Effect of amide-triazole linkers on the electrochemical and biological properties of ferrocene-carbohydrate conjugates

Article abstract of DOI:10.1039/c2dt31927f

Amide-triazole linker incorporated ferrocene-carbohydrate conjugates were prepared by adopting a regiospecific copper(ii)-catalysed 1,3-cycloaddition of ferrocenoyl propargylamide and isopropylidene/acetyl protected carbohydrate azides. Hydrophilic ferrocene glycoside with an amide-triazole linker was synthesised by deacetylation of the hydroxyl groups. All the new compounds were characterised by UV-visible and electrochemical studies and they were found to be stable in organic solvents as well as in the buffer system under physiological conditions (pH = 7.0). The diffusion coefficient (D_f) of the conjugates was also calculated by means of cyclic voltammetric studies. It was observed that while the molecular weight of the compounds had no significant effect on the diffusion coefficient, the hydrophobic/hydrophilic nature of the carbohydrate scaffold displayed varied diffusion coefficient values. Stabilization of the compounds in buffer solution under physiological pH led to almost identical diffusion coefficient values. The compounds derived from xylose and ribose exhibited cytotoxicity on hormone-dependent and hormone-independent breast cancer cell lines, whereas the conjugates derived from glucose and galactose were found to be non-toxic in nature. The compounds did not show any antimicrobial activity against Gram-positive and Gram-negative pathogens. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2dt31927f

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Effect of amide-triazole linkers on the electrochemical
and biological properties of ferrocene-carbohydrate
conjugates†
Cite this: Dalton Trans., 2013, 42, 1180
Sadanala Bhavya Deepthi,a Rajiv Trivedi,*a Lingamallu Giribabu,a Pombala Sujithab
and C. Ganesh Kumarb
Amide-triazole linker incorporated ferrocene-carbohydrate conjugates were prepared by adopting a
regiospecific copper(II)-catalysed 1,3-cycloaddition of ferrocenoyl propargylamide and isopropylidene/
acetyl protected carbohydrate azides. Hydrophilic ferrocene glycoside with an amide-triazole linker was
synthesised by deacetylation of the hydroxyl groups. All the new compounds were characterised by UV-
visible and electrochemical studies and they were found to be stable in organic solvents as well as in the
buffer system under physiological conditions (pH = 7.0). The diffusion coefficient (Df ) of the conjugates
was also calculated by means of cyclic voltammetric studies. It was observed that while the molecular
weight of the compounds had no significant effect on the diffusion coefficient, the hydrophobic/hydro-
philic nature of the carbohydrate scaffold displayed varied diffusion coefficient values. Stabilization of
the compounds in buffer solution under physiological pH led to almost identical diffusion coefficient
values. The compounds derived from xylose and ribose exhibited cytotoxicity on hormone-dependent
and hormone-independent breast cancer cell lines, whereas the conjugates derived from glucose and
galactose were found to be non-toxic in nature. The compounds did not show any antimicrobial activity
against Gram-positive and Gram-negative pathogens.
Received 23rd August 2012,
Accepted 3rd October 2012
DOI: 10.1039/c2dt31927f
www.rsc.org/dalton
several organic and organometallic compounds containing a
triazole functionality, a number of them often displaying excel-
Introduction
In recent years a plethora of potential biological applications lent biological activity.21–27
of ferrocene and its derivatives has been reported.1–8 These
Moreover, several biologically active compounds with tria-
include some of the well known ferrocene analogues of con- zole as a peptide surrogate have displayed substantial inhibi-
ventional drugs for the treatment of breast cancer and malaria, tory activities.28–34 An amide-triazole linker was identified as
such as ferrocifens, hydroxyferrocifens, ferrocenophanes and one of the components in these compounds responsible for
ferroquine respectively.9–14 These examples have provided the inhibition properties (Fig. 1). Metzler-Nolte and co-workers
impetus for the synthesis of ferrocene conjugates for biological
applications.15–17 Facile synthetic procedures using click reac-
tion conditions have made triazoles an attractive functional
group for the construction of bioconjugates.18 Triazoles
exhibit several features of biological importance, for instance,
non-susceptibility to hydrolytic cleavage or redox modifi-
cations, mimicking the hydrogen bond acidity and peptide
bond basicity.19,20 These features have led to the synthesis of
aInorganic and Physical Chemistry Division, CSIR-Indian Institute of Chemical
Technology (CSIR-IICT), Hyderabad 500007, Andhra Pradesh, India.
E-mail: trivedi@iict.res.in, rtrajiv401@gmail.com; Fax: (+) 91-40-27160921;
Tel: (+) 91-40-27191667
bChemical Biology Laboratory, Natural Products Chemistry Division, CSIR-IICT,
Hyderabad 500007, Andhra Pradesh, India
†Electronic supplementary information (ESI) available: 1H and 13C NMR spectra,
UV-Visible and CV spectra of compounds. See DOI: 10.1039/c2dt31927f
Fig. 1 Some biologically active compounds with amide-triazole linkers.
1180 | Dalton Trans., 2013, 42, 1180–1190
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were among the first to report the synthesis of biologically been calculated by means of the Randles–Ševčik equation. The
important PNA oligomers and peptides connected through anticancer activity of the compounds was determined against
amide-triazole linkers.35–37 In recent times, click JAHA type A549 (human alveolar adenocarcinoma cells), HeLa (cervical
ligands consisting of ferrocene conjugates with amide-triazole carcinoma cancer cells), MDA-MB-231 (human breast adeno-
linkers have been shown to exhibit cytotoxicity towards both carcinoma cells) and MCF-7 (human breast adenocarcinoma
hormone-dependent and hormone-independent breast cancer cells) cell lines. The antimicrobial activity of the compounds
cell lines.38,39
was screened against both Gram-positive and Gram-negative
Low cost, ready availability, non-toxicity and facile modifi- pathogens.
cation into several functional groups of carbohydrate moieties
have led to the development of novel synthetic routes for tran-
sition metal complexes containing glycoconjugates.40–44 In
Experimental section
General information
addition to these advantages, the hydrophilicity of ferrocene-
carbohydrate conjugates can be tuned by protecting and
deprotecting the hydroxyl group on the carbohydrate skeleton.
Ferrocene-carbohydrate complexes have been at the forefront
of scientific research for their use as electrochemical
probes45–48 and biological activity.49–59 Most of the biologically
active ferrocene-carbohydrate conjugates have been prepared
from glucose and glucosamine sugars and they have shown
significant anti-malarial activities.54–58
Our recent study on ferrocene-carbohydrate conjugates con-
nected through triazole and amide linkers has shown reason-
able cytotoxicity and antimicrobial activity.60,61 These results
motivated us to study the effect of the amide-triazole linker on
the electrochemical and biological properties of such ferrocene
conjugates. Herein, we report on the synthesis, characteris-
ation, electrochemical properties of various pentose and
hexose sugar ferrocene amide-triazoles 3a–f (Scheme 1). A
detailed electrochemical study has been carried out on all the
compounds and the electrochemical diffusion coefficient has
Optical rotations were determined on a JASCO J-120 polari-
meter. 1H spectra were recorded for CDCl3 and DMSO solu-
tions on 300 MHz (Avance 300) and 500 MHz (Innova 500)
instruments. Chemical shifts for protons were reported using
tetramethylsilane (TMS) as the internal reference. 13C NMR
spectra were recorded at 75.5 MHz on an Avance 300 instru-
ment and the carbon shifts are referenced to the 13C signal of
CDCl3 at 77.0 ppm. Coupling constants (J) were expressed in
Hz. Infrared spectra were recorded on a Thermo Nicolet Nexus
670 spectrometer using KBr discs. Melting points were deter-
mined on a Toshniwal melting point apparatus and are un-
corrected. Ferrocenoyl propargylamide was prepared according
to literature procedures.62 Water soluble amide-triazole 3g was
prepared by the deacetylation of 3f according to the literature
procedure. The UV-visible spectra were recorded on a Varian
Cary 500 spectrophotometer over the range 200–550 nm using
1 cm path length cuvettes. Cyclic voltammetry (CV) was per-
formed with a conventional three-electrode configuration con-
sisting of glassy carbon as a working electrode, platinum as an
auxiliary electrode and an Ag/AgCl reference electrode. The
cyclic voltammograms were recorded in anhydrous methanol
in the presence of 0.1 M tetrabutylammonium perchlorate
(TBAP) as the supporting electrolyte with a scan rate of 0.1 V
s−1, using a CHI620 model electrochemical analyzer at room
temperature.
General procedure for the preparation of ferrocene-
carbohydrate amide-triazoles
Ferrocenoyl propargyl amide (0.20 g, 1.39 mmol) and azido
sugars (3a–f) (1.39 mmol) were suspended in a 1 : 1 mixture of
water and tert-butyl alcohol (8 mL). To this solution, sodium
ascorbate (0.05 g, 0.2 mmol) and CuSO4·5H2O (0.02 g,
0.09 mmol) were added. The heterogeneous mixture was
stirred at 40 °C, until the disappearance of the starting
materials. The reaction mixture was quenched with saturated
NH4Cl solution and extracted with CH2Cl2 (2 × 5 mL). The
combined organic layers were dried over Na2SO4 and the crude
mass, obtained by the removal of the solvent under vacuum,
was purified by column chromatography on alumina.
5-Deoxy-1,2-O-isopropylidene-5-(4-methyl amido ferrocenyl-
1H-1,2,3-triazole-1-yl)-α-D-xylofuranose (3a). Treatment of 5-
azido-5-deoxy-1,2-O-isopropylidene-α-D-xylofuranose (0.3 g,
1.39 mmol) (2a) and ferrocenoyl propargylamide (0.2 g,
Scheme 1 Synthesis of ferrocene-carbohydrate amide-triazoles by the click
reaction of sugar azide and ferrocenoylpropargylamide.
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1.39 mmol) resulted in 3a as an orange solid. Yield: 0.41 g m/z = 603 [M + H]+; UV-Vis in CH3OH: λmax [ε(dm3 mol−1
(69%); mp: 70 °C; [α]D34 (c 0.2, CH3OH) = −13.14; IR (KBr, νmax
/
cm−1)] = 264 (4016), 306 (938), 442 (162) nm; Anal. calc. for
cm−1) = 3434, 3107, 2982, 1656, 1069; 1H NMR (300 MHz, C30H34FeN4O6 (602.18): C 59.81, H 5.69, N 9.30, Found: C
CDCl3, Me4Si): δ (ppm) = 1.30 (s, 3H, CH3 of CMe2), 1.40 (s, 59.09, H 5.47, N 8.91.
3H, CH3 of CMe2), 4.13 (s, 5H, C5H5), 4.19–4.23 (m, 1H, H-5),
Methyl-5-deoxy-2,3-O-isopropylidene-5-(4-methyl
amido
4.34 (t, 2H, J = 1.70 Hz, C5H4), 4.46–4.50 (m, 1H, H-5), ferrocenyl-1H-1,2,3-triazol-1-yl)-β-D-ribofuranoside (3d). Treat-
4.57–4.60 (m, 4H, CH2NH, H-3 and H-4), 4.71–4.80 (m, 3H, ment of 5-azido-5-deoxy-1-O-methyl-2,3-O-isopropylidene-β-D-
C5H4, H-2), 5.30 (s, 1H, OH), 5.99 (d, 1H, J = 3.21 Hz, H-1), 6.93 ribofuranoside (2d) (0.23 g, 1.39 mmol) and ferrocenoyl pro-
(t, 1H, J = 5.09 Hz, NH), 7.85 (s, 1H, triazole); 13C (75.5 MHz, pargylamide (0.2 g, 1.39 mmol) resulted in 3d as an orange
CDCl3): δ (ppm) = 26.14, 26.77, 34.56, 48.67, 68.15, 68.24, solid. Yield: 0.49 g (80%); mp: 180–185 °C; [α]3D4 (c 0.2, CH3OH)
69.73, 70.69, 74.35, 75.06, 79.19, 85.3, 105.1, 111.93, 124.02 (C5 = −12.06; IR (KBr, νmax/cm−1) = 3399, 3135, 2989, 2945, 1635,
1
triazole), 144.86 (C4 triazole), 171.09 ppm; ESI-MS (in CH3OH): 1519, 1106; H NMR (300 MHz, CDCl3, Me4Si): δ (ppm) = 1.28
m/z = 483 [M + H]+; UV-Vis in CH3OH: λmax [ε(dm3 mol−1 (s, 3H, CH3 of CMe2), 1.44 (s, 3H, CH3 of CMe2), 3.39 (s, 3H,
cm−1)] = 264 (5244), 307 (1196), 442 (220) nm; Anal. calc. for OCH3), 4. 13 (s, 5H, C5H5), 4.34 (t, 2H, J = 2.26 and 1.51 Hz,
C22H26FeN4O5 (482.1): C 54.79, H 5.43, N 11.62; found: C5H4), 4.41 (m, 2H, CH2NH), 4.52–4.57 (m, 2H, H-5), 4.62–4.67
C 54.66, H 5.29, N 11.47.
3-O-Benzyl-5-deoxy-1,2-O-isopropylidene-5-(4-methyl amido (s, 1H, H-1), 6.39 (t, 1H, J = 5.28 and 3.77 Hz, NH), 7.71 (s, 1H,
ferrocenyl-1H-1,2,3-triazol-1-yl)-α-D-xylofuranose
(3b). Treat- triazole); 13C NMR (75.5 MHz, CDCl3 + DMSO): δ (ppm) =
(m, 4H, C5H4, H-3 and H-4), 4.75 (d, 1H, J = 5.28 Hz, H-2), 5.0
ment of 5-azido-3-O-benzyl-5-deoxy-1,2-O-isopropylidene-α-D- 24.92, 26.35, 34.88, 53.16, 55.60, 68.13, 69.68, 70.51, 75.51,
xylofuranose (2b) (0.42 g, 1.39 mmol) and ferrocenoyl propar- 81.74, 84.95, 85.18, 110.01, 112.92, 122.77 (C5 triazole), 145.18
gylamide (0.2 g, 1.39 mmol) resulted in 3b as an orange solid. (C4 triazole), 170.49; ESI-MS (in CH3OH): m/z = 497 [M + H]+;
Yield: 0.56 g (79%); mp: 65 °C; [α]D34 (c 0.2, CH3OH) = −20.37; UV-Vis in CH3OH: λmax [ε(dm3 mol−1 cm−1)] = 264 (4038), 308
IR (KBr, νmax/cm−1) = 3417, 3090, 2984, 2931, 1730, 1641, (908), 449 (134) nm; Anal. calc. for C23H28FeN4O5 (496.14):
1
1530,1076; H NMR (300 MHz, CDCl3, Me4Si): δ (ppm) = 1.30 C 55.66, H 5.69, N 11.29; found: C 55.26, H 5.14, N 10.93.
(s, 3H, CH3 of CMe2), 1.40 (s, 3H, CH3 of CMe2), 3.97 (d, 1H,
J = 3.02 Hz, H-2), 4.14 (s, 5H, C5H5), 4.33 (t, 2H, J = 2.26 and cenyl-1H-1,2,3-triazol-1-yl)-α-D-galactopyranose (3e). Treatment
1.62 Hz, C5H4), 4.51–4.54 (m, 3H, CH2NH and H-5), 4.57–4.62 of 6-azido-6-deoxy-1,2 : 3,4-O-diisopropylidene-α-D-galacto-
6-Deoxy-1,2 : 3,4-di-O-isopropylidene-6-(4-methyl amido ferro-
(m, 3H, C5H4 and H-5), 4.65–4.7 (m, 4H, H-3, H-4 and CH2Ar), pyranose (2e) (0.28 g, 1.39 mmol) and ferrocenoyl propargyl-
5.90 (d, 1H, J = 3.02 Hz, H-1), 6.43 (t, 1H, J = 6.04 and 5.28 Hz, amide (0.2 g, 1.39 mmol) resulted in 3e as an orange solid.
NH), 7.32–7.41 (m, 5H, Ar), 7.61 (s, 1H, triazole) ppm; 13C Yield: 0.38 g (76%); mp: 140 °C; [α]D34 (c 0.2, CH3OH) = −31.7;
NMR (75.5 MHz, CDCl3): δ = 26.10, 26.65, 34.82, 49.17, 66.05, IR (KBr, νmax/cm−1) = 3109, 2984, 2921, 1641, 1379, 1219, 1080,
1
68.19, 69.62, 70.39, 71.85, 78.69, 81.40, 81.85, 105.12, 111.98, 1007, 814; H NMR (500 MHz, CDCl3, Me4Si): δ (ppm) = 1.27
123.39, 127.89, 128.25, 128.61, 136.71 (C5 triazole), 144.85 (C4 (s, 3H, CH3 of CMe2), 1.34 (s, 3H, CH3 of CMe2), 1.39 (s, 3H,
triazole), 170.39 ppm; ESI-MS (in CH3OH): m/z = 573 [M + H]+; CH3 of CMe2), 1.48 (s, 6H, CH3 of CMe2), 4.14 (s, 5H, C5H5),
UV-Vis in CH3OH: λmax [ε(dm3 mol−1 cm−1)] = 262 (5610), 305 4.16–4.20 (m, 2H, CH2NH), 4.30–4.32 (m, 3H, C5H4 and H-6),
(960), 447 (220) nm; Anal. calc. for C29H32FeN4O5 (572.17): 4.45 (dd, 1H, J = 5.9 and 7.9 Hz, H-6), 4.57 (d, 1H, J = 3.99 Hz,
C 60.85, H 5.63, N 9.79; found: C 60.51, H 5.84, N 9.67.
H-5), 4.60–4.64 (m, 3H, H-4, H-3 and H-2), 4.66–4.68 (t, 2H, J =
3-O-Benzyl-6-deoxy-1,2-O-isopropylidene-6-(4-methyl amido 2.0 Hz, C5H4), 5.48 (d, 1H, J = 4.9 Hz, H-1), 6.43 (m, 1H, NH),
ferrocenyl-1H-1,2,3-triazol-1-yl)-α-D-glucofuranose (3c). Treat- 7.75 (s, 1H, triazole); 13C NMR (75.5 MHz, CDCl3): δ (ppm) =
ment of 3-O-benzyl-6-azido-6-deoxy-1,2-O-isopropylidene-α-D- 24.43, 24.82, 25.93, 34.94, 50.42, 67.04, 68.04, 68.17, 69.67,
glucofuranose (2c) (0.33 g, 1.39 mmol) and ferrocenoyl propar- 70.23, 70.43, 70.73, 71.05, 96.19, 108.97, 109.88, 123.59 (C5 tri-
gylamide (0.2 g, 1.39 mmol) resulted in 3c as an orange solid. azole), 156.25 (C4 triazole), 170.32; ESI-MS (in CH3OH): m/z =
Yield: 0.42 g (77%); mp: 65 °C; [α]D34 (c 0.2, CH3OH) = −17.86; 553 [M + H]+; UV-Vis in CH3OH: λmax [ε(dm3 mol−1 cm−1)] =
IR (KBr, νmax/cm−1) = 3384, 3090, 2984, 2931, 1743, 1635, 1531, 262 (4500), 307 (966), 447 (162) nm; Anal. calc. for
1
1076, 1024; H NMR (300 MHz, CDCl3, Me4Si): δ (ppm) = 1.31 C26H32FeN4O6 (552.16): C 56.53, H 5.84, N 10.14; found:
(s, 3H, CH3 of CMe2), 1.43 (s, 3H, CH3 of CMe2), 3.25 (d, 1H, C 56.91, H 5.42, N 9.98.
J = 5.28 Hz, OH), 3.87–3.91 (m, 1H, H-5), 4.13 (s, 5H, C5H5),
2,3,4,6-Tetra-O-acetyl-6-(4-methyl amido ferrocenyl-1H-1,2,3-
4.15 (d = 2H, J = 5.28 Hz, CH2Ar), 4.33 (t, 3H, J = 2.26 Hz, triazol-1-yl)-β-D-glucopyranoside (3f). Treatment of 1-deoxy-
C5H4), 4.40–4.55 (m, 2H, CH2NH), 4.58–4.65 (m, 3H, C5H4, azido-2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (2f) (0.28 g,
H-4), 4.65–4.67 (m, 2H, H-3 and H-2) 4.71–4.74 (m, 2H, H-6), 1.39 mmol) and ferrocenoyl propargylamide (0.2 g,
5.93 (d, 1H, J = 3.02 Hz, H-1), 6.36 (t, 1H, J = 6.04 Hz, NH) 1.39 mmol) resulted in 3f as an orange solid. Yield: 0.38 g
7.28–7.46 (m, 5H, Ar), 7.69 (s, 1H, triazole); 13C NMR (76%); mp: 95 °C; [α]3D4 (c 0.2, CH3OH) = −36.4; IR (KBr, νmax
/
(75.5 MHz, CDCl3): δ (ppm) = 26.25, 26.77, 34.79, 50.14, 53.84, cm−1) = 3109, 2984, 2921, 1641, 1379, 1219, 1080, 1007, 814;
67.67, 68.27, 69.67, 70.51, 72.16, 80.23, 81.05, 81.54, 82.15, 1H NMR (300 MHz, CDCl3, Me4Si): δ (ppm) = 1.86 (s, 3H,
105.19, 111.98, 123.92, 127.80, 128.29, 128.63. 137.08 (C5 OCH3), 2.03 (s, 6H, OCH3), 2.06 (s, 3H, OCH3), 3.97–4.01
triazole), 144.61 (C4 triazole), 170.43; ESI-MS (in CH3OH): (m, 1H, H-5), 4.11 (s, 5H, C5H5), 4.24–4.30 (m, 1H, H-6), 4.35
1182 | Dalton Trans., 2013, 42, 1180–1190
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(d, 2H, J = 1.77 Hz, C5H4), 4.53–4.60 (m, 1H, H-6), 4.67–4.74 incubation for 24 h the cells were treated for 2 days with test
(m, 4H, C5H4 and CH2NH), 5.23 (t, 1H, H-4), 5.39–5.46 (m, 2H, compounds at concentrations ranging from 0.1–100 μm in
H-2 and H-3), 5.86 (d, 1H, J = 8.49 Hz, H-1), 6.49 (t, 1H, J = 5.09 DMSO (1% final concentration) and were assayed at the end of
and 5.28 Hz, NH), 7.90 (s, 1H, triazole); 13C NMR (75.5 MHz, the second day. Each assay was performed with two internal
CDCl3): δ (ppm) = 20.12, 20.44, 20.58, 34.72, 61.46, 67.58, controls: (1) an IC0 with cells only, (2) an IC100 with media
68.01, 68.23, 69.61, 70.32, 70.50, 72.59, 74.97, 75.29, 85.66, only. After incubation for 24 h, the cells were subjected to the
121.15 (C5 triazole), 145.75 (C4 triazole), 168.68, 169.20, MTT colorimetric assay (5 mg mL−1). The effect of the
169.88, 170.43; ESI-MS (in CH3OH): m/z = 641 [M + H]+; UV-Vis different test compounds on the viability of the tumour
in CH3OH: λmax [ε(dm3 mol−1 cm−1)] = 225 (5100), 307 (1168), cell lines was measured at 540 nm on a multimode reader
447 (220) nm; Anal. calc. for C28H32FeN4O10 (640.14): C 52.51, (Infinite® M200, Tecan, Switzerland). The IC50 values (50%
H 5.04, N 8.75. Found: C 52.19, H 5.30, N 8.87.
inhibitory concentration) were calculated from the plotted
6-(4-Methyl amido ferrocenyl-1H-1,2,3-triazol-1-yl)-β-D-gluco- absorbance data for the dose–response curves. The assay was
pyranoside (3g). The acetylated 1,2,3-triazole 3f was dissolved performed using doxorubicin and cisplatin as standards and
in dry MeOH (10 mL) and the solution was made alkaline with 1% DMSO as a vehicle control. To avoid DMSO toxicity, the
a freshly prepared solution of NaOMe in methanol (1 M, values obtained for the DMSO control were subtracted from
1 mL). The resulting solution was stirred at room temperature those of the test compounds. IC50 values (in μM) are expressed
until the formation of the product, as indicated by TLC (4 h). as the average of two independent experiments.
After completion of the reaction, the solvent was removed
Antimicrobial activity
under vacuum and the crude product was purified by column
chromatography using a methanol–ethyl acetate mixture. The
pure compound was dissolved in water and lyophilised. Yield:
0.632 g (76%); mp: 95 °C; [α]3D4 (c 0.2, CH3OH) = −5.7; IR (KBr,
νmax/cm−1) = 3423, 2925, 1633, 1381, 1290, 1099, 1047, 814;
1H NMR (300 MHz, DMSO, Me4Si) δ (ppm) = 1.25 (bs, 4H, OH),
3.46–3.66 (m, 4H, H-6, H-5, H-4), 3.76–3.86 (m, 2H, H-3,H-2),
4.15 (s, 5H, C5H5), 4.35 (t, 2H, J = 1.7 Hz, C5H4), 4.55 (d, 2H,
J = 1.7 Hz CH2NH), 4.82 (t, 2H, J = 1.7 Hz, C5H4), 5.5 (d, 1H, J =
9.1 Hz, H-1), 7.91 (s, 1H, triazole), 8.19 (m, 1H, NH); 13C NMR
(75.5 MHz, CDCl3): δ (ppm) = 27.76, 28.47, 29.21, 37.56, 66.85,
67.37, 68.57, 68.71, 69.72, 77.42, 127.65, 130.30 (C5 triazole),
131.23 (C4 triazole), 166.44; ESI-MS (in CH3OH): m/z = 495
[M + Na]+; UV-Vis in CH3OH: λmax [ε(dm3 mol−1 cm−1)] = 264
(2760), 307 (644), 444 (132) nm; Anal. calc. for C20H24FeN4O6
(472.10): C 50.36, H 5.12, N 11.86. Found: C 49.98, H 5.31,
N 11.15.
The antimicrobial activity of the compounds was determined
by a modified microtiter broth dilution method64 against
different pathogenic reference strains, including Gram-positive
bacteria (Bacillus subtilis MTCC 121, Staphylococcus aureus
MTCC 96, S. aureus MLS16 MTCC 2940, Micrococcus luteus
MTCC 2470), Gram-negative bacteria (Klebsiella planticola
MTCC 530, Escherichia coli MTCC 739 and Pseudomonas aerugi-
nosa MTCC 2452) and Candida albicans MTCC3018, procured
from the Microbial Type Culture Collection (MTCC), CSIR-In-
stitute of Microbial Technology, Chandigarh, India. Sterile
blank discs (6.0 mm dia., HiMedia Laboratories Pvt. Ltd.,
Mumbai, India) were impregnated with the test compounds at
a dose of 1.4–300 μg mL−1 and allowed to dry at room tempera-
ture in a laminar air flow chamber. The sterile discs contain-
ing different test compounds were placed individually on the
surface of the medium in Petri plates, containing Müller-
Hinton agar seeded with 0.1 mL previously prepared microbial
suspensions individually containing 1.5 × 108 cfu mL−1 (equal
to 0.5 McFarland). Standard antibiotic solutions of neomycin
(for bacterial strains), fluconazole (for Candida albicans) and
the well containing methanol (vehicle control) served as posi-
tive and negative controls, respectively. The plates were incu-
bated for 24 h at 37 °C and the minimum inhibitory
concentration (MIC) values were determined as the lowest con-
centration of the test compound, which resulted in a signifi-
cant decrease in the inoculum viability (>95%) at which no
coloration was observed. All experiments were carried out in
duplicate and the mean values were considered.
Biological studies
In vitro cytotoxicity testing. The cytotoxicity of the test com-
pounds was assessed according to literature procedures.63 Cell
lines used for testing in vitro cytotoxicity included HeLa
derived from human cervical cancer cells (ATCC No. CCL-2),
A549 derived from human alveolar adenocarcinoma epithelial
cells (ATCC No. CCL-185), MDA-MB-231 derived from human
breast adenocarcinoma cells (ATCC No. HTB-26) and MCF7
derived from human breast adenocarcinoma cells (ATCC No
HTB-22) were obtained from the American Type Culture Collec-
tion, Manassas, VA, USA. All the tumour cell lines were main-
tained in a modified DMEM medium supplemented with 10%
fetal bovine serum, along with 1% non-essential amino acids
without L-glutamine, 0.2% sodium hydrogen carbonate, 1%
sodium pyruvate and 1% antibiotic mixture (10 000 units peni-
Results and discussion
cillin and 10 mg streptomycin per mL). The cells were washed The ferrocene-carbohydrate amide-triazoles were prepared by
and resuspended in the above medium and 100 μL of this sus- the click reaction between ferrocenoyl propargylamide (1) and
pension was seeded in 96 well-bottom plates. The cells were various sugar azides (2a–f) in the presence of sodium ascor-
t
maintained at 37 °C in a humidified 5% CO2 incubator (Model bate and copper(II) sulfate in a 1 : 1 mixture of BuOH and
2406 Shellab CO2 incubator, Sheldon, Cornelius, OR). After water at 40 °C for 4 h (Scheme 1). The reaction of per-O-acetyl
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glucoside azide (2f) with ferrocenoyl propargylamide was slow
compared to the terminal carbohydrate azides (2a–e). The
water soluble amide-triazole (3g) was prepared by the deacety-
lation of compound 3f using sodium methoxide in
methanol.45,46
1
All the compounds were characterised by H and 13C NMR
spectroscopy. The protons of the unsubstituted cyclopenta-
diene ring appeared as a singlet at 4.1 ppm, while those for
the substituted Cp ring appeared as a triplet/multiplet around
1
4.3 ppm and 4.6 ppm in the H NMR spectra. Except for the
compound 3d, the anomeric proton of the sugar moieties was
observed as a doublet around 5.7–5.9 ppm. In the case of com-
pound 3d, a singlet was observed for the anomeric carbon at
5.6 ppm. The characteristic peaks corresponding to the amide
proton and the triazole protons were observed at around
6.4 ppm and 7.9 ppm, respectively. For the compound 3e, the
amide and the triazole protons were observed at 6.9 and
8.1 ppm, respectively.
Fig. 3 Cyclic voltammograms of ferrocene conjugates 3a, 3f and 3g in 1.0 mM
methanol solution at 25 °C.
In the 13C NMR spectra, the gem-dimethyl groups appeared
as singlets around 24 ppm and 26 ppm. The amide carbonyl
carbon appeared at 170 ppm and the triazole carbons were
observed in the regions 120 ppm and 140 ppm.
DMSO and DMSO-buffer solutions under physiological con-
ditions (pH 6.0–7.4). No significant change in the intensity of
the spectral band in the region 250 and 335 nm indicates the
stability of the compounds in aqueous solutions beyond 8 h.
Except for 3g, all the compounds were characterised by the
[M + H] peak in the mass spectra. For the compound 3g, the
Electrochemistry
[M + Na] peak was observed in the mass spectra. For all The electrochemical characterisation of the compounds was
the compounds, the characteristic IR bands for –NH and –CO performed in methanol by means of cyclic voltammetry (CV)
stretching were observed around 3385–3417 cm−1 and in 1.0 mM methanol solution using a three electrode cell con-
1635 cm−1 respectively.
sisting of a glassy carbon working electrode, platinum wire
The UV-visible spectra of ferrocene conjugates were auxiliary electrode and Ag/AgCl reference electrode with a scan
recorded in methanol over a wavelength range 250–550 nm rate of 0.1 V s−1. The cyclic voltammograms of the compounds
(Fig. 2). All the compounds exhibited two absorption maxima are shown in Fig. 3. All the ferrocene-carbohydrate conjugates
around 260 nm and 310 nm corresponding to the Fe(a1g) → exhibited one electron reversible oxidation wave with respect
Cp(e1g) charge transfer due to the amide-triazole function- to the Ag/AgCl electrode in the presence of 0.1 M tetrabutyl
ality.60,61 In addition to these two absorption maxima, a ammonium perchlorate (TBAP) as the supporting electrolyte.
characteristic broad band was observed around 450 nm due to The compounds exhibited peak separation potentials
the symmetry forbidden Fe(a1g) → Fe(e1g) transitions.
ranging from 89–97 mV and current ratios equal to unity
The stability of the compounds in aqueous media was (Table 1).
determined by recording the UV-visible spectra in aqueous
The cyclic voltammetry experiments were also conducted in
DMSO and DMSO buffer solutions (phosphate buffer pH =
7.0). It was observed that in DMSO solution, the compounds
exhibited a quasi-reversible wave (Fig. 4) with an oxidation
potential ranging from 623–626 mV. Except for 3g, all the com-
pounds exhibited an additional reversible wave around
110–280 mV, resulting from the expulsion of the Cp ring due
to the attack of the nucleophile at the Fe(III) centre.65 In the
case of 3g, only the quasi-reversible wave for the ferrocenium
ion was observed, the absence of the reversible wave may be
due to the stabilization of the molecule by the free hydroxyl
groups of the compound.66,67
On addition of 0.1 M phosphate buffer solution to a
1.0 mM DMSO solution (1 : 1) of the compounds, a reversible
oxidation wave corresponding to the ferrocenium ion was
observed with E1/2 values ranging from 496–561 mV (Fig. 5). It
was also observed that, in the buffer solution, the compounds
were unaffected by the nucleophilic solvent molecules as indi-
cated by the absence of an extra oxidation peak. The peak
Fig. 2 UV-Vis spectrum of ferrocene conjugates 3a, 3d, 3f and 3g in 0.5 mM
methanol solution.
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Table 1 Electrochemical data of compounds 3a–f
Compound
Epa(mV)
Epc(mV)
ΔEp(mV)
ΔE1/2(mV)
ia/ic
In methanol
Ferrocene
477
638
645
644
644
647
644
651
406
549
551
550
552
550
555
559
71
89
94
94
92
97
89
92
441
594
598
597
598
599
600
605
0.99
1.07
1.05
1.05
1.06
1.07
1.07
1.02
3a
3b
3c
3d
3e
3f
3g
In DMSO + buffer
3a
3b
3c
3d
3e
3f
555
461
466
464
452
464
473
512
92
90
92
88
92
83
99
509
510
510
496
510
514
561
1.06
1.01
1.02
1.05
1.06
1.01
1.01
556
556
540
556
556
611
3g
Fig. 5 Cyclic voltammograms of ferrocene conjugates 3a, 3e, 3f and 3g in
1.0 mM DMSO solution containing 0.1 M phosphate buffer (pH = 7.4) at 25 °C.
ΔEP = (Epa − Epc) and ΔE1/2 = 0.5(Epa + Epc), where Epa and Epc are the
anodic and cathodic peak potentials, respectively. The electrode
system consists of
auxiliary electrode and Ag/AgCl reference electrode. Scan rate: 100 mV
s−1
a glassy carbon working electrode, platinum
geometries and interaction of these molecules with the solvent
system.46 It has been mentioned by Vargas-Berenguel and co-
workers that the diffusion coefficients of the carbohydrate
derived ferrocene conjugates were independent of the mole-
cular weight of the compound.46 The objective of the present
study was to observe the trend of the diffusion coefficient of
ferrocene conjugates containing various sugars linked through
amide-triazole linkers. Another point of interest originates
from the lack of substantial literature evidence on the study of
the diffusion coefficient of ferrocene-carbohydrate conjugates
with different sugar moieties in polar and buffer solutions.
Hence, the diffusion coefficient (Df) was calculated, based
on the reversible redox behaviour, in methanol and in buffer
solutions, according to literature procedures.68 In order to cal-
culate the diffusion coefficient, the corresponding anodic peak
currents (ia) were obtained by recording the cyclic voltammo-
grams at various scan rates ranging from 20–100 mV (see
ESI†). The straight line obtained (Fig. 6) due to the diffusion-
controlled electrochemical process shows that the current is
directly proportional to the scanning rate υ1/2. In this case, the
diffusion coefficients (Df) of 3a–g were obtained from the
Randles–Ševčik relationship:68
.
Fig. 4 Cyclic voltammograms of ferrocene conjugates 3a, 3e, 3f and 3g in
1.0 mM DMSO solution at 25 °C.
ia ¼ 0:4463 ꢀ n3=2F 3=2 A ðRTÞꢁ1=2Df 1=2Cυ1=2
separation values were obtained in the range 88–99 mV and
the current ratios approached unity. The stabilization of the
conjugates in buffer solutions leads to a slight decrease in
the electrode potential compared to the DMSO solvent.66,67 In
the case of compound 3g, a slight variation in the E1/2 value
was observed due to the interaction of solvent molecules with
the free hydroxyl groups of the compound.
At 25 °C, the RT value becomes 2480 J mol−1, and by using
the Faraday constant of F = 96 500 C mol−1, the above equation
can be written as
ia ¼ 2:69 ꢀ 105 ꢀ ADf1=2Cυ1=2
where ia is the anodic peak current, A is the area of the elec-
trode (m2), C is the concentration of the electrochemically
active species (mol m−3), n is the number of electrons (for
Determination of diffusion coefficients
In general, it is observed that the diffusion coefficient ferrocene, n = 1), υ expresses the sweep rate (V s−1), and the
decreases with an increase in the molecular weight and size of diffusion coefficient, Df (m2 s−1) of 3a–g thus calculated.
the compound.68 However, a similar trend was not observed in
It was observed from the calculations that the compounds
the case of carbohydrate conjugates due to their different display variable diffusion coefficients with an increase in the
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In DMSO-buffer solution, under physiological pH, the
diffusion coefficient of the compound was found to be almost
ten times lower than in the methanol. For all the compounds,
the Df value was found to be around 0.1–0.18 × 10–10 m2 s−1
,
which indicated a slow diffusion rate due to the stabilization
of the compounds in the buffer system. In conclusion, it was
observed that in the buffer solution, the geometry, the spatial
arrangement and the type of carbohydrate scaffold had no
appreciable effect on the diffusion coefficient of the amide-
triazole linked ferrocene-carbohydrate conjugates.
Biological evaluation
In vitro cytotoxicity (MTT assay). The cytotoxicity of the
ferrocene-carbohydrate amide-triazoles 3a–f was assessed on
the basis of the measurement of the in vitro growth in 96 well
plates by cell-mediated reduction of tetrazolium salt to form
water insoluble formazan crystals according to the literature
procedures.63 These compounds were tested for cytotoxicity
against the different test cell lines up to a concentration of
100 μM. Ferrocene was also tested on these cell lines for com-
parison purposes. The concentration of the compounds at
which 50% of the cell growth was inhibited (IC50) was calcu-
lated and shown in Table 3.
From the literature, it is known that the compounds with
amide-triazole linkers exhibited cytotoxicity towards hormone-
dependent and hormone-independent breast cancer cell
lines.39 However, it was observed that in the case of ferrocene-
carbohydrate amide-triazoles, except for compounds 3b and
3d, all the compounds were non-toxic towards the tested cell
lines. It was interesting to note that although ferrocene exhib-
ited cytotoxicity towards the cell lines, the ferrocene conjugates
containing amide-triazoles derived from galactose (3e) and
glucose (3c, 3f and 3g) did not show any cytotoxicity on the cell
lines.
The amide-triazole 3a, derived from xylofuranose, did not
show cytotoxicity on any cell lines, whereas compound 3b,
derived from the benzylated xylofuranose, exhibited cytotoxi-
city on the hormone-independent breast cancer cell line,
MDA-MB-231, with an IC50 value of 2.82 μM, (so far this
happens to be the best IC50 value reported for ferrocene-carbo-
hydrate conjugates). In addition to this, the compound also
exhibited cytotoxicity on the hormone-dependent breast
cancer cell lines like MCF-7 and A549 with IC50 values of
11.31 μM and 7.01 μM, respectively.
Ribofuranoside amide-triazole (3d) was selectively active on
the breast cancer cell lines. This compound exhibited cytotoxi-
city on the hormone-dependent breast cancer cell line, MCF-7,
with an IC50 value of 3.18 μM. The compound exhibited very
good cytotoxicity on the hormone-independent breast cancer
cell line, MBA-MD-231, with an IC50 value of 7.31 μM. More-
over, it is interesting to note that, irrespective of the linker
used, the ferrocene-carbohydrate conjugates containing the
ribofuranoside moiety displayed almost similar IC50 values for
the MBA-MD-231 cell lines (Table 4).
Fig. 6 Plots of anodic peak current of ferrocene-carbohydrate conjugates in
(a) methanol and (b) DMSO-buffer solutions.
molecular weight of the compound (Table 2). In methanol, the
compounds with free hydroxyl groups (3a and 3g) exhibited
low diffusion coefficients compared to the other compounds
and the Df value was further decreased as the number of free
hydroxyl groups increased, which is evident from the low Df
value of compound 3g. The compounds containing an
O-benzyl group (3b and 3c), showed significantly high Df
values. Compounds 3d, 3e and 3f with completely protected
sugar skeletons (other than benzyl groups) showed Df values in
the range 4.0–5.8 × 10–10 m2 s−1. The compound with an acetyl
protected sugar (3f) exhibited a high Df value compared to the
compound with the free hydroxyl group. The lower Df value of
3g may be due to stabilization by the non-covalent interactions
such as hydrogen-bonding with the solvent molecules. From
this, we can conclude that the electrochemical diffusion
of the ferrocene-carbohydrate conjugates depends on the
nature of the protecting groups as well as the structure of the
carbohydrate moiety.
It was found that among all the sugar derivatives
screened for the present study, the amide-triazole linked
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Table 2 Influence of the molecular weight of compounds on the diffusion coefficient (Df ) in methanol and DMSO-buffer solutions
Compound
Molecular weight (mg mmol−1
)
Carbohydrate scaffold
Df in methanol (×10−10 m2 s−1
)
Df in DMSO-buffer (×10−10 m2 s−1
)
a
Ferrocene
3g
186
472
None
164
1.0
—
0.10
3a
3d
482
496
3.0
4.9
0.18
0.11
3e
3b
552
572
5.8
6.8
0.18
0.22
3c
602
7.0
0.23
3f
640
4.0
0.09
a Not determined.
Table 3 Cytotoxicity of ferrocene amide-triazoles
IC50 (μM)b
Table 4 Effect of linker on IC50 values against MDA-MB-231 in the case of ribo-
furanoside containing ferrocene conjugates
Linker
IC50 (μM) for MDA-MB-231
Compounda A549
HeLa
MDA-MB-231 MCF-7
Amidea
7.29
7.35
7.31
Triazoleb
Ferrocene
21.3 ( 0.13) 28.3 ( 0.09) 35.2 ( 0.15)
54.8 ( 0.05)
11.31 ( 0.03)
3.18 ( 0.10)
3b
3d
7.01 ( 0.11) >100
>100 >100
2.82 ( 0.14)
7.31 ( 0.13)
Amide-triazole
Doxorubicinc 1.21 ( 0.10) 0.45 ( 0.07) 0.50 ( 0.11)
1.05 ( 0.04) a Ref. 61. b Ref. 60.
0.41 ( 0.10)
Cisplatinc
0.11 ( 0.07) 0.20 ( 0.13) 0.21 ( 0.05)
a For compounds 3a, 3c, 3e–g, IC50 values of >100 μM was observed.
b Values are the mean of two independent experiments, and the
standard deviation values are given in brackets. c Standard.
Antimicrobial activity
It is well known that various microbial strains often develop
resistance to antibiotics.69,70 A metal-specific mode of action
of some metallocene drugs has been envisaged to counter
such microbial resistance.16 Edwards and co-workers have syn-
thesised and studied the antimicrobial activity of ferrocenyl
ferrocene-carbohydrate
protected furanose ring structure displayed significant
cytotoxicity.
conjugates
with
a
hydroxyl
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penicillin and ferrocenyl cephalosporin derivatives.71–74 In
recent times, the group of Metzler-Nolte have reported
Acknowledgements
the antimicrobial activity of metallocene and half-metallocene This work was financially supported by the in-house project
derivatives of the conventional drug platensimycin.75–78 In MLP-0008. Dr. L.G. thanks the Department of Science and
addition, certain metallocene-peptide conjugates have also Technology (DST), New Delhi, for the funding. S. B. D. and
exhibited antimicrobial activity.78–81 These findings have P. S. are grateful to the Council of Scientific and Industrial
motivated us to determine the antimicrobial activity of the Research (CSIR), New Delhi for the award of research
ferrocene-carbohydrate conjugates. During the course of fellowships.
our study, we observed that some of the ferrocene-
carbohydrate conjugates displayed antimicrobial proper-
ties.60,61 In the continuation of the study, we decided to extend
our investigation towards finding the effect of the amide-
Notes and references
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