Trapping para-quinone methide intermediates with ferrocene: Synthesis and preliminary biological evaluation of new phenol-ferrocene conjugates

Article abstract of DOI:10.3390/molecules23061335

The reaction of para-hydroxybenzyl alcohols with ferrocene in the presence of a catalytic amount of InCl₃ provided ferrocenyl phenol derivatives, an interesting class of organometallic compounds with potential applications in medicinal chemistry. This transformation exhibited a reasonable substrate scope delivering the desired products in synthetically useful yields. Evidence of involvement of a para-quinone methide intermediate in this coupling process was also provided. Preliminary biological evaluation demonstrated that some of the ferrocene derivatives available by this methodology exhibit significant cytotoxicity against several cancer cell lines with IC₅₀ values within the range of 1.07–4.89 μM.

Full text of DOI:10.3390/molecules23061335

molecules
Article
Trapping para-Quinone Methide Intermediates with
Ferrocene: Synthesis and Preliminary Biological
Evaluation of New Phenol-Ferrocene Conjugates
Silvia González-Pelayo 1, Enol López 1, Javier Borge 2 ID , Noemí de-los-Santos-Álvarez 3 and
ID
Luis A. López 1,
*
1
Departamento de Química Orgánica e Inorgánica and Instituto Universitario de Química Organometálica
“Enrique Moles”, Universidad de Oviedo, Julián Clavería 8, 33006 Oviedo, Spain;
gonzalezpelayos@gmail.com (S.G.-P.); enol.lopezh@gmail.com (E.L.)
Laboratorio de Compuestos Organometálicos y Catálisis (Unidad Asociada al CSIC), Centro de Innovación
en Química Avanzada (ORFEO-CINQA) and Departamento de Química Física y Analítica, Universidad de
Oviedo, Julián Clavería 8, 33006 Oviedo, Spain; jborge@uniovi.es
2
3
Departamento de Química Física y Analítica, Universidad de Oviedo, Julián Clavería 8, 33006 Oviedo, Spain;
santosnoemi@uniovi.es
*
Correspondence: lalg@uniovi.es; Tel.: +34-985-106-223
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 9 May 2018; Accepted: 29 May 2018; Published: 1 June 2018
Abstract: The reaction of para-hydroxybenzyl alcohols with ferrocene in the presence of a catalytic
amount of InCl3 provided ferrocenyl phenol derivatives, an interesting class of organometallic
compounds with potential applications in medicinal chemistry. This transformation exhibited a
reasonable substrate scope delivering the desired products in synthetically useful yields. Evidence
of involvement of a para-quinone methide intermediate in this coupling process was also provided.
Preliminary biological evaluation demonstrated that some of the ferrocene derivatives available by
this methodology exhibit significant cytotoxicity against several cancer cell lines with IC50 values
within the range of 1.07–4.89 µM.
Keywords: ferrocene; phenol; para-quinone methides; cytotoxic activity
1. Introduction
Since the discovery of ferrocene in the 1950s [1,2], the interest in this organometallic compound
has not declined. In fact, its chemistry remains one of the most active areas of research. Very likely,
this enduring interest resides in the fact that many functionalized ferrocene derivatives display a wide
number of applications in a diverse range of fields [
demonstrated the potential of some ferrocene derivatives in medicinal chemistry [10
some ferrocene-containing phenols have proved to be of great interest in cancer therapeutics because of
their antitumoral activity [12 16]. Among them, a family of ferrocene analogues of hydroxytamoxifen,
the so-called ferrocifens (Figure 1a), have been the subject of in-depth investigations showing
exceptional cytotoxic activities against some types of breast cancer [17 19]. The mode of action of
3–
9]. For example, recent investigations have
,
11]. Particularly,
–
–
these organometallic drug candidates has been elucidated by electrochemical and chemical oxidation
methods. According to these studies, ferrocenyl quinone methides have been suggested to play a key
role in the antiproliferative activity [20–24]. The antitumoral activity of some unconjugated bisphenol
derivatives of ferrocene (Figure 1b) has also been evaluated [25,26].
Molecules 2018, 23, 1335; doi:10.3390/molecules23061335
www.mdpi.com/journal/molecules

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Figure 1. Phenol-ferrocene conjugates: (a) Ferrocifen family; (b) ferrocene bisphenol derivatives;
(c) ortho-substituted ferrocenyl phenols previously developed in our group; (d) para-substituted
ferrocenyl phenols reported in this study.
In connection with our studies on C–H bond functionalization of ferrocene based on the trapping
of highly electrophilic species [27 29], we have recently described the trapping of ortho-quinone
–
methide intermediates with ferrocene [30]. Interestingly, some of the ferrocene-containing monophenol
derivatives available by this methodology (Figure 1c) display remarkable cytotoxic activity against
various cancer cell lines.
In order to elucidate the structural requirements for cytotoxicity and, eventually, to identify more
bioactive derivatives, we decided to develop a synthetic methodology for the synthesis of the isomeric
para-substituted ferrocenylphenol analogues (Figure 1d). Herein, we report the results of this study;
specifically, we describe the generation of para-quinone methide intermediates and their trapping
with ferrocene. Preliminary biological evaluation of some of the functionalized ferrocene derivatives
prepared in this study is also disclosed.
2. Results and Discussion
The present study was carried out using easily available p-hydroxybenzyl alcohols 1a
in Figure 2.
–l outlined
Figure 2. Starting p-hydroxybenzyl alcohols 1 used in this work.
For our initial study, p-hydroxybenzyl alcohol 1a was chosen as the model substrate (Scheme 1).
On the basis of our previous investigations in the ortho series, InCl3 in dichloroethane (DCE) was
selected as the catalytic system. Pleasingly, we found that heating a mixture of 1a (1 equiv.), ferrocene
◦
(2
, 3 equiv.), and InCl3 (10 mol%) in DCE at 60 C led to complete disappearance of the starting
p-hydroxybenzyl alcohol after 2 h (checked by thin layer chromatography, TLC). Chromatographic
purification (SiO2, 5:1 hexane ethyl acetate) provided the desired functionalized ferrocene derivative
3a in a remarkable 75% yield. Interestingly, under otherwise similar conditions, benzydryl alcohol 1a’
was found to be a fruitless reaction partner, thus demonstrating the key role of the phenolic OH group
in the reaction course [31,32].

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Scheme 1. Trapping of p-quinone methides with ferrocene: proof of concept. DCE: dichloroethane.
Ferrocenyl phenol 3a was characterized by Nuclear Magnetic Resonance (NMR) spectroscopy.
Moreover, crystals of compound 3a were obtained from diffusion of pentane into a dichloromethane
solution at
−
20 ◦C and its molecular structure in the solid state has been determined by single-crystal
X-ray diffraction (Figure 3 and Appendix A) [33
,34]. The electrochemical behavior of compound 3a
was investigated by cyclic voltammetry (Appendix B).
Figure 3. X-ray structure of ferrocene derivative 3a. Thermal ellipsoids are drawn at the 30% probability
level. Hydrogen atoms are excluded, except those bonded to C7 (H7) and O4 (H4O).
With suitable reaction conditions in hand (10 mol% of InCl3, DCE as solvent, 60 ◦C), a
variety of p-hydroxybenzyl alcohols were then evaluated for their suitability for this C–H bond
functionalization process (Table 1). First, some p-hydroxybenzyl alcohols substituted with various aryl
groups were investigated (entries 2–5). As shown, all three isomeric 4-[hydroxy(tolyl)methyl]phenol
derivatives 1b
functionalized ferrocene derivatives 3b
–
d
(R = tolyl) served as suitable reaction partners for this process furnishing the desired
in acceptable isolated yields (51–82%, entries 2–4). Similarly,
–
d
para-methoxy substituted substrate 1e (R = p-MeOC6H4) delivered the corresponding product 3e in
moderate isolated yield (48%, entry 5).

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Table 1. InCl3-catalyzed reaction of p-hydroxybenzyl alcohols 1 and ferrocene (2).
Entry
Substrate
R
3
Yield (%) a
1
2
3
4
5
6
7
8
9
1a
1b
1c
1d
1e
1f
1g
1h
1i
C6H5
o-MeC6H4
m-MeC6H4
p-MeC6H4
p-MeOC6H4
Me
3a
3b
3c
3d
3e
3f
3g
3h
3i
75
76
51
82
48
62
60
42
62
43
44
64
Et
n-Bu
i-Pr
t-Bu
Allyl
H
10
11
12
1j
1k
1l
3j
3k
3l
a
Isolated yield after chromatographic purification.
Next, substrates 1f–j with alkyl groups in the benzylic position were tested (entries 6–10). As
shown, primary, secondary and tertiary alkyl groups were well tolerated delivering functionalized
ferrocene derivatives 3f–j in moderate yields (42–62%).
This transformation was compatible with unsaturated functional groups in the benzylic position
as demonstrated by the synthesis of ferrocene derivative 3k in moderate yield when substrate 1k
(R = allyl) was subjected to the standard reaction conditions. Finally, we found that the parent
p-hydroxybenzyl alcohol 1l (R = H) is also a viable substrate affording the desired product 3l in 64%
yield (entry 12).
Next, to provide further evidence for the involvement of p-quinone methide intermediates
in the present coupling, we performed an experiment with a stable p-quinone methide. Thus,
4-benzylidene-2,6-di-tert-butylcyclohexa-2,5-dienone (
standard reaction conditions (10 mol% of InCl3, DCE, 60 C). However, a very low conversion
4
) an◦d ferrocene (
2) were subjected to the
was observed after 24 h very likely due to steric hindrance by the tert-butyl groups. Gratifyingly,
performing the reaction in toluene at 100 ◦C enabled the preparation of ferrocene derivative
yield (Scheme 2). Notably, in the absence of InCl3, no reaction occurred at all.
5 in 80%
Scheme 2. InCl3-catalyzed reaction of ferrocene and stable p-quinone methide 4.

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Based on these control experiments and on previous related literature precedents, a mechanistic
proposal for the reaction of hydroxybenzyl alcohols and ferrocene ( ) is outlined in Scheme 3. In the
present process, the Lewis acid is proposed to play a dual role. Firstly, it would promote the generation
of the key quinone methide intermediate through dehydration of hydroxybenzyl alcohol . Subsequent
activation of the quinone methide by Lewis acid complexation would provide intermediate . This
1
2
1
I
intermediate, with a high electrophilic character at the exocyclic C=C bond, may be involved in a
Friedel-Crafts type electrophilic aromatic substitution [35]. Indeed, 1,6-addition of ferrocene would
provide the
σ-complex intermediate II, which would evolve to the final product with regeneration of
the catalyst [36].
Scheme 3. Mechanism for the InCl3-catalyzed reaction of phenol derivatives 1 and ferrocene (2).
Some of the functionalized ferrocene derivatives prepared were evaluated for their cytotoxicity
against several cancer cell lines (Table 2). In this preliminary study, ferrocene derivatives 3a and 3g
were identified as the most active ones [37]. For example, 3a displayed significant toxicity against
A2780 ovarian cancer cell line (IC50 of 1.07
ortho-isomer (IC50 of 2.68 M), ferrocene derivative 3a has superior characteristics. Ferrocene 3g also
displayed toxicity against this cell line (IC50 of 2.23 M), although somewhat lower than that found
for the ortho-analogue (IC50 of 1.86 M). We have also studied the cytotoxicity profile of ferrocene
derivatives 3a and 3g against A549 lung cancer cells. Both derivatives exhibited moderate cytotoxicity
with IC50 values of 3.55 and 4.89 M, respectively. These values are comparable to that previously
µM). Compared with the value previously reported for the
µ
µ
µ
µ
measured for the ortho-isomers (IC50 of 2.77 and 5.96 µM, respectively).
Table 2. IC50 [µM] values for selected ferrocenyl compounds on different cell lines a.
3a
3g
A2780
A549
1.07
3.55
2.23
4.89
a
Measured after 72 h of culture.

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3. Materials and Methods
3.1. General
NMR spectra were recorded at room temperature in CDCl3 on a Bruker DPX-300 or Bruker
AVANCE-300 MHz instruments (Bruker, Billerica, MA, USA). Chemical shifts are given in ppm relative
to TMS (1H, 0.0 ppm) or CDCl3 (13C, 77.0 ppm). High-resolution mass spectra were determined on a
VG Autospec M mass spectrometer (Waters Corporation, Milford, MA, USA). Cyclic voltammetric
studies were performed using a µ-AutoLab type II equipped with GPES 4.9 software (EcoChemie,
Utrecht, The Netherlands). All measurements were carried out using a conventional three electrode
system in phosphate saline buffer (pH 7.4). A modified carbon paste acted as the working electrode and
a Pt wire as a counter electrode. All potentials were referred to a Ag|AgCl|KCl(sat) reference electrode.
Experiments were carried out under nitrogen using standard Schlenck techniques.
1,2-Dichloroethane was distilled from CaH2. Toluene was distilled from sodium-benzophenone
ketyl prior to use. TLC was performed on aluminum-backed plates coated with silica gel 60 with F254
indicator. Flash column chromatography was carried out on silica gel (230–240 mesh). The solvents
used in column chromatography, hexane and ethyl acetate, were obtained from commercial suppliers
and used without further purification.
p-Hydroxybenzyl alcohols 1a
the corresponding Grignard reagents following a literature procedure [38]. p-Hydroxybenzyl
alcohol 1l was obtained by the reaction of 4-hydroxybenzaldehyde with NaBH4 39].
4-Benzylidene-2,6-di-tert-butylcyclohexa-2,5-dienone ( ) was prepared from 2,6-di-tert-butylphenol
–k were prepared by reaction of 4-hydroxybenzaldehyde with
[
4
and benzaldehyde according to a literature procedure [40]. Ferrocene (2) was commercially available
and used without further purification.
3.2. General Procedure for the Synthesis of Ferrocene Derivatives 3a–l
InCl3 (4.4 mg, 0.02 mmol, 10 mol%) was added to a solution of p-hydroxybenzyl alcohols
0.2 mmol) and ferrocene (111.6 mg, 0.6 mmol) in 1,2-dichloroethane (2 mL). The mixture was stirred
at 60 ◦C for 2–14 h (disappearance of
checked by TLC). The solvent was removed under reduced
1
(
2
1
pressure and the resulting residue was purified by flash chromatography (silica gel, mixtures of
hexanes/ethyl acetate). Two fractions were collected. The first fraction was unreacted ferrocene and
the second one was the corresponding functionalized ferrocene derivative 3. Crystals of compound 3a
suitable for X-ray analysis were obtained from diffusion of pentane into dichloromethane at
Copies of 1H- and 13C-NMR spectra are provided in the Supplementary Materials.
−
20 ◦C.
◦
1
4-[(Ferrocenyl)(phenyl)methyl]phenol (3a): yellow solid; melting point (m.p.) 63–64 C; H-NMR
300 MHz, CDCl3): 4.02 (s, 2H, Cp), 4.05 (s, 5H, Cp), 4.19 (s, 2H, Cp), 4.83 (s, 1H, OH), 5.13 (s,
1H, CH), 6.76 (d, J = 8.1 Hz, 2H, Ar), 7.07 (d, J = 8.1 Hz, 2H, Ar), 7.19–7.33 (m, 5H, Ar); 13C-NMR
(
(
75 MHz, CDCl3): 51.0 (CH), 67.59 (CH), 67.63 (CH), 68.7 (CH), 92.0 (C), 114.9 (CH), 126.1 (CH), 128.1
(CH), 128.7 (CH), 129.9 (CH), 137.6 (C), 145.3 (C), 153.7 (C); HRMS (EI) calculated for [C23H20FeO]+
(M+): 368.0858, found 368.0856.
◦
1
4-[(Ferrocenyl)(2-methylphenyl)methyl]phenol (3b): yellow solid; m.p. 66–67 C; H-NMR (300 MHz,
CDCl3): 2.25 (s, 3H, CH3), 3.81 (s, 1H, Cp), 4.07 (s, 5H, Cp), 4.16–4.12 (m, 1H, Cp), 4.20–4.16 (m, 1H,
Cp), 4.76 (s, 1H, Cp), 5.25 (s, 1H, CH), 6.78 (d, J = 8.6 Hz, 2H, Ar), 6.96 (s, 1H, OH), 7.09–7.14 (m, 6H,
Ar); 13C-NMR (75 MHz, CDCl3): 20.3 (CH3), 47.9 (CH), 67.4 (CH), 68.5 (CH), 68.8 (CH), 69.1 (CH), 69.9
(CH), 93.4 (C), 115.1 (CH), 125.9 (CH), 126.5 (CH), 128.6 (CH), 130.5 (CH), 130.9 (CH), 135.5 (C), 136.0
(C), 144.7 (C), 154.1 (C); HRMS (EI) calculated for [C24H22FeO]+ (M+): 382.1015, found 382.1009.
◦
1
4-[(Ferrocenyl)(3-methylphenyl)methyl]phenol (3c): yellow solid; m.p. 72–73 C; H-NMR (300 MHz,
CDCl3): 2.32 (s, 3H, CH3), 4.02–4.05 (m, 7H, Cp), 4.18 (m, 2H, Cp), 4.62 (s, 1H, OH), 5.06 (s, 1H, CH),
6.75 (d, J = 8.4 Hz, 2H, Ar), 7.00–7.17 (m, 6H, Ar); 13C-NMR (75 MHz, CDCl3): 21.5 (CH3), 50.9 (CH),

Molecules 2018, 23, 1335
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67.7 (CH), 68.9 (CH), 92.3 (C), 114.7 (CH), 120.5 (CH), 125.6 (CH), 126.8 (CH), 127.9 (CH), 129.4 (CH),
129.9 (CH), 137.5 (C), 145.2 (C), 153.7 (C); HRMS (EI) calculated for [C24H22FeO]+ (M+): 382.1015,
found 382.1011.
◦
1
4-[(Ferrocenyl)(4-methylphenyl)methyl]phenol (3d): yellow solid; m.p. 64–65 C; H-NMR (300 MHz,
CDCl3): 2.33 (s, 3H, CH3), 3.99 (m, 2H, Cp), 4.03 (s, 5H, Cp), 4.17 (m, 2H, Cp), 4.73 (s, 1H, OH), 5.08 (s,
1H, CH), 6.74 (d, J = 8.5 Hz, 2H, Ar), 7.04–7.11 (m, 6H, Ar); 13C-NMR (75 MHz, CDCl3): 21.4 (CH3),
51.0 (CH), 67.9 (CH), 69.1 (CH), 92.6 (C), 115.2 (CH), 128.9 (CH), 129.1 (CH), 130.2 (CH), 135.9 (C), 138.2
(C), 142.7 (C), 154.1 (C); HRMS (EI) calculated for [C24H22FeO]+ (M+): 382.1015, found 382.1009.
4-[(Ferrocenyl)(4-methoxyphenyl)methyl]phenol (3e): yellow oil; 1H-NMR (300 MHz, CDCl3): 3.80 (s, 3H,
OMe), 3.98 (s, 2H, Cp), 4.04 (s, 5H, Cp), 4.17 (s, 2H, Cp), 4.72 (s, 1H, CH), 5.06 (s, 1H, OH), 6.79–6.73
(m, 2H, Ar), 6.80–6.84 (m, 2H, Ar), 7.03–7.10 (m, 4H, Ar); 13C-NMR (75 MHz, CDCl3): 50.5 (CH), 55.7
(CH3), 68.0 (CH), 69.2 (CH), 92.9 (C), 113.8 (CH), 115.2 (CH), 130.0 (CH), 130.2 (CH), 138.1 (C), 138.3
(C), 154.1 (C), 158.2 (C); HRMS (EI) calculated for [C24H22FeO2]+ (M+): 398.0964, found 398.0972.
4-(1-Ferrocenylethyl)phenol (3f): yellow solid; m.p. = 95–96 ◦C; 1H-NMR (300 MHz, CDCl3): 1.57 (d,
J = 7.2 Hz, 3H, CH3), 3.90 (q, J = 7.2 Hz, 1H, CH), 4.09 (s, 1H, Cp), 4.10–4.18 (m, 8H, Cp), 4.63 (s, 1H,
OH), 6.74 (d, J = 8.6 Hz, 2H, Ar), 7.05 (d, J = 8.6 Hz, 2H, Ar); 13C-NMR (75 MHz, CDCl3): 23.1 (CH3),
39.3 (CH), 66.7 (CH), 67.3 (CH), 67.9 (CH), 68.2 (CH), 69.0 (CH), 95.1 (C), 115.4 (CH), 128.6 (CH), 140.4
(C), 153.9 (C); HRMS (EI) calculated for [C18H18FeO]+ (M+): 306.0702, found 306.0701. Ferrocene 3f is
a known compound; our characterization data match those previously reported in the literature [25].
4-(1-Ferrocenylpropyl)phenol (3g): yellow solid; m.p. = 87–88 ◦C; 1H-NMR (300 MHz, CDCl3): 0.85 (t,
J = 7.4 Hz, 3H, CH3), 1.67–1.83 (m, 1H, CH2), 2.07–2.14 (m, 1H, CH2), 3.41–3.46 (dd, J = 10.7 and 4.3 Hz,
1H, CH), 4.06 (s, 1H, Cp), 4.05–4.10 (m, 7H, Cp), 4.17 (s, 1H, Cp), 4.73 (s, 1H, OH), 6.78 (d, J = 8.5 Hz,
2H, Ar), 7.06 (d, J = 8.5 Hz, 2H, Ar); 13C-NMR (75 MHz, CDCl3): 12.7 (CH3), 30.0 (CH2), 47.0 (CH),
66.8 (CH), 66.9 (CH), 67.3 (CH), 67.4 (CH), 68.6 (CH), 94.8 (C), 114.9 (CH), 129.0 (CH), 137.8 (C), 153.6
(C); HRMS (EI) calculated for [C19H20FeO]+ (M+): 320.0858, found 320.0853.
4-(1-Ferrocenylpentyl)phenol (3h): yellow oil; 1H-NMR (300 MHz, CDCl3): 0.89 (t, J = 7.0 Hz, 3H, CH3),
1.16–1.40 (m, 4H, CH2), 1.70–1.82 (m, 1H, CH2), 2.01–2.08 (m, 1H, CH2), 3.53 (dd, J = 10.8 and 4.3 Hz,
1H, CH), 3.95 (s, 1H, Cp), 4.05–4.11 (m, 7H, Cp), 4.18 (s, 1H, Cp), 4.80 (s, 1H, OH), 6.76 (d, J = 8.3 Hz,
2H, Ar), 7.06 (d, J = 8.3 Hz, 2H, Ar); 13C-NMR (75 MHz, CDCl3): 14.1 (CH3), 22.7 (CH2), 30.2 (CH2),
36.8 (CH2), 45.1 (CH), 66.8 (CH), 66.9 (CH), 67.3 (CH), 67.4 (CH), 68.6 (CH), 95.1 (C), 114.9 (CH), 128.9
(CH), 138.1 (C), 153.6 (C); HRMS (EI) calculated for [C21H24FeO]+ (M+): 348.1171, found 348.1184.
◦
4-[1-(Ferrocenyl)(2-methyl)propyl]phenol (3i): yellow solid; m.p. = 95–96 C; 1H-NMR (300 MHz, CDCl3):
0.74 (d, J = 6.6 Hz, 3H, CH3), 0.92 (d, J = 6.6 Hz, 3H, CH3), 1.97–2.03 (m, 1H, CH), 3.08 (d, J = 8.7 Hz,
1H, CH), 3.94 (s, 5H, Cp), 4.13–4.27 (m, 4H, Cp), 4.74 (s, 1H, OH), 6.75 (d, J = 8.3 Hz, 2H, Ar), 7.05 (d,
J = 8.3 Hz, 2H, Ar); 13C-NMR (75 MHz, CDCl3): 21.7 (CH3), 22.2 (CH3), 53.1 (CH), 66.7 (CH), 67.2
(CH), 68.4 (CH), 69.3 (CH), 70.4 (CH), 94.7 (C), 114.9 (CH), 129.6 (CH), 137.8 (C), 153.5 (C); HRMS (EI)
calculated for [C20H22FeO]+ (M+): 334.1015, found 334.1012.
4-[1-(Ferrocenyl)(2,2-dimethyl)propyl]phenol (3j): yellow solid; m.p. = 105–106 ◦C; 1H-NMR (300 MHz,
CDCl3): 0.84 (s, 9H, CH3), 3.23 (s, 1H, CH), 3.73 (s, 5H, Cp), 4.07 (s, 1H, Cp), 4.14 (d, J = 6.2 Hz, 2H,
Cp), 4.24 (s, 1H, Cp), 4.93 (s, 1H, OH), 6.84 (d, J = 8.4 Hz, 2H, Ar), 7.27–7.29 (m, 2H, Ar); 13C-NMR
(
75 MHz, CDCl3): 28.7 (CH3), 35.2 (C), 57.3 (CH), 65.7 (CH), 68.0 (CH), 68.1 (CH), 68.5 (CH), 69.2 (CH),
72.1 (CH), 90.4 (C), 114.2 (CH), 132.0 (CH), 136.9 (C), 153.6 (C); HRMS (EI) calculated for [C21H24FeO]+
(M+): 348.1171, found 348.1182.

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4-[1-(Ferrocenyl)but-3-enyl]phenol (3k): yellow oil; 1H-NMR (300 MHz, CDCl3): 2.53–2.62 (m, 1H, CH2),
2.80–2.89 (m, 1H, CH2), 3.65 (dd, J = 10.5 and 4.7 Hz, 1H, CH), 4.08 (s, 1H, Cp), 4.13–4.10 (m, 7H, Cp),
4.19 (s, 1H, Cp), 4.67 (s, 1H, OH), 4.93–5.04 (m, 2H, =CH2), 5.76–5.77 (m, 1H, =CH), 6.76 (d, J = 8.6 Hz,
2H, Ar), 7.06 (d, J = 8.6 Hz, 2H, Ar); 13C-NMR (75 MHz, CDCl3): 12.7 (CH3), 30.0 (CH2), 47.0 (CH),
66.8 (CH), 66.9 (CH), 67.3 (CH), 67.4 (CH), 68.6 (CH), 94.8 (C), 114.9 (CH), 129.0 (CH), 137.8 (C), 153.6
(C); HRMS (EI) calculated for [C20H20FeO]+ (M+): 332.0858, found 332.0855.
4-(Ferrocenylmethyl)phenol (3l): yellow oil; 1H-NMR (300 MHz, CDCl3): 3.64 (s, 2H, CH2), 4.11 (s, 4H,
Cp), 4.15 (s, 5H, Cp), 4.84 (s, 1H, OH), 6.75 (d, J = 8.2 Hz, 2H, Ar), 7.02 (d, J = 8.2 Hz, 2H, Ar); 13C-NMR
(75 MHz, CDCl3): 45.2 (CH2), 77.6 (CH), 78.7 (CH), 78.8 (CH), 98.7 (C), 125.1 (CH), 139.6 (CH), 144.0
(C), 163.7 (C); HRMS (EI) calculated for [C17H16FeO]+ (M+): 292.0545, found 292.0524. Ferrocene 3l is a
known compound [25].
3.3. Synthesis of Ferrocene Derivative 5
InCl3 (4.4 mg,
0.02 mmol,
10 mol%) was added to
a
solution of
4-benzylidene-2,6-di-tert-butylcyclohexa-2,5-dienone
(111.6 mg, 0.6 mmol) in toluene (2 mL). The mixture was stirred at 100 C for 6 h (disappearance
4
(58.9 mg, 0.2 mmol) and ferrocene
2
◦
of
residue was purified by flash chromatography (silica gel, hexanes/ethyl acetate 5:1) to yield ferrocene
derivative
(76.9 mg, 80%) as a yellow oil; 1H-NMR (300 MHz, CDCl3): 1.44 (s, 18H, CH3), 3.98–3.99
4 checked by TLC). Then, the solvent was removed under reduced pressure and the resulting
5
(m, 1H, Cp), 4.01 (s, 5H, Cp), 4.03–4.04 (m, 1H, Cp), 4.16–4.17 (m, 1H, Cp), 4.91 (s, 1H, Cp), 5.06 (s, 1H,
CH), 5.09 (s, 1H, OH), 7.04 (s, 2H, Ar), 7.18–7.21 (m, 3H, Ar), 7.26–7.29 (m, 2H, Ar); 13C-NMR (75 MHz,
CDCl3): 30.0 (CH3), 34.4 (CH), 51.8 (CH), 67.3 (CH), 67.6 (CH), 68.6 (CH), 68.7 (CH), 68.8 (CH), 92.8
(C), 125.4 (CH), 125.8 (C), 127.9 (CH), 128.6 (CH), 135.1 (C), 135.4 (C), 143.8 (C), 151.9 (C); HRMS (EI)
calculated for [C31H36FeO]+ (M+): 480.2110, found 480.2124.
3.4. Cytotoxic Assays
Cell Counting Kit-8 (CCK-8) from Sigma-Aldrich (Madrid, Spain) was used according to the
protocol provided by the company. The A2780 and A549 cell lines were used in this preliminary study.
First, cell lines were cultured for 7 days in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% (v/v) fetal bovine serum (FBS). Then, cells were seeded into a 96-well flat-bottom culture plate
at a cell density of 500–2000 cells/well and incubated for 24 h in the same medium (DMEM/10% FBS).
After that, 10
µL of a solution of the corresponding ferrocene derivative at different concentrations were
added and the cells were incubated for 72 h. Then, 10
µL of the CCK-8 solution were added to each
well of the plate. After 2 h of incubation the absorbance at 450 nm was recorded using a BioTek ELx800
Absorbance Microplate Reader (BioTek, Bad Friedrichshall, Germany). Measurements were performed
in triplicate, and each experiment was repeated three times. The IC50 values (µm) were estimated by
treatment of the data obtained with the statistical program GraphPad Prism5 (version 5.04).
4. Conclusions
Guided by earlier work from our group, we have developed a convenient synthesis of
para-substituted phenol derivatives containing a ferrocenyl moiety. Salient features of our protocol
include (i) easy availability of the required starting materials, (ii) synthetically useful yields, and
(iii) mild reaction conditions. This C–H bond functionalization of ferrocene relies on the generation
of a para-quinone methide intermediate that, activated by Lewis acid complexation, would serve
as electrophilic partner in an aromatic electrophilic substitution. Preliminary biological evaluation
revealed that some of the ferrocene derivatives available by this protocol display significant cytotoxicity
against ovarian and lung cancer cell lines. Further studies aimed at the preparation of new ferrocene
derivatives with enhanced antiproliferative properties are being pursued in our laboratory.

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Supplementary Materials: Supplementary Materials available online: Copies of 1H- and 13C-NMR spectra, X-Ray
crystallography (printcif and checkcif).
Author Contributions: L.A.L. conceived the experiments; S.G.-P. and E.L. designed and performed the
experiments; J.B. performed the X-ray crystallographic study; N.d.-l.-S.-A. performed the voltammetry study;
L.A.L., J.B. and N.d.-l.-S.-A. wrote the paper.
Acknowledgments: Financial support from the Ministerio de Economía y Competitividad (MINECO, grants
CTQ2013-41511-P and CTQ2015-63567-R), Agencia Estatal de Investigación (AEI) and Fondo Europeo de
Desarrollo Regional (FEDER) (Grants CTQ2016-76840-R and CTQ2016-75986-P), and Principado de Asturias
(grants GRUPIN14-013 and GRUPIN14-006) is gratefully acknowledged. E.L. thanks the Principado de Asturias
for a predoctoral grant (Severo Ochoa Program). We also thank J.M. González for interesting discussions.
Conflicts of Interest: The authors declare no conflict of interest.
Appendix A. Crystal Data for Ferrocene Derivative 3a
Crystal Data for C23H20FeO (Mr = 368.24 g/mol):◦monoclinic, space group P21/n (No. 14), a =
15.662(1) Å, b = 6.0219(3) Å, c = 18.786(1) Å,
β µ(CuKα) =
= 96.990(6) , V = 1758.6(2) Å3, Z = 4, T = 299 K,
◦
◦
6.91 mm−1, Dx = 1.391 g/cm3, 8336 measured reflections (3.5 <
θ < 69.6 ), 3254 independent reflections,
2641 observed reflections (I > 2σ(I)), Rint = 0.045. Final R[F2 > 2σ(F2)] was 0.079 and wR(F2) = 0.255.
Appendix B. Electrochemical Study for Ferrocene Derivative 3a
Compound 3a was studied by cyclic voltammetry (CV). All potentials were referred to a
Ag|AgCl|KCl(sat) reference electrode. The ferrocene/ferrocenium couple (Ia/Ic) is clearly observed at
a formal potential, E◦0 = 0.352 V (Figure A1a). The anodic peak current is higher than the cathodic
one indicating that some ferrocenium ions diffuse from the carbon paste electrode to the bulk solution
due to the positive charge of the cation. Nucleation at potentials ~0.450 V points out to a dissimilar
electrochemical behavior with other structurally related compounds. When the potential is swept
up to +0.6 V, a second redox process (IIa/IIc) appears causing the decrease of Ia (Figure A1b). The
reduction of ferrocenium species (Ic) remains visible but progressively shifted towards less positive
potentials in subsequent scans. Process IIa at about +0.570 V is clear and well-shaped but process IIc
at 0.426 V is partially overlapped with Ic. Of note, a notably increase in the non-faradaic current is
observed. When the potential is extended to +1.3 V, additional oxidation reactions take place (see
the rising anodic current) probably associated to phenolic compounds. Only one reduction process
is observed as a result of Ic and IIc overlapping (Figure A1c). The potential of both oxidation and
reduction peaks shift to more extreme potentials which indicates that the process is irreversible. The
origin of the irreversibility might be the formation of non-conducting products on the electrode surface
that hinder the electron transfer. The appearance of resistance affecting the CV shape strongly supports
this explanation.

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Figure A1. Cyclic voltammetry of compound 3a incorporated to the carbon paste electrode in phosphate
saline buffer (pH 7.4). Scan rate = 50 mV/s. Potential scan from 0 V to +0.5 V; ( ) +0.6 (first scan, black;
a
second scan, blue) and +0.7 (first scan, green; second scan, red) (b) and +1.3 V (c).
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Sample Availability: Samples of the compounds are available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).