Synthesis, cyclization, and evaluation of the anticancer activity against hela S-3 cells of ethyl 2-acetylamino-3-ethynylazulene-1-carboxylates

Article abstract of DOI:10.3987/COM-12-S(N)4

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).

Full text of DOI:10.3987/COM-12-S(N)4

HETEROCYCLES, Vol. 86, No. 1, 2012
233
HETEROCYCLES, Vol. 86, No. 1, 2012, pp. 233 - 244. © 2012 The Japan Institute of Heterocyclic Chemistry
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, Noda, Chiba 278-8510, Japan
E-mail noriabe@rs.noda.tus.ac.jp
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
(1b) gave
of
ethyl
ethyl
2-acetylamino-3-iodoazulene-1-carboxylate
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 [M] : 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
†Dedicated to Prof. Ei-ichi Negishi on the occasion of his 77th birthday.

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HETEROCYCLES, Vol. 86, No. 1, 2012
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
1
was deduced from its H 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 
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  5.62 (4H, br s) and ethyl protons. The molecular
1
formula of 4 was deduced to C39H35N3O6 from its HRMS spectrum. In the H NMR spectrum of 4, six
doublet signals for eight protons, at  7.30 (2H, d, J 10.2), 7.56 (2H, d, J 10.2), 7.94 (1H, d, J 10.2), 8.99

HETEROCYCLES, Vol. 86, No. 1, 2012
235
(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  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
CO2Et
NHR
CO2Et
NHR
I
1a
2a
: R = H
: R = H
2b : R = Ac
1b : R = Ac
CO2Et
NH2
CO2Et
NH2
CO2Et
NH2
NIS/CHCl3
NH2
+
o
2 C
H2N
EtO2C
CO2Et
H2N
EtO2C
2a
4
3
NIS/Et3N/CH2Cl2
- 7 oC
CO2Et
NH2
I
1a
Scheme 1
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

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HETEROCYCLES, Vol. 86, No. 1, 2012
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  6.99, and methyl
signal was appeared at  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  6.87,19 therefore the signal at  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.
CO2Et
NHAc
CO2Et
NHAc
CO2Et
N
R / PdCl2(PPh3)2
CuI / Et3N / THF
Ac
Ph
+
I
5a : R = Ph
5b
1b
: R = TMS
R
Scheme 2
N
N
Cu-catalyst
MsCl
N
NH2
NHMs
Ms
Ph
N
8a
8b
Ph
Ph
Scheme 3

HETEROCYCLES, Vol. 86, No. 1, 2012
237
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
1
its H NMR spectrum, phenyl protons were seen at  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.
CO2Et
NHAc
CO2Et
NHAc
Cu(OTf)2
C O
Ph
H2C
Pd-C/Na2CO3/DMF
9
5a
Ph
or PdCl2(PPh3)2/Et3N/DMF
CO2Et
N
H
Ph
Scheme 4
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.

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HETEROCYCLES, Vol. 86, No. 1, 2012
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 [M] 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.
Table 1. Cytotoxic evaluation of compounds (1b, 3, 5a, 5b and 8a); IC50 were expressed in M.
1b
3
5a
5b
8a
2.9 ± 0.2
13.4 ± 1.1
99.0 ± 1.1
IC50
> 52.2
> 55.8
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 [M] : 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
1
for H 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

HETEROCYCLES, Vol. 86, No. 1, 2012
239
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  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  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; max/cm-1
3485, 3335 (NH2), 1669 (C=O). MS (ESI+) m/z 451 ([M + Na]+). Calcd. for C26H24N2O4: M = 428;
HRMS (ESI+): Calcd. for C26 H2414N2 Na1 O4: 451.1634. Found: m/z 451.1627. Anal. Calcd. for
12
1
23
16
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  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); max/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;
1
23
16
HRMS (ESI+): Calcd. for 12C39 H3514N3 Na1 O6: 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  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),

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HETEROCYCLES, Vol. 86, No. 1, 2012
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)  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; max/cm-1 3473, 3313 (NH2), 1669 (C=O). MS (ESI+)
m/z 363 ([M + Na]+). Calcd. for C13H12NO2: M = 340; HRMS (ESI+): Calcd. for
C H1214N1 Na1127I1 O2: 363.9810. Found: m/z 363.9795.
12
1
23
16
13
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)  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); max/cm-1 3260 (NH), 1693 and 1646 (C=O). MS (ESI+) m/z 280 ([M + Na]+). Calcd.
1
23
16
for C15H15NO3: M = 257; HRMS (ESI+): Calcd. for 12C15 H1514N1 Na1 O3: 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) 
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
13
(1H, br s, NH); C NMR  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; max/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
C H1414N1127I1 Na1 O3: 405.9916. Found: 405.9888.
12
1
23
16
15
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%)

HETEROCYCLES, Vol. 86, No. 1, 2012
241
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  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,
1
H-4), and 9.24 (1H, d, J 9.9, H-8), and 10.20 (1H, br s, NH); H NMR (DMSO-d6)  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),
13
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); C NMR  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; max/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+):
1
23
16
Calcd. for 12C23 H1914N1 Na1 O3: 380.1249. Found: m/z 380.1267.
1
6a: Dark green needles (from hexane-CHCl3), mp 142.0-143.1 °C; H NMR  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); max/cm-1 1693, 1678, and
1659. MS (ESI+) m/z 380 ([M + Na]+): Calcd. for C23H19NO3: M = 357; HRMS (ESI+): Calcd. for
C H1914N1 Na1 O3: 380.1263. Found: m/z 380.1249.
12
1
23
16
23
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)  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
 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,

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HETEROCYCLES, Vol. 86, No. 1, 2012
139.93, 144.97, 166.15, and 167.21; max/cm-1 3250 (NH), 2140 (C C), 1693 and 1650 (C=O). MS
(ESI+) m/z 376 ([M + Na]+). Calcd. for C20H23NO3Si: M = 353; HRMS (ESI+): Calcd. for
C H2314N1 Na1 O3 Si1: 376.1345. Found: m/z 376.1330.
12
1
23
16
28
20
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  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); max/cm-1 1703, 1686, and 1649 (C=O). MS (ESI+) m/z 398 ([M + Na]+). Calcd.
for C23H21NO4: M = 375; HRMS (ESI+): Calcd. for C23 H2114N1 Na1 O4: 398.1368. Found: m/z
12
1
23
16
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%).
1
6b: Dark green needles (from hexane-CHCl3), mp 123.7-124.6 °C (lit.,13 mp 124 °C); H NMR  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,
13
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); C NMR  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; max/cm-1 3486 (NH) and 1687 (C=O). MS (ESI+) m/z 380
([M + Na]+). Calcd. for C23H21NO3: M = 357; HRMS (ESI+): Calcd. for C23 H2114N1 Na1 O3:
12
1
23
16
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

HETEROCYCLES, Vol. 86, No. 1, 2012
243
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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