Synthesis of the ferrocenyl analogue of clotrimazole drug

Article abstract of DOI:10.1016/j.jorganchem.2016.12.009

The ferrocenyl analogue of clotrimazole, in which the metallocene fragment replaces one of the phenyl rings in the triphenylmethane system, and a related isomer were prepared through the direct substitution of a methoxygroup in the α-position to ferrocene with imidazole in the key step. The obtained ferrocenyl derivatives were spectroscopically characterized and in a preliminary assay for bioactivity their cell growth inhibitory activity on two different human cancer cell lines was evaluated. In comparison with the parent drug the ferrocene analogues displayed about two-fold increase of cytotoxicity on HT29 colorectal cancer cells, whereas comparable activity was displayed against MCF-7 breast cancer cell line.

Full text of DOI:10.1016/j.jorganchem.2016.12.009

Accepted Manuscript
Synthesis of the ferrocenyl analogue of clotrimazole drug
Sonia Pedotti, Martina Ussia, Angela Patti, Nicolò Musso, Vincenza Barresi, Daniele
Filippo Condorelli
PII:
S0022-328X(16)30560-5
10.1016/j.jorganchem.2016.12.009
JOM 19732
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 7 October 2016
Revised Date: 6 December 2016
Accepted Date: 8 December 2016
Please cite this article as: S. Pedotti, M. Ussia, A. Patti, N. Musso, V. Barresi, D.F. Condorelli, Synthesis
of the ferrocenyl analogue of clotrimazole drug, Journal of Organometallic Chemistry (2017), doi:
10.1016/j.jorganchem.2016.12.009.
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ACCEPTED MANUSCRIPT
Graphical abstract
The introduction of a ferrocene unit in the clotrimazole scaffold led to increased biological activity compared to the
parent drug
N
N
N
Increased growth
inhibitory activity on
colorectal cancer cells
N
R
R1
Fe
Cl
Clotrimazole
Ferrocenyl analogues

ACCEPTED MANUSCRIPT
Synthesis of the ferrocenyl analogue of clotrimazole drug
Sonia Pedotti,a Martina Ussia, a Angela Patti, a,*
Nicolò Musso,b Vincenza Barresi,b Daniele Filippo Condorelli b
aCNR – Istituto di Chimica Biomolecolare, via Paolo Gaifami 18, I-95126, Catania
bDipartimento Scienze Biomediche e Biotecnologiche, Sez. Biochimica Medica, via S. Sofia 64, I-95125 Catania
E-mail: angela.patti@cnr.it
Keywords: ferrocenes, clotrimazole, synthesis, cytotoxicity.
Abstract
The ferrocenyl analogue of clotrimazole, in which the metallocene fragment replaces one of the
phenyl rings in the triphenylmethane system, and a related isomer were prepared through the direct
substitution of a methoxygroup in the α-position to ferrocene with imidazole in the key step. The
obtained ferrocenyl derivatives were spectroscopically characterized and in a preliminary assay for
bioactivity their cell growth inhibitory activity on two different human cancer cell lines was
evaluated. In comparison with the parent drug the ferrocene analogues displayed about two-fold
increase of cytotoxicity on HT29 colorectal cancer cells, whereas comparable activity was
displayed against MCF-7 breast cancer cell line.
1. Introduction
Since the discovery of the anticancer activity of platinum(II) complexes, metal-containing
compounds have attracted growing interest as drug candidates due to their peculiar physico-
chemical properties and structural diversity.1 Following the observations that the use of cisplatin
and its derivatives in cancer therapy is associated with severe drawbacks,2 the search for alternative
coordination complexes3 and organometallic compounds4 with different metals was increasingly
stimulated and several highly cytotoxic compounds were disclosed, many of which also showed
innovative and metal-specific modes of action.5 The potential of organometallic compounds for
enzyme-inhibitors6 and non-cancer therapeutics has been also explored,7 leading to promising
results in the field of anti-malarial drugs.8
In this context, ferrocene, the prototype of metallocenes, provides an useful platform in bio-
organometallic chemistry for the synthesis of a large variety of derivatives with medicinal
applications9 and conjugates with biomolecules10 due to its stability in aqueous aerobic media, non-
toxicity, redox activity and chemical versatility. For its aromatic character and metabolism related
to benzene,11 the ferrocene can be considered a “bio-isosteric” fragment in replacement of a phenyl

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ring in known drugs. On the basis of this approach, the Jaouen’s group developed a series of
ferrocenyl analogues (the “ferrocifen” family) of tamoxifen,12 a chemotherapeutic agent for patients
with hormone-dependent breast cancer, and some of them not only displayed higher
antiproliferative effects with respect to the parent drug, but were also active on tumor cell lines
lacking the overexpression of estrogen receptor and hence not susceptible to treatment with
tamoxifen. A specific mechanism whereby redox activation induces anticancer activity in ferrocifen
has been proposed13 and it seems that the redox properties of iron in the ferrocene moiety also
plays a role in ferroquine, an antimalarial drug candidate at clinical stage II derived by the
incorporation of the metallocene fragment in the molecule of chloroquine (Scheme 1).14
R
OH
Fe
NMe2
NMe2
O
O
n
R = H, OH
n = 2 - 8
Tamoxifens
Ferrocifens
NEt2
HN
NMe2
HN
Fe
Cl
N
Cl
N
Chloroquine
Ferroquine
Scheme 1. Ferrocenyl analogues of active drugs
Other established drugs, as the antiandrogen nilutamide,15 anticancer paclitaxel,16 antimalarial
artemisinin,17 have been modified by using ferrocene as a substituent in their core structure, and the
biologically activity is usually retained or enhanced.
Clotrimazole is a broad-spectrum antimycotic drug containing an imidazole ring linked to a
triphenylmethyl group, a structural motif that has recently considered as pharmacophore in the
design of novel antimalarial agents.18 Clotrimazole also displays antiproliferative effects on
different cancer cells, related with its activity as potent inhibitor of glycolysis.19 Coordination
compounds of clotrimazole with different metals20 and ruthenium(II) organometallic derivatives21
are reported, many of them with increased biological activity with respect to the original drug.
Although ferrocenylphenylimidazole22 and some ferrocenyl(alkyl)azoles have been prepared and
evaluated for their antitumor activity,23 ferrocenyl derivatives of clotrimazole have not previously
considered.

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Here we describe the synthesis of two ferrocenyl analogues of clotrimazole, 1a and 1b (Scheme 2),
in which the metallocene fragment replaces one of the phenyl rings in the triphenylmethane system,
and the evaluation of their cell growth inhibitory effect on two different human cancer cell lines.
Although the inspiring molecule of clotrimazole contains an atom of chlorine in position 2, the
isomeric compound 1b was also prepared in order to detect possible effects on the biological
activity related with the position of the chlorine.
Scheme 2. Ferrocenyl analogues of clotrimazole
2. Results and discussion
In the synthesis of clotrimazole and related derivatives the introduction of the heteroaromatic ring
on the quaternary carbon center is usually accomplished by reaction of a suitable triphenylchloride
with imidazole in the presence of a base and, according to this route, the ferrocenyl carbinols 2a-b
were then identified as key intermediate in the synthesis of the target compounds 1a-b. Among the
different pathways deduced from retrosynthetic analysis (Scheme 3), we firstly resorted into (a) as a
more convenient way to the tertiary alcohols 2a-b for the commercial availability of the starting
benzophenones.
Scheme 3. Retrosynthetic analysis for ferrocenyl analogues of clotrimazole

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Metalation of ferrocene with tert-BuLi in THF followed by reaction with 2-chloro- or 4-
chlorobenzophenone for 12 h gave the expected alcohols 2a-b in moderate yield (55-60%), that did
not increase changing the reaction solvent or the reagent stoichiometry. In spite of the additional
step required for the preparation of the starting ferrocenylketones (by Friedel-Crafts acylation of
ferrocene24), the alternative reaction of 2- or 4-chlorobenzoylferrocene with a phenyl anion source
(route (b) in Scheme 1) was also evaluated. The use of phenylmagnesium bromide was not
effective, but the reaction in the presence of phenyllithium proceeded smoothly resulting in good
yield (72-78%) of target alcohols (Scheme 4).
1
Ferrocenyl alcohols 2a-b were characterized by their spectroscopic properties and in the H-NMR
spectra the metallocenic moiety was easily identified from the usual pattern for monosubstituted
ferrocenes, with a singlet accounting for five protons on the unsubstituted cyclopentadienyl ring and
distinct narrow multiplets for the protons on the other ring, in the typical region between 4.0 and 4.5
ppm. In CDCl3 the resonances of phenyl protons appeared as complex multiplets for 2a while only
two singlets were observed for 2b, indicating a magnetic near equivalence for both the substituted
and unsubstituted rings in the diarylmethane moiety of the molecule. However, when the spectrum
of 2b was registered in (CD3)2SO the expected pattern for a para-disubstituted benzene was
observed, partially overlapped to the resonances of the other aromatic protons.
Scheme 4. Synthesis of the ferrocenyl analogues of clotrimazole

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The conversion of alcohols 2a-b into the ferrocenyl analogues of clotrimazole 1a-b was attempted
by treatment with SOCl2 followed by in situ reaction with imidazole according a procedure reported
for triphenylmethane derivatives,18 but extensive degradation of the starting alcohols was observed.
Alternative ferrocene substrates for direct reaction with imidazole were then considered since it is
well known that α-ferrocenylalkylderivatives with suitable leaving groups easily undergo the
displacement of such groups by a broad variety of nucleophiles with retention of configuration and
in mild conditions. This peculiar reactivity, originally demonstrated for α-ferrocenylalkyl
trimethylammonium salts and α-ferrocenylacetates,25 has been also reported for α-ferrocenyl
alcohols with phosphines26 and some C- and N-nucleophiles.27
The reaction of 2a or 2b with imidazole in water at 100 °C27b or in the presence of acid
catalysis26,27a did not gave any product and the use of carbonyldiimidazole as alternative source of
the nitrogen ring22 was also unsuccessful.
The conversion of 2a or 2b into the corresponding acetates was then tried, without success, in
standard conditions using acetic anhydride in combination with different bases (pyridine, Et3N,
Et3N/DMAP) and reactions of esterification in the presence of Lewis acids,28 with microwave
irradiation or in solvent-free conditions29 were also carried out, but the formation of the expected
products was not detected in any case.
These discouraging results, otherwise in agreement with the reported unreactivity of sterically
hindered tertiary ferrocenyalcohols,27a,30 prompted us to look for different derivatives and,
pleasantly, we were able to obtain the methyl ethers 3a and 3b in nearly quantitative yield by simple
treatment of 2a or 2b with a mixture of acetic acid and methanol.
In spite of the exchange reaction of ferrocenyl α-methoxy substituent by nucleophiles is limited, at
the best of our knowledge, to just two examples31,32 on ferrocenes bearing the ether functionality on
a secondary carbon, we succeeded into the synthesis of the target compounds 1a and 1b by
treatment of ethers 3a and 3b with imidazole under acid catalysis. Although in acidic medium the
protonation of the N-basic center of imidazole involves the use of large excess of this reagent, the
acid catalysis was found strictly necessary. Attempts to decrease the reaction time using high-
boiling solvents led to degradation of the substrate. Alcohols 2a or 2b in about 18-20 % yield were
detected in the reaction mixture unless dry CH2Cl2 is used, so confirming the reported sensitivity of
α-methoxyferrocenes to hydrolysis, further increased under acid catalysis.32
The described synthesis of the ferrocenyl analogues of clotrimazole, though moderately efficient in
the last step (44% yield), provides an additional example in the short list of nucleophilic
substitutions on fully α-substituted ferrocenes. The presence of the imidazole ring in the target
compounds 1a and 1b was associated to three additional resonances in the aromatic region of the

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1H-NMR spectrum and to a sensible upfield shift for the 13C-NMR signal of the quaternary carbon
with respect to the oxygenated parent compounds. In the ESI-MS spectra of 1a and 1b the base
peak derived from the preferential loss of the imidazole fragment from the molecule and molecular
ion was hardly detected (<1%). Acquisition of MS spectra in SIM mode allowed us to better detect
the molecular ion, so confirming the identity of the products.
A careful inspection of the reaction mixture of 3a or 3b with imidazole revealed the presence of
side products identified as previously unreported diphenylfulvene derivatives 5a or 5b, resulting
from Nesmeyanov fragmentation33 of ferrocenyl carbenium ions 4a-b, whose formation is favoured
1
by the acidic medium (Scheme 5). The identification of 5a an 5b was mainly based on their H-
NMR spectra that displayed characteristic resonances with a complex AA’XX’ coupling pattern for
protons in the cyclopentadiene ring.34
Scheme 5. Fulvenes from ferrocenyl derivatives
Ferrocenyl analogues of clotrimazole 1a and 1b were preliminarly assayed for their cell growth
inhibition activity on two different human cancer cell lines in comparison with clotrimazole. The
cell models were chosen on the basis of the reported antiproliferative effects of clotrimazole19 on
breast cancer and colon cancer cell lines. As shown in Table 1 clotrimazole was more active in
breast cancer cell MCF-7 than in colon cancer cell line HT29, although its activity (GI50 21.44 µM)
was lower than a classical anticancer chemotherapeutic drug, such as 5-fluorouracil (5-FU, GI50
2.98 µM).
The growth inhibitory effect of clotrimazole on MCF-7 is in agreement with reported data19a and the
present result on HT29 is comparable with that observed in murine colon adenocarcinoma cells.19b
Compounds 1a and 1b were active on MCF-7 cell line with a potency similar to that of the parent
drug. Conversely, GI50 values more than halved compared to clotrimazole (Table 1) were
determined for ferrocenyl derivatives 1a and 1b on HT29 cancer cell line and no significant
difference related with the isomeric position of the chlorine atom in the molecules was observed.
The structural modification with ferrocene resulted in the enhancement of cytotoxic activity of the
organometallic analogues in comparison with the parent drug in HT29 cells. This effect might be

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related with the redox properties of iron in the ferrocene moiety12,14 and its ability to generate
cytotoxic reactive oxygen species (ROS) in proper cellular conditions.
Indeed, it has been suggested that cancer cells are more vulnerable to treatment with agents that
generate ROS due to altered metabolism and mitochondrial functions.35 Colon cancer cell lines
accumulate high level of redox cycling metals and show high level of endogenous oxidative
stress.36 Further studies are necessary to evaluate the role of oxidative stress in induction of
cytotoxicity by ferrocene derivatives.
Table 1. Growth inhibition assay on different cancer cell linesa
GI50 (µM)b,c
Compound
HT29 (HTB-38)
MCF-7(HTB-22)
Clotrimazole
64.19
27.51
28.13
5.71
21.44
23.84
20.44
2.98
1a
1b
5-FUd
aGrowth inhibition was measured with colorimetric MTT assay (see
b
experimental). Values are reported as GI50, the concentration of the
compound required to cause 50% inhibition of cell growth.
cConcentration-response curves are reported in supplemental
material.d5-Fluorouracil.
3. Conclusion
Two ferrocenyl analogues of clotrimazole, in which the metallocene unit replaces one of the phenyl
ring in the triarylmethane system, were prepared and their growth inhibitory activity assayed on two
different human cancer cell lines MCF-7 and HT29. In spite of some chemical inertness of the
intermediate tertiary ferrocenylalcohol, the corresponding ferrocenyl methyl ether was identified as
a suitable substrate for the direct nucleophilic substitution with imidazole. In comparison with the
parent drug the ferrocene derivatives displayed about two-fold increase of cytotoxicity on HT29
colorectal cancer cells, whereas comparable activity was displayed against MCF-7 breast cancer
cell line and further studies are in progress to elucidate the molecular basis of the observed
selectivity.
4. Experimental
4.1. General
1H- and 13C-NMR spectra were recorded on Bruker AvanceTM 400 spectrometer at 400.13 and
100.62 MHz, respectively. Chemical shifts (δ) are given as ppm relative to the residual solvent
peak and coupling constants (J) are in Hz. In the NMR assignment Cp and Cp’ refers to substituted

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and unsubstituted cyclopentadienyl ring, respectively. Ferrocene, 2-chlorobenzophenone and 4-
chlorobenzophenone were purchased from Aldrich. Column chromatography was performed on
silica gel 60 (Merck, 40-63 µm) using the specified eluents. High resolution mass spectra (HR-MS)
were obtained on a Thermofisher Orbitrap QExactive instrument with ESI ionization mode, using
3.2V cone voltage and 300 °C source temperature. Melting point are uncorrected. Curve-fitting and
GI50 analysis was performed with the GraphPad Prism software (San Diego, California, USA).
4.2. Friedel-Craft acylation of ferrocene
To a suspension of AlCl3 (413 mg, 3.1 mmol) and ferrocene (500 mg, 2.7 mmol) in CH2Cl2 (10
mL) at 0 °C, 2-chlorobenzoylchloride (353 µL, 2.8 mmol) or 4-chlorobenzoylchloride (350 µL, 2.7
mmol) in CH2Cl2 (3 mL) was added dropwise within 20 min. The mixture was warmed to room
temperature and stirred overnight. After quenching at 0 °C by dropwise addition of ice-cold water
(5 mL, Caution: gas evolution), the reaction mixture was diluted with CH2Cl2 (100 mL) and
washed with satd. NaHCO3 (2 × 50 mL) and brine (50 mL). The organic layer was dried over
Na2SO4 and taken to dryness to give a residue which was purified by column chromatography on
silica gel (n-hexane:AcOEt 90:10 v/v). 2-Chlorobenzoylferrocene: (715 mg, 2.2 mmol, 82%
1
yield),red crystals (from n-hexane), mp 111-112 °C, H-NMR (CDCl3): δ 4.28 (s, 5H, Cp’H), 4.60
(t, J = 2.0, 2H, CpH), 4.75 (t, J = 2.0, 2H, CpH), 7.35-7.40 (m, 2H, ArH), 7.46 (dd, J = 8.0 and 1.2,
13
1H, ArH), 7.51 (dd, J = 7.4 and 1.6, 1H, ArH),; C-NMR (CDCl3): δ 70.13 (Cp’H), 71.10 (CpH),
72.89 (CpH), 78.54 (Cp), 126.20 (ArH), 128.64 (ArH), 130.28 (ArH), 130.70 (ArH), 131.05 (Ar),
139.51 (Ar), 198.66 (CO). HRMS calcd for C17H14ClFeO [M+H]+ 325.00826, found 325.00791.
4-Chlorobenzoylferrocene: (702 mg, 2.2 mmol, 80% yield), red crystals (from n-hexane), mp 119-
1
120 °C, H-NMR (CDCl3): δ 4.21 (s, 5H, Cp’H), 4.62 (t, J = 1.6, 2H, CpH), 4.89 (t, J = 1.6, 2H,
13
CpH), 7.46 (d, J = 8.4, 2H, ArH), 7.87 (d, J = 8.4, 2H, ArH); C-NMR (CDCl3): δ 70.34 (Cp’H),
71.51 (CpH), 72.80 (CpH), 78.42 (Cp), 128.59 (ArH), 129.58 (ArH), 137.81 (Ar), 138.11 (Ar),
197.81 (CO). HRMS calcd for C17H14ClFeO [M+H]+ 325.00826, found 325.00848.
4.3. Synthesis of 1-[(2-chlorophenyl)]-1-phenyl-1-hydroxymethylferrocene, 2a
Procedure A: Ferrocene (500 mg, 2.68 mmol) was dissolved in anhydrous THF (10 mL) under
argon atmosphere and 1.7 M pentane solution of t-BuLi (1.6 ml, 2.68 mmol) was slowly added at
0°C under stirring. After 1 h 2-chlorobenzophenone (550 mg, 2.54 mmol) was added and the
reaction mixture left to stand overnight at room temperature under stirring. The reaction was then
quenched with H2O and extracted with AcOEt (10 mL × 3). The organic layer was washed with
brine, dried over Na2SO4 and taken to dryness under vacuum. The residue was purified by column
chromatography on silica gel (n-hexane-AcOEt 97:3 v/v) to give 2a (565 mg, 1.40 mmol, 55%
yield) as a yellow solid, mp = 129-130 °C; 1H-NMR (CDCl3): δ 3.78 (bs, 1H, CpH), 4.00 (s, 1H, -

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OH), 4.19 (s, 5H, Cp’H), 4.26 (s, 1H, CpH), 4.33 (s, 2H, CpH), 7.16-7.20 (m, 3H, ArH), 7.27-7.38
(m, 3H, ArH); 13C-NMR (CDCl3): δ 67.78 (CpH), 68.05 (CpH), 68.64 (CpH), 68.90 (Cp’H), 70.30
(CpH), 78.54 (C-q), 98.12 (Cp), 125.96 (ArH), 126.86 (ArH), 127.02 (ArH), 127.27 (ArH), 128.82
(ArH), 130.36 (ArH), 131.28 (ArH), 133.29 (Ar), 144.28 (Ar), 145.06 (Ar). HRMS calcd for
C23H19ClFeO 402.04738, found 402.04758.
Procedure B: To a solution of 2-chlorobenzoylferrocene (200 mg, 0.62 mmol) in 10 mL of
anhydrous THF, PhLi (350 µl of 1.8M solution in Et2O, 0.63 mmol) was added at –78 °C and the
mixture was allowed to stir for 2 h at this temperature. The reaction was quenched with H2O and
extracted with AcOEt (6 mL × 3). The organic layers were pooled, washed with brine and dried
over Na2SO4. Evaporation of the solvent gave a residue that was purified by column
chromatography on silica gel (n-hexane-AcOEt 97:3 v/v) to give pure 2a (180 mg, 0.45 mmol, 72%
yield).
4.4. Synthesis of 1-[(4-chlorophenyl)]-1-phenyl-1-hydroxy-methylferrocene, 2b
Reaction of ferrocene (500 mg, 2.68 mmol) and 4-chlorobenzophenone (550 mg, 2.54 mmol)
according procedure A described above gave title compound 2b as a yellow solid (543 mg, 1.35
1
mmol, 53%yield), mp = 115-116 °C ; H-NMR (d6-DMSO): δ 3.99 (s, 1H, CpH), 4.01 (s, 5H,
Cp’H), 4.05 (s, 1H, CpH), 4.21 (s, 2H, CpH), 5.89 (s, 1H, -OH), 7.19-7.28 (m, 7H, ArH), 7.33 (d, J
13
= 8.4, 2H, ArH); C-NMR (d6-DMSO): δ 67.96 (CpH), 67.99 (CpH), 69.00 (Cp’H), 77.03 (C-q),
97.63 (Cp), 127.03 (ArH), 127.46 (ArH), 127.56 (ArH), 129.38 (ArH), 131.50 (Ar), 147.84 (Ar),
148.52 (Ar). HRMS calcd for C23H19ClFeO 402.04738, found 402.04746.
4.5. Synthesis of 1-[(2-chlorophenyl)]-1-phenyl-1-methoxy-methylferrocene, 3a
A sample of 2a (200 mg, 0.50 mmol) was dissolved in 20 mL of MeOH/AcOH 8:1 v/v mixture and
heated at 80°C until complete conversion of the substrate was obtained (2-3 h). The reaction
mixture was taken to dryness to give 3a as a yellow solid (184 mg 0.44 mmol, 88% yield) used
1
without further purification. Mp = 121-122 °C; H-NMR (CDCl3): δ 3.38 (s, 3H, -OMe), 3.76 (bs,
1H, CpH), 4.16 (s, 5H, Cp’H), 4.17-4.20 (m, 2H, CpH), 4.24-4.26 (m, 1H, CpH), 7.22-7.24 (m, 2H,
ArH), 7.26-7.34 (m, 6H, ArH), 7.58 (m, 1H, ArH); 13C-NMR (CDCl3): δ 52.68 (-OMe), 66.97
(CpH), 67.98 (CpH), 69.12 (CpH), 69.23 (Cp’H), 71.05 (CpH), 84.18 (C-q), 94.87 (Cp), 125.66
(ArH), 126.75 (ArH), 127.07 (ArH), 127.81 (ArH), 128.89 (ArH), 131.65 (ArH), 132.21 (ArH),
134.42 (Ar), 141.43 (Ar), 144.33 (Ar). HRMS calcd for C24H21ClFeO 416.06303, found 416.06280.
4.6. Synthesis of 1-[(4-chlorophenyl)]-1-phenyl-1-methoxy-methylferrocene, 3b
Treatment of 2b with MeOH/AcOH mixture as described above gave title compound 3b in 90% as
1
a yellow solid, mp = 111-112 °C; H-NMR (CDCl3): δ 3.21 (s, 3H, -OMe), 3.91 (bs, 2H, CpH),
4.08 (s, 5H, Cp’H), 4.21 (bs, 2H, CpH), 7.26-7.41 (m, 9H, ArH); 13C-NMR (CDCl3): δ 52.19 (-

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OMe), 67.69 (CpH), 67.83 (CpH), 69.23 (Cp’H), 69.53 (CpH), 69.64 (CpH), 83.75 (C-q), 93.38
(Cp), 127.19 (ArH), 127.43 (ArH), 128.60 (ArH), 130.00 (ArH), 132.78 (Ar), 143.79 (Ar), 144.39
(Ar). HRMS calcd for C24H21ClFeO 416.06303, found 416.06083.
4.7. Synthesis of 1-[(2-chlorophenyl) -1-phenyl-1-ferrocenylmethyl]-1H-imidazole, 1a
To a solution of 3a (180 mg, 0.43 mmol) in anhydrous CH2Cl2 (20 mL) imidazole (290 mg, 4.26
mmol) and glacial acetic acid (400 µl) were added and the mixture was stirred under reflux for 5
days. The reaction mixture was diluted with H2O and extracted with CH2Cl2 (5 mL × 3). Collected
organic layers were dried on Na2SO4, filtered off and concentrated in a rotary evaporator to give a
residue that was purified by column chromatography on silica gel. Elution of the column with n-
hexane gave 6-phenyl-6’-(2-chlorophenyl)fulvene, 5a (16 mg, 0.06 mmol, 14% yield) as a pale
orange oil, 1H-NMR (CDCl3): δ 5.95 (bdt, 1H), 6.47 (bdt, 1H), 6.52 (bdt, 1H), 6.63 (bdt, 1H), 7.33-
7.40 (m, 9H).
Further elution of the column with n-hexane-AcOEt 60:40 v/v afforded pure 1a (85 mg, 0.19 mmol,
44% yield) as yellow solid, mp = 139-140°C; 1H-NMR (CDCl3): δ 3.87 (bs, 1H, CpH), 4.04 (s, 6H,
Cp’H and CpH), 4.25 (bs, 1H, CpH), 4.35 (bs, 1H, CpH), 6.44 (dd, J = 1.2 and 8.0, 1H, ArH), 7.13
(m, 2H, ArH and Im-H), 7.23 (bs, 1H, Im-H), 7.26 (dd, J = 1.2 and 8.0, 1H, ArH), 7.31 (dd, J = 1.6
and 8.4, 1H, ArH), 7.40-7.42 (m, 3H, ArH), 7.49-7.51 (m, 2H, ArH), 7.89 (s, 1H, Im-H); 13C-NMR
(CDCl3): δ 67.22 (CpH), 68.91 (CpH), 69.74 (Cp’H), 70.14 (CpH), 71.28 (C-q), 72.64 (CpH), 95.25
(Cp), 121.38 (Im-H), 126.72 (ArH), 127.27 (Im-H), 127.83 (ArH), 128.30 (ArH), 128.79 (Ar-H),
129.59 (ArH), 130.47 (ArH), 131.83 (Ar-H), 134.96 (Ar), 139.10 (Im-H), 140.18 (Ar), 142.24 (Ar).
HRMS calcd for C23H18ClFe [M-Im]+ (100% relative intensity) 385.04464, found 385.03906; calcd
for C26H21ClFeN2 [M]+ (SIM mode) 452.07426, found 452.07370.
4.8. Synthesis of 1-[(4-chlorophenyl)-1-phenyl-1-ferrocenylmethyl]-1H-imidazole, 1b
A solution of 3b (200 mg, 0.48 mmol) in anhydrous CH2Cl2 (20 mL) was reacted with imidazole
(325 mg, 4.78 mmol) and acetic acid (480 µl) for 5 days and the reaction mixture worked up as
above to give a residue that was purified by column chromatography on silica gel. Elution of the
column with n-hexane gave 6-phenyl-6’-(4-chlorophenyl)fulvene, 5b (20 mg, 0.08 mmol, 16%
1
yield) as a pale orange oil, H-NMR (CDCl3): δ 6.25-6.28 (m, 2H), 6.60-6.62 (m, 2H), 7.25-7.31
(m, 4H), 7.35-7.40 (m, 5H).
Further elution of the column with n-hexane-AcOEt 60:40 v/v afforded pure 1b (95 mg, 0.21 mmol,
44% yield) as a yellow solid, mp = 135-136°C; 1H-NMR (CDCl3): δ 3.96 (bs, 1H, CpH), 3.98 (bs,
1H, CpH), 4.00 (s, 5H, Cp’H), 4.32 (bs, 2H, CpH), 7.00-7.03 (m, 3H, 2 × ArH and 1 × Im-H), 7.09
(dd, J = 2.0 and 8.0, 2H, ArH), 7.11 (s, 1H, Im-H), 7.30 (d, J = 8.4, 2H, ArH), 7.33-7.35 (m, 3H,
13
ArH), 7.59 (s, 1H, Im-H); C-NMR (CDCl3): δ 68.51 (CpH), 68.68 (CpH), 69.62 (Cp’H), 70.53

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(CpH), 70.72 (C-q), 70.77 (CpH), 94.19 (Cp), 121.14 (Im-H), 127.86 (ArH), 127.96 (ArH), 127.98
(ArH), 128.34 (ArH), 128.48 (ArH), 129.95 (ArH), 134.08 (Ar), 138.77 (Im-H), 142.62 (Ar),
143.42 (Ar). HRMS calcd for C23H18ClFe [M-Im]+ (100% relative intensity) 385.04464, found
385.03898; calcd for C26H21ClFeN2 [M]+ (SIM mode) 452.07426, found 452.07371.
4.9. Human Cell Cultures
Human colorectal adenocarcinoma cell line HT29 (ATCC number: HTB-38), and human
mammary adenocarcinoma MCF-7 (ATCC number: HTB22) were cultured as previously
reported.37
4.10. Cell growth inhibition assay
Stock solutions of compounds 1a, 1b and 5-fluorouracil (5-FU, positive control) were prepared in
9:1 dimethylsulfoxide (DMSO)/ Dulbecco Modified Eagle Medium (DMEM). Cancer cell lines
(2.5-3.0 x 103 cells/0.33cm2) were plated in 96 well plates "Nunclon TM Microwell TM" (Nunc),
and incubated at 37 °C in the culture medium (DMEM + 10% fetal bovine serum). After 24 h, 10
µl of a solution of 1a, 1b or 5-FU at the suitable concentration were added to each culture well
containing 90 µl of culture medium, so that the final concentration of DMSO was 1%.
Concentrations of in the range 0.01 - 100µM (0.01, 0.1, 1.0, 10 and 100 µM) were tested.
Cells treated with 1% of DMSO were used as negative control. Microplates were incubated at
37 °C in humidified atmosphere of 5% CO2, 95% air for 3 days, and cytotoxicity was measured
with colorimetric assay based on the use of tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT).38 Optical densities were read on a multiwell scanning
spectrophotometer (PlateReader AF2200, Eppendorf, Milan, Italy), using a wavelength of 570 nm.
Each value was the average of 6-8 wells. The GI50 value was calculated according to NCI39 thus,
GI50 is the concentration of test compound when 100 x (T - T0)/(C - T0) = 50 (where T is the optical
density of the test well after a 72 h period of exposure to test compound, T0 is the optical density at
time zero, C is the DMSO control optical density). The cytotoxicity effect was calculated according
to NCI when the optical density of treated cells was lower than the T0 value using the following
formula: 100 x (T-T0) /T0.
Acknowledgements. Training grant (Ussia M.) from FSE PO 2007-2013 (avviso 1/2012 Regione
Sicilia) is gratefully acknowledged. Thanks are also due to Dr. Giuseppe Di Natale, Institute of
Biostructure and Bioimaging of CNR, for helpful assistance in HR-ESI-MS spectra.
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The ferrocenyl analogue of clotrimazole and an isomeric compound were synthesized
The ferrocenyl derivatives were spectroscopically characterized
The key synthetic step involves nucleophilic substitution of a tertiary alcohol
Compounds were tested on two cancer cell lines for antiproliferative activity