Synthesis, characterization, and pharmacological studies of ferrocene-1H-1,2,3-triazole hybrids

Article abstract of DOI:10.1016/j.molstruc.2017.06.027

A series of ferrocene-1H-1,2,3-triazole hybrids namely 1-(4-nitrophenyl)-4-ferrocenyl-1H-1,2,3-triazole (1), 1-(4,4′-dinitro-2-biphenyl)-4-ferrocenyl-1H-1,2,3-triazole (2), 1-(3-chloro-4-fluorophenyl)-4-ferrocenyl-1H-1,2,3-triazole (3), 1-(4-bromophenyl)-

Full text of DOI:10.1016/j.molstruc.2017.06.027

Accepted Manuscript
Synthesis, characterization, and pharmacological studies of ferrocene-1H-1,2,3-
triazole hybrids
Ashanul Haque, Ming-Fa Hsieh, Syed Imran Hassan, Md. Serajul Haque Faizi,
Anannya Saha, Necmi Dege, Jahangir Ahmad Rather, Muhammad S. Khan
PII:
S0022-2860(17)30806-2
10.1016/j.molstruc.2017.06.027
MOLSTR 23913
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 20 March 2017
Revised Date: 6 June 2017
Accepted Date: 6 June 2017
Please cite this article as: A. Haque, M.-F. Hsieh, S.I. Hassan, M.S. Haque Faizi, A. Saha, N. Dege, J.A.
Rather, M.S. Khan, Synthesis, characterization, and pharmacological studies of ferrocene-1H-1,2,3-
triazole hybrids, Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.06.027.
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ACCEPTED MANUSCRIPT
Ferrocene-Triazole hybrid

ACCEPTED MANUSCRIPT
Synthesis, characterization, and pharmacological studies of ferrocene-1H-
1,2,3-triazole hybrids
Ashanul Haque*a, Ming-Fa Hsieh*,b, Syed Imran Hassana, Md. Serajul Haque Faizia, Anannya
Sahac, Necmi Deged, Jahangir Ahmad Rathera, Muhammad S. Khan*a
aDepartment of Chemistry, Sultan Qaboos University, P.O. Box 36, Al Khod 123, Sultanate of
Oman.
bDepartment of Biomedical Engineering, Chung Yuan Christian University, 200 Chung Pei
Road, Chung Li District, Taoyuan City, Taiwan 32023, R.O.C.
cDepartment of Chemical Sciences, Indian Institute of Science Education and Research Kolkata,
Mohanpur 741 246, India.
dDepartment of Physics, Faculty of Arts and Sciences, Ondokuz Mayis University, 55139
Atakum, Samsun, Turkey.
ABSTRACT
A series of ferrocene-1H-1,2,3-triazole hybrids namely 1-(4-nitrophenyl)-4-ferrocenyl-1H-1,2,3-
triazole (1), 1-(4,4′-dinitro-2-biphenyl)-4-ferrocenyl-1H-1,2,3-triazole (2), 1-(3-chloro-4-
fluorophenyl)-4-ferrocenyl-1H-1,2,3-triazole (3), 1-(4-bromophenyl)-4-ferrocenyl-1H-1,2,3-
triazole (4) and 1-(2-nitrophenyl)-4-ferrocenyl-1H-1,2,3-triazole (5) were designed and
synthesized by copper-catalyzed azide alkyne cycloaddition (CuAAC) reaction. All the new
hybrids were characterized by microanalyses, NMR (1H and 13C), UV-vis, IR, ESI-MS and
electrochemical techniques. Crystal structure of the compound (3) was solved by single crystal
X-ray diffraction method. The structural (single crystal) and spectroscopic (UV-Vis. and IR)
properties of the compound 3 have been analyzed and compared by complementary quantum
modeling. Hybrids 1-5 exhibited low toxicity and demonstrated neuroprotective effect.
Keywords: Anti-inflammatory; Click-chemistry; Copper catalyzed azide alkyne cycloaddition
reaction; Ferrocene; Viability; Toxicity.
*Corresponding authors:
E-mail address:
haque_chem@yahoo.com, ashanul@squ.edu.om (AH)
mfhsieh@cycu.edu.tw (MH), msk@squ.edu.om (MSK)
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1. Introduction
A diverse range of natural and synthetic heterocyclic compounds endowed with unique chemical
and biological properties have been serving to the humankind since many centuries.[1-4]
Scaffolds based on N/O/S heteroatoms are perhaps the most studied class of chemicals
worldwide. Especially, N-containing fragment, termed as azo, is the most commonly studied
moiety by the medicinal and materials chemists. [5-8] Basically, the presence of a heteroatom
(viz nitrogen) profoundly modulates the physico-chemical, electronic and metabolic
characteristics of a molecule leading to significant change in the biological activity. In this
context, the versatile application of azo-based compounds in medicinal chemistry is well known.
Among them, 1,2,3-triazole is one of the promising molecular scaffolds present in numerous
biologically active agents.[9] Due to its high metabolic reactivity and propensity to biological
receptors via polar and non-polar interactions, last few decades have seen huge research effort in
the pursuit of developing new 1,2,3-triazoles-based pharmaceutical agents possessing anti-
cancer, anti-fungal, anti-malarial, anti-diabetic properties.[4, 10-13] Furthermore, easy synthetic
methodologies and control over purpose-specific functionality installation make this class of
molecules an obvious choice for the design and development of new drugs.[14]
Recent drug discovery programs have also seen a tremendous interest in the development of
metal-based drugs.[15-17] In addition to classical platinum and ruthenium-based
pharmaceuticals, transition metal-ion based stable sandwich metallocenes (like ferrocene,
ruthenocene, titanocene, osmocene, etc) also emerged as promising candidate for drug
development. [18-20]
Among them, ferrocene is the most popular and offers easy
functionalization or modification to prepare a library of compounds.[21-23]. Well known
examples of ferrocene-based therapeutic agents in clinical use are hydroxyferrocifen for breast
cancer and ferroquine for malaria treatment.[24] As a consequence of unique reactivity and
diverse biological potential, several studies have been undertaken to demonstrate the usefulness
of ferrocene grafting in an organic backbone.[25-34] Chart 1 shows some of the recently
reported potent ferrocene-triazole hybrids. Supuran and co-workers [35] reported triazole-
ferrocene-conjugates as carbonic anhydrase (CA) inhibitors with Ki in nanomolar range. These
inhibitors, in which 3- or 4-aminobenzenesulfonamides were connected to ferrocene unit through
a triazole ring, displayed high selectivity for cancer-associated isoenzyme CA IX and CA XII.
Kumar and co-workers [26, 30] found 1H-1,2,3-triazole tethered isatin-ferrocene conjugates as
2

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promising candidate for tuberculosis therapy. The same group reported next generation anti-
malarial agents composed of functionalized isatin and ferrocene connected through 1H-1,2,3
triazole. These hybrids were active against both chloroquine-susceptible (3D7) and chloroquine-
resistant (W2) strains of P. falciparum.[36] Recently, some ferrocene-cinchona hybrids
connected via triazole and chalcone linkers have been reported.[37] The reported hybrids
showed substantial antiproliferative activity against human malignant cell lines (HepG-2 and
HT-29 cells).
Chart 1: Some ferrocene and ferrocene-1H-1,2,3-triazole based pharmacophores.
Although a large body of publications on ferrocene, 1H-1,2,3-triazole and their conjugate is
available, but, as per our best of the knowledge, no report ever been published dealing with the
biological effects ferrocene-1H-1,2,3-triazole hybrids on microglial cells. A search on web of
science using keywords “triazole and microglial cells” resulted in only three publications and
none of them reported the anti-inflammatory potential of ferrocene-1H-1,2,3-triazole conjugates.
This notion motivated us to design and develop new hybrid molecules with different
functionalities and study their anti-inflammatory potential on microglial BV-2 cells.
Herein, we report on the synthesis, characterization, electrochemical and in-vitro
pharmacological (cell viability and anti-inflammatory) properties of five new ferrocene-1H-
1,2,3-triazole hybrids 1-5. (Scheme 1). The compounds were obtained through click-chemistry
using ethynyl ferrocene and substituted anilines as starting precursors. Crystal structures of the
compound (3) was solved by single crystal X-ray diffraction method. The structural (single
crystal) and spectroscopic (UV-Vis. and IR) properties of the compound 3 was analyzed and
compared by complementary quantum modeling. The compounds were examined in in-vitro
model of microglial cells derived from central neural system to elucidate how the substitution
pattern on the triazole ring affected the biological properties.
2. Experimental
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All chemicals, except where stated otherwise, were obtained from Sigma Aldrich and used as
received. Sodium ascorbate was synthesized from L-ascorbic acid in good yield following
procedure reported in the literature.[38] Column chromatography was performed on alumina
(Brockman Grade II-III) using dichloromethane/hexane as eluent.
2.1. Characterization
Melting points were determined by using open capillary method and are uncorrected. 1H and 13C
NMR were recorded on Bruker Avance III HD 700 MHz spectrometer equipped with a 5 mm
TCI H/C/N cryoprobe. The chemical shifts values were recorded on δ scale (expressed in ppm)
and the coupling constants (J) in Hertz. Tetramethylsilane (TMS) was used as internal standard.
The following abbreviations were used in reporting spectra: s = singlet, d = doublet, dd = double
doublet, m = multiplet. Mass spectra were obtained using a VG Autospec magnetic sector
instrument using ESI technique in positive/negative ion modes. Attenuated-total-reflectance IR
spectra were recorded on pure samples on diamond using a Cary 630 FT-IR spectrometer.
Absorption spectra were recorded on a Varian-Cary 50 UV-visible spectrophotometer using a
quartz cuvette with a 1 cm path length. Microanalyses were performed by EuroEA Elemental
Analyser. Single crystal X-ray diffraction for compound 3 was carried out on a Stoe IPDS 2
diffractometer equipped with a graphite crystal monochromator situated in the incident beam for
data collection at room temperature.[39] The determination of unit-cell parameters and data
collections were performed with Mo-Kα radiation (λ= 0.71073 Å).[40] Unit cell dimensions
were obtained with least-squares refinements, and all structures were solved by direct methods
with SHELXT2015.[41, 42] All H atoms were located from difference-Fourier maps but in the
final cycles of refinement they were included in calculated positions and treated as riding
atoms: C—H = 0.93–0.98 A˚ with Uiso(H) = 1.2Ueq(C). The crystal data and structure
refinement of compound 3 are listed in Table ST1. Electrochemical measurements of the
compounds were performed using potentiostat (Gamery Instruments, USA) connected to a
conventional three electrode cell system consists of glassy carbon (GCE), platinum wire (Pt) and
Ag/AgCl (saturated KCl) used as working, counter and reference electrodes, respectively. The
measurements were carried out at a concentration of (3.0 mg mL-1 solution) in tetrahydrofuran
containing 0.1 M tetrabutylammonium perchlorate [nBu4N]ClO4 as a supporting electrolyte. All
the measurements were performed at room temperature in the potential range from −0.1 to +0.9
V with a scan rate of 50 mV s−1. The onset oxidation potentials of synthesized compounds (1, 3-
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5) were calculated from the intersection of two tangents drawn at the rising and background
currents of the cyclic voltammogram. The LUMO energy levels of the compounds was
calculated by equation ELUMO = [EHOMO+ Eg (opt.)] eV, where optical band gap (Eg = 1240/λonset
is estimated from the onset of the absorption spectra of synthesized compounds.
2.2. General protocol for the synthesis of azides (A1-A5)
)
The intermediates and final products have been obtained via following a recently reported
literature method.[13] Briefly aromatic azides were obtained as follows: aromatic amines (2.0
mmol) were dissolved in minimum amount of hydrochloric acid (6N) at room temperature
followed by cooling in ice-water and addition of aqueous solution of sodium nitrite (2.0 mmol)
with continuous stirring. After 10 min. of stirring at same temperature, sodium azide (2.2 mmol)
was added to the reaction mixture. This mixture was further stirred at room temperature for 2-3
h. The product was extracted using chloroform and the organic layer was washed with brine
solution and dried over anhydrous Na2SO4. After evaporation of the solvent, the crude products
(A1-A5) were pure enough for further reactions.
2.3. General protocol for the synthesis of ferrocene-1H-1,2,3-triazole hybrids
Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is a well-known regioselective
method for making 1H-1,2,3-triazole bearing building blocks for medicinal and material
chemistry applications. The high popularity of this method is due to its simplicity, large
functional group tolerability, eco-friendly nature and high selectivity (1,2,3-triazole functionality
rather than 1,2,4-triazole). [43, 44] Ferrocene-based 1,2,3-triazoles hybrids 1-5 were synthesized
in good yields following procedure reported in the literature. [13] In a typical method,
ethynylferrocene (2.3 mmol) and substituted aromatic azide A1-A5 (2.6 mmol) were suspended
in N,N-dimethylformamide in one neck RBF. To this mixture, the solution of sodium ascorbate
(0.3 mmol in minimum amount of water) and Cu(II) sulfate pentahydrate (0.03 mmol in
minimum amount of water) were added and stirred vigorously at room temperature. The progress
of the reaction was monitored by TLC. After completion of the reaction, the mixture was poured
into water-ice and the precipitate was collected by filtration. The desired products were purified
by alumina column chromatography using gradient dichloromethane/hexane.
1-(4-Nitrophenyl)-4-ferrocenyl-1H-1,2,3-triazole (1)
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1
Red solid, Yield: 81%, M.P: Decompose at > 230 ºC; H NMR (700 MHz, CDCl3): δ/ppm 8.44
(d, J = 8.7 Hz, 2H, H-3 & 5), 8.02 (d, J = 8.7 Hz, 2H, H-2 & 6), 7.97 (s, 1H, H-7), 4.81 (m, 2H,
13
H-10 & 11), 4.38 (m, 2H, H-12 & 13), 4.13 (m, 5H, H-14 - 18); C NMR (176 MHz, CDCl3):
δ/ppm 148.9 (C-8), 147.0 (C-4), 141.2 (C-1), 125.7 (C-3 & 5), 120.1 (C-2 & 6), 115.9 (C-7), 74.0
(C-9), 69.7 (C-14 – 18), 69.2 (C-10 & 11), 66.9 (C-12 & 13). ESI-MS: m/z 374.07 (M+). Anal.
calc. for C18H14FeN4O2: C 57.78 %, H 3.77 %, N 14.97 %; found: C 58.54 %, H 3.91 %, N 14.28
%.
1-(4,4′-Dinitro-2-biphenyl)-4-ferrocenyl-1H-1,2,3-triazole (2)
Orange solid, Yield: 75%, M.P: 216-217 ºC; 1H NMR (700 MHz, CDCl3): δ/ppm 8.38 (d, J = 8.7
Hz, 2H, H-3’ & 5’), 7.70 (s, 1H, H-7), 7.70 (m, 1H, H-4), 7.69 (d, J = 8.7 Hz, 2H, H-2’ & 6’),
7.65 (d, J = 2.1 Hz, H-6), 7.27 (d, J = 8.3 Hz, H-3), 4.71 (m, 2H, H-10 & 11), 4.34 (m, 2H, H-12
& 13), 4.12 (m, 5H, H-14 - 18); 13C NMR (176 MHz, CDCl3): δ 149.0 (C-8), 148.9 (C-5), 147.7
(C-4’), 144.4 (C-2), 144.2 (C-1’), 131.0 (C-3), 130.5 (C-1), 129.8 (C-2’ & 6’), 124.5 (C-3’ & 5’),
115.8 (C-7), 113.4 (C-4), 110.2 (C-6), 74.1 (C-9), 69.6 (C-14 – 18), 69.1 (C-10 & 11), 66.8 (C-
12 & 13). ESI-MS: m/z 495.06 (M+). Anal. calc. for C24H17FeN5O4: C 58.20 %, H 3.46 %, N
14.14 %; found: C 55.54 %, H 3.57 %, N 14.90 %.
1-(3-Chloro-4-fluorophenyl)-4-ferrocenyl-1H-1,2,3-triazole (3)
Yellowish solid, Yield: 60%, M.P: 245 ºC; 1H NMR (700 MHz, CDCl3): δ 7.91 (dd, J = 8.7, 2.5
Hz, 1H, H-6), 7.86 (s, 1H, H-7), 7.71 (m, 1H, H-2), 7.34 (m, 1H, H-5), 4.80 (m, 2H, H-10 & 11),
4.38 (m, 2H, H-12 & 13), 4.14 (m, 5H, H-14 - 18); 13C NMR (176 MHz, CDCl3): δ 158.5 (C-3),
148.2 (C-8), 133.6 (C-1), 122.7 (C-6), 122.6 (C-4), 120.0 (C-2), 117.7 (C-5), 116.4 (C-7), 74.5
(C-9), 69.6 (C-14 – 18), 69.0 (C-10 & 11), 66.8 (C-12 & 13). ESI-MS: m/z 380.01 (M-1)+. Anal.
calc. for C18H13ClFFeN3: C 56.65 %, H 3.43 %, N 11.01 %; found: C 56.05 %, H 3.36 %, N
10.01 %.
1-(4-Bromophenyl)-4-ferrocenyl-1H-1,2,3-triazole (4)
Creamy solid, Yield: 72%, M.P: 262 ºC; 1H NMR (700 MHz, CDCl3): δ 7.88 (s, 1H, H-7), 7.70
(m, 2H, H-2 & 6), 7.69 (m, 2H, H-3 & 5), 4.80 (m, 2H, H-10 & 11), 4.37 (m, 2H, H-12 & 13),
4.14 (m, 5H, H-14 - 18); 13C NMR (176 MHz, CDCl3): δ 148.0 (C-8), 136.0 (C-1), 132.9 (C-3 &
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5), 122.1 (C-4), 121.7 (C-2 & 6), 116.2 (C-7), 74.7 (C-9), 69.6 (C-14 – 18), 68.9 (C-10 & 11),
66.8 (C-12 & 13). ESI-MS: m/z 409.9744 (M + 3H)+. Anal. calc. for C18H14BrFeN3: C 52.98 %,
H 3.46 %, N 10.30 %; found: C 52.47 %, H 3.33 %, N 9.25 %.
1-(2-Nitrophenyl)-4-ferrocenyl-1H-1,2,3-triazole (5)
Brown solid, Yield: 69%, M.P: 184 ºC; 1H NMR (700 MHz, CDCl3): δ 8.11 (m, 1H, H-3), 7.82
(m, 1H, H-5), 7.76 (s, 1H, H-7), 7.72 (m, 1H, H-4), 7.71 (m, 1H, H-6), 4.82 (m, 2H, H-10 & 11),
4.37 (m, 2H, H-12 & 13), 4.15 (m, 5H, H-14 - 18); 13C NMR (176 MHz, CDCl3): δ 147.8 (C-8),
144.4 (C-2), 133.8 (C-5), 130.6 (C-4), 130.4 (C-1), 127.9 (C-6), 125.6 (C-3), 119.9 (C-7), 74.2
(C-9), 69.7 (C-14 – 18), 69.0 (C-10 & 11), 66.8 (C-12 & 13). ESI-MS: m/z 374.04 (M+). Anal.
calc. for C18H14FeN4O2: C 57.78 %, H 3.77 %, N 14.97 %; Found: C 57.07 %, H 3.74 %, N
12.86 %.
3. Computational Details
All DFT calculations were performed using the Gaussian 09 program [45] with a hybrid
exchange-correlation functional Coulomb-Attenuating Method-B3LYP (CAM-B3LYP).[46]
Here ECP (effective core potential) quality basis set proposed by Hay and Wadt, LANL2DZ
pseudopotential[47, 48] has been employed for iron center. For carbon and hydrogen; 6-
311+G(d) [49] (a valence double-zeta polarized basis set) was used. While atomic orbitals of all
other atoms are described by 6-31G(d,p) basis set. [50] In order to substantiate the nature of the
ground-state geometries of each optimized-structure, frequency calculations were performed at
the same level of theory, as that of geometry-optimization. TD-DFT calculations were done
employing the CAM-B3LYP functional and the polarizable continuum model, CPCM with
dicholoromethane (ε = 8.93) as the surrounding solvent in order to account for the bulk solvent
effects.[51] TD-DFT-derived electronic spectra were plotted using GaussSum [52], Chemcraft
[53] visualization program was used to generate the corresponding orbitals of the optimized
structure.
4. Pharmacological Studies
4.1.1. In-vitro cell viability assay:
Murine microglial cell line (BV-2) was cultured in 10% Fetal bovine serum buffered with high
glucose Dulbecco's Modified Eagle Medium in the incubator kept at 37 °C and 5% CO2. BV-2
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cells in the culture T-flask were passaged for every 2 days. The cell viability was determined by
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. For cell viability
assay, BV2 cells were seeded in 96-well plate at a density of 1 ×104 cell per well and incubated
for 24 hr. The cells were then treated with different concentrations of drug for 24hr. After
incubation for 24 hr, the culture medium was removed and a mixture of culture medium and
MTT reagent (5 mg/mL) at a volume ratio of 9:1 was added in each well. Following an
additional 4 hr incubation at 37 °C, DMSO (100 µL) was added in each well to dissolve
formazan crystals. The absorbance was then measured at 570 nm using ELISA reader. The cell
viability was determined using the equation:
ꢋꢆꢌ. ꢍꢎ ꢏꢐꢑꢒꢌ
ꢉ ꢊ
ꢀꢁꢂꢂ ꢃꢄꢅꢆꢄꢂꢄꢇꢈ % =
ꢕ 100%
ꢋꢆꢌ. ꢍꢎ ꢀꢓꢔ
4.1.2. In-vitro inflammatory response of ferrocene-1H-1,2,3-triazole hybrids:
BV-2 cells were seeded in a 96-well plate (2 × 104 cells/well) and incubated in 37◦C, 5% CO2 for
1 day. To test the inflammatory response, BV-2 cells were incubated in medium containing
various concentrations of compounds 1-5 including ferrocene (10 µg/mL) and 50 ng/mL of LPS
for the activation of inflammation for 24 hr. The supernatants were collected after 24 h and NO
production was determined by Greiss reagent (1% sulfanilamide, 2.5% H3PO4, 0.1%
naphthylethylene diamine dihydrochloride). Briefly, 50µL culture medium and 50 µL Greiss
reagent were incubated for 10 min. The absorbance was then measured at 540 nm using ELISA
reader.
5. Results and discussion
5.1. Synthesis and Characterization
Target ferrocene-1H-1,2,3-triazole hybrids (1-5) were obtained in a two-step reaction procedure
as reported in the literature [13] and outlined in Scheme 1.
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Scheme 1. Synthesis of ferrocene-triazole hybrids. Reagents and conditions: (a) HCl (6N), 0 ºC
→ RT, 3-4 h. (b) DMF, Sod. Ascorbate, CuSO4.5H2O, RT, 4-5 h.
The key precursor compounds ethynylferrocene and substituted aniline were commercially
obtained. The aromatic azides (A1-A5) were obtained by treatment of substituted aniline with
sodium azide and sodium nitrite in acidic aqueous medium. Note that the azides (A1-A5) were
used as prepared without further purification. Ferrocene-1H-1,2,3-triazole hybrids (1-5) were
obtained by copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction of equimolar
mixture of substituted azides and ethynylferrocene. The crude products were purified by alumina
column chromatography using hexane-dichloromethane as eluents. The products were fully
characterized by UV-visible, IR and NMR spectroscopy, mass spectrometry, and elemental
analysis. UV-vis. absorption spectra of the compounds in DCM (vide infra) showed almost
similar absorption pattern in the UV region (226 nm-275 nm), except for compound 2, which
also showed comparatively stronger absorption in near visible region (385 nm), due to extended
conjugation. The IR spectra lacked peaks in the region of ~ 2110 cm-1 (azide) and 2105-2109 cm-
1 (alkynes), indicating the formation of products. (Figure S1, supplementary file) Furthermore,
the presence of peaks at ~1650 and 1430 cm-1 was attributed to triazole rings.[54] The proton
attached to C-7 of the 1,4-disubstituted-1H-1,2,3-triazole ring appeared as a singlet between δ
7.97-7.70 ppm. (Figure S2-S11, supplementary file) Moreover, the presence of 13C peak of C-7,
between δ115-120 ppm further confirmed the formation of 1,4-disubstituted-1H-1,2,3-triazoles
(Figure S2-S11, supplementary file) instead of isomeric 1,5-disubstituted-1H-1,2,3-triazoles,
which shows shift at higher values.[55] The mass spectrometry results confirm the molecular
masses expected for the compounds. The molecular structure of compound (3) was confirmed X-
ray crystallography (vide-infra).
5.2. X-ray crystal structure
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To underpin the results of spectroscopic characterization, we were able to grow single crystal of
compound 3 suitable for X-ray diffraction. The crystallographic data is summarized in Table 1
while experimental details and crystal structure data is shown in Table ST1 (supplementary file).
As clear from the structure (Fig. 1), compound 3 contain one benzene ring (C1-C6) attached to
the central triazole ring (N1/N2/N3/C7/C8) which is nearly planar [r.m.s. deviation = 0.0031 Å].
In the title molecule, the benzene ring and cyclopentadienyl ring (C9-C13) form dihedral angles
of 29.764 (1) and 15.084 (1)°, respectively, with the triazole ring (Fig. 1). The cyclopentadienyl
rings of the ferrocene moiety are parallel to each other, with a dihedral angle of 0.629 (2)°.
Figure 1: The molecular structure of compound 3, with atom labeling. Displacement ellipsoids
are drawn at the 40% probability level.
Table 1: Selected geometric parameters of compound 3 (Å, º)
Fe1—C18
Fe1—C13
Fe1—C14
C9—C8
Fe1—C17
C8—N3
Fe1—C15
N1—N2
Fe1—C16
N1—C7
2.011 (3)
2.042 (2)
2.016 (3)
1.460 (3)
2.023 (3)
1.368 (3)
2.031 (3)
1.349 (2)
2.033 (3)
1.350 (3)
2.037 (2)
1.426 (3)
2.0397 (19)
1.723 (2)
Fe1—C11
C3—F1
Fe1—C12
2.040 (2)
1.347 (3)
2.042 (2)
120.93 (16)
120.68 (19)
108.95 (17)
119.4 (2)
107.37 (16)
119.0 (2)
119.63 (18)
165.5 (2)
0.3 (2)
N2—N1—C6
C1—C2—Cl1
N2—N3—C8
F1—C3—C4
N3—N2—N1
F1—C3—C2
C3—C2—Cl1
C13—C9—C8—C7
C8—N3—N2—N1
C10—C9—C8—C7
C9—C8—C7—N1
Fe1—C10
N1—C6
Fe1—C9
C2—Cl1
−15.4 (3)
179.22 (19)
The dihedral angle between N-phenyl (C1-C6) ring and triazole (N1-N3/C7/C8) ring is 29.77(3)°
which indicate non planarity of the molecule. The dihedral angle between the cyclopentadienyl
(Cp) (C9-C13) ring and the triazolyl (N1-N3/C7/C8) ring is 15.05 (3)°. The N-phenyl (C1-C6)
and cyclopentadienyl (Cp) (C9-C13) rings form a dihedral angle of 43.77(3)°. In the ferrocene
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group of the molecule, the Fe—C bond distances in unsubstituted cyclopentadienyl (Cp) ring
[C14–C18] are in the range of 2.011 (3)–2.033 (3) Å and 2.037 (2)–2.042 (2) Å for the
substituted Cp ring [C9–C13]. Both substituted and unsubstituted Cp rings are in eclipsed form.
The distance from iron centre to centroid of unsubstituted and substituted Cp rings are 1.649 (9)
and 1.645 (9) Å respectively. The angle between [Cg1-Fe1-Cg2] shows 179.04 (19)°. The C8
atom is almost in the plane of their carrier Cp ring. (See Fig. 2 and Table 1 for details). The
crystal structure is stabilized by intermolecular C-H···π interactions between Hydrogen (H4) and
unsubstituted cyclopenta-dienyl ring (Cg1 = 2.519 Å).
Figure 2: A view of the C—H⋯π interactions, shown as dashed lines in the crystal structure of
compound 3.
6. DFT Calculations
The structure of the 3 with substituted ƞ5- cyclopentadienyl ligand has been well described by
DFT calculations at the CAM-B3LYP level of theory (see Computational Details in the
Experimental Section). Geometry-optimization of compound 3 was done using X-ray
coordinates. The DFT calculated bond-lengths and bond-angles of the optimized structure are
found to be in good agreement with those extracted from the experimental analysis. (Figure 3
and Table ST2 (supplementary file).
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Figure 3: Crystal structure (A) and DFT optimized structure (B) of compound 3.
To get more insight about the electronic transitions of the given complex time dependent (TD)-
DFT calculation has been performed. The calculated and experimental UV-Vis. spectra is shown
in Fig. 4(a-b). Experimentally, dichloromethane solution of 3 showed intense absorptions in UV
region at 275 nm and shoulder at ~ 350 nm (Figures 4b). On the other hand, theoretical
calculation indicated absorption around 250 nm and 350 nm. Calculation on 3 also revealed that
all transitions are mainly due to intraligand charge-transfer from ƞ5-cyclopentadienyl moiety to
benzene ring via 1H-1,2,3-triazole group. (Table ST3, supplementary file).
Figure 4: (a) TD-DFT simulated electronic spectrum of 3 and (b) Absorption spectra of
compounds 1-5 in DCM solution.
The observed and calculated IR spectra of compound 3 are depicted in Figure 5(a,b). The
frequencies
(observed
and
calculated)
using
DFT
CAM-B3LYP/6-31+G(d,p)/
6311+G(d)/631G(d,p) force field along with their relative intensities and probable assignments
are listed in Table ST4 (Supplementary file). Here we found that aromatic compounds exhibited
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multiple weak bands in the region 3100–3000 cm−1 due to aromatic C–H stretching
vibrations.[56]The strongest absorptions in the region 1085–850 cm−1 are due to the C–H
vibrations out of the plane(wagging) to the aromatic ring.[57-59] Based on the literature survey,
we can explain that the FT-IR bands at 1517 and 1440 cm−1 arise due to the in-plane and out of-
plane bending as well as stretching vibrations of C-N bond of the compound.[60]Again, the C–Cl
and C-F stretching vibrations generally give strong bands in the region 1300 cm-1 and 750 cm−1
respectively.[61] These results provide a good agreement with the experimental result.
Figure 5:
(a) ATR in solid state and (b) DFT calculated spectrum of 3.
Analysis of orbitals reveals that HOMO has major ligand character and based mainly on
cyclopentadienyl moiety. (Fig. 6) Thus during oxidation electron is supposed to be removed
from the cyclopentadienyl moiety. The LUMO has also major ligand character and it is mainly
based on benzene-triazole ring. Therefore, reduction will take place at the above-mentioned
benzene-triazole moiety.
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Figure 6: Representative molecular orbitals involved in TD-DFT transitions of the compound 3.
7. Electrochemical properties
The redox properties of newly synthesized compounds (1-5) were studied at the glassy carbon
electrode using cyclic voltammetry (CV) to estimate the HOMO and LUMO energy levels. The
CV curves and the electrochemical parameters of compounds (1-5) are shown in Fig. 7 and
Table 2. The external standard potential of the ferrocene/ferricenium ion couple (EFc/Fc+) was
estimated under the same experimental condition and the value was located at 0.41 V vs
Ag/AgCl electrode. As shown in Fig. 7 and Table 2, the synthesized compounds 3-5 depicts a
well-defined ferrocene-based one-electron reversible wave with the half-wave potentials (E1/2) of
320 mV, 450 mV and 320 mV respectively compared to compound 1 which has E1/2 = 444 mV.
However, the redox properties of the compound 2 was inhibited due to unknown reason.
The oxidation potentials of synthesized compounds (1, 3, 4 and 5) are characterized by an
irreversible wave with onset oxidation potentials at 0.39, 0.34, 0.32, and 0.31 V respectively. The
HOMO energy levels of compounds was calculated using the equation
EHOMO = −[Eox(onset)−EFc/Fc++ 4.8] eV, where Eox represents the onset oxidation potential. [62] The
HOMO energy levels of the synthesized compounds (1, 3, 4 and 5) were estimated to be −4.78,
−4.73, −4.71, and −4.70 eV, respectively. The LUMO energy levels of (1, 3, 4 and 5) compounds
were found to be −2.53, −2.36, −2.36, and −2.34 eV, respectively. The value of band-gap
Eg(electro.) calculated by CV ranges from 2.25-2.37 eV, similar to calculated using optical
absorption Eg(op.). The role of frontier molecular orbitals in affecting biological and material
properties is well established [4, 63] as small interfrontier orbitals favors high chemical reactivity
and low kinetic stability.[64-66] In our case, compounds 1-5 have moderate interfrontier orbitals
energy level, indicating towards the stable nature of the compounds.
Table 2: Electrochemical and optical parameters of synthesized compounds.
b
Comp.
E1/2
(V)
0.44
-
0.32
0.45
0.32
∆
E
Eox(onset)
(V)
0.39
-
0.34
0.32
0.31
cEHOMO
(eV)
-4.78
-
-4.73
-4.71
-4.70
dELUMO
(eV)
-2.53
-
-2.36
-2.36
-2.36
eEg(electro.)
(eV)
2.25
-
2.37
2.35
2.34
eEg(op)
(eV)
2.25
2.63
2.37
2.35
2.34
(mV)
85
-
71
82
72
1
2
3
4
5
aE1/2 = Epa + Epc/2; b
ꢀE
Epa - Epc; cE HOMO - [Eox (onset) - EFc/Fc (E1/2) + 4.80] eV; dE LUMO = [E HOMO
+
Eg (Opt.)]; eEg (electro.) = E HOMO - E LUMO,; f=Eg (Opt) = 1240/λonset,
.
=
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Figure 7: CV of ferrocene and ferrocene-1H-1,2,3-triazole hybrids (1-5) at a concentration of 3
mgmL-1 in tetrahydrofuran.
8. Pharmacology: Cytotoxicity and inflammatory response assays
The cytotoxicity and anti-inflammatory properties of the synthesized compounds were
determined (vide-infra). In-vitro cell viability (cytotoxicity) was assessed by the MTT assay,
while anti-inflammatory study by Greiss assay to detect nitric oxide (NO).
8.1. In-vitro cell viability assay:
In-vitro cell viability assay using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) is an established preliminary method to evaluate the toxicity of new compounds. In this
method, succinate dehydrogenase present in living mitochondrion reduces MTT to generate
water-insoluble purple formazan crystals. The generated crystals are then dissolved in organic
solvent (DMSO) and measured spectrophotometrically.[67, 68] To explore the anti-inflammatory
effect of synthesized compounds on the mammalian cells, the ferrocene-triazole hybrids (1-5)
were assessed on murine microglia cell lines (BV-2 cells) at different concentration (0.1, 1, 10
and 100 ug/mL). The cell viability (%) obtained with continuous exposure for 24 h is depicted in
Fig. 8. The cytotoxicity of all compounds was found to be concentration-dependent. Up to
concentration of 1 µg/mL, the viability was found > 90 % and the half lethal dose (IC50) for all
compounds were found between 10 to 100 µg/mL. Except compound 1, BV-2 cells showed
similar level of viability against all other compounds including the reference material ferrocene
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(F). Compound 1 showed relative high cytotoxicity to BV-2 cells. It contains a nitro group while
two nitro groups are present in compound 2 which is not as cytotoxic as that of compound 1. To
know what happened when the cells interact with these compounds, biochemical analysis is
required. In addition, computational study might also give cues to answer this question. In
summary, the synthesized compounds have low toxicity level, and hence have potential for safe
utilization.
Figure 8: Percentage of viable cells after 24 h treatment of murine microglia cell lines (BV-2
cell) with compounds 1-5 and reference (ferrocene, F) evaluated by the MTT assay.
8.2. In-vitro inflammatory response of BV-2 cells to the synthesized compounds
Microglial cells are macrophage cells in central nerve system for primary immune function. In
diseased brain, microglial cells are activated to damage other surrounding cells such as neurons.
Therefore, the present study aims to explore how microglial cells respond to the triazole
molecules synthesized. The relationship between over reactive glial cells and neurodegenerative
diseases is well established. [69-73] The hyperactivity of microglia produces inflammatory
mediators which triggers inflammation leading to neuronal damage.[74] In the inflammatory
disorders in the central nerve system, nitric oxide is released from microglial cells along with
other inflammatory molecules such as Tumor necrosis factor-α and Interleukin-1β.[75] The
present assay is used to investigate how lipopolysaccharide (LPS)-activated BV-2 cells respond
to the synthesized compounds. As the first step to evaluate the synthesized compounds, the
amount of nitric oxide (NO) produced by the microglial cells could be an indicator for the
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potency of anti-inflammation. Furthermore, inhibition of over-reactive glial cells would be a
promising strategy for the treatment of neuron-inflammation related diseases like disorders such
as Alzheimer’s disease (AD), and Parkinson’s disease (PD) etc. Herein, BV-2 cells were co-
cultured with compounds 1-5 and lipopolysaccharide (LPS). A separate group of LPS was used a
positive control to indicate the activation of inflammation in BV-2 cells. The effect of
compounds on NO production in microglial cells (BV-2) is shown in Figure 4. Experimental
results indicated that that compound 1-5 were mostly non-inflammatory since they did not
increase the NO production (Fig. 9). The NO production remained as low as that of the control
group. Conversely, LPS (50 ng/ml) significantly increased the NO production of BV-2 cells
(about 3 times to control group, P < 0.01) indicating that the cells can be activated by LPS. The
calibration curve of nitrite showed linear increase in absorbance with increase in concentration of
NO. (Fig. 9b) The results shown here indicates that the compounds endow BV-2 cells a potency
of neuroprotection to the stimulus of LPS. The exact mechanism of anti-inflammatory effects
requires further detailed study. Furthermore, Cyclooxygenase is a typical enzyme responsible for
the inflammation and pain. In a report of inflammation inhibitor study for 1,2,3-triazole
compounds [76], it was found that the derivatives of 1,2,3-triazoles can inactivate isoenzyme
COX-1 which is predominately localized in microglia. Compounds 1-5 will be further
investigated as inhibitors of neuro-inflammation and neuro-degeneration.
Figure 9: (A) Microglial cells (BV-2 cells) treated with Ferrocene (F) and compounds 1-5 (X-1
to X-5) for 24 hr in the presence of 50 ng/mL of LPS. LPS group (50 ng/mL) was used to treat
BV-2 cells to confirm the activation of cells. Then the medium was removed and analyzed by
Griess Reagent. Data represent the mean ±S.D. The results shown were obtained from 3
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independent experiments. ****P<0.001 as compared with the LPS group. (B) Calibration curve
of Nitrite.
9. Conclusion
A series of ferrocene-triazole hybrid compounds has been synthesized via copper-catalyzed azide
alkyne cycloaddition reaction (CuAAC) between substituted anilines and ethynylferrocene. The
compounds have been characterized by analytical and spectroscopic techniques, including X-ray
single crystal for compound 3. In-vitro pharmacological (cell viability and anti-inflammatory)
studies indicated that the compounds exhibit low toxicity and have non-inflammatory effect. The
results of this study will be a valuable addition to the growing body of work on biological
activities of metallocenes.
Acknowledgement
We thank the Department of Chemistry, SQU and the Central Analytical and Applied Research
Unit (CAARU) facility at Sultan Qaboos University, Oman for analytical data. AH
acknowledges the Research Council (TRC), Oman for a Post-doctoral fellowship. MFH
acknowledges financial support from the Ministry of Science and Technology, Taiwan under
grant number MOST-102-2632-M-033-001-MY3.
Data-access statement
Crystallographic data from the structural analysis have been deposited with the Cambridge
Crystallographic Data Centre under the CCDC codes 1532260, and are available free of charge
from https://summary.ccdc.cam.ac.uk/structure-summary-form. Any other data may be obtained
on request from the authors.
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3-triazoles and validation of a predictive pharmacometric PLS model, European journal of
medicinal chemistry 94 (2015) 252-264.
Captions to Illustration
Caption to Chart
Chart 1:
Some ferrocene and ferrocene-triazole based pharmacophores.
Caption to Scheme
Scheme 1. Synthesis of ferrocene-triazole hybrids. Reagents and conditions: (a) HCl (6N), 0
ºC → RT, 3-4 h. (b) DMF, Sod. Ascorbate, CuSO4.5H2O, RT, 4-5 h.
Caption to Table
Table 1:
Table 2:
Selected geometric parameters of compound 3 (Å, º).
Electrochemical and optical parameters of synthesized compounds.
Caption to Figures
Figure 1:
The molecular structure of compound 3, with atom labeling. Displacement
ellipsoids are drawn at the 40% probability level.
Figure 2:
A view of the C—H⋯π interactions, shown as dashed lines in the crystal structure
of compound 3.
Figure 3:
Figure 4:
Crystal structure (A) and DFT optimized structure (B) of compound 3.
(a) TD-DFT simulated electronic spectrum of 3 and (b) Absorption spectra of
compounds 1-5 in DCM solution.
Figure 5:
(a) ATR in solid state and (b) DFT calculated spectrum of 3.
Figure 6:
Figure 7:
Representative molecular orbitals involved in TD-DFT transitions of the
compound 3.
CV of ferrocene and ferrocene-1H-1,2,3-triazole hybrids (1-5) at a concentration
of 3 mgmL-1 in tetrahydrofuran.
Figure 8:
Percentage of viable cells after 24 h treatment of murine microglia cell lines (BV-
2 cell) with compounds 1-5 and reference (ferrocene, F) evaluated by the MTT
assay.
23

ACCEPTED MANUSCRIPT
Figure 9:
(A) Microglial cells (BV-2 cells) treated with Ferrocene (F) and compounds 1-5
(X-1 to X-5) for 24 hr in the presence of 50 ng/mL of LPS. LPS group (50 ng/mL)
was used to treat BV-2 cells to confirm the activation of cells. Then the medium
was removed and analyzed by Griess Reagent. Data represent the mean ±S.D. The
results shown were obtained from 3 independent experiments. ****P<0.001 as
compared with the LPS group. (B) Calibration curve of Nitrite.
24

ACCEPTED MANUSCRIPT
Highlights
•
•
•
•
•
Five new ferrocene-triazole hybrids were synthesized.
Hybrids were characterized by different spectroscopic techniques.
The structure of one of the hybrid was characterized by X-ray crystallography.
DFT studies were conducted to further support the structure.
In-vitro cell viability and inflammatory responses of the hybrids were studied.