The synthesis, structural characterization and in vitro anti-cancer activity of novel 1-alkyl-1′-N-para-(ferrocenyl) benzoyl dipeptide esters

Article abstract of DOI:10.1016/j.jorganchem.2014.01.031

1-Alkyl-1′-N-para-(ferrocenyl) benzoyl dipeptide esters 4-12 were prepared by coupling the alkyl ferrocenyl benzoic acids 1-3 to the dipeptide ethyl esters Gly-L-Ala(OEt), Gly-L-Leu(OEt) and Gly-L-Phe(OEt) using the standard N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole (HOBt) protocol. All the compounds were fully characterized using a combination of ¹H NMR, ¹³C NMR, DEPT-135 and ¹H-¹³C COSY (HMQC) spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and cyclic voltammetry (CV). Selected compounds showed micromolar activity in the H1299 NSCLC cell line, with IC ₅₀ values in the range of 4.5-6.6 μM.

Full text of DOI:10.1016/j.jorganchem.2014.01.031

Journal of Organometallic Chemistry 757 (2014) 28e35
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
The synthesis, structural characterization and in vitro anti-cancer
activity of novel 1-alkyl-10-N-para-(ferrocenyl) benzoyl dipeptide
esters
,
Andy G. Harry a, William E. Butler a b, Jennifer C. Manton a, Mary T. Pryce a,
,b,
Norma O’Donovan b, John Crown c, Dilip K. Rai d, Peter T.M. Kenny a
*
a School of Chemical Sciences, Dublin City University, Dublin 9, Ireland
b National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland
c Dept. of Medical Oncology, St. Vincent’s University Hospital, Dublin 4, Ireland
d Dept. of Food Biosciences, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland
a r t i c l e i n f o
a b s t r a c t
1-Alkyl-10-N-para-(ferrocenyl) benzoyl dipeptide esters 4e12 were prepared by coupling the alkyl fer-
rocenyl benzoic acids 1e3 to the dipeptide ethyl esters Gly-L-Ala(OEt), Gly-L-Leu(OEt) and Gly-L-
Phe(OEt) using the standard N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), 1-
hydroxybenzotriazole (HOBt) protocol. All the compounds were fully characterized using a combina-
tion of 1H NMR, 13C NMR, DEPT-135 and 1He13C COSY (HMQC) spectroscopy, electrospray ionization
mass spectrometry (ESI-MS) and cyclic voltammetry (CV). Selected compounds showed micromolar
Article history:
Received 22 November 2013
Received in revised form
27 January 2014
Accepted 28 January 2014
Keywords:
Ferrocene
Bioorganometallic chemistry
Dipeptides
activity in the H1299 NSCLC cell line, with IC50 values in the range of 4.5e6.6 mM.
Ó 2014 Elsevier B.V. All rights reserved.
Cytotoxicity
Lung cancer
1. Introduction
potential applications [9e13]. In vitro toxicity testing showed that
N-(ferrocenyl)benzoyl and N-(ferrocenyl)naphthoyl amino acid and
peptide derivatives were potential anticancer agents [14e21].
These ferrocenyl-peptide bioconjugates are composed of three key
moieties, namely, (i) an electroactive core, (ii) a conjugated aro-
matic linker, and (iii) an amino acid or peptide derivative that can
interact with other molecules via hydrogen bonds. One key feature
of ferrocene is the ease by which it undergoes oxidation to form the
ferricenium cation (Fc / Fcþ). This occurs in a reversible manner
and is accommodated readily by the loss/gain of an electron from a
non-bonding high energy molecular orbital. In the case of our
ferrocenyl-peptide bioconjugates, the presence of the conjugating
aromatic linker between the ferrocene and peptide units lowers the
redox potential to within the range of biologically accessible po-
tentials, allowing for the interconversion between the ferrocene
and ferricenium species. Ferricenium salts that are known to inhibit
tumour growth, have been shown to produce hydroxyl (HOꢀ) rad-
icals under physiological conditions, leading to oxidatively
damaged DNA [5]. The catalytic generation of intracellular reactive
oxygenated species (ROS) such as the HOꢀ radical offers an attrac-
tive and alternative method to target and kill cancer cells [22]. The
anticancer activity of the ferrocenyl bioconjugates is possibly due at
Organometallic compounds have been incorporated in a wide
variety of materials that have diverse applications. Ferrocene is a
particularly attractive candidate for incorporation into biologically
active compounds due to its aromaticity, stability, redox properties
and low toxicity [1,2]. The medicinal application of ferrocene de-
rivatives is an active area of research, with applications in the area
of cancer research being the most popular and well-researched
[3,4].
The reversible redox properties of ferrocene have been strongly
associated with its biological activity [5]. To date, the most prom-
ising ferrocene-based drug candidates have been reported by
Jaouen and co-workers [6]. These anticancer compounds contain a
[3]-ferrocenophane motif and have
a potent in vitro anti-
proliferative effect in breast and prostate cancer cells lines [7,8].
This research group have prepared N-ferrocenoyl and N-ferro-
cenyl amino acid and peptide derivatives and investigated their
* Corresponding author. School of Chemical Sciences, Dublin City University,
Dublin 9, Ireland. Tel.: þ353 1 7005689; fax: þ353 1 7005503.
E-mail address: peter.kenny@dcu.ie (P.T.M. Kenny).
0022-328X/$ e see front matter Ó 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jorganchem.2014.01.031

A.G. Harry et al. / Journal of Organometallic Chemistry 757 (2014) 28e35
29
least in part to their low redox potentials, which are within the
range of biologically accessible potentials. In order to explore this
hypothesis further, alkyl groups were introduced on the previously
unsubstituted cyclopentadiene ring to further modify the redox
potentials.
All compounds gave spectroscopic data in accordance with the
proposed structures. The 1-alkyl-10-N-para-(ferrocenyl)benzoyl
dipeptide esters 4e12 were characterized by a combination of 1H
NMR, 13C NMR, DEPT-135 and 1He13C COSY (HMQC) spectroscopy,
cyclic voltammetry (CV) and electrospray ionization mass spec-
trometry (ESI).
We now report the synthesis and structural characterization of
novel 1-alkyl-10-N-para-(ferrocenyl)benzoyl dipeptide esters
which consist of four key moieties, namely (i) an electroactive core
(ii) a conjugate linker (iii) an alkyl group which further lowers the
oxidation potential of the ferrocene moiety and (iv) a dipeptide
ester. These novel derivatives differ from the previously reported N-
para-(ferrocenyl)benzoyl dipeptide esters by having an alkyl group
on the previously unsubstituted cyclopentadiene ring. The 1-alkyl-
10-N-para-(ferrocenyl)benzoyl dipeptide esters were prepared by
coupling the alkyl ferrocenyl benzoic acids 1e3 to the dipeptide
ethyl esters using the conventional N-(3-dimethylaminopropyl)-N0-
ethylcarbodiimide (EDC), 1-hydroxybenzotriazole (HOBt) protocol.
The dipeptide esters employed in the synthesis were Gly-L-
Ala(OEt), Gly-L-Leu(OEt) and Gly-L-Phe(OEt). All the compounds
were fully characterized using a combination of 1H NMR, 13C NMR,
DEPT-135 and 1He13C COSY (HMQC) spectroscopy, electrospray
ionization mass spectrometry (ESI-MS) and cyclic voltammetry
(CV). In addition, we present the in vitro anti-cancer activity of
selected compounds against the human lung carcinoma cell line
H1299.
2.2. 1H and 13C spectroscopic analysis
All the proton and carbon chemical shifts for compounds 4e12
were unambiguously assigned by a combination of DEPT-135 and
1He13C COSY (HMQC). The 1H and 13C NMR spectra for compounds
4e12 showed peaks in the ferrocene region characteristic of a
disubstituted ferrocene moiety [23e25]. For the disubstituted
ferrocene moiety, 3 splitting patterns are observed. For the cyclo-
pentadiene ring attached to the benzoyl spacer moiety (
benzoyl), the protons appear as either singlets or triplets between
4.88 and 4.27, whilst the protons on the alkylated cyclo-
pentadiene ring (
5-C5H4-alkyl) overlap with the signals of the
methylene groups of the glycine moiety, resulting in a multiplet
being observed between 3.97 to 3.79. For the alkyl groups
attached to the cyclopentadiene ring, the methyl (eCH3) group
proton signals are present between 1.63 to 1.4. The methylene
and methyl protons of the ethyl group appear as a quartet
between 2.1 to 2.0 and as a triplet between 0.98 to 0.76
respectively. For the propyl group the protons on the methylene
groups (eCH2CH2CH3) appear as a triplet between 2.07 to 1.9
and as a multiplet between 1.4 to 1.27 whilst, the protons on the
methyl group (eCH2CH2CH3) appear as a triplet between 0.74 to
0.66.
The 13C NMR spectra of compounds 4e12 show signals in the
region between 93.1 to 66.0 indicative of a disubstituted
ferrocene unit. The ipso carbon atom on the (
5-C5H4-alkyl) ring
appears between 93.1 to 89.8 whilst the ipso on the (
5-C5H4-
benzoyl) ring appears in the range 84.2 to 82.0. These signals are
absent in the DEPT-135 spectra. The carbon atoms on the benzoyl
linker appear between 142.5 and 124.0 for compounds 4e12.
h
5C5H4-
d
d
h
d
d
d
d
d
d
d
d
d
d
2. Results and discussion
d
d
d
2.1. Synthesis
d
2.1.1. Synthesis of 1-alkyl-10-N-para-(ferrocenyl)benzoyl dipeptide
esters 4e12
d
d
h
1-Alkyl-10-N-para-(ferrocenyl) benzoic acids 1e3 were prepared
by coupling either methyl, ethyl or propyl ferrocene to 4-
aminobenzoic acid via diazonium coupling. The 1H NMR spectra
d
d
h
d
d
showed signals for the aromatic ring protons between
d
7.86 and
d
d
d
7.62. The carboxylic acid proton was present in the narrow range
d 12.85e12.8. For the disubstituted ferrocene moiety, 3 splitting
The quaternary carbon atoms of the aromatic rings and the meth-
ylene carbon atoms of derivatives 4e12 were identified by DEPT-
135. Complete spectroscopic data for all the compounds is pre-
sented in the experimental section.
of
patterns are observed. For the cyclopentadiene ring attached to the
benzoyl spacer moiety (
5C5H4-benzoyl), the protons appear as
either singlets or triplets between 4.89 and 4.31, whilst the
protons on the alkylated cyclopentadiene ring (
5-C5H4-alkyl)
appear as doublets of doublets between 3.92e3.9.
h
d
d
h
2.3. Mass spectrometry
d
The free N-terminal dipeptide ethyl esters Gly-L-Ala(OEt), Gly-L-
Leu(OEt) and Gly-L-Phe(OEt), were coupled to the 1-alkyl-10-para-
(ferrocenyl) benzoic acids 1e3 using EDC and HOBt in the presence
of excess triethylamine in dichloromethane (Scheme 1). EDC was
used in preference to the less expensive coupling reagent N, N0-
dicyclohexylcarbodiimide (DCC) as its reaction by-products are
easier to remove compared to those of DCC, namely dicyclohex-
ylurea (DCU). Purification by column chromatography furnished
the pure products in yields of 14e25%. The relatively low yields of
the products is partly due to the coupling procedure. The first step
in amide bond formation in the coupling protocol is formation of
the O-acylisourea ester intermediate. This intermediate is highly
reactive and thus, side-reactions can pose a serious problem and
can result in extensive racemisation resulting in low yields. The
addition of HOBt stabilizes the O-acylisourea ester intermediate
thus supressing side reactions, however the addition does not
result in 100% suppression. As the reaction proceeds, upon addition
of the amino acid and dipeptide ethyl esters the HOBt is displaced
resulting in the formation of compounds 4e12. As a result of the
complexity of the reaction which is associated with the competing
reactions, low yields for compounds 4e12 were obtained.
Since the introduction of soft ionization techniques such as
matrix assisted laser desorption ionization (MADLI) and electro-
spray ionization (ESI), a wide range of thermolabile and non-
volatile compounds can be subjected to mass spectrometric anal-
ysis [26e28]. Compounds 4e12 were not amenable to electron
ionization studies, therefore electrospray ionization (ESI) mass
spectrometry was employed in the analysis and confirmed the
correct relative molecular mass for all the compounds. The ESI mass
spectra displayed both radical cation and [M þ H]þ species and
adducts due to sodium and potassium were present 22 Da and
38 Da higher than the protonated molecular ion species.
2.4. Electrochemistry
The CV curves for compounds 4e12 exhibit quasi-reversible
beþhaviour similar to the ferrocene/ferricenium redox couple (Fc/
Fc ). The Eꢁ0 (oxidation potential) values for compounds 4 (20 mV
versus Fc/Fcþ), 7 (30 mV versus Fc/Fcþ) and 10 (30 mV versus Fc/
Fcþ) are lower than that reported for N-{para(ferrocenyl)-benzoyl}-
glycine-
considerably lower than the ferrocenoyl dipeptide ester derivatives.
L
-alanine ethyl ester (73 mV versus Fc/Fcþ) [29]. It is also

30
A.G. Harry et al. / Journal of Organometallic Chemistry 757 (2014) 28e35
Scheme 1. Synthesis of 1-alkyl-10-N-para-(ferrocenyl) benzoyl dipeptide esters (i) NaNO2, HCl (ii) EDC, HOBt, triethylamine, dipeptide ethyl esters, R ¼ CH3; R0 ¼ Gly-L-Ala(OEt) 4,
Gly-L-Leu(OEt) 5, Gly-L-Phe(OEt) 6, R ¼ CH2CH3; R0 ¼ Gly-L-Ala(OEt) 7, Gly-L-Leu(OEt) 8, Gly-L-Phe(OEt) 9, R ¼ CH2CH2CH3; R0 ¼ Gly-L-Ala(OEt) 10, Gly-L-Leu(OEt) 11, Gly-L-
Phe(OEt) 12.
Foþr example, Fc-Ala-Ala-OMe, was reported as Eꢁ0 ¼ 230 mV (vs Fc/
Fc ) [30]. The benzoyl linker of the 1-methyl-10-N-{para-(ferro-
cenyl)-benzoyl}dipeptide esters provides extended conjugation to
the p-electrons of the Cp rings making initial oxidation of the iron
centre easier, relative to N-ferrocenoyl dipeptide esters. The methyl
phosphatase is an enzyme which dephosphorylates the p-nitro-
phenyl phosphate substrate converting it to p-nitrophenol which in
the presence of strong alkali can be quantified colorimetrically. The
acid phosphatase assay is highly sensitive and is easier to perform
than the neutral red assay as it involves fewer steps and fewer re-
agents. It is also more convenient than the MTT assay because of the
inherent problem of removal of the medium from the insoluble
crystals. The reproducibility between replicate wells is also excel-
lent in the acid phosphatase assay. To determine the IC50 values of
the three compounds, individual 96-well plates containing H1299
cells were treated with the test compound at concentrations
substituent on the Cp ring further lowers the oxidation potential.
2.5. In vitro anti-cancer activity of 4e12
The in vitro anti-proliferative effect of compounds 4e12 was
studied in the H1299 non-small cell lung cancer (NSCLC) cell line.
Proliferation was measured using the acid phosphatase assay as
previously described [31]. In a preliminary screen the cells were
treated with the 1-alkyl-10-N-para-(ferrocenyl) benzoyl dipeptide
ranging from 0.1 mM to 100 mM. The cells were then incubated for
5e6 days, until cell confluency was reached. Cell survival was
determined by performing the acid phosphatase assay. The IC50
value for each compound was calculated using Calcusyn software,
and standard deviations have been calculated using data obtained
from three independent experiments. The values obtained are lis-
ted in Table 2.
esters 4e12 at a concentration of 10 mM and were incubated for 5e6
days until cell confluency reached 80e90%. Cell survival was
established through determination of the acid phosphatase activity
of surviving cells and growth inhibition calculated relative to con-
trols (untreated cells). The results for the preliminary screen of
compounds 4e12 are depicted in Table 1. Of the nine compounds
screened the three most active compounds which showed %
growth inhibition values ꢂ70%, were selected for further evalua-
tion. A general trend was also observed in these derivatives, that is,
the Gly-L-Ala(OEt) derivatives are more active than the Gly-L-Leu
and Gly-L-Phe ethyl esters which display % growth inhibition
values ꢃ 55%. For instance 1-ethyl-10-N-{para-(ferrocenyl)-ben-
From the IC50 values of the 1-alkyl-10-N-{para-(ferrocenyl)-
benzoyl}-glycine- -alanine ethyl esters 4, 7 and 10 it can be seen
L
Table 1
% Growth inhibition values for compounds 4e12 against human lung carcinoma cell
line H1299.
Compound name
% Growth inhibition
at 10
m
M
zoyl}-glycine-L-alanine ethyl ester 7 displayed a % growth
1-Methyl-10-N-{para-(ferrocenyl)-benzoyl}-
83 ꢄ 1.0
28 ꢄ 18.6
20 ꢄ 9.9
84 ꢄ 6.3
34 ꢄ 10.6
19 ꢄ 13.6
80 ꢄ 4.5
55 ꢄ 10.6
34 ꢄ 10.6
inhibition value of 84 ꢄ 6.3% whereas the 1-ethyl-10-N-{para-(fer-
glycine-
1-Methyl-10-N-{para-(ferrocenyl)-benzoyl}-
glycine- -leucine ethyl ester 5
1-Methyl-10-N-{para-(ferrocenyl)-benzoyl}-
glycine- -phenylalanine ethyl ester 6
1-Ethyl-10-N-{para-(ferrocenyl)-benzoyl}-
glycine- -alanine ethyl ester 7
1-Ethyl-10-N-{para-(ferrocenyl)-benzoyl}-
glycine- -leucine ethyl ester 8
1-Ethyl-10-N-{para-(ferrocenyl)-benzoyl}-
glycine- -phenylalanine ethyl ester 9
1-Propyl-10-N-{para-(ferrocenyl)-benzoyl}-
glycine- -alanine ethyl ester 10
1-Propyl-10-N-{para-(ferrocenyl)-benzoyl}-
glycine- -leucine ethyl ester 11
1-Propyl-10-N-{para-(ferrocenyl)-benzoyl}-
glycine- -phenylalanine ethyl ester 12
L-alanine ethyl ester 4
rocenyl)-benzoyl}-glycine- -leucine ethyl ester 8 and the 1-ethyl-
L
10-N-{para-(ferrocenyl)-benzoyl}-glycine-
L-phenylalanine
ethyl
L
ester 9 displayed % growth inhibition values of 34 ꢄ 10.6% and
L
19 ꢄ 13.6% respectively. Therefore the 1-methyl, 1-ethyl and 1-
propyl-10-N- {para-(ferrocenyl)-benzoyl}-glycine-
L-leucine ethyl
L
esters and the 1-methyl, 1-ethyl and 1-propyl-10-N-{para-(ferro-
cenyl)-benzoyl}-glycine- -phenylalanine ethyl ester derivatives
were not investigated further. It can be concluded that when chiral
-amino acids with bulky side chains are used as the second amino
L
L
L
a
acid in the dipeptide moiety a loss of anti-proliferative activity is
observed. The IC50 values were determined for compounds 4, 7 and
10 using the acid phosphatase assay. This colorimetric end-point
assay is an indirect measure of cytotoxicity which evaluates the
enzyme activity of cells after a given treatment period. Acid
L
L
L

A.G. Harry et al. / Journal of Organometallic Chemistry 757 (2014) 28e35
31
Table 2
against the human lung carcinoma cell line H1299. The compounds
showed micromolar activity in the H1299 NSCLC cell line, with IC50
values of 4.5, 5.6 and 6.6 mM respectively. Alkylation of the previ-
ously unsubstituted cyclopentadiene ring increases the cytotoxicity
of the ferrocenyl bioconjugates. The cytotoxicity of the derivatives
decreases with an increase of the size of the alkyl group on the
previously unsubstituted cyclopentadiene ring.
IC50 values for compounds 4, 7, 10, 15, carboplatin 13 and cisplatin 14 against human
lung carcinoma cell line H1299.
Compound
IC50 value
M)
(
m
Carboplatin
Cisplatin
1-Methyl-10-N-{para-(ferrocenyl)-benzoyl}-
glycine-
N-{para-(ferrocenyl)-benzoyl}-
glycine- -alanine ethyl ester 15
1-Ethyl-10-N-{para-(ferrocenyl)-benzoyl}-
glycine- -alanine ethyl ester 7
1-Propyl-10-N-{para-(ferrocenyl)-benzoyl}-
glycine- -alanine ethyl ester 10
10.0 ꢄ 1.6
1.5 ꢄ 0.1
4.5 ꢄ 0.4
L-alanine ethyl ester 4
6.6 ꢄ 0.7
5.6 ꢄ 1.6
6.6 ꢄ 2.1
4. Experimental
L
4.1. General procedures
L
L
All chemicals were purchased from SigmaeAldrich, Lennox
Chemicals, Fluorochem Limited or Tokyo Chemical Industry UK
Limited; and used as received. Commercial grade reagents were
used without further purification. When necessary, all solvents
were purified and dried prior to use. Riedel-Haën silica gel was
used for thin layer and column chromatography. Melting points
were determined using a Stuart melting point (SMP3) apparatus
and are uncorrected. Optical rotation measurements were made on
a Perkin Elmer 343 Polarimeter and are quoted in units of
10ꢅ1 deg cm2 gꢅ1. Infrared spectra were recorded on a Perkin Elmer
Spectrum 100 FT-IR with ATR. UVeVis spectra were recorded on a
Hewlett Packard 8452 A diode array UVeVis spectrophotometer. 1H
and 13C NMR spectra were recorded in deuterated solvents on a
Bruker Avance 400 NMR. The 1H and 13C NMR chemical shifts are
reported in ppm (parts per million). Tetramethylsilane (TMS) or the
residual solvent peaks have been used as an internal reference. All
coupling constants (J) are in Hertz. Electrospray ionisation mass
spectra were performed on a Micromass LCT mass spectrometer.
Cyclic voltammograms were recorded in anhydrous acetonitrile
that the cytotoxicity of the derivatives decreases with an increase of
the size of the alkyl group incorporated (propyl < ethyl < methyl).
The in vitro cytotoxicity of the platinum(II)-based anti-cancer drug
carboplatin 13 was also evaluated against the H1299 cell line, and
was found to have an IC50 value of 10.0 ꢄ 1.6
mM. Thus compounds
4, 7 and 10 are more cytotoxic in vitro than the clinically employed
anti-cancer drug carboplatin. 1-Methyl-10-N-{para-(ferrocenyl)-
benzoyl}-glycine-
4.5 M (Fig. 1). This is lower than N-{para-(ferrocenyl)-benzoyl}-
glycine- -alanine ethyl ester 15 which displayed an IC50 value of
6.6 M [16]. Therefore alkylation of the previously unsubstituted
L-alanine ethyl ester 4 displayed an IC50 value of
m
L
m
cyclopentadiene ring increases the cytotoxicity of the ferrocenyl
bioconjugates. However compounds 4, 7 and 10 are less active
compared to cisplatin 14 which displays an IC50 value of 1.5 mM
against human H1299 lung carcinoma cells. A potential mechanism
by which these novel organometallic anticancer compounds may
induce DNA damage is by the catalytic generation of reactive
oxygenated species (ROS). This is possible via a Fenton-type reac-
tion, in which HOꢀ radicals are generated from the superoxide
dismutation product, hydrogen peroxide (H2O2). It was shown that
the generation of 8-oxo-7,8-dihydroguanine (8-oxoGua) by a
compound prepared in a previous SAR study, N-(6-ferrocenyl-2-
naphthoyl)-glycine-glycine ethyl ester, that the oxidation was
occurring by Fenton chemistry and that N-(6-ferrocenyl-2-
naphthoyl)-glycine-glycine ethyl ester is generating oxidative
(SigmaeAldrich), with 0.1
M
tetrabutylammonium hexa-
fluorophosphate (TBAPF6) as a supporting electrolyte, using a CH
Instruments 600a electrochemical analyzer (Pico-Amp Booster and
Faraday Cage). The experiments were carried out at room tem-
perature. A three-electrode cell consisting of a glassy carbon
working-electrode, a platinum wire counter-electrode and an
AgjAgCl reference electrode was used. The glassy carbon electrode
was polished with 0.3 mm alumina followed by 0.05 mm alumina,
between each experiment to remove any surface contaminants. The
scan rate was 0.1 V sꢅ1. The concentration range of the ferrocene
compounds was 1.0 mM in acetonitrile. The E00 values obtained for
the test samples were referenced relative to the ferrocene/ferrice-
nium redox couple.
damage via
a ROS-mediated mechanism. Therefore guanine
oxidation studies confirmed that N-(6-ferrocenyl-2-naphthoyl)-
glycine-glycine ethyl ester was capable of causing oxidative dam-
age to guanine, and it does so by the generation of HOꢀ radicals
from H2O2 [20].
4.2. General procedure for the synthesis of the starting materials
3. Conclusion
4.2.1. 1-Methyl-10-para-ferrocenyl benzoic acid 1
In conclusion, the novel 1-alkyl-10-N-para-(ferrocenyl)benzoyl
dipeptide esters were synthesized and fully characterized by a
range of NMR spectroscopic techniques, mass spectrometry and
cyclic voltammetry. Compounds 4, 7 and 10 were tested in vitro
4-Aminobenzoic acid (3.43 g, 25.00 mmol) was dissolved in
deionised water (30 ml) at 0 ꢁC followed by the addition of
concentrated HCl (7 ml). Sodium nitrite (1.72 g, 25.00 mmol) was
dissolved in deionised water (20 ml) at 0 ꢁC and was then added to
the 4-aminobenzoic acid solution with stirring at a temperature
less than 5 ꢁC. The resulting pale yellow diazonium salt was then
added to a solution of methylferrocene (5.00 g, 25.00 mmol) in dry
diethyl ether (100 ml) and the reaction mixture was allowed to stir
at room temperature for 48 h. The reaction mixture was then
washed with water and brine and the organic layer was dried over
MgSO4. The solvent was removed in vacuo to yield the crude
product. The crude product was purified by column chromatog-
raphy {eluant 1:1 hexane: ethyl acetate} yielding the title com-
pound as a red oil (1.65 g, 21%).
1H NMR (400 MHz)
d, J ¼ 7.2 Hz, ArH), 7.67 (2H, d, J ¼ 7.2 Hz, ArH), 4.89 (2H, s, ortho on
d (DMSO-d6): 12.8 (1H, s, eCOOH), 7.79 (2H,
Fig. 1. 1-Methyl-10-N-{para-(ferrocenyl)-benzoyl}-glycine-L-alanine ethyl ester 4.

32
A.G. Harry et al. / Journal of Organometallic Chemistry 757 (2014) 28e35
h
dd,
5-C5H4-benzoyl), 4.41 (2H, s, meta on
h
5-C5H4-benzoyl), 3.9 (4H,
J ¼ 8.4 Hz, ArH), 4.81 (2H, s, ortho on
meta on
5-C5H4-benzoyl), 4.31e4.24 ( 1H, m, eCHCH3), 4.05 (2H, q,
5-C5H4-alkyl), (e
h
5-C5H4-benzoyl), 4.36 (2H, s,
h
5-C5H4-alkyl), 1.62 (3H, s, eCH3).
h
13C NMR (100 MHz)
d
(DMSO-d6): 174.3 (C]O), 140.1 (Cq), 132.4
J ¼ 6.8 Hz, eOCH2CH3), 3.96e3.85 {6H, m, (
h
(Cq), 126.5, 123.9, 91.6 (Cipso h5-C5H4-alkyl), 80.7 (Cipso h5-C5H4-ben-
NHCH2COe)}, 1.6 (3H, s, eCH3), 1.31 (3H, d, J ¼ 6.2 Hz, eCHCH3), 1.18
zoyl), 73.2 (Cmeta h h
5-C5H4-benzoyl), 69.8(Cmeta 5-C5H4-alkyl), 68.0
(3H, t, J ¼ 6.8 Hz, eOCH2CH3).
(Cortho h5-C5H4-alkyl), 67.5 (Cortho h5-C5H4-benzoyl), 13.1(eCH3).
13C NMR (100 MHz)
d (DMSO-d6): 172.5 (C]O), 169.0 (C]O),
166.5 (C]O), 142.5 (Cq), 130.8 (Cq), 127.4, 125.2, 92.3 (Cipso h5-C5H4-
alkyl), 82.0 (Cipso
5-C5H4-benzoyl), 71.0 (Cmeta 5-C5H4-benzoyl),
69.8 (Cmeta
4.2.2. 1-Ethyl-10-para-ferrocenyl benzoic acid 2
h
h
4-Aminobenzoic acid (3.20 g, 23.36 mmol) and ethylferrocene
(5.00 g, 23.36 mmol) were used as starting materials. Recrystalli-
sation from chloroform furnished the desired product as an orange
solid (1.80 g, 23%).
h h
5-C5H4-alkyl), 68.5 (Cortho 5-C5H4-alkyl), 66.9 (Cortho
h
5-C5H4-benzoyl), 60.9 (eOCH2CH3, ꢅve DEPT), 47.6 (eCHCH3),
42.0 (eNHCH2CHOe, ꢅve DEPT), 17.0 (eCHCH3), 14.5 (eOCH2CH3),
14.0 (eCH3).
1H NMR (400 MHz)
d (DMSO-d6): 12.85 (1H, s, eCOOH), 7.86
(2H, d, J ¼ 7.6 Hz, ArH), 7.62 (2H, d, J ¼ 7.6 Hz, ArH), 4.82 (2H, t,
4.3.2. 1-Methyl-10-N-{para-(ferrocenyl)-benzoyl}-glycine-L-leucine
ethyl ester 5
J ¼ 1.6 Hz, ortho on
h
5-C5H4-benzoyl), 4.31 (2H, t, J ¼ 1.6 Hz, meta on
h
5-C5H4-benzoyl), 3.91 (4H, dd,
h
5-C5H4-alkyl), 2.04 (2H, q,
Glycine-L-leucine ethyl ester hydrochloride (0.17 g, 0.78 mmol)
J ¼ 7.2 Hz, eCH2CH3), 0.97 (3H, t, J ¼ 7.2 Hz, eCH2CH3).
13C NMR (100 MHz)
(DMSO-d6): 167.3 (C]O), 144.3 (Cq), 129.4
(Cq), 127.5, 125.4, 91.6 (Cipso
5-C5H4-alkyl), 81.3 (Cipso h5
C5H4-benzoyl), 70.2 (Cmeta
5-C5H4-benzoyl), 68.8 (Cmeta 5-C5H4-
alkyl), 68.6 (Cortho
5-C5H4-alkyl), 67.0 (Cortho 5-C5H4-benzoyl),
was used as a starting material. The crude product was purified by
column chromatography {eluant 1:1 hexane: ethyl acetate} and
recrystallisation from hexane: ethyl acetate yielded the desired
d
h
-
h
h
product as an orange solid (0.07 g, 17%), m.p 142e144 ꢁC;
20
h
h
Eꢁ0 ¼ 25 mV (vs Fc/Fcþ); [
a
]
D
¼ ꢅ17ꢁ (c 0.1, EtOH).
20.7 (eCH2CH3, ꢅve DEPT), 14.4 (eCH2CH3).
Mass spectrum: [M þ Na]þ found: 541.1884.
C
28H34N2O4FeNa requires: 541.1888.
4.2.3. 1-Propyl-10-para-ferrocenyl benzoic acid 3
I.R. ymax (KBr): 3267 (NH), 1742 (C]Oester), 1640 (C]Oamide),
1610 (C]Oamide) cmꢅ1
UVeVis lmax EtOH: 358, 450 nm.
1H NMR (400 MHz)
(DMSO-d6): 8.65 (1H, t, J ¼ 6 Hz, eCONHe),
8.33 (1H, d, J ¼ 7.6 Hz, eCONHe), 7.8 (2H, d, J ¼ 8.0 Hz, ArH), 7.6 (2H,
d, J ¼ 8.0 Hz, ArH), 4.82 (2H, s, ortho on
5-C5H4-benzoyl), 4.37e
4.29 {3H, m, (meta on
5-C5H4-benzoyl), (eCH(CH2CH(CH3)2)}, 4.08
(2H, q, J ¼ 7.2 Hz, eOCH2CH3), 3.96e3.85 {6H, m, (
5-C5H4-alkyl),
(eNHCH2COe)}, 1.63e1.4 {6H, m, (eCH3), (eCH(CH2CH(CH3)2)},
1.18 (3H, t, 7.2 Hz, eOCH2CH3), 0.91e0.89 {6H, m,
CH(CH2CH(CH3)2};
13C NMR (100 MHz)
166.2 (C]O), 142.2 (Cq), 130.8 (Cq), 127.4, 125.2, 90.4 (Cipso h5
-
4-Aminobenzoic acid (3.01 g, 21.93 mmol) and propyl ferrocene
(5.00 g, 21.93 mmol)) were used as starting materials. Recrystalli-
sation from chloroform furnished the desired product as an orange
solid (1.61 g, 21%).
.
d
1H NMR (400 MHz)
d
(DMSO-d6): 12.85 (1H, s, eCOOH), 7.86
h
(2H, d, J ¼ 7.4 Hz, ArH), 7.62 (2H, d, J ¼ 7.4 Hz, ArH), 4.82 (2H, s, ortho
h
on
(4H, dd,
h
5-C5H4-benzoyl), 4.38 (2H, s, meta on
h
5-C5H4-benzoyl), 3.92
h
h
5-C5H4-alkyl), 1.94 (2H, t, J ¼ 7.6 Hz, eCH2CH2CH3), 1.4e
1.27 (2H, m, eCH2CH2CH3), 0.75 (3H, t, J ¼ 7.6 Hz, eCH2CH2CH3).
J
¼
e
13C NMR (100 MHz)
(Cq), 127.5, 125.4, 89.3 (Cipso
C5H4-benzoyl), 70.1 (Cmeta
5-C5H4-benzoyl), 68.8 (Cmeta
alkyl), 68.5 (Cortho
5-C5H4-alkyl), 67.0 (Cortho 5-C5H4-benzoyl),
d
( DMSO-d6): 167.3 (C]O), 144.3 (Cq), 129.3
5-C5H4-alkyl), 81.7 (Cipso h5
5-C5H4-
h
-
d (DMSO-d6): 172.5 (C]O), 169.2 (C]O),
h
h
h
h
C5H4-alkyl), 84.2 (Cipso
benzoyl), 70.0 (Cmeta
h
5-C5H4-benzoyl), 70.1 (Cmeta
h
h
5-C5H4-
5-C5H4-
32.3 (eCH2CH2CH3, ꢅve DEPT), 22.0 (eCH2CH2CH3, ꢅve DEPT), 14.4
h
5-C5H4-alkyl), 68.4 (Cortho
(eCH2CH2CH3).
alkyl), 66.9 (Cortho
h
5-C5H4-benzoyl), 60.4 (eOCH2CH3, ꢅve DEPT),
50.3 {eCH(CH2CH(CH3)2}, 42.0 (eNHCH2CHOe, ꢅve DEPT), 39.4 {e
CH(CH2CH(CH3)2), ꢅve DEPT}, 24.2 {eCH(CH2CH(CH3)2}, 22.7 (e
CH(CH2CH(CH3)2}, 21.4 {eCH(CH2CH(CH3)2},14.0 (eOCH2CH3), 13.2
(eCH3).
4.3. General procedure for the synthesis of 1-alkyl-10-N-ferrocenyl
benzoyl dipeptide ethyl esters
4.3.1. 1-Methyl-10-N-{para-(ferrocenyl)-benzoyl}-glycine-L-alanine
ethyl ester 4
4.3.3. 1-Methyl-10-N-{para-(ferrocenyl)-benzoyl}-glycine-
L-
phenylalanine ethyl ester 6
1-Methyl-10-N-para-ferrocenyl benzoic acid (0.25 g, 0.78 mmol)
was dissolved in dichloromethane (100 ml) at
0
ꢁC. N-
Glycine-L-phenylalanine ethyl ester hydrochloride (0.20 g,
(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride
0.78 mmol) was used as a starting material. The crude product was
purified by column chromatography {eluant 1:1 hexane: ethyl ac-
etate} and recrystallisation from hexane: ethyl acetate yielded the
(0.15 g, 0.78 mmol), 1-hydroxybenzotriazole (0.11 g, 0.78 mmol),
glycine-L-alanine ethyl ester hydrochloride (0.14 g, 0.78 mmol). and
triethylamine (3 ml) were added and the reaction mixture was
allowed to stir at 0 ꢁC for 45 min. The reaction mixture was then
washed with water and brine. The organic layer was then dried
over MgSO4. The solvent was removed in vacuo to yield the crude
product. The crude product was purified by column chromatog-
raphy {eluant 1:1 hexane: ethyl acetate} yielding the title com-
desired product as an orange solid (0.07 g, 16%), m.p 82e83 ꢁC;
20
[a
]
¼ þ6ꢁ (c 0.1, EtOH).
D
Mass spectrum: [M þ Na]þ found: 575.1794.
C
31H32N2O4FeNa requires: 575.1796.
I.R. ymax (KBr): 3263 (NH), 1735 (C]Oester), 1663 (C]Oamide),
1609 (C]Oamide) cmꢅ1
UVeVis lmax EtOH: 358, 448 nm.
1H NMR (400 MHz)
.
pound as a red oil (0.08 g, 22%), Eꢁ0 ¼ 20 mV (vs Fc/Fcþ);
20
[
a]
¼ ꢅ10ꢁ (c 0.1, EtOH).
d
( DMSO-d6): 8.68 (1H, t, J ¼ 6.4 Hz, e
D
Mass spectrum: [M þ Na]þ found: 499.1394.
CONHe), 8.34 (1H, d, J ¼ 7.6 Hz, eCONHe), 7.81 (2H, d, J ¼ 8.4 Hz,
ArH), 7.60 (2H, d, J ¼ 8.4 Hz, ArH), 7.27e7.21 {5H, m, eCH(CH2Ph)},
C25H28N2O4FeNa requires: 499.1396.
I.R. ymax (KBr): 3295 (NH), 1743 (C]Oester), 1636 (C]Oamide),
1608 (C]Oamide) cmꢅ1
UVeVis lmax EtOH: 360, 451 nm.
1H NMR (400 MHz)
(DMSO-d6):8.67 (1H, t, J ¼ 6.4 Hz, eCONHe),
8.41(1H, d,J¼ 7.0 Hz, eCONHe),7.8(2H, d,J¼ 8.4Hz, ArH),7.61(2H,d,
4.8 (2H, s, ortho on h
5-C5H4-benzoyl), 4.47e4.45 {1H, m, e
.
CH(CH2Ph)}, 4.35 (2H, s, meta on
h
5-C5H4-benzoyl), 4.04 (2H, q,
5-C5H4-alkyl), (e
J ¼ 7.2 Hz, eOCH2CH3), 3.96e3.85 {6H, m, (
h
d
NHCH2COe)}, 3.06e2.93 {2H, m, eCH(CH2Ph)}, 1.57 (3H, s, eCH3),
1.12 ( 3H, t, J ¼ 7.2 Hz, eOCH2CH3).

A.G. Harry et al. / Journal of Organometallic Chemistry 757 (2014) 28e35
33
13C NMR (100 MHz)
166.3 (C]O), 142.0 (Cq) 136.9 (Cq), 130.7 (Cq), 129.1, 128.0, 127.4,
126.0, 125.2, 90.2 (Cipso
5-C5H4-alkyl), 82.0 (Cipso 5-C5H4-ben-
zoyl), 71.4 (Cmeta
5-C5H4-benzoyl), 71.0 (Cmeta 5-C5H4-alkyl),
68.0 (Cortho
5-C5H4-alkyl), 66.4 (Cortho 5-C5H4-benzoyl), 61.4 (e
OCH2CH3, ꢅve DEPT), 53.6 {eCH(CH2Ph)}, 42.9 (eNHCH2CHOe
, ꢅve DEPT), 36.7 {eCH(CH2Ph), ꢅve DEPT}, 14.0 (eOCH2CH3), 13.2
(eCH3).
d
(DMSO-d6): 172.4 (C]O), 169.2 (C]O),
{eCH(CH2CH(CH3)2)}, 23.4 (eCH2CH3, ꢅve DEPT), 22.9 {e
CH(CH2CH(CH3)2)}, 21.0 {eCH(CH2CH(CH3)2)},15.1 (eOCH2CH3), 13.0
(eCH2CH3).
h
h
h
h
h
h
4.3.6. 1-Ethyl-10-N-{para-(ferrocenyl)-benzoyl}-glycine-
L-
phenylalanine ethyl ester 9
Glycine-L-phenylalanine ethyl ester hydrochloride (0.19 g,
0.75 mmol) was used as a starting material. The crude product
was purified by column chromatography {eluant 1:1 hexane:
ethyl acetate} and recrystallisation from hexane: ethyl
4.3.4. 1-Ethyl-10-N-{para-(ferrocenyl)-benzoyl}-glycine-
L-alanine
ethyl ester 7
Glycine-L-alanine ethyl ester hydrochloride (0.13 g, 0.75 mmol)
was used as a starting material. The crude product was purified by
acetate yielded the desired product as an orange solid (0.07 g,
20
17%), m.p 92e94 ꢁC; Eꢁ0 ¼ ꢅ30 mV (vs Fc/Fcþ); [
(c 0.1, EtOH).
a
]
¼ þ12ꢁ
D
column chromatography {eluant 1:1 hexane: ethyl acetate}
Mass spectrum: [M þ Na]þ found: 589.1770.
yielding the title compound as a red oil (0.09 g, 25%), Eꢁ0 ¼ 30 mV
C32H34N2O4FeNa requires: 589.1769.
20
(vs Fc/Fcþ); [
a
]
D
¼ ꢅ16ꢁ (c 0.1, EtOH).
I.R. ymax (KBr): 3303 (NH), 1742 (C]Oester), 1629 (C]Oamide),
1608 (C]Oamide) cmꢅ1
UVeVis lmax EtOH: 356, 450 nm.
1H NMR (400 MHz)
Mass spectrum: [M þ Na]þ found: 513.1454.
.
C
26H30N2O4FeNa requires: 513.1453.
I.R. ymax (KBr): 3290 (NH), 1753 (C]Oester), 1626 (C]Oamide),
1608 (C]Oamide) cmꢅ1
UVeVis lmax EtOH: 359, 449 nm.
1H NMR (400 MHz)
(DMSO-d6): 8.68 (1H, t, J ¼ 6 Hz, eCONHe),
8.4 (1H, t, J ¼ 7.2 Hz, eCONHe), 7.81 (2H, d, J ¼ 7.8 Hz, ArH), 7.6 (2H,
d, J ¼ 7.8 Hz, ArH), 4.82 (2H, t, J ¼ 1.6 Hz, ortho on
5-C5H4-benzoyl),
d
(DMSO-d6): 8.6 (1H, t, J ¼ 6.0 Hz, eCONHe
.
), 8.37 (1H, d, J ¼ 7.6 Hz, eCONHe), 7.85 (2H, d, J ¼ 7.4 Hz, ArH), 7.63
(2H, d, J ¼ 7.4 Hz, ArH), 7.27e7.21 {5H, m, eCH(CH2Ph)}, 4.83 (2H, t,
d
J ¼ 1.6 Hz, ortho on
h
5-C5H4-benzoyl), 4.48e4.46 {1H, m, e
CH(CH2Ph)}, 4.37 (2H, t, J ¼ 1.6 Hz, meta on
h
5-C5H4-benzoyl), 4.00
5-C5H4-alkyl), (e
h
(2H, q, J ¼ 6.8 Hz, eOCH2CH3), 3.9e3.83 {6H, m, (
h
4.37 (2H, t, J ¼ 1.6 Hz, meta on
h
5-C5H4-benzoyl), 4.28e4.25 (1H, m,
NHCH2COe)}, 3.06e2.93 {2H, m, eCH(CH2Ph)}, 2.08 (2H, q,
J ¼ 7.6 Hz, eCH2CH3), 1.12 (3H, t, J ¼ 6.8 Hz, eOCH2CH3), 0.98 (3H, t,
J ¼ 7.6 Hz, eCH2CH3).
eCHCH3), 4.15 (2H, q, J ¼ 6.8 Hz, eOCH2CH3), 3.9e3.85 {6H, m, (h5
-
C5H4-alkyl), (eNHCH2COe)}, 2.1 (2H, q, J ¼ 7.6 Hz, eCH2CH3), 1.31
(3H, d, J ¼ 6.4 Hz, eCHCH3), 1.18 (3H, t, J ¼ 6.8 Hz, eOCH2CH3), 0.98
(3H, t, J ¼ 7.6 Hz, eCH2CH3).
13C NMR (100 MHz)
d (DMSO-d6): 170.8 (C]O), 168.2 (C]O),
166.7 (C]O), 137.0 (Cq), 132.7 (Cq), 130.1 (Cq), 128.9, 127.2, 126.9,
124.0, 120.2, 93.1 (Cipso h5-C5H4-alkyl), 84.0 (Cipso h5-C5H4-benzoyl),
71.4 (Cmeta h5-C5H4-benzoyl), 68.4 (Cmeta h5-C5H4-alkyl), 67.5 (Cortho
13C NMR (100 MHz)
d (DMSO-d6): 171.7 (C]O), 168.7 (C]O),
167.2 (C]O), 142.5 (Cq), 130.8 (Cq), 127.4, 125.2, 92.3 (Cipso h5-C5H4-
alkyl), 82.0 (Cipso
5-C5H4-benzoyl), 71.0 (Cmeta 5-C5H4-benzoyl),
69.8 (Cmeta
h
h
h
5-C5H4-alkyl), 66.4 (Cortho h5-C5H4-benzoyl), 61.2 (eOCH2CH3, ꢅve
h
5-C5H4-alkyl), 68.7 (Cortho 5-C5H4-alkyl), 66.0 (Cortho
h
DEPT), 51.6 (eCH(CH2Ph)), 43.0 (eNHCH2CHOe, ꢅve DEPT), 38.0 {e
CH(CH2Ph), ꢅve DEPT}, 24.5 (eCH2CH3, ꢅve DEPT), 13.3
(-OCH2CH3), 13.0 (eCH2CH3).
h
5-C5H4-benzoyl), 61.4 (eOCH2CH3, ꢅve DEPT), 48.0 (eCHCH3),
43.0 (eNHCH2CHOe, ꢅve DEPT), 22.8 (eCH2CH3, ꢅve DEPT), 18.0
(eCHCH3), 14.5 (eOCH2CH3), 13.9 (eCH2CH3).
4.3.5. 1-Ethyl-10-N-{para-(ferrocenyl)-benzoyl}-glycine-L-leucine
4.3.7. 1-Propyl-10-N-{para-(ferrocenyl)-benzoyl}-glycine-L-alanine
ethyl ester 8
ethyl ester 10
Glycine-
L
-leucine ethyl ester hydrochloride (0.16 g, 0.75 mmol)
Glycine-L-alanine ethyl ester hydrochloride (0.13 g, 0.72 mmol)
was used as a starting material. The crude product was purified by
column chromatography {eluant 1:1 hexane: ethyl acetate} and
recrystallisation from hexane: ethyl acetate yielded the desired
was used as a starting material. The crude product was purified by
column chromatography {eluant 1:1 hexane: ethyl acetate}
yielding the title compound as a red oil (0.08 g, 22%), Eꢁ0 ¼ 30 mV
20
product as an orange solid (0.07 g, 18%), m.p 97e99 ꢁC; Eꢁ0 ¼ 10 mV
(vs Fc/Fcþ); [
a]
¼ ꢅ14ꢁ (c 0.1, EtOH).
D
20
(vs Fc/Fcþ); [
a
]
D
¼ ꢅ12ꢁ (c 0.1, EtOH).
Mass spectrum: [M þ Na]þ found: 527.1632.
Mass spectrum: [M þ Na]þ found: 555.1919.
C26H30N2O4FeNa requires: 527.1609.
C
29H36N2O4FeNa requires: 555.1922.
I.R. ymax (KBr): 3295 (NH), 1743 (C]Oester), 1636 (C]Oamide),
1608 (C]Oamide) cmꢅ1
UVeVis lmax EtOH: 360, 451 nm.
1H NMR (400 MHz)
(DMSO-d6): 8.68 (1H, t, J ¼ 6 Hz, eCONHe),
8.41 (1H, d, J ¼ 7.0 Hz, eCONHe), 7.78 (2H, d, J ¼ 7.4 Hz, ArH), 7.64
(2H, d, J ¼ 7.4 Hz, ArH), 4.82 (2H, s, ortho on
5-C5H4-benzoyl), 4.37
(2H, s, meta on
5-C5H4-benzoyl), 4.3e4.25 (1H, m, eCHCH3), 4.08
5-C5H4-alkyl),
I.R. ymax (KBr): 3280 (NH), 1740 (C]Oester), 1641 (C]Oamide),
1615 (C]Oamide) cmꢅ1
UVeVis lmax EtOH: 357, 446 nm.
1H NMR (400 MHz)
(DMSO-d6): 8.68 (1H, t, J ¼ 6 Hz, eCONHe),
8.34 (1H, d, J ¼ 7.6 Hz, eCONHe), 7.82 (2H, d, J ¼ 8.2 Hz, ArH), 7.61
(2H, d, J ¼ 8.2 Hz, ArH), 4.81 (2H, t, J ¼ 1.6 Hz, ortho on
5-C5H4-
benzoyl), 4.36 (2H, t, J ¼ 1.6 Hz, meta on
5-C5H4-benzoyl), 4.3e4.25
{1H, m, eCH(CH2CH(CH3)2)}, 4.15 (2H, q, J ¼ 7.2 Hz, eOCH2CH3),
3.97e3.87 {6H, m, (
5-C5H4-alkyl), (eNHCH2COe)}, 2.0 (2H, q,
;
.
d
d
h
h
h
h
(2H, q, J ¼ 6.8 Hz, eOCH2CH3), 3.95e3.88 {6H, m, (
h
(eNHCH2COe)}, 2.07 (2H, t, J ¼ 7.6 Hz, eCH2CH2CH3), 1.4e1.27 {5H,
m, (eCH2CH2CH3), (eCHCH3)}, 1.18 (3H, t, J ¼ 6.8 Hz, eOCH2CH3),
0.74 (3H, t, J ¼ 7.2 Hz, eCH2CH2CH3).
h
J ¼ 7.2 Hz, eCH2CH3),1.69e1.5 {3H, m, eCH(CH2CH(CH3)2)}, 1.15 (3H,
t, J ¼ 7.2 Hz, eOCH2CH3), 0.9e0.85 {6H, m, eCH(CH2CH(CH3)2)}, 0.76
(3H, t, J ¼ 7.2 Hz, eCH2CH3).
13C NMR (100 MHz)
d (DMSO-d6): 171.0 (C]O), 169.2 (C]O),
168.6 (C]O), 141.5 (Cq), 132.8 (Cq), 128.4, 124.2, 90.3 (Cipso h5-C5H4-
alkyl), 83.0 (Cipso
5-C5H4-benzoyl), 71.5 (Cmeta 5-C5H4-benzoyl),
68.7 (Cmeta
13C NMR (100 MHz)
d
(DMSO-d6): 173.4 (C]O),170.0 (C]O),166.9
h
h
(C]O), 140.1 (Cq), 130.8 (Cq), 127.4, 125.2, 92.1 (Cipso h5-C5H4-alkyl),
83.1 (Cipso h5-C5H4-benzoyl), 71.0 (Cmeta h5-C5H4-benzoyl), 69.9 (Cmeta
h h
5-C5H4-alkyl), 68.2 (Cortho 5-C5H4-alkyl), 67.9 (Cortho
h
5-C5H4-benzoyl), 62.4 (eOCH2CH3, ꢅve DEPT), 49.6 (eCHCH3),
h
5-C5H4-alkyl), 68.1 (Cortho
h
5-C5H4-alkyl), 67.2 (Cortho
h
5-C5H4-ben-
43.0 (eNHCH2CHOe, ꢅve DEPT), 32.0 (eCH2CH2CH3, eve DEPT),
22.8 (eCH2CH2CH3, eve DEPT), 17.6 (eCHCH3), 14.8 (eOCH2CH3),
13.9 (eCH2CH2CH3).
zoyl), 61.4 (eOCH2CH3, ꢅve DEPT), 51.3 {eCH(CH2CH(CH3)2)}, 42.6 (e
NHCH2CHOe, ꢅve DEPT), 39.3 {eCH(CH2CH(CH3)2), ꢅve DEPT}, 28.1

34
A.G. Harry et al. / Journal of Organometallic Chemistry 757 (2014) 28e35
4.3.8. 1-Propyl-10-N-{para-(ferrocenyl)-benzoyl}-glycine-leucine
4.4. General procedure for in vitro cytotoxicity assays
ethyl ester 11
Glycine-
L
-leucine ethyl ester hydrochloride (0.16 g,
4.4.1. Biological assays e cell line
0.72 mmol) was used as a starting material. The crude product
was purified by column chromatography {eluant 1:1 hexane:
ethyl acetate} and recrystallisation from hexane: ethyl acetate
yielded the desired product as an orange solid (0.06 g, 15%), m.p
H1299 was obtained from the American Tissue Culture Centre
(ATCC). The cell line was grown in RPMI-1640 supplemented with
10% foetal calf serum (FCS) at 37 ꢁC in a 5% CO2 humidified chamber.
76e78 ꢁC; Eꢁ0 ¼ 20 mV (vs Fc/Fcþ); Eꢁ0 ¼ 20 mV (vs Fc/Fcþ);
4.4.2. In vitro proliferation assays
20
[
a
]
¼ ꢅ15ꢁ (c 0.1, EtOH).
Cells in the exponential phase of growth were harvested by
D
Mass spectrum: [M þ Na]þ found: 569.2197.
trypsinisation and a cell suspension of 1 ꢆ 104 cells per ml was
C30H38N2O4FeNa requires: 569.2181.
prepared in fresh culture medium. The cell suspension (100 mL) was
I.R. ymax (KBr): 3275 (NH), 1749 (C]Oester), 1629 (C]Oamide),
1613 (C]Oamide) cmꢅ1
UVeVis lmax EtOH: 360, 451 nm.
1H NMR (400 MHz)
(DMSO-d6): 8.67 (1H, t, J ¼ 6 Hz, eCONHe),
8.33 (1H, d, J ¼ 7.6 Hz, eCONHe), 7.85 (2H, d, J ¼ 7.6Hz, ArH), 7.66 (2H,
d, J ¼ 7.6 Hz, ArH), 4.88 (2H, s, ortho on
5-C5H4-benzoyl), 4.37 (2H, s,
meta on
5-C5H4-benzoyl), 4.33e4.27 {1H, m, eCH(CH2CH(CH3)2)},
5-C5H4-alkyl),
added to a flat bottom 96-well plate (Costar, 3599), plates were
agitated gently in order to ensure even dispersion of cells over the
surface of the wells, and then cells were incubated for an initial 24 h
in a 37 ꢁC, 5% CO2 incubator, to allow cell attachment to the wells. A
10 mM stock solution of a test sample was prepared in dimethyl
sulfoxide; dilute solutions of the test sample were prepared at 2ꢆ
final concentration by spiking the cell culture medium with a
.
d
h
h
4.14 (2H, q, J ¼ 7.2 Hz, eOCH2CH3), 3.97e3.86 {6H m, (
h
calculated amount of the stock solution. 100
mL aliquot of each
(eNHCH2COe)}, 2.0(2H,t,J¼ 7.6 Hz, eCH2CH2CH3),1.69e1.45 {3H, m,
eCH(CH2CH(CH3)2)}, 1.40e1.27 (2H, m, eCH2CH2CH3), 1.15 (3H, t,
J ¼ 7.2 Hz, eOCH2CH3), 0.91e0.83 {6H, m, eCH(CH2CH(CH3)2)}, 0.72
(3H, t, J ¼ 7.2 Hz, eCH2CH2CH3).
dilute solution was added to each well of the plate, the plate was
gently agitated, and then incubated at 37 ꢁC, 5% CO2 for 5e6 days,
until cell confluency reached 80e90%. Assessment of cell survival in
the presence of the compounds 4e12 was determined by the acid
phosphatase assay [31]. The percentage cell growth in the presence
of each compound was determined relative to the control cells. The
concentration of compounds causing a 50% growth inhibition (IC50
of the compound) was determined using Calcusyn (Biosoft, UK).
13C NMR (100 MHz)
d (DMSO-d6): 171.4 (C]O), 169.4 (C]O),
168.1 (C]O), 140.1 (Cq), 131.8 (Cq), 129.4, 126.2, 89.8 (Cipso h5-C5H4-
alkyl), 84.1 (Cipso
5-C5H4-benzoyl), 71.2 (Cmeta 5-C5H4-benzoyl),
69.9 (Cmeta
5-C5H4-alkyl), 68.5 (Cortho 5-C5H4-alkyl), 67.9 (Cortho
5-C5H4-benzoyl), 62.8 (eOCH2CH3, eve DEPT), 52.1 {e
h
h
h
h
h
CH(CH2CH(CH3)2)}, 42.5 (eNHCH2CHOe, ꢅve DEPT), 40.0 {e
CH(CH2CH(CH3)2), ꢅve DEPT}, 32.0 (eCH2CH2CH3, ꢅve DEPT), 26.4
{eCH(CH2CH(CH3)2)}, 23.5 (eCH2CH2CH3, ꢅve DEPT), 23.7 {e
CH(CH2CH(CH3)2)}, 22.4 {eCH(CH2CH(CH3)2)}, 14.9 (eOCH2CH3),
14.0 (eCH2CH2CH3).
Acknowledgements
The authors thank James Murphy for assistance with the bio-
logical testing.
References
4.3.9. 1-Propyl-10-N-{para-(ferrocenyl)-benzoyl}-glycine-L-
phenylalanine ethyl ester 12
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0.72 mmol) was used as a starting material. The crude product
was purified by column chromatography {eluant 1:1 hexane:
ethyl acetate} and recrystallisation from hexane: ethyl acetate
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[
a
]D20 ¼ þ11ꢁ (c 0.1, EtOH).
Mass spectrum: [M þ Na]þ found: 603.2046.
C33H36N2O4FeNa requires: 603.2034.
I.R. ymax (KBr): 3292 (NH), 1737 (C]Oester), 1630 (C]Oamide),
1609 (C]Oamide) cmꢅ1
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A.G. Harry et al. / Journal of Organometallic Chemistry 757 (2014) 28e35
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