Synthesis, characterization and in vitro anti-cancer activity of N-(ferrocenyl)benzoyl tri- and tetrapeptide esters

Article abstract of DOI:10.1016/j.ica.2009.01.021

N-ortho, N-meta and N-para-(ferrocenyl)benzoyl tri- and tetrapeptide esters (2-7) were prepared by coupling ortho, meta and para-ferrocenyl benzoic acids to the tri- and tetrapeptide ethyl esters of GlyGlyGly(OEt) and GlyGlyGlyGly(OEt) in the presence of

Full text of DOI:10.1016/j.ica.2009.01.021

Inorganica Chimica Acta 362 (2009) 2957–2961
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis, characterization and in vitro anti-cancer activity
of N-(ferrocenyl)benzoyl tri- and tetrapeptide esters
Alan J. Corry a, Áine Mooney a, Dermot O’Sullivan b, Peter T.M. Kenny a,b,
*
a School of Chemical Sciences, Dublin City University, Dublin 9, Ireland
b National Institute for Cellular Biotechnology, Dublin 9, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
N-ortho, N-meta and N-para-(ferrocenyl)benzoyl tri- and tetrapeptide esters (2–7) were prepared by cou-
pling ortho, meta and para-ferrocenyl benzoic acids to the tri- and tetrapeptide ethyl esters of GlyGly-
Gly(OEt) and GlyGlyGlyGly(OEt) in the presence of N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride and 1-hydroxybenzotriazole. The compounds were characterized by a range of NMR spec-
troscopic techniques, mass spectrometry and cyclic voltammetry. The anti-proliferative effects of the
ortho derivatives 2 and 5 were measured in vitro against H1299 lung cancer cells and both gave IC50 val-
Received 19 November 2008
Received in revised form 5 January 2009
Accepted 21 January 2009
Available online 31 January 2009
Keywords:
Ferrocene
Anti-cancer
Bioorganometallic chemistry
Peptides
ues greater than 50
activity, relative to N-(ferrocenyl)benzoyl amino acid and dipeptide derivatives.
Ó 2009 Elsevier B.V. All rights reserved.
lM. Therefore, extending the length of the peptide chain had a negative effect on
1. Introduction
redox potentials when compared to the corresponding ferrocenoyl
dipeptide derivatives. This fact can be explained in terms of substi-
tuent effects. The benzoyl moiety offers extended conjugation to
the pi electrons of the ferrocene rings making these derivatives eas-
ier to oxidise to the ferricenium species thus making them suitable
as anti-cancer agents. The novel ferrocenyl benzoyl dipeptide deriv-
atives were shown to be cytotoxic. An ortho-ferrocenylbenzoyl ami-
no acid derivative of glycine, N-{ortho-(ferrocenyl)-benzoyl}-
glycine ethyl ester was initially tested for its in vitro anti-cancer
activity towards H1299 lung cancer cells. This compound was
The metallocene ferrocene has several novel applications due
to its ease of derivatization, stability, spectroscopic properties
and redox activity. Research in the area of ferrocenyl derivatives
has focused on their potential as sensors, peptide mimetic models
and unnatural drugs [1–7]. Amino acids and peptides play diverse
roles in biological systems, hence the synthesis of N-ferrocenyl
and N-ferrocenoyl amino acid and peptide derivatives has been
extensively reported [8–21]. The medicinal application of ferro-
cene is currently an active area of research with many reports
showing the activity of ferrocene derivatives in vivo and in vitro.
The main attention has focused on their use as anti-malarial
and anti-cancer drugs [22]. It has also been shown that N-(ferr-
ocenylmethyl)fluorobenzene-carboxamide derivatives display
anti-cancer activity against ER (+) MDA-MB-435-S-F breast cancer
cells [23].
We have previously reported the synthesis and structural char-
acterization of N-ortho, N-meta and N-para-(ferrocenyl)benzoyl
derivatives incorporating natural amino acids and dipeptide deriv-
atives [24–30,32,33]. The compounds are composed of three key
moieties, namely, (i) an electroactive core, (ii) a conjugated aro-
matic linker that lowers the redox potential and (iii) an amino acid
or peptide derivative that can interact with other molecules via
hydrogen bonding. The ferrocenyl benzoyl derivatives have lower
found to be cytotoxic and had an IC50 value of 48
lM, whereas
the starting material, ortho-ferrocenyl ethyl benzoate, was com-
pletely inactive against the cell lines. This indicates that the amino
acid or dipeptide derivative of these compounds is essential for bio-
logical activity. Therefore, other derivatives were evaluated for
their anti-cancer activity against H1299 lung cancer cells. The
dipeptide derivative N-{ortho-(ferrocenyl)-benzoyl}-glycine-gly-
cine ethyl ester was shown to have an IC50 value of approximately
20 lM. From this it may be assumed that the glycine residue of the
dipeptide that is attached to the benzoyl group is important for
activity. As the dipeptide derivative was more active than the ami-
no acid derivative, a logical extension of this study was the prepa-
ration of longer peptide chains. Therefore, the peptide moiety was
chain extended by additional glycine residues. Herein, we now
report the synthesis and structural characterization of N-ortho,
N-meta and N-para-(ferrocenyl)benzoyl tri- and tetrapeptide esters.
The in vitro anti-proliferative activity for N-ortho-(ferrocenyl)ben-
zoyl tri- and tetrapeptide esters 2 and 5 against H1299 lung cancer
cells is also presented.
* 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).
0020-1693/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2009.01.021

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A.J. Corry et al. / Inorganica Chimica Acta 362 (2009) 2957–2961
that forms the amide bond between the benzoyl group and the pep-
2. Results and discussion
2.1. Synthesis
tide chain was present between d 8.9 and d 8.5 for the tripeptidesand
d 8.9 and d 8.6 for the tetrapeptides. For example in the case of N-
{ortho-(ferrocenyl)-benzoyl}-glycyl-glycyl-glycyl-glycine ethyl es-
ter 5 the amide protons appear downfield between d 8.50 and d
8.17. There are two triplets that integrate for one proton each and
a multiplet that integrates for two protons. The signals in the aro-
matic region confirm the presence of four protons, observed as a
doublet, triplet and multiplet between d 7.85 and d 7.50. The multi-
plet integrates for two protons while the triplet and doublet both
integrate for one proton. The protons in the ortho position of the
The arylation of ferrocene is readily achieved by reacting ferro-
cene with an aryl diazonium salt. For the synthesis of the N-(ferr-
ocenyl)benzoyl peptide derivatives (2–7) ethyl-2, ethyl-3 and
ethyl-4-aminobenzoate were used in order to generate the starting
materials, ortho, meta and para-ferrocenyl ethyl benzoates, respec-
tively. These compounds were isolated as viscous oils. The ethyl es-
ter group was efficiently cleaved to yield the three ferrocenyl
benzoic acids by saponification using 10% sodium hydroxide. This
procedure is outlined in Scheme 1.
Coupling reactions were used to facilitate the introduction of
the ferrocenyl benzoyl group onto the peptide esters. Ortho, meta
and para-ferrocenyl benzoic acids 1 were treated with N-(3-
dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC),
1-hydroxybenzotriazole (HOBt), and triethylamine (TEA) in dichlo-
romethane at 0 °C in the presence of glycyl-glycyl-glycine ethyl es-
ter hydrochloride and glycyl-glycyl-glycyl-glycine ethyl ester
hydrochloride (Scheme 1). The resulting N-(ferrocenyl)benzoyl
peptide derivatives (2–7) gave spectroscopic data in accordance
with the proposed structures in yields ranging from 29% to 55%.
(g
5-C5H4) ring appear at d 4.71 and the meta protons occur at d
4.33. The singlet at d 4.1 represents the unsubstituted cyclopentadi-
enyl (
5-C5H5) ring. This peak overlaps with the methylene protons
g
of the ethyl ester. The methylene groups of the peptide chain appear
as a triplet at d 3.91 and two doublets at d 3.86 and 3.81, respectively.
The doublets integrate for two protons each, while the triplet inte-
grates for four protons. The methyl group of the ethyl ester appears
as a triplet at d 1.25.
In the 13C NMR spectra of the N-(ferrocenyl)benzoyl peptide es-
ters the amide and ethyl ester carbonyl carbon atoms appear be-
tween d 170.1 and d 166.4. In the aromatic region the pattern
observed depended on whether the derivatives were ortho, meta
or para substituted. The ortho and meta derivatives give rise to six
carbon peaks as all six carbon atoms are non-equivalent. The para
derivatives displays four carbon signals, two of these being quater-
nary carbon atoms that were easily identified by DEPT-135.
The ferrocenyl carbon atoms are present between d 84.4 and d
68.2 indicative of a monosubstituted ferrocene unit. The ipso carbon
2.2. Characterization
The N-(ferrocenyl)benzoyl peptide derivatives (2-7) were char-
acterized by a combination of 1H NMR, 13C NMR and DEPT-135 spec-
troscopy, mass spectrometry and cyclic voltammetry. All the proton
and carbon chemical shifts for compounds 2–7 were unambiguously
assigned by a combination of DEPT-135 and 1H–13C–COSY (HMQC).
Thearomaticsignalsin the1H NMRspectra of theN-(ferrocenyl)ben-
zoylpeptideesters(2–7)varieddependingonwhetherortho, metaor
para-ferrocenyl benzoic acid was used as a starting material. For the
ortho derivatives the aromatic region showed a doublet, triplet, mul-
tiplet splitting pattern, integrating for one, one and two protons,
respectively. In the meta derivatives the pattern observed was a sin-
glet that integrated for one proton, a multiplet that integrated for
two protons and a triplet that integrated for one proton. The para
substituted splitting pattern consisted of two doublets that each
integrated for two protons. The chemical shift of the amide proton
on the substituted cyclopentadienyl (
narrow range of d 84.3 to d 83.1. The unsubstituted cyclopentadi-
enyl (
5-C5H5) ring occurs as an intense peak at approximately d
69, while the ortho and meta carbon atoms of the substituted cyclo-
pentadienyl ( -C5H4) ring have chemical shifts between d 68 and d
g-C5H4) ring appears in the
g
g
66. The methylene group of the ethyl ester appears at d 60.4 in all
the spectra. This methylene group of the ethyl ester and the meth-
ylene groups of the peptide chain are easily recognised by their neg-
ative resonance in DEPT-135 spectra. The methylene carbon atoms
of the tripeptide chain appear in the range d 42.7 and d 40.0 and are
in the range of d 42.2 and d 39.9 for the tetrapeptide derivatives. The
methyl group of the ethyl ester appears at d 14.0 in all the spectra. A
summary of selected chemical shifts (d) for the 13C NMR spectra of
compounds (2–7) is presented in Table 1.
NH2
All N-(ferrocenyl)benzoyl peptide derivatives (2–7) exhibit a
one electron reversible redox process in the cyclic voltammograms
(CVs) similar to ferrocene, under the same conditions. The E°0 val-
ues range from 39 to 75 mV versus the ferrocene/ferricenium re-
dox couple (Fc/Fc+). A notable trend is observed whereby the
orientation around the central benzoyl moiety effects the redox
potentials in the order ortho < meta < para. Oxidation of the ferr-
ocenyl unit in the ortho derivatives occurs more readily compared
to the meta and para derivatives. It is possible that the ortho orien-
tation around the benzoyl moiety imparts electron density to the
ferrocene and therefore makes the iron centre more susceptible
to oxidation. This electron density is less pronounced in the meta
and para derivatives.
i
Fe
+
Fe
O
O
O
O
ii
iii
Soft ionization techniques such as electrospray ionization (ESI)
mass spectrometry permit the analysis of thermolabile and non-
volatile analytes [31]. Electrospray ionization (ESI) was employed
in the analysis of compounds (2–7) and confirmed the correct rel-
ative molecular mass for all the compounds. Examination of the
mass spectra revealed the presence of both radical-cations [M]+Å
as well as [M+H]+ species. Intense adducts due to sodium were also
present 22 Da higher than the protonated molecular ion species.
Similar observations were made in the analysis of the N-(ferroce-
nyl)benzoyl dipeptide ester derivatives [27–30,32,33]. Sequence
2-7
Fe
Fe
O
O
NH
HO
1
EtO
O
n
Scheme 1. Synthesis of N-ortho, N-meta and N-para ferrocenyl benzoyl tri- and
tetrapeptide esters 2–7. i = NaNO2, HCl, 5 °C, ii = NaOH/MeOH, HCl, iii = EDC, HOBt,
TEA, GlyGlyGlyOEt.HCl (2–4) n = 3, GlyGlyGlyGlyOEt.HCl (5–7) n = 4.

A.J. Corry et al. / Inorganica Chimica Acta 362 (2009) 2957–2961
2959
Table 1
13C spectroscopic data for compounds (2–7).
Compound
C@O
Ipso (
g
-C5H4)
(g
-C5H5)
O-CH2CH3
Peptide CH2
2
3
4
5
6
7
170.1–169.1
170.0–166.6
169.7–166.5
170.1–169.1
169.6–166.6
169.6–166.5
84.4
83.9
83.1
84.4
84.0
83.2
69.4
69.4
69.5
69.4
69.4
69.5
60.4, 14.0
60.4, 14.0
60.4, 14.0
60.4, 14.0
60.4, 14.0
60.4, 14.0
42.3–40.6
42.8–40.6
42.7–40.6
42.3–40.6
42.8–40.6
42.8–41.2
specific fragment ions were not observed or were of low intensity
in the mass spectra.
3. Conclusion
In conclusion N-(ferrocenyl)benzoyl tri-and tetrapeptide esters
have been prepared using standard peptide chemistry. The
products (2–7) gave spectroscopic data in accordance with the pro-
posed structures in yields ranging from 29% to 55%. The ortho tri-
and tetrapeptide derivatives 2 and 5 were tested in vitro against
H1299 lung cancer cells and showed IC50 values greater than those
of N-(ferrocenyl)benzoyl amino acid and dipeptide esters. There-
fore, extending the length of the peptide chain had a negative ef-
fect on anti-cancer activity, relative to N-(ferrocenyl)benzoyl
amino acid and dipeptide derivatives.
2.3. In vitro anti-proliferation activity
In previous biological testing it was observed that the IC50 de-
creased as the peptide chain increased from one to two amino acid
residues [32]. Therefore, it was of interest to prepare longer pep-
tide chains of three and four amino acid residues. The in vitro cyto-
toxicity of the N-(ferrocenyl)benzoyl derivatives 2 and 5 against
the human lung carcinoma cell line H1299 was evaluated by the
acid phosphatase assay. A plot of cell survival versus compound
concentration for compounds 2, 5, the dipeptide N-{ortho-(ferroce-
nyl)-benzoyl}-glycine-glycine ethyl ester and N-{ortho-(ferroce-
nyl)-benzoyl}-glycine ethyl ester is presented in Fig. 1. The
synthesis of N-{ortho-(ferrocenyl)-benzoyl}-glycine-glycine ethyl
ester and N-{ortho-(ferrocenyl)-benzoyl}-glycine ethyl ester has
previously been reported [32]. Compounds 2 and 5 both have
4. Experimental
4.1. General procedures
All chemicals were purchased from Sigma/Aldrich and used as
received. Commercial grade reagents were used without further
purification; however, solvents were purified prior to use. Melting
points were determined using a Griffin melting point apparatus
and are uncorrected. Infrared spectra were recorded on a Nicolet
405 FT-IR spectrometer and UV–Vis spectra on a Hewlett-Packard
8452A diode array UV–Vis spectrophotometer. NMR spectra were
obtained on a Bruker AC 400 NMR spectrometer operating at
400 MHz for 1H NMR and 100 MHz for 13C NMR. The 1H and 13C
NMR chemical shifts (ppm) are relative to TMS and all coupling
constants (J) are in Hertz. Electrospray ionization mass spectra
were obtained on a Brüker Daltonics 3000 Esquire-LC ion trap mass
spectrometer. Elemental Analysis was carried out by the Microan-
alytical Laboratory at University College Dublin. Cyclic voltammo-
grams were recorded in acetonitrile (Sigma–Aldrich), with 0.1 M
tetrabutylammonium perchlorate (TBAP) as a supporting electro-
lyte, using a CH Instruments 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
Ag/Ag+ reference electrode was used. The scan rate was 0.1 V/s.
The concentration range of the ferrocene compounds was
1.0 mMol in acetonitrile. The E°0 values obtained for the test sam-
ples were referenced to the Fc/Fc+ couple.
IC50 values >50
has a negative effect on the anti-proliferative effect of the ferroce-
nyl derivatives. Compound 2 had an IC50 value of 63 M (RSD 8%),
lM, thus increasing the length of the peptide chain
l
whereas compound 5 did not register an IC50 value in the concen-
tration range used against H1299 lung cancer cells. From Fig. 1 it
can be concluded that a dipeptide chain is required for optimum
activity. The production of Reactive Oxygen Species (ROS) has been
implicated in the biological activity of ferrocenyl compounds, these
species result from the easily accessible ferrocene/ferricenium re-
dox couple. As compounds 2–7 have very similar redox potentials
to N-(ferrocenyl)benzoyl dipeptide esters that are highly active
in vitro, it may be assumed that the peptide chain imparts a sec-
ondary mechanism of action to these compounds [32,33]. How-
ever, further studies regarding the mode of action must be
undertaken, before any definitive conclusions may be drawn.
100
90
80
5
70
60
50
40
30
20
10
0
2
N-ortho-Fc-Gly-Gly-OEt
N-ortho-Fc-Gly-OEt
4.2. General procedure for the preparation
of N-(ferrocenyl)benzoyl tripeptide esters
0
5
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
4.2.1. N-{ortho-(ferrocenyl)-benzoyl}-glycyl-glycyl-glycine
ethyl ester (2)
1-Hydroxybenzotriazole (0.189 g, 1.4 mmol) was added to a
solution of ortho-ferrocenyl benzoic acid (0.245 g, 0.8 mmol),
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride
(0.267 g 1.4 mmol) and triethylamine (2 mls) in dichloromethane
(40 mls) at 0 °C. After 30 min glycyl-glycyl-glycine ethyl ester
hydrochloride (0.229 g, 0.9 mmol) was added and the reaction
was stirred at room temperature for 48 h. The reaction mixture
was washed with water, 10% potassium hydrogen carbonate and
Concentration (µM)
Fig. 1. IC50 plot of compounds 2, 5 and selected N-(ferrocenyl)benzoyl derivatives
in vitro vs. H1299 lung cancer cells.

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A.J. Corry et al. / Inorganica Chimica Acta 362 (2009) 2957–2961
5% citric acid. The organic layer was dried over MgSO4 and the sol-
vent was removed in vacuo. The product was purified by column
chromatography (eluant: ethyl acetate). Recrystallization from
ethyl acetate furnished the product as an orange solid. (0.24 g,
59%). m.p. 138–140 °C; E°0 = 39 mV (vs Fc/Fc+).
Anal. Calc. for C25H27FeN3O5: C, 59.42; H, 5.39; N, 8.32. Found: C,
59.32; H, 5.47; N, 7.91%.
Mass spectrum: [M+Na]+ found: 528.20.
C25H27N3O5FeNa requires: 528.12.
UV–Vis kmax MeCN: 335, 450 nm.
4.3. General procedure for the preparation of N-(ferrocenyl)benzoyl
tetrapeptide esters
I.R.
mmax (KBr): 3293, 2927, 2851, 1737, 1691, 1516, 1426, 1277,
1104 cmꢀ1
.
1H NMR (400 MHz) d (DMSO): 8.54 (1H, t, J = 6 Hz, –CONH–), 8.32
(1H, t, J = 5.6 Hz, –CONH–), 8.13 (1H, t, J = 6 Hz, –CONH–), 7.85 (1H, d,
J = 7.6 Hz ArH), 7.42 (1H, t, J = 4.8 Hz ArH), 7.22–7.38 (2H, m, ArH),
4.3.1. N-{ortho-(ferrocenyl)-benzoyl}-glycyl-glycyl-glycyl-glycine
ethyl ester (5)
1-Hydroxybenzotriazole (0.09 g, 0.65 mmol) was added to a
solution of ortho-ferrocenyl benzoic acid (0.18 g, 0.6 mmol),
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride
(0.267 g 1.4 mmol) and triethylamine (2 mls) in dichloromethane
(40 mls) at 0 °C. After 30 min glycyl-glycyl-glycyl-glycine ethyl
ester hydrochloride (0.18 g, 0.6 mmol) was added and the reac-
tion was stirred at room temperature for 48 h. The reaction mix-
ture was washed with water, 10% potassium hydrogen carbonate
and 5% citric acid. The organic layer was dried over MgSO4 and
the solvent was removed in vacuo. The product was purified by
column chromatography (eluant 9:1 ethyl acetate:methanol).
Recrystallization from ethyl acetate furnished the product as an
orange solid. (0.10 g, 30%). m.p. 168–170 °C; E°0 = 44 mV (vs Fc/
Fc+).
4.65 {2H, t, J = 1.6 Hz, ortho on (
g
-C5H4)}, 4.26 {2H, t, J = 1.6 Hz, meta
-C5H5), –OCH2CH3}, 3.78–3.83
on ( -C5H4)}, 4.00–4.05 {7H, m, (
g
g
(6H, m, –NHCH2CO–), 1.17 (3H, t, J = 7.2 Hz, –OCH2CH3).
13C NMR (100 MHz) d (DMSO): 170.1, 169.6, 169.3, 169.1, 136.2,
136.0, 130.6, 128.7, 127.4, 125.4, 84.4, 69.4, 68.8, 68.2, 60.4 (ꢀve
DEPT), 42.3 (ꢀve DEPT), 41.7 (ꢀve DEPT), 40.6 (ꢀve DEPT), 14.0.
Anal. Calc. for C25H27FeN3O5: C, 59.42; H, 5.39; N, 8.32. Found: C,
59.72; H, 5.56; N, 8.75%.
Mass spectrum: [M+Na]+ found: 528.20.
C25H27N3O5FeNa requires: 528.12.
4.2.2. N-{meta-(ferrocenyl)-benzoyl}-glycyl-glycyl-glycine
ethyl ester (3)
For compound
3
meta-ferrocenyl benzoic acid (0.24 g,
UV–Vis kmax MeCN: 332, 445 nm.
0.8 mmol) was used as a starting material. Recrystallization from
ethyl acetate furnished the product as a yellow solid. (0.26 g,
64%). m.p. 166–168 °C; E°0 = 55 mV (vs Fc/Fc+).
I.R.
mmax (KBr): 3293, 3083, 2346, 1522, 1430, 1407, 1211, 1105,
1011 cmꢀ1
.
1H NMR (400 MHz) d (DMSO): 8.50 (1H, t, J = 6 Hz, –CONH–),
8.29–8.35 (2H, m, –NHCO–), 8.17 (1H, t, J = 5.6 Hz, –NHCO–), 7.87
(1H, d, J = 7.6 Hz, ArH), 7.47 (1H, t, J = 4.8 Hz, ArH), 7.31–7.35
(2H, m, ArH), 4.71 {2H, J = 2 Hz, ortho on (
J = 2 Hz, meta on (g
UV–Vis kmax MeCN: 325, 450 nm.
I.R.
mmax (KBr): 3229, 3079, 1831, 1725, 1740, 1603, 1335, 1118,
1105 cmꢀ1
.
g
5-C5H4)}, 4.33 {2H, t,
5-C5H5),
1H NMR (400 MHz) d (DMSO): 8.89 (1H, t, J = 6 Hz, –CONH–),
8.29–8.32 (2H, m, –CONH–), 8.01 (1H, s, ArH), 7.75 (2H, t,
J = 1.6 Hz, ArH), 7.43 (1H, t, J = 8 Hz, ArH), 4.86 {2H, t, J = 2 Hz, ortho
(
g
5-C5H4)}, 4.11–4.17 {7H, m,
–OCH2CH3}, 3.91 (4H, t, J = 4.8 Hz, –NHCH2CO–), 3.86 (2H, d,
J = 5.6 Hz, –NHCH2CO–), 3.81 (2H, d, J = 6 Hz, –NHCH2CO–), 1.25
(3H, t, J = 7.2 Hz, –OCH2CH3).
on (g
-C5H4)}, 4.40, {2H, t, J = 1.6 Hz, meta on (
g
5-C5H4)}, 4.12, (2H,
q, J = 7.2 Hz, –OCH2CH3), 4.04 {5H, s, (
g
5-C5H5)}, 3.95 (2H, d,
13C NMR (100 MHz) d (DMSO): 170.1, 169.6, 169.3, 169.2, 169.1,
136.2, 136.0, 130.0, 128.7, 127.4, 125.4, 84.4, 69.4, 68.7, 68.2, 60.4
(ꢀve DEPT), 42.3 (ꢀve DEPT), 42.1 (ꢀve DEPT), 41.7 (ꢀve DEPT),
40.6 (ꢀve DEPT), 14.0.
J = 6 Hz, –NHCH2CO–), 3.86 (2H, d, J = 6 Hz, –NHCH2CO–), 3.77
(2H, d, J = 6 Hz, –NHCH2CO–), 1.20 (3H, t, J = 7.2 Hz, –OCH2CH3).
13C NMR (100 MHz) d (DMSO): 170.0, 169.7, 169.4, 166.6, 139.2,
133.9, 128.8, 128.4, 124.9, 124.3, 83.9, 69.4, 69.1, 66.4, 60.4
(ꢀve DEPT), 42.8 (ꢀve DEPT), 41.7 (ꢀve DEPT), 40.6 (ꢀve DEPT),
14.0.
Anal. Calc. for C27H30FeN4O6 requires: C, 57.66; H, 5.38; N, 9.96.
Found: C, 57.18; H, 5.53; N, 9.75%.
Mass spectrum: [M+Na]+ found: 585.20.
Anal. Calc. for C25H27FeN3O5: C, 59.42; H, 5.39; N, 8.32. Found: C,
59.08; H, 5.22; N, 8.64%.
C27H30N4O6FeNa requires: 585.14.
Mass spectrum: [M+Na]+ found: 528.20.
4.3.2. N-{meta-(ferrocenyl)-benzoyl}-glycyl-glycyl-glycyl-glycine
ethyl ester (6)
C25H27N3O5FeNa requires: 528.12.
For compound
6
meta-ferrocenyl benzoic acid (0.18 g,
4.2.3. N-{para-(ferrocenyl)benzoyl}-glycyl-glycyl-glycine
ethyl ester (4)
For compound 4 para-ferrocenyl benzoic acid (0.21 g, 0.7 mmol)
was used as a starting material. Recrystallization from ethyl ace-
tate furnished the product as an orange solid. (0.22 g, 62%). m.p.
206–208 °C; E°0 = 73 mV (vs Fc/Fc+).
0.6 mmol) was used as a starting material. Recrystallization from
ethyl acetate furnished the product as a yellow solid. (0.09 g,
27%). m.p. 171–173 °C; E°0 = 58 mV (vs Fc/Fc+).
UV–Vis kmax MeCN: 330, 450 nm.
I.R.
mmax (KBr): 3280, 3084, 2366, 1735, 1559, 1458, 1376, 1283,
1204, 1148 cmꢀ1
.
UV–Vis kmax MeCN: 355, 450 nm.
1H NMR (400 MHz) d (DMSO): 8.95 (1H, t, J = 4.4 Hz, –CONH–),
8.34 (2H, q, J = 3.2 Hz, –CONH–), 8.29 (2H, t, J = 6 Hz, –CONH–),
8.01 (1H, s, ArH), 7.79–7.87 (2H, m, ArH), 7.47 (1H, t, J = 7.6 Hz,
I.R.
mmax (KBr): 3275, 3090, 2987, 2345, 1751, 1607, 1519, 1378,
1249, 1028, 993 cmꢀ1
.
1H NMR (400 MHz) d (DMSO): 8.79 (1H, t, J = 6 Hz, –CONH–),
8.30 (2H, t, J = 6.4 Hz, –CONH–), 7.83 (2H, d, J = 8.4 Hz, ArH), 7.65
ArH), 4.93 {2H, t, J = 1.6 Hz, ortho on (
J = 1.6 Hz, meta on (
5-C5H4)}, 4.15 (2H, q, J = 7.2 Hz, –OCH2CH3),
4.10 {5H, s, (
5-C5H5)}, 4.02 (2H, d, J = 5.6 Hz, –NHCH2CO–),
g
5-C5H4)}, 4.46 {2H, t,
(2H, d, J = 8.4 Hz, ArH), 4.90 {2H, t, J = 2 Hz, ortho on (
4.41 {2H, t, J = 1.6 Hz, meta on (
5-C5H4)}, 4.10 (2H, q, J = 7.2 Hz,
–OCH2CH3), 4.02 {5H, s,
5-C5H5)}, 3.92 (2H, d, J = 5.6 Hz,
g
5-C5H4)},
g
g
g
3.82–3.89 (6H, m, –NHCH2CO–), 1.25 (3H, t, J = 7.2 Hz,
(
g
–OCH2CH3).
–NHCH2CO–), 3.86 (2H, d, J = 6 Hz, –NHCH2CO–), 3.76 (2H, d, J =
6 Hz, –NHCH2CO–), 1.19 (3H, t, J = 6.8 Hz –OCH2CH3).
13C NMR (100 MHz) d (DMSO): 169.6, 169.5, 169.3, 169.1, 166.6,
139.2, 133.9, 128.8, 128.4, 124.9, 124.3, 84.0, 69.4, 69.1, 66.4, 60.4
(ꢀve DEPT), 42.8 (ꢀve DEPT), 42.1 (ꢀve DEPT), 41.7 (ꢀve DEPT),
40.6 (ꢀve DEPT), 14.0.
13C NMR (100 MHz) d (DMSO): 169.7, 169.5, 169.4, 166.5, 142.8,
130.9, 127.5, 125.3, 83.1, 69.5, 66.6, 66.4, 60.4 (ꢀve DEPT), 42.7
(ꢀve DEPT), 41.7 (ꢀve DEPT), 40.6 (ꢀve DEPT), 14.0.

A.J. Corry et al. / Inorganica Chimica Acta 362 (2009) 2957–2961
2961
Anal. Calc. for C27H30FeN4O6: C, 57.66; H, 5.38; N, 9.96. Found: C,
Acknowledgements
57.57; H, 5.74; N, 10.09%.
Mass spectrum: [M+Na]+ found: 585.20.
C27H30N4O6FeNa requires: 585.14.
A.J.C. and A.M. would like to thank the Embark Initiative and
IRCSET for all their support.
This research was partly supported by the National Institute for
Cellular Biotechnology under the Programme for Research in Third
Level Institutions (PRTLI, round 3, 2001–2006).
4.3.3. N-{para-(ferrocenyl)-benzoyl}-glycyl-glycyl-glycyl-glycine ethyl
ester (7)
For compound 7 para-ferrocenyl benzoic acid (0.18 g, 0.6 mmol)
was used as a starting material. Recrystallization from ethyl ace-
tate furnished the product as an orange solid. (0.10 g, 30%). m.p.
160–162 °C; E°0 = 75 mV (vs Fc/Fc+).
References
[1] G. Jaouen (Ed.), J. Organomet. Chem. 589 (1999) 1. Special Issue on
Bioorganometallic Chemistry.
UV–Vis kmax MeCN: 349, 450 nm.
[2] R.D. Adams (Ed.), J Organomet Chem. 637–639 (2001) 1. Special Issue on
Ferrocene Chemistry.
[3] R.H. Fish, G. Jaouen, Organometallics 22 (2003) 2166.
[4] E.W. Neuse, J. Inorg. Organomet. Poly. Mat. 15 (1) (2005) 3.
[5] A. Nomoto, T. Moriuchi, S. Yamazaki, A. Ogawa, T. Hirao, J. Chem. Soc. Chem.
Commun. (1998) 1963.
I.R.
mmax (KBr): 3270, 2937, 2739, 2345, 1719, 1542, 1474, 1283,
1120, 1035 cmꢀ1
.
1H NMR (400 MHz) d (DMSO): 8.83 (1H, t, J = 5.6 Hz, –CONH–),
8.21–8.29 (3H, m, –CONH–), 7.83 (2H, d, J = 8.4 Hz ArH), 7.64 (2H,
[6] T. Itoh, S. Shirakami, N. Ishida, Y. Yamashita, T. Yoshida, H.-S. Kim, Y. Wataya,
Bioorg. Med. Chem. Lett. 10 (2000) 1657.
d, J = 8.4 Hz ArH), 4.90 {2H, t, J = 1.6 Hz, ortho on (
g
5-C5H4)}, 4.41
{2H, t, J = 1.6 Hz, meta on (
g
5-C5H4)}, 4.01 (2H, q, J = 6.8 Hz,
[7] T. Moriuchi, A. Nomoto, K. Yoshida, T. Hirao, Organometallics 20 (2001) 1008.
[8] J.F. Gallagher, P.T.M. Kenny, M.J. Sheehy, Inorg. Chem. Commun. 2 (1999) 200.
[9] J.F. Gallagher, P.T.M. Kenny, M.J. Sheehy, Inorg. Chem. Commun. 2 (1999) 327.
[10] H.-B. Kraatz, D.M. Leek, A. Houmam, G.D. Enright, J. Lusztyk, D.D.M. Wayner, J.
Organomet. Chem. 589 (1999) 38.
[11] T. Moriuchi, A. Nomoto, K. Yoshida, T. Hirao, J. Organomet. Chem. 589 (1999)
50.
[12] P. Saweczko, H.-B. Kraatz, Coordin. Chem. Rev. 192 (1999) 185.
[13] O. Brosch, T. Weyhermuller, N. Metzler-Nolte, Eur. J. Inorg. Chem. 2 (2000) 323.
[14] A. Hess, J. Sehnert, T. Weyhermuller, N. Metzler-Nolte, Inorg. Chem. 39 (2000)
5437.
[15] T. Moriuchi, K. Yoshida, T. Hirao, Organometallics 20 (2001) 3101.
[16] Y.M. Xu, H.-B. Kraatz, Tetrahedron Lett. 42 (2001) 2601.
[17] T. Moriuchi, K. Yoshida, T. Hirao, J. Organomet. Chem. 637 (2001) 75.
[18] H. -B. Kraatz, Y.M. Xu, P. Saweczko, J. Organomet. Chem. 637 (2001) 335.
[19] S. Maricic, U. Berg, T. FreJd, Tetrahedron 58 (2002) 3085.
[20] D.R. van Staveren, T. Weyhermuller, N. Metzler-Nolte, J. Chem. Soc., Dalton
Trans. (2003) 210.
[21] M.J. Sheehy, J.F. Gallagher, M. Yamashita, Y. Ida, J. White-Colangelo, J. Johnson,
R. Orlando, P.T.M. Kenny, J. Organomet. Chem. 689 (2004) 1511.
[22] M.F.R. Fouda, M.M. Abd-Elzaher, R.A. Abdelsamaia, A.A. Labib, Appl.
Organomet. Chem. 21 (2007) 613.
[23] P.N. Kelly, A. Pretre, S. Devoy, I. O’Reilly, R. Devery, A. Goel, J.F. Gallagher, A.J.
Lough, P.T.M. Kenny, J. Organomet. Chem. 692 (2007) 1327.
[24] D. Savage, J.F. Gallagher, Y. Ida, P.T.M. Kenny, Inorg. Chem. Commun. 5 (2002)
1034.
[25] D. Savage, G. Malone, J.F. Gallagher, Y. Ida, P.T.M. Kenny, J. Organomet. Chem.
690 (2005) 383.
[26] D. Savage, N. Neary, G. Malone, S.R. Alley, J.F. Gallagher, P.T.M. Kenny, Inorg.
Chem. Commun. 8 (2005) 429.
[27] 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.
[28] D. Savage, S.R. Alley, J.F. Gallagher, A. Goel, P.N. Kelly, P.T.M. Kenny, Inorg.
Chem. Commun. 9 (2006) 152.
[29] A. Goel, D. Savage, S.R. Alley, T. Hogan, P.N. Kelly, S.M. Draper, C.M. Fitchett,
P.T.M. Kenny, J. Organomet. Chem. 691 (2006) 4686.
[30] 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.
[31] J.B. Fenn, J. Am. Soc. Mass Spectrom. 4 (1993) 524.
[32] A.J. Corry, A. Goel, S.R. Alley, P.N. Kelly, D. O’Sullivan, D. Savage, P.T.M. Kenny, J.
Organomet. Chem. 692 (2007) 1405.
[33] A. Goel, D. Savage, S.R. Alley, P.N. Kelly, D. O’Sullivan, H. Mueller-Bunz, P.T.M.
Kenny, J. Organomet. Chem. 692 (2007) 1292.
–OCH2CH3), 3.93 (2H, d, J = 5.6 Hz –NHCH2CO–), 3.82 (2H, d,
J = 6 Hz, –NHCH2CO–), 3.76 (4H, t, J = 6.4 Hz, –NHCH2CO–) 1.18
(3H, t, J = 7.2 Hz, –OCH2CH3).
13C NMR (100 MHz) d (DMSO): 169.60, 169.57, 169.3, 169.2,
166.5, 142.8, 131.1, 127.6, 125.3, 83.2, 69.53, 69.47, 66.6, 60.4
(ꢀve DEPT), 42.8 (ꢀve DEPT), 42.1 (ꢀve DEPT), 41.7 (ꢀve DEPT),
41.2 (ꢀve DEPT), 14.0.
Anal. Calc. for C27H30FeN4O6: C, 57.66; H, 5.38; N, 9.96. Found: C,
57.96; H, 5.77; N, 10.18%.
Mass spectrum: [M+Na]+ found: 585.20.
C27H30N4O6FeNa requires: 585.14.
4.4. General in vitro anti-proliferation assay procedure
Compounds 2 and 5 were dissolved in 100
dilutions at two times their final concentrations were prepared in
cell culture media. 100 l of these drug dilutions were added to
ll of DMSO and drug
l
plates containing cells that had been incubated for 24 h in a
37 °C, 5% CO2 incubator. The plates were then incubated in the
same conditions for 6–7 days or until cell confluency reached
80–90%. The assessment of cell survival in the presence of the drug
was then measured using an acid phosphatase assay. Media was
removed from the plates and each well on the plate was washed
with 100 ll PBS. This was removed and 100 ll of freshly prepared
phosphatase substrate in 0.1 M sodium acetate was added to each
well. The plates were then incubated in the dark for 2 h at 37 °C.
Colour development was monitored during this time then the
enzymatic reaction was stopped by adding 50 ll of 1 N NaOH.
The fluorescence of the plate was measured at 405 nm with a ref-
erence wavelength of 620 nm. The IC50 value was determined by
plotting cell survival percentage (relative to control cells) against
drug concentration.