Synthesis and evaluation of 3-methyl-4-oxo-6-phenyl-4,5,6,7- tetrahydrobenzofuran-2-carboxylic acid ethyl ester derivatives as potent antitumor agents

Article abstract of DOI:10.1248/cpb.53.638

For the construction of new combinatorial libraries, a lead compound was created by replacing the core structure of a hit compound discovered by screening for cytotoxic agents against a tumorigenic cell line. The newly designed compound maintained biologi

Full text of DOI:10.1248/cpb.53.638

638
Chem. Pharm. Bull. 53(6) 638—640 (2005)
Vol. 53, No. 6
Synthesis and Evaluation of 3-Methyl-4-oxo-6-phenyl-4,5,6,7-
tetrahydrobenzofuran-2-carboxylic Acid Ethyl Ester
Derivatives as Potent Antitumor Agents
a
Ichiro HAYAKAWA, ,a Rieko SHIOYA,a Toshinori AGATSUMA,b and Yuichi SUGANO
*
a Lead Discovery Research Laboratories, Sankyo Co., Ltd.; and b Biomedical Research Laboratories, Sankyo Co., Ltd.;
Shinagawa-ku, Tokyo 140–8710, Japan. Received February 1, 2005; accepted March 11, 2005
For the construction of new combinatorial libraries, a lead compound was created by replacing the core
structure of a hit compound discovered by screening for cytotoxic agents against a tumorigenic cell line. The
newly designed compound maintained biological activity and allowed alternative library construction for antitu-
mor drugs.
Key words antitumor; anticancer; cytotoxicity; 4,5,6,7-tetrahydrobenzofuran
In the process of new drug discovery, it is one of the most Results and Discussion
significant steps to generate lead compounds from hit com-
As the most accessible compounds, 4,5,6,7-tetrahydroben-
pounds selected by random screening. Structurally-optimized zofuran derivatives 7, which was readily prepared from inter-
derivatives are obtained from libraries constructed by refer- mediates of benzofuran derivatives, were synthesized (Chart
ring to each lead compound, and the derivatives from each li-
brary are usually expected to possess various biological prop-
erties, which may sometimes show advantage as a desired
drug.
Fig. 1. Structures of a Hit Compound and a Metabolically Stable Deriva-
tive
Chart 1. Synthesis of 4,5,6,7-Tetrahydrobenzofuran and 4,5,6,7-Tetrahy-
dro-1H-indole Derivatives
Reagents: (a) ethyl 2-chloroacetoacetate, KOH in H2O–MeOH; (b) AcOH in
H2O–MeOH; (c) ethyl acetylacetate, NaNO2 in AcOH–H2O; (d) zinc powder in AcOH;
(e) see ref. 3.
From hit compound 1, which was obtained by screening
for anticancer agents displaying selective cytotoxicity against
a tumorigenic cell line, optimized compounds 2 and 3 were
designed (Fig. 1).1—3) The phenyl substituent at the 6-posi-
tion of the benzofuran core was tuned to introduce a fluorine
substituent (2), and the biologically labile ethyl ester group
was replaced with 2,4-dimethoxyphenyl ketone (3).
Fig. 2. Derivatives Consisting of an Alternative Core Structure
Thieno[2,3-b]pyridine derivative 5, which has moderate
similarity to 4-hydroxy-3-methylbenzofuran (3), was discov-
ered and studied as a lead compound possessing a different
core structure. The structure activity relationship (SAR),
however, was significantly different from that of benzofuran
derivatives; the corresponding ethyl ester 4 completely lost
biological activity.4) This result induced interest in the bio-
logical activities of the derivatives having a much more simi-
lar core structure to that of compound 1.
Chart 2. Conversion of Ethyl Ester to 2,4-Dimethoxyphenyl ketone
Reagents: (a) NaBH4, CeCl3·7H2O in MeOH; (b) t-BuMe2SiCl, imidazole in DMF;
(c) NaOH in EtOH; (d) N,O-dimethylhydroxylamine, 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole hydrate (HOBt), Et3N
in DMF; (e) 2,4-dimethoxybromobenzene, n-BuLi in THF; (f) n-Bu4NF, AcOH in
THF; (g) Dess–Martin periodinane6) in CH2Cl2.
∗ To whom correspondence should be addressed. e-mail: hayaka@sankyo.co.jp
© 2005 Pharmaceutical Society of Japan

June 2005
639
Table 1. Cytotoxicity of Synthesized Derivatives and Related Compounds
Cytotoxicity of structure A
Cytotoxicity of structure B
Entry
R1
R2
X
Compd.
EC50, ng/mla)
Compd.
EC50, ng/mla)
1
2
3
4
H
F
H
H
OEt
OEt
OEt
O
O
NH
O
7a
7b
8a
15
22
78
72
1
2
9a
3
40
17
470
40
2,4-dimethoxyphenyl
ꢄ4000
a) Selectivity against tumor cell was confirmed.
After cultivation for two or three days, test compounds dissolved in DMSO
were diluted with fresh growth medium and then added to the plates. The
final concentrations of DMSO were always kept at less than 0.5%. The
plates were incubated for 24 h. Then the culture medium was removed and
washed. Finally, 150 ml/well of fresh medium was added to the cells. After
cultivation for three days, 50 ml/well of MEM-E containing 1 mg/ml of 2,3-
bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanide (XTT:
X-4251, Sigma) and 25 mM of phenazine methosulfate (P-9625, Sigma) was
added and the plates were incubated for 2.5 h. OD450 was measured by a
SPECTRA MAX 250 (Molecular Devices) and cell viability was calculated
using the following formula:
1). The corresponding dimethoxyphenyl ketone (15), which
was expected to be metabolically stable,1) was converted
from 7a (Chart 2). For the purpose of comparing the SAR of
indole derivative 9a, 4,5,6,7-tetrahydro-1H-indole derivative
8a was also synthesized as shown in Chart 1.
The cytotoxicity of the synthesized derivatives and related
compounds are summarized in Table 1. The potency of com-
pound 7a was stronger than corresponding benzofuran com-
pound 1. A derivative with a fluorine substituent (7b) also
possessed significant biological activity. These results indi-
cate that 4,5,6,7-tetrahydrobenzofuran is an alternative core
structure that shows selective cytotoxicity as benzofuran
core. Nitrogen analogues (8a)5) maintained cytotoxicity, nev-
ertheless dimethoxyphenyl ketone (15) completely lost bio-
logical activity.
cell viability (%)ꢁ(WCꢂBG)ꢀ100/(RFꢂBG)
WC: OD450 for well containing cells treated with compounds
RF: OD450 for well containing cells and no compounds
BG: OD450 for well containing neither cells nor compounds
A dose–response curve was drawn and the 50% effective concentration
(EC50) was determined as an indicator of compound cytotoxicity.
3-Methyl-4-oxo-6-phenyl-4,5,6,7-tetrahydrobenzofuran-2-carboxylic
Acid Ethyl Ester (7a) To a solution of KOH (7.21 g, 110 mmol) in H2O
(20 ml), was added 5-phenyl-1,3-cyclohexanedione (20.8 g, 110 mmol), and
the mixture was stirred at room temperature for 15 min. Ethyl 2-chloroace-
toacetate (15.2 ml, 110 mmol) in MeOH (40 ml) was added, and the mixture
was stirred for 20 h at room temperature. AcOH (62 ml) was added, and the
reaction mixture was heated under reflux for 3 h and then the solvent was re-
moved. The mixture was diluted with EtOAc, and washed with saturated
aqueous NaHCO3 and brine. The organic phase was dried over Na2SO4, and
concentrated in vacuo. The residue was purified by flash column chromatog-
Although ketone derivative 15 was designed to avoid
metabolic deactivation by the enzymatic hydrolysis of the
ethyl ester and this was successful in the benzofuran deriva-
tives, this approach was unfortunately revealed to be inap-
plicable to 4,5,6,7-tetrahydrobenzofuran derivatives.
In conclusion, 4,5,6,7-tetrahydrobenzofuran was recog-
nized to be a new core structure for compound libraries tar-
geting antitumor drugs. The derivatives based on this differ-
ent core structure are expected to have different biological raphy (hexane : EtOAcꢁ2 : 1 as a solvent) to give 19.1 g (58%) of 7a as a
1
colorless solid. mp 101—102 °C; H-NMR (CDCl3) d: 7.45—7.36 (2H, m),
7.33—7.27 (3H, m), 4.40 (2H, q, Jꢁ7.1 Hz), 3.63—3.50 (1H, m), 3.24 (1H,
dd, Jꢁ5.1, 17.6 Hz), 3.09 (1H, dd, Jꢁ10.8, 17.6 Hz), 2.80 (2H, d, Jꢁ8.3 Hz),
2.60 (3H, s), 1.41 (3H, t, Jꢁ7.1 Hz); IR (KBr) cmꢂ1: 1709, 1677, 1604,
1456, 1321, 1244; HR-MS (EI) Calcd for C18H18O4 (M)ꢃ: 298.1205, Found:
298.1207.
properties, which expand the possibilities to be new antitu-
mor drugs.
Experimental
Melting points (mp) were determined with a Yanaco melting point appara-
tus and not corrected. Infrared (IR) spectra were measured with a Nic 5SXC
6-(3-Fluorophenyl)-3-methyl-4-oxo-4,5,6,7-tetrahydrobenzofuran-2-
FT-IR spectrophotometer. NMR spectra were recorded on a JEOL JNM-GX carboxylic Acid Ethyl Ester (7b) Compound 7b was prepared using a
270 FT-NMR or a Varian Mercury 400 spectrophotometer. Chemical shifts
similar procedure as for 7a (25%, colorless solid) from 6b (503 mg,
are expressed in d ppm from the internal standard tetramethylsilane (TMS). 2.44 mmol). mp 98—100 °C; 1H-NMR (CDCl3) d: 7.38—7.30 (1H, m),
Mass spectra were obtained on a JEOL HX-100, SX-102A or JMS-AX-
505H mass spectrometer instruments by applying an electric ionization (EI)
method, a fast atom bombardment (FAB) ionization method, or an electro-
spray ionization (ESI) method. Column chromatography was carried out
7.06—6.96 (3H, m), 4.39 (2H, q, Jꢁ7.1 Hz), 3.60—3.50 (1H, m), 3.25 (1H,
dd, Jꢁ5.0, 17.0 Hz), 3.06 (1H, dd, Jꢁ11.0, 17.0 Hz), 2.79—2.75 (2H, m),
2.59 (3H, s), 1.40 (3H, t, Jꢁ7.1 Hz); IR (KBr) cmꢂ1: 1710, 1673, 1589,
1450, 1244; HR-MS (ESI) Calcd for C18H18FO4 (MꢃH)ꢃ: 317.1189, Found:
using SK-85 (230—400 mesh). Preparative thin-layer chromatography 317.1179.
(PTLC) was performed using 60 F254 plates (Merck art. 5744). Yields were
not optimized.
3-Methyl-4-oxo-6-phenyl-4,5,6,7-tetrahydro-1H-indole-2-carboxylic
Acid Ethyl Ester (8a) To a solution of ethyl acetoacetate (15.0 g,
Evaluation of Biological Activity Human tumorigenic cell line VA13 93.8 mmol) in AcOH (30 ml), was added a solution of NaNO2 (8.00 g,
(CCL-75.1) and its parental normal cell line WI-38 (CCL-75) were obtained
116 mmol) in H2O (25 ml) below 12 °C, and the mixture was stirred at such
from the American Tissue Culture Collection (ATCC) and were maintained a rate that the temperature remained below 12 °C for 3 h and was left at
and assayed in Minimum Essential Medium-Eagle (MEM-E) with Earl’s room temperature overnight. A solution of 5-phenyl-1,3-cyclohexanedione
salts (11095-080, Gibco BRL) with 100 U/ml of penicillin and 100 mg/ml of (24.0 g, 128 mmol) in AcOH (60 ml) was added. Then the zinc dust (16.7 g,
streptomycin supplemented with 10% fetal calf serum (Hyclone). Exponen-
255 mmol) was added at such a rate that the temperature did not rise above
tially growing VA-13 cells and WI-38 cells were collected and distributed 60 °C. After stirring for 0.5 h and refluxing for 3 h, the solution was sepa-
into 96-well plates (3598, Corning-Coaster) at a density of 1.5—3.0ꢀ103
cells and 4.8—9.6ꢀ103 cells, respectively, at a final volume of 200 ml/well.
rated from the excess zinc dust and poured into iced water (500 ml) to give a
yellow solid. The crude product was purified by recrystallization from ben-

640
Vol. 53, No. 6
zene to give 9.45 g (34%) of 8a as a colorless solid. mp 207—208 °C (dec.);
1H-NMR (CDCl3) d: 9.26 (1H, s), 7.38—7.26 (5H, m), 4.34 (2H, q,
Jꢁ7.1 Hz), 3.56—3.44 (1H, m), 3.09 (1H, dd, Jꢁ5.0, 16.0 Hz), 3.00 (1H,
dd, Jꢁ11.0, 16.0 Hz), 2.77—2.73 (2H, m), 2.63 (3H, s), 1.37 (3H, t,
Jꢁ7.1 Hz); IR (KBr) cmꢂ1: 3194, 1692, 1635, 1488, 1282, 1184; HR-MS
(ESI) Calcd for C18H20NO3 (MꢃH)ꢃ: 298.1443, Found: 298.1429.
4-Hydroxy-3-methyl-6-phenyl-4,5,6,7-tetrahydrobenzofuran-2-car-
boxylic Acid Ethyl Ester (10) To a solution of 7a (100 mg, 0.335 mmol)
and CeCl3·7H2O (150 mg, 0.402 mmol) in MeOH (2 ml), was added NaBH4
(13 mg, 0.335 mmol), and the mixture was stirred at room temperature for
30 min. The reaction mixture was diluted with EtOAc, and washed with
aqueous 5% HCl, saturated aqueous NaHCO3 and brine. The organic phase
was dried over Na2SO4, and concentrated in vacuo. The residue was purified
by PTLC (hexane : EtOAcꢁ3 : 1 as a solvent) to give 86 mg (85%) of 10 as a
colorless amorphous foam. 1H-NMR (CDCl3) d: 7.38—7.32 (2H, m),
7.29—7.19 (3H, m), 4.99—4.88 (1H, m), 4.37 (2H, q, Jꢁ7.3 Hz), 3.18—
3.08 (1H, m), 2.98—2.75 (2H, m), 2.50—2.37 (1H, m), 2.46 (3H, s), 1.99—
1.87 (1H, m), 1.53—1.50 (1H, m), 1.39 (3H, t, Jꢁ7.3 Hz); EI-MS m/z: 300
(M)ꢃ.
4-(tert-Butyldimethylsilanyloxy)-3-methyl-6-phenyl-4,5,6,7-tetrahy-
drobenzofuran-2-carboxylic Acid Ethyl Ester (11) tert-Butyldimethyl-
silyl chloride (80 mg, 0.533 mmol) was added to a stirred solution of 10
(79 mg, 0.267 mmol) and imidazole (55 mg, 0.800 mmol) in DMF (1.0 ml) at
room temperature. The mixture was stirred for 40 h at room temperature.
The reaction mixture was diluted with EtOAc, and washed with 10% aque-
ous KHSO4, saturated aqueous NaHCO3 and brine. The organic phase was
dried over Na2SO4, and concentrated in vacuo. The residue was purified by
flash column chromatography (hexane : EtOAcꢁ4 : 1 as a solvent) to give
104 mg (95%) of 11 as a colorless oil. 1H-NMR (CDCl3) d: 7.39—7.34 (2H,
m), 7.29—7.25 (3H, m), 5.04—4.96 (1H, m), 4.36 (2H, q, Jꢁ7.3 Hz),
3.09—2.92 (1H, m), 2.88—2.70 (2H, m), 2.42 (3H, s), 2.37—2.30 (1H, m),
2.00—1.92 (1H, m), 1.38 (3H, t, Jꢁ7.3 Hz), 0.92 (9H, s), 0.17 (3H, s), 0.13
(3H, s); FAB-MS m/z: 415 (MꢃH)ꢃ.
0.566 mmol) in THF (1.0 ml) at ꢂ78 °C under N2. The mixture was stirred
for 30 min at the same temperature. After 12 (81 mg, 0.189 mmol) in THF
(1.0 ml) was added to the reaction mixture over 5 min at ꢂ78 °C, the reac-
tion mixture was stirred for 30 min at the same temperature. The reaction
mixture was quenched with saturated aqueous NH4Cl, and extracted with
EtOAc. The extract was washed with brine, dried over Na2SO4, and concen-
trated in vacuo. The residue was purified by PTLC (hexane : EtOAcꢁ3 : 1 as
a solvent) to give 64 mg (67%) of 13 as a colorless solid. 1H-NMR (CDCl3)
d: 7.38—7.33 (3H, m), 7.28—7.23 (3H, m), 6.54—6.49 (2H, m), 5.06—
5.00 (1H, m), 3.85 (3H, s), 3.79 (3H, s), 3.10—3.03 (1H, m), 2.90—2.69
(2H, m), 2.38—2.34 (1H, m), 2.29 (3H, s), 2.02—1.89 (1H, m), 0.92 (9H,
s), 0.17 (3H, s), 0.12 (3H, s); FAB-MS m/z: 507 (MꢃH)ꢃ.
(2,4-Dimethoxyphenyl)-(4-hydroxy-3-methyl-6-phenyl-4,5,6,7-tetrahy-
drobenzofuran-2-yl)methanone (14) To a stirred solution of 13 (62 mg,
0.122 mmol) in THF (1.0 ml) was added n-Bu4NF (0.244 ml, 1.0 M solution
in THF) and AcOH (14 ml, 0.244 mmol) at room temperature. The mixture
was stirred for 14 h at room temperature. The n-Bu4NF (0.488 ml, 1.0 M so-
lution in THF) and AcOH (28 ml, 0.488 mmol) were added to the reaction
mixture, and stirred for 1 h at 60 °C. The reaction mixture was diluted with
EtOAc, and washed with H2O, saturated aqueous NaHCO3 and brine. The
organic phase was dried over Na2SO4, and concentrated in vacuo. The
residue was purified by flash column chromatography (hexane : EtOAcꢁ1 : 1
as a solvent) to give 45 mg (94%) of 14 as a yellow oil. 1H-NMR (CDCl3) d:
7.39—7.32 (3H, m), 7.28—7.24 (3H, m), 6.55—6.50 (2H, m), 4.98—4.95
(1H, m), 3.86 (3H, s), 3.80 (3H, s), 3.17—3.10 (1H, m), 2.94—2.73 (2H,
m), 2.51—2.45 (1H, m), 2.33 (3H, s), 2.00—1.88 (1H, m), 1.60—1.57 (1H,
m); HR-MS (ESI) Calcd for C24H25O5 (MꢃH)ꢃ: 393.1702, Found:
393.1700.
2-(2,4-Dimethoxybenzoyl)-3-methyl-6-phenyl-4,5,6,7-tetrahydroben-
zofuran-4-one (15) To a solution of 14 (42 mg, 0.107 mmol) in CH2Cl2
(1.0 ml) was added Dess–Martin periodinane (68 mg, 0.160 mmol), and the
mixture was stirred for 2 h at room temperature. The reaction mixture was
diluted with EtOAc, washed with saturated aqueous NaHCO3 and brine. The
organic phase was dried over Na2SO4, and concentrated in vacuo. The
residue was purified by flash column chromatography (hexane : EtOAcꢁ2 : 1
as a solvent) to give 33 mg (79%) of 15 as a colorless amorphous foam. 1H-
NMR (CDCl3) d: 7.43—7.35 (3H, m), 7.32—7.26 (3H, m), 6.56 (1H, dd,
Jꢁ2.2, 8.6 Hz), 6.51 (1H, d, Jꢁ2.2 Hz), 3.87 (3H, s), 3.77 (3H, s), 3.62—
3.52 (1H, m), 3.21 (1H, dd, Jꢁ5.0, 17.8 Hz), 3.07 (1H, dd, Jꢁ10.9,
17.8 Hz), 2.79 (2H, d, Jꢁ8.2 Hz), 2.42 (3H, s); IR (KBr) cmꢂ1: 1682, 1604,
1456, 1211, 1029, 944; HR-MS (ESI) Calcd for C24H23O5 (MꢃH)ꢃ:
391.1546, Found: 391.1544.
4-(tert-Butyldimethylsilanyloxy)-3-methyl-6-phenyl-4,5,6,7-tetrahy-
drobenzofuran-2-carboxylic Acid N-Methoxy-N-methylamide (12)
A
mixture of 11 (103 mg, 0.251 mmol) and 1 N NaOH (1.0 ml) in EtOH
(2.0 ml) was heated under reflux for 2 h. The mixture was acidified
(pHꢁ3.0) with 10% aqueous HCl, and extracted with EtOAc. The extract
was washed with brine, dried over Na2SO4, and concentrated in vacuo. To a
solution of residue, 1-hydroxybenzotriazole (34 mg, 0.251 mmol), N,O-di-
methylhydroxylamine hydrochloride (49 mg, 0.502 mmol) and Et3N (105 ml,
0.753 mmol) in DMF (1.5 ml), was added 1-(3-dimethylaminopropyl)-3-eth-
ylcarbodiimide hydrochloride (96 mg, 0.502 mmol) under ice cooling. After
stirring the mixture for 14 h at room temperature. The reaction mixture was
diluted with EtOAc, and washed with 5% aqueous HCl, saturated aqueous
NaHCO3 and brine. The organic phase was dried over Na2SO4 and concen-
trated in vacuo. The residue was purified by flash column chromatography
(hexane : EtOAcꢁ2 : 1 as a solvent) to give 84 mg (79%) of 12 as a colorless
amorphous foam. 1H-NMR (CDCl3) d: 7.39—7.34 (2H, m), 7.29—7.26
(3H, m), 5.05—4.99 (1H, m), 3.78 (3H, s), 3.29 (3H, s), 3.12—3.00 (1H,
m), 2.90—2.71 (2H, m), 2.37 (3H, s), 2.37—2.31 (1H, m), 2.02—1.89 (1H,
m), 0.92 (9H, s), 0.18 (3H, s), 0.13 (3H, s); FAB-MS m/z: 430 (MꢃH)ꢃ.
[4-(tert-Butyldimethylsilanyloxy)-3-methyl-6-phenyl-4,5,6,7-tetrahy-
drobenzofuran-2-yl]-(2,4-dimethoxyphenyl)methanone (13) n-Butyl-
lithium (0.360 ml, 0.548 mmol, 1.53 M in hexane) was added dropwise
to the stirred solution of the 1-bromo-2,4-dimethoxybenzene (123 mg,
Acknowledgements The authors are grateful to Ms. Youko Oda for her
technical support in the cytotoxicity test.
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