HETEROCYCLES

Paper | Special issue | Vol. 86, No. 1, 2012, pp. 233-244
Received, 10th March, 2012, Accepted, 10th April, 2012, Published online, 17th April, 2012.
DOI: 10.3987/COM-12-S(N)4

■ Synthesis, Cyclization, and Evaluation of the Anticancer Activity against HeLa S-3 Cells of Ethyl 2-Acetylamino-3-ethynylazulene-1-carboxylates

Marie Hyoudou, Hajime Nakagawa, Takahiro Gunji, Yoshino Ito, Yu Kawai, Reiko Ikeda, Takeo Konakahara, and Noritaka Abe*

Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan

Abstract

Reaction of ethyl 2-aminoazulene-1-carboxylate with NIS in CHCl3 gave 2,2’-diamino-3,3’-diethoxycarbonyl-1,1’-biazulene (3) and a terazulene derivative. The coupling of azulenes was catalyzed by acid. Operation of the reaction in the presence of Et3N in CH2Cl2 for 7 min at -7 °C retarded the coupling of the azulene nuclei to give ethyl 2-amino-3-iodoazulene-1-carboxylate (1a) in 95% yield. Sonogashira cross-coupling of ethyl 2-acetylamino-3-iodoazulene-1-carboxylate (1b) gave ethyl 2-acetylamino-3-ethynylazulene-1-carboxylates (5a and 5b) in good yields. Cyclization of ethyl 2-acetylamino-3-phenylethynylazulene-1-carboxylate with Pd-catalyst gave azuleno[2,1-b]pyrrole derivatives. Compounds (3 and 5b) showed potent cytotoxic activity against HeLa S-3 cells (IC50 [μM] : 3: 2.9 ± 0.2, 5b: 13.4 ± 1.1).

INTRODUCTION
The chemistry of azulenes began as natural product chemistry at first, and is attracted attention from peculiarities of their structures and reactions, as well as their numerical pharmaceutical utilities.1 As the potential application of azulenes, very recently, antigastric ulcer activity of guaiazulene derivatives was reported.2 Heterocycle-fused azulenes have also been described from the early studies of azulene chemistry,1e,1f and recently reviewed.3 Some heterocycle-fused azulenes also attracted from their pharmaceutical utilities; especially, the reports about linderazulene and its derivatives, such as gorgiallylazulene, were often seen.4 Pharmaceutical utilities of heterocycles-fused azulenes other than furan ring were also reported; e.g., azuleno[5,6-b]indole displays antineoplastic activity,5 a 3,4-dihydroazuleno[4,3a,3-bc]pyridin-5(5H)-one derivative inhibits protein kinases (serine/threonine kinases),6 azuleno[4,3-f]pyrazoles show antiphlogistic, sedative, and analgestic effects,7 and azuleno[2,1-b]pyridines are anti-inflammatory.8
As a synthetic method for heterocycles, the electrophilic cyclization and the transition-metal-catalyzed annulation of alkynes with preferentially situated substituents were efficient methods; e.g., Sonogashira cross-coupling of 2-haloanilines and successive base-mediated (or electrophilic) cyclization; Pd-mediated cyclization of 2-ethynylanilines; the reaction of 2-haloanilines with Cu-acetylide known as Cacci reaction; 2-iodoaniline with internal alkynes being referred as the Larock heteroannulation, etc.9
Ethynylazulenes mainly have been investigated from the respect to the construction of advanced materials for electronic and photonic applications.1f,10,11 In spite of the synthetically and pharmaceutical potentiality of (amino substituted)-ethynylazulenes, attempts of synthesis and reactions of the ethynylazulene were little; only related report was Cacci reaction of ethyl 2-acetylamino-3-iodoazulene-1-carboxylate12 with Cu-phenylacetylide; where azuleno[2,1-b]pyrrole derivative was obtained, but ethyl 2-acetylamino-3-ethynylazulene-1-carboxylates were not isolated.13 For extension of the synthesis of heterocycle-fused azulenes and the inquiry of bioactivity of azulenes, we investigated the synthesis and reactions of ethyl 2-acetylamino-3-ethynylazulene-1-carboxylate.

RESULTS AND DISCUSSION
For the synthesis of ethyl 2-amino-3-ethynylazulene-1-carboxylate, we examined the synthesis of ethyl 2-amino-3-iodoazulene-1-carboxylate (1a), at first. According to Morita’s methods,12 we treated ethyl 2-aminoazulene-1-carboxylate12,14 (2a) with N-iodosuccinimide (NIS) in CH2Cl2 for 15 min at 5 °C. From the reaction mixture, 1a was not obtained, so we reinvestigated about the synthesis of 1a. When 2a was treated with NIS in CHCl3 for 15 min at 2 °C, 2,2’-diamino-3,3’-diethoxycarbonyl-1,1’-biazulene (3) and terazulene derivative (4) were isolated in 46% and 6% yields, respectively (Scheme 1). The structure of 3 was deduced from its 1H NMR, 13C NMR, IR, and HRMS spectra. The molecular formula of 3 was C26H24N2O4 from its HRMS spectrum. The 1H NMR spectrum of 3 showed seven-membered protons at δ 7.19 (dd, J 10.1 and 9.6, H-5,5’), 7.29 (t, J 10.1, H-7,7’), 7.41 (t, J 10.1, H-6,6’), 7.42 (d, J 10.1, H-4,4’), and 9.03 (d, J 9.6, H-8,8’), together with NH protons at δ 5.62 (4H, br s) and ethyl protons. The molecular formula of 4 was deduced to C39H35N3O6 from its HRMS spectrum. In the 1H NMR spectrum of 4, six doublet signals for eight protons, at δ 7.30 (2H, d, J 10.2), 7.56 (2H, d, J 10.2), 7.94 (1H, d, J 10.2), 8.99 (1H, d, J 9.6), 9.04 (1H, d, J 9.6), and 9.07 (1H, d, J 9.6), were seen together with other six aromatic protons, three ethyl ester protons, and amino protons. Existence of couples of doublets at δ 7.30 (2H, d, J 10.2) and 7.56 (2H, d, J 10.2) suggested that the centered azulene nucleus was substituted at the 6-position by another azulene.
It was described that the bromination of azulene derivatives with NBS affords dimer, trimers and oligomers, and the reaction was suggested to proceed by radical mechanism.15 Shoji et al. reported that 1-azulenyl sulfoxides gave 3,3’-bis(methylthio)-1,1’-biazulene

derivatives in the presence of acid.16 They proposed a radical mechanism with reference to reports of Razus.17 Apart from this, forming of 2,2’-diamino-3,3’-diethoxycarbonyl-8,8’-diphenyl-1,1’-biazulene by radical coupling of ethyl 2-amino-4-phenylazulene-1-carboxylate in the presence of FeCl3 was reported.18 It seems that our results resemble to Shoji’s report. So we examined the behavior of 1a in the presence of acid. The solution of 1a in CH2Cl2, which was prepared from 2a with NIS, did not convert at rt for 5 h under tracing the reaction with TLC. Addition of a small amount of TsOH to the solution, and stirring the mixture for 24 h led to consume 1a. From the reaction mixture, 3 was obtained in 61% yield together with a small amount of 1a.
For considering the instability of haloazulenes with acid,15 we achieved the synthesis of 1a in 95% yield by treating of 2a with NIS in CH2Cl2 for 7 min at -7 °C in the presence of Et3N.
Because 1a was rather labile, we decided to use 2-acetylamino-3-iodoazulene-1-carboxylate12 (1b) as a raw material for Sonogashira cross-coupling. Thus 1b was synthesized in 96% yield by the treatment of 2b with NIS in CH2Cl2 for 15 min at 5 °C.
The reaction of 1b with phenylacetylene was carried out in the presence of PdCl2(PPh3)2, CuI, and Et3N in THF for 24 h at rt, and ethyl 2-acetylamino-3-phenylethynylazulene-1-carboxylate (5a) was obtained in 76% yield. When the reaction operated in the presence of a large excess of Et3N, ethyl 1-acetylamino-2-phenylazuleno[2,1-b]pyrrole-9-carboxylate (6a) was obtained in 10% yield together with 5a (63%) (Scheme 2). The molecular formula of 5a was C23H19NO3 from the HRMS spectrum. In its IR spectrum, peaks at 3300 (NH), 2200 and 2140 cm-1 (C≡C) were seen. The molecular formula of 6a was C23H19NO3 from its HRMS spectrum. In its 1H NMR spectrum, 1H-singlet was seen at δ 6.99, and methyl signal was appeared at δ 2.36 but NH signal was not observed. It is known that 1H-singlet in the 1H NMR spectrum of 2a-aza-2H-cyclopent[cd]azulene (7) was seen at δ 6.87,19 therefore the signal at δ 6.99 of 6b is considered to be reasonable.
In a similar manner, treatment of 1b with trimethylsilylacetylene gave ethyl 2-acetylamino-3- trimethylsilylethynylazulene-1-carboxylate (5b) in 83% yield.

It was reported that cyclization of 2-mesylamino-3-phenylethynyl-1-azaazulene (8b), prepared from 8a, underwent in the presence of Cu-catalyst and gave 2a-aza-2H-cyclopent[cd]azulene (7) (Scheme 3).19 As for cyclization of 5a, we adopted similar condition at first. Thus we treated 5a with an equivalent of Cu(OTf)2 in toluene for 3 h at 110 °C in a sealed tube. The reaction showed complex feature, and no identified product was isolated. When the reaction was performed in CH2Cl2 for 3 h at 40 oC, the reaction showed complex feature again, but ethyl 2-acetylamino-3-phenacylazulene-1-carboxylate (9) was isolated in 9% yield from the mixture. The molecular formula of 9 was C23H21NO4 from the HRMS spectrum. In its 1H NMR spectrum, phenyl protons were seen at δ 7.10-7.16 (5H, m), and o-situated hydrogens of phenyl group did not shift to low field. From the results, we assigned the structure. Then we treated 5a with an equivalent of CuI in toluene for 4 h at 110 °C in a sealed tube, but the reaction did not undergo and 5a was recovered.

Next, we examined the cyclization of 5a by Pd-catalyst. Treatment of 5a with 2 mol% of 5% Pd-C and Na2CO3 in DMF for 24 h at 120 °C gave ethyl 2-phenyl-3H-azuleno[2,1-b]pyrrole-4-carboxylate (6b)13 in 33% yield. The reaction of 5a with PdCl2(PPh3)2 and Et3N in DMF for 24 h at 100 °C gave 6b in 72% yield. Thus cyclization of 5a was achieved by the treatment of Pd-catalyst, but in the reaction deacetylation occurred.
On the contrary of the case of 5a, treatment of 5b with Cu(OTf)2 in CH2Cl2 for 3 h at 40 °C gave a complex mixture and no distinct product was obtained. The reaction of 5b with CuI did not undergo at all. Reaction of 5b with Pd-C gave a complex mixture, and recovered 5b (24%) was only identified compound.

Evaluation of cytotoxic activity
Compounds (1b, 3, 5a, 5b, and 8a) were evaluated for their cytotoxic activity against HeLa S-3 cells. The IC50 values [µM] are summarized in Table 1. The compounds (3 and 5b) showed potent cycotoxic activity. On the other hand the compound (1b and 5a) showed scarcely cytotoxic activity. From the facts as above and that 2-amino-3-phenylethynyl-1-azaazulene (8a) has scarcely cytotoxic activity against HeLa S-3 cells, it seems that the phenyl group obstructed the activity.

CONCLUSION
Reaction of ethyl 2-aminoazulene-1-carboxylate (2a) with NIS was reinvestigated. Treatment of 2a with NIS in CHCl3 at 5 °C gave 2,2’-diamino-3,3’-diethoxycarbonyl-1,1’-biazulene (3: 46%) and terazulene derivative (4: 6%). The reaction was catalyzed by acid and the reaction of 1a with TsOH gave 3 in 61%. The synthesis of ethyl 2-amino-3-iodoazulene-1-carboxylate (1a) was achieved in the presence of Et3N in CH2Cl2 for 7 min at -7 °C and 1a was obtained in 95% yield. Ethyl 2-acetylamino-3- iodoazulene-1-carboxylate (1b) was obtained in 96% yield by the reaction of 2-acetylaminoazulene-1-carboxylate (2b) with NIS. Sonogashira cross-coupling of 1b with terminal acetylenes gave corresponding ethyl 2-acetylamino-3-ethynylazulene-1-carboxylates (5a and 5b) in good yields. Cyclization of ethyl 2-acetylamino-3-phenylethynylazulene-1-carboxylate (5a) with Pd-catalyst gave azuleno[2,1-b]pyrrole derivatives. Compounds (3 and 5b) showed potent cytotoxic activity against HeLa S3 cells (IC50 [µM] : 3: 2.9 ± 0.2, 5b: 13.4 ± 1.1).

EXPERIMENTAL
Melting points were determined with a Bibby Sterilin melting point SMP-3 apparatus and were uncorrected. 1H NMR spectra and 13C NMR spectra were recorded on a JEOL JNM ECP-500 (500 MHz for 1H and 125 MHz for 13C) 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 JASCO FT/IR-6100 unless otherwise stated. Mass specra were taken with JEOL JMS-T100LC. ESI-MS was measured using dried methanol as a solvent. High-resolution mass spectra (HRMS) were found to be with in ±5% of the theoretical values. Merck Kieselgel 60 was used for silica gel column chromatography, and Wako activated Alumina (300 mesh) was used for alumina column chromatography.

Reaction of ethyl 2-aminoazulene-1-carboxylate (2a) with NIS
a) A mixture of 2a12,14 (0.215 g, 1.00 mmol) and NIS (0.225 g, 1.00 mmol) in CHCl3 (10 mL) was stirred at 2 °C for 15 min, then evaporated. Chromatography of the residue on silica gel with CH2Cl2 gave 3 (0.0984 g, 46%) and 4 (0.0155 g, 6%), successively.
3: Red needles (from hexane-CH2Cl2), mp 176-179 °C; 1H NMR δ 1.40 (6H, t, J 7.1, CH3), 4.50 (4H, q, J 7.1, OCH2), 5.62 (4H, br s, NH2), 7.19 (2H, dd, J 10.1 and 9.6, H-5, 5’), 7.29 (2H, t, J 10.1, H-7, 7’), 7.41 (2H, t, J 10.1, H-6, 6’), 7.42 (2H, d, J 10.1, H-8, 8’), 9.03 (2H, d, J 9.6, H-4, 4’); 13C NMR δ 14.7, 59.6, 98.40, 107.08, 128.41, 129.08, 129.55, 129.62, 131.32, 142.58, 143.42, 158.93, and 166.86; νmax/cm-1 3485, 3335 (NH2), 1669 (C=O). MS (ESI+) m/z 451 ([M + Na]+). Calcd. for C26H24N2O4: M = 428; HRMS (ESI+): Calcd. for 12C261H2414N223Na116O4: 451.1634. Found: m/z 451.1627. Anal. Calcd. for C26H24N2O4: C, 72.88; H, 5.65; N, 6.64. Found: C, 72.65; H, 5.41; N, 6.47.
4: Dark red powders (from hexane-CH2Cl2), mp 206-208 °C; 1H NMR δ 1.49 (3H, t, J 6.9), 1.51 (3H, t, J 7.2), 1.52 (3H, t, J 6.9), 4.49 (2H, q, J 6.9), 4.50 (2H, q, J 7.2), 4.53 (2H, q, J 6.9), 5.5-7.0 (6H, br), 7.17 (1H, like t, J 9.4), 7.19 (1H, like t, J 9.6), 7.20 (1H, like t, J 9.6), 7.30 (2H, d, J 10.2), 7.31 (1H, like t, J 9.6), 7.39 (1H, like t, J 9.6), 7.43 (1H, like t, J 10.5), 7.56 (2H, d, J 10.2), 7.94 (1H, d, J 10.2), 8.99 (1H, d, J 9.6), 9.04 (1H, d, J 9.6), and 9.07 (1H, d, J 9.6); νmax/cm-1 3490, 3484, 3450, 3352, 3336, and 3308 (NH2), 1657 (C=O), 1593 (C=N). MS (ESI+) m/z 664 ([M + Na]+). Calcd. for C39H35N3O6: M = 641; HRMS (ESI+): Calcd. for 12C391H3514N323Na116O6: 664.2424. Found: m/z 664.2415.
b) A mixture of 2a (0.304 g, 1.41 mmol) and NIS (0.320 g, 1.42 mmol) in CH2Cl2 (10 mL) was stirred at -10 °C for 10 min, then the temperature was elevated to rt and stirred for 5 h. Tracing the reaction with TLC showed that only 1a was forming. To the mixture TsOH (0.01 g) was added, and the mixture was stirred for 24 h at rt, then Et3N (0.1 mL) was added. The mixture was evaporated, and silica gel chromatography of the residue with CH2Cl2 gave 3 (0.182 g, 61%) and 1a (0.003 g, 0.5%).

Synthesis of ethyl 2-amino-3-iodoazulene-1-carboxylate (1a)
A mixture of 2a (0.641 g, 2.98 mmol) and NIS (0.671 g, 2.97 mmol) in the presence of Et3N (3 drops) in CH2Cl2 (10 mL) was stirred at -7 °C for 7 min, then evaporated. Chromatography of the residue on silica gel with CH2Cl2 gave 1a (0.968 g, 95%).
1a: Dark red needles (from EtOH), mp 119.6 °C (decomp.) (lit.,12 mp 121-122 °C); 1H NMR δ 1.48 (3H, t, J 7.1, CH3), 4.71 (4H, q, J 7.1, OCH2), 6.34 (2H, br s, NH2), 7.37 (2H, tdd, J 9.6, 9.1, and 1.4, H-7), 7.38 (1H, td, J 9.1 and 1.7, H-5), 7.41 (1H, ddd, J 9.1, 8.7, and 1.4, H-6), 7.92 (1H, ddd, J 8.7, 1.7, and 1.4, H-4), 8.93 (1H, d, J 9.6, H-8); 13C NMR (CDCl3) δ 14.7, 59.6, 98.40, 107.08, 128.41, 129.08, 129.55, 129.62, 131.32, 142.58, 143.42, 158.93, and 166.86; νmax/cm-1 3473, 3313 (NH2), 1669 (C=O). MS (ESI+) m/z 363 ([M + Na]+). Calcd. for C13H12NO2: M = 340; HRMS (ESI+): Calcd. for 12C131H1214N123Na1127I116O2: 363.9810. Found: m/z 363.9795.

Synthesis of ethyl 2-acetylaminoazulene-1-carboxylate (2b)
A mixture of 2a12,14 (0.511 g, 2.40 mmol), Ac2O (5.0 mL), and 2 drops of pyridine was stirred for 3 h at rt, then the precipitate was collected by filtration and recrystallization from benzene gave 2b (0.408 g, 66%).
2b: Red needles (from benzene), mp 143.6-144 °C (lit.,14 mp 140-141.5 °C); 1H NMR (DMSO-d6) δ 1.43 (3H, t, J 7.0, CH3), 2.26 (3H, s, COCH3), 4.44 (2H, q, J 7.0, OCH2), 7.60 (1H, t, J 9.9, H-7), 7.65 (1H, t, J 9.9, H-5), 7.77 (1H, t, J 9.9, H-6), 7.99 (1H, s, H-3), 8.46 (1H, d, J 9.9, H-4), 9.20 (1H, d, J 9.9, H-8), and 10.65 (1H, br s, NH); νmax/cm-1 3260 (NH), 1693 and 1646 (C=O). MS (ESI+) m/z 280 ([M + Na]+). Calcd. for C15H15NO3: M = 257; HRMS (ESI+): Calcd. for 12C151H1514N123Na116O3: 280.0950. Found: 280.0933.

Synthesis of ethyl 2-acetylamino-3-iodoazulene-1-carboxylate (1b)
A mixture of 2b (0.200 g, 0.77 mmol) and NIS (0.170 g, 0.77 mmol) in CH2Cl2 (10 mL) was stirred 15 min at 5 °C, then evaporated. Chromatography of the residue on alumina with CH2Cl2 (added small amount of Et3N) gave 1b (0.29 g, 96%).
1b: Violet needles (from EtOH-acetone), mp 187-189 °C (lit.,12 mp 188-189 °C); 1H NMR (DMSO-d6) δ 1.29 (3H, t, J 7.0, CH3), 2.13 (3H, s, COCH3), 4.24 (2H, q, J 7.0, OCH2), 7.68 (1H, t, J 9.9, H-7), 7.71 (1H, t, J 9.9, H-5), 7.92 (1H, t, J 9.9, H-6), 8.42 (1H, d, J 9.9, H-4), 9.10 (1H, d, J 9.9, H-8), and 10.14 (1H, br s, NH); 13C NMR δ 14.51, 24.51, 60.54, 73.99, 109.03, 128.63, 129.15, 135.76, 137.90, 138.80, 140.24, 142.71, 150.09, 165.28, and 167.61; νmax/cm-1 3273 (NH), 1689 and 1671 (C=O), 595 (C-I). MS (ESI+) m/z 406 ([M + Na]+). Calcd. for C15H14NIO3: M = 383; HRMS (ESI+): Calcd. for 12C151H1414N1127I123Na116O3: 405.9916. Found: 405.9888.

Synthesis of ethyl 2-acetylamino-3-phenylethynylazulene-1-carboxylate (5a)
a) Under argon atmosphere, a mixture of 1b (0.200 g, 0.52 mmol), phenylacetylene (0.09 mL, 0.53 mmol), PdCl2(PPh3)2 (0.01 g, 0.016 mmol), CuI (0.01 g, 0.05 mmol), and Et3N (2 mL) in THF (3 mL) was stirred for 24 h at rt, then water was added. The mixture was extracted with CHCl3. The extract was dried over Na2SO4 and evaporated. Chromatography of the residue on alumina with hexane-AcOEt (5 : 1) gave 5a (0.139 g, 76%).
b) Under argon atmosphere, a mixture of 1b (0.200 g, 0.52 mmol), phenylacetylene (0.09 mL, 0.53 mmol), PdCl2(PPh3)2 (0.010 g, 0.016 mmol), CuI (0.010 g, 0.05 mmol), and Et3N (7 mL) in THF (6 mL) was stirred for 24 h at rt, then water was added. The mixture was extracted with CHCl3. The extract was dried over Na2SO4 and evaporated. Chromatography of the residue on alumina with hexane-AcOEt (5 : 1) gave 5a (0.120 g, 63%) and 6a (0.019 g, 10%), successively.
5a: Green needles (from hexane-CHCl3), mp 138-139.5 °C; 1H NMR δ 1.50 (3H, t, J 7.0, CH3), 2.36 (3H, s, COCH3), 4.49 (2H, q, J 7.0, OCH2), 7.30-7.35 (3H, m, H-m, p-Ph), 7.55 (1H, t, J 9.9, H-7), 7.58 (1H, dd, J 9.9 and 9.5, H-5), 7.64 (2H, dd, J 7.3 and 1.5, H-o-Ph), 7.71 (1H, t, J 9.9, H-6), 8.75 (1H, d, J 9.5, H-4), and 9.24 (1H, d, J 9.9, H-8), and 10.20 (1H, br s, NH); 1H NMR (DMSO-d6) δ 1.32 (3H, t, J 7.0, CH3), 2.20 (3H, s, COCH3), 4.28 (2H, q, J 7.0, OCH2), 7.43 (2H, dd, J 7.3 and 6.6, H-m-Ph), 7.45 (1H, td, J 6.6 and 1.1, H-p-Ph), 7.65 (2H, t, J 9.9, H-5, 7), 7.69 (2H, t, J 7.3, H-o-Ph), 7.91 (1H, t, J 9.9, H-6), 8.68 (1H, d, J 9.9, H-4), 9.10 (1H, d, J 9.9, H-8), and 10.44 (1H, br s, NH); 13C NMR δ 14.48, 24.55, 60.50, 83.68, 98.72, 103.93, 104.55, 124.14, 127.71, 128.21, 129.05, 129.90, 131.32, 135.63, 135.69, 137.50, 139.92, 144.43, 149.43, 166.27, and 167.35; νmax/cm-1 3300 (NH), 2200 and 2140 (C≡C), 1692, 1678, and 1652 (C=O). MS (ESI+) m/z 380 ([M + Na]+). Calcd. for C23H19NO3: M = 357; HRMS (ESI+): Calcd. for 12C231H1914N123Na116O3: 380.1249. Found: m/z 380.1267.
6a: Dark green needles (from hexane-CHCl3), mp 142.0-143.1 °C; 1H NMR δ 1.53 (3H, t, J 7.0, CH3), 2.36 (3H, s, COCH3), 4.49 (2H, q, J 7.0, OCH2), 6.99 (1H, s, H-1), 7.31 (2H, dd, J 7.7 and 7.4, H-m-Ph), 7.56 (2H, t, J 9.9, H-6, 8), 7.60 (1H, dd, J 7.7 and 1.2, H-p-Ph), 7.61 (2H, dd, J 7.4 and 1.2, H-o-PH), 7.72 (1H, t, J 9.9, H-7), 8.72 (1H, d, J 9.9, H-9), and 9.26 (1H, d, J 9.9, H-5); νmax/cm-1 1693, 1678, and 1659. MS (ESI+) m/z 380 ([M + Na]+): Calcd. for C23H19NO3: M = 357; HRMS (ESI+): Calcd. for 12C231H1914N123Na116O3: 380.1263. Found: m/z 380.1249.

Synthesis of ethyl 2-acetylamino-3-trimethylsilylethynylazulene-1-carboxylate (5b)
Under argon atmosphere, a mixture of 1b (0.200 g, 0.52 mmol), trimethylsilyllacetylene (0.07 mL, 0.62 mmol), PdCl2(PPh3)2 (0.010 g, 0.016 mmol), CuI (0.010 g, 0.05 mmol), and Et3N (3 mL) in THF (3 mL) was stirred for 24 h at rt, then water was added. The mixture was extracted with CHCl3. The extract was dried over Na2SO4 and evaporated. Chromatography of the residue on alumina with hexane-AcOEt (5 : 1) gave 5b (0.091 g, 53%).
5b: Green needles (from hexane-CHCl3), mp 123-124.5 °C; 1H NMR (DMSO-d6) δ 0.28 (9H, s, SiCH3), 1.31 (3H, t, J 7.0, CH3), 2.14 (3H, s, COCH3), 4.27 (2H, q, J 7.0, OCH2), 7.69 (2H, t, J 9.9, H-5, 7), 7.90 (1H, t, J 9.9, H-6), 8.50 (1H, d, J 9.9, H-4), 9.11 (1H, d, J 9.9, H-8), and 10.27 (1H, br s, NH); 13C NMR δ 0.26, 14.57, 24.37, 60.50, 85.91, 98.56, 1004.01, 104.68, 128.49, 129.18, 135.74, 135.80, 137.55, 139.93, 144.97, 166.15, and 167.21; νmax/cm-1 3250 (NH), 2140 (C[イメージ]C), 1693 and 1650 (C=O). MS (ESI+) m/z 376 ([M + Na]+). Calcd. for C20H23NO3Si: M = 353; HRMS (ESI+): Calcd. for 12C201H2314N123Na116O328Si1: 376.1345. Found: m/z 376.1330.

Reaction of ethyl 2-acetylamino-3-phenylethynylazulene-1-carboxylate with Cu(OTf)2
A mixture of 5a (0.100 g, 0.28 mmol), Cu(OTf)2 (0.100 g, 0.28 mmol) in CH2Cl2 (6 mL) was heated at 40 °C for 3 h, then water was added. The mixture was extracted with CH2Cl2. The extract was dried over Na2SO4 and evaporated. Chromatography of the residue on silica gel with CHCl3 gave 9 (0.009 g, 9%).
9: Red powder (from hexane-CHCl3), mp 126-127.5 °C; 1H NMR δ 1.52 (3H, t, J 7.0, CH3), 2.36 (3H, s, COCH3), 4.00 (2H, s, COCH2Ph), 4.53 (2H, q, J 7.0, OCH2), 7.10-7.16 (5H, m, Ph), 7.47 (1H, t, J 9.9, H-7), 7.57 (1H, t, J 9.9, H-5), 7.70 (1H, t, J 9.9, H-6), 8.62 (1H, d, J 9.9, H-4), 9.35 (1H, d, J 9.9, H-8), and 11.14 (1H, br s, NH); νmax/cm-1 1703, 1686, and 1649 (C=O). MS (ESI+) m/z 398 ([M + Na]+). Calcd. for C23H21NO4: M = 375; HRMS (ESI+): Calcd. for 12C231H2114N123Na116O4: 398.1368. Found: m/z 398.1353.

Reaction of ethyl 2-acetylamino-3-phenylethynylazulene-1-carboxylate with Pd/C
A mixture of 5a (0.100 g, 0.28 mmol), 5% Pd/C (0.007 g, 0.0056 mmol), Na2CO3 (0.089 g, 0.84 mmol) in DMF (2 mL) was heated at 120 °C for 24 h, then water was added. The mixture was extracted with AcOEt. The extract was dried over Na2SO4 and evaporated. Chromatography of the residue on silica gel with benzene gave 6b (0.030 g, 33%).
6b: Dark green needles (from hexane-CHCl3), mp 123.7-124.6 °C (lit.,13 mp 124 °C); 1H NMR δ 1.53 (3H, t, J 6.8, CH3), 4.53 (2H, q, J 7.0, OCH2), 7.17 (1H, s, H-1), 7.31 (1H, t, J 9.9, H-8), 7.45-7.49 (3H, m, H-m, p-Ph), 7.51 (1H, t, J 9.9, H-7), 7.65 (1H, dd, J 9.9 and 9.6, H-6), 7.69 (2H, d, J 7.0 and 1.5, H-o-Ph), 8.68 (1H, d, J 9.9, H-9), 9.09 (1H, br s, NH), and 9.48 (1H, d, J 9.6, H-5); 13C NMR δ 14.11, 59.75, 97.45, 98.21, 124.35, 124.61, 126.51, 127.24, 127.36, 129.09, 131.99, 132.67, 133.46, 134.55, 134.96, 139.76, 144.59, 148.81, and 165.24; νmax/cm-1 3486 (NH) and 1687 (C=O). MS (ESI+) m/z 380 ([M + Na]+). Calcd. for C23H21NO3: M = 357; HRMS (ESI+): Calcd. for 12C231H2114N123Na116O3: 380.1263. Found: m/z 380.1240.

Reaction of ethyl 2-acetylamino-3-phenylethynylazulene-1-carboxylate with PdCl2(PPh3)2
A mixture of 5a (0.0728 g, 0.207 mmol), PdCl2(PPh3)2 (0.0098 g, 0.0014 mmol), and Et3N (0.02 g, 0.28 mmol) in DMF (2 mL) was heated at 100 °C for 24 h, then water was added. The mixture was extracted with AcOEt. The extract was dried over Na2SO4 and evaporated. Chromatography of the residue silica gel with benzene gave 6b (0.0526 g, 72%).

Biological assay
HeLa S3 cells were obtained from AIST and used after cultivation. The cultivated HeLa S3 cells were cellcounted and the culture fluid was prepared to the cell consistency of 2 x 104 cell/mL. The　compounds were added to the medium in DMSO solutions. To the aliquot of the culture fluid, which was incubated for 3 h at 37 °C, 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 °C, and the solvent was evaporated. Then DMSO was added to 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 cytotoxic assay was replicated four times in order to define the IC values.

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