HETEROCYCLES

Paper | Special issue | Vol. 80, No. 1, 2010, pp. 427-437
Received, 19th June, 2009, Accepted, 27th July, 2009, Published online, 29th July, 2009.
DOI: 10.3987/COM-09-S(S)39

■ Synthesis and Some Reactions of 11-Azacyclohept[a]azulen-3(3H)-ones and Evaluation of Their Cytotoxic Activity against HeLa S3 Cells

Tomoyuki Ariyoshi, Kengo Yoshinaga, Kazuya Koizumi, Hiroyuki Fujii, Reiko Ikeda, Takeo Konakahara, and Noritaka Abe*

Graduate School of Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8512, Japan

Abstract

Aldol condensation of 2,3-diformyl-1-azaazulene with acetone, methoxyacetone, and benzylacetone in aq. NaOH solution gave corresponding 11-azacyclohept[a]azulen-3(3H)-one (2a), 2-methoxy-derivative (2b) and 2-benzyl-derivative (2d) in good yields. Hydrolysis of 2b using hot HBr gave 2-hydroxy-11-azacyclohept[a]azulen-3(3H)-one (2f). Acetylation of 2f gave 2-acetoxy-11-azacyclohept[a]azulen-3(3H)-one (2g). Reaction of the compounds (2a, 2b, 2f) with diethyl malonate in Ac2O under heating gave 1,9(11bH)-11b-azaazuleno[1,2,3-cd]azulenedione derivatives (4a, 4b, 4f). Compounds (2b, 2f) showed weak cytotoxic activity against HeLa S3 cells.

INTRODUCTION
Tricyclic and tetracyclic systems of containing azulene nucleus such as azulenotropylium ions, azulenotropones and azulenoazulenes are attracted attentions form the view of aromaticity and bioactivity.1-3 Several azulenotropones and azulenoazulenes have been synthesized notably by K. Takase,4-6 C. Jutz,7 T. Toda,8 Z. Yoshida,9 and S. Kuroda,10 and M. Nitta.11 Although many researches of fused azaazulene chemistry were proceeding,12 researches about aza-analogs of azulenotropylium ions, azulenotropones, and azulenoazulenes were few,13-16 and the report was not observed about the reaction of azaazulenotropones except the N-protonation13,14 and the N-methylation14. Recently, we reported the synthesis of 2,3-diformyl-1-azaazulene,17 which would be a key product for construction of fused 1-azaazulene systems. We now report the facile synthesis 11-azacyclohept[a]azulen-3(3H)-ones (11-azaazuleno[1,2-d]tropones) (2) and the addition-cyclization reaction of 2 with diethyl malonate.

RESULTS AND DISCUSSION
Treatment of 2,3-diformyl-1-azaazulene (1) with acetone in aq. NaOH solution for 10 min at rt gave 11-azacyclohept[a]azulen-3(3H)-one (2a) in 48% yield. It is reported that 2a was prepared by the oxidation of dicyclohepta[b,d]pyrrole with MnO2 by Nitta, but the yield low (9%) and the reaction of 2a was scarcely investigated.14) In the 1H NMR spectrum of 2a, seven-membered ring protons in the azaazulene moiety were seen at δ 7.96 (ddd, J 9.8, 9.6, and 0.4, H-7), 8.03 (dd, J 10.0 and 9.8, H-9), 8.15 (t, J 9.8, H-8), 8.88 (dd, J 10.0 and 0.4, H-10), and 8.93 (d, J 9.6, H-6), where bond-alternation was not observed. On the other hand, seven-membered ring protons of tropone moiety were seen at δ 6.95 (dd, J 12.1 and 2.4, H-4), 7.25 (d, J 12.4 and 2.4, H-2), 8.07 (d, J 12.1, H-5), and 8.08 (d, J 12.4, H-1). The results suggest that 2a would have a 1,4-pentadien-3-one fused with 1-azaazulene structure. Unfortunately, we could not obtain a favorable crystal of 2a for the X-ray analysis. Therefore, to clarify the consideration, we performed the molecular orbital calculation by Gaussian ’03 using RHF/6-31G*18. The calculated bond lengths for 2a, shown in Figure 1, supported our consideration.

The condensation of 1 with acetone underwent in good yield. Therefore, our method is superior to Nitta’s method. The method could be extended to the reaction with other acetone derivatives. Thus 1 was treated with methoxyacetone in aq. NaOH solution for 10 min at rt to give only 2-methoxy-11-azacyclohept[a]azulen-3(3H)-one (2b) in 63% yield, and 4-methoxy-11-azacyclohept[a]azulen-3(3H)-one13 (2c) was not obtained. Melting point of obtained compound (mp 214-215 ℃) differed from that of the reported 2c (mp 209.5-211.5 ℃)13. The electronic spectra of 2b and 2c13 were distinctly different as shown in Figure 2. In the 1H NMR spectrum of 2b, methoxy proton was seen at δ 4.11, and a singlet signal assigned to H-1 proton was seen at δ 7.68 and AB-doublet protons were seen at δ 7.13 (1H, d, J 12.2, H-4) 8.17 (1H, d, J 12.2, H-5), except for the seven-membered ring protons of the azaazulene moiety. From the results, we assigned the structure.

When 1 was treated with benzylacetone for 10 min at rt, 2d (34%) and 3 (22%) were obtained. In the 1H NMR spectrum of 3, two methine protons were observed at δ 3.56 (1H, dddd, J 8.1, 7.7, 6.0, and 1.2, H-2), 5.21 (1H, d, J 6.0, H-1) and benzyl protons were seen at δ 2.60 (1H, dd, J 13.8 and 7.7) and 2.77 (1H, dd, J 13.8 and 8.1), except for the seven-membered ring protons of the azaazulene moiety. In the 13C NMR spectrum of 3, two methine carbons were observed at δ 59.1 and 68.8 together with benzyl carbon at δ 34.3. In the IR spectrum of 3, characteristic peaks were observed at 3444 (OH), 1623 (C=O), and 1603 cm-1 (C=C). From the results, we assigned the structure. Prolonged treatment of 1 with benzylacetone (for 2h at rt) gave 2d in 74% yield and none of 3. It is considered that intermediate (3) was obtained at first, then successive dehydration afforded 2d.
Similar treatment of 1 with diethyl 1,3-acetonedicarboxylate gave 2e in 13% yield. For improvement of the yield, 1 was treated under azeotropic conditions in the presence of NHEt2 in benzene under reflux for 4 h, but 2e was obtained only in 5% yield.
Hydrolysis of 2b was achieved by treatment with 48% HBr under reflux for 3 h and 2-hydroxy-11-azacyclohept[a]azulen-3(3H)-one (2f) was obtained in 88% yield. Compound 2f was acetylated with Ac2O in the presence of pyridine for 1 h at rt, and 2-acetoxy-11-azacyclohept[a]azulen-3(3H)-one (2g) was obtained in 61% yield, where the isomeric acetate could not be isolated.

It is well known that 8,8-dicyanoheptafulvene was obtained by the condensation of tropone with malononitrile in refluxing Ac2O.19 Therefore we examined the reaction of 2a with malononitrile, but distinct product was not isolated. When 2a was treated with diethyl malonate in refluxing Ac2O for 1.5 h gave 11b-azaazuleno[1,2,3-cd]azulene-1,9(11bH)-dione derivative (4a) in 24 % yield. In the 1H NMR spectrum of 4a, one ethyl ester signals were observed at δ 1.45 (J 7.1, Me) and 4.43 (J 7.1, OCH2) and four proton signals owing to seven-membered ring of azaazulene moiety were seen at δ 7.51 (dd, J 10.4 and 10.3, H-5), 7.81 (dd, J 11.0 and 10.3, H-4), 8.04 (d, J 10.4, H-6), and 8.71 (d, J 11.0, H-3), and tropone moiety at δ 7.07 (dd, J 12.2 and 2.5, H-8), 7.22 (dd, J 12.4 and 2.5, H-10), 7.73 (dd, J 12.2, H-7), and 8.48 (d, J 12.4, H-11). In the IR spectrum of 4a, three carbonyl signals were seen at 1738, 1674, and 1619 cm-1, owing to ester, γ-lactim, and tropone carbonyls, respectively. From the results, we assigned the structure. Michael addition of the enol form of diethyl malonate to the C-10 of 2a would generate intermediate A, and successive cyclization and dehydrogenation furnished to 4a.
Similar treatment of 2b and 2d with diethyl malonate in refluxing Ac2O gave 4b (39%) and 4f (25%), respectively.
As 2a was not reacted at the carbonyl of tropone moiety as above, we examined the reaction of 2a with 2,4-dinitrophenyl hydrazine to substantiate the reactivity. But we could not obtain distinct products, and failed to elicit the reactivity of the carbonyl group.

Evaluation of cytotoxic activity
Compounds (2b and 2f) were evaluated for their cytotoxic activity against HeLa S3 cells. The IC50 values [µM] were 41 and 19 for 2b and 2f, respectively. The results revealed that the compounds (2b and 2f) showed weak activity against HeLa S3 cells.

EXPERIMENTAL
Mps are measured using a Yanagimoto micro-melting apparatus and uncorrected. 1H NMR spectra (including HH-COSY and CH-COSY NMR) were recorded on a Bruker AVANCE 400S (400 MHz) and 13C NMR spectra were recorded on a Bruker AVANCE 400S (100.6 MHz) using CDCl3 as a solvent with tetramethylsilane as an internal standard unless otherwise stated; J values are recorded in Hz. IR spectra were recorded for KBr pellets on a Nicolet FT-IR AVTAR 370DTGS unless otherwise stated. Electronic spectra were recorded with JASCO V-570 spectrophotometer. Elemental analyses were taken with a Perkin Elmer 2400II. Kieselgel 60 and activated alumina C300 were used for column chromatography and Kieselgel 60G was used for thin-layer chromatography.

Synthesis of 11-azacyclohept[a]azulen-3(3H)-one (2a)
A solution of 2,3-diformyl-1-azaazulene (1) (0.0988 g, 0.534 mmol) and Me2CO (0.06 mL, 0.82 mmol) in 0.11 M NaOH solution (100 mL, 1.1 mmol) was stirred for 10 min at room temperature. The mixture was extracted with CH2Cl2. The extract was dried over Na2SO4, and evaporated. The residue was chromatographed with EtOAc to give 2a (0.0531 g, 48%) as purple powders.
2a: Purple micro needles (from EtOH), mp 195 ℃ (decomp.); δH 6.95 (1H, dd, J 12.1 and 2.4, H-4), 7.25 (1H, d, J 12.4 and 2.4, H-2), 7.96 (1H, ddd, J 9.8, 9.6, and 0.4, H-7), 8.03 (1H, dd, J 10.0 and 9.8, H-9), 8.07 (1H, d, J 12.1, H-5), 8.08 (1H, d, J 12.4, H-1), 8.15 (1H, t, J 9.8, H-8), 8.88 (1H, dd, J 10.0 and 0.4, H-10), and 8.93 (1H, d, J 9.6, H-6); δH(TFA-d) 7.71 (1H, dd, J 12.0 and 2.0, H-4), 7.92 (1H, d, J 12.1 and 2.0, H-2), 8.47 (1H, d, 12.1, H-1), 8.83 (1H, dd, J 10.0 and 9.9, H-7), 8.86 (1H, dd, J 10.1 and 9.8, H-9), 8.88 (1H, d, J 12.0, H-5), 8.98 (1H, dd, J 10.0 and 9.8, H-8), 9.39 (1H, d, J 10.1, H-10), and 9.87 (1H, d, J 9.9, H-6); δC 107.5, 120.0, 130.4, 131.9, 132.5, 135.8, 138.7, 139. 9, 134.0, 141.0, 141.4, 143.6, 149.7, and 184.9; νmax / cm-1 1592 (C=O); λmax(CHCl3) nm (log ε) 286 (4.11), 328 (4.52), 342 (4.66), 360 (3.97, sh), 385 (3.86), 406 (3.42), 500 (2.82, sh), 541 (3.03, sh), 568 (3.07), and 616 (2.84, sh); λmax(MeOH) nm (log ε) 286 (4.16), 328 (4.60), 342 (4.72), 358 (4.06, sh), 384 (3.93), 406 (3.49), 541 (3.15, sh), 566 (3.17), and 607 (2.97, sh). Anal. Calcd for C14H9NO·H2O: C, 74.65; H, 4.92; N, 6.22. Found: C, 74.44; H, 4.98; N, 6.19.

Synthesis of 2-methoxy-11-azacyclohept[a]azulen-3(3H)-one (2b)
A solution of 1 (0.0981 g, 0.530 mmol) and methoxyacetone (0.07 mL, 0.76 mmol) in 0.055 M NaOH solution (200 mL, 1.1 mmol) was stirred for 10 min at rt. The mixture was extracted with CH2Cl2. The extract was dried over Na2SO4, and evaporated. The residue was chromatographed with EtOAc to give 2b (0.0795 g, 63%) as reddish purple leaflets.
2b: Reddish purple leaflets (from hexane-CH2Cl2), mp 214-215 ℃; δH 4.11 (3H, s, OMe), 7.13 (1H, d, J 12.2, H-4), 7.68 (1H, s, H-1), 7.95 (1H, td, J 9.4 and 1.2, H-7), 7.99 (1H, ddd, J 9.6, 9.4, and 1.2, H-9), 8.10 (1H, ddd, J 9.6, 9.4, and 1.4, H-8), 8.17 (1H, d, J 12.2, H-5), 8.77 (1H, dd, J 9.6 and 1.4, H-10), and 8.86 (1H, d, J 9.4, H-6); δH(TFA-d) 4.35 (3H, s, OMe), 7.92 (1H, d, J 11.9, H-4), 8.13 (1H, s, H-1), 8.81-8.96 (3H, m, H-7, 8, and 9), 9.06 (1H, d, J 11.9, H-5), 9.36 (1H, d, J 9.9, H-10), and 9.82 (1H, d, J 9.8, H-6); δC 56.7, 110.1, 121.9, 128.2, 129.4, 130.9, 132.9, 133.1, 137.1, 139.0, 144.9, 160.0, 161.3, 162.4, and 181.7; νmax / cm-1 1614 (C=O); λmax(CHCl3) nm (log ε) 259 (3.99), 284 (4.19, sh), 296 (4.22), 322 (4.34, sh), 343 (4.64), 358 (4.43, sh), 382 (4.05, sh), 409 (4.00), 528 (3.14, sh), 555 (3.19), and 592 (2.96, sh); λmax (MeOH) nm (log ε) 258 (3.88), 285 (4.19, sh), 294 (4.22), 318 (4.29, sh), 341 (4.63), 376 (4.07, sh), 400 (3.98), and 535 (3.12). Anal. Calcd for C15H11NO2·2H2O: C, 70.02; H, 5.88; N, 5.44. Found: C, 70.12; H, 5.88; N, 5.34.

Reaction of 1 with benzylacetone
a) A solution of 1 (0.093 g, 0.502 mmol) and benzylacetone (0.113 g, 0.762 mmol) in 0.048 M NaOH solution (100 mL, 4.80 mmol) was stirred for 10 min at rt. The mixture was extracted with CH2Cl2. The extract was dried over Na2SO4, and evaporated. The residue was chromatographed with EtOAc to give 2d (0.0471 g, 34%) and 3 (0.0334 g, 22%), successively.
b) A solution of 1 (0.0370 g, 0.200 mmol) and benzylacetone (0.113 g, 0.762 mmol) in 0.0445 M NaOH solution (100 mL, 4.45 mmol) was stirred for 2 h at rt. The mixture was extracted with CH2Cl2. The extract was dried over Na2SO4, and evaporated. The residue was chromatographed with EtOAc to give 2d (0.0442 g, 74%).
2d: violet powders (from hexane-CH2Cl2), mp 208-210 ℃; δH 4.14 (2H, s, CH2), 7.01 (1H, d, J 12.2, H-4), 7.23 (1H, tt, J 7.2 and 2.2, H-p-Ph), 7.31 (2H, dd, J 7.7 and 7.2, H-m-Ph), 7.37 (2H, dd, J 7.7 and 2.2), H-o-Ph), 7.91 (1H, dd, J 9.8 and 9.5, H-7), 7.97 (1H, dd, J 9.9 and 9.6, H-9), 8.01 (1H, d, J 12.2, H-5), 8.08 (1H, dd, J 9.9 and 9.8, H-8), 8.12 (1H, s, H-1), 8.79 (1H, d, J 9.6, H-10), and 8.86 (1H, d, J 9.5, H-6); δC 40.9, 124.0, 126.5, 128.3, 128.6, 129.6, 130.5, 131.1, 132.3, 124.5, 134.6, 138.6, 139.2, 140.1, 144.5, 152.1, 159.1, 161.8, and 187.3; νmax / cm-1 1597 (C=O). Anal. Calcd for C21H15NO·H2O: C, 79.98; H, 5.43; N, 4.44. Found: C, 79.66; H, 5.32; N, 4.64.
3: Red powders (from hexane-CH2Cl2), mp 202 ℃ (decomp.); δH 2.60 (1H, dd, J 13.8 and 7.7, CH2), 2.77 (1H, dd, J 13.8 and 8.1, CH2), 3.56 (1H, dddd, J 8.1, 7.7, 6.0, and 1.2, H-2), 5.21 (1H, d, J 6.0, H-1), 6.24 (1H, d, J 12.3 and 1.2, H-4), 6.94 (2H, dt, J 7.5 and 1.5, H-o-Ph), 7.11 (1H, tt, J 7.0 and 1.5, H-p-Ph), 7.16 (2H, dd, J 7.5 and 7.0, H-m-Ph), 7.56 (1H, d, J 12.3, H-5), 7.76 (1H, dd, J 10.0 and 9.7, H-7), 7.80 (1H, dd, J 9.8 and 9.7, H-9), 7.97 (1H, ddd, J 9.8, 9.7, and 0.8, H-8), 8.57 (1H, d, J 10.0, H-6), and 8.63 (1H, dd, J 9.5 and 0.8, H-10), (OH was not observed); δC 34.3, 59.1, 68.8, 119.5, 125.9, 126.3, 128.3, 128.9, 130.5, 131.6, 133.8, 137.6, 138.3, 139.7, 144.5, 157.0, 169.9, and 196.9; νmax / cm-1 3444 (OH), 1623 (C=O), and 1603 (C=C). Anal. Calcd for C21H17NO2: C, 79.98; H, 5.43; N, 4.44. Found: C, 80.06; H, 5.52; N, 4.28.

Reaction of 1 with diethyl acetone-1,3-dicarboxylate
a) A solution of 1 (0.0951 g, 0.514 mmol) and diethyl acetone-1,3-dicarboxylate (0.20 mL, 1.20 mmol) in 0.25 M NaOH solution (50 mL, 1.25 mmol) was stirred for 10 min at rt. The mixture was extracted with CH2Cl2. The extract was dried over Na2SO4, and evaporated. The residue was chromatographed with EtOAc to give 2e (0.0215 g, 13%) as violet powders.
b) To the solution of diethyl acetone-1,3-dicarboxylate (0.20 mL, 1.20 mmol) and NHEt2 (0.15 mL) in dry benzene (50 mL) was added benzene solution (20 mL) of 1 (0.1149 g, 0.625 mmol), and the mixture was refluxed for 4 h using Dean-Stark trap. The mixture was evaporated and the residue was chromatographed with EtOAc to give 2e (0.0015 g, 5%).
2e: Violet powders (from AcOEt), mp 148-149 ℃; δH 1.41 (3H, t, J 7.0, Me), 1.42 (3H, t, J 7, Me), 4.43 (4H, q, J 7.0, CH2), 8.05 (1H, dd, J 10.1 and 9.9, H-9), 8.10 (1H, dd, J 9.8 and 9.5, H-7), 8.23 (1H, dd, J 9.9 and 9.5, H-8), 8.55 (1H, s, H-1), 8.77 (1H, s, H-5), 8.93 (1H, d, J 10.1, H-10), and 9.05 (1H, d, J 9.5, H-6); δC 14.1, 14.2, 61.9, 62.1, 122.9, 130.9, 131.5, 131.6, 133.3, 134.2, 136.3, 140.3, 141.1, 141.8, 145.9, 159.8, 160.8, 166.2, 167.1, and 184.2; νmax / cm-1 1736, 1695, and 1626 (C=O); λmax(CHCl3) nm (log ε) 296 (4.32, sh), 334 (4.59), 341 (4.58), 376 (4.12, sh), 523 (3.00, sh), 553 (3.04), and 592 (2.84, sh). Anal. Calcd for C20H17NO5: C, 68.37; H, 4.88; N, 3.99. Found: C, 68.57; H, 4.93; N, 3.75.

Synthesis of 2-hydroxy-11-azacyclohept[a]azulen-3(3H)-one (2f)
A mixture of 2b (0.0317 g, 0.134 mmol) and 48% HBr (10 mL) was stirred for 3 h under heating at 160 ℃. The mixture was neutralized with Na2CO3 and extracted with CHCl3. The extract was dried over Na2SO4, and evaporated. The compound 2f (0.0263 g, 88%) was obtained as purple powders.
2f: Purple scales (from EtOH-EtOAc), mp 178-180 ℃; δH 5.11 (1H, br, OH), 7.31 (1H, d, J 12.0, H-4), 7.97 (1H, ddd, J 9.6, 9.5 and 1.4, H-7), 8.04 (1H, ddd, J 9.6, 9.3, and 1.4, H-9), 8.07 (1H, s, H-1), 8.08 (1H, dd, J 9.6 and 9.5, H-8), 8.43 (1H, d, J 12.0, H-5), 8.82 (1H, dd, J 9.3 and 1.8, H-10), and 8.88 (1H, d, J 9.5, H-6); δC 11.9, 122.8, 123.8, 125.0, 131.9, 132.7, 133.4, 135.2, 137.4, 139.0, 145.5, 160.4, 161.0, 162.6, and 180.3; νmax / cm-1 3249 (OH) and 1625 (C=O); λmax(CHCl3) nm (log ε) 276 (4.04), 292 (4.06), 300 (4.09), 321 (4.16), 339 (4.32, sh), 350 (4.38), 391 (3.80, sh), 396 (3.78, sh), 410 (3.68, sh), 428 (3.44, sh), 510 (3.20), 533 (3.18), and 581 (2.91, sh); λmax(MeOH) nm (log ε) 259 (3.77), 265 (3.82), 285 (3.95, sh), 297 (4.05, sh), 320 (4.23), 360 (4.29), 409 (3.65), 506 (3.79), and 531 (3.78); λmax(MeOH + 1 drop of aq. NaOH) nm (log ε) 259 (3.77), 265 (3.82), 285 (3.94, sh), 298 (4.05, sh), 321 (4.25), 359 (4.28), 424 (3.65), 508 (3.79), and 532 (3.78). Anal. Calcd for C14H9NO2·EtOAc: C, 69.44; H, 5.51; N, 4.50. Found: C, 69.41; H, 5.35; N, 4.24.

Acetylation of 2f
A mixture of 2f (0.0185 g, 0.083 mmol), Ac2O (1 mL), and a drop of pyridine was stirred for 1 h at rt. To the mixture water was added, and the mixture was extracted with CHCl3. The extract was dried over Na2SO4, and evaporated. The residue was chromatographed with EtOAc to give 2g (0.0134 g, 61%) as violet powders.
2g: Violet needles (from MeOH-CH2Cl2), mp 188 ℃; δH 2.44 (1H,s, Me), 7.10 (1H, d, J 12.2, H-4), 7.99 (1H, td, J 10.0 and 9.8, H-9), 8.05 (1H, dd, J 9.8 and 9.6, H-7), 8.10 (1H, s, H-1), 8.14 (1H, d, J 12.2, H-5), 8.17 (1H, t, J 9.8, H-8), 8.82 (1H, d, J 10.0, H-10), and 8.88 (1H, d, J 9.6, H-6); δC 20.8, 124.0, 126.3, 129.4, 139.5, 131.0, 132.9, 135.0, 139.2, 140.8, 144.6, 155.6, 159.2, 159.5, 168.8, and 180.0; νmax / cm-1 1754 and 1603 (C=O); λmax(CHCl3) nm (log ε) 259 (3.95), 288 (4.23, sh), 312 (4.36, sh), 327 (4.45), 340 (4.53), 360 (4.43), 384 (3.94, sh), 494 (3.82, sh), 503 (3.82), and 525 3.81, sh). Anal. Calcd for C16H11NO3: C, 72.45; H, 4.18; N, 5.28. Found: C, 72.41; H, 4.33; N, 5.24.

Reaction of 11-azacyclohept[a]azulen-3(3H)-ones (2a, 2b, 2f) with diethyl malonate
A mixture of 2a (0.0872 g, 0.42 mmol) and diethyl malonate (0.35 mL, 2.3 mmol) in Ac2O (10 mL) was refluxed for 1.5 h. To the mixture was added water, then the mixture was extracted with CHCl3. The extract was dried over Na2SO4, and evaporated. The residue was chromatographed with EtOAc to give 4a (0.318g, 24%) as red powders.
4a: Red powders (from CH2Cl2), mp 137-138 ℃; δH 1.45 (3H, t, J 7.1, Me), 4.43 (2H, q, J 7.1, OCH2), 7.07 (1H, dd, J 12.2 and 2.5, H-8), 7.22 (1H, dd, J 12.4 and 2.5, H-10), 7.51 (1H, dd, J 10.4 and 10.3, H-5), 7.73 (1H, dd, J 12.2, H-7), 7.81 (1H, dd, J 11.0 and 10.3, H-4), 8.04 (1H, d, J 10.4, H-6), 8.48 (1H, d, J 12.4, H-11), and 8.71 (1H, d, J 11.0, H-3); δC 14.5, 60.4, 125.0, 126.5, 128.6, 129.4, 130.3, 130.9, 131.4, 135.2, 136.7, 136.8, 139.0, 140.7, 141.8, 149.6, 162.3, 163.4 and 187.3; νmax / cm-1 1738, 1674, 1619 (C=O), and 1588 (C=C); λmax(CHCl3) nm (log ε) 296 (4.32, sh), 334 (4.59), 341 (4.58), 376 (4.12, sh), 418 (3.23, sh), 518 (2.99, sh), 553 (3.04), and 604 (2.77, sh). Anal. Calcd for C19H13NO4: C, 71.47; H, 4.10; N, 4.39. Found: C, 71.53; H, 3.85; N, 4.21.

Similar treatment of 2b and 2f gave 4b (39%) and 4f (21%), respectively.
4b: Reddish brown powders (from cyclohexane), mp 223-224 ℃; δH 1.46 (3H, t, J 7.1, Me), 4.12 (3H, s, OMe), 4.45 (2H, q, J 7.1, OCH2), 7.24 (1H, d, J 12.4, H-8), 7.58 (1H, dd, J 10.3 and 9.8, H-5), 7.83 (1H, d, J 12.4, H-7), 7.85 (1H, dd, J 11.0 and 9.8, H-4), 8.11 (1H, d, J 10.3, H-6), 8.27 (1H, s, H-11), and 8.79 (1H, d, J 11.0, H-3); νmax / cm-1 1721, 1690, 1613 (C=O), and 1585 (C=C). Anal. Calcd for C20H15NO5: C, 68.76; H, 4.33; N, 4.01. Found: C, 69.02; H, 4.55; N, 3.86.
4f: Red powders (from CH2Cl2), mp 132-133 ℃; δH 1.45 (3H, t, J 7.1, Me), 4.43 (2H, q, J 7.1, OCH2), 7.25 (1H, d, J 12.4, H-8), 7.52 (1H, dd, J 10.4 and 9.6, H-5), 7.82 (1H, d, J 12.4, H-7), 7.83 (1H, dd, J 11.0 and 9.6, H-4), 8.05 (1H, d, J 10.4, H-6), 8.56 (1H, s, H-11), and 8.71 (1H, d, J 11.0, H-3) (OH was not observed); νmax / cm-1 3294 (OH), 1720, 1690, 1613 (C=O), and 1585 (C=C). Anal. Calcd for C19H13NO5: C, 68.06; H, 3.91; N, 4.18. Found: C, 67.72; H, 3.86; N, 4.28.

Biological assay
HeLa S3 cells were obtained from AIST and used after cultivation. The cultivated HeLa S3 cells were cell counted and the culture fluid was prepared to the cell consistency of 2 ☓ 104 cells/ml. The compounds added to the medium in DMSO solutions. To the aliquot of the culture fluid, which was incubated for 3 h at 37 ℃, the test sample was added and then the culture fluid was incubated for 72 h. To the culture fluid, MTT (3-[4,5-dimethylthiazol]-2-yl-2,5-diphenyltetrazolium bromide) solution was added, and incubated for 4 h. Then the sample was centrifuged at 3000 rpm for 10 min at 4 ℃, and the solvent was evaporated. Then DMSO was added to the obtained mixture. The MTT-formazan was dissolved by plate-mixing and OD540 was measured. The rate of outlive determined to refer with un-dosed control. Dose-response curve was drawn up and IC50 was pursued. Every experiment in the cycotoxic assay was replicated twice in order to define the IC values.

References

1. B. A. Hess, Jr. and L. J. Schaad, J. Org. Chem., 1971, 36, 3418. CrossRef
2. T. Okazaki and K. K. Laali, Org. Biomol. Chem., 2003, 1, 3078. CrossRef
3. A. Toyata and T. Nakajima, Tetrahedron, 1981, 37, 2575. CrossRef
4. M. Saito, T. Morita, and K. Takase, Chem. Lett., 1974, 955. CrossRef
5. M. Saito, T. Morita, and K. Takase, Chem. Lett., 1975, 441. CrossRef
6. M. Yasunami, T. Amemiya, and K. Takase, Tetrahedron Lett., 1983, 24, 69. CrossRef
7. C. Jutz, H. G. Peuker, and W. Kosbahn, Synthesis, 1976, 673. CrossRef
8. T. Toda, N. Shimazaki, T. Mukai, and C. Kabuto, Tetrahedron Lett., 1980, 21, 4001. CrossRef
9. Z. Yoshida, M. Shibata, E. Ogino, and T. Sugimoto, Tetrahedron Lett., 1984, 24, 3343. CrossRef
10. S. Kuroda, S. Hirooka, H. Iwaki, M. Ikeda, T. Nakao, M. Ogisu, M. Yasunami, and K. Takase,
Chem. Lett., 1986, 2039. CrossRef
11. T. Takayasu and M. Nitta, J. Chem. Soc., Perkin Trans. 1, 1997, 3537. CrossRef
12. For reviews see, N. Abe, ‘Recent Research Developments in Organic and Bioorganic Chemistry’
2001, 4, 14, Transworld Research Network; T. Nishiwaki and N. Abe, Heterocycles, 1981, 15, 547; CrossRef M. Kimura, Yuki Gosei Kagaku Kyokai Shi, 1981, 39, 690.
13. N. Hirata, M. Yasunami, and K. Takase, Tetrahedron Lett., 1975, 1849. CrossRef
14. Y. Iino and M. Nitta, J. Chem. Soc., Perkin Trans. 1, 1994, 2579. CrossRef
15. M. Nitta, Y. Iino, T. Sugiyama, and A. Toyota, Tetrahedron Lett., 1993, 34, 835. CrossRef
16. M. Nitta, Y. Iino, T. Sugiyama, and A. Akaogi, Tetrahedron Lett., 1993, 34, 831. CrossRef
17. K. Koizumi, C. Miyake, T. Ariyoshi, K. Umeda, N. Yamauchi, S. Tagashira, Y. Murakami, H.
Fujii, and N. Abe, Heterocycles, 2007, 73, 325. CrossRef
18. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, J. A. Montgomery, Jr., T. Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rege, G. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian, J. B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador, J. J. Dnnenberg, V. G. Zakrezewski, S. Dapprich, A. D. Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S. Clifford, I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, M. Chllacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W. Wong, C, Gonzalez, and J. A. Pople, Gaussian 03, Gaussian, Inc., Pittsburgh, PA (2003).
19. M. Oda, M. Funamizu, and Y. Kitahara, Bull. Chem. Soc. Jpn., 1969, 42, 2386. CrossRef