Studies on quinones. Part 40: Synthesis and cytotoxicityevaluation of anthraquinone epoxides and

isomerization products*

Jaime A. Valderrama,a,* Omar Espinoza,a M. Florencia Gonzalez,a Ricardo A. Tapia,a

Jaime A. Rodrıguez,b Cristina Theodulozb and Guillermo Schmeda-Hirschmannb

aFacultad de Quımica, Pontificia Universidad Catolica de Chile, Casilla 306, Santiago 6094411, ChilebLaboratorio de Cultivo Celular, Facultad de Ciencias de la Salud, and Instituto de Quımica de Recursos Naturales,

Universidad de Talca, Casilla 747, Talca, Chile

Received 6 September 2005; revised 14 November 2005; accepted 15 December 2005

Available online 18 January 2006

Abstract—Aerobic oxidation of 1,4,4a,10a-tetrahydro-1,4-alkano-5,10-anthraquinones and thiophene-analogues in dichloromethane–DBUyielded the corresponding dihydroalkanoquinones which, depending on their structures, react with in situ generated hydroperoxide anion togive quinone epoxides and/or hydroperoxides. The calcium hydroxide-induced rearrangement of quinone epoxides yielded furan-containingangular quinones. The cytotoxic activities of quinone epoxides and their isomerization products were evaluated in vitro against normalhuman lung fibroblasts (MRC-5) and human cancer gastric epithelial cells (AGS).q 2005 Elsevier Ltd. All rights reserved.

1. Introduction

Epoxidation of quinone double bonds is of great interestbecause of the synthetic utility of the resulting epoxides forfurther functionalization of the quinones. Aqueous hydro-gen peroxide is the usual common epoxidation reagentbecause it is both inexpensive and safe, yielding water as thebyproduct. Nevertheless, this epoxidation procedure is notapplicable to base-sensitive quinones due to the stronglyalkaline conditions required (NaOH; KOH; K2CO3).2–5

We have recently reported a quinone epoxidation procedureunder non-aqueous conditions using the urea–hydrogenperoxide complex (UHP) in basic media (DBU; K2CO3),which is potentially useful for the epoxidation of base-sensitive quinones.6

In 1972 Giles4a reported that treatment of 1,4-naphthoqui-none-spirocyclopentadiene adduct 1 with aqueous metha-nolic sodium hydroxide in the presence of air yielded amixture of isomeric quinone epoxides 3C5 together with a

0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.tet.2005.12.038

* For Part 39, see Ref. 1.

Keywords: Diels–Alder adducts; Aerobic epoxidation; Rearrangement;Angular quinones; Cytotoxicity.* Corresponding author. Tel.: C56 2 6864432; fax: C56 2 6864744;

e-mail: jvalderr@uc.cl

minor quantity of angular quinone 7. Their findings showthat compound 7 arises from epoxides through a photo-induced rearrangement. Later, Marchand7 described theformation of quinone epoxides 4C6, together with smallamounts of quinone 8, by aerobic oxidation of adduct 2under conditions similar to those reported by Giles.4b

Taking into account the aerobic oxidation of phenols inaqueous sodium hydroxide reported by Hewgill,8 theprobable oxidation mechanism of adducts 1 and 2 involvessemiquinone radical and hydroperoxide anion species

Tetrahedron 62 (2006) 2631–2638

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generated by redox reactions of oxygen with phenolateanion intermediates.

As part of our continuous interest in the synthesis andbiological evaluation of quinones,9 we decided to study theoxidation of Diels–Alder adducts such as 2 aimed at thefollowing objectives: (i) to explore a quinone epoxidationprocedure by aerobic oxidation under non-aqueous basicconditions, potentially useful for base-sensitive quinoneepoxides, and (ii) to use the resulting quinone epoxides inthe synthesis of tetracyclic angular quinones such as 7, forcytotoxicity evaluation. The synthesis of new angularquinones is of current interest due to the fact that a widevariety of compounds containing an angular tetracyclicskeleton arrangement have antitumoral activities.10

In this work, we report the results of our studies on aerobicoxidation of Diels–Alder adducts of naphthoquinones andbenzothiophenequinones with cyclopentadiene and cyclo-hexadiene, and their base-induced isomerization to angularquinones. We also report on the in vitro evaluation of theproductsagainstnormalfibroblasts(MRC-5)andgastriccancercells (AGS).

2. Results and discussion

In agreement with reports on the generation of superoxideanions and perhydroxyl radicals by electron-transfer reductionof molecular oxygen with phenolate anion11,12 and semi-quinone radicals,13,14 we examined the eventual formation ofhydroperoxide anions by aerobic oxidation of phenolates inorganic solvents. Menadione 9 was used for trapping thehydroperoxide anion formed in situ, which was generated viareduction of oxygen in these basic media.

The trials were run in an open flask containing a vigorouslystirred solution of menadione 9, with 1 equiv of thecorresponding phenol and 1 equiv of DBU in organic solution(CH2Cl2, MeCN, MeOH). The assays were conducted at rt andthe following phenols were tested: phenol, hydroquinone,resorcinol and 4-tert-butyl-2 methoxyphenol. In theseexperiments, resorcinol exhibited the best capability to givemenadione epoxide 10 in CH2Cl2 and MeCN. Thus, theaerobic treatment of menadione with 1 equiv of resorcinol and1 equiv of DBU in dichloromethane or acetonitrile for 30 and

Figure 1. Molecular models showing the nucleophilic attack of the hydroperoxide8 and 28.

80 min, respectively, produced menadione epoxide 10 in 35and 22% isolated yields. These experiments demonstrate theepoxidation capability of organic aerobic solutions containingphenolate anions.

Onthebasisoftheseresults,weattemptedtoextendthisaerobicprocedure to the synthesis of quinone epoxides from Diels–Alder adducts 2, 14–18. These compounds were prepared byreactionofeither cyclopentadiene orcyclohexa-1,3-diene withdienophiles 11–13 using standard procedures. It is appropriateto point out that base-induced enolization of Diels–Alderadducts with DBU in organic media would provide thephenolate anions required for the molecular oxygen reduction.

Aerobic oxidation of Diels–Alder adducts 2, 14–18 wereconducted under standard conditions using 2 equiv of DBU indichloromethane. We first examined the behavior of Diels–Alder adducts 2 and 15, derived from cyclopentadiene.Oxidation of adduct 2 for 4 h yielded a 4.6:1 mixture of exo-and endo-epoxides 4C6 in 68% yield, together with smallamounts of hydroperoxide 25. The exo/endo ratio wasdetermined by 1H NMR using the vinylic proton signals ofthe isomers at d 6.53 and 6.10 ppm, respectively. Quinoneepoxides 4 and 6 were identified by comparing their 1H and 13CNMR spectra with those reported by Giles4b and Marchand.7

The behavior of adduct 2 to aerobic oxidation in dichloro-methane–DBU is similar to the oxidation of 2 with aqueoushydrogen peroxide in ethanol–Na2CO3 reported by Paquette,5

whichyieldeda3:1mixtureofexo-andendo-quinoneepoxides4C6 along with trace amounts ofhydroperoxide 25. Oxidationof adduct 15 gave a 20:1 mixture of exo- and endo-epoxides21C22 in 62% yield, together with trace amounts ofhydroperoxides 26C27 (detected by 1H NMR). The exo/endo ratio was determinedby 1H NMR using the vinylic protonsignals of the isomers at d 6.52 and 6.15 ppm, respectively.

The formation of exo-epoxides as the main isomer in the airoxidations of adducts 2 and 15 implies that the endo faceof the quinone intermediates are more sterically crowded

anion on the quinone double bond through the less hindered face of quinone

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towards the approach of the hydroperoxide anion. Figure 1shows the favorable attack of the hydroperoxide anion onthe quinone double bond through the less hindered exoface of quinone intermediate 8.

Aerobic oxidation of Diels–Alder adducts 14 and 16 derivedfrom cyclohexadiene was examined. The reaction ofcompound 14 gave a mixture of quinone 28 and endo-epoxide 20 in approximately equal amounts (1H NMR),together with trace amounts of exo-epoxide 19. The structureof compounds 19, 20 and 28 were established by comparingtheir spectral data with those reported in literature.5 Due toour interest in the synthesis of angular quinones byisomerization of quinone epoxides, the reaction mixturearising from the aerobic oxidation of 14 was treated withurea hydrogen peroxide (UHP) to give endo-epoxide 20 in84% isolated yield.

A similar result was seen in the aerobic oxidation ofadduct 16, which yielded a mixture of quinone 29 andendo-epoxide 24 in nearly equal amounts, together withtrace quantities of exo-epoxide 23. Further treatment ofthe reaction mixture resulting from the aerobic oxidationof 16 with UHP yielded endo-epoxide 24 in 72%isolated yield. It should be noted that aerobic oxidationof 16 in refluxing dichloromethane gave quinone 29 in87% yield and no endo-epoxide 24 was detected.Apparently, under these conditions, formation of 29 byaerobic oxidation of 16 is much faster than itssubsequent epoxidation reaction to give 24.

The aerobic oxidation of Diels–Alder adduct 17showed a different reactivity with respect to adducts2 and 15. Thus, treatment of adduct 17 gave acomplex mixture of highly polar products. The 1HNMR of the crude product displays signals for themain product in agreement with hydroperoxide 30(unstable orange oil). Assignment of the regiochemistryof 30 was established by 2D NMR experiments(HMBC, 400 MHz) that displayed 3JC,H and 4JC,H

couplings for the proton at C-3 (d 7.90) with carbonatoms at C-4 (d 188.2) and C-4a (d 82.2), respectively.Further reaction of 30 with 2 equiv of DBU indichloromethane did not result in the formation ofquinone epoxides.

A different reactivity was seen for the aerobic oxidationof 18 with respect to 17. In fact, the reaction of adduct18 gave a 1:11 mixture of the exo/endo quinone epoxides31C32. The exo/endo ratio was determined by 1H NMRanalysis using the vinylic proton signals of the isomers atd 6.44 and 6.06 ppm, respectively.

According to our results on the aerobic oxidation of Diels–Alder adducts 2, 14–18 in dichloromethane–DBU, theprobable mechanism for the formation of quinone epoxidesis initiated by a sequence of two electron transfer processesmediated by phenoxide anion and semiquinone radical

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intermediates. The hydroperoxide anion generated by theseredox reactions undergoes conjugate addition to give ahydroperoxide intermediate that, by further cyclisation,yielded quinone epoxides (Scheme 1). The formation ofendo-epoxides as the main isomers in the aerobic oxidationsof adducts 14, 16 and 18 indicates that the endo-face of thequinone intermediates is less sterically crowded towards theapproach of the hydroperoxide anion (Fig. 1).

O

O

DBU

CH2Cl2

O

OH

O2 O2

O

OH

+ H+ HO2

O

OHO2

O

O

O2HDBU

4 + 6

2 phenolate anion

semiquinone

25

O2

8

Scheme 1. Probable mechanism of the aerobic oxidation of Diels–Alderadduct 2 in DBU–dichloromethane.

Table 1. Cytotoxic activity of quinones and quinone epoxides* againstMRC-5 fibroblasts and AGS gastric cells

Ent-ry

Compoundb IC50 (mM)a

Human normal lungfibroblasts (MRC-5)

Human gastric cancerepithelial cells (AGS)

1 20 O100 O1002 24 O100 O1003 29 41.6G1.4 15.0G2.34 32 O100 O1005 33 53.5G2.2 29.3G1.66 34 6.3G0.2 4.1G0.37 35 13.3G0.3 4.9G0.29 Camptothecin O100 O10010 Vinblastine 76.6G2.7 O100

a Values are meansGstandard error of the mean.b All compounds were quite stable in DMSO solution.

During the isolation of compounds 4C6 by columnchromatography, angular quinone 33 was detected. Thiscompound was also detected when the 4C6 mixture wasanalyzed by thin-layer chromatography. These evidencessuggest that quinone epoxides 4C6 undergo rearrange-ments to quinone 33 via photochemical4 or ionic pathways.

In order to verify the first assumption, solutions of epoxides4C6 in CDCl3 contained in NMR tubes were exposed tosunlight. The 1H NMR analyses of the samples showed that noreaction had occurred after 1 week of irradiation. Interestingly,when epoxides 4C6 were stirred with silica gel/gypsum indichloromethane at rt, they underwent smooth rearrangementto produce, after 7 days, quinone 33 in 45% isolated yield, thusconfirming an ionic process involved in the rearrangement ofquinone epoxides 4 and 6. Conversion of epoxides 4C6 toquinone 33 was improved (68% yield) by treatment of 4C6with calcium hydroxide in CH2Cl2 for 3 days at rt.

Quinone epoxide 21 was subjected to rearrangement withcalcium hydroxide, yielding angular quinones 34 and 35 in 9and 19% isolated yields, respectively. The structure of themajor regioisomer 35 was established by HMBC experi-ments, which showed 3JC,H between the carbon atom of thehydrogen-bonded carbonyl group at C-10 (d 188.4 ppm)with the proton at C-10b (d 4.17 ppm).

We tried to induce the rearrangement of quinone epoxides20 and 32 by microwave irradiation of the epoxides loaded

on calcium hydroxide and on silica gel/gypsum. However,no rearrangement products were detected.

The stable epoxides 20, 24, 32 and quinones 29, 33, 34 and35 were evaluated in vitro on normal fibroblast and gastriccancer cell lines. The results are reported in Table 1 as IC50

(concentration of compound expressed in mg/mL required toinhibit cell growth by 50% after 24 h of drug exposure).Vinblastine and camptothecin were used as reference drugs.

The screening indicates that quinone epoxides 20, 24, and32 (entries 1, 2 and 4) do not display cytotoxic effects onthese cell lines. Quinones 29, 33, 34 and 35 showedcytotoxic effects at micromolar concentrations, lowselectivity (i.e., ratio between cytotoxic effect on normalfibroblast and on gastric cancer cells) and were morecytotoxic than the reference drugs.

Comparison of the activities of angular quinones 33, 34 and35 (entries 6 and 7) indicates that the presence of a hydroxylgroup on the aromatic ring induces a significant increase incytotoxicity. It seems possible that the influence of thehydroxyl groups on the biological activity is due tohydrophobic interactions with biological targets and/or tothe increase of the redox potential of the quinone system.

The cytotoxic activities of compounds 29, 33, 34 and 35 areprobably due to the generation of reactive oxygen speciesafter redox cycling and/or alkylation of cellular nucleo-philes.15 In the case of angular quinones 33, 33 and 34,intercalation in DNA or binding to a DNA–enzyme complexcould also be important aspects involved in thebioactivity.10,16

In summary, we have reported the aerobic oxidation of1,4,4a,9a-tetrahydro-1,4-alkano-9,10-anthra-quinones indichloromethane–DBU solution, which yields the corres-ponding 1,4-alkanoanthraquinones, which by further epox-idation with in situ generated hydroperoxide anion or withUHP yielded the corresponding quinone epoxides. Thisapproach to the synthesis 1,4,4a,10a-tetrahydro-1,4-alkano-5,10-anthraquinone epoxides from Diels–Alder adductsoffers an alternative to previously reported preparationprocedures for this class of quinone epoxides. The salient

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aspects of this new procedure are simple workup, in situgeneration of the epoxidation reagent (i.e., 4, 6, 21, 22) andcompatibility with base-sensitive functional groups (OH,CO2CH3).3,6

Furthermore, we have shown that quinone epoxides with themethano bridge undergo isomerization with calciumhydroxide to the corresponding angular quinones, butquinone epoxides containing the ethano bridge are inertto this rearrangement. Anthraquinone 29 and the furan-containing angular quinones 33, 34 and 35 showed cytotoxicactivities, in the 4–54 mM concentration range, againstnormal fibroblasts and gastric cancer AGS cells. Sincecompounds 29, 33 and 35 showed selective activity, beingmore cytotoxic towards AGS cells than to fibroblasts theymight be good prototypes for the development of newanticancer drugs.

3. Experimental

All reagents were of commercial quality reagent grade andwere used without further purification. Melting points weredetermined on a Kofler hot-stage apparatus and areuncorrected. IR spectra were recorded on an FT Brukerspectrophotometer using KBr discs, and wave numbers aregiven in cmK1. 1H NMR spectra were measured on BrukerAM-200 and AM-400 equipment in CDCl3. Chemical shiftsare expressed in ppm downfield relative to TMS (d scale)and coupling constants (J) are reported in Hz. 13C NMRspectra were obtained in deuteriochloroform at 50 and100 MHz. 2D NMR techniques (COSY, HMBC) and DEPTwere used for signal assignment. Chemical shifts arereported in d ppm downfield from tetramethylsilane(TMS), and J values are given in Hz. Silica gel 60(70–230 mesh), and TLC aluminiun foil 60 F254 (Merck)were used for preparative column and analytical TLC,respectively. Heterocyclic quinone 13 was prepared fromcommercially available 2,5-dimethoxybenzaldehyde asreported previously.17

3.1. Aerobic epoxidation of menadione 9 in basic organicmedia. Typical procedure

A solution of quinone 9 (200 mg, 1.16 mmol), resorcinol(128 mg, 1.16 mmol) DBU (176 mg, 1.16 mmol) indichloromethane (15 mL) was vigorously stirred at rt in anopen flask for 30 min. The mixture was evaporated underreduced pressure and the dark residue chromatographedon silica gel (CH2Cl2) to give 2,3-epoxy-2-methyl-2,3-dihydro-1,4-naphthoquinone 10 (76 mg, 35%). The spectralproperties were in full agreement with those reported in theliterature.6

3.1.1. endo-1,4,4a,9a-Tetrahydro-1,4-methano-9,10-anthraquinone 2. A solution of quinone 11 (180 mg,1.14 mmol), cyclopentadiene (100 mg, 1.52 mmol) inCH2Cl2 (15 mL) was left for 2 h at rt. The mixture wasevaporated under reduced pressure and the residuechromatographed on silica gel, eluting with CH2Cl2, togive endo-cycloadduct 2 (220 mg, 86%) as a yellow solid,mp 119–120 8C (exo-cycloadduct: lit.18 mp 160 8C); IR:nmax 1675 (C]O); 1H NMR: d 1.53 (m, 2H, 11-H), 3.44

(m, 2H, 4a- and 9a-H), 3.63 (s, 2H, 1- and 4-H), 5.95 (s, 2H,2- and 3-H), 7.68 (m, 2H, 6- and 7-H), 8.00 (m, 2H, 5- and8-H); 13C NMR: d 49.2, 49.5, 126.8, 134.1, 135.5, 135.8,197.8. Anal. Calcd for C15H12O2: C, 80.34; H, 5.39. Found:C, 79.99; H, 5.39.

3.1.2. endo-5-Hydroxy-1,4,4a,9a-tetrahydro-1,4-methano-9,10-anthraquinone 15. A solution of quinone12 (250 mg, 1.44 mmol), cyclopentadiene (100 mg,1.52 mmol) in CH2Cl2 (15 mL) was left for 2 h at rt. Themixture was evaporated under reduced pressure and theresidue chromatographed on silica gel, eluting with CH2Cl2to give adduct 15 (330 mg, 95%) as a yellow solid, mp 132–133 8C; IR: nmax 3449 (OH), 1673 and 1632 (C]O); 1HNMR: d 1.54 (m, 2H, 11-H), 3.42 (m, 2H, 4a- and 9a-H),3.65 (s, 2H, 1- and 4-H), 6.00 (s, 2H, 2- and 3-H), 7.20 (m,1H, 6-H), 7.55 (m, 2H, 7- and 8-H), 12.59 (s, 1H, OH); 13CNMR: d 48.9, 49.3, 49.7, 49.9, 118.2, 123.5, 135.1, 135.8,137.0, 162.1, 196.9, 204.8. Anal. Calcd for C15H12O3: C,74.99; H, 5.03. Found: C, 74.76; H, 5.01.

3.1.3. endo-4,9-Dioxo-4,4a,5,8,8a,9-hexahydro-5,8-methano-2-methoxycarbonylnaphthto[2,3-b]tiophene17. Following the procedure described for the adducts ofcyclopentadiene, compound 17 was obtained (150 mg,96%) from 4,7-dihydro-2-methoxycarbonyl-4,7-dioxo-benzo[b]thiophene 13 (120 mg, 0.54 mmol), cyclopenta-diene (100 mg, 2.28 mmol) in CH2Cl2 (15 mL), mp 129–131 8C; IR: nmax 1725, 1712 and 1666 (C]O); 1H NMR: d1.55 (m, 2H, 10-H), 3.44 (m, 2H, 5- and 8-H), 3.62 (t, 2H,JZ1.7 Hz, 4a- and 9a-H), 3.91 (s, 3H, OCH3), 6.01 (t, 2H,JZ1.7 Hz, 6- and 7-H), 8.00 (s, 1H, 3-H); 13C NMR: d 49.2,49.3, 49.5, 51.0, 53.0, 131.0, 134.1, 135.2, 141.3, 145.2,151.9, 161.4, 1925, 192.6. Anal. Calcd for C15H12O4S: C,62.49; H, 4.20; S, 11.12. Found: C, 62.18; H, 4.31; S, 10.94.

3.1.4. endo-1,4,4a,9a-Tetrahydro-1,4-ethano-9,10-anthraquinone 14. A solution of 1,4-naphthoquinone 11(250 mg, 1.58 mmol), 1,3-cyclohexadiene (170 mg,2.1 mmol) in CH2Cl2 (15 mL), was left for 9 days at rt.Removal of the solvent followed by column chromatog-raphy (CH2Cl2/petroleum ether, 1:1) of the residue yieldedadduct 14 as yellow solid (110 mg, 29%), mp 132.5–133.0 8C; IR: nmax 1679 (C]O); 1H NMR: d 1.24 (m, 2H,12-H), 1.64 (m, 2H, 11-H), 3.07 (d, 2H, JZ1.1 Hz, 1- and4-H), 3.17 (t, 2H, JZ1.3 Hz, 4a- and 9a-H), 5.97 (c, 2H, JZ1.36, 3.1 Hz, 2- and 3-H), 7.52 (m, 2H, 6- and 7-H), 7.84 (m,2H, 5- and 8-H); 13C NMR: d 22.8, 23.9, 38.8, 81.5, 122.6,126.0, 126.3, 127.8, 131.6, 133.0, 133.2, 134.2, 135.2,160.0, 178.4, 182.4. Anal. Calcd for C16H14O2: C, 80.65; H,5.92. Found: C, 80.67; H, 5.99.

3.1.5. endo-1,4,4a,9a-Tetrahydro-5-hydroxy-1,4-ethano-9,10-anthraquinone 16. A solution of 12 (310 mg,1.78 mmol), cyclohexadiene (250 mg, 3.1 mmol) inCH2Cl2 (15 mL) was refluxed for 12 days. The mixturewas evaporated under reduced pressure and the residuechromatographed on silica gel (CH2Cl2/petroleum ether,1:1) to give adduct 16 (220 mg, 49%) as an orange solid, mp90–91 8C (lit.19 mp 89–90 8C); IR: nmax 3438 (OH), 1671,1623 (C]O); 1H NMR: d 1.38 (dd, 2H, JZ1.52, 6.0 Hz, 12-H), 1.76 (d, 2H, JZ7.2 Hz, 11-H), 3.22 (m, 2H, 1- and 4-H),3.35 (s, 2H, 4a- and 9a-H), 6.18 (t, 2H, JZ3.78 Hz, 2- and

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3-H), 7.19 (q, 1H, JZ1.8, 5.7 Hz, 7-H), 7.57 (q, 2H, JZ3.1,6.8 Hz, 6- and 8-H), 12.58 (s, 1H, OH); 13C NMR: d 24.8,25.0, 36.0, 36.1, 49.8, 50.2, 118.2, 123.3, 133.3, 134.0,135.4, 137.0, 161.9, 197.2, 204.6. Anal. Calcd C16H14O3: C,75.57; H, 5.55. Found: C, 75.29; H, 5.31.

3.1.6. endo-4,9-Dioxo-4,4a,5,8,8a,9-hexahydro-5,8-ethano-2-methoxycarbonylnaphtho[2,3-b]thiophene 18.A solution of 4,7-dihydro-2-methoxycarbonyl-4,7-dioxo-benzo[b]thiophene (130 mg, 0.59 mmol), cyclohexadiene(250 mg, 3.15 mmol), in CH2Cl2 (15 mL) was left for 4 daysat rt. Removal of the solvent and purification bychromatography (CH2Cl2) yielded adduct 18 (180 mg,95%) as a yellow solid, mp 102.5–103.5 8C; IR: nmax

1727, 1713, 1667 (C]O); 1H NMR: d 1.37 (m, 2H, 11-H),1.73 (m, 2H, 10-H), 3.15 (c, 2H, JZ2.15, 2.54 Hz, 5- and8-H), 3.31 (s, 2H, 4a- and 8a-H), 3.90 (s, 3H, OCH3), 6.15(m, 2H, 6- and 7-H), 8.00 (s, 1H, 3-H); 13C NMR: d 21.0,24.9, 35.8, 35.9, 51.6, 53.0, 60.4, 131.2, 133.3, 133.7, 141.1,144.9, 151.5, 161.4, 192.7, 192.9. Anal. Calcd forC16H14O4S: C, 63.56; H, 4.67; S, 10.61. Found: C, 63.77;H, 4.03; S, 9.98.

3.1.7. Aerobic oxidation of adduct 2 in dichlomethane–DBU. A solution of adduct 2 (220 mg, 0.98 mmol), DBU(298 mg, 1.96 mmol) in CH2Cl2 (15 mL) was vigorouslystirred in an open flask for 4 h. The mixture was evaporatedunder reduced pressure and the residue was chromato-graphed on silica gel. Elution with petroleum ether yielded a4.6:1 mixture of exo- and endo-1,4,4a,9a-tetrahydro-4a,9a-epoxy-1,4-methano-9,10-anthraquinone 4C6 (160 mg,68%) as a pale yellow solid, mp 120–123 8C. The spectralproperties of epoxides 4C6 were in good agreement withthose reported in the literature.7

Further elution with CH2Cl2–EtOAc (1/1) gave 1,4,4a,9a-tetrahydro-4a-hydroperoxy-1,4-methano-9,10-anthraqui-none 25 (18 mg, 7%) as a viscous orange oil; 1H NMR: d1.95 (m, 2H, 11-H), 2.90 (s, 1H, 1-H), 3.22 (s, 1H, 4-H),3.77 (d, 1H, JZ1.4 Hz, 4a-H), 3.97 (s, 1H, OH), 6.28(q, 1H, JZ2.67, 2.91 Hz, 3-H), 6.50 (q, 1H, JZ2.7, 2.9 Hz,2-H), 7.45 (m, 2H, 6- and 7-H), 7.84 (m, 2H, 5- and 8-H).

3.1.8. Aerobic oxidation of adduct 15 in DBU–dichloro-methane solution. A solution of adduct 15 (230 mg,0.96 mmol), DBU (292 mg, 1.92 mmol) in CH2Cl2

(15 mL) was vigorously stirred under aerobic conditionsfor 4 h at rt. The mixture was evaporated under reducedpressure and the residue subjected to column chromato-graphy. Elution with CH2Cl2 yielded a 20:1 mixture of exo-and endo-5-hydroxy-1,4,4a,9a-tetrahydro-4a,9a-epoxy-1,4-methano-9,10-anthraquinone 21C22. Further elution withAcOEt yielded a mixture of 8-hydroxy-1,4,4a,9a-tetra-hydro-4a-hydroperoxy- and 8-hydroxy-1,4,4a,9a-tetrahy-dro-9a-hydroperoxy-1,4-methano-9,10-anthraquinone 26C27 (27 mg, 10%), as an unstable viscous orange oil. Columnchromatography of the mixture 21C22 yielded 21 (150 mg,62%) as a yellow solid, mp 103–105 8C; IR: nmax 3190 br(OH), 1692 and 1642 (C]O), 1294 and 910 (C–O epoxide);1H NMR: d 1.71 (m, 2H, 11-H), 3.59 (t, 2H, JZ1.5 Hz, 1-and 4-H), 3.68, 6.59 (dd, 2H, JZ3.6, 1.5 Hz, 2- and 3-H),7.22 (m, 1H, 6-H), 7.53 (m, 2H, 7- and 8-H), 11.09 (s, 1H,OH); 13C NMR: d 41.6, 42.0, 42.6, 72.6, 115.9, 119.5,

124.1, 134.5, 136.6, 141.4, 142.4, 161.5, 190.4, 196.3.Anal. Calcd for C15H10O4: C, 70.86; H, 3.96. Found: C,70.53; H, 3.77.

3.1.9. endo-1,4,4a,9a-Tetrahydro-4a,9a-epoxy-1,4-ethano-9,10-anthraquinone 20. A solution of 14(119 mg, 0.5 mmol), DBU (152 mg, 1.00 mmol) andCH2Cl2 (15 mL) was vigorously stirred for 4 h at rt. UHP(100 mg, 1.06 mmol) was added to the solution and themixture was allowed to stand for 24 h at rt. The solventwas removed, yielding a 1:18 mixture of exo- and endo-1,4,4a,9a-tetrahydro-4a,9a-epoxy-1,4-ethano-9,10-anthra-quinone 19C20. The exo/endo ratio was determinedusing the vinylic proton signals of the isomers at d6.52 and 6.06 ppm, respectively. Column chromatographyof the mixture over silica gel yielded endo-epoxide 20(106 mg, 84%), mp 143–144 8C (lit.5 144–145 8C). Thespectral properties of epoxide 20 were in full agreementwith those reported in the literature.5

3.1.10. endo-5-Hydroxy-1,4,4a,9a-tetrahydro-4a,9a-epoxy-1,4-ethano-9,10-anthraquinone 24. A solution of16 (120 mg, 0.47 mmol), DBU (143 mg, 0.94 mmol) andCH2Cl2 (15 mL) was vigorously stirred for 4 h at rt. UHP(100 mg, 1.06 mmol) was added to the solution and themixture was left for 24 h at rt. Evaporation of the solventyielded a 1:20 mixture of exo- and endo-5-hydroxy-1,4,4a,9a-tetrahydro- 4a,9a-epoxy-1,4-ethano-9,10-anthra-quinone 23C24. The ratio of isomers 23C24 was evaluatedusing the vinylic proton signals of the isomers at d 6.55 and6.06 ppm, respectively. The mixture was column chromato-graphed on silica gel to give endo-epoxide 24 (90 mg, 72%)as a yellow solid, mp 132.5–133 8C; IR: nmax 1687 (C]O),1246, 887, 814 (C–O epoxide); 1H NMR: d 11.27 (s, 1H,OH), 7.59 (m, 2H, 6- and 8-H), 7.25 (dd, 1H, JZ1.5, 7.5 Hz,7-H), 6.06 (t, 2H, JZ4.0 Hz, 2- and 3-H,), 3.85 (s, 2H, 3a-and 10a-H), 1.49 (m, 4H, 11- and 12-H); 13C NMR: d 21.3,21.4, 29.0, 29.5, 60.3, 60.8, 115.7, 119.4, 124.2, 129.1,129.4, 133.8, 136.9, 161.4, 190.1, 196.3. Anal. Calcd forC16H12O4: C, 71.64; H, 4.51. Found: C, 71.23; H, 4.12.

3.1.11. 1,4-Dihydro-5-hydroxy-1,4-ethano-9,10-anthra-quinone 29. A solution of adduct 16 (120 mg,0.47 mmol), DBU (143 mg, 0.94 mmol) in CH2Cl2

(15 mL) was refluxed for 24 h. Removal of the solventfollowed by column chromatography of the residue(CH2Cl2) yielded quinone 29 (103 mg, 87%) as an orangesolid, mp 158–159 8C; IR: nmax 3449 (OH), 1658 and 1638(C]O); 1H NMR: d 12.12 (s, 1H, OH), 7.57 (m, 2H, 6- and8-H), 7.23 (t, 1H, JZ7 Hz, 7-H), 6.45 (m, 2H, 2- and 3-H),4.54 (s, 2H, 1- and 4-H), 1.49 (m, 4H, 11- and 12-H); 13CNMR: d 24.57, 24.65, 33.54, 34.32, 114.91, 119.12, 124.18,132.49, 133.70, 133.81, 135.85, 150.44, 151.92, 161.52,180.60, 186.60. Anal. Calcd for C16H12O3: C, 76.18; H,4.79. Found: C, 76.28; H, 4.95.

3.1.12. 3b,10a-Dihydronaphtho[2,3-b]-1H-cyclopenta[d]-furan-5,10-dione 33. Method A. A suspension of epoxides4C6 (151 mg, 0.64 mmol), silica gel/gypsum (1 g) indichloromethane (20 mL) was stirred for 7 days at rt. Themixture was filtered and the solid washed thoroughly withdichloromethane. The filtrate was concentrated underreduced pressure and the residue chromatographed over

J. A. Valderrama et al. / Tetrahedron 62 (2006) 2631–2638 2637

silica gel. Elution with CH2Cl2 yielded quinone 33 (70 mg,45%) as a yellow solid, mp 190–191 8C (lit.4,20 189–190 8C;191–193 8C). The 1H and 13C NMR properties were inagreement with those reported for 33.4,20

Method B. A suspension of epoxides 4C6 (106 g,0.44 mmol), calcium hydroxide (1.0 g) in dichloromethane(20 mL) was stirred for 3 days at rt. Work-up followed bycolumn chromatography gave quinone 33 (71 mg, 68%).

3.1.13. 6- and 9-Hydroxy-3b,10a-dihydronaphtho[2,3-b]-1H-cyclopenta[d]furan-5,10-dione 34 and 35. A suspen-sion of quinone epoxide 22 (126 mg, 0.5 mmol), silica gel/gypsum (1 g) in dichloromethane (15 mL) was stirred for7 days. Work up and column chromatography (CH2Cl2)gave compound 35 (25 mg, 19%) as orange solid, mp137.5–138 8C; IR (KBr): nmax 3443 (OH), 1694 and 1682(C]O); 1H NMR: d 2.80 (m, 1H, 1-H), 2.98 (m, 1H, 1-H),4.17 (dt, 1H, JZ2.2, 8.6 Hz, 10b-H), 5.98 (m, 1H, 2- or 3-H), 6.17 (m, 1H, 3a-H), 7.19 (dd, 1H, JZ1.2, 8.4 Hz, 3- or2-H), 7.53 (t, 1H, JZ7.9 Hz, 6-H), 7.63 (dd, 2H, JZ1.2,7.5 Hz, 7- and 8-H), 12.31 (s, 1H, OH); 13C NMR: d 38.2,41.7, 96.6, 115.0, 119.4, 125.7, 126.5, 127.7, 131.9, 135.0,137.6, 159.4, 161.2, 177.8, 188.4. Anal. Calcd forC15H10O4: C, 70.86; H, 3.96. Found: C, 70.67; H, 3.80.

Further elution with dichloromethane yielded quinone 34(12 mg, 9%) as a yellow solid, mp 145–146 8C; IR (KBr):nmax 3443 (OH), 1694 and 1682 (C]O); 1H NMR: d 2.79(m, 1H, 1-H), 2.97 (m, 1H, 1-H), 4.08 (dt, 1H, JZ2.2,8.6 Hz, 4-H), 5.98 (s, 1H, 10a-H), 6.12 (d, 1H, JZ8.9 Hz, 2-H), 6.21 (m, 1H, 3-H), 7.23 (dd, 1H, JZ2.1, 7.6 Hz, 6-H),7.64 (m, 2H, 7- and 8-H), 11.70 (s, 1H, OH); 13C NMR: d38.2, 42.7, 96.3, 114.8, 119.0, 124.0, 127.7, 127.8, 133.5,136.9, 137.7, 158.4, 162.0, 181.5, 183.3.

3.1.14. Aerobic oxidation of adduct 18 in dichloro-methane–DBU. A solution of adduct 18 (113 mg,0.43 mmol), DBU (130 mg, 0.86 mmol) in dichloromethane(15 mL) was vigorously stirred under aerobic conditions for48 h at rt. Column chromatography of the residue (CH2Cl2)yielded a 1:11 mixture of exo- and endo-4,9-dioxo-4,4a,5,8,8a,9-hexahydro-4a,8a-epoxy-5,8-ethano-2-meth-oxycarbonylnaphtho-[2,3-b]thiophene 31C32. Further col-umn chromatography of the crude product gave pure endo-epoxide 32 (110 mg, 81%) as a yellow solid, mp 129.5–130.5 8C; IR: nmax 1728 (C]O ester), 1710 and 1670(C]O enone); 1H NMR: d 1.24 (d, 2H, JZ5 Hz, 11-H),1.46 (s, 2H, 10-H), 3.80 (s, 2H, 5- and 8-H), 3.95 (s, 3H,OCH3), 6.06 (dd, 2H, JZ1.37, 3.49 Hz, 6- and 7-H), 8.07 (s,1H, 3-H); 13C NMR: d 21.2, 21.3, 29.4, 29.7, 53.1, 61.1,61.3, 129.2, 129.3, 131.0, 140.8, 141.6, 161.1, 184.9, 185.3.Anal. Calcd for: C16H12O5S: C, 60.75; H, 3.82; S, 10.14.Found: C, 60.69; H, 3.80; S, 9.98.

3.2. Cytotoxicity screening

MRC-5 cell culture. Human normal lung fibroblasts MRC-5(ATCC CCL-171) were grown as monolayers in minimumessential Eagle medium, with Earle’s salts, 2 mML-glutamine and 2.2 g/L sodium hydrogencarbonate, sup-plemented with 10% heat-inactivated fetal bovine serum(FBS), 100 IU/mL penicillin and 100 mg/mL streptomycin

in a humidified incubator with 5% CO2 in air at 37 8C. Cellpassage was maintained between 10 and 16, and the mediumwas changed every 2 days.

AGS cell culture. Human gastric cancer epithelial cells AGS(ATCC CRL-1739) were grown as monolayers in Ham F-12medium containing 1 mM L-glutamine and 1.5 g/L sodiumhydrogencarbonate, supplemented with 10% heat-inacti-vated FBS, 100 IU/mL penicillin and 100 mg/mL strepto-mycin in a humidified incubator with 5% CO2 in air at37 8C. Cell passage was maintained between 42–48, and themedium was changed every 2 days.

Cytotoxicity assay. Confluent cultures of MRC-5 as well asAGS cells were treated during 24 h with medium containingthe compounds or the reference compounds at concen-trations ranging from 0 up to 100 mM. The substances werefirst dissolved in DMSO (1% final concentration) and thenin the corresponding culture medium supplemented with 2%FBS. Untreated cells were used as controls. At the end ofthe incubation, the neutral red assay was carried out asdescribed previously.21 To calculate the concentration thatproduces a 50% inhibitory effect on the cell viability (IC50),results were converted to percentage of controls and the IC50

values were graphically obtained from the dose-responsecurves.

Acknowledgements

We thank Fondo Nacional de Ciencia y Tecnologıa (Grant1020885) for financial support of this study.

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