New iron(II) cyclopentadienyl derivative complexes: Synthesis and antitumor activity against human leukemia cancer cells

Article abstract of DOI:10.1016/j.jorganchem.2014.01.027

A new family of Fe^II(η⁵-C₅H ₅) half sandwich compounds bearing a N-heteroaromatic ligand coordinated to the iron center by a nitrile functional group has been synthesized and fully characterized by NMR and UV-Vis spectroscopy. X-ray analysis of single crystal was achieved for complexes 1 and 3, which crystallized in the monoclinic P2₁/c and monoclinic P2_1/n space groups, respectively. Studies of interaction of these five new complexes with plasmid pBR322 DNA by atomic force microscopy showed very strong and different types of interaction. Antiproliferative tests were examined on human leukemia cancer cells (HL-60) using the MTT assay, and the IC₅₀ values revealed excellent antiproliferative activity compared to cisplatin.

Full text of DOI:10.1016/j.jorganchem.2014.01.027

Accepted Manuscript
New iron(II) cyclopentadienyl derivative complexes: synthesis and antitumor activity
against human leukemia cancer cells
Andreia Valente, Ana Margarida Santos, Leonor Côrte-Real, M. Paula Robalo,
Virtudes Moreno, Mercè Font-Bardia, Teresa Calvet, Julia Lorenzo, M. Helena Garcia
PII:
S0022-328X(14)00042-4
10.1016/j.jorganchem.2014.01.027
JOM 18468
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 31 July 2013
Revised Date: 10 December 2013
Accepted Date: 20 January 2014
Please cite this article as: A. Valente, A.M. Santos, L. Côrte-Real, M.P. Robalo, V. Moreno, M. Font-
Bardia, T. Calvet, J. Lorenzo, M.H. Garcia, New iron(II) cyclopentadienyl derivative complexes:
synthesis and antitumor activity against human leukemia cancer cells, Journal of Organometallic
Chemistry (2014), doi: 10.1016/j.jorganchem.2014.01.027.
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ACCEPTED MANUSCRIPT
New organometallic half sandwich Fe(II)Cp compounds bearing a coordinated nitrile
group bonded to one or two N-heteroaromatic rings. IC50 values for HL-60 leukemia
cancer cells and AFM images of the interaction with pBR322 DNA revealing
interesting cytotoxic behaviour.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
New iron(II) cyclopentadienyl derivative complexes: synthesis and antitumor
activity against human leukemia cancer cells
Andreia Valentea, Ana Margarida Santosa, Leonor Côrte-Reala, M. Paula Robalob,
Virtudes Morenoc, Mercè Font-Bardiad, Teresa Calvetd, Julia Lorenzoe, M. Helena
Garcia*a
aCentro de Ciências Moleculares e Materiais, Facultade de Ciências da Universidade de
Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
bÁrea Departamental de Engenharia Química, Instituto Superior de Engenharia de
Lisboa, Rua Conselheiro Emídio Navarro, 1, 1959-007 Lisboa, Portugal; Centro de
Química Estrutural, Complexo I, Instituto Superior Técnico, Universidade de Lisboa, Av.
Rovisco Pais, 1049-001 Lisboa, Portugal.
cDepartment de Química Inorgànica, Universitat de Barcelona, Martí y Franquès 1-11,
08028, Barcelona, Spain
dCristal.lografia, Mineralogia i Dipòsits Minerals, Universitat de Barcelona, Martí y
Franquès s/n, 08028, Barcelona, Spain; Centre Cientific i Tecnològic (CCiTUB)
Universitat de Barcelona Sole Sabaris 1-3, 08028 Barcelona, Spain
eInstitut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193,
Bellaterra, Barcelona, Spain.
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Abstract
A new family of “FeII(η5-C5H5)” half sandwich compounds bearing a N-heteroaromatic
ligand coordinated to the iron centre by a nitrile functional group has been synthesized
and fully characterized by NMR and UV–Vis spectroscopy. X-ray analysis of single
crystal was achieved for complexes 1 and 3, which crystallized in the monoclinic P21/c
and monoclinic P21/n space groups, respectively. Studies of interaction of these five new
complexes with plasmid pBR322 DNA by atomic force microscopy showed very strong
and different types of interaction. Antiproliferative tests were examined on human
leukemia cancer cells (HL-60) using the MTT assay, and the IC50 values revealed
excellent antiproliferative activity compared to cisplatin.
Keywords
Iron(II); Cyclopentadienyl; Atomic Force Microscopy (AFM); Leukemia cancer cells;
Apoptosis.
1. Introduction
Organometallic chemistry emerged in the recent years as an attractive field for the search
of new compounds as potential drugs for medicinal chemistry, in particular for
chemotherapy. In this frame, metallocene derivatives have appeared at the end of the 1970s
with the pioneering work of Köpf and Köpf-Maier involving the antitumor activity of early
transition-metal cyclopentadienyl complexes.[1] The promising results obtained for
dichloride metallocenes (Cp2MCl2, where M = Ti, V, Nb, Mo; Cp = η5-cyclopentadienyl)
showing antitumor activity against numerous tumors, such as Ehrlich ascites tumor, B16
melanoma, colon 38 carcinoma and Lewis lung carcinoma, as well as against several
human tumors heterotransplanted to athymic mice, certainly constitute an important
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impulsion for the interest of this area.[2] Titanium dichloride, (η5-C5H5)2TiCl2, was the
first of such species in clinical trials.[3] Nevertheless, problems related with formulation
led to the abandonment of titanocene dichloride in Phase II clinical trials.[4,5,6] Ferrocene
derivatives also appeared with promising results showing activity against Rauscher
leukemia virus and EAT in CF1 mice[7,8] and in P388 leukemia cells[9] reinoculated
tumors.[10] Particularly, the ferrocifens family, which is a ferrocene derivative of
tamoxifen (Astra Zeneca, London, UK - the drug used for treating breast cancer), has
revealed good cytotoxicity activities. However, these molecules suffer from poor
bioavailability restricting them from entering clinical trials. In order to overcome this
limitation and advance toward clinical studies, several formulations are being tested, such
as nanoparticles, lipid nanocapsules and cyclodextrins.[11]
The half- sandwich family of compounds emerged more recently using different central
metals and has revealed potentialities as anticancer drugs. The particular geometry of
piano stool compounds provides a good scaffold for building new molecules by changing
the coordinated arene, which can be η6 or η5-bonded, the chelated active ligand and also
the coligands. In this context, [Ru(η6-arene)(X)(Y–Z)] complexes (where Y–Z is a
chelating ligand, and X is monoanionic ligand) revealed high cytotoxicity against human
ovarian tumor cells[12-15] and they are thought to act through covalent Ru–DNA
interactions.[16,17]
Related
compounds
incorporating
the
1,3,5-triaza-7-
phosphaadamantane (PTA) ligand, such as [Ru(η6-p-cymene)(PTA)Cl2] (RAPTA-C),
have shown activity against metastases and although their mechanism of action has not
been established, a pH dependent interaction with DNA may be a key component.[18]
During the last years, our research group has been exploring a third family of half-
sandwich compounds based on the “RuII(η5-C5H5)” fragment, with the general formula
[Ru(η5-Cp)(P–P)(L)][X] (where P–P is a chelating phosphane or two phosphane ligands,
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L is a N-heteroaromatic sigma ligand mono or bidentate ligand and X is a
counterion).[19-24] Our studies showed significant toxicity for a variety of cancer cell
lines, namely LoVo and HT29 human colon adenocarcinoma, MiaPaCa pancreatic cancer
cell lines, HL-60 human leukemia cancer cells, A2780 human ovarian cancer cells (and
the resistant form A2780CisR), MCF7 and MDAMDB human breast cancer cells
(estrogen dependent and independent, respectively) and PC3 human prostate cancer cells,
with IC50 values lower than those of cisplatin in most cases.[19-24] One important
advantage of ruthenium based compounds for therapeutic uses compared to other metal
complexes, is pointed out on its ability to mimic iron in binding biologically relevant
molecules such as albumin and transferrin and consequently to show much lower toxicity
than that of platinum therapies.[25] The success of the coordination of N heteroaromatic
ligands to the fragment ‘RuCp’ in terms of finding new compounds with important
cytotoxicity against several cancer cell lines led us to extend our studies to the analogue
´FeCp` derivatives. In this context, we have recently published our first results
concerning a new family of compounds with the general cationic structure [Fe(η5-Cp)(P–
+
P)(L)] , where L is coordinated to the iron center by the N atom of the heteroaromatic
ligand.[26] These new compounds showed values of cytotoxicity against MCF7 and
HeLa much lower than the ones found for cisplatin in the same experimental conditions.
Having in mind to exploit the effect of cytotoxicity of other ligands coordinated by a
different group than a N-heteroaromatic atom, we had previously studied two new
[Ru(η5-Cp)(PPh3)2(N≡CL)]+ derived compounds where N≡CL was coordinated by a
nitrile functional group (benzo[1,2-b;4,3-b’] dithio-phen-2-carbonitrile and [5-(2-
thiophen-2-yl)-vinyl]-thiophene-2-carbonitrile]) which were tested against HL-60
cells.[20] The IC50 values obtained after 24 h of incubation were 1.46 ± 0.25 and 5.89 ±
0.67 µM, respectively, while cisplatin in the same experimental conditions presented a
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higher IC50 value of 15.61 + 1.15 µM. These motivating results obtained with ruthenium
coordinated nitrile ligands together with our interest to continue the exploitation of the
cytotoxic properties of ‘FeCp’ compounds led us to the synthesis of a new family of iron
nitrile compounds of general formula [Fe(η5-Cp)(NCL)(P–P)]+. In the present paper we
report the synthesis of compounds of the general formula [Fe(η5-Cp)(NCL)(P–P)]+,
where the NCL ligands, 2-quinolinecarbonitrile (L1), 3-quinolinecarbonitrile (L2), 2-
pyrazinecarbonitrile (L3) or 2,3-pyrazinedicarbonitrile (L4), present on their structures
one or two N-heteroaromatic rings. These new compounds were fully characterized and
their interaction with plasmid pBR322 DNA was studied by atomic force microscopy.
Moreover, their potentialities as cytotoxic agents against human leukemia cancer cells
(HL-60 cells) were evaluated. Remarkably, our studied compounds revealed IC50 values
lower than those of cisplatin. Apoptotic behaviour was also evaluated and compared with
cisplatin.
2. Experimental
2.1. General procedures
All syntheses were carried out under dinitrogen atmosphere using current Schlenk
techniques and the solvents used were dried using standard methods.[27] [Fe(η5-
C5H5)(dppe)I] was prepared following the method described in literature.[28] FT-IR
spectra were recorded in a Mattson Satelite FTIR spectrophotometer with KBr pellets;
only significant bands are cited in text. 1H, 13C and 31P NMR spectra were recorded on a
1
Bruker Avance 400 spectrometer at probe temperature. The H and 13C chemical shifts
31
are reported in parts per million (ppm) downfield from internal Me4Si and the P NMR
spectra are reported in ppm downfield from external standard, 85% H3PO4. Elemental
analyses were obtained at Centro de Apoio Científico Tecnológico Á Investigación
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(C.A.C.T.I.), at Universidade de Vigo, using a Fisons Instruments EA1108 system.
Electronic spectra were recorded at room temperature on a Jasco V-560 spectrometer in
the range of 200-900 nm.
2.2. Complexes synthesis
2.2.1. General procedure applied to the synthesis of the complexes 1 to 5.
To a stirred suspension of 0.5 mmol of [FeCp(dppe)I] in dichloromethane (25 mL) was
added 0.6 mmol of the adequate ligand (L1 = 2-quinolinecarbonitrile; L2 = 3-
quinolinecarbonitrile; L3 = 2-pyrazinecarbonitrile; L4 = 2,3-pyrazinedicarbonitrile
followed by addition of 0.6 mmol of TlPF6 (for complexes 1, 2, 3 and 4) or AgCF3SO3
(for complex 3). After refluxing for a period of 5 - 6 h the colour changed from grey to
orange reddish. The reaction mixture was cooled down to room temperature and the
solution was filtered to eliminate the TlCl or the AgCl precipitate. The solvent was then
removed under vacuum and the residue was washed with n-hexane (2 × 10 mL). Dark red
crystals were obtained after recrystalization from dichloromethane/n-hexane solutions.
Compound 5 was obtained by stirring for 90 min, a suspension of 0.32 g (0.5 mmol) of
[FeCp(dppe)I] and TlPF6 (0.21g, 0.6 mmol), in dichloromethane (25 mL) to which a
solution of 2,3-dicianopyrazine in dichloromethane (0.078g; 0.6 mmol) was slowly
added. The obtained purple solution was dried and washed with n-hexane, giving a
powder, which after recrystalization from dichloromethane/n-hexane, gave needle shaped
purple crystals.
2.2.2. [FeCp(dppe)(2-cq)][PF6], 1
-
1
Dark red; Yield = 81%. IR (KBr, cm-1): ν(C≡N, stretch) 2208, ν(PF6 ) 837 and 557. H
NMR ((CD3)2CO, Me4Si, δ/ppm): 8.30 (d, 1, H10); 8.15 (t, 4, dppe); 7.96 (dd, 2, H4+H7);
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7.88 (t, 1, H6); 7.73 (t, 1, H5); 7.53 (m, 16, dppe); 6.78 (d, 1, H9) 4.75 (s, 5, η5- C5H5);
2.80 (m, 4, CH2-dppe). 13C NMR ((CD3)2CO, Me4Si, δ/ppm): 148.3 (C8); 138.0 (C9);
137.6-137.0 (Cq, dppe); 133.8 (CH-, dppe); 133.1 (C≡N); 132.5 (CH-, dppe); 132.2 (C6);
131.6-131.4 (CH-, dppe); 130.3 (C5); 130.0 (C4); 129.9 (CH-, dppe + C3); 129.4 (C2);
128.9 (C7); 123.8 (C10); 81.4 (Cp); 28.4 (-CH2-, dppe). 31P((CD3)2CO, δ/ppm): 96.2 (s,
dppe); -144.2 (setp, PF6). UV-Vis. in CH2Cl2, λmax/nm (ε/M-1cm-1): 240 (73195), 385
(6049), 441 (6893). UV-Vis. in DMSO, λmax/nm (ε/M-1cm-1): 392 (Sh), 455 (7669).
Elemental analysis (%) Found: C, 59.50; H, 4.30; N, 3.40; Calc. for
C41H35N2P3F6Fe.0.1CH2Cl2 (826.9): C, 59.70.16; H, 4.30; N, 3.40.
2.2.3. [FeCp(dppe)(3-cq)][PF6], 2
-
1
Dark red; Yield: 80%. IR (KBr, cm-1): ν(C≡N, stretch) 2210, ν(PF6 ) 837 and 557. H
RMN ((CD3)2CO, Me4Si, δ/ppm): 8.15 (t, 4, C6H5-dppe); 8.00 (d, 1, H5); 7.92 (m, 2,
H3+H7); 7.85 (d, 1, H8); 7.71 (t, 1, H6); 7.68 (s, 1, H10); 7.58 (m, 16, C6H5-dppe); 4.73 (s,
5, η5- C5H5); 2.82 (m, 4, CH2). 13C NMR ((CD3)2CO, Me4Si, δ/ppm): 149.9 (C3); 148.8
(C2); 142.3 (C10); 137.7-137.1 (Cq, dppe); 134.0 (CH-, dppe); 133.8 (C7); 132.4 (CH-,
dppe); 131.7-131.4 (CH-, dppe); 130.1 (C5); 130.0 (CH-, dppe); 129.4 (C8); 129.3 (C6);
126.5 (C4); 107.1 (C9); 81.1 (Cp); 28.5 (-CH2-, dppe); C1 is overlapped by dppe signals.
31P RMN ((CD3)2CO, δ/ppm): 97.1 (s, dppe); -144.2 (setp, PF6). UV-Vis in CH2Cl2,
λmax/nm (ε/M-1cm-1): 239 (75639), 376 (7169), 429 (5136). UV-Vis in DMSO,
λmax/nm (ε/M-1cm-1): 386 (6559), 442 (7292). Elemental analysis (%) Found: C, 59.10;
H, 4.30; N, 3.40; C41H35N2P3F6Fe (826.9): C, 59.2; H, 4.30; N, 3.30.
2.2.4. [FeCp(dppe)(3-cq)][CF3 SO3], 3
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-
Dark red; Yield: 80 %. IR (KBr, cm-1): ν(C≡N, stretch) 2212, ν(CF3SO3 ) 1269. 1H NMR
((CD3)2CO, Me4Si, δ/ppm): 8.15 (t, 4, C6H5-dppe); 8.01 (d, 1, H5); 7.92 (m, 2, H3+H7);
7.87 (d, 1, H8); 7.71 (m, 2, H10+H6); 7.63 (t, 4, C6H5-dppe); 7.57(m, 12, C6H5-dppe); 4.74
(s, 5, η5-C5H5); 2.80 (m, 4, CH2). 13C NMR ((CD3)2CO, Me4Si, δ/ppm): 149.7 (C3); 148.6
(C2); 142.6 (C10); 137.7-137.4 (Cq, dppe); 134.0 (CH-, dppe); 133.8 (C7); 132.4 (CH-,
dppe); 132.1 (C1); 131.7-131.4 (CH-, dppe); 130.2 (C5); 130.0 (CH-, dppe); 129.4 (C8);
129.3 (C6); 126.6 (C4); 107.1 (C9); 81.1 (Cp); 28.5 (-CH2-, dppe). 31P RMN (CD3Cl3,
δ/ppm): 97.1 (s, dppe). UV-Vis in CH2Cl2, λmax/nm (ε/M-1cm-1): 239 (51562), 369
(3559), 428 (2371). UV-Vis in DMSO, λmax/nm (ε/M-1cm-1): 386 (4380), 442 (4798).
Elemental analysis (%) Found: C, 57.4; H 4.2; N, 3.08; C42H37N2P2F3SO3FeꢀCH2Cl2: C,
56.8; H, 4.32; N, 3.08.
2.2.5. [FeCp(dppe)(cpz)][PF6], 4
-
1
Red; Yield: 85%. IR (KBr, cm-1): ν(C≡N, stretch) 2218, ν(PF6 ) 837 and 557. H NMR
((CD3)2CO, Me4Si, δ/ppm) 8.67 (d,1, H4); 8.55 (d, 1, H5), 8.05 (m, 4, C6H5), 7.88 (s, 1,
H3), 7.56-7.48 (m, 16, C6H5), 4.75 (s, 5, η5- C5H5); 2.79 (m, 4, CH2). 13C NMR
((CD3)2CO, Me4Si, δ/ppm): 148.9 (C3), 147.8 (C4), 146.2 (C5), 137.4-137.0 (Cq, dppe);
133.8 (CH-, dppe); 132.5 (CH-, dppe); 131.5 (CH-, dppe); 130.0 (CH-, dppe); 81.7 (Cp);
-
28.4 (-CH2-, dppe).31P NMR ((CD3)2CO, δ/ppm): 96.10 (s, dppe); -144.2 (setp, PF6 ).
UV-Vis in CH2Cl2, λmax/nm (ε/M-1cm-1): 264 (20282), 388 (2564), 445 (2895). UV-Vis
in DMSO, λmax/nm (ε/M-1cm-1): 446 (5225). Elemental analysis (%) Found: C, 55.75;
H, 4.1; N, 5.35; Calc. for C36H32N3P3F6Feꢀ1/10CH2Cl2: C, 55.74; H, 4.17; N, 5.40.
2.2.6. [FeCp(dppe)(2,3-dcpz)][PF6], 5
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-
Purple; Yield: 86%. IR (KBr, cm-1): ν(C≡N, stretch) 2198, ν(PF6 ) 837 and 559. 1H RMN
(CDCl3, Me4Si, δ/ppm): 8.84-8.81 (m, 2, H4+H5); 8.00 (t, 4, C6H5); 7.48 (m, 16, C6H5);
4.86 (s, 5, η5-C5H5); 2.85 (m, 4, CH2). 13C NMR ((CD3)2CO, Me4Si, δ/ppm): 148.8 (C4);
147.0 (C5); 137.1-136.6 (Cq, dppe); 133.8 (CH-, dppe); 133.2 (C2+C3); 131.7 (CH-,
dppe); 130.1 (CH-, dppe); 120.0 (CH-, dppe); 128.7 (C1); 114.6 (C6); 82.7 (Cp); 28.6 (-
CH2-, dppe).31P RMN ((CD3)2CO, δ/ppm): 95.4 (s, dppe); -144.27 (setp, PF6). UV-Vis in
CH2Cl2, λmax/nm (ε/M-1cm-1): 266 (12201), 521 (2521). UV-Vis in DMSO, λmax/nm (ε/M-
1cm-1): 512 (5710).
Elemental analysis (%) Found: C, 55.10; H, 4.0; N, 6.90; Calc. for
C41H35N2P3F6Fe.0.2CH2Cl2 (811.40): C, 55.94; H, 3.93; N, 7.05.
2.3. Crystal structure determination of [FeCp(dppe)(2-cq)][PF6] 1 and
[FeCp(dppe)(3-cq)][CF3SO3] 3
Prismatic crystals (0.1x0.1x0.2 mm and 0.2x0.1x0.1 mm respectively) were selected and
mounted on a MAR345 diffractometer with an image plate detector. Intensities were
collected with graphite monochromatized Mo Kα radiation. Lorentz-polarization and
absorption corrections were made. The structures were solved by Direct methods, using
SHELXS computer program[29] and refined by full-matrix least-squares method with
SHELX93 computer program,[30] (very negative intensities were not assumed). The
function minimized was Σ w ││Fo│2 - │Fc│2 │2, where w = [σ2(I)
+(0.0566P)2+0.4472P]-1, and P = (│Fo│2 + 2 │Fc│2 )/3, f, f' and f" were taken from
International Tables of X-Ray Crystallography.[31] All H atoms were located from a
difference synthesis and refined with an overall isotropic temperature factor. CCDC
939633 and 939634 contain the supplementary crystallographic data for this paper. These
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data can be obtained free of charge from the Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
2.4. Electrochemical experiments
The electrochemical experiments were performed on an EG&G Princeton Applied
Research Model 273A potentiostat/galvanostat and monitored with a personal computer
loaded with Electrochemistry PowerSuite v2.51 software from Princeton Applied
Research. Cyclic voltammograms were obtained in 0.1 M or 0.2 M solutions of
[NBu4][PF6] in CH3CN or CH2Cl2 respectively, using a three-electrode configuration cell
with a platinum-disk working electrode (1.0 mm diameter) probed by a Luggin capillary
connected to a silver-wire pseudo-reference electrode and a Pt wire counter electrode.
The electrochemical experiments were performed under a dinitrogen atmosphere at room
temperature. The redox potentials were measured in the presence of ferrocene as the
internal standard and the redox potential values are normally quoted relative to the SCE
by using the ferrocenium/ferrocene redox couple (E1/2 = 0.46 or 0.40 V vs. SCE for
CH2Cl2 or CH3CN, respectively).[32] The supporting electrolyte was purchased from
Fluka, electrochemical grade) was dried under vacuum for several hours and used without
further purification. Reagent grade acetonitrile and dichloromethane were dried over P2O5
and CaH2, respectively, and distilled under dinitrogen atmosphere before use.
2.5. DNA Interaction studies
2.5.1. Formation of Drug-DNA complexes
Deionised Milli-Q water (18.2 MΩ) was filtered through 0.2-nm FP030/3 filters
(Schleicher & Schuell) and centrifuged at 4.000 g prior to use. pBR322 DNA was heated
at 60 ºC for 10 min to obtain open circular (OC) form. To stock aqueous solutions of
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plasmid pBR322 DNA in Hepes buffer (4 mM Hepes, pH 7.4/ 2 mM MgCl2) were added
aqueous solutions (with 4% of DMSO) of complexes 1 to 5 in a relationship DNA base
pair to complex 10:1. In parallel experiments, blank sample of free DNA and DNA
complex solutions were equilibrated at 37 ºC for 4h in the dark shortly thereafter.
2.5.2. AFM imaging
Atomic Force Microscopy (AFM) samples were prepared by casting a 3-µL drop of test
solution onto freshly cleaved green mica disks as support. The drop was allowed to stand
undisturbed for 3 min to favour the adsorbate-substrate interaction. Each DNA-laden disk
was rinsed with Milli-Q water and was blown dry with clean compressed argon gas
directed normal to the disk surface. Samples were stored over silica prior to AFM
imaging. All AFM observations were made with a Nanoscope III Multimode AFM
(Digital Instrumentals, Santa Barbara, CA). Nano-crystalline Si cantilevers of 125-nm
length with a spring constant of 50 N/m average ended with conical-shaped Si probe tips
of 10-nm apical radius and cone angle of 35° were used. High-resolution topographic
AFM images were performed in air at room temperature (relative humidity < 40%) on
different specimen areas of 2x2 µm operating in intermittent contact mode at a rate of 1-3
Hz.
2.6. Growth inhibition assays
Antiproliferative activity of these new iron complexes, and cisplatin, was tested in a cell
culture system using the human acute promyelocytic leukemia cell line HL-60 (American
Type Culture Collection (ATCC)). The cells were grown in RPMI-1640 medium
supplemented with 10% (v/v) heat inactivated fetal bovine serum, 2 mmol/L glutamine,
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(Invitrogen, Inc.) in a highly humidified atmosphere of 95% air with 5% CO2 at 37 ºC.
Growth inhibitory effect was measured by the microculture tetrazolium [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT assay.[33] Cells were
seeded at density 104 cells/well in 100 µL of culture medium and after that cells were
treated with different concentrations ranging from 0 (culture medium) to 200 µM of
compounds in 100 µl of culture medium. The exact concentrations assayed were 0.1,
0.24, 0.5, 0.75, 1, 2.5, 5, 10, 25, 50, 100 and 200 µM. All the assays were done in
quadruplicate and three independent assays were realized. After incubation at 37 ºC
during 24 h or 72 h, without washing, 20 µl of soluble tetrazolium salt was added to each
well and incubated 3 additional hours. As we used soluble tretrazolium salts we
determined the amount of formazan directly reading the absorbance at 490 nm in a
spectrophotometric plate reader (Labsystems iEMS Reader MF). Cytotoxicity was
evaluated in terms of cell growth inhibition in treated cultures versus that in untreated
controls. IC50, the concentration of compound at which cell proliferation was 50% of that
observed in control cultures, were obtained by GraphPad Prism software, version 4.0.
Experiments were repeated at least three times to get the mean values.
2.7. Apoptosis assays
Induction of apoptosis in vitro by iron compounds was determined by a flow cytometric
assay with Annexin V-FITC by usingan Annexin V-FITC Apoptosis Detection Kit
(Roche).[34] Exponentially growing HL-60 cells in 6-well plates (5x105cells/well) were
exposed to concentrations equal to the IC50 of the platinum and iron drugs for 24 h. After,
the cells were subjected to staining with the Annexin V-FITC and propidium iodide. The
amount of apoptotic cells was analyzed by flow cytometry (BD FACSCalibur).
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3. Results and Discussion
3.1. Synthesis of Fe(II) complexes
Five new cationic iron(II) complexes (1–5) of the general type [Fe(η5-C5H5)(dppe)L][X]
where
L
=
2-quinolinecarbonitrile (L1), 3-quinolinecarbonitrile (L2), 2-
-
-
pyrazinecarbonitrile (L3) or 2,3-pyrazinedicarbonitrile (L4) and X = PF6 or CF3SO3
were prepared by σ coordination of the functional nitrile N≡C group of the L1-L4 ligands
(Scheme 1). Compounds were obtained in good yields (80-86 %), by halide abstraction
from [Fe(η5-C5H5)(dppe)I] with thallium hexafluorophosphate or silver triflate, in
dichloromethane, in the presence of a slight excess of the adequate ligand and
recrystallized from dichloromethane/n-hexane solutions. The new compounds are stable
in cellular media for several hours (Figure S1, in SI) and were all fully characterized by
1
13
31
FT-IR, H, C and P NMR spectroscopies; the elemental analyses were in accordance
with the proposed formulations. The structures of compounds 1 and 3 were also
characterized by X-ray diffraction studies (see below).
Scheme 1. Reaction scheme for the synthesis of the new Fe(II) complexes and the ligand
structures numbered for NMR purposes
Solid state FT-IR spectra (KBr pellets) of the complexes present the characteristic bands
-
-
of the cyclopentadienyl ligand (≈ 3050 cm-1), the PF6 (840 and 550 cm-1) or CF3SO3
(1250 cm-1) anion and the characteristic stretching vibration of the nitrile functional
group in the range 2200 to 2220 cm-1. The coordination of the ligand to the metal centre
lead to a weakness of the νN≡C of ~ -20 cm-1 for compounds 1−4 being this value
somehow higher (-47 cm-1) for compound 5, probably due to the presence of the second
nitrile acceptor group. These negative shifts observed on νN≡C are in good agreement with
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the values found before for other related η5-monocyclopentadieny iron compounds[35-
37] and show an enhanced π-backdonation from the metal d orbitals to the π* orbital of
the N≡C group leading to a decreased N≡C bond order.
1H NMR chemical shifts of the cyclopentadienyl ring are displayed in the characteristic
range of monocationic iron(II) complexes (4.70 – 4.90 ppm, Table 1). The effect of
coordination on the nitrile ligands is observed through the shielding of the ortho protons
relatively to N≡C coordination position (≈ 1.20 ppm) in compounds 2-4 indicating an
electronic flow towards the heteroaromatic ligand due to π-backdonation involving the
metal centre. Furthermore, an increased electronic density was also found in compound 1
in both ortho (≈ 0.30 ppm) and meta (≈ 1.20 ppm) protons with special relevance for the
meta position (opposite to N in the heteroaroamtic ring) probably due to a higher
contribution of the corresponding resonance form. The electronic flow in compound 1 is
still observed in the second fused ring (≈ 0.20 ppm). This shielding effect on the second
fused ring was also observed for compounds 2 and 3. Here, the difference in the anion did
not cause any additional effect. Relatively to the pyrazinecarbonitrile complexes (4 and 5)
both protons suffered a shielding of about 0.30 ppm. 13C NMR data confirm the evidence
found for proton spectra. The Cp ring chemical shifts are in the range usually observed
for Fe(II) cationic derivatives, a significant deshielding (up to ≈ 14 ppm) being observed
on the carbon of the N≡C functional group upon coordination. All the other carbons of
the chromophore ligand were only slightly deshielded or remained almost unchanged for
31
the studied compounds. P NMR data of the complexes showed the typical septuplet of
-
the PF6 anion at approximately - 144 ppm (with the exception of compound 3 where the
-
-
PF6 anion was replaced by CF3SO3 ). Moreover, a single sharp signal corresponding to
the phosphine coligand (≈ 96 ppm) was observed for all the complexes, revealing the
equivalency of the two phosphorus atoms, together with the expected deshielding upon
14

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1
coordination, in accordance with its σ donor character. Table 1 displays the H NMR
chemical shifts of the ligands (L1-L4) and corresponding complexes (1-5) in (CD3)2CO.
Table 1. 1H NMR data for the ligands (L1-L4) and the complexes (1-5), in (CD3)2CO.
3.2. UV-Vis studies
The optical absorption spectra of these five new Fe(II) complexes and all the ligands
were recorded in 10-3 – 10-5 M dichloromethane solutions in order to identify any MLCT
absorption and π-π* absorption bands expected for these complexes (Table 2). The
electronic spectra of all the compounds showed intense absorption bands in the UV
region, which can be assigned to electronic transitions occurring both in the
organometallic fragment {FeCp(dppe)}+ (λ ≈ 235-260 nm) and in the coordinated
chromophore (λ ≈ 260 – 450 nm) (Figure 1). Additional charge transfer (CT) bands were
also observed in all studied complexes. In fact, all complexes presented one band
compatible with a MLCT nature, which was confirmed by solvatochromism studies in
DMSO (example given in Figure 2 for complex 1).
Figure 1. Electronic spectra of [FeCp(dppe)(L)]+ (1 – 5) in dichloromethane solutions:
── 1; - - - - 2; ꢀꢀꢀꢀꢀꢀ 3; ─ ─ 4; ─ ꢀ ─ ꢀ 5.
Figure 2. Electronic spectra of [Fe(η5-C5H5)(dppe)(2-cq)][PF6] 1 in dichloromethane
(ꢀꢀꢀꢀꢀꢀ) and dimethylsulfoxide (──) showing solvatochromism of the MLCT transition.
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Table 2. Optical spectra data for complexes [Fe(η5-C5H5)(dppe)(L)]+ (1 - 5) in
dichloromethane and dimethylsulfoxide solutions
Sh: shoulder
3.3. X-ray structural studies of the complexes [Fe(η5-C5H5)(dppe)(2-cq)][PF6] 1
and [Fe(η5-C5H5)(dppe)(3-cq)][CF3SO3] 3
Suitable crystals for X-ray diffraction studies of the complexes [Fe(η5-C5H5)(dppe)(2-
cq)][PF6] 1 and [Fe(η5-C5H5)(dppe)(3-cq)][CF3SO3] 3, crystallized in different crystalline
systems and space groups (monoclinic P21/c and monoclinic P21/n space groups,
respectively). Crystal data and structure refinement for both complexes are collected in
Table 3. In Figure 3 and Figure 4 the molecular structure of both complexes 1 and 3 are
respectively presented. Both complexes present the usual distorted three-legged piano
stool geometry for η5-monocyclopentadienyl complexes confirmed by P-Fe-P angles of
86.93 to 87.11º and N-Fe-P angles varying from 90.02 to 92.64º, with the remaining η5-
Cp(centroid)-Fe-X (with X = N or P) angles between 120.40 and 128.39º (see Table 4).
These values are within the range found for η5-monocyclopentadienylmetal nitrile
derivatives with coordinated phosphanes.[38-40]
The distances Fe-η5-Cp(centroid) are very similar in both complexes (1.7153 Å for
complex 1, and 1.7164 Å for complex 3) and in good agreement with the donor/acceptor
nature and number of other ligands bound to iron atom. The distances Fe-N≡C range
from 1.8670 and 1.8865 Å are well within the values expected for this family of
compounds and their bond angles present only a slight deviation of the linearity, with
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values in accordance to those found for related compounds [38,39,40]. Different spatial
orientation of the two isomeric ligands in both complexes can be observed. This fact can
have some biological importance since it might determine the way of interaction of each
complex with DNA or other biological molecules. In Table 4, the main bond lengths and
angles are presented.
Figure 3. ORTEP drawing of [Fe(η5-C5H5)(dppe)(2-cq)][PF6] 1 with atomic numbering
scheme.
Figure 4. ORTEP drawing of [Fe(η5-C5H5)(dppe)(3-cq)][CF3SO3] 3 with atomic
numbering scheme.
Table 3. Crystal data and structure refinement for [Fe(η5-C5H5)(dppe)(2-cq)][PF6] 1 and
[Fe(η5-C5H5)(dppe)(3-cq)][CF3SO3] 3
Table 4. Selected bond lenghts and torsion angles for [Fe(η5-C5H5)(dppe)(2-cq)][PF6] 1
and [Fe(η5-C5H5)(dppe)(3-cq)][CF3SO3] 3
3.4. Electrochemical studies
In order to obtain an insight on the electron richness of the organometallic fragment and
on the coordinated ligands, the electrochemical properties of the ligands L1-L4 and the
new iron(II) complexes were studied by cyclic voltammetry in acetonitrile and
dichloromethane solutions (1 x 10-3 M) using 0.1 M or 0.2 M tetrabutylammonium
hexafluorophosphate (TBAPF6) as supporting electrolyte, between the limits imposed by
the solvents. The electrochemical data measured for the studied compounds at the scan
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rate of 0.200 V/s, are reported in Table 5 and Table 6. The redox behaviour of the
ligands L1-L3 was characterised by an irreversible reductive process near -1.70 V,
whereas for the 2,3-pyrazinedicarbonitrile ligand (L4) this process is observed at ~ -1.15
V, for both solvents. The electrochemical responses of the iron(II) compounds 1, 2, 4 and
5 in acetonitrile were characterized by the presence of an irreversible redox process in the
positive potential range and two or three reductive processes at negative potentials. This
behaviour is also expected for compound 3 since its cation is isostructural of compound
2. The cyclic voltammogram of complex [FeCp(dppe)(2,3-dcpz)][PF6] 5 is showed on
Figure 5 and typifies the behaviour found for all the complexes in this solvent. The
irreversible oxidation placed in the range 0.80 - 0.92 V can be attributed to the metal
centered process (FeII/FeIII). The correspondent reductive wave was observed at 0.62 V
for all the complexes and no changes in this potential were observed at different scan
rates. This behavior can be related with an FeII→FeIII oxidation, leading to the 17-
electron species [FeCp(dppe)(L)]2+, formed on the electrode surface which undergo fast
substitution of the cyanoquinoline or pyrazine ligands by an acetonitrile molecule. The
formed [FeCp(dppe)(NCCH3)]+ species is responsible for the observed reductive process
when the scan direction is reverted. Moreover, the presence of a small reductive wave in
the free ligand position (Epc = -1.16 V) (Figure 5) confirms the ligand exchange process.
This result is consistent with the redox behavior of the isolated complex
[FeCp(dppe)(NCCH3)][PF6] (Figure 5) studied before in an independent experiment for
related
monocyclopentadienyliron(II)dppe
derivatives[39]
where
the
same
electrochemical ligand exchange process was observed. In fact, for complexes
[FeCp(dppe)(NC{SC4H2}nNO2)][PF6], the substitution of the thiophene ligands by the
acetonitrile solvent was observed during the oxidative process in the electrochemical
experiments. The reductive processes found at negative potentials were attributed to
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ligand-based processes occurring at the coordinated cyanoquinoline (L1 and L2) or
pyrazine (L4) ligands, which became easier upon coordination.
Figure 5. Cyclic voltammetry of complexes [Fe(η5-C5H5)(dppe)( 2,3-dcpz)][PF6] 5 (──)
and [Fe(η 5-C5H5)(dppe)(NCMe)][PF6] (- - - - -) in acetonitrile (v = 200 mV/s)
The electrochemical response of compounds 1, 2 and 4 in dichloromethane is slightly
different. Figure 6 shows the cyclic voltammogram of complex [Fe(η5-C5H5)(dppe)(3-
cq)][PF6] 2 which typifies the electrochemical behavior of complexes 1 and 2 with the
cyanoquinoline ligands. No substitution processes involving solvent molecules were
observed and the redox behavior is characterized by the presence of a single quasi-
reversible process (I) attributed to the FeII/FeIII redox pair in the range 0.84 – 0.90 V at
positive potentials and one or two irreversible reductive waves at negative range (II and
III, see Figure 6) derived from processes occurring at the coordinated cyanoquinoline
ligands. Nevertheless, for complex 4 a distinct behavior was observed and only an
irreversible metal centered oxidation at 0.66 V was observed, indicating a complete
decomposition process for the complex after iron(II) oxidation. Moreover, the instability
in the electrochemical cell of compound 5 did not allow further studies.
Figure 6. Cyclic voltammetry of complex [Fe(η5-C5H5)(dppe)(3-cq)][PF6] 2 (──) and 3-
cq ligand (- - - - -) in dichloromethane (v = 200 mV/s)
Table 5. Electrochemical data for complexes [Fe(η5-C5H5)(dppe)L][PF6] (1-5) in
-1
I
acetonitrile at scan rate of 200mV s
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Table 6. Electrochemical data for complexes [Fe(η5-C5H5)(dppe)L][PF6] (1-6) in
dichloromethane at scan rate of 200 mV s-1
3.5. Preliminary biological studies
3.5.1. Atomic Force Microscopy
Compounds 1–5 were incubated for 4 h at 37 ºC in the molar relationship
compound:DNA = 1:2. The images obtained by AFM are presented in Figure 7. These
images show that all the compounds modify the free DNA forms. In Figure 7 (a) the free
pBR322 plasmid DNA shows the usual open and supercoiled forms. The image (b)
shows the modifications caused by compound 1 after incubation with pBR322 DNA
clearly showing broken chains and a strong interaction of the complex on DNA. In image
(c) it can be mainly observed an aggregation of the forms on the mica surface and
modifications in the supercoling caused by interaction of compound 2 on pBR322 DNA.
Compounds 3 and 4 produce similar effects on DNA (images (d) and (e), respectively):
the number of supercoiled forms deposited on the mica has increased. Finally, image (f)
reveals that complex 5 causes strong supercoling in some DNA forms and kinks in those
forms that remain open. The authors have observed similar effects in compounds with
planar ligands which probably intercalate on DNA.[42]
Figure 7. AFM images of (a) plasmid pBR322 DNA and plasmid pBR322 DNA
incubated with complex (b) [Fe(η5-C5H5)(dppe)(2-cq)][PF6] 1, (c) [Fe(η5-C5H5)(dppe)(3-
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cq)][PF6] 2, (d) [Fe(η5-C5H5)(dppe)(3-cq)][CF3SO3] 3, (e) [Fe(η5-C5H5)(dppe)(cpz)][PF6]
4 and (f) [Fe(η5-C5H5)(dppe)(2,3-dcpz)][PF6] 5
3.5.2. Cytotoxicity of the iron complexes against HL-60 cells
The cytotoxic effect of the iron complexes was examined on human leukemia cancer cells
(HL-60) using the MTT assay, a colorimetric determination of cell viability during in
vitro treatment with a drug. The assay, developed as an initial stage of drug screening,
measures the amount of MTT reduction by mitochondrial dehydrogenase and assumes
that cell viability (corresponding to the reductive activity) is proportional to the
production of purple formazan that is measured spectrophotometrically. A low IC50 is
desired and implies cytotoxicity or antiproliferation at low drug concentrations. All the
new Fe(II) complexes were tested, together with cisplatin (CDDP) as a positive control.
Cells were exposed to each compound continuously for a 24 h or a 72 h period of time
and then assayed for growth using the MTT endpoint assay. Table 7 presents the IC50
values against HL-60 cells. The IC50 values at 24 h are lower than that for the reference
drug, cisplatin. At 72 h, compounds 2 and 4 show higher IC50 values than cisplatin,
although compounds 1, 3 and 5 present an excellent antiproliferative behavior with IC50
values lower than cisplatin forecasting interesting structure-activity relationships.
Table 7. IC50 values of iron compounds (1-5) and cisplatin against HL-60 cells.
3.7 Quantification of Apoptosis by Annexin V Binding and Flow Cytometry
We have also analyzed, by Annexin V-PI flow cytometry, whether complexes 1 - 5 are
able to induce apoptosis in HL-60 cells after 24 hours of incubation at equitoxic
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concentrations (IC50 values). Annexin V binds phosphatidyl serine residues, which are
asymmetrically distributed towards the inner plasma membrane but migrate to the outer
plasma membrane during apoptosis.[43]
Table 8. Percentage of HL-60 cells in each state after treatment with complexes 1 – 5 at
IC50 concentration for 24 hours of incubation
As it can be seen in Table 8, complex 3 is able to induce apoptosis death in a 26.21 %,
close to that of cisplatin. Compounds 1, 4 and 5, induce cell death by apoptosis in a lower
percentage. Complex 2 presents only a discrete percentage of apoptosis at IC50 dose,
being the major death process caused by necrosis.
Conclusions
A new family of five half sandwich compounds derived from “FeII(η5-C5H5)” bearing a
coordinated nitrile ligand, which structure comprises one or two N-heteroaromatic rings,
has been synthesized and successfully characterized. Spectroscopic evidence shows a
strong π-backdonation involving the metal centre. X-ray studies for two of these new
compounds revealed crystallization in the monoclinic P21/c and monoclinic P21/n space
groups.
In a preliminary approach to evaluate the cytotoxic behavior of these new compounds
against cancer cells, some studies were carried out involving human leukemia cancer
cells (HL-60) by MTT assay. Also their interaction AFM images with pBR322 DNA
plasmid show different behaviors that can be related with the NCL ligand. Indeed, IC50
values together with the apoptosis results show significant differences between the whole
series of compounds. There are some main conclusions that can be drawn: i) compound
1, bearing the 2-quinolinecarbonitrile, presents the best cytotoxicity and its AFM image
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showed the most relevant modifications in pBR322 DNA; ii) structural difference in the
position of the nitrile group in compounds 1 and 2 (ortho vs. meta) leads to a decrease
on the cytotoxicity (4-fold); iii) comparison of results of compounds 2 and 3 (bearing in
-
both cases the 3-quinolinecarbonitrile ligand) shows that the replacement of PF6 by
-
CF3SO3 leads to a more cytotoxicity compound and the principal mechanism of death is
changed from necrosis to apoptosis; iv) 2-pyrazinecarbonitrile (L3) leads to the less
cytotoxic compound (4); and finally v) the introduction of a second nitrile group in L3
leads to a 3-fold increase in the cytotoxic behavior (compound 5). The overall results
show that after 24h of incubation all the compounds are more cytotoxic than cisplatin.
Thus, this is a potentially interesting family of compounds to be studied in the frame of
anticancer drugs.
Acknowledgments
The authors thank financial support from the Portuguese Foundation for Science and
Technology for the projects PEst-OE/QUI/UI0536/2011, PTDC/QUI-QUI/118077/2010,
PTDC/QUI-QUI/101187/2008 and SFRH/BPD/80459/2011 (Andreia Valente’s
postdoctoral scholarship) and Spanish Ministry of Ciencia e Innovacion, projects
CTQ2008-02064 and BIO2010-22321-C02-01.
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