Synthesis and antitumor evaluation of thiophene analogs of kigelinone

Article abstract of DOI:10.2174/157017809788490006

The synthesis of kigelinone thiophene analogs and related naphtho[2,3-b]thiophene-4,9-quinones from 2-substituted 4,7-dimethoxybenzo[b] thiophenes via an oxidative deprotection, Diels-Alder, and oxidative aromatization reaction sequence is reported. The 2-substituted naphtho[2,3-b]thiophene-4,9-quinones display significant antitumor activity in the range IC₅₀ 1.1-47 μM on a panel of four distinct human cancer cell lines.

Full text of DOI:10.2174/157017809788490006

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278
Letters in Organic Chemistry, 2009, 6, 278-281
Synthesis and Antitumor Evaluation of Thiophene Analogs of Kigelinone
Jaime A. Valderrama*,a, Omar Espinozaa, Jaime Rodriguezb and Cristina Theodulozb
aFacultad de Quimica, Pontificia Universidad Católica de Chile, Casilla 306, Santiago, Chile
bFacultad de Ciencias de la Salud, Universidad de Talca, Santiago, Chile
Received December 04, 2008: Revised March 12, 2009: Accepted March 29, 2009
Abstract: The synthesis of kigelinone thiophene analogs and related naphtho[2,3-b]thiophene-4,9-quinones from 2-
substituted 4,7-dimethoxybenzo[b]thiophenes via an oxidative deprotection, Diels-Alder, and oxidative aromatization re-
action sequence is reported. The 2-substituted naphtho[2,3-b]thiophene-4,9-quinones display significant antitumor activity
in the range IC50 1.1-47 μM on a panel of four distinct human cancer cell lines.
Keywords: Quinones, Benzo[b]thiophenes, Oxidative deprotection, Diels-Alder reaction, Regioisomers, Citotoxicity.
Among the broad variety of biological active naturally
occurring heterocyclic quinones [1] a series of naphthoqui-
nones based on the naphtho[2,3-b]furan-4,9-quinone skele-
ton display potent cytotoxic activity against numerous tumor
cell lines [2, 3]. Kigelinone 1 and its positional isomers 2
(Fig. (1)), isolated from Tabebuia avellanedae [3], are two
representative members of this series.
SK-MES-1 lung cancer, J82 bladder carcinoma, and HL-60
leukemia in 72-h drug exposure assays. The cytotoxicity of
the compounds was measured using a conventional micro-
culture tetrazolium reduction assay [8], using etoposide as
positive control.
The synthesis of 5- and/or 8-hydroxy-2-(1-
hydroxyethyl)-naphtho[2,3-b]thiophene-4,9-quinones
was
O
OH
O
explored following the retrosynthetic sequence shown in
Scheme 1, which is based on heterocyclization and Diels-
Alder reactions as the key steps.
OH
Me
OH
Me
Benzothiophene 4 was prepared from 6-nitro-2,5-
dimethoxybenzaldehyde (3) and methyl thioglycolate
according to a previously reported method [9].
O
O
OH
O
O
2
1
Two synthetic approaches to the target benzothiophene 5
via the addition reaction of methylmagnesium chloride to a
carbonyl group were planned using the precursors 4 or 8
(Scheme 2). Our attempts with the first precursor were un-
successful. Therefore aldehyde 8 was prepared from 4 [10]
via alcohol 7 [9]. Further reaction of 8 with methylmagne-
sium chloride in THF gave the expected benzothiophene 9 in
90% yield.
Fig. (1). Structure of kigelinone and its positional isomer.
These naphthofuranquinones exhibit similar cytotoxic
activities against leukemia cells P-388 (IC50: 1 = 16
μmol/mL; 2 = 12 μmol/mL), suggesting that location of the
phenolic hydroxyl substituent, at C-5 or C-8, does not have a
significant influence on the biological activity in these assays
[4]. Kigelinone 1, exhibits remarkably potent inhibition
against Epstein-Barr virus early antigen (EBV-EA) activa-
tion induced by the tumor promoter, 12-O-tetra-
decanoylphorbol-13-acetate (TPA) [5]. Furthermore, com-
pound 1 acts as a cancer chemopreventive agent by strongly
inhibiting TPA-induced tumor promotion on mouse skin
initiated with 7,12 dimethylbenz[a]-anthracene in two stage
carcinogenesis tests [5, 6].
Next, compound 9 was reacted with acetic anhydride to
give benzothiophene 5 in 95% yield, which by a subsequent
oxidative demethylation with CAN in acetonitrile-water pro-
duced the target benzothiophenequinone 6 in 94% yield [11].
Diels-Alder reaction of benzothiophenequinone 6 with (E)-1-
trimethylsilyloxy-1,3-butadiene in dichloromethane at room
temperature yields a nearly 50:50 mixture of regioisomers
10a+10b. The ratio between these isomers was determined
from the thiophene proton signals at ꢀ 8.06 and 8.08 ppm
(400 MHz).
Following our interest in developing novel anticancer
agents [7], a series of naphtho[2,3-b]thiophene-4,7-quinones
isosterically related to the antitumor keligenone 1 and its
isomer 2, were designed and synthesized. The novel thio-
phene analogs were evaluated for in vitro anticancer activity
against MRC-5 human lung fibroblast cells, and four distinct
human tumor cell lines: AGS gastric adenocarcinoma,
The lack of regioselectivity of the cycloaddition between
quinone 6 and the silyloxydiene can be attributed to the
small difference between the LUMO eigenvectors at C-5
(0.26244 eV) and C-6 (-0.24576 eV) of dienophile 6 [12].
Acid-induced hydrolysis of cycloadduct 10a+10b fol-
lowed by aromatization of the alcohol intermediates with
PCC gave naphthothiophenequinones 11a+11b in 80% yield.
Further hydrolysis of acetates 11a+11b produced a 50:50
*Address correspondence to this author at the Facultad de Quimica, Pontifi-
cia Universidad Católica de Chile, Casilla 306, Santiago, Chile; Tel: +56-
02-6864432; Fax: +56-02-6864744; E-mail: jvalderr@uc.cl
1570-1786/09 $55.00+.00
© 2009 Bentham Science Publishers Ltd.

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Synthesis and Antitumor Evaluation of Thiophene Analogs
Letters in Organic Chemistry, 2009, Vol. 6, No. 4
279
OH
OH
O
O
O
O
OMe
OH
Me
DA
OAc
OAc
Me
+
S
S
Me
S
Me3SiO
OMe
6
5
OMe
OMe
CHO
NO2
heterocyclisation
HS
CO2Me
CO2Me
S
OMe
OMe
3
4
Scheme 1. Strategy to prepare kigelinone thiophene-analogues.
OMe
OMe
OMe
OMe
CHO
NO2
c
b
a
CH2OH
CHO
CO2Me
S
S
S
OMe
OMe
OMe
OMe
8
3
4
7
OMe
OMe
O
O
OMe
OAc
Me
OH
Me
OAc
Me
d
g
e
f
S
S
S
OMe
6
5
9
R1
O
O
R1
O
O
R1
R1
O
O
O
O
OAc
OAc
Me
OH
Me
h
i
c
S
S
S
S
Me
Me
R2
R2
11a,b
R2
O
R2
12a,b
13a,b.
10a,b
Scheme 2. Synthesis of compounds 13a-b: substituents: R1 = H(OSiMe3), R2 = OSiMe3 (H); conditions: a) HSCH2CO2Me, K2CO3, DMF,
60°C; b) LiAlH4, Et2O, reflux; c) PCC, AcONa, CH2Cl2; d) MeMgCl, THF, reflux; e) Ac2O, 2h, rt; f) CAN, MeCN, H2O, rt; g) (E)-Me3SiO-
CH=CH-CH=CH2, CH2Cl2, 2d, rt; h) HCl, H2O, rt; i) HCl, H2O, reflux.
mixture of hydroxynaphthothiophene-quinones 12a+12b in
96% yield [13]. Quinones 12a/12b were converted into the
corresponding 2-acetylnaphthothiophenequinones 13a/13b
in 95% yield by oxidation with PCC in dichloromethane
[14].
regioisomer, in each mixture, on the basis of 2D NMR
experiments. Calculation of the HOMO and LUMO eigen-
vectors of the silyloxydiene and dienophiles 14 and 15 [16]
indicates that the regiochemistry of these cycloadditions pro-
ceed in accordance with that predicted by FMO theory
[17, 18].
Since our efforts to isolate the regioisomers from the
corresponding 12a,b and 13a,b mixtures by column and
preparative TLC chromatography were unsuccessful, the
antitumor evaluation was performed with the isomeric
mixtures.
Table 1 shows the in vitro cytotoxic evaluation of naph-
thothiophenequinone mixtures 12, 13, 18 and 19 against
normal MRC-5 human lung fibroblasts and four human
tumor cells: AGS gastric adenocarcinoma cell lines,
SK-MES-1 lung cancer cells, J82 bladder carcinoma cells,
and HL-60 leukemia cells. Data for the etoposide, as positive
control, are included for comparison. The results are ex-
pressed as IC50-values, that is, as the micromolar concentra-
tion of a compound that achieves 50% cellular growth reduc-
tion after 72 h of drug exposure.
Compounds 18a,b and 19a,b [15], prepared as 70:30 re-
gioisomeric mixtures from benzothiophenequinones 14 [9]
and 15, as shown in Scheme 3, were also included into the
biological screening. The ratio between regioisomers
18a/18b and 19a/19b was established from the signals of the
1
phenolic protons in H NMR and the structure of the major

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280 Letters in Organic Chemistry, 2009, Vol. 6, No. 4
Valderrama et al.
OH
O
O
R3
O
O
O
O
O
O
R
O
O
R
b,c
a
+
R1
S
R
S
S
S
OH
R2
O
O
18b. R = OMe (30%)
19b. R = H (30%)
16a. R1 = OMe, R2 = OSiMe3, R3 = H
16b. R1 = OMe, R2 = H, R3 = OSiMe3
17a. R1 = R3 =H, R2 = OSiMe3
18a. R = OMe (70%)
19a. R = H (70%)
14. R = OMe
15. R = H
17b. R1 = R2 = H, R3 = OSiMe3
Scheme 3. Preparation of compounds 18 and 19: a) (E)-1-Me3SiO-CH=CH-CH=CH2, CH2Cl2, 2d, rt; b) HCl, H2O, rt; c) PCC, AcONa,
CH2Cl2.
Table 1. In Vitro Antitumor Activity of Naphthothiophenequinones 12, 13, 18 and 19
IC50 (μM)a
SK-MES-1d
Structure, N° (% in the mixture)
MRC-5b
AGSc
J82e
HL-60f
O
OH
OH
OH
OH
O
OH
Me
OH
Me
4.9 ± 0.2
2.7 ±0.1
3.0 ± 0.2
1.6 ± 0.05
10.9 ± 0.7
S
S
OH
OH
OH
O
O
12a (50%)
12b (50%)
O
O
O
O
8.1 ± 0.4
2.6 ± 0.2
2.0 ± 0.2
1.3 ± 0.07
46.9 ± 2.5
S
Me
S
Me
O
O
13a (50%)
13b (50%)
O
O
O
O
10.0 ± 0.6
5.1 ± 0.3
1.8 ± 0.08
1.1 ± 0.06
42.7 ± 2.2
S
OMe
S
OMe
O
O
18a (30%)
18b (70%)
O
O
O
H
O
H
5.9 ± 0.3
3.9 ± 0.3
1.2 ± 0.07
0.36 ± 0.1
3.1 ± 0.2
2.5 ± 0.1
2.2 ± 0.2
2.8 ± 0.2
15.5 ± 0.9
0.80 ± 0.1
S
S
OH
O
O
19a (30%)
19b (70%)
etoposide
aData represent mean values (±SEM) for six independent determination; bhuman lung fibroblasts cells; chuman gastric adenocarcinoma cell line; dhuman lung cancer cell line; ehuman
bladder carcinoma cell line; fhuman leukemia cell line.
Comparison of the IC50 values obtained with compounds
12, 13, 18 and 19 against human lung fibroblast cells indi-
cates that these thiophene analogs are less cytotoxic than the
antitumor drug etoposide. The 12 and 13 mixtures, which
contain equal amount of their regioisomers, displayed com-
parable antitumor activity on the cell lines, except for HL-60
leukemia cells.
It should be noted that among the evaluated naphthotio-
phenequinones, compounds 18a+18b showed the highest
antitumor potencies and selectivities against SK-MES-1
human lung cancer and J82 bladder carcinoma cell lines.

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Synthesis and Antitumor Evaluation of Thiophene Analogs
Letters in Organic Chemistry, 2009, Vol. 6, No. 4
281
Rodríguez, J. A.; Theoduloz, C.; Schmeda-Hirschmann, G.
Tetrahedron, 2006, 62, 2631; e) Valderrama, J. A.; Zamorano, C.;
González, M. F.; Prina, E.; Fournet, A. Bioorg. Med. Chem., 2005,
13, 4153; f) Valderrama, J. A.; Benites, J.; Cortés, M.; Pessoa-
Mahana, D.; Prina, E.; Fournet, A. Bioorg. Med. Chem., 2003, 11,
4713.
Rodríguez, J. A.; Haun, M. Planta Med., 1999, 65, 522.
Valderrama, J. A.; Valderrama, C. Synth. Commun., 1997, 27,
2143.
Tapia, R.A.; Alegría, L.; Pessoa, C.D.; Salas, C.; Cortés, M.;
Valderrama, J. A.; Sarciron, M.-E.; Pautet, F.; Walchshofer, N.;
Fillon, H. Bioorg. Med. Chem., 2003, 11, 2175.
Experimental procedure: A solution of cerium ammonium nitrate
(300 mg, 0.55 mmol) in water (10 mL) was dropwise added to a
stirred solution of 5 (50 mg, 0.18 mmol) in water (5 mL). The
mixture was left for 10 min at rt and then extracted with ethyl
acetate (3x15 ml). The organic layer was dried over magnesium
sulfate and evaporated under reduced pressure. Column
chromatography of the crude over silica gel (CH2Cl2) yield quinone
6 (42 mg, 94%) as a yellow solid mp 112-113°C. IR (KBr): ꢀ 1745
(C=O, ester), 1670-1635 (C=O quinone); 1H NMR (200 MHz,
CDCl3): ꢁ 1.65 (d, 3H, J = 6.5 Hz, CHCH3), 2.10 (s, 3H, COCH3),
6.11 (q, 1H, J = 6.5 Hz, CH), 6.77 (d, 1H, J = 10 Hz, 5- or 6-H),
6.82 (d, 1H, J = 10 Hz, 6- or 5-H), 7.41 (s, 1H, 3-H). 13C NMR (50
MHz, CDCl3): ꢁ 14.3, 52.6, 76.4, 133.9, 136.9, 137.9, 139.5, 145.8,
147.0, 162.1, 180.3, 182.1; Anal Calcd for C12H10O4S: C, 57.59; H,
4.03; S, 12.81. Found: C, 57.21; H, 3.99; S, 12.76.
The LUMO eigenvector coefficients (eV) were determined using
the semiempirical AM1 method implemented in the PC Spartan Pro
Inc package.
Spectral and analytical data for compounds 12a+12b: IR (KBr): ꢀ
3400-3200 (OH), 1660, 1635 (C=O quinone). 1H NMR (200 MHz,
CDCl3): ꢁ 1.66 (d, 3H, J = 6.5 Hz, CH3), 2.13 (s, 1H, OH), 5.08 (m,
1H, CH), 7.30-7.80 (m, 3H, arom.), 8. 09 (s, 0.5H, 3-H), 8.11 (s,
0.5H, 3-H), 12.07 (s, 0,5H, OH), 12.17 (s, 0,5H, OH); Anal Calcd
for C14H10O4S: C, 61.30; H, 3.67; S, 11.69. Found: C, 61.17; H,
3.53; S, 11.76.
These compound along with isomers 13a+13b were less
cytotoxic on MRC-5 human lung fibroblasts and more
potent than the reference etoposide on SK-MES-1 lung
cancer and J82 bladder carcinoma cell lines. Furthermore,
the kigelinone thiophene-analogs 12a,12b and their acetyl
congeners 13a,13b exhibited less cytotoxic effect and more
antitumor potencies against J82 bladder carcinoma cells than
etoposide
[8]
[9]
[10]
In conclusion, we have described the synthesis of kigeli-
none thiophene-analogs from 2- substituted 4,7-dimethoxy-
benzo[b]thiophenes. The reported synthesis involves easily
available precursors and an oxidative deprotection, a Diels-
Alder, and oxidative aromatization reaction sequence. This
approach may be used to make a large number of such thio-
phene analogs from diverse 2-substituted 4,7-dimethoxy-
benzo[b]-thiophenes. The described biological evaluation
provides significant information about the design of novel
thiophene-containing quinones as potential antitumor agents
based on bioisosteric replacement. Further studies on the
preparation of 2-substituted naphthothiophenequinones and
isolation of regioisomers 12a and 12b via derivatization are
in progress.
[11]
[12]
[13]
ACKNOWLEDGEMENTS
We thank the Fondo Nacional de Ciencia y Tecnología
(Grant N˚ 8980003 and Nº1020885) for financial support of
this study.
[14]
[15]
Spectral and analytical data for compounds 13a+13b: IR (KBr):
3300 (OH), 1683 (C=O), 1646 (C=O quinone). 1H NMR (200
MHz, CDCl3): ꢁ 2.66 (s, 3H, CH3), 7.30-7.85 (m, 3H, arom.); 8.29-
8.30 (2s br, 2H, 3-H), 12.01(s, 0.5H, OH); 12.15 (s, 0.5H, OH).
Anal Calcd for C14H8O4S: C, 61.76, H, 2.96, S, 11.79. Found: C,
61.56; H, 3.08, S, 11.67.
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1680 (C=O aldehyde), 1645 (C=O quinone); H NMR (400 MHz,
CDCl3): ꢁ7.30-7.85 (m, 3H, arom.), 8.40 (s, 0.7H, 3-H), 8.42 (s,
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eigenvectors (eV): silyloxydiene: C-1 = -0.4744 , C-4 = 0.4929.
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