Synthesis, structural characterisation and biological activity of novel N-(ferrocenylmethyl)benzene-carboxamide derivatives

Article abstract of DOI:10.1016/j.jorganchem.2006.11.012

A series of N-(ferrocenylmethyl)benzene-carboxamide derivatives (4a-f) have been synthesised by coupling ferrocenylmethyl amine 3 with benzoic acid and various substituted fluorobenzoic acids using the standard 1,3-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt) protocol. All compounds were fully characterised using a combination of ¹H NMR, ¹³C NMR, ¹⁹F NMR, DEPT-135, ¹H-¹H COSY and ¹H-¹³C COSY (HMQC) spectroscopy and electrospray ionisation mass spectrometry (ESI-MS). The compounds 4a, 4d, 4e and 4f exhibited cytotoxic effects on the MDA-MB-435-S-F breast cancer cell line. Single crystal X-ray crystallographic data for 4d is also presented.

Full text of DOI:10.1016/j.jorganchem.2006.11.012

Journal of Organometallic Chemistry 692 (2007) 1327–1331
www.elsevier.com/locate/jorganchem
Synthesis, structural characterisation and biological activity of novel
N-(ferrocenylmethyl)benzene-carboxamide derivatives
a,b
c
d
a,d
ˆ
Paula N. Kelly , Adeline Pretre , Siobhan Devoy , Isobel O’Rielly
,
Rosaleen Devery a,d, Alok Goel a,b, John F. Gallagher , Alan J. Lough ,
b
e
a,b,
*
Peter T.M. Kenny
a
National Institute for Cellular Biotechnology, DCU, Dublin 9, Ireland
School of Chemical Sciences, Dublin City University, Dublin 9, Ireland
b
c
University of Nantes, Nantes, France
School of Biotechnology, DCU, Dublin 9, Ireland
Department of Chemistry, 80 St. George St., University of Toronto, Ont., Canada M5S 3H6
d
e
Received 31 August 2006; received in revised form 7 November 2006; accepted 7 November 2006
Available online 16 November 2006
Abstract
A series of N-(ferrocenylmethyl)benzene-carboxamide derivatives (4a–f) have been synthesised by coupling ferrocenylmethyl amine 3
with benzoic acid and various substituted fluorobenzoic acids using the standard 1,3-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzo-
1
triazole (HOBt) protocol. All compounds were fully characterised using a combination of H NMR, 13C NMR, 19F NMR, DEPT-135,
1H–1H COSY and 1H–13C COSY (HMQC) spectroscopy and electrospray ionisation mass spectrometry (ESI-MS). The compounds 4a,
4d, 4e and 4f exhibited cytotoxic effects on the MDA-MB-435-S-F breast cancer cell line. Single crystal X-ray crystallographic data for 4d
is also presented.
ꢀ 2006 Elsevier B.V. All rights reserved.
Keywords: Ferrocene; Bioorganometallic chemistry; Breast cancer; MDA-MB-435-S-F; X-ray crystal structure
1. Introduction
cological properties of the tamoxifen derivative, ferrocifen,
have been reported [8–10]. The incorporation of C–F
bonds is a recognised strategy often employed in the devel-
opment of pharmaceuticals [11]. The success of fluorine
substitution is evident from the large number of fluorinated
drugs approved by the FDA and currently being used as
anticancer, antidepressant, antiviral and anaesthetic
agents.
The synthesis, characterisation and cytotoxicity of a
series of N-(ferrocenylmethyl)fluorobenzene-carboxamide
derivatives which combine the ferrocene moiety with a sub-
stituent containing one or more C–F bonds is reported in
this communication. The parent compound ferrocenylm-
ethylamine 3 was synthesised from ferrocenecarboxalde-
hyde 1 via the ferrocene oxime 2 intermediate (Scheme 1)
[12].
We are interested in the synthesis of ferrocene deriva-
tives with a view to finding new cytotoxic agents to aid in
the fight against one of the most prolific diseases facing
medical science, cancer [1,2]. In the past two decades the
use of organometallic compounds in the treatment of can-
cer has been an active field of research. As early as 1984 the
antineoplastic properties of a variety of ferrocenium salts
was reported [3,4]. Several reviews on the bioorganometal-
lic chemistry and structural features of ferrocene have
recently been published [5–7]. More recently, the pharma-
*
Corresponding author. Address: School of Chemical Sciences, Dublin
City University, Dublin 9, Ireland. Tel.: +353 1 7005689; fax: +353 1
7005503.
E-mail address: peter.kenny@dcu.ie (P.T.M. Kenny).
0022-328X/$ - see front matter ꢀ 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2006.11.012

1328
P.N. Kelly et al. / Journal of Organometallic Chemistry 692 (2007) 1327–1331
O
O
HO
N
H
N
H2N
H
i
ii
iii
Fe
Fe
Fe
Fe
X
4a-f
1
2
3
Scheme 1. Synthesis of N-(ferrocenylmethyl)benzene-carboxamide derivatives (i) NH2OH Æ HCl, Pyridine, EtOH (ii) LiAlH4, THF (iii) DCC, HOBt,
Benzoic acid derivative, CH2Cl2, 48 h. 4a X = H, 4b X = 2-F, 4c X = 3-F, 4d X = 4-F, 4e X = 2-F and 6-F, 4f X = 2-F, 3-F, 4-F, 5-F and 6-F.
The condensation of ferrocenylmethylamine with ben-
zoic acid and fluorobenzoic acids in the presence of DCC
and HOBt yielded the corresponding N-(ferrocenylm-
ethyl)benzene-carboxamides 4a–4f as orange/red coloured
crystals. All compounds gave analytical and spectroscopic
data in accordance with the proposed structures. All of
the proton and carbon chemical shifts for compounds
4a–4f were unambiguously assigned by a combination of
ethyl-carboxamide moiety are relatively rare. We have pre-
viously reported single crystal X-ray structures of a number
of ferrocene benzoyl amino acid derivatives [17,18,20] and
a ferrocene benzoyl dipeptide [2]. Herein, we report the sin-
gle crystal structure of an N-(ferrocenylmethyl)benzene-
carboxamide derivative 4d. Molecular drawings using
ORTEP are shown in Fig. 1 and the crystallographic details
given in Section 2.
DEPT-135 and H–13C COSY (HMQC). The H NMR
and the 13C NMR spectra for the compounds showed
peaks in the ferrocene region characteristic of a mono-
substituted ferrocene moiety [13–20]. The protons in the
ortho position appear in the region d 4.24–4.25, whereas
those in the meta position occur in the range d 4.09–4.12.
The five protons of the unsubstituted Cp ring (g5-C5H5)
appear as a strong singlet in the range d 4.18–4.20. The pro-
tons of the methylene unit adjacent to the ferrocene moiety
appear as a doublet (J = 6.4 to 7.2 Hz) in the range d 4.18–
4.20 often overlapping with the singlet of the (g5-C5H5)
ring. The aromatic protons of the benzene ring appear in
the range d 7.45–7.87 for the 4a derivative. For the mono-
and difluorobenzene derivatives, the protons of the fluoro-
benzene rings exhibit 1H–19F coupling interactions and
appear as complex multiplets in the range d 7.28–7.94.
The amide protons appear between d 8.55 and 8.84 as trip-
lets in DMSO-d6. The 13C NMR spectra of compounds
4a–4f show signals typical of a monosubstituted ferrocene
moiety in the range d 67.2–86.7. The Cipso of the (g5-
C5H4) ring appears between d 85.9 and 86.7 which are in
good agreement with previous results [21]. This signal is
absent in the DEPT 135 spectra. The 13C–19F coupling
can be seen in the 13C NMR and DEPT-135 spectra of
the fluorinated compounds. The ESI mass spectra of com-
pounds 4a–4f revealed the formation of the cation adducts
due to sodium in addition to radical cation species, proton-
ated molecular ion species and potassium cation adducts.
In each case an intense fragment ion was observed at m/z
199 and is due to the [FcCH2]+ cation. Fragment ions cor-
responding to [M-65]+ were also noted in the spectra of the
4a and 4e derivatives. This corresponds to loss of the
unsubstituted (g5-C5H5) ring.
Red/orange block shaped crystals of compound 4d, of a
quality suitable for crystallographic determination, were
grown from dichloromethane. The compound 4d crystal-
lises in the orthorhombic space group P212121 (No. 19),
with one molecule per asymmetric unit. The principle inter-
action is an intermolecular amide–amide hydrogen bond-
ing interaction which is orientated along the a-axis
(Fig. 2). The carbon atoms of the two Cp rings of the fer-
1
1
A number of crystal structures of ferrocene derivatives
have been reported in the Cambridge Structural Database
(CSD); however, structures incorporating a ferrocenylm-
Fig. 1. A view of 4d using PLATON [22], with atomic displacement ellipsoids
depicted at the 30% probability level.

P.N. Kelly et al. / Journal of Organometallic Chemistry 692 (2007) 1327–1331
1329
two concentrations (1 lM and 10 lM) for the preliminary
study. A previous study with a ferrocene pyrazole had
shown significant cytotoxicity at concentrations as low as
10 lM in a MCF-7 breast cancer cell line [23]. The MDA-
MB-435-S-F cells were incubated for 3 days with the com-
pounds to be evaluated. Cell survival was established
through determination of the acid phosphatase activity of
surviving cells and growth inhibition calculated relative to
controls (untreated cells).
It can be seen from Fig. 1 that all compounds tested
exerted inhibitory effects on the growth of MDA-MB-
435-S-F cells. Overall, compound 4d was the most cyto-
toxic, with 41 3% inhibition achieved at 10 lM. The
remaining three compounds demonstrated relatively good
inhibition at the same concentration ranging from
27 5% to 37 4%.
Compound 4d was also tested over a wider range of con-
centrations (1–40 lM). At a concentration of 40 lM 67%
growth inhibition was recorded. The IC50 was found to
be in the range of 11–14 lM. The results demonstrate that
cytotoxicity increased significantly as the concentration of
compound 4d increased, resulting in a dose-dependant rela-
tionship (see Fig. 3).
This ease of synthesis and general stability of these new
compounds coupled with their encouraging cytotoxic activ-
ity against the hormone independent breast cancer cell line
MDA-MB-435-S-F clearly promotes further research in
this area. We are currently in the process of synthesising
other derivatives with the aim of improving cytotoxicity
and identifying derivatives with lower IC50 values.
Fig. 2. The primary N–H. . .O@C amide hydrogen bonding interactions in
4d showing the one-dimensional chain of rings (marked as , with graph
*
set R127) along [100] (symmetry operations: # = 1/2 + x, 3/2 ꢀ y, 2 ꢀ z
and $ = ꢀ1/2 + x, 3/2 ꢀ y, 2 ꢀ z).
rocene moiety are essentially eclipsed with the five C1n–
Cg1–Cg2–C2n torsion angles ranging from 1.95ꢁ (for the
C13/C23 pair) to 2.29ꢁ (for the C12/C22 pair). The Fe1–
C bond lengths for the substituted Cp ring are in the range
2.041(3)–2.053(2) and those for the unsubstituted Cp ring
are quite similar, being [2.034(2)–2.055(2)]. The C–C bonds
of the substituted Cp ring are in the range 1.415(4)–
1.434(3) while those for the unsubstituted Cp ring are
slightly shorter in the range 1.404(4)–1.415(4). The C1–
C11–Fe1 angle is 127.51(14)ꢁ. The ferrocenyl moiety lies
approximately orthogonal to the amide moiety, while the
angle between the amide plane and the phenyl ring is
9.43ꢁ. The key molecular geometry of the molecule is
described by the N1–C1-C11–C12 and the N1–C2–C31–
C32 torsion angles (74.61(3)ꢁ and 169.54(2)ꢁ, respectively).
The angle between the two Cp rings is calculated to be
2.96ꢁ. The C–C bonds of the fluorinated phenyl ring range
2. Experimental
2.1. General procedure for the synthesis of compounds 4a–f
2.1.1. Synthesis of N-(Ferrocenylmethyl)-4-fluorobenzene-
carboxamide 4d
Ferrocenylmethyl amine (0.44 g, 2.5 mmol) was added
to a stirred solution of 4-fluorobenzoic acid (0.33 g,
2.5 mmol), DCC (0.55 g, 2.5 mmol) and HOBt (0.35 g,
2.5 mmol) in CH2Cl2 (40 ml) at 0 ꢁC. After 30 min the tem-
perature was allowed to rise to room temperature and stir-
ring continued for 48 h. The precipitated N,N0-
dicyclohexylurea was removed by filtration and the solvent
removed in vacuo. The mixture was purified on a silica-gel
column using a mobile phase of hexane-ethyl acetate (2:1).
Recrystallisation form ethyl acetate-petroleum ether fur-
nished 4d as orange/red crystals (0.45 g, 54% yield). m.p.
150–152 ꢁC (uncorrected). UV–Vis kmax (435.0 nm,
e = 720); I.R. (KBr, cmꢀ1): mC@O 1644; 1H NMR
(400 MHz) d (DMSO-d6): 4.09 (2H, t, J = 1.6 Hz, g5-
C5H4 meta), 4.18–4.20 (7H, m, g5-C5H5 and CH2Fc),
4.25 (2H, t, J = 1.6 Hz, g5-C5H4 ortho), 7.28–7.35 (2H,
m, ArH), 7.92–7.95 (2H, m, ArH), 8.78 (1H, t,
J = 5.8 Hz, NH). 13C NMR (100 MHz) d (DMSO-d6):
38.5 (ꢀve DEPT), 67.7, 68.6, 68.7, 86.4, 115.5–115.7,
130.1–130.2, 131.2–131.3, 162.9, 165.3. 19F NMR
˚
from 1.369(4) to 1.394(3) A. The C@O (C2–O1) distance is
1.240(3) while the C2–N1 distance is 1.332(3), demonstrat-
ing significant double bond character.
The in vitro activity of four of the compounds (4a, 4d, 4e
and 4f) against the hormone independent MDA-MB-435-S-
F breast cancer cell line was evaluated using the acid phos-
phatase assay as previously described [22]. This colorimetric
end-point assay is an indirect measure of cytotoxicity which
evaluates the enzyme activity of cells after a given treatment
period. Acid phosphatase is an enzyme which dephospho-
rylates p-nitrophenyl phosphate substrate converting it to
p-nitrophenol which in the presence of strong alkali can
be quantified colorimetrically. These four compounds were
selected as a preliminary study of the cytotoxic properties of
this series of compounds. The compounds were chosen as a
representative sample each containing a different number of
fluorine substituents. The cells were treated with the N-
(ferrocenylmethyl)benzene-carboxamide compounds at

1330
P.N. Kelly et al. / Journal of Organometallic Chemistry 692 (2007) 1327–1331
Percentgrowth inhibition of Breast Cancer Cells following incubation
with
N
-(Ferrocenylmethyl)benzene-carboxamide Derivatives
60
50
40
30
20
10
0
1µm
10µm
4(a)
4(d)
4(e)
4(f)
Compound
Fig. 3. Bar chart illustrating % growth inhibition of the N-(ferrocenylmethyl)benzene-carboxamide compounds at concentrations of 1 lM and 10 lM,
n = 3.
(376 MHz) d (DMSO-d6): ꢀ34.5 (broad m). Anal. Calc. for
C18H16NOFFe requires: C, 64.12; H, 4.78; N, 4.15. Found:
C, 64.01; H, 4.85; N, 4.06%. Mass spectrum: found:
[M + Na]+ 360.1. C18H16NOFFeNa requires: 360.17.
Crystallographic data 4d: chemical formula C18H16-
and 10 mM sodium pyruvate and seeded at a density of
5 · 104 cells/ml in 96-well plates. After 24 hours incubation
in a 37ꢁC, 5% CO2 incubator 100 lL of medium containing
dilute solutions of the ferrocenyl compounds at concentra-
tions of 1 lM and 10 lM from stock solutions in ethanol
were added. Controls were supplemented with medium
containing ethanol (0.7%). Treatments were set up in six
well replicates. Cells were then incubated at 37ꢁC, 5%
CO2 for 3 days. Cell survival was evaluated using the acid
phosphatase assay. This assay was shown to be linear with
respect to cell number up to 5 · 104 cells/ml (5000 cells/
well). The cytotoxic conjugated linoleic acid (CLA) mixture
of isomers (NuChek-Prep, Elysian MN, USA) (0–40 lM)
[28–30] was used as a reference.
NOFFe, red/orange block, molecular weight 337.17 g molꢀ1
orthorhombic, space group P212121, unit cell dimensions
,
˚
˚
˚
a = 10.0181(2) A, b = 11.6928(2) A, c = 12.2586(2) A, V =
1435.97(4) A , Z = 4, density = 1.56 g cmꢀ3
,
l = 1.061
3
˚
mmꢀ1, extinction coefficient = 0.0051(8), BASF compo-
nent = 0.1429, 10539 reflections collected in the range 2.7–
27.5ꢁ, 3255 independent reflections, 2983 > 2r(I), 205 param-
eters, R factor = 0.0306, wR2 = 0.0653.
Crystal data were collected using KappaCCD Server
Software [24]. The structures were solved by direct methods
using SHELXS-97 [25] and refined using SHELXL-97 [26]. All H
atoms attached to C atoms were treated as riding atoms
using the SHELXL97 defaults for 150 K in the refinement
and the N–H H atom was allowed to refine freely with iso-
Acknowledgements
P.T.M.K. and P.N.K. thank the National Institute of
Cellular Biotechnology, DCU, for a studentship under
the Programme for Research in Third Level Institutions
(PRTLI, round 3, 2001–2006). A.J.L. thanks the University
of Toronto for funds in aid of research.
˚
tropic temperature factors to a bond length of 0.79(3) A.
Anisotropic temperature factors were used for all non-
hydrogen atoms. Molecules of 4d are not chiral, however,
it crystallises in the chiral space group P212121. The Flack
parameter was determined to be 0.141(17) in the final struc-
ture factor calculations and indicating a small but signifi-
cant fractional contribution from the inverted component
of a ‘racemic twin’. Subsequent refinement using the
TWIN/BASF SHELXL97 commands of (ꢀ1000ꢀ1000ꢀ1)
and 0.143 yielded a final molecular structure. Molecular
graphics were produced using PLATON [27].
Appendix A. Supplementary data
CCDC 619909 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free
of charge via http://www.ccdc.cam.ac.uk/conts/retriev-
ing.html, or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax:
(+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.jorganchem.
2006.11.012.
2.2. General procedure for in vitro cytotoxicity assays
MDA-MB-435-S-F breast cancer cells were grown in
RPMI medium supplemented with 10% foetal calf serum

P.N. Kelly et al. / Journal of Organometallic Chemistry 692 (2007) 1327–1331
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