Synthesis and in vitro anticancer activity of ferrocenyl-aminoquinoline- carboxamide conjugates

Article abstract of DOI:10.1016/j.ica.2012.06.039

The syntheses and characterization of new multifunctional aminoquinoline-carboxamides and their ferrocene derivatives are reported, as well as their cytotoxicity against human colon adenocarcinoma (Caco-2, HTB-37), human breast carcinoma (HTB-129) and a normal cell line as a control (human normal breast epithelial cells MCF-10A, CRL-10317). All tested compounds showed higher activity against HTB-129 cells than against Caco-2 cells. The ferrocenyl-chloroquine amide conjugates displayed higher activity against both cancer cells than did their parent organic compounds.

Full text of DOI:10.1016/j.ica.2012.06.039

Inorganica Chimica Acta 393 (2012) 276–283
Contents lists available at SciVerse ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis and in vitro anticancer activity of ferrocenyl-aminoquinoline-carboxamide
conjugates
⇑
Adriana Esparza-Ruiz, Christoph Herrmann, Jessie Chen, Brian O. Patrick, Elena Polishchuk, Chris Orvig
Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, Canada V6T 1Z1
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 22 July 2012
The syntheses and characterization of new multifunctional aminoquinoline-carboxamides and their fer-
rocene derivatives are reported, as well as their cytotoxicity against human colon adenocarcinoma (Caco-
2, HTB-37), human breast carcinoma (HTB-129) and a normal cell line as a control (human normal breast
epithelial cells MCF-10A, CRL-10317). All tested compounds showed higher activity against HTB-129 cells
than against Caco-2 cells. The ferrocenyl-chloroquine amide conjugates displayed higher activity against
both cancer cells than did their parent organic compounds.
Metals in Medicine Special Issue
Keywords:
Aminoquinoline
Carboxamides
Ó 2012 Elsevier B.V. All rights reserved.
Ferrocene
Ferrocenyl-chloroquine conjugates
Cytotoxicity
1. Introduction
and characterization of new aminoquinoline-carboxamides and
ferrocenyl-chloroquineamides (Scheme2)and theirin vitroantican-
Organometallic compounds that incorporate a ferrocene have
attracted considerable attention in medicinal organometallic
chemistry; ferrocene has shown itself to be an excellent choice to
design new drugs [1–3] due to its small size, aromaticity, hydro-
phobicity, stability toward air and moisture, and redox behavior.
Ferrocene derivatives have shown antimalarial [1,4–17], antican-
cer [1,2,18–23], antimycobacterial [18,19], antiviral [19], and anti-
bacterial activity [24]. One strategy for improving the activity of
biologically active molecules is to incorporate ferrocene into the
framework. Some relevant examples are ferrocifen [25,26] and fer-
roquine derivatives [27,28] (Scheme 1) that have shown higher
anticancer and antimalarial activity, respectively, than do their
precursor compounds. The biological activity of ferrocenium salts
[29] and ferrocene derivatives [1] may be augmented by its capac-
ity to generate hydroxyl radicals (OHÅ) in physiological solutions,
inducing oxidative damage to DNA.
Our general aim is the development of methods that provide easy
access to anticancer and antimalarial drugs that include ferrocene in
their structure and aminoquinolineamide conjugates. Recently, we
reported the cytotoxic and antimalarial activities of various ferroce-
nyl carbohydrate chloroquine conjugates in MDA-MB-435S breast
cancer and HTB-37 colon carcinoma cells [17]. The IC50 values
showed higher anticancer activity in the ferrocene functionalized
with chloroquine and carbohydrate groups than in their compounds
without chloroquine. In this current work, we report the synthesis
cerandcytotoxicityactivity. Weexpectthattheinsertionofamideor
ferrocenyl amide groups into the 2-position of chloroquine should
improve the biological activity against cancer.
2. Experimental
2.1. Materials and instrumentation
Reagents and analytical grade materials were obtained from
commercial suppliers and used without further purification Sol-
vents were dried and distilled prior to use. 3-Chloroaniline,
dimethylacetylene dicarboxylate, ferrocene carboxaldehyde, phos-
phorous(V) oxychloride (POCl3), sodium borohydride (NaBH4),
anhydrous ethylenediamine (ETD), anhydrous 1,3-diaminopropane
and diphenyl ether (Ph2O) were purchased from Sigma–Aldrich.
Reactions in the microwave reactor were performed in a Biotage
version 2.5. The following instruments were used for physical
characterization of the compounds: elemental analyses, Carlo Erba
Elemental Analyser EA 1108; electrospray ionization mass spec-
trometry (ESI-MS) spectra, Micromass LCT and Waters LC–MS;
and NMR spectra, Bruker Avance 300 (1H: 300 MHz and 13C:
75 MHz). Some compounds were separated using a CombiFlash
Rf system, Teledyne Isco.
2.2. X-ray crystallography
⇑
The crystal of 5 was obtained from ethyl acetate. The crystal
was mounted on glass fiber, and measurement was made on a
Corresponding author. Tel.: +1 604 822 4449; fax: +1 604 822 2847.
E-mail address: orvig@chem.ubc.ca (C. Orvig).
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.06.039

A. Esparza-Ruiz et al. / Inorganica Chimica Acta 393 (2012) 276–283
277
are bonded. The O–H hydrogens were added using the HFIX 147
command. All other hydrogen atoms were placed in calculated
positions.
2.3. Synthesis of compounds in Scheme 2
2.3.1. Synthesis of quinoline ester – methyl 4,7-dichloroquinoline-2-
carboxylate 1
Compound 1 was prepared using procedures previously reported
[29,30]. 3-Chloroaniline (20.4 mL, 194.3 mmol) was dissolved in
MeOH (200 mL), and dimethylacetylene dicarboxylate (25 mL,
203.4 mmol) was then added dropwise (exothermic reaction). The
red-brown mixture was heated at reflux for 2 h, and then cooled
at rt. After evaporating methanol in vacuum, the solid was dissolved
in Ph2O (50 mL), and added dropwise over 40 min into a solution of
Ph2O (250 °C) with further stirring for 4 h. After, it was cooled at rt.
The reaction mixture was filtered and washed with hexane and
ether. A yellow solid was obtained (24 g), redissolved in pyridine
(150 mL) and refluxed for 1.5 h. The solution was then cooled and
the resulting precipitate was filtered, washed with ether, dried un-
der vacuum, and then refluxed with POCl3 (9 mL) for 1 h. After cool-
ing, the excess POCl3 was removed under vacuum, and 1 M NaOH
solution was then added to adjust the reaction mixture to pH 8,
Scheme 1. Ferrocene derivatives with biological activity.
Bruker X8 APEX II instrument using graphite-monochromated Mo
Ka radiation. The data were collected at 100 K. The structure was
solved with direct methods using SHELX-97 [30]. The material crys-
tallized with two crystallographically independent molecules in
the asymmetric unit; the N37-C38-C39-N40 fragment of one mol-
ecule is disordered and was modeled in two orientations. Addition-
ally, the material crystallizes with one disordered molecule of
methanol in the asymmetric unit. All non-hydrogen atoms were
refined anisotropically. All N–H hydrogen atoms were located in
difference maps and refined isotropically; however the isotropic
thermal parameters were linked to the nitrogens to which they
Scheme 2. Compounds 1–9.

278
A. Esparza-Ruiz et al. / Inorganica Chimica Acta 393 (2012) 276–283
resulting in the formation of a precipitate. This was triturated and
washed with water, and dissolved in chloroform to recrystallize.
White very thin needles were obtained (yield: 6.7 g, 14%). 1H
NMR (300 MHz, CDCl3, 300 K, d, ppm): 8.36 (1H, H8), 8.29 (1H,
H3), 8.25 (1H, H5), 7.72 (1H, H6), 4.11 (3H, CH3). 13C NMR
(75 MHz, CDCl3, 300 K, d, ppm): 164.9 (C@O), 148.9 (C2), 148.7
(C10), 144.2 (C4), 137.6 (C7), 130.8 (C6), 130.0 (C8), 126.1 (C9),
125.6 (C5), 121.6 (C3), 53.7 (CH3). HRMS (ESI): (C11H8NO2Cl2)+ Calc.:
255.9932, found: 255.9934. Anal. Calc. for C11H7Cl2NO2: C, 51.59; H,
2.76; N, 5.47. Found: C, 51.88; H, 2.90; N, 5.77%.
evaporation of the solvent in air, a precipitate was formed and re-
moved by filtration. The resulting yellow solution was evaporated
to yield a yellow oil (101 mg, 37%). 1H NMR (300 MHz, CDCl3,
300 K, d, ppm): 8.37 (1H, NH17), 7.73 (1H, H8), 7.65 (1H, H5),
7.51 (1H, NH11), 7.18 (1H, H6), 7.10 (1H, H3), 3.34 (2H, H13),
2.68 (2H, H12), 2.61 (4H, H14, H20), 3.43 (2H, H19), 3.10 (2H,
H18), 1.56 (4H, NH15, NH21). 13C NMR (75 MHz, CDCl3, 300 K, d,
ppm): 165.6 (O@C), 151.9 (C2), 151.5 (C4), 147.9 (C10), 135.1
(C7), 128.6 (C8), 125.8 (C5), 122.4 (C6), 118.0 (C9), 96.0 (C3), 43.5
(C18), 41.3 (C12), 39.6 (C20), 37.1 (C14), 33.4 (C19), 30.0 (C13).
HRMS (ESI): (C16H23N5OCl)+ Calc.: 336.1591, found: 336.1578.
2.3.2. General method for the preparation of aminoquinolin-
carboxamides 2–4 and carbamic acid 5
2.3.2.3. N-(2-Aminopropyl)-4-((2-aminopropyl)amino)-7-chloroquin-
Compound 1 and the corresponding diamine were dissolved in
a 2–5 mL microwave vial using a heat gun to achieve complete dis-
solution. The reaction was carried out in the microwave with dif-
oline-2-carboxamide 4.
ferent reaction times and temperatures to yield
a yellow
solution. After cooling to room temperature, the microwave vial
was opened. In a separation funnel the reaction mixture and 1 M
NaOH solution were mixed. The aqueous phase was extracted with
CHCl3 (3 Â 20 mL). The combined organic phases were dried over
MgSO4 and after filtration, the solvent was removed in vacuum.
Yellow oils were obtained in all cases.
2.3.2.1. N-(2-Aminoethyl)-4-((2-aminoethyl)amino)-7-chloroquino-
line-2-carboxamide 2.
Compound 1 (220 mg, 0.86 mmol) and 1,2-diaminepropane (1 mL,
11.7 mmol) were used with microwave conditions 140 °C and
15 min. Adding 10 mL of 1 M NaOH yielded a yellow oil (245 mg,
85%). 1H NMR (300 MHz, CDCl3, 300 K, d, ppm): 8.46 (1H, NH17),
7.77 (1H, H8), 7.75 (1H, H5), 7.20 (1H, H6), 7.12 (1H, H3), 6.39
(1H, NH11), 4.70 (4H, 2NH2), 3.33–2.92 (6H, H12, H13, H18, H19),
1.04 (6H, 2CH3). 13C NMR (75 MHz, CDCl3, 300 K, d, ppm): 165.5
(O@C), 151.5 (C2), 151.3 (C4), 147.8 (C10), 135.2 (C7), 128.4 (C8),
125.9 (C5), 122.0 (C6), 117.8 (C9), 96.6 (C3), 54.2 (C12), 50.2
(C18), 47.5 (C13), 46.9 (C19), 22.4 (C14), 21.4 (C20). HRMS (ESI):
(C16H23N5OCl)+ Calc.: 336.1591, found: 336.1589.
Compound 1 (200 mg, 0.78 mmol) and ethylenediamine (1 mL,
15.0 mmol) were used with microwave conditions 140 °C and
30 min. Adding 10 mL of 1 M NaOH yielded a yellow oil (178 mg,
74%). 1H NMR (300 MHz, CDCl3, 300 K, d, ppm): 8.30 (1H, NH16),
7.56 (1H, H5), 7.47 (1H, H8), 6.94 (1H, H6), 6.88 (1H, H3), 6.51
(1H, NH11), 3.13 (2H, H17), 2.98 (2H, H12), 2.63 (2H, H18), 2.56
(2H, H13), 2.11 (4H, NH14, NH19). 13C NMR (75 MHz, CDCl3,
300 K, d, ppm): 165.4 (O@C), 151.5 (C2), 151.3 (C4), 147.7 (C10),
134.9 (C7), 128.3 (C8), 125.5 (C5), 122.3 (C6), 117.8 (C9), 96.3
(C3), 45.2 (C17), 42.5 (C12), 41.6 (C18), 40.1 (C13). HRMS (ESI):
(C14H19N5OCl)+ Calc.: 308.1278, found: 308.1286.
2.3.2.4.
(2-((2-((2-Aminoethyl)carbamoyl)-7-chloroquinolin-4-
yl)amino)ethyl)carbamic acid 5. 4,7-Dichloroquinoline-2-carboxyl-
ate (300 mg, 1.2 mmol) and ethylenediamine (15 mL, 224.4 mmol)
were used with microwave conditions 165 °C and 30 min. Adding
30 mL of 1 M NaOH yielded a yellow oil that was dissolved in a
small volume of hot ethyl acetate. When this solution was cooled
to RT, a precipitate formed to which methanol:hexane (4:6) was
added to form a yellow solution. This was evaporated at RT and
light yellow crystals were obtained (yield: 292 mg, 71%) and ana-
lyzed by X-ray diffraction. The yellow crystals were scarcely solu-
ble in methanol and insoluble in CDCl3 and DMSO-d6; they were
soluble enough in MeOH to be characterized in solution by low
mass spectrometry: MS ES+ (MeOH): [C15H18ClN5O3]+ (350 m/z,
20%). Anal. Calc. C15H18ClN5O3Á2H2O: C, 46.46; H, 5.72; N, 18.06.
Found: C, 46.06; H, 5.63; N, 18.14%.
2.3.2.2. N-(3-Aminopropyl)-4-((3-aminopropyl)amino)-7-chloroquin-
oline-2-carboxamide 3.
2.3.3. Synthesis of ferrocenyl-chloroquine conjugates 6 and 7
Compound 2 (170 mg, 0.55 mmol) and ferrocene carboxalde-
hyde (118 mg, 0.55 mmol) were dissolved in dry methanol
(20 mL) in Schlenk-type glassware under Ar; a red-orange solution
was obtained and stirred for 16 h, after which time a red-brown
solution was obtained. NaBH4 (31 mg, 0.82 mmol) was added.
The solution changed color to dark brown, and was stirred for
1.5 h at RT. The reaction was quenched by addition of 1 M NaOH
solution (10 mL). Brine (NaCl conc., 20 mL) was added and the
aqueous phase was extracted using chloroform (3 Â 40 mL). The
combined organic phases were dried over MgSO4 and after filtra-
tion, the solvent was removed in vacuum. The crude product was
Compound 1 (210 mg, 0.82 mmol) and 1,3-diaminopropane (1 mL,
12.3 mmol) were used with microwave conditions 140 °C and
15 min. Adding 10 mL of 1 M NaOH yielded an impure yellow oil
(197 mg) that was redissolved in a small volume of CHCl3. After

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279
a colorless suspension which was then heated at 70 °C for 30 min.
The light yellow mixture, after 10 min of heating, changed to
green-yellow. Then, with stirring and no heating, under Ar, dry
methanol (8 mL) was added dropwise over 30 min (exothermic
reaction) to give a dark brown reaction mixture. This was stirred
at 70 °C for 4 h. The reaction mixture was then cooled, quenched
with aq. NH4Cl (15 mL) and stirred for an additional 2 h. The organic
layer was separated and the aqueous phase extracted with ethyl
acetate (30 mL). The organic phases were dried over MgSO4 and
concentrated to give 8 as a white solid (yield: 370 mg, 70%). 1H
NMR (300 MHz, CDCl3, 300 K, d, ppm): 8.99 (1H, H5), 7.82 (1H,
H8), 7.65 (1H, H3), 7.45 (1H, H6), 4.78 (1H, OH), 4.73 (2H, CH2).
13C NMR (75 MHz, CDCl3, 300 K, d, ppm): 162.9 (C2), 147.3 (C10),
143.1 (C4), 136.2 (C7), 127.5 (C8), 126.4 (C6), 124.9 (C5), 123.3
(C9), 118.5 (C3), 63.7 (C11). HRMS (ESI): (C10H8Cl2NO)+ Calc.:
227.9983, found: 227.9983.
purified by column chromatography on silica with a 3:2 mixture of
chloroform/methanol as eluent (Rf = 0.17) for 6 and in 4:1 chloro-
form/methanol (Rf = 0.41) for 7.
2.3.3.1. N-(2-Aminoethyl)-7-chloro-4-((2-((ferrocenylmethyl)amino)
ethyl)amino)quinoline-2-carboxamide 6.
An orange-red oil was obtained (yield: 64 mg, 23%). In the 1H NMR
spectrum (300 MHz, CDCl3, 300 K), the signals were very broad and
not available to be assignable: 8.54, 7.91–7.78, 7.39–7.29, 6.25,
4.30–2.96, 2.25. 13C NMR (75 MHz, CDCl3, 300 K, d, ppm): 165.7
(O@C), 151.7 (C2, C4), 148.1 (C10), 135.5 (C7), 128.8 (C8), 126.4
(C5), 122.2 (C6), 118.4 (C9), 96.8 (C3), 86.8 (C16), 68.7 (C17, C19),
68.3 (C18), 50.8 (C22), 48.9 (C15), 47.1 (C12), 42.7 (C23), 39.6
(C13). HRMS (ESI): (C25H29ClFeN5O)+ Calc.: 504.1457, found:
504.1449.
2.3.5. (4-((2-Aminoethyl)amino)-7-chloroquinolin-2-yl)methanol 9
2.3.3.2. 7-Chloro-N-(2-((ferrocenylmethyl)amino)ethyl)-4-((2-((ferr-
ocenylmethyl)amino)-ethyl))amino)quinolone-2-carboxamide 7.
Compound 9 was obtained using the general method to prepare the
aminoquinoline-carboxamides 2–4, using a mixture of compound 8
(83 mg, 0.36 mmol) and ethylenediamine (1 mL, 15 mmol) at 140 °C
for 30 min. A dark red oil was obtained. The target product was not
soluble in CHCl3 and therefore the crude mixture was freed of impu-
rities by washing with CHCl3; the obtained solid was dried under
vacuum (yield: 37 mg, 41%). It was very soluble in methanol. 1H
NMR (300 MHz, CD3OD, 300 K, d, ppm): 8.00 (1H, H5), 7.71 (1H,
H8), 7.30 (1H, H6), 6.69 (1H, H3), 4.68 (2H, H15), 4.66 (NH2), 3.56
(1H, NH), 3.42 (OH), 3.40 (2H, H12), 2.97 (2H, H13). 13C NMR
(75 MHz, CDCl3, 300 K, d, ppm): 163.3 (C2), 152.0 (C4), 148.2
(C10), 135.2 (C7), 126.1 (C8), 124.5 (C5), 122.8 (C6), 116.9 (C9),
94.4 (C3), 64.9 (C15), 45.3 (C12), 39.8 (C13). HRMS (ESI): (C12H15-
ClN3O)+ Calc.: 252.0904, found: 252.0902.
An orange-yellow oil was obtained (yield: 34 mg, 9%). 1H NMR
(300 MHz, CDCl3, 300 K, d, ppm): 8.58 (1H, HN21), 7.95 (1H, H5),
7.73 (1H, H8), 7.40 (1H, H6), 7.32 (1H, H3), 6.12 (1H, HN11), 4.24
(4H, H18, H28), 4.20 (4H, H17, H27), 4.14 (10H, H19, H29), 3.62
(2H, H25), 3.61 (2H, H15), 3.59 (2H, H22), 3.58 (1H, HN24), 3.41
(2H, H12), 3.05 (2H, H23), 2.98 (2H, H13), 1.89 (1H, HN14). 13C
NMR (75 MHz, CDCl3, 300 K, d, ppm): 165.6 (O@C), 151.6 (C2),
151.4 (C4), 148.0 (C10), 135.5 (C7), 129.1 (C8), 126.3 (C5), 121.8
(C6), 117.9 (C9), 97.1 (C3), 87.0 (C26), 86.6 (C16), 68.7 (C29), 68.6
(C19), 68.2 (C17, C27), 68.0 (C18, C28), 48.6 (C15), 48.5 (C22),
48.4 (C25), 46.9 (C12), 42.2 (C13), 42.2 (C23). HRMS (ESI): (C36H39-
ClFe2N5O)+ Calc.: 702.1589, found: 702.1583.
2.4. Biological activity evaluation (in vitro)
2.4.1. Cell viability assays
The MTT assay measures the reduction of a tetrazolium compo-
nent by the mitochondria into insoluble purple formazan crystals
in metabolically active cells [33]. The amount of color produced
is directly proportional to the number of viable cells. Cellular re-
sponses of two tumour cell lines (human colon adenocarcinoma
Caco-2, ATCC #HTB-37 and human breast carcinoma, ATCC
#HTB-129) and one normal cell line as a control (human normal
breast epithelial cells MCF-10A, ATCC CRL-10317) after exposure
to compounds 1–8 were determined.
2.3.4. Reduction of 1 to (4,7-dichloroquinolin-2-yl)methanol 8
The cells were purchased from ATCC. Caco-2 cells were incu-
bated in Eagle Minimum Essential Medium (MEM-
vine serum (FBS), 50 IU/mL penicillin, 50 g/mL streptomycin at
37 °C; 5% CO2. The HTB-129 cells were grown in Leibovitz’s L-15
medium with 2 mM -glutamine, supplemented with 0.01 mg/mL
human recombinant insulin, Zn solution (GIBCO 12585-014); 10%
FBS; 50 IU/mL penicillin, 50 g/mL streptomycin at 37 °C with
atmospheric air. The MCF-10A cells were grown in Dulbecco’s
a), 10% fetal bo-
l
L
Compound 1 (600 mg, 2.3 mmol) and finely powdered NaBH4
(530 mg, 14.0 mmol) were stirred together in THF (30 mL) to give
l

280
A. Esparza-Ruiz et al. / Inorganica Chimica Acta 393 (2012) 276–283
modified Eagle medium F12, 5% FBS, 50 IU/mL penicillin, 50
streptomycin, 2 mM glutamine, 0.01 g/mL human epidermal
growth factor, 0.5 g/mL hydrocortisone, 10 g/mL insulin at
lg/mL
l
l
l
37 °C, 5% CO2. The cell lines were grown in Falcon BD T-25 vented
flasks. All cell culture reagents were purchased from Invitrogen.
Cell viability assays were performed in 96-well flat bottom plates
(Becton Dickinson, Franklin Lakes, NJ). Each cell line monolayer
was detached from flasks with 0.25% trypsin and 0.38 g/L EDTA
and the cells were re-suspended in fresh culture medium at a den-
sity of 1 Â 105 cells/mL. An aliquot of 100
l
L of cell suspension was
L of medium without
L of sterile distilled
used in each well. For blank control 200
l
cells was used. To prevent evaporation 200
l
water was added to the plate’s outer wells. Each plate was incu-
bated for 24 h in correspondence with cell line growth require-
ment before treatment with experimental compounds.
Compounds 1–8 were dissolved in DMSO and serially diluted in
culture medium in concentrations from 100 to 0.1
added to the cells (compound 9 was not available in large enough
quantities for testing. For ‘‘no treatment’’ control 100 L of culture
medium was used instead. For the ‘‘DMSO effect’’ control 100
lg/mL and
Fig. 1. Displacement ellipsoid plot (50% probability) showing one of the two
independent molecules in the crystal of 5. Intramolecular hydrogen bonds found in
5 are depicted.
l
lL
0.5% DMSO dissolved in medium was added to the control wells.
The plate was incubated as previously described for 72 h. Then
50 lL of MTT solution (2.5 mg/mL) was added to all of the plate’s
experimental wells, except those containing water. The plates were
incubated for 3 h and medium was carefully aspirated from each
pound was obtained in each case. The reaction of compound 1 with
the asymmetric 1,2-diaminopropane gave structural isomers, ob-
served by NMR in a 5:1 ratio, isomer 4 the major product; they
were analyzed as a mixture. In all cases the products were obtained
as yellow oils, in moderate yields. The amides 2–4 were very solu-
ble in CHCl3, and stable towards air and moisture.
well. DMSO (150 lL) was added to lyse the cells and solubilize
the formazan crystals and the plate was placed on an Orbit P4 sha-
ker for 3 min (Labnet International, Woodbridge, NJ). The samples
were analyzed on a DTX 880 (Beckman Coulter Inc.) plate reader
at a wavelength of 570 nm. Cell viability at each drug concentra-
tion was expressed as a percentage of untreated controls and IC50
values were determined by non-linear regression analysis using
Prism5 (GraphPad, CA, USA).
These new amides were reacted with ethyl acetate (EtAcO) in
alkaline conditions to form carbamic acids, as was probed with
the amide 2. Amide 2 was converted into its acid derivative by
ammonolysis and the ester was hydrolysed in alkaline solution
to give 5. The solid product formed in ethyl acetate was completely
solubilized by addition of MeOH and hexane (4:6), and then further
purified by recrystallization from these solvents. Crystals of com-
pound 5 were characterized in the solid state by X-ray diffraction
and elemental analysis. It too poorly soluble for characterization
in solution; only the low resolution mass spectrum was obtained
from a solution in MeOH, from which the crystals were obtained.
The solid state structure of 5 is shown in Fig. 1, and selected
bond distances and angles are listed in Table 1. Compound 5 is a
zwitterionic species in the solid state, with N16 protonated and
the carboxylate group deprotonated. The delocalization of elec-
trons in the fragment O–C–O is deduced from the similar C–O bond
lengths (C21–O22 = 1.271(5) Å and C21–O23 = 1.272(5) Å). The po-
sitive charge on nitrogen N16 can be deduced from the covalent
bond length C15–N16 (1.485(5) Å) being longer than other N–C
bonds in the molecule (from 1.326(5) to 1.459(5) Å).
3. Results and discussion
3.1. Synthesis and spectroscopic characterization
Methyl 4,7-dichloroquinoline-2-carboxylate 1 was prepared
from 3-chloroaniline and dimethylacetylene dicarboxylate, using
procedures modified from the literature [31,32]. The target amino-
quinoline-carboxamides 2–4 were prepared by substitution reac-
tion of compound 1 with an excess of the corresponding diamine
via microwave-assisted conditions, as shown in Scheme 3. The
reactions were carried out at 140 °C for 15–30 min, and the purifi-
cation of the products was accomplished by addition of 1 M NaOH
and extraction with chloroform. As expected, the chlorine was not
the only substituent in the quinoline ring (4-position) subject to
nucleophilic displacement, but the carboxylate group also suffered
a nucleophilic attack, given the formation of aminoquinoline car-
boxamides 2–4. When symmetrical bidentate amines were used,
such as ethylenediamine or 1,3-diaminopropane, just one com-
Table 1
Selected bond distances and angles for zwitterion 5.
Bond distance (Å)
C2–N1
C2–C3
C2–C11
C3–C4
C4–N17
C4–C9
C10–N1
C11–O12
1.331(4)
1.392(5)
1.519(5)
1.382(4)
1.347(4)
1.440(4)
1.375(4)
1.225(4)
C11–N13
C14–N13
C15–N16
C18–N17
C19–N20
C21–O22
C21–O23
C21–N20
1.326(5)
1.459(5)
1.485(5)
1.451(4)
1.431(5)
1.271(5)
1.272(5)
1.365(5)
Bond angles (°)
N1–C2–C11
N17–C4–C3
N17–C4–C9
O12–C11–N13
O12–C11–C2
N13–C11–C2
116.4(3)
122.3(3)
120.4(3)
124.3(3)
120.5(3)
115.1(3)
N16–C15–C14
N20–C19–C18
O22–C21–O23
O22–C21–N20
O23–C21–N20
C4–N17–C18
113.1(3)
113.4(3)
123.0(3)
119.3(4)
117.6(4)
123.8(3)
Scheme 3. Synthesis of diamides 2–4. Microwave conditions: (i) 140 °C, 30 min; (ii
and iii) 140 °C, 15 min.

A. Esparza-Ruiz et al. / Inorganica Chimica Acta 393 (2012) 276–283
281
Table 2
13C NMR data (ppm) of compounds 1–4 and 6–9.
Fig. 2.
p-Stacking in 5, the distance between the ring centroids is shown (3.60 Å).
Compound
1
2
3
4
6
7
8
9
C@O
C2
C3
164.9 165.4 165.6 165.5 165.7 165.6
148.9 151.5 151.9 151.5 151.7 151.6 162.9 163.3
121.6 96.3 96.0 96.6 96.8 97.1 118.5 94.4
144.2 151.3 151.5 151.3 151.7 151.4 143.1 152.0
–
–
The crystal structure of 5 shows four weak cooperative intramo-
lecular interactions: two short contacts (O22Á Á ÁH17 = 1.99(5) Å and
P
C4
N1Á Á ÁH13 = 2.29(5) Å;
rvdW = 2.8 Å) [34], and another OÁ Á ÁH–C
hydrogen bond with an aromatic proton (O12Á Á ÁH14 = 2.47 Å). In
the lattice structure of 5, the molecules are linked through
p stack-
ing, Fig. 2, between quinoline moieties (distance of 3.6 Å), which is
considered a weak interaction [35]. This kind of arrangement has
been observed in similar structures of quinoline derivatives
[36,37]. Also, dimers of 5 were observed in the lattice, formed by
hydrogen bonding interactions of the amide with the carboxylate
oxygen atoms.
3.2. Cytotoxicity activity evaluation (in vitro)
The in vitro antiproliferative effect of compounds 1–8 were
evaluated in two different cancer cell lines, namely human colon
carcinoma (Caco-2, HTB-37), and human breast cancer (MDA-
MB-435S, HTB-129), while the determination of compound cyto-
toxicity was done on non-cancerous cell lines (human normal
breast epithelial cells (MCF-10A). Six wells per condition for each
of the three cell lines. Cisplatin was used as reference in the cell
proliferation assay. Also, a selectivity index (SI) was determined,
based on the ratio of IC50 values obtained against the normal cells
(cytotoxicity) and the IC50 values obtained against the cancer cells.
The ferrocenyl-aminoquinoline-carboxamide conjugates
6
and 7 result from the reaction of aminoquinoline-carboxamides 2
and ferrocene carboxaldehyde in a 1:1 M ratio, Scheme 4. They
were obtained as a mixture and separated by column chromatog-
raphy on silica gel using chloroform and methanol as eluent.
Ferrocene conjugate 6 was obtained as the main product. The
ferrocene conjugate 6 is orange-red and 7 is orange-yellow oil. In
order to avoid the double amine substitution in 1 to have only
one ferrocene unit, the ester 1 was converted into alcohol. The
reduction of 1 was carried out with NaBH4 in dry THF, the reaction
product was the (4,7-dichloroquinolin-2-yl)methanol 8. Then, it
was treated with an excess of ethyleneamine, as solvent at
140 °C for 30 min, giving the (4-((2-aminoethyl)amino)-7-chloro-
quinolin-2-yl)methanol 9.
The structures of compound 1–4 and 6–9 were deduced in solu-
tion by 1H and 13C NMR spectroscopy and confirmed by mass spec-
trometry. The 1H and 13C NMR spectra of compounds 1–4 and 6–9
presented all the expected chemical shifts (vide supra), with the
exception of the 1H NMR spectrum of 6, which showed very broad
signals. The most relevant chemical shifts are shown in Table 2. In
the 13C NMR spectra of these compounds, the chemical shifts
around 165.0 ppm correspond to carbonyl groups. In compounds
2–4 and 6–9, the C3 signal was found at lower frequencies than
in the starting material 1 due to the interchange of chlorine atom
by amine group (ꢀ25 ppm). The signal of C4 appeared ꢀ7 ppm at
high frequency with respect to compound 1, corroborated the
presence of amine groups. A similar effect was observed in C2
(ꢀ2 ppm at high frequencies). The high-resolution mass spectra
of the compounds showed the corresponding molecular ions, fur-
ther confirming their identity.
The IC50 values (lM) are listed in Table 3.
The data revealed that compounds 1–8 were more active
against HTB-129 cells than against Caco-2 cells. A general trend
in the IC50 values of HTB-129 was that compounds containing fer-
rocene moieties were more active than their organic parent com-
pound, chloroquinecarboxamide 2. Compound
7 with two
ferrocene groups in its structure was slightly more potent than
was compound 6, with one ferrocene. In compounds 2 and 3, which
differ in the side chain length between the amine groups by one
CH2 unit, the cytotoxicity was ethyl-2 > propyl-3. For the organic
compound 2, replacement of the OCH3 of 1 by an ethylenediamine
chain improved the cytotoxicity of the compound, 2 > 1. In this cell
line, compounds 4 and 8 were more potent than cisplatin, which
was used as reference (compound 4 was a racemate).
When compounds 1–8 were tested against Caco-2 cells, it was
found that only 1, 6–8 (143.16, 0.53, 0.33 and 28.5
tively) possessed greater potencies in comparison to cisplatin
(395.8 M). A similar trend in activity against HTB-129 cells of
lM, respec-
l
the ferrocenyl-aminoquinoline-carboxamide conjugates 6 and 7
was observed against Caco-2 cells, the presence of two ferrocene
groups in the structure of 7 improved its activity by a factor of
two in comparison with its analog with one ferrocene unit. So, both
compounds 6–7 were again the most potent against Caco-2 of all
Scheme 4. Synthesis of ferrocenyl derivatives 6 and 7. Conditions: (i) MeOH, RT, 16 h, then NaBH4, RT, 1.5 h.

282
A. Esparza-Ruiz et al. / Inorganica Chimica Acta 393 (2012) 276–283
Table 3
In vitro cytotoxicity results.
Compound
IC50
(lM)
SIa
HTB-129
Caco-2
MCF-10A
HTB-129
Caco-2
Cisplatin
50.50( 0.04)
16.83( 0.01)
3.31( 0.01)
16.73
2.11( 0.01)
5.12( 0.02)
0.59( 0.02)
0.28( 0.02)
23.50( 0.02)
39.1
395.80( 0.19)
143.16( 1.46)
984.14
3739.99
–
12.80( 0.02)
170.02( 0.97)
16.51( 0.01)
93.08( 0.04)
4.50( 0.02)
54.44( 0.02)
0.32( 0.01)
0.24( 0.02)
8.55( 0.14)
69.7
0.25
10.11
5.00
5.57
2.14
10.66
0.54
0.85
0.36
0.03
1.19
0.02
0.02
–
1b
2
3
4
5
6
7
8
–
–
0.53( 0.02)
0.33( 0.02)
28.50( 0.02)
112
0.60
0.72
0.30
c
CQ(PO4)2
FQc
8.7
101
34.3
a
Selectivity index (SI) = IC50 MCF-10A/IC50 cancer cell line.
Compound 1 was slightly soluble in DMSO and the maximum concentration was 50 lg/mL.
IC50 values reported by Herrmann et al. [17].
b
c
the studied compounds. Also, they were more active than chloro-
quine diphosphate (112 M) and ferroquine (101 M) [17]. Ferr-
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