Synthesis and characterization of new organometallic benzo[b]thiophene derivatives with potential antitumor properties

Article abstract of DOI:10.1021/om9003835

The incorporation of organometallic moieties into the structure of known active drugs to improve their therapeutic properties has gained considerable interest in recent years. The benzo[b]thiophene derivative raloxifene is a selective estrogen receptor mo

Full text of DOI:10.1021/om9003835

5412 Organometallics 2009, 28, 5412–5423
DOI: 10.1021/om9003835
Synthesis and Characterization of New Organometallic
Benzo[b]thiophene Derivatives with Potential Antitumor Properties
†
Andre P. Ferreira, Joao L. Ferreira da Silva, M. Teresa Duarte,
ꢀ
†
†
ꢀ
~
M. Fatima Minas da Piedade,†,‡ M. Paula Robalo,†, § Shrika G. Harjivan,†
Cristina Marzano,^ Valentina Gandin,^ and M. Matilde Marques*,†
†
´
Centro de Quımica Estrutural, Instituto Superior Tecnico, Universidade Tecnica de Lisboa,
Avenida Rovisco Pais, 1049-001 Lisboa, Portugal , Departamento de Quımica e Bioquımica,
ꢀ
ꢀ
‡
´
´
§
Faculdade de Cie^ncias, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal , Departamento
´
´
de Engenharia Quımica, Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emıdio Navarro 1,
^
ꢁ
1959-007, Lisboa, Portugal, and Dipartimento di Scienze Farmaceutiche, Universita di Padova,
Via F. Marzolo 5, 35131 Padova, Italy
Received May 12, 2009
The incorporation of organometallic moieties into the structure of known active drugs to
improve their therapeutic properties has gained considerable interest in recent years. The benzo-
[b]thiophene derivative raloxifene is a selective estrogen receptor modulator (SERM) that has been
found to decrease breast cancer risk in postmenopausal women compared to placebo. The current
data suggest that, in the postmenopausal setting, raloxifene may have the benefits of the widely used
tamoxifen with fewer side effects. As part of a program designed toward the synthesis and biological
screening of organometallic benzo[b]thiophene derivatives inspired by the structure of raloxifene,
we have prepared a series of 2-benzoyl-3-ferrocenylbenzo[b]thiophenes where the benzoyl sub-
stituent contains terminal tertiary alkylamino groups, expected to ensure affinity to the estrogen
receptor. The synthetic strategy and full characterization (NMR, MS, X-ray diffraction, cyclic
voltammetry) of the new ferrocenylbenzo[b]thiophenes is reported herein. Moreover, the new 2-
benzoyl-3-ferrocenylbenzo[b]thiophene derivatives were tested for their cytotoxic properties
against several human tumor cell lines. All the test compounds showed considerable cytotoxic
activity; among these, [3-ferrocenyl-6-methoxybenzo[b]thiophen-2-yl][4-(piperazin-1-yl)methyl-
phenyl]methanone (compound 13) is of note, showing IC50 values in the low-micromolar range
and more than 1 order of magnitude lower than those of the reference compound, cisplatin. In
addition, chemosensitivity tests on resistant phenotypes indicated that compound 13 elicited no
cross-resistance with cisplatin, besides not being a potential multidrug-resistant (MDR) substrate.
Moreover, caspase-3 activation analyses revealed that 13 induced a caspase-3-dependent apoptotic
cell-death mechanism. Taken together, these data suggest that the new 2-benzoyl-3-
ferrocenylbenzo[b]thiophenes, in particular compound 13, have potentially useful antitumor
properties.
Introduction
sitic, viral, and microbial diseases.2 One such example is the
chloroquine analogue ferroquine, a new antimalarial drug
candidate with a low toxicity profile and no cross-resistance
with chloroquine, which reached phase IIb clinical trials in
the fall of 2007.3
The crucial event in the launching of metal-based drugs is
widely considered to be the discovery of the antitumoral
effects of cisplatin.4 Ever since its approval in 1978 for the
treatment of testicular and ovarian cancer, this compound
has been used extensively in cancer therapy. However,
In the last two decades, bioorganometallic chemistry has
become a rapidly growing field, which has expanded the
classical scope of organometallic chemistry into biology and
medical applications.1 In particular, the topic of organome-
tallic pharmaceuticals is of emergent significance, and
although efforts have predominantly been directed toward
the evaluation of organometallics as anticancer agents, other
targets for organometallic therapeutic agents include para-
*To whom correspondence should be addressed. Tel: 351-21-8419200.
Fax: 351-21-8464457. E-mail: matilde.marques@ist.utl.pt.
(3) (a) Dive, D.; Biot, C. ChemMedChem 2008, 3, 383. (b) Kreidenweiss,
A.; Kremsner, P. G.; Dietz, K.; Mordm€uller, B. Am. J. Trop. Med. Hyg.
2006, 75, 1178.
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Metzler-Nolte, N. Angew. Chem., Int. Ed. 2001, 40, 1040.
(2) Allardyce, C. S.; Dorcier, A.; Scolaro, C.; Dyson, P. J. Appl.
Organomet. Chem. 2005, 19, 1.
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r
pubs.acs.org/Organometallics
Published on Web 09/02/2009
2009 American Chemical Society

Article
Organometallics, Vol. 28, No. 18, 2009 5413
cisplatin has numerous shortcomings, including induced
cellular resistance, severe toxicity, and a spectrum of activity
limited to a narrow range of tumor types.5 This has
prompted considerable efforts to develop novel metal-based
anticancer agents that could circumvent the limitations of
cisplatin. As a result, thousands of platinum-based com-
pounds have been synthetized and screened for toxicity and
anticancer activity, although very few have been approved
for clinical use.5,6 The search for other metal-based anti-
cancer compounds that could circumvent the drawbacks
associated with platinum drugs has been pursued concur-
rently; representative examples include titanocene and fer-
rocene derivatives,2 as well as ruthenium complexes.2,7
Ferrocene derivatives, in particular, due to their stability in
aerobic aqueous media, synthetic versatility, and favorable
electrochemistry, are among the most widely considered
species for biological applications.8
A pioneering strategy regarding the medicinal use of
organometallic species involves the incorporation of orga-
nometallic moieties into the structures of known active drugs
to improve their therapeutic properties.9 One of the most
intriguing examples of this approach is the chemical mod-
ification of the estrogen antagonist tamoxifen (1, Scheme 1)
to contain a series of metallocene fragments, which resulted
in varied, and in some instances unforeseen, biological
properties.10 In particular, the inclusion of a ferrocenyl
fragment into the structure of the active tamoxifen metabo-
lite, 4-hydroxytamoxifen (2, Scheme 1), imparted cytotoxic
characteristics to the final product, hydroxyferrocifen (3,
Scheme 1), without hampering the intrinsic antiestrogenicity
of the tamoxifen vector.11 In fact, hydroxyferrocifen dis-
played antiproliferative effects in vitro on both estrogen
receptor (ER)-positive and ER-negative breast cancer cell
lines, which indicated an ability to act through both hor-
mone-dependent and hormone-independent mechanisms.11
Tamoxifen is currently an important adjuvant chemother-
apeutic agent for the treatment of all stages of hormone-
dependent breast cancer12 and also a chemoprotective agent
for the prevention of the disease in high-risk women.13
Despite being beneficial for breast cancer treatment and
prevention, tamoxifen is known to increase the risk of
endometrial cancer and thromboembolic events in women.14
These limitations have prompted the search for additional
selective estrogen receptor modulators (SERMS). An ideal
SERM should have improved effects on breast cancer,
osteoporosis, and coronary heart disease, but fewer toxic
side effects, such as endometrial cancer induction.
The 2-arylbenzothiophene raloxifene (4, Scheme 1) is a
SERM originally developed, and approved, for the preven-
tion and treatment of postmenopausal osteoporosis. While
raloxifene is an estrogen agonist in bone tissue, resulting in
potent inhibition of the loss of volumetric bone mineral
density, it acts as an antiestrogen in mammary tissue and
has minimal effects on the endometrium.15 Raloxifene ad-
ministration has been found to decrease breast cancer risk in
postmenopausal women compared to placebo, and the cur-
rent data suggest that, in the postmenopausal setting, it may
have the benefits of tamoxifen with fewer side effects.16
Based upon these observations, and with the aim of explor-
ing the concept of target-oriented organometallic drugs, we
have recently undertaken a program designed toward the
synthesis and biological screening of organometallic benzo-
[b]thiophene derivatives inspired by the structure of ralox-
ifene. These species have been designed to assess the possible
combination of SERM properties associated with the benzo-
[b]thiophene backbone with potential cytotoxicity imparted
by the organometallic fragment. We report herein the synth-
esis and characterization of the first representatives of this
class, a series of 2-benzoyl-3-ferrocenylbenzo[b]thiophenes
where the benzoyl substituent contains terminal tertiary
alkylamino groups, expected to ensure affinity to the ER.17
As an initial step in ascertaining the antitumor potential of
the new compounds, their cytotoxic properties were evalu-
ated against several human tumor cell lines, with all test
compounds displaying considerable cytotoxic activity. In
addition, we report the results of chemosensitivity tests on
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5414 Organometallics, Vol. 28, No. 18, 2009
Scheme 1. Structures of Tamoxifen (1), 4-Hydroxytamoxifen (2), Ferrocifen (3), and Raloxifene (4)
Ferreira et al.
resistant phenotypes conducted with all the new ferrocenyl-
benzo[b]thiophenes, as well as caspase-3 activation analyses,
performed with the most active compound, [3-ferrocenyl-
6-methoxybenzo[b]thiophen-2-yl][4-(piperazin-1-yl)methyl-
phenyl]methanone (compound 13). The test compounds
elicited no cross-resistance with cisplatin and gave no indica-
tion of being potential multidrug-resistant (MDR) sub-
strates; moreover, compound 13 was found to induce a
caspase-3-dependent apoptotic cell-death mechanism.
(HSQC) and heteronuclear multiple bond correlation (HMBC)
experiments, performed with standard pulse programs.
Electrochemical experiments were performed on an EG&G
Princeton Applied Research potentiostat/galvanostat model
273A and controlled by a personal computer using Electro-
chemistry PowerSuite v2.51 data acquisition software from
Princeton Applied Research. The voltammetric experiments
were performed at room temperature, using a three-electrode
configuration with a platinum-disk working electrode (1.0 mm
diameter) probed by a Luggin capillary connected to a silver-
wire pseudoreference electrode; a platinum wire auxiliary elec-
trode was employed. The solutions under study were 1 mM in
acetonitrile and 0.1 M in the supporting electrolyte (tetra-n-
butylammonium hexafluorophosphate). The redox potentials
of the ferrocenylbenzo[b]thiophenes were measured in the pre-
sence of ferrocene as the internal standard and are reported
relative to the value of the ferrocenium/ferrocene redox couple
[Ep/2 = 0.40 V versus the saturated calomel electrode (SCE) in
acetonitrile].19 Ep/2 values were determined as (Epa þ Epc)/2.
The solutions were purged with nitrogen and kept under an inert
atmosphere throughout the measurements. Reagentgrade aceto-
nitrile was dried over CaH2 and distilled under nitrogen before
use.
Experimental Section
Chemicals and General Procedures. All commercially avail-
able chemicals were acquired from Sigma-Aldrich Quımica, S.
´
A. (Madrid, Spain). All synthetic procedures were conducted in
air. Whenever necessary, solvents were purified by standard
methods.18
Microwave-assisted syntheses were conducted in a CEM
Discover Benchmate microwave reactor, operating at 300 W.
Melting temperatures were measured with a Leica Galen III
hot stage apparatus and are uncorrected. Microanalyses (C,
ꢀ
H, N, and S) were performed at Laboratorio de Analises,
Instituto Superior Tecnico. Infrared (IR) spectra were re-
ꢀ
ꢀ
Syntheses. 2-Chloro-1-ferrocenylethanone (5). Ferrocene (15 g,
81 mmol) and chloroacetyl chloride (6 g, 53 mmol) were dissolved in
100 mL of dry, nitrogen-purged methylene chloride, and the solu-
tion was cooled to -15 ꢀC. Anhydrous aluminum trichloride (7.15
g, 54 mmol) was then added slowly, for a period of 4 h, while
maintaining the temperature, and the mixture was subsequently
allowed to reach room temperature and stirred overnight. The
reaction was then quenched with ice, and the mixture extracted
with methylene chloride. The organic extracts were dried with
anhydrous magnesium sulfate and evaporated to dryness, yielding
a brown solid. This solid was homogenized with Celite and then
purified by column chromatography on silica, using methylene
chloride/n-hexane (1:2) as the eluent, to yield a red crystalline
product (9.6 g, 37 mmol, 69%). Mp: 88-90 ꢀC [mp 94-96 ꢀC;20
92-93 ꢀC21]. IR (KBr): 1679 (s, CdO) cm-1. 1H NMR δ 4.90 (2H,
bs, Fc-H2þH5), 4.68 (2H, s, CH2Cl), 4.66 (2H, bs, Fc-H3þH4),
4.31 (5H, s, Fc-H10 to H50). 13C NMR δ 195.0 (CO), 77.2 (Fc-C1),
73.5 (Fc-C2þC5), 70.7 (Fc-C10 to C50), 70.1 (Fc-C3þC4), 47.1
(CH2Cl).
corded on a Perkin-Elmer 683 FT-IR spectrometer. Group
frequencies are reported in cm-1; the symbols s, m, w, and br
represent strong, medium, weak, and broad bands, respec-
tively.
Mass spectra (MS) were recorded on either a GC-TOF
Micromass mass spectrometer, operated in the electron ioniza-
tion (EI) mode, or a Varian 500-MS LC ion trap mass spectro-
meter, operated in the electrospray ionization (ESI) mode. For
ESI analyses, the spray voltage was set at (5 kV and the
capillary voltage was set at 10 V. Fragment percent intensities
and fragment assignments are indicated in parentheses and
square brackets, respectively. High-resolution mass spectra
(HRMS) were recorded on a Finnigan FT/MS 2001-DT spec-
trometer.
1H NMR spectra were recorded in acetone-d6, on a Bruker
Avance III 300 or a Bruker Avance III 400 spectrometer,
operating at 300 or 400 MHz, respectively. 13C NMR spectra
were recorded on the same instruments, operating at 75.47 and
100.62 MHz, respectively. Chemical shifts are reported in ppm
downfield from tetramethylsilane, and coupling constants (J)
are reported in Hz; the subscripts ortho and meta refer to ortho
and meta couplings, respectively. The abbreviations used to
assign specific protons and carbons include Ar (aryl, for benzo-
thiophene), Fc (ferrocene), and Ph (para-substituted phenyl).
Resonance and structural assignments were based on the ana-
lysis of coupling patterns, including the 13C-1H coupling
profiles obtained in heteronuclear single quantum coherence
1-Ferrocenyl-2-[3-(methoxyphenyl)thio]ethanone (6). Potas-
sium tert-butoxide (1.5 equiv) was added to a solution of 3-
methoxybenzenethiol (2 g, 14 mmol) in 30 mL of anhydrous
diethyl ether, and the suspension was stirred at room tempera-
ture for 1 h. 2-Chloro-1-ferrocenylethanone (5, 5.6 g, 1.5 equiv)
was added, and the mixture was stirred overnight. The solid
materials were then removed by filtration over Celite. The
(19) Connelly, N. G.; Geiger, W. E. Chem. Rev. 1996, 96, 877.
(20) Fang, J.-X.; Jin, Z.; Li, Z.-M.; Liu, W. Appl. Organomet. Chem.
2003, 17, 145.
(18) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory
Chemicals, 3rd ed.; Pergamon: Oxford, U.K., 1988.
€
(21) Schlogl, L.; Egger, M. Monatsh. Chem. 1962, 95, 376.

Article
Organometallics, Vol. 28, No. 18, 2009 5415
thioether (6) precipitated upon cooling, and red crystals (3.33 g,
9.1 mmol, 64%) were collected. Mp: 124-126 ꢀC (diethyl ether).
IR (KBr): 1659 (s, CdO), 1588 (m), 1454 (m), 1247 (m), 1032 (m)
cm-1. 1H NMR: δ 7.24 (1H, t, J 7.8, Ar-H5), 7.02 0(2H, m, Ar-
H2þH4 or H6), 6.79 (1H, ddd, Jortho 8.4, Jmeta 2.3, J meta 0.9, Ar-
H4 or Ar-H6), 4.90 (2H, t, J 2.0, Fc-H2þH5), 4.61 (2H, t, J 2.0,
Fc-H3þH4), 4.28 (2H, s, CH2S), 4.27 (5H, s, Fc-H10 to H50),
3.81 (3H, s, CH3O). 13C NMR: δ 197.9 (CO), 160.8 (Ar-C3),
138.5 (Ar-C1), 130.4 (Ar-CH), 121.5 (Ar-CH), 114.9 (Ar-CH),
112.4 (Ar-CH), 78.7 (Fc-C1), 73.1 (Fc-C2þC5), 70.5 (Fc-C10 to
C50 or Fc-C3þC4), 70.3 (Fc-C10 to C50 or Fc-C3þC4), 55.4
(CH3O), 41.5 (CH2S). MS (EI): m/z 366 (50%) [Mþ], 213
(100%) [FcCOþ], 185 (58%) [Fcþ], 129 (27%) [Fcþ - Fe], 121
(9%) [CpFeþ], 56 (4%) [Feþ]. Anal. Calcd for C19H18Fe-
8.8, Jmeta 2.4, Ar-H5), 4.69 (2H, s, CH2Cl), 4.39 (2H, t, J 1.8, Fc-
H2þH5 or Fc-H3þH4), 4.16 (2H, t, J 1.8, Fc-H3þH4 or Fc-
H2þH5), 4.11 (5H, s, Fc-H10 to H50), 3.99 (3H, s, CH3O). 13C
NMR: δ 192.3 (CO), 161.0 (Ar-C6), 144.0 (Ph-C4 or Ar-C7a),
143.8 (Ar-C7a or Ph-C4), 139.3 (Ar-C3 or Ph-C1), 139.1 (Ph-C1
or Ar-C3), 135.6 (Ar-C2), 134.6 (Ar-C3a), 131.5 (Ph-C2þC6),
130.0 (Ph-C3þC5), 128.8 (Ar-C4), 116.7 (Ar-C5), 106.1 (Ar-C7),
82.0 (Fc-C1), 71.7 (Fc-C2þC5 or Fc-C3þC4), 71.0 (Fc-C10 to
C50), 70.2 (Fc-C2þC5 or Fc-C3þC4), 56.9 (CH3O), 46.8
(CH2Cl). MS (ESI): m/z 502 [Mþ(37Cl)], 500 (97%) [Mþ(35Cl)],
437 [Mþ(37Cl) - Cp], 435 (49%) [Mþ(35Cl) - Cp], 390 (26%)
[MHþ - Cl - CH3OH - CdS], 348 (100%) [MHþ - ClCH2-
C6H4CO]. Anal. Calcd for C27H21ClFeO2S LiCl: C, 59.70; H,
3
3.90; S, 5.90. Found: C, 59.56; H, 4.06; S, 6.30.
O2S 0.5Et2O: C, 62.54; H, 5.75; S, 7.95. Found: C, 62.54; H,
3
[3-Ferrocenyl-6-methoxybenzo[b]thiophen-2-yl][4-(dimethyl-
aminomethyl)phenyl]methanone (9). Dimethylamine (2 M in
THF, 0.8 mL, 1.6 mmol) was added to a solution of the
benzo[b]thiophene 8 (250 mg, 0.5 mmol) in DMF (10 mL),
and the mixture was stirred overnight. Following dilution with
approximately 10 volumes of water and extraction with
methylene chloride, the organic extracts were dried with
anhydrous sodium sulfate, and the ensuing dark red oil was
purified by plc (silica, methylene chloride) to yield a red
crystalline solid (110 mg, 0.22 mmol, 43%). Mp: 97-98 ꢀC.
IR (KBr): 2939 (m), 1705 (m), 1624 (s, CO), 1601 (s), 1521 (m),
5.59; S, 8.28.
3-Ferrocenyl-6-methoxybenzo[b]thiophene (7). 1-Ferrocenyl-
2-[3-(methoxyphenyl)thio]ethanone (6, 3.33 g, 9.1 mmol) was
dissolved in dry p-xylene (30 mL) and refluxed for 4 h with HCl-
activated Amberlyst 15 resin (10% w/w). The reaction mixture
was then filtered over Celite, and the solvent was evaporated
under vacuum. The slightly oily orange residue was purified by
flash chromatography on silica, by eluting with methylene
chloride/n-hexane (1:4). The pure benzo[b]thiophene derivative
(7) was obtained as a crystalline orange product (2.28 g, 6.5
mmol, 72%). In subsequent experiments, the reaction was
1
1453 (s), 1338 (m), 1280 (s), 1231 (s) cm-1. H NMR: δ 8.75
further optimized by subjecting solutions of
6
(40-60
(1H, d, Jortho 9.1, Ar-H4), 7.52 (2H, d, Jortho 7.9, Ph-H2þH6),
7.59 (1H, d, Jmeta 2.2, Ar-H7), 7.28 (1H, dd, Jortho 9.1, Jmeta 2.2,
Ar-H5), 7.22 (2H, d, Jortho 7.9, Ph-H3þH5), 4.36 (2H, bs, Fc-
H2þH5 or Fc-H3þH4), 4.16 (2H, bs, Fc-H3þH4 or Fc-
H2þH5), 4.13 (5H, s, Fc-H10 to H50), 3.98 (3H, s, CH3O),
3.40 (2H, s, CH2N), 2.22 (6H, s, (CH3)2N). 13C NMR: δ 191.0
(CO), 159.2 (Ar-C6), 144.2 (Ph-C4), 142.2 (Ar-C7a), 137.2
(Ar-C3 or Ph-C1), 136.7 (Ar-C3 or Ph-C1), 134.4 (Ar-C2),
133.0 (Ar-C3a), 129.5 (Ph-C2þC6), 128.4 (Ph-C3þC5), 127.0
(Ar-C4), 115.0 (Ar-C5), 104.4 (Ar-C7), 80.5 (Fc-C1), 70.0
(Fc-C2þC5 or Fc-C3þC4), 69.0 (Fc-C10 to C50), 68.4
(Fc-C2þC5 or Fc-C3þC4), 63.2 (CH2N), 55.2 (CH3O), 44,5
[(CH3)2N)]. MS (ESI): m/z 510 (100%) [MH]þ, 465 (47%) [MH
- (CH3)2NH]þ, 444 (19%) [MH - CpH]þ, 359 (17%) [MH -
mg mL-1) in dry p-xylene, in the presence of Amberlyst 15
3
resin (ca. 60 mg mL-1), to microwave irradiation for 20 min at
3
300 W; under these conditions, 7 was isolated in 90-99% yield.
Mp: 85-87 ꢀC. IR (KBr): 3091 (m), 2930 (m), 1602 (s), 1471 (s),
1266 (s), 1233 (s) cm-1. 1H NMR: δ 8.31 (1H, d, Jortho 9.0, Ar-
H4), 7.53 (1H, d, Jmeta 2.4, Ar-H7), 7.41 (1H, s, Ar-H2), 7.15
(1H, dd, Jortho 9.0, Jmeta 2.4, Ar-H5), 4.73 (2H, t, J 1.8, Fc-
H2þH5 or Fc-H3þH4), 4.38 (2H, t, J 1.8, Fc-H3þH4 or Fc-
H2þH5), 4.15 (5H, s, Fc-H10 to H50), 3.92 (3H, s, CH3O). 13C
NMR: δ 157.4 (Ar-C6), 142.1 (Ar-C7a), 133.6 (Ar-C3), 131.8
(Ar-C3a), 124.9 (Ar-C4), 118.7 (Ar-C2), 113.8 (Ar-C5), 105.1
(Ar-C7), 81.3 (Fc-C1), 68.9 (Fc-C10 to C50), 68.1 (Fc-C3þC4 or
Fc-C2þC5), 67.1 (Fc-C2þC5 or Fc-C3þC4), 54.8 (CH3O). MS
(EI): m/z 348 (100%) [Mþ], 333 (29%) [Mþ - CH3], 305 (17%)
[Mþ - CH3 - CO], 239 (5%) [Mþ - CH3 - CO - Cp - H], 227
(6%) [Mþ - FeCp], 215 (6%) [Mþ - CH3 - CO - Cp - C2H],
184 (7%) [Mþ - CH3 - CO - FeCp], 152 (10%) [Mþ - CH2O -
FeCp - CHdS], 139 (6%) [Mþ - CH3 - CO - FeCp - CHdS],
121 (7%) [FeCpþ], 56 (2.5%) [Feþ]. Anal. Calcd for C19H16-
(CH3)2NH - CH3 - CO - Cp þ 2H], 269 (17%) [MH2
C6H4CH2N(CH3)2 - CH3 - CO - Cp]. Anal. Calcd for
29H27FeNO2S 0.5HCl: C, 66.01; H, 5.25; N, 2.65; S, 6.08.
-
C
3
Found: C, 66.23; H, 5.47; N, 2.44; S, 6.33.
[3-Ferrocenyl-6-methoxybenzo[b]thiophen-2-yl][4-(piperidin-
1-yl)methylphenyl]methanone (10). This compound was pre-
pared and purified as described for 9, using benzo[b]thiophene
8 (250 mg, 0.5 mmol) and 200 μL (2 mmol) of piperidine.
Compound 10 was isolated as a red crystalline solid (170 mg,
0.31 mmol, 62%). Mp: 97-99 ꢀC. IR (KBr): 2933 (m), 1627 (s,
CO), 1602 (s), 1521 (m), 1451 (s), 1341 (m), 1268 (s), 1233 (s)
cm-1. 1H NMR: δ 8.76 (1H, d, Jortho 9.1, Ar-H4), 7.60 (1H, d,
Jmeta 2.4, Ar-H7), 7.51 (2H, d, Jortho 8.3, Ph-H2þH6), 7.28
(1H, dd, Jortho 9.1, Jmeta 2.4, Ar-H5), 7.22 (2H, d, Jortho 8.3, Ph-
H3þH5), 4.36 (2H, t, J 1.9, Fc-H2þH5 or Fc-H3þH4), 4.13
(2H, t, J 1.9, Fc-H3þH4 or Fc-H2þH5), 4.11 (5H, s, Fc-H10
to H50), 3.99 (3H, s, CH3O), 3.41 (2H, s, CH2N), 2.31 (4H, m,
piperidine-C2H2þC6H2), 1.55 (4H, m, piperidine-C3H2þ
C5H2), 1.44 (2H, m, piperidine-C4H2). 13C NMR: δ 191.7
(CO), 160.0 (Ar-C6), 145.0 (Ph-C4), 142.9 (Ar-C7a), 137.9
(Ar-C3 or Ph-C1), 137.2 (Ar-C3 or Ph-C1), 135.2 (Ar-C2),
133.7 (Ar-C3a), 130.1 (Ph-C2þC6), 129.1 (Ph-C3þC5), 127.8
(Ar-C4), 115.6 (Ar-C5), 105.1 (Ar-C7), 81.2 (Fc-C1), 70.7
(Fc-C2þC5 or Fc-C3þC4), 70.0 (Fc-C10 to C50), 69.0
(Fc-C2þC5 or Fc-C3þC4), 63.5 (CH2N), 55.7 (CH3O), 54.8
(piperidine-C2þC6), 26.6 (piperidine-C3þC5), 24.9 (piperi-
dine-C4). MS (ESI): m/z 550 (100%) [MH]þ, 465 (17%) [MH
- piperidine]þ, 437 (8%) [MH - C5H10NCH2 - CH3]þ. Anal.
FeOS 0.5MeOH: C, 64.30; H, 4.98; S, 8.80. Found: C, 64.33; H,
3
5.06; S, 8.47.
[4-(Chloromethyl)phenyl][3-ferrocenyl-6-methoxybenzo[b]thio-
phen-2-yl]methanone (8). The benzo[b]thiophene 7 (2.5 g, 7.2
mmol) was dissolved in anhydrous diethyl ether and lithiated at
-70 ꢀC with a stoichiometric amount of n-butyllithium (2 M
solution in n-pentane). The reaction mixture was allowed to
reach room temperature, stirred overnight, and then evaporated
to dryness and washed with n-hexane; the resulting yellow salt
was used without further purification. The lithiated benzo-
[b]thiophene was redissolved in anhydrous diethyl ether (20
mL) and added slowly to a stirred solution of 4-chloromethyl-
benzoyl chloride (1.35 g, 7.1 mmol) in 50 mL of dry diethyl ether,
kept at -60 ꢀC. An immediate color change, from yellow to dark
red, was observed. The reaction mixture was allowed to warm
slowly to room temperature, and stirring was continued for one
additional hour. The mixture was then filtered and the solution
evaporated to dryness. The dark red oily residue was purified by
column chromatography (silica, methylene chloride) to yield
dark red crystals of compound 8 (1.8 g, 3.6 mmol, 51%). Mp:
70-72 ꢀC. IR (KBr): 3090 (w), 2933 (w), 1626 (m), 1602 (s), 1452
(m), 1267 (m), 1229 (s) cm-1. 1H NMR: δ 8.77 (1H, d, Jortho 8.8,
Ar-H4), 7.60 (1H, Jmeta 2.4, Ar-H7), 7.59 (2H, d, Jortho 8.4, Ph-
H2þH6), 7.36 (2H, d, Jortho 8.4, Ph-H3þH5), 7.29 (1H, dd, Jortho
Calcd for C32H31FeNO2S 0.2HCl: C, 69.03; H, 5.65; N, 2.52;
3
S, 5.76. Found: C, 68.72; H, 5.78; N, 2.53; S, 6.19.

5416 Organometallics, Vol. 28, No. 18, 2009
Ferreira et al.
[3-Ferrocenyl-6-methoxybenzo[b]thiophen-2-yl][4-(pyrrolidin-
1-yl)methylphenyl]methanone (11). This compound was pre-
pared and purified as described for 9, using benzo[b]thiophene
8 (250 mg, 0.5 mmol) and 170 μL (2 mmol) of pyrrolidine.
Compound 11 was isolated as a red crystalline solid (180 mg,
0.35 mmol, 67%). Mp: 82-84 ꢀC. IR (KBr): 2963 (m), 2778 (m),
1636 (s, CO), 1602 (s), 1525 (m), 1456 (m), 1340 (m), 1264 (s),
1231 (s), 1107 (s), 1019 (s), 806 (s) cm-1. 1H NMR: δ 8.76 (1H, d,
Jortho 9.0, Ar-H4), 7.60 (1H, d, Jmeta 2.4, Ar-H7), 7.52 (2H, d,
Jortho 8.4, Ph-H2þH6), 7.28 (1H, dd, Jortho 9.0, Jmeta 2.4, Ar-H5),
7.24 (2H, d, Jortho 8.4, Ph-H3þH5), 4.37 (2H, t, J 1.8, Fc-
H2þH5 or Fc-H3þH4), 4.15 (2H, t, J 1.8, Fc-H3þH4 or Fc-
H2þH5), 4.11 (5H, s, Fc-H10 to H50), 3.99 (3H, s, CH3O), 3.58
(2H, s, CH2N), 2.41 (4H, m, pyrrolidine-C2H2þC5H2), 1.76
(4H, m, pyrrolidine-C3H2þC4H2). 13C NMR: δ 191.4 (CO),
159.6 (ArC6), 145.3 (Ph-C4), 142.6 (Ar-C7a), 137.5 (Ar-C3 or
Ph-C1), 136.9 (Ar-C3 or Ph-C1), 134.7 (Ar-C2), 133.4 (Ar-C3a),
129.9 (Ph-C2þC6), 128.5 (Ph-C3þC5), 127.5 (Ar-C4), 115.4
(Ar-C5), 104.9 (Ar-C7), 80.9 (Fc-C1), 70.4 (Fc-C2þC5 or
Fc-C3þC4), 69.7 (Fc-C10 to C50), 68.8 (Fc-C2þC5 or Fc-C3
þC4), 60.0 (CH2N), 55.6 (CH3O), 54.0 (pyrrolidine-C2þC5),
23.7 (pyrrolidine-C3þC4). MS (ESI): m/z 536 (100%) [MH]þ,
465(21%) [MH- pyrrolidine]þ, 437(8.5%) [MH- C4H8NCH2 -
CH3]þ, 262 (9%) [MH2 - C4H8NCH2C6H4CO - CdS - CH3 -
CO]þ. HRMS Calcd for C31H2956FeNO232S: 535.127387. Found:
535.12827.
Ph-C1), 136.6 (Ph-C1 or Ar-C3), 134.5 (Ar-C2), 133.0 (Ar-C3a),
129.5 (Ph-C2þC6), 128.5 (Ph-C3þC5), 127.1 (Ar-C4), 115.0
(Ar-C5), 104.4 (Ar-C7), 80.5 (Fc-C1), 70.0 (Fc-C2þC5 or Fc-C3
þC4), 69.3 (Fc-C10 to C50), 68.4 (Fc-C2þC5 or Fc-C3þC4),
62.8 (CH2N), 55.2 (CH3O), 54.2 (piperazine-C2þC6), 45.9
(piperazine-C3þC5). MS (ESI): m/z 551 (100%) [MH]þ, 485
(26%) [MH - CpH]þ, 465 (13%) [MH - piperazine]þ, 437 (6%)
[MH-NHC4H8NCH2-CH3]þ. HRMS Calcd for C31H3056Fe-
N2O232S: 550.138266. Found: 550.137497.
X-ray Crystallographic Analyses. X-ray crystallographic data
for compounds 6, 11, and 12 were collected using an area
detector diffractometer (Bruker AXS-KAPPA APEX II)
equipped with an Oxford Cryosystem open-flow nitrogen cryo-
stat at 150 K and graphite-monochromated Mo KR (λ = 0.710
73 A) radiation. Cell parameters were retrieved using Bruker
SMART software and refined using Bruker SAINT22 on all
observed reflections. Absorption corrections were applied using
SADABS.23 The structures were solved by direct methods using
SIR 9724 and refined using full-matrix least-squares refinement
against F2 using SHELXL-97.25 All the programs are included
in the WINGX package (version 1.64.05).26 All non-hydrogen
atoms were refined anisotropically; the hydrogen atoms were
inserted in idealized positions, riding on the parent C atom,
except for compound 6, where they were localized in the electron
density map. Drawings were made with ORTEP3 for Win-
dows.27 Intermolecular interactions were drawn using Mercury
1.4.2 (Build 2).28 Crystals had good quality and diffrac-
ting power, presenting low Rint (0.0416 for 6, 0.0991 for 11,
and 0.0330 for 12) values that allowed obtaining low R values
[3-Ferrocenyl-6-methoxybenzo[b]thiophen-2-yl][4-(morpholin-
4-yl)methylphenyl]methanone (12). This compound was pre-
pared and purified as described for 9, using benzo[b]thiophene
8 (250 mg, 0.5 mmol) and 350 μL (4 mmol) of morpholine.
Compound 12 was isolated as a red crystalline solid (200 mg,
0.36 mmol, 73%). Mp: 141-142 ꢀC. IR (KBr): 2961 (m), 2941
(m), 2860 (m), 1622 (s, CO), 1601 (s), 1453 (s), 1340 (m), 1277 (s),
1265 (s), 1231 (s), 1105 (s), 1008 (s), 830 (s) cm-1. 1H NMR: δ
8.76 (1H, d, Jortho 9.0, Ar-H4), 7.60 (1H, d, Jmeta 2.4, Ar-H7),
7.51 (2H, d, Jortho 8.1, Ph-H2þH6), 7.28 (1H, dd, Jortho 9.0, Jmeta
2.4, Ar-H5), 7.23 (2H, d, Jortho 8.1, Ph-H3þH5), 4.36 (2H, t,
J 1.8, Fc-H2þH5 or Fc-H3þH4), 4.12 (2H, t, J 1.8, Fc-H3þH4
or Fc-H2þH5), 4.10 (5H, s, Fc-H10 to H50), 3.99 (3H, s, CH3O),
3.62 (4H, t, J 4.5, morpholine-C2H2þC6H2), 3.46 (2H, s,
CH2N), 2.35 (4H, t, J 4.5, morpholine-C3H2þC5H2). 13C
NMR: δ 191.0 (CO), 159.2 (ArC6), 143.2 (Ph-C4), 142.2
(Ar-C7a), 137.3 (Ar-C3 or Ph-C1), 136.7 (Ar-C3 or Ph-C1),
134.4 (Ar-C2), 133.0 (Ar-C3a), 129.4 (Ph-C2þC6), 128.5
(Ph-C3þC5), 127.0 (Ar-C4), 114.9 (Ar-C5), 104.4 (Ar-C7),
80.5 (Fc-C1), 70.0 (Fc-C2þC5 or Fc-C3þC4), 69.3 (Fc-C10
to C50), 68.3 (Fc-C2þC5 or Fc-C3þC4), 66.5 (morpho-
line-C2þC6), 62.4 (CH2N), 55.2 (CH3O), 53.4 (morpho-
line-C3þC5). MS (ESI): m/z 552 (100%) [MH]þ, 486 (10%)
[MH - CpH]þ, 465 (23%) [MH - morpholine]þ, 437 (7%) [MH
- OC4H8NCH2 - CH3]þ, 334 (13%) [MH2 - CH3OH -
FeCp2]þ, 278 (70%) [MH - OC4H8NCH2C6H4 - CH3OH -
(R1all=0.0401 and R1 obs=0.0279 for 6; R1all=0.0845 and R1obs.
=
0.0423 for 11; R1all=0.0324 and R1obs.=0.0273 for 12) after refin-
ing. Relevant details of the X-ray data analysis are presented as
Supporting Information.
Biology. Experiments with Human Tumor Cell Lines. The 2-
benzoyl-3-ferrocenylbenzo[b]thiophene derivatives were dis-
solved in DMSO just before the experiment, and a calculated
amount of drug solution was added to the growth medium,
containing cells to a final solvent concentration of 0.5%. This
had no discernible effect on cell killing. MTT [3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide], cisplatin, and
doxorubicin were obtained from Sigma Chemical Co., St. Louis,
MO.
Cell Cultures. Human lung (A549), cervical (A431), and colon
(HCT-15) carcinoma cell lines were obtained from ATCC,
Rockville, MD. The 2008 cell line, as well as its cisplatin-
resistant (C13*) and -revertant (RH4) variants, are human
ovarian cancer cell lines and were kindly provided by Prof. G.
Marverti (Dept. of Biomedical Science, University of Modena,
Italy). The MCF-7 human breast cancer cell line and its multi-
drug-resistant subline derivative (MCF-7 ADR) were kindly
provided by Prof. N. Colabuffo (Dept. Chimico-Biologico,
University of Bari, Italy). Cell lines were maintained in the
CpH]þ. Anal. Calcd for C31H29FeNO3S 0.2HCl: C, 66.63; H,
3
5.27; N, 2.51; S, 5.74. Found: C, 66.99; H, 5.30; N, 2.50; S, 5.93.
[3-Ferrocenyl-6-methoxybenzo[b]thiophen-2-yl][4-(piperazin-
1-yl)methylphenyl]methanone (13). This compound was pre-
pared and purified as described for 9, using benzo[b]thiophene
8 (250 mg, 0.5 mmol) and 170 mg (2 mmol) of piperazine.
Compound 13 was isolated as a red crystalline solid (150 mg,
0.27 mmol, 54%). Mp: 138-140 ꢀC. IR (KBr): 3434 (br, NH),
2933 (m), 2813 (m), 1619 (s, CO), 1602 (s), 1453 (m), 1384 (s),
1340 (m), 1276 (m), 1230 (s), 1106 (m), 824 (m) cm-1. 1H NMR:
δ 8.75 (1H, d, Jortho 9.1, Ar-H4), 7.59 (1H, d, Jmeta 2.4, Ar-H7),
7.50 (2H, d, Jortho 8.4, Ph-H2þH6), 7.28 (1H, dd, Jortho 9.1, Jmeta
2.4, Ar-H5), 7.22 (2H, d, Jortho 8.4, Ph-H3þH5), 4.35 (2H, t,
J 1.9, Fc-H2þH5 or Fc-H3þH4), 4.12 (2H, t, J 1.9, Fc-H3þH4
or Fc-H2þH5), 4.10 (5H, s, Fc-H10 to H50), 3.98 (3H, s, CH3O),
3.41 (2H, s, CH2N), 2.91 (4H, bs, piperazine-C3H2þC5H2), 2.80
(4H, bs, piperazine-C2H2þC6H2). 13C NMR: δ 191.0 (CO),
159.2 (ArC6), 143.8 (Ph-C4), 142.3 (Ar-C7a), 137.3 (Ar-C3 or
(22) SMART and SAINT, Area Detector Control and Integration
Software; Bruker AXS: Madison, WI, 2004.
(23) : Sheldrick G. M., SADABS, Program for Empirical Absorption
€
Correction of Area Detectors (Version 2.10); University of Gottingen:
€
Gottingen, Germany, 2004.
(24) Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G. L.;
Giacovazzo, C.; Guagliardi, A.; Moliterni, A. G. G.; Polidori, G.;
Spagna, R. J. Appl. Crystallogr. 1999, 32, 115.
(25) Sheldrick, G. M. SHELXL-97, a Computer Program for the
€
€
Refinement of Crystal Structures; University of Gottingen: Gottingen,
Germany, 1997.
(26) Farrugia, L. J. WinGX (v1.64.03b). J. Appl. Crystallogr. 1999,
32, 837.
(27) Farrugia, L. J. ORTEP-3 for Windows (v1.076), based on
ORTEP-III (v1.03) by: Johnson, C. K.; Burnett, M. N. J. Appl.
Crystallogr. 1997, 30, 565.
(28) Bruno, I. J.; Cole, J. C.; Edgington, P. R.; Kessler, M.; MacRae,
C. F.; McCabe, P.; Pearson, J.; Taylor, R. Acta Crystallogr. 2002, B58,
389.

Article
Organometallics, Vol. 28, No. 18, 2009 5417
Figure 1. Synthetic sequence leading to the 3-ferrocenyl raloxifene analogues (9-13) reported in this work. Reaction conditions: (a)
AlCl3/ClCOCH2Cl; (b) t-BuOK/3-methoxybenzenethiol; (c) Amberlyst 15; (d) 1. n-BuLi; 2. 4-chloromethylbenzoyl chloride; (e)
R2NH. R2NH = dimethylamine (9), piperidine (10), pyrrolidine (11), morpholine (12), and piperazine (13).
logarithmic phase at 37 ꢀC in a 5% carbon dioxide atmosphere
with the following culture media containing 10% fetal calf
fluoromethylcoumarin [DEVD-AFC], Clontech). The fluores-
cence was determined on a Perkin-Elmer 550 spectrofluoro-
meter (excitation 440 nm, emission 505 nm). The caspase-3
activity was expressed as the increase in AFC-emitted fluores-
cence. Student’s t-test was used for data analysis.
serum (Euroclone, Milan, Italy), antibiotics (50 units mL-1
3
penicillin and 50 μg mL-1 streptomycin), and 2 mM L-gluta-
3
mine: (i) RPMI-1640 medium (Euroclone) with 25 mM HEPES
buffer for HCT-15, MCF-7, MCF-7 ADR, 2008, C13*, RH4,
and A431 cells; (ii) D-MEM medium (Euroclone) for A549 cells.
Results and Discussion
The MCF-7 ADR culture medium also contained 0.1 μg mL-1
3
doxorubicin.
Syntheses and Spectral Characterization. The synthetic
sequence toward the 2-acyl-3-ferrocenyl benzo[b]thiophenes
(9-13) is outlined in Figure 1. Following a standard Friedel-
Crafts-type acylation of ferrocene with chloroacetyl chloride
to give 5, the thioether 6 was obtained by quantitative
nucleophilic substitution with 3-methoxybenzenethiolate.
The key step in the synthetic sequence was the acid-catalyzed
cyclization of 6, which allowed the construction of the
benzo[b]thiophene system in a regioselective manner. Pre-
sumably, steric constraints determined the predominant ring
closure para to the methoxy substituent, to give 7, which was
isolated in 72% yield under conventional reflux conditions,
as opposed to cyclization ortho to both pre-existing substit-
uents in the phenyl ring. In subsequent experiments, the
reaction was found to be much faster (20 min versus 4 h) and
efficient (>90% yield of the isolated product) when con-
ducted under microwave irradiation at 300 W. Ring closure
was readily apparent from the absence of methylene protons
Cytotoxicity Assays. The growth inhibitory effect toward
tumor cell lines was evaluated by means of the MTT
(tetrazolium salt reduction) assay.29 Briefly, (3-5) ꢀ 103 cells/
well, depending upon the growth characteristics of the cell line,
were seeded in 96-well microplates in growth medium (100 μL)
and then incubated at 37 ꢀC in a 5% carbon dioxide atmosphere.
After 24 h, the medium was removed and replaced with a fresh
one containing the compound to be studied at the appropriate
concentration. Triplicate cultures were established for each
treatment. After 72 h, each well was treated with 10 μL of a
5 mg mL-1 MTT saline solution, and after 5 h of incubation,
3
100 μL of a sodium dodecylsulfate (SDS) solution in 0.01 M HCl
was added. Following overnight incubation, the inhibition of
cell growth induced by the tested derivatives was detected by
measuring the absorbance of each well at 570 nm with a Bio-Rad
680 microplate reader. The mean absorbance for each drug dose
was expressed as a percentage of the untreated control well
absorbance and plotted versus drug concentration. IC50 values,
which represent the drug concentrations that reduced the mean
absorbances at 570 nm to 50% of those in the untreated control
wells, were calculated by probit analysis (P < 0.05, χ2 test).
Whenever appropriate, resistance factors (RF) were calculated
as the ratio of the IC50 values obtained for each compound in
resistant versus parent cell lines.
1
in the H NMR spectrum, which were replaced by a one-
proton aromatic singlet at 7.41 ppm, assigned to H2 of the
benzo[b]thiophene. Likewise, the methylene and carbonyl
carbons were absent from the 13C NMR spectrum, and two
new aromatic carbons, at 133.6 (quaternary, C3) and 118.7
(methine, C2) ppm, were present. The regioselectivity of ring
closure was unequivocally established from the presence of a
meta-coupled doublet in the 1H NMR spectrum at 7.53 ppm,
assigned to H7 of the benzo[b]thiophene framework, in
addition to an ortho-coupled doublet at 8.31 ppm (H4) and
a doublet of doublets (Jortho 9.0, Jmeta 2.4) at 7.15 ppm (H5).
Lithiation of 7 was then accomplished at C2 of the benzo-
[b]thiophene ring, and subsequent acylation with chloro-
methylbenzoyl chloride yielded 8, which served as the
parent compound for a series of derivatives (9-13) contain-
ing tertiary amino substituents.
Caspase-3 Activity. Caspase-3 activity was detected with the
ApoAlert Caspase-3 fluorescent assay kit (Clontech, Mountain
View, CA) according to the manufacturer’s recommended
procedures. In the amount of 106, 2008 cells were collected
following 12 or 24 h of incubation with the test compounds (at
concentrations corresponding to IC50 values), lysed on ice for 10
min in 50 μL of lysis buffer, and then treated with 50 μL of
reaction buffer containing dithiothreitol (DTT) and 5 μL of
caspase-3 substrate solution (Asp-Glu-Val-Asp-7-amino-4-tri-
(29) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.;
Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker,
R. H.; Boyd, M. R. Cancer Res. 1988, 48, 589.

5418 Organometallics, Vol. 28, No. 18, 2009
Ferreira et al.
Figure 2. ORTEP diagram, drawn with 50% probability ellip-
soids, showing the crystallographic labeling scheme for 1-ferro-
cenyl-2-[3-(methoxyphenyl)thio]ethanone (6).
All compounds described in this work were stable in air.
Full characterization was achieved by standard spectro-
scopic techniques (IR, 1H and 13C NMR, and MS), as well
as elemental analyses or HRMS, whenever the compounds
retained irreproducible fractional amounts of solvents. All
spectral data were fully consistent with the proposed struc-
tures. With few exceptions (typically, the methine carbons of
the substituted cyclopentadienyl group, for which 3-bond
1H-13C connectivities did not exist) carbon resonances
could be assigned from the HSQC and HMBC cross-peak
patterns. Prominent characteristic mass spectral fragmenta-
tions for compounds 9-13 in the ESI mode involved loss
of the neutral amines from the protonated molecule, with
concomitant formation of a tropylium ion; the loss of cyclo-
pentadiene was also observed in some instances. X-ray
quality crystals were grown for the benzothiophenes 11
and 12 and their thioether precursor 6, which allowed further
unequivocal characterization by X-ray diffraction (see
below).
Crystal Structures. The structures of 1-ferrocenyl-2-[3-
(methoxyphenyl)thio]ethanone (6) and of two representative
benzo[b]thiophenes, [3-ferrocenyl-6-methoxybenzo[b]thio-
phen-2-yl][4-(pyrrolidin-1-yl)methylphenyl]methanone (11)
and [3-ferrocenyl-6-methoxybenzo[b]thiophen-2-yl][4-(mor-
pholin-4-yl)methylphenyl]methanone (12), were obtained by
X-ray diffraction analysis and found to be in agreement with
the remaining spectral characterization data. Both com-
pounds 6 and 11 crystallized in the monoclinic P21/c space
group, the latter with two crystallographically independent
molecules in the asymmetric unit, while compound 12 crys-
tallized in the triclinic P1 space group. The corresponding
ORTEP diagrams are displayed in Figures 2, 3, and 4,
respectively. Selected bond lengths and angles for 6, 11,
and 12 are presented in Table 1.
Figure 3. ORTEP diagram, drawn with 50% probability
ellipsoids, showing the crystallographic labeling scheme for
[3-ferrocenyl-6-methoxybenzo[b]thiophen-2-yl][4-(pyrrolidin-1-yl)-
methylphenyl]methanone (11, molecule 1).
Figure 4. ORTEP diagram, drawn with 50% probability
ellipsoids, showing the crystallographic labeling scheme for
[3-ferrocenyl-6-methoxybenzo[b]thiophen-2-yl][4-(morpholin-4-yl)-
methylphenyl]methanone (12).
Both the organometallic moieties and the organic frag-
ments in these three compounds present bond lengths and
angles within the expected ranges.30,31 As anticipated, and
due to the relative positioning of the cyclic amino substitu-
ents in relation to the ferrocenyl and benzo[b]thiophene
systems, all the equivalent bonds and angles of molecules
11 and 12 display very similar values.
In all molecules the Cp rings are nearly parallel to each
other and their relative conformation is almost fully eclipsed,
although molecule 6 shows a larger deviation angle (9.41ꢀ)
than 11 (2.01ꢀ, 1.69ꢀ) or 12 (2.16ꢀ); this presumably results
Figure 5. Dimeric arrangement for compound 6, showing the
2
C-H O interactions that define a R2 (8) synthon.
3 3 3
from a greater mobility of the Cp rings in 6 compared to the
benzo[b]thiophene derivatives 11 and 12, where the bulkier
groups bound to the ferrocenyl moiety are likely to hinder Cp
rotation. The smaller steric hindrance in molecule 6, and
particularly the higher mobility associated with the presence
of a sequence of four single bonds (between C6 and C13),
may also explain the value for the angle measured between
the Cp(C6-10) plane and the phenyl plane (83.57ꢀ). The
absence of this long sequence of single bonds forces mole-
cules 11 and 12 to display smaller angles (38.32ꢀ and 44.06ꢀ;
36.84ꢀ) between the Cp(C6-10) plane and the closest planar
group in the molecule (the fused benzo[b]thiophene). It
(30) Orpen, A. G.; Brammer, L.; Allen, F. H.; Kennard, O.; Watson,
D. G.; Taylor, R. J. Chem. Soc., Dalton Trans. 1989, S1–S83.
(31) Allen, F. H.; Kennard, O.; Watson, D. G.; Brammer, L.; Orpen,
A. G.; Taylor, R. J. Chem. Soc., Perkin Trans. 2 1987, S1–S19.

Article
Organometallics, Vol. 28, No. 18, 2009 5419
Table 1. Selected Bond Lengths and Angles for Compounds 6, 11, and 12
compound 11
molecule 1
compound 11
molecule 2
compound 6
compound 12
Bonds (A)
Fe(1)-Centroid Cp(C1-5)
Fe(1)-Centroid Cp(C6-C10)
average Fe-C Cp’s
average C-C Cp’s
C(6)-C(11)
1.649
1.640
2.040
1.420
1.467(3)
1.507(3)
1.820(2)
1.776(2)
1.625
1.619
2.011
1.399
1.450(5)
1.351(5)
1.711(4)
1.709(4)
1.457(5)
1.465(5)
1.490(5)
1.433(5)
1.632
1.629
2.018
1.399
1.441(5)
1.355(5)
1.715(4)
1.710(3)
1.462(5)
1.470(5)
1.491(5)
1.427(4)
1.656
1.653
2.050
1.423
1.4678(18)
1.3806(19)
1.7431(15)
1.7319(16)
1.489(2)
1.4897(19)
1.5127(19)
1.4667(19)
C(11)-C(12)
C(12)-S(1)
S(1)-C(13)
C(12)-C(20)
C(20)-C(21)
C(24)-C(27)
C(27)-N(1)
Angles (deg)
C(6)-C(11)-C(12)
C(6)-C(11)-C(14)
C(11)-C(12)-S(1)
C(12)-S(1)-C(13)
C(12)-C(20)-C(21)
C(24)-C(27)-N(1)
117.57(19)
121.9(3)
126.6(3)
113.9(3)
90.35(18)
119.5(3)
112.1(3)
123.3(3)
125.9(3)
113.9(3)
90.90(17)
119.5(3)
111.9(3)
124.23(12)
124.29(11)
112.94(11)
91.62(7)
122.93(12)
113.45(11)
111.16(14)
105.05(10)
Torsion Angles (deg)
tilt anglea
9.41
2.01
Angles between Planes (deg)
3.05
1.69
-2.16
Cp(C1-C5)-Cp(C6-C10)
Cp(C6-10)-phenyl
Cp(C6-10)-benzothiophene
benzothiophene-phenyl
2.10
83.57
4.16
3.45
38.32
54.08
44.06
59.36
36.84
48.27
a C(1)-Cp(C1-5)centroid-Cp(C6-10)centroid-C(6).
Figure 7. Asymmetric unit of [3-ferrocenyl-6-methoxybenzo-
[b]thiophen-2-yl][4-(pyrrolidin-1-yl)methylphenyl]methanone (11),
showing the S S and C-Hmethoxy πCp contacts that ensure
3 3 3 3 3 3
Figure 6. 3D assembly in compound 6 by way of C-H
interactions.
S
the dimeric arrangement.
3 3 3
remains to be established if this preferred geometry is a factor
in the overall balance of thermodynamic interactions
that will determine whether or not compounds 11 and
12 and their analogues are capable of favorable supramole-
cular interactions with specific biological receptors (e.g., the
ERs).
In a preliminary study, the supramolecular array of each
molecule was analyzed on the basis of the respective inter-
molecular interactions. Compound 6 forms dimers through a
C-H S interactions [distance C-S = 3.778 A; angle
3 3 3
C-H S = 151.42ꢀ; Figure 6 and Figure S1 (Supporting
3 3 3
Information)]. The two molecules of the asymmetric unit
of compound 11 form a dimer involving S S (distance
3 3 3
S
S = 3.530 A) and C-Hmethoxy
π
Cp contacts (distance
3 3 3
3 3 3
C-Cpcentroid = 3.500 and 3.577 A), respectively (Figure 7).
These dimers form 2D layers using π-π interactions between
Cp rings (distance = 3.712 A) aided by C-Hpyrrolidine πCp
3 3 3
contacts (distance C-Cpcentroid = 3.536 A) (Figure S2,
Supporting Information). Compound 12 also arranges its
C-H O interaction involving the O atom of the methoxy
group and a carbon of the phenyl ring, in a R2 (8) synthon
3 3 3
2
2
molecules in dimers, which are based on a R2 (6) synthon
(distance C O = 3.461 A; angle C-H O = 139.3ꢀ;
3 3 3 3 3 3
involving C-H O methoxy group interactions (distance
3 3 3
3 3 3
Figure 5); these dimers self-organize in a 3D assembly using
C
O = 3.215 A); these dimers self-arrange in a linear

5420 Organometallics, Vol. 28, No. 18, 2009
Ferreira et al.
Figure 8. Self-arrangement of [3-ferrocenyl-6-methoxybenzo[b]thiophen-2-yl][4-(morpholin-4-yl)methylphenyl]methanone (12), show-
2
ing the dimer units based on a R2 (6) synthon involving C-H O methoxy group interactions and the assembly of dimers in a linear
3 3 3
3 3 3
2
chain by means of a second R2 (6) synthon formed by C-H O morpholine interactions.
2
chain by means of another R2 (6) synthon formed by
C-H O morpholine interactions (distance C O =
3 3 3 3 3 3
3.387 A; angle C-H O = 144.29ꢀ) (Figure 8).
3 3 3
Electrochemical Studies. It is generally accepted that the
ferrocene/ferrocenium couple is central to the antitumor
activity of ferrocene-based drug molecules. Furthermore,
ferrocenium salts have been reported in some instances to
be more potent anticancer agents than their neutral counter-
parts, a property thought to be associated in part with
increased oxidative DNA damage stemming from the gen-
eration of reactive oxygen species via Fenton chemistry.32
Representative examples regarding the relevance of the
redox properties of ferrocene derivatives to their biological
activity are the electrochemical studies conducted by Jaouen
and co-workers on a series of ferrocenyl diphenol complexes,
which have demonstrated a positive correlation between
the ease of oxidation of the ferrocene group and the
ensuing cytotoxicity for prostate and breast cancer cell
lines.33 Taking these observations into account, we sought to
Figure 9. Cyclic voltammogram for [3-ferrocenyl-6-methoxy-
benzo[b]thiophen-2-yl][4-(piperazin-1-yl)methylphenyl]methanone
(13), in acetonitrile, at a scan rate of 200 mV/s. Waves I and II are
discussed in the text.
characterize the redox properties of the ferrocene derivatives
synthesized in the current study.
The electrochemical behavior of compounds 9-13 was
studied by cyclic voltammetry in acetonitrile between the
limits imposed by the solvent, i.e., ca. -2.2 and 1.4 V. All
measurements were carried out at room temperature, at a
€
(32) (a) Kopf-Maier, P. Z. Naturforsch. C 1985, 40, 843. (b) Neuse, E.
W.; Kanzawa, F. Appl. Organomet. Chem. 1990, 4, 19. (c) Osella, D.;
Ferrali, M.; Zanello, P.; Laschi, F.; Fontani, M.; Nervi, C.; Cavigiolio, G.
Inorg. Chim. Acta 2000, 306, 42. (d) Tabbì, G.; Cassino, C.; Cavigiolio, G.;
Colangelo, D.; Ghiglia, A.; Viano, I.; Osella, D. J. Med. Chem. 2002, 45,
5786.
(33) (a) Hillard, E.; Vessieres, A.; Thouin, L.; Jaouen, G.; Amatore,
C. Angew. Chem., Int. Ed. 2006, 45, 285. (b) Hillard, E.; Vessieres, A.; Le
Bideau, F.; Plaz_uk, D.; Spera, D.; Huche, M.; Jaouen, G. ChemMedChem
2006, 1, 551. (c) Hillard, E. A.; Pigeon, P.; Vessieres, A.; Amatore, C.;
Jaouen, G. Dalton Trans. 2007, 5073.
scan rate of 200 mV s-1, in a solution containing 0.1 M tetra-
3
ꢁ
n-butylammonium hexafluorophosphate as the supporting
electrolyte. A representative example of the electrochemical
response is shown in Figure 9 for compound 13, and the most
relevant parameters for the redox properties exhibited by the
ꢁ
ꢀ
ꢁ

Article
Organometallics, Vol. 28, No. 18, 2009 5421
Table 2. Electrochemical Data for Compounds 9-13 in Acetoni-
trile at Room Temperaturea
Figure 10. Induction of caspase-3 by 3-ferrocenyl-6-methoxy-
benzo[b]thiophen-2-yl][4-(piperazin-1-yl)methylphenyl]methanone
(13). Human 2008 ovarian cancer cells were incubated with 13 or
cisplatin at the IC50 concentration and then submitted to the test on
caspase-3 induction, as described in the Experimental Section. The
data are the means of at least three independent experiments and
express the measured increases in 7-amino-4-trifluoromethylcou-
marin (AFC) fluorescence. The error bars represent the standard
deviations. The asterisks (*) indicate statistically significant in-
creases (P < 0.01) compared to untreated cells.
amino group,37 though also conceivable for compounds
9-13, was not detected in our experiments.
Cytotoxic Activity. The newly synthesized compounds
were evaluated for their cytotoxic activity toward a panel
of human tumor cell lines containing examples of ovarian
(2008, C13*, RH4), cervical (A431), lung (A549), colon
(HCT-15), and breast (MCF-7, MCF-7 ADR) cancers.
Cytotoxicity was evaluated by means of MTT tests after
72 h of treatment with increasing concentrations of the test
compounds. For comparison purposes, the cytotoxicity of
cisplatin was evaluated in the same experimental conditions.
The IC50 values, calculated from dose-survival curves, are
shown in Table 3. All organometallic benzo[b]thiophene
derivatives showed a marked inhibitory potency toward
the different types of tumor cells investigated.
Compound 12, containing a morpholine substituent, was
the least potent but still showed detectable cytotoxic activity;
this activity was slightly lower than that of the reference
drug, cisplatin, with the mean IC50 (μM) values across the
tested cell lines being 11.75 (9.35-15.27) and 8.76
(2.56-18.13, excluding the C13* IC50 value) for 12 and
cisplatin, respectively. Compounds 9, 10, and 11, containing
dimethylamine, piperidine, and pyrrolidine substituents,
respectively, showed quite similar patterns of response
across the various cancer cell lines. A comparison of the
mean IC50 (μM) values for 9, 10, and 11 [5.28 (2.15-7.13),
5.46 (3.01-7.43), and 4.44 (2.27-5.41), respectively] to that
of cisplatin (8.76, excluding the C13* IC50 value) is indicative
of an in vitro antitumor activity roughly 2-fold higher than of
cisplatin itself.
a The potentials are referenced to the ferrocene/ferrocenium (Fc/Fcþ)
pair (Ep/2 = 0.40 V versus SCE).19 b pKa of the parent amine.34
compounds under study are summarized in Table 2. The
electrochemical behavior of this family of compounds is
predominantly characterized by a quasi-reversible redox
wave (process I), in the range 0.07 to 0.11 V, with compound
13 showing the lower value, and an irreversible redox process
(process II), in the range -2.02 to -2.11 V, mostly apparent
for compounds 9, 11, and 13. The quasi-reversible wave (I) is
consistent with a one-electron redox process involving the
FeII/FeIII couple; this wave is up to 110 mV more anodic than
that observed for ferrocene,19 presumably as a result of the
electron-withdrawing effect of the 2-acyl substituent(s),
which offers extended conjugation to the π-electrons of the
cyclopentadienyl ring, thereby decreasing the electron den-
sity around the iron atom. Although an increased basicity of
the amino group34 appears to correlate with a small anodic
shift of the FeII/FeIII oxidation peak (Table 2), there is no
significant effect as expected from the remoteness of the
tertiary amine in relation to the metal center. The irreversible
cathodic wave (II) can be attributed to redox processes
occurring within the acyl benzo[b]thiophene moiety, such
as the reduction of the carbonyl group35 or of the benzo-
[b]thiophene skeleton.36 Anodic oxidation at the tertiary
Among all 2-benzoyl-3-ferrocenylbenzo[b]thiophene com-
pounds tested, the piperazinyl derivative (13) showed the
greatest in vitro antitumor efficacy, about 10-fold higher
(34) (a) Bjerrum, J.; Schwarzenbach, G.; Sillen, L. G. Stability
Constants; The Chemical Society: London, U.K., 1958. (b) Hall, H. K. Jr.
J. Am. Chem. Soc. 1957, 79, 5441.
€
(35) Maurice, C.; Schollhorn, B.; Canet, I.; Mousset, G.; Mousty, C.;
Guilbot, J.; Plusquellec, D. Eur. J. Org. Chem. 2000, 813.
(36) Garcia, M. H.; Florindo, P.; Piedade, M. F. M.; Duarte, M. T.;
Robalo, M. P.; Heck, J.; Wittenburg, C.; Holtmann, J.; Licandro, E.
J. Organomet. Chem. 2008, 693, 2987.
(37) (a) Shono, T.; Toda, T.; Oshino, N. J. Am. Chem. Soc. 1982, 104,
2639. (b) Jurva, U.; Bissel, P.; Isin, E. M.; Igarashi, K; Kuttab, S.; Castagnoli,
N. Jr. J. Am. Chem. Soc. 2005, 127, 12368.

5422 Organometallics, Vol. 28, No. 18, 2009
Ferreira et al.
Table 3. Cytotoxicity Studies
IC50 (μM) ( SDa
compound
MCF-7
MCF-7 ADR
A431
HCT-15
A549
2008
C13*
RH4
9
10
11
12
13
cisplatin
5.21 ( 1.11
5.01 ( 0.96
4.12 ( 0.23
11.64 ( 1.28
1.09 ( 0.79
10.32 ( 1.21
4.53 ( 1.37 (0.86)b 7.13 ( 1.46
6.67 ( 0.91
5.82 ( 1.62
3.30 ( 0.68
6.21 ( 1.11
6.98 ( 0.84
4.45 ( 0.73
5.52 ( 0.13
6.32 ( 1.06
5.29 ( 1.57
4.85 ( 1.54 (0.87)
4.91 ( 1.04 (1.28)
5.41 ( 0.96 (1.02)
9.35 ( 1.23 (0.67)
1.22 ( 1.06 (0.96)
2.15 ( 0.33
7.43 ( 1.28
5.01 ( 1.49
12.65 ( 2.32
0.73 ( 0.39
6.43 ( 0.68
4.21 ( 1.35 (0.84)
5.35 ( 0.67 (1.29)
10.17 ( 1.24 (0.87)
1.04 ( 0.41 (0.95)
8.41 ( 1.43 (0.81)
3.01 ( 0.74
2.27 ( 0.96
9.71 ( 1.02 10.43 ( 2.12
1.01 ( 0.46 2.95 ( 0.95
3.21 ( 1.72 12.31 ( 1.26 18.13 ( 1.51
15.27 ( 1.84 14.43 ( 2.12
3.85 ( 1.09 1.26 ( 1.21
2.56 ( 1.13 39.21 ( 1.92 (15.31)
a IC50 values toward the indicated cell lines were calculated by probit analysis (P < 0.05, χ2 test). Cells ((5-8) ꢀ 104 mL-1) were treated for 72 h with
increasing concentrations of the test compounds. Cytotoxicity was assessed by the MTT test. SD = standard deviation. b The numbers in parentheses are
the RF (resistance factor) values, calculated as [IC50 (resistant cell line)/IC50 (parent cell line)]. The RF for doxorubicin in the multidrug-resistant MCF-7
ADR cell line was 30.75.
than that of cisplatin and with a mean IC50 (μM) of 1.64
(0.73-3.85). Moreover, it is noteworthy that the growth
inhibitory activity displayed by 13 was more marked against
breast, cervical, and ovarian cancer cells (Table 3), which
are all particularly sensitive to estrogenic/antiestrogenic
effects.38
organometallic benzo[b]thiophenes are not potential MDR
substrates.
To characterize the cell death pathway triggered by the
most active derivative, 13, we examined its effect in terms of
apoptosis induction. Apoptosis is generally considered as
one of the main mechanisms of the antitumor effect of
cisplatin. Caspase-3 is a well-known executor enzyme in
the apoptosis pathway, and cisplatin may cause apoptosis
associated with increased caspase-3 activity.42 Compound 13
was tested for its ability to activate caspase-3 in 2008 human
ovarian cancer cells. As shown in Figure 10, a 24 h treatment
using IC50 concentrations resulted in a significant enhance-
ment of enzyme activity, about 10- and 1.3-fold higher than
those detected in untreated and cisplatin-treated cells, re-
spectively. Albeit preliminary, these results, which are con-
sistent with the cytotoxicity data, are encouraging because it
is particularly advantageous to develop new agents capable
of inducing tumor cell death by means of an apoptotic
mechanism, as opposed to a necrotic one.
With all benzo[b]thiophenes, very interesting results were
obtained against cisplatin-resistant (C13*) and -revertant
(RH4) cells, as well as against multidrug-resistant (MCF-7
ADR) phenotypes. In ovarian carcinoma C13* cells, cispla-
tin resistance has been correlated with reduced cell drug
uptake, high cellular thioredoxin reductase and glutathione
levels, and enhanced repair of DNA damage.39 From the
tests on the 2008/C13* cell pair, the estimated resistance
factor of all benzo[b]thiophene derivatives 9-13 was found
to be about 15-fold lower than that of cisplatin (Table 3),
clearly revealing no cross-resistance phenomena. RH4 re-
vertant cells were obtained from C13* cells by selection with
the lipophilic rhodamine 123 (Rh123) cation. Compared to
C13* cells, the RH4 cells lost a substantial portion of their
cisplatin resistance, maintaining only a 2- to 3-fold resis-
tance. Despite this major loss of resistance, they retained a
number of the phenotypic features related to cisplatin resis-
tance that are observed in C13* cells.40 As a whole, the
behavior of the benzo[b]thiophenes 9-13 against RH4 cis-
platin-revertant cells was comparable to that elicited in the
cisplatin-sensitive 2008 cell line (Table 3), which further
emphasizes the lack of cross-resistance properties in the
new compounds.
Acquired multidrug resitance (MDR), whereby cells be-
come refractory to multiple drugs, poses a most important
challenge to the success of anticancer chemotherapy. MCF-7
ADR cells are resistant to doxorubicin, a drug belonging to
the MDR spectrum, and this property has been associated
with overexpression of multispecific drug transporters.41
Cytotoxicity assays testing all the benzo[b]thiophene deriva-
tives 9-13 against the MCF-7/MCF-7 ADR cell line pair
showed a similar pattern of response across the parental and
resistant subline (Table 3) and allowed the calculation of RF
values (0.84-1.29) about 30-fold lower than that obtained
with doxorubicin (RF = 30.75), suggesting that these new
Conclusions
We have synthesized and characterized a series of 2-
benzoyl-3-ferrocenylbenzo[b]thiophenes (9-13) bearing ter-
tiary alkylamino groups linked to the benzoyl C4 through a
methylene spacer. These are the first representatives of a
class of ferrocenylbenzo[b]thiophenes designed as part of a
program intended to explore the antitumor and antiestro-
genic potential of organometallic compounds having a
structural resemblance to the SERM raloxifene. Although
the new benzo[b]thiophenes 9-13 display a 2-acyl-3-ferro-
cenyl substitution pattern, as opposed to the 2-aryl-3-acyl
substitution of raloxifene, they all showed cytotoxic activity
in the low micromolar range toward a series of human cancer
cell lines, including cisplatin-resistant (C13*) and -revertant
(RH4) human ovarian cells and multidrug-resistant (MCF-7
ADR) breast cancer cells. Of particular interest is the piper-
azinyl derivative 13, with a mean IC50 about 10-fold lower
than that of cisplatin across the tested cell lines. Although
potential SERM properties have yet to be evaluated, it is
noteworthy that the highest growth inhibitory activities
displayed by compound 13 were measured against human
gynecological cancer cells (breast, cervix, ovary), which are
well known to be sensitive to estrogenic/antiestrogenic ef-
fects. Finally, it is also worth mentioning that preliminary
caspase-3 induction tests indicated a significant ability of
compound 13 to induce cell death by way of an apoptotic
mechanism. Taken together, these in vitro data suggest that
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Article
Organometallics, Vol. 28, No. 18, 2009 5423
the new ferrocenylbenzo[b]thiophene derivatives, and com-
pound 13 in particular, may have good antitumor potency.
Our observation that the more easily oxidized of the new
compounds (13) was the one with the lowest mean IC50 value
is in line with the findings of Jaouen and co-workers on the
cytotoxicity of conjugated and unconjugated ferrocenyl
phenols33 and has encouraged us to increase the number
and diversity of 2-acyl-3-ferrocenylbenzo[b]thiophenes
available for biological screening. We are currently applying
the synthetic strategy reported herein to expand the library of
members of this structural series by varying the spacer size
and type and by including different substituents on the
benzo[b]thiophene and/or organometallic fragments, to-
ward increased intramolecular electron transfer to the ferro-
cenyl moiety33 and H-bond donation,43 which may be at the
core of the higher activity observed for the piperazinyl
derivative 13.
and the Portuguese MS Network (IST-UTL Center) for
providing access to the facilities. This work was sup-
~
^
ported in part by Fundac-ao para a Ciencia e a Tecnologia
(FCT), Portugal, through a research grant (PTDC/QUI/
67522/2006) and a postdoctoral fellowship to A.P.F.
(SFRH/BPD/21014/2004), by the University of Padova
(Progetto di Ateneo CPDA065113/06), and by Ministero
ꢁ
dell’Istruzione dell’Universita e della Ricerca, Italy (PRIN
20078EWK9B). We are also grateful to CIRCMSB
(Consorzio Interuniversitario di Ricerca in Chimica dei
Metalli nei Sistemi Biologici), Italy.
Supporting Information Available: Table S1, summarizing the
crystal data and structure refinement parameters for com-
pounds 6, 11, and 12; crystallographic data of the same com-
pounds in CIF format; Figure S1, showing the 3D supra-
molecular assembly of compound 6 along the c axis; Figure
S2, showing the 2D layers formed by dimers of compound 11.
These materials are available free of charge via the Internet
at http://pubs.acs.org. Crystallographic data for compounds 6,
11, and 12 are also deposited with the Cambridge Crystallo-
graphic Data Centre (CCDC 692699, 692700, and 692701,
respectively) and can be obtained free of charge from CCDC,
12 Union Road, Cambridge CB2 1EZ, UK (fax: 44-1223-
336033; e-mail: deposit@ccdc.cam.ac.uk; http://www.ccdc.
cam.ac.uk/deposit).
Acknowledgment. We thank Alexandra Antunes for
~
the EI mass spectra and Conceic-ao Oliveira for the ESI
and HRMS analyses. Thanks are also due to Jorge
Morgado, for allowing use of the microwave reactor,
and to the Portuguese NMR Network (IST-UTL Center)
(43) Mukherjee, S.; Nagar, S.; Mullick, S.; Mukherjee, A.; Saha, A.
J. Mol. Graph. Model. 2008, 26, 884.