere for 10 min, at which time TLC showed no starting material. The reaction was quenched with saturated aqueous NaHCO3 (1 mL), and the cooling bath was removed. The mixture was poured into MTBE (90 mL)/H2O (20 mL), and the phases were shaken and separated. The organic phase was washed with 2 N HCl (3 × 10 mL) and brine (2 × 10 mL) and dried over Na2SO4. Removal of the solvent under vacuum afforded a crude product, which was purified by flash chromatography on silica gel (10% MTBE/hexanes) to yield 444 mg of 34 (90%) as a colorless syrup: [α]25D +20 (c 0.1, CHCl3); IR (film) νmax 1741, 1375, 1239, 1063, 1034, 861 cm−1; 1H NMR (CDCl3, 500 MHz) δ 0.90 (d, J = 6.9 Hz, 3H), 0.97 (s, 3H), 1.01 (d, J = 6.9 Hz, 3H), 1.07 (s, 3H), 1.20− 1.30 (m, 2H), 1.33 (s, 3H), 1.36 (d, J = 4.6 Hz, 3H), 1.38 (d, J = 4.6 Hz, 1H), 1.41 (s, 3H), 1.45−1.47 (m, 1H), 1.59−1.66 (m, 3H), 1.68 (d, J = 10.8 Hz, 1H), 1.81−1.91 (m, 2H), 1.99 (h, J = 6.9 Hz, 1H), 2.05 (s, 3H), 2.91 (d, J = 10.5 Hz, 1H), 3.80 (d, J = 11.0 Hz, 1H), 3.88 (d, J = 11.0 Hz, 1H), 4.23 (dd, J = 11.9, 3.8 Hz, 1H), 4.34 (s, 1H), 4.43 (dd, J = 11.9, 3.8 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ: 33.8 (C1), 17.2 (C-2), 35.9 (C-3), 33.4 (C-4), 54.7 (C-5), 63.6 (C-6), 73.7 (C-7), 131.9 (C-8), 150.2 (C-9), 46.8 (C-10), 24.7 (C-11), 18.9 (C12), 85.6 (C-13), 77.2 (C-14), 36.8 (C-15), 18.2 (C-16)*, 18.6 (C17)*, 73.5 (C-18), 18.9 (C-19), 16.3 (C-20), 108.5 (O−C−O), 28.8 (CH3−C−O), 28.9 (CH3−C−O), 170.92 (CH3COO), 170.98 (CH3COO), 20.92 (CH3COO), 20.95 (CH3COO); HRESIMS m/z 485.2867 [M + Na]+ (calcd for C27H42O6Na, 485.2879); *interchangeable signals. (4R,5R,7R,10S,13S,14S)-6,18-Bis(acetyloxy)-13,14-di-O-isopropyliden-5(6→7)abeo-abiet-8-en-11-one (35). PCC (800 mg, 3.72 mmol), pyridine (0.17 mL, 2.9 mmol), and Celite (2 g) were added to a stirred solution of 34 (407 mg, 0.88 mmol) in dry benzene (40

(C-18), 20.6 (C-19), 17.0 (C-20), 52.3 (COOCH3), 61.1 (OCH3); HRESIMS m/z 413.1931 [M + Na]+ (calcd for C22H30O6Na, 413.1940); *interchangeable signals. Methyl (4R,5R,7R,10S)-12-methoxy-6,11,14-trioxo-5(6→7)abeoabieta-8,12-dien-18-oate (30). PDC (240 mg, 0.64 mmol) was added to a stirred solution of 29 (50 mg, 0.13 mmol) in dry CH2Cl2 (15 mL), and the mixture was stirred at room temperature under an Ar atmosphere for 30 h, at which time TLC showed no remaining starting material. The reaction was worked up by the addition of MTBE (10 mL), and the resulting mixture was filtered through a silica gel pad and washed with MTBE (2 × 15 mL). The solvent was evaporated, and the mixture was separated by chromatography (30% MTBE/hexanes) to give 38 mg (76%) of 30 as yellow syrup: [α]25D = −52 (c 0.1, CHCl3); IR (film) νmax 1727, 1723, 1658, 1556, 1427, 1247, 1217, 1131, 935 cm−1; 1H NMR (CDCl3, 500 MHz) δ 1.05−1.12 (m, 1H), 1.16 (d, J =7.0 Hz, 3H), 1.17 (d, J =7.0 Hz, 3H), 1.18 (s, 3H), 1.26−1.30 (m, 1H), 1.37 (s, 3H), 1.64−1.73 (m, 1H), 1.76−1.86 (m, 1H), 2.33 (d, J =11.9 Hz, 1H), 2.71 (d, J =11.5 Hz, 1H), 3.17 (h, J = 7.0 Hz, 1H), 3.56 (s, 3H), 3.68 (dd, J = 6.9, 2.9 Hz, 1H), 3.70 (dd, J = 6.9, 2.9 Hz, 1H), 3.94 (s, 3H), 9.59 (d, J = 4.0 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ 33.9 (C-1), 18.4 (C-2), 35.8 (C-3), 44.3 (C-4), 53.6 (C-5), 198.6 (C6), 54.6 (C-7), 146.0 (C-8), 156.4 (C-9), 48.5 (C-10), 181.3 (C-11), 154.1 (C-12), 137.1 (C-13), 185.7 (C-14), 24.7 (C-15), 20.37 (C16)*, 20.45 (C-17)*, 177.1 (C-18), 20.47 (C-19), 17.1 (C-20), 52.1 (COOCH3), 61.2 (OCH3); HRESIMS m/z 411.1769 [M + Na]+ (calcd for C22H28O6Na, 411.1784); *interchangeable signals. Methyl (4R,5R,7R,10S,12R,13S,14S)-6,12-bis(acetyloxy)-13,14-diO-isopropyliden-11-oxo-5(6→7)abeo-abiet-8-en-18-oate (31). Pb(OAc)4 (328 mg, 0.74 mmol) was added to a solution of ketone 22 (114 mg, 0.758 mmol) in dry benzene (25 mL), and the mixture was stirred at reflux for 3 days, at which time TLC showed complete consumption of 22. The mixture was filtered through a silica gel pad, washed with MTBE (50 mL). The organic phase was washed with 5% aqueous NaHSO3 (10 mL), saturated aqueous NaHCO3 (3 × 10 mL), and brine, and dried over Na2SO4. Removal of the solvent under vacuum gave a crude product, which was purified by flash chromatography on silica gel (25% MTBE hexanes) to afford pure 31 (108 mg, 84%) as a colorless oil: [α]25D −3 (c 0.1, CHCl3); IR (film) νmax 1744, 1695, 1372, 1224, 1089, 1046, 756 cm−1; 1H NMR (CDCl3, 500 MHz) δ 0.81 (d, J = 7.1 Hz, 3H), 1.02 (d, J = 7.1 Hz, 3H), 1.11 (s, 3H), 1.36 (s, 3H), 1.41 (s, 3H), 1.53 (s, 3H), 1.59−1.77 (m, 5H), 1.79−1.91 (m, 1H), 2.07 (s, 3H), 2.24 (s, 3H), 2.42 (h, J = 7.1 Hz, 1H), 2.48 (d, J = 11.7 Hz, 1H), 3.10 (dt, J = 11.6, 3.9 Hz, 1H), 3.63 (s, 3H), 4.06 (dd, J = 12.2, 3.9 Hz, 1H), 4.24 (dd, J = 12.2, 4.0 Hz, 1H), 4.54 (s, 1H), 5.70 (s, 1H); 13C NMR (CDCl3, 125 MHz) δ 34.1 (C-1), 18.4 (C-2), 36.4 (C-3), 44.4 (C-4), 53.2 (C-5), 60.9 (C-6), 48.2 (C-7), 153.3 (C-8), 153.3 (C-9), 45.7 (C-10), 189.1 (C-11), 69.7 (C-12), 87.8 (C-13), 78.2 (C-14), 30.0 (C-15), 18.5 (C-16)*, 19.7 (C17)*, 178.1 (C-18), 16.9 (C-19), 17.3 (C-20), 110.5 (O−C−O), 27.0 (CH 3 −C-O), 27.9 (CH 3 −C-O), 170.1 (CH 3 COO), 170.7 (CH3COO), 20.7 (CH3COO), 20.8 (CH3COO), 52.2 (COOCH3); HRESIMS m/z 543.2584 [M + Na]+ (calcd for C28H40O9Na, 543.2570); *interchangeable signals. Methyl (4R,5R,7R,10S)-7,12-dihydroxy-11,14-dioxo-5(6→7)abeoabieta-8,12-dien-18-oate (32). Concentrated HCl (1 mL) was added to a stirred solution of 31 (95 mg, 0.18 mmol) in MeOH (10 mL) under an O2 atmosphere, and the reaction mixture was heated at 40 °C for 14 h, at which time TLC showed no starting material remaining. The solvent was removed under vacuum, and MTBE (60 mL)/H2O (20 mL) was added. The organic phase was washed with H2O (2 × 15 mL) and brine (2 × 15 mL) and dried over anhydrous Na2SO4. Removal of the solvent under vacuum afforded a crude product, which was purified by flash chromatography (25% MTBE/hexanes) to yield 56 mg of 32 (81%) as a yellow syrup: [α]25D −107 (c 0.1, CHCl3); IR (film) νmax 3375, 1724, 1644, 1454, 1311, 1284, 1248, 1132, 1045, 986, 759 cm−1; 1H NMR (CDCl3, 500 MHz) δ 1.16 (s, 3H), 1.20 (d, J = 7.1 Hz, 3H), 1.22 (d, J = 7.1 Hz, 3H), 1.37 (s, 3H), 1.63−1.83 (m, 6H), 2.28 (dd, J = 8.1, 3.8 Hz, 1H), 2.50 (d, J = 11.7 Hz, 1H), 2.95− 3.05 (m, 1H), 3.17 (h, J = 7.1 Hz, 1H), 3.38 (dd, J = 12.2, 5.8 Hz, 1H), 3.70 (s, 1H), 4.00 (d, J = 12.2 Hz, 1H), 6.97 (s, 1H); 13C NMR 316



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performed using Spearman and Pearson correlation tests, with p < 0.05 considered significant.

mL), and the mixture was stirred at reflux under an Ar atmosphere for 3 days, at which time TLC showed no remaining starting material. The reaction was worked up by the addition of MTBE (50 mL), and the resulting mixture was filtered through a silica gel pad and washed with MTBE (30 mL). The solvent was evaporated to yield a crude product, which was chromatographed on silica gel (25% MTBE/hexanes) to yield 35 (302 mg, 72%) as white solid: mp 141 °C; [α]25D +24.3 (c 0.1, CHCl3); IR (KBr) νmax 1740, 1674, 1374, 1233, 1029, 754 cm−1; 1 H NMR (CDCl3, 500 MHz) δ 0.90 (d, J = 6.8 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H), 1.06 (s, 3H), 1.12 (s, 3H), 1.25−1.45 (m, 6H), 1.28 (s, 6H), 1.41 (s, 3H), 1.82 (d, J = 11.6 Hz, 1H), 2.05 (s, 3H), 2.07 (s, 3H), 2.28 (dd, J = 13.0, 4.1 Hz, 1H), 2.52 (d, J = 16.5 Hz, 1H), 2.63 (d, J = 16.5 Hz, 1H), 3.07 (dd, J = 13.0, 4.1 Hz, 1H), 3.77 (d, J = 11.1 Hz, 1H), 3.85 (d, J = 11.1. Hz, 1H), 4.33 (dd, J = 12.2, 3.7 Hz 1H), 3.55 (dd, J = 12.2, 3.7 Hz, 1H), 4.59 (s, 1H); 13C NMR (CDCl3, 125 MHz) δ 35.2 (C-1), 18.7 (C-2), 41.1 (C-3), 36.6 (C-4), 53.8 (C-5), 62.4 (C-6), 42.9 (C-7), 146.9 (C-8), 156.8 (C-9), 46.1 (C-10), 194.5 (C-11), 33.9 (C-12), 87.8 (C-13), 72.8 (C-14), 34.7 (C-15), 19.0 (C16)*, 19.4 (C-17)*, 72.9 (C-18), 17.9 (C-19), 15.9 (C-20), 109.6 (Cacetonide), 28.7 (CH3-acetonide), 28.9 (CH3-acetonide), 170.6 (CH 3 COO), 170.8 (CH 3 COO), 20.88 (CH 3 COO), 20.90 (CH3COO); HRESIMS m/z 499.2660 [M + Na]+ (calcd for C27H40O7Na, 499.2672); *interchangeable signals. (4R,5R,7R,10S)-12-Bromo-6,18-bis(acetyloxy)-5(6→7)abeo-abieta-8,12-diene-11,14-dione (36). Bromine (0.11 mL, 4.2 mmol) was added to a solution of 35 (237 mg, 0.5 mmol) in CH2Cl2 (12 mL), and the mixture was stirred at room temperature for 1 h, at which time TLC showed the disappearance of 35. A 5% NaHSO3 solution (1 mL) was added, and the mixture was diluted with MTBE (60 mL). The organic phase was washed with H2O (2 × 15 mL) and brine (2 × 15 mL) and dried over anhydrous Na2SO4. Removal of the solvent under vacuum afforded the resulting crude residue, which was purified by flash chromatography on silica gel (35% MTBE/hexanes), giving the bromoquinone derivative 36 (202 mg, 82%) as a yellow syrup: [α]25D −57 (c 0.1, CHCl3); IR (film) νmax 1738, 1661, 1221, 1033, 772 cm−1; 1 H NMR (CDCl3, 500 MHz) δ 1.07 (s, 3H), 1.15 (s, 3H), 1.24 (d, J = 7.0 Hz, 3H), 1.32 (d, J = 7.0 Hz, 3H), 1.38−1.49 (m, 2H), 1.52 (ddd, J = 15.4, 11.6, 5.6 Hz, 2H), 1.65−1.85 (m, 2H), 1.98 (s, 3H), 2.02 (s, 3H), 2.30−2.41 (m, 1H), 3.25 (ddd, J = 11.3, 3.5, 2.0 Hz, 1H), 3.37 (h, J = 7.0 Hz, 1H), 3.81 (d, J = 11.2 Hz, 1H), 3.87 (d, J = 11.2 Hz, 1H), 4.33 (dd, J = 11.8, 1.9 Hz, 1H), 4.82 (dd, J = 11.8, 3.6 Hz, 1H); 13 C NMR (CDCl3, 125 MHz) δ 33.8 (C-1), 18.5 (C-2), 35.3 (C-3), 36.9 (C-4), 52.4 (C-5), 62.5 (C-6), 42.4 (C-7), 146.5 (C-8), 154.1 (C9), 47.8 (C-10), 183.5 (C-11), 152.9 (C-12), 134.7 (C-13), 177.4 (C14), 34.1 (C-15), 19.6 (C-16)*, 20.1 (C-17)*, 72.4 (C-18), 20.8 (C19), 18.1 (C-20), 170.4 (CH3COO−), 170.7 (CH3COO), 20.73 (CH3COO), 20.78 (CH3COO); HRESIMS m/z 517.1193 [M + Na]+ (calcd for C24H31O6BrNa, 517.1202); *interchangeable signals. Biological Assays. Cell Lines and Culture. Human breast adenocarcinoma line MCF-7, human lung tumor line A-549, and human colorectal carcinoma line T-84 were supplied by the Department of Cell Cultures of the Granada University Scientific Instrumentation Centre. All lines were cultured at 37 °C in 5% CO2 and 90% humidity with Dulbecco’s modified Eagle medium, supplemented with 10% heat inactivated fetal bovine serum, 10 mL/ L penicillin−streptomycin 100×, and 2 mM L-glutamine. Culture media and respective supplements were supplied by Sigma-Aldrich (Madrid, Spain). In Vitro Antiproliferative Assay. In order to calculate the IC50 values of the compounds, 1 × 104 cells/cm2 were seeded in quadruplicate. After 24 h, the cells were induced with increasing compound concentrations for 3 days. Subsequently, the cells were fixed with 10% cold trichloroacetic acid (4 °C) and stained with 0.4% sulforhodamine in 1% HOAc. The colorant was solubilized with 10 mM Tris-base pH 10.5, and optical density values were determined by colorimetry at 492 nm (Multiskan EX, Thermo Electron Corporation). IC50 values were calculated from the semilogarithmic dose− response curve by linear interpolation. Statistical Analysis. SPSS 14 for Windows (SPSS, Chicago, IL, USA) was used for the statistical analysis. The correlation analysis was

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00700. Copies of 1H and 13C NMR spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel (R. Chahboun): (+34) 958 248 089. E-mail: rachid@ugr. es. *E-mail (E. Alvarez-Manzaneda): [email protected]. ORCID

Enrique Alvarez-Manzaneda: 0000-0002-3659-4475 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Spanish Ministry of Science and Innovation (Project CTQ2014-56611-R/BQU), the Regional Government of Andalusia (Project P11-CTS-7651 and assistance for the FQM-348 and CTS-107 groups), and the Erasmus Mundus−Al Idrisi predoctoral fellowship granted to S.M. This paper is related to the Ph.D. thesis of N.M.-S.

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REFERENCES

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