The synthesis and structural characterization of novel N-meta-ferrocenyl benzoyl dipeptide esters: The X-ray crystal structure and in vitro anti-cancer activity of N-{meta-ferrocenyl)benzoyl}-l-alanine-glycine ethyl ester

Article abstract of DOI:10.1016/j.jorganchem.2006.09.057

A series of N-meta-ferrocenyl benzoyl dipeptide esters 2-5 have been prepared by coupling meta-ferrocenyl benzoic acid 1b to the dipeptide ethyl esters using the conventional 1,3-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt) protocol. The dipeptides employed in the synthesis were AlaGly(OEt) (2), AlaAla(OEt) (3), AlaLeu(OEt) (4) and AlaPhe(OEt) (5). The compounds were fully characterized by a range of NMR spectroscopic techniques, mass spectrometry (MALDI-MS, ESI-MS), and cyclic voltammetry (CV). In addition, the X-ray crystal structure and cytotoxicity of N-{meta-(ferrocenyl)-benzoyl}-l-alanine-glycine ethyl ester (2) towards lung cancer cells has been determined.

Full text of DOI:10.1016/j.jorganchem.2006.09.057

Journal of Organometallic Chemistry 692 (2007) 1292–1299
www.elsevier.com/locate/jorganchem
The synthesis and structural characterization of novel
N-meta-ferrocenyl benzoyl dipeptide esters: The X-ray crystal
structure and in vitro anti-cancer activity of
N-{meta-ferrocenyl)benzoyl}-L-alanine-glycine ethyl ester
a
b
Alok Goel a,b, David Savage , Steven R. Alley a,b, Paula N. Kelly a,b, Dermot O’Sullivan ,
c
a,b,
*
Helge Mueller-Bunz , Peter T.M. Kenny
a
School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland
National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland
b
c
Department of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland
Received 22 August 2006; received in revised form 25 September 2006; accepted 25 September 2006
Available online 6 October 2006
Abstract
A series of N-meta-ferrocenyl benzoyl dipeptide esters 2–5 have been prepared by coupling meta-ferrocenyl benzoic acid 1b to the
dipeptide ethyl esters using the conventional 1,3-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt) protocol. The dipep-
tides employed in the synthesis were AlaGly(OEt) (2), AlaAla(OEt) (3), AlaLeu(OEt) (4) and AlaPhe(OEt) (5). The compounds were
fully characterized by a range of NMR spectroscopic techniques, mass spectrometry (MALDI-MS, ESI-MS), and cyclic voltammetry
(CV). In addition, the X-ray crystal structure and cytotoxicity of N-{meta-(ferrocenyl)-benzoyl}-L-alanine-glycine ethyl ester (2) towards
lung cancer cells has been determined.
ꢀ 2006 Elsevier B.V. All rights reserved.
Keywords: Ferrocene; Bioorganometallic chemistry; ESI; CV; Dipeptides; X-ray; Cytotoxicity; Lung cancer
1. Introduction
gated with biologically important compounds due to its
stability, electrochemical properties, and ease of use [1,2].
Ferrocenyl derivatives with low oxidation potentials are
attracting increasing attention due to their ability to cata-
lyze the production of reactive oxygenated species (ROS),
under physiological conditions, that generates cytotoxic
effects [2]. Some ferrocene derivatives have shown cytotox-
icity against lung tumours [15].
In our laboratory, we have synthesized various ferrocene
derivatives incorporating natural amino acids and dipep-
tides [16–22]. We have incorporated three key moieties in
the synthesis of these unusual biological materials, namely:
(i) an electroactive core, (ii) a conjugated linker that lowers
the oxidation potential of the ferrocene moiety, and (iii) an
amino acid or peptide derivative that can interact with
other molecules via hydrogen bonding. The ferrocene
Bioorganometallic chemistry has recently developed as a
rapidly growing area, the main focus being the preparation
of peptide mimetic models, macromolecular assemblies for
molecular recognition, and anti-cancer drugs [1–13]. The
conjugation of redox active organometallic compounds to
biological assemblies has also allowed for the design of
materials for biomolecular sensors and switches [14]. The
organometallic compound ferrocene is a promising candi-
date for biological applications. Ferrocene can be conju-
*
Corresponding author. Address: School of Chemical Sciences, Dublin
City University, Glasnevin, 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.2006.09.057

A. Goel et al. / Journal of Organometallic Chemistry 692 (2007) 1292–1299
1293
moiety is linked to the peptide derivative via a disubstituted
benzoyl group. Herein, we report the synthesis and electro-
chemical properties of N-meta-ferrocenyl benzoyl dipeptide
esters. In addition, we present a molecular and crystal
structural study, and an anti-cancer activity study of N-
{meta-(ferrocenyl)-benzoyl}-L-alanine-glycine ethyl ester
(2).
is envisaged that the phenyl group of the dipeptide chain
on the meta position causes steric hindrance to the unsub-
stituted cyclopentadienyl ring and makes this compound
slightly unstable.
Compounds 2–5 were fully characterized by a range of
NMR spectroscopic techniques, cyclic voltammetry, and
mass spectrometry. Crystals of sufficient quality for X-
ray diffraction studies were also obtained for compound
2. However, it is important to note that the crystals decom-
posed if completely dried. Hence, crystals were kept in the
mother liquor until ready for crystallographic analysis.
Thus, it is not surprising that compound 2 co-crystallizes
with a molecule of ethyl acetate (vide infra).
2. Results and discussion
2.1. Synthesis
The conventional 1,3-dicyclohexylcarbodiimide (DCC),
1-hydroxybenzotriazole (HOBt) protocol was employed
in the preparation of the dipeptide ethyl esters and to also
couple meta-ferrocenyl benzoic acid (1b) to the dipeptides
AlaGly(OEt) (2), AlaAla(OEt) (3), AlaLeu(OEt) (4) and
AlaPhe(OEt) (5) as previously reported [18,20] and is out-
lined in Scheme 1. The compounds 2–5 were purified by
column chromatography, using 2:3 petroleum ether (40–
60 ꢁC): ethyl acetate as the eluant, to obtain yellow/orange
colored crystals. The yields obtained were in the 51–56%
range, and all compounds gave analytical and spectro-
scopic data in accordance to the proposed structures. Com-
pounds 2–4 were reasonably stable, however compound 5
underwent slow decomposition over a period of time. It
1
2.2. H and 13C NMR spectroscopic analysis
All the proton and carbon chemical shifts for com-
pounds 2–5 were unambiguously assigned by a combina-
1
1
tion of DEPT-135 and H–13C-COSY (HMQC). The H
and 13C NMR spectra for compounds 2–5 showed peaks
in the ferrocene region characteristic of a mono substituted
ferrocene benzoyl moiety [16–22]. The protons in the ortho
position of the (g5-C5H4) ring appear in the region d 4.62
to d 4.67 whereas the protons in the meta position occur
in the range d 4.17 to d 4.31. The (g5-C5H5) ring appears
in the region d 3.8 to d 4.0. The protons of the meta-disub-
i
+
H2N
Fe
Fe
O
1a
O
MeO
MeO
ii
Fe
iii
O
Fe
HN
O
HO
O
1b
2-5
NH
R
O
EtO
Scheme 1. Synthesis of N-meta-ferrocenyl benzoyl dipeptide esters 2–5. (i) NaNO2, HCl, 5 ꢁC; (ii) NaOH/MeOH; (iii) DCC, HOBt, triethylamine,
Ala-dipeptide ethyl esters.

1294
A. Goel et al. / Journal of Organometallic Chemistry 692 (2007) 1292–1299
stituted benzoyl group appear as a triplet, doublet, doublet
and singlet in the region d 7.18 to d 7.8. For example, in the
case of N-{meta-(ferrocenyl)-benzoyl}-L-alanine-glycine
ethyl ester 2, the aromatic protons are present as a triplet,
two doublets and a singlet at d 7.27, d 7.48, d 7.54, and d
7.8, respectively. The meta and ortho protons on the (g5-
C5H4) ring are present at d 4.31 and d 4.67. The L-alanine
methyl group is present as a doublet at d 1.48 and the ethyl
ester methyl group appears as a triplet at d 1.14.
The N-meta-ferrocenyl benzoyl dipeptide esters were not
amenable to electron ionization (EI) or chemical ionization
(CI) studies, therefore ESI was employed in the analysis of
compounds 2 and 4 whereas compound 3 and 5 were ana-
lyzed by MALDI. Both ionization techniques confirmed
the correct relative molecular mass for the compounds
and examination of the mass spectra revealed the presence
of intense radical-cation species. The formation of the rad-
ical-cation molecular ion species was further confirmed by
the detection of the sodium adducts [M+23]+ and potas-
sium adducts [M+39]+ for each of the compounds ana-
lyzed. Similar observations were made in the analysis of
related ferrocenyl benzoyl amino acid and peptide deriva-
tives. Fragment ions were not observed in the MALDI
spectra of compounds 3 and 5. Sequence specific fragment
ions were also not observed or were of low intensity in the
ESI mass spectra of compounds 2 and 4 and therefore tan-
dem mass spectrometry was employed to confirm the integ-
rity of the structures. Sequence specific ions were observed
in the MS/MS spectrum for compound 4 confirming that
the alanine residue was linked to the benzoyl spacer group.
Important product ions were present at m/z 261, m/z 289,
m/z 331 and m/z 359. The ions at m/z 261 and m/z 289 were
previously observed in the FAB, MALDI and ESI mass
spectra of related ferrocenyl benzoyl derivatives and are
due to the ferrocenylphenyl and ferrocenylbenzoyl sub-
units, respectively [16–19]. However, the expected a1 and
b1 product ions at m/z 332 and m/z 360 were not observed,
instead a1 ꢀ 1 and b1 ꢀ 1 product ions were observed at
m/z 331 and m/z 359, respectively. Accurate mass measure-
ment of the MS/MS spectrum for compound 4 gave an ele-
mental composition of C19H17N1O1Fe1 and a molecular
mass of m/z 331.0662 for the ion at nominal mass m/z
331. This corresponds to a ppm error of 0.8. This indicates
that a hydrogen atom has been lost during the fragmenta-
tion process. A similar observation was made in the analy-
sis of the related para derivatives [21,22].
The 13C NMR spectra of compounds 2–5 show signals
in the region d 66.8 to d 84.5 indicative of a monosubsti-
tuted ferrocene benzoyl subunit. The ipso carbon of the
(g5-C5H4) ring appears in a very narrow range of d 84.4
to d 84.5. This signal is absent in the DEPT 135 spectra.
The carbon atoms of the aromatic ring are non-equivalent
and therefore six signals are visible in the region d 124.5–
140.8. The quaternary carbons of the aromatic ring and
the methylene carbon atoms of derivatives 2–5 were identi-
1
fied by DEPT-135. A complete assignment of the H and
13C NMR spectra of N-{meta-(ferrocenyl)-benzoyl}-L-ala-
nine-glycine ethyl ester (2) is presented in Table 1.
2.3. Mass spectrometry
Since the introduction of soft ionization techniques such
as matrix assisted laser desorption ionization (MADLI)
and electrospray ionization mass spectrometry (ESI-MS),
a wide range of thermolabile and non-volatile compounds
can be subjected to mass spectrometric analysis [23,24].
Table 1
1H and 13C spectroscopic data for 2
12
13
4
2
11
1
5
3
14
17
19
18
O
Fe
16 15
H
23
20
N
O
N
21 22
2.4. Electrochemistry
H
6-10
24
O
O
1H NMR
13C NMR
HMQC
Compounds 2–5 exhibited quasi-reversible cyclic vol-
Site
tammograms, similar to ferrocene under the same condi-
1
84.5
tions, and E0 values were in 46–59 mV (vs Fc/Fc+)
0
2,3
4,5
6–10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
4.67
67
range. Lower oxidation potential values as compared to
4.31
3.8–4.0
69.8
70.1
the corresponding dipeptide derivatives lacking a benzoyl
+
moiety Fc-Ala-Ala-OMe, E0 = 230 mV (vs Fc/Fc ) [25];
0
140.8
7.48
7.27
7.54
124.5
129.0
129.8
Fc-Ala-Ph-OMe, E00 = 190 mV (vs Fc/Fc+) [26] are expli-
cable in terms of substituent effects. The benzoyl moiety
offers extended conjugation to the pi electrons of the Cp
rings and makes these derivatives easier to oxidize, thus
making them suitable as anti-cancer agents (vide infra).
134.2
167.8
7.8
125.1
4.74
1.48
49.5
18.8
2.5. X-ray crystallographic studies of 2
170.0
173.0
3.8–4.0
41.8
A number of crystal structures of ferrocene peptide con-
jugates have been reported in the Cambridge Structural
Database (CSD); however, structures incorporating a fer-
4.14
1.14
62
14.5

A. Goel et al. / Journal of Organometallic Chemistry 692 (2007) 1292–1299
1295
Table 3
Hydrogen bonds
C47
˚
C48
D–H. . .A
H. . .A (A)
DHA (ꢁ)
O8
N1–H1N1. . .O5
N2–H1N2. . .O6
N3–H1N3. . .O1
N4–H1N4. . .O2
1.96
1.99
1.97
2.03
164.3
151.1
164.1
145.3
O7
C46
C45
N4
C26
C27
O6
C44
C25
C29
Table 4
C28
Short contacts
O5
N3
Fe2
C32
C42
˚
˚
Contact
(Molecules)
Length (A)
Length-VdW (A)
C36
C43
C33
C37
C41
C35
O1. . .H38
(A. . .B)
(A. . .C)
(B. . .D)
(B. . .C)
(D. . .C)
2.38
2.51
2.70
2.51
2.44
ꢀ0.34
ꢀ0.21
ꢀ0.20
ꢀ0.21
ꢀ0.28
O3. . .H53A
C35. . .H49B
O6. . .H55A
O9. . .H56C
C38
C39
C34
C31
C40
C30
Fig. 1. Molecular drawing of B using ORTEP: displacement ellipsoids are
drawn at the 50% probability level.
rocene benzoyl moiety and peptides are relatively rare. We
have previously reported single crystal X-ray structures of
a number of ferrocene benzoyl amino acid derivatives
[16,17,19] and a ferrocene benzoyl dipeptide [22]. Herein,
we report a single crystal structure of another ferrocene
benzoyl dipeptide derivative 2. A molecular drawing of
molecule B using ORTEP is shown in Fig. 1 with selected
structural parameters such as torsion angles, hydrogen
bonds and short contacts listed in Tables 2–4 and the crys-
tallographic details given in the footnote.
N2
O6
O5
C41
2.5.1. Molecular and crystal structure study of 2
Compound 2 crystallizes in the orthorhombic space
group P212121 (no. 19). The unit cell has two crystallo-
graphically independent molecules of N-{meta-(ferroce-
nyl)benzoyl}-L-alanine-glycine ethyl ester (A and B) and
two crystallographically independent molecules of ethyl
acetate (C and D). Molecules A are packed along the crys-
tallographic c-axis and molecules B are packed along the a-
axis; ethyl acetate molecules C and D are packed along the
c and a-axis, respectively. Each A molecule forms four
intermolecular amideꢁ ꢁ ꢁamide hydrogen bonds with two
B molecules (and vice versa) and that generates a helical
structure along the b-axis with alternating B and A mole-
cules packed in a head to tail arrangement along their
own crystallographic axis (Fig. 2). The helix has a pitch
O1
N2
O5
Fig. 2. Hydrogen bonding interactions between molecules B and A; [meta-
ferrocenyl] phenyl groups are represented by their ipso-carbons only.
It is interesting to note that the corresponding para
derivative [22] and the ferrocene dipeptide conjugates lack-
ing the benzoyl moiety (Fc-Ala-Phe-OMe and Fc-Leu-Phe-
OMe) also form helical structures [25,26]; however, Fc-
Gly-Gly-OMe forms parallel b-sheet-like structures [27].
It is envisaged that the ferrocenyl benzoyl groups have a
minimal effect on the molecular assembly of these conju-
gates, and even a small group like the methyl group on
the peptide chain has a significant effect on the molecular
assembly in the solid state. The helical assembly in the Ala-
Gly(OEt) 2 derivative may have applications in recognition
of biomolecular species by electrochemical methods, as the
˚
of 16.7111(14) A, which is equivalent to the length of the
crystallographic b-axis.
Table 2
Torsion angles (ꢁ)
A
B
C6–C10–C11–C16
C30–C34–C35–C40
C20–N2–C21–C22
C44–N4–C45–C46
20.7(6)
25.9(7)
102.1(4)
65.0(5)

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A. Goel et al. / Journal of Organometallic Chemistry 692 (2007) 1292–1299
pendant ferrocenyl benzoyl group is redox active and can
provide an electrical signal.
125.00
100.00
75.00
50.00
25.00
0.00
In addition to hydrogen bonding interactions, molecules
˚
B and A also have a short contact O1. . .H38, 2.38 A; also
ethyl acetate molecules make short contacts with molecules
B and A and with each other. Hydrogen bonding interac-
tions are summarized in Tables 3 and 4 shows a list of
selected intermolecular contacts that stabilize the structure.
The cyclopentadienyl (Cp) rings in both molecules B
and A are nearly eclipsed and the centroids of the Cp rings
are equidistant from the iron core; the peptide chains are
directed towards their unsubstituted Cp rings and the ester
groups are pointing away from the unsubstituted rings
(transoid), similar to the L-alanine derivative [16]. The key
difference between molecules B and A is in the degree of
rotation around the bond between the methylene carbon
of the glycine subunit and the amide nitrogen of the alanine
subunit. The dihedral angle C20–N2–C21–C22 between the
substituents in molecule B is 65.0(5)ꢁ as compared to the
corresponding dihedral angle C44–N4–C45–C46 of
102.1(4)ꢁ in molecule A. The phenyl rings in both mole-
cules B and A are twisted out of the plane of the Cp rings,
with torsion angles C6–C10–C11–C16 and C30–C34–C35–
C40 of 20.7(6)ꢁ and 25.9(7)ꢁ, respectively. It is likely that
the rigid arrangement created by the hydrogen bonds
between B and A (vide supra) forces the phenyl group
out of the Cp ring’s plane.
0
10
20
30 40
50
60 70
80 90 100
Concentration (uM)
meta-FC-Bz-Ala-Gly-OEt
ortho-Fc-Bz-Ala-Gly-OEt
ortho-Fc-Bz-Gly-OEt
Fig. 3. Cytotoxicity of compound 2, the ortho analog of 2, and the
corresponding ortho-glycine derivative.
and their ability to catalyze the generation of reactive oxy-
genated species (ROS), under physiological conditions,
that can oxidatively modify cellular components (e.g.,
DNA), disturb the redox balance in the cell, and/or inter-
fere with the redox-related cellular signalling pathways.
Several mechanisms may be involved in the production of
ROS, including a Fenton-type reaction that generates
hydroxyl radicals from the superoxide dismutation prod-
uct, hydrogen peroxide [28]:
The amide C@O bond lengths in B and A are in the
FcðIIÞ ꢀ Bz ꢀ AG ꢀ OEt þ H2O2
˚
range of 1.243(4)–1.251(4) A as expected, whereas all ester
! FcþðIIIÞ ꢀ Bz ꢀ AG ꢀ OEtþÅOH þ OHꢀ:
˚
C@O bonds are of same length, 1.199(5) A. The amide
CO–NH bond lengths are similar in all the cases and are
Since, preliminary results imply some structure activity
relationship, it is envisaged that the peptide chain of
these derivatives may have a secondary mode of action.
The actual mechanism is not clear, however it is plausi-
ble that the lipophilic ferrocenyl benzoyl moiety anchors
the cell membrane and the peptide chain blocks the
openings of the channels in the cell membrane, leading
to cell death [29]; the extent of blocking may depend
on the chain length or steric factors, or both. These pre-
liminary results are encouraging and further studies are
in progress to elucidate the mechanism of action and
for the selection of a series that has maximum cytotoxic-
ity against lung cancer cells.
˚
in the 1.319(5)–1.346(5) A range. The amide groups are
twisted in all molecules with respect to the phenyl groups,
the torsion angles are in the 13.7(5)–14.9(6)ꢁ range. Twist-
ing of the amide groups is likely due to the rigid framework
of strong intermolecular hydrogen bonds present in the
structure. Twisting of the amide groups is not uncommon
and has been observed previously in rigid systems [26].
2.6. In vitro anti-cancer activity of 2
We had previously tested an ortho-ferrocenylbenzoyl
amino acid derivative of glycine for its in vitro anti-can-
cer activity towards lung cancer cells (H1299, highly
invasive/superinvasive; H1299 carboplatin resistant vari-
ant). This compound was found to be cytotoxic and
had IC50 value of 48 lM. Therefore other derivatives,
including compound 2 that represents the Ala-dipeptide
series, were evaluated for their anti-cancer activity
against lung cancer cell lines. Preliminary results show
that the cytotoxicity of compound 2 is ca. 2 times higher
than the ortho-glycine derivative, the IC50 value being
26 lM (RSD 20%); the corresponding ortho analog of
2 has an IC50 value of 21 lM (RSD 20%). The results
are depicted in Fig. 3.
3. Conclusion
A series of novel N-meta-ferrocenyl benzoyl dipeptide
esters 2–5 were synthesized and fully characterized. The
potential applications of these derivatives are in the anti-
cancer and the biosensor area. Preliminary in vitro bioassay
results show that these derivatives are cytotoxic and their
cytotoxicity is higher than the corresponding amino acid
derivatives. The helical molecular assembly of 2 coupled
with its low oxidation potential may be useful in sensing
biomolecular species and may have applications as materi-
als for biomolecular sensor devices.
The possible mechanism for their anti-cancer activity
originates from the low redox potential of these derivatives

A. Goel et al. / Journal of Organometallic Chemistry 692 (2007) 1292–1299
1297
4. Experimental
4.2. General procedure for the synthesis of the starting
materials
4.1. General procedures
4.2.1. meta-Ferrocenyl ethyl benzoate (1a)
All chemicals were purchased from Sigma/Aldrich and
used as received. Commercial grade reagents were used
without further purification; however, solvents were puri-
fied prior to use. Melting points were determined using a
Griffin melting point apparatus and are uncorrected. Infra-
red spectra were recorded on a Nicolet 405 FT-IR spec-
trometer and UV–Vis spectra on a Hewlett-Packard
8452 A diode array UV–Vis spectrophotometer. NMR
spectra were obtained on a Bruker AC 400 NMR spec-
trometer operating at 400 MHz for 1H NMR and
Concentrated hydrochloric acid (6 ml) was added with
intermittent cooling to a solution of ethyl-3-aminobenzoate
(3 g, 18.2 mmol) in 15 ml of water. A solution of sodium
nitrite (1.4 g, 20.3 mmol) in 15 ml of water was then added
slowly to this mixture with stirring, keeping the tempera-
ture below 5 ꢁC furnishing a pale yellow solution. The
resulting diazo salt was added to a solution of ferrocene
(3.8 g, 20.4 mmol) in diethyl ether (90 ml) and allowed to
react for 12 h. The reaction mixture was then washed with
water, the ether layer was dried over MgSO4, and the sol-
vent was removed in vacuo to yield the crude product.
The crude product was purified using column chromatog-
raphy {eluant, 2:3 petroleum ether (40–60 ꢁC): diethyl
ether} to obtain an orange solid 1a, m.p. 74–76 ꢁC; I.R.
mmax (KBr) 1719 cmꢀ1; UV–Vis kmax (MeCN): 354 (e
1
100 MHz for 13C NMR. The H and 13C NMR chemical
shifts (ppm) are relative to TMS and all coupling constants
(J) are in Hertz. Matrix assisted laser desorption ionization
(MALDI) mass spectra were obtained on a Bruker Ultra-
flex TOF/TOF mass spectrometer employing a nitrogen
laser at 337 nm. Electrospray ionization mass spectra were
performed on either a Bruker Esquire ion trap mass spec-
trometer or a Micromass Q-ToF Ultima quadrupole time
of flight mass spectrometer.
1
1289), 453 (e 240) nm. H NMR (400 MHz) d DMSO-
d6): 8.03 (1H, s, ArH), 7.84 (1H, d, J = 8 Hz, ArH), 7.79
(1H, d, J = 8 Hz, ArH), 7.46 (1H, t, J = 8 Hz, ArH), 4.84
{2H, s, ortho on (g5-C5H4)}, 4.40 {2H, s, meta on (g5-
C5H4)}, 4.34 (2H, q, J = 7.2 Hz, –OCH2CH3), 4.03 {5H,
s, (g5-C5H5)}, 1.34 (3H, t, J = 7.2 Hz, –OCH2CH3). 13C
NMR (100 MHz) d DMSO-d6): 165.8, 138.8, 131.4,
129.5, 129.2, 127.2, 125.8, 83.1, 68.6, 68.2, 67.0, 65.5 (ꢀve
DEPT), 13.3.
Cyclic voltammograms were recorded in acetonitrile
(Sigma-Aldrich), with 0.1 M tetraethylammonium perchlo-
rate (TEAP) as a supporting electrolyte, using a CH Instru-
ments electrochemical analyzer (Pico-Amp Booster and
Faraday Cage). The experiments were carried out at room
temperature. A three-electrode cell consisting of a glassy car-
bon working-electrode, a platinum wire counter-electrode
and an AgjAg+ reference electrode was used. The glassy car-
bon electrode was polished with 0.3 lm alumina followed by
0.05 lm alumina, between each experiment to remove any
surface contaminants. Sample solutions (1 mM) containing
tetraethylammonium perchlorate (0.1 mol, TEAP) were
prepared in acetonitrile. Typically, the sample solution
(5 mL) was pipetted into an electrochemical cell, the elec-
trodes were inserted into the receptor solution and the cyclic
voltammograms was recorded by scanning voltage in a pre-
4.2.2. meta-Ferrocenyl benzoic acid (1b)
Sodium hydroxide (0.3 g, 7.5 mmol) was added to meta-
ferrocenyl ethyl benzoate (2.4 g, 7.2 mmol) in a 1:1 mixture
of water/methanol and was refluxed for 3 h. Concentrated
HCl was added until pH 2 was reached. The solution was
allowed to cool and filtered to obtain an orange solid 1b,
m.p. 159–161ꢁC; I.R. mmax(KBr): 3450, 1688 cmꢀ1; UV–
1
Vis kmax (CH2Cl2): 350 (e 720), 436 (e 290) nm; H NMR
(400 MHz) d DMSO-d6): 13.2 (1H, s, –COOH), 8.03 (1H,
s, ArH), 7.80(1H, d, J = 8 Hz, ArH), 7.77 (1H, d,
J = 8 Hz, ArH), 7.43 (1H, t, J = 8 Hz, ArH), 4.84 {2H, s,
ortho on (g5-C5H4)}, 4.39 {2H, t, meta on (g5-C5H4)},
4.03 {5H, s, (g5-C5H5)}; 13C NMR (100 MHz) d DMSO-
d6): 167.8, 140.0, 131.2, 130.7, 129.1, 127.1, 126.5, 84.0,
69.8, 69.6, 68.1, 66.8.
defined range (e.g., 0.0–0.5 V) at a scan rate of 100 mV sꢀ1
.
A CV scan of a sample of ferrocene (1 mM) with tetraethy-
lammonium perchlorate (0.1 mol) in acetonitrile was also
recorded before each experiment to obtain E0 (Fc/Fc+),
0
and the E0 values obtained for the test samples were refer-
0
enced to the Fc/Fc+ couple.
Crystal data were collected at 100 (2) K using a Bruker
¨
4.3. General procedure for the synthesis of N-{meta-
(ferrocenyl)benzoyl} dipeptide esters (2)–(5)
SMART APEX CCD area detector diffractometer. A full
sphere of the reciprocal space was scanned by /–x scans.
Pseudo-empirical absorption correction based on redun-
dant reflections was performed by the program SADABS
[30]. The structures were solved by direct methods using
SHELXS-97 [31] and refined by full-matrix least-squares on
F2 for all data using SHELXL-97 [32]. All hydrogen atoms
were located in the difference Fourier map and allowed
to refine freely with isotropic temperature factors. Aniso-
tropic temperature factors were used for all non-hydrogen
atoms.
4.3.1. N-{meta-(ferrocenyl)-benzoyl}-L-alanine-glycine
ethyl ester (2)
L-Alanine-glycine ethyl ester hydrochloride (0.2 g,
0.95 mmol) was added to a solution of meta-ferrocenoyl
benzoic acid (0.3 g, 1.0 mmol), 1-hydroxybenzotriazole
(0.2 g, 1.5 mmol), triethylamine (0.5 ml), and dicyclohexyl-
carbodiimide (0.45 g, 2.1 mmol) in 50 ml of dichlorometh-
ane at 0 ꢁC. After 30 min, the temperature was raised to
room temperature and the reaction was allowed to proceed

1298
A. Goel et al. / Journal of Organometallic Chemistry 692 (2007) 1292–1299
for 48 h. The precipitated N,N0-dicyclohexylurea was
removed by filtration and the filtrate was washed with
water, 10% potassium hydrogen carbonate, 5% citric acid,
dried over MgSO4, and the solvent was removed in vacuo.
The product was purified by column chromatography {elu-
ant, 2:3 petroleum ether (40–60 ꢁC): ethyl acetate}. Recrys-
tallization from petroleum ether (40–60 ꢁC): ethyl acetate
furnished the title compound as a yellow solid (0.236 g,
51%). The crystals were of sufficient quality for0 an X-ray
diffraction study and had m.p. 151–153 ꢁC; E0 = 50 mV
4.3.3. N-{meta-(ferrocenyl)-benzoyl}-L-alanine-L-leucine
ethyl ester (4)
For the compound 4 L-alanine-L-leucine ethyl ester
hydrochloride (0.2 g, 0.75 mmol) was used as starting
material. The product was purified by column chromatog-
raphy {eluant 2:3 petroleum ether (40–60 ꢁC): ethyl ace-
tate}. Recrystallization from petroleum ether (40–60 ꢁC):
ethyl acetate furnished the title compound 0 as an orange
solid (0.203 g, 56%), m.p. 110–112 ꢁC; E0 = 46 mV (vs
20
Fc/Fc+); ½aꢂD ¼ ꢀ25ꢃ (c1.8, EtOH); UV–Vis kmax EtOH;
20
(vs Fc/Fc+); ½aꢂD ¼ ꢀ18ꢃ (c2, EtOH); UV–Vis kmax MeCN;
323 (e1430), 442 (e 410) nm; I.R. mmax(KBr): 3286, 2963,
1
234 (e 580), 445 (e 100) nm; I.R. mmax(KBr): 3300, 2937,
1750, 1637, 1537, 1450, 1180 cmꢀ1; H NMR (400 MHz)
1751, 1657, 1632, 1580, 1543, 1199 cmꢀ1
;
1H NMR
d DMSO-d6): 8.32 (1H, d, J = 7.6 Hz, –CONH–), 8.08
(1H, d, J = 7.6 Hz, –CONH–), 7.77 (1H, s, ArH), 7.50
(2H, t, J = 8 Hz, ArH), 7.18 (1H, t, J = 8 Hz, ArH), 4.65
{2H, t, J = 2 Hz, ortho on (g5-C5H4)}, 4.34 {1H, quint,
J = 6.4 Hz, –CH(CH3)}, 4.17 {2H, t, J = 1.6 Hz, meta on
(g5-C5H4)}, 3.97–4.05 [1H, m, CH{CH2CH(CH3)2}],
3.83–3.87 (2H, m, –OCH2CH3), 3.81 {5H, s, (g5-C5H5)},
1.24–1.52 [3H, m, CH{CH2CH(CH3)2}], 1.15 {3H, d,
J = 7.2 Hz, CH(CH3)}, 0.95 (3H, t, J = 7.2 Hz,
–OCH2CH3), 0.69 [3H, d, J = 6.4 Hz, CH{CH2CH(CH3)2}],
0.64 [3H, d, J = 6.4 Hz, CH{CH2CH(CH3)2}]; 13C NMR
(100 MHz) d (DMSO-d6): 173.1, 172.8, 166.3, 139.5,
134.5, 129.1, 128.6, 125.5, 124.7, 84.4, 69.8, 69.4, 66.8,
60.7 (ꢀve DEPT), 50.8, 48.9, 40.0 (ꢀve DEPT), 24.6,
23.1, 21.8, 18.2, 14.4.
(400 MHz) d (CDCl3): 7.80 (1H, s, ArH), 7.54 (1H, d,
J = 8 Hz, ArH), 7.48 (1H, d, J = 8 Hz, ArH), 7.27 (1H, t,
J = 8 Hz, ArH), 6.83–6.87 (2H, m, –CONH–), 4.74 {1H,
quint, J = 7.2 Hz, –CH(CH3)}, 4.67 {2H, s, ortho on (g5-
C5H4)}, 4.31 {2H, s, meta on (g5-C5H4)}, 4.14 (2H, q,
J = 7.2 Hz, -OCH2CH3), 3.80–4.00 {7H, m, (g5-C5H5),
–NHCH2CO–}, 1.48 {3H, d, J = 7.2 Hz, –CH(CH3)},
1.14 (3H, t, J = 7.2 Hz, –OCH2CH3); 13C NMR
(100 MHz) d (CDCl3): 173.0, 170.0, 167.8, 140.8, 134.2,
129.8, 129.0, 125.1, 124.5, 84.5, 70.1, 69.8, 67.1, 67.0, 62.0
(ꢀve DEPT), 49.5, 41.8 (ꢀve DEPT), 18.8, 14.5.
Analysis: found: C, 62.25; H, 5.85; N, 6.36.
C24H26N2O4Fe requires: C, 62.35; H, 5.67; N, 6.06.
Mass spectrum: found: [M+Na]+ 485.20.
C24H26N2O4FeNa requires: 485.12.
Analysis: found: C, 64.68; H, 6.67; N, 5.46.
C28H34N2O4Fe requires: C, 64.87; H, 6.61; N, 5.40.
Mass spectrum: found: [M]+ 518.1860.
C28H34N2O4Fe requires: 518.1868.
4.3.2. N-{meta-(ferrocenyl)-benzoyl}-L-alanine-L-alanine
ethyl ester (3)
For compound 3, L-alanine-L-alanine ethyl ester hydro-
chloride (0.2 g, 0.89 mmol) was used as a starting material.
The product was purified by column chromatography {elu-
ant, 2:3 petroleum ether (40–60 ꢁC): ethyl acetate}. Recrys-
tallization from petroleum ether (40–60 ꢁC): ethyl acetate
furnished the title compound as yellow needles. (0.224 g,
4.3.4. N-{meta-(ferrocenyl)-benzoyl}-L-alanine-L-
phenylalanine ethyl ester (5)
For compound 5, L-alanine L-phenylalanine ethyl ester
hydrochloride (0.2 g, 0.67 mmol) was used as a starting
material. The product was purified by column chromatog-
raphy ({eluant, 2:3 petroleum ether (40–60 ꢁC): ethyl ace-
tate}. Recrystallization from petroleum ether (40–60 ꢁC):
ethyl acetate furnished the title compound as an orange
53%), m.p. 58–60 ꢁC; E0 = 50 mV (vs Fc/Fc+);
0
20
½aꢂD ¼ ꢀ37ꢃ (c1.9, EtOH); UV–Vis kmax EtOH; 325 (e
1540), 443 (e 430) nm; IR mmax(KBr):3276, 2986, 2939,
1739, 1637, 1584, 1543, 1400, 1128 cmꢀ1
;
1H NMR
solid (0.203 g, 52%), m.p. 61–63 ꢁC; E0 = 59 mV (vs Fc/
0
20
(400 MHz) d (CDCl3): 7.83 (1H, s, ArH), 7.50–7.56 (2H,
m, ArH), 7.26 (1H, t, J = 8 Hz, ArH), 7.06 (2H, t,
J = 7.6 Hz, –CONH–), 4.78 {1H, quint, J = 7.2 Hz,
–CH(CH3)}, 4.62 {2H, t, J = 1.2 Hz, ortho on (g5-
C5H4)}, 4.50 [1H, quint, J = 7.2 Hz, –CH(CH3)], 4.26
{2H, t, J = 1.6 Hz, meta on (g5-C5H4)}, 4.14 (2H, q,
J = 7.2 Hz, –OCH2CH3), 3.96 {5H, s, (g5-C5H5)}, 1.49
{3H, d, J = 6.8 Hz, –CH(CH3)}, 1.37 {3H, d, J = 6.8 Hz,
–CH(CH3)}, 1.22 (3H, t, J = 7.2 Hz, –OCH2CH3); 13C
NMR (100 MHz) d (DMSO-d6): 173.1, 172.5, 167.6,
140.7, 134.3, 129.8, 128.9, 125.1, 124.6, 84.4, 70.1, 69.7,
67.1, 67.0, 62.0 (ꢀve DEPT), 49.6, 48.7, 19.4, 18.5, 14.5.
Analysis: found: C, 62.96; H, 6.13; N, 5.62.
Fc+); ½aꢂD ¼ ꢀ19ꢃ (c1.9, EtOH); UV–Vis kmax (EtOH);
327 (e 1440), 442 (e 390) nm; I.R. mmax(KBr): 3325, 2930,
1739, 1637, 1579, 1532, 1499, 1208 cmꢀ1
;
1H NMR
(400 MHz) d (CDCl3): 7.82 (1H, s, ArH), 7.58 (1H, d,
J = 8 Hz, ArH), 7.46 (1H, d, J = 8 Hz, ArH), 7.29 (1H, t,
J = 8 Hz, ArH), 7.10–7.12 (3H, m, ArH), 7.02–7.04 (2H,
m, ArH), 6.67 (1H, d, J = 7.2 Hz, –CONH–), 6.50 (1H,
d,
J = 7.2 Hz,
–CONH–),
4.78–4.80
[1H,
m,
–NHCH(CH3)], 4.63– 4.66 {3H, m, ortho on (g5-C5H4),
–NHCH(CH2Ph)}, 4.29 {2H, s, meta on (g5-C5H4)}, 4.14
(2H, q, J = 7.2 Hz, –OCH2CH3), 3.98 {5H, s, (g5-
C5H5)}, 3.08 {1H, dd, Jax = 2.0 Hz, Jab = 5.6 Hz,
CH(CH2Ph)}, 3.03 {1H, dd, Jmax = 2.0 Hz, Jab = 5.6 Hz,
CH(CH2Ph)}1.62 {3H, d, J = 6.4 Hz, –CH(CH3)}, 1.21
(3H, t, J = 7.2 Hz, –OCH2CH3); 13C NMR (100 MHz) d
(CDCl3): 172.2, 171.6, 167.5, 140.8, 136.1, 134.2, 129.8,
C25H28N2O4Fe requires: C, 63.04; H, 5.92; N, 5.88.
Mass spectrum: found: [M]+ 476.14.
C25H28N2O4Fe requires: 476.14.

A. Goel et al. / Journal of Organometallic Chemistry 692 (2007) 1292–1299
1299
[5] H. Fink, N.J. Long, A.J. Martin, G. Opromolla, A.J.P. White, D.J.
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(2001) 1008.
129.7, 129.0, 127.5, 125.1, 124.5, 84.5, 70.1, 69.8, 67.1, 67.0,
62.1 (ꢀve DEPT), 53.8, 49.6, 38.3 (ꢀve DEPT), 18.8, 14.5.
Analysis: found: C, 65.96; H, 5.85; N, 5.14.
C31H32N2O4Fe requires: C, 67.40; H, 5.84; N, 5.07.
Mass spectrum: found: [M]+ 552.169.
C31H32N2O4Fe requires: 552.171.
4.3.5. Crystallographic footnotes for 2
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Chem., Int. Ed. 45 (5) (2006) 751.
Crystallographic data: chemical formula C28H34N2-
O6Fe, formula weight 550.42 g molꢀ1, orange needle, crys-
tal size = 0.60 · 0.20 · 0.10 mm3, tetragonal, space group
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(2006) 348.
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Organomet. Chem. 690 (2005) 383.
˚
P43, unit cell dimensions a = 16.0466(14) A (a = 90ꢁ),
˚
˚
b = 16.7111(14) A (b = 90ꢁ), c = 20.3902(17) A (c = 90ꢁ),
3
V = 5467.8(8) A , Z = 8, density = 1.337 g cmꢀ3
,
l =
˚
0.595 mmꢀ1, 36839 reflections collected in the range
1.58–26.00ꢁ, 10588 independent reflections, 675 parame-
ters, R factor = 0.0604, wR2 = 0.1471, Flack parameter =
0.031(17).
5. Supplementary material
[18] D. Savage, N. Neary, G. Malone, S.R. Alley, J.F. Gallagher, P.T.M.
Kenny, Inorg. Chem. Commun. 8 (2005) 429.
[19] D. Savage, G. Malone, S.R. Alley, J.F. Gallagher, A. Goel, P.N.
Kelly, H. Mueller-Bunz, P.T.M. Kenny, J. Organomet. Chem. 691
(2006) 463.
[20] D. Savage, S.R. Alley, J.F. Gallagher, A. Goel, P.N. Kelly, P.T.M.
Kenny, Inorg. Chem. Commun. 9 (2006) 152.
[21] D. Savage, S.R. Alley, A. Goel, T. Hogan, Y. Ida, P.N. Kelly, L.
Lehmann, P.T.M. Kenny, Inorg. Chem. Commun. 9 (2006) 1267.
[22] A. Goel, D. Savage, S.R. Alley, T. Hogan, P.N. Kelly, S. Draper,
C.M. Fitchett, P.T.M. Kenny, J. Organomet. Chem. 691 (2006)
4686.
CCDC 616784 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free
of charge via www.ccdc.cam.ac.uk/conts/retrieving.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.
Acknowledgements
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D.S. thanks the Irish American Partnership and Dublin
City University for the funding of a studentship award
1999–2002. This research was partly supported by the Na-
tional Institute for Cellular Biotechnology under the Pro-
gramme for Research in Third Level Institutions (PRTLI,
round 3, 2001–2006).
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