Fluorescent-fipronil: Design and synthesis of a stable conjugate

Article abstract of DOI:10.1016/j.bmcl.2018.06.036

Fipronil is a phenyl pyrazole molecule widely used across the world as both insecticide and veterinary drug. The main goal of this work was to synthesize a fluorescently labeled fipronil derivative for cellular imaging without affecting its intrinsic prop

Full text of DOI:10.1016/j.bmcl.2018.06.036

Accepted Manuscript
Fluorescent-Fipronil: Design and Synthesis of a Stable Conjugate
Adriano Aloisi, Adrien Franchet, Dominique Ferrandon, Alberto Bianco,
Cécilia Ménard-Moyon
PII:
S0960-894X(18)30527-4
https://doi.org/10.1016/j.bmcl.2018.06.036
BMCL 25917
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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14 May 2018
17 June 2018
Please cite this article as: Aloisi, A., Franchet, A., Ferrandon, D., Bianco, A., Ménard-Moyon, C., Fluorescent-
Fipronil: Design and Synthesis of a Stable Conjugate, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://
doi.org/10.1016/j.bmcl.2018.06.036
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Fluorescent-Fipronil: Design and Synthesis of a Stable Conjugate
Adriano Aloisi,1 Adrien Franchet,2 Dominique Ferrandon,2 Alberto Bianco,1 Cécilia Ménard-
Moyon1
1University of Strasbourg, CNRS, Immunology, Immunopathology and Therapeutic
Chemistry, UPR 3572, 67000 Strasbourg, France. E-mail: c.menard@ibmc-cnrs.unistra.fr
2CNRS, M3I UPR 9022, Université de Strasbourg, Strasbourg, France
Abstract
Fipronil is a phenyl pyrazole molecule widely used across the world as both insecticide and
veterinary drug. The main goal of this work was to synthesize a fluorescently labeled fipronil
derivative for cellular imaging without affecting its intrinsic properties. We selected
fluorescein as fluorescent probe and we investigated different strategies for stable chemical
ligation between both entities, such as thiourea and direct peptide bond. While thiourea bond
displayed low stability, direct peptide bond was difficult to achieve due to problems of steric
hindrance. The best result was obtained by conjugation using click chemistry, which allowed
to obtain fipronil stably labeled with the fluorescent probe.
Keywords
Fipronil; fluorescein; azide-alkyne cycloaddition; stability
1

Fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)-phenyl]-4-(trifluoromethylsufinyl)-1-
H-pyrazole-3-carbonitrile) is a phenyl pyrazole insecticide used to protect crops with good
selectivity between insects and mammals.1 It became recently famous due to the egg scandal
(millions of eggs have been contaminated with fipronil despite being banned by the European
Union for use on animals intended for human consumption). Fipronil has been investigated
for its insecticidal activity, toxicology, and impact on the environment.2-7 In 2011 Vidau et al.
showed that sublethal doses of fipronil highly increased mortality of honeybees previously
infected by the microsporidian parasite N. ceranae.2 However, uninfected honeybees exposed
to fipronil were significantly less affected (mortality or behavior), meaning that uninfected
honeybees would be able to respond to insecticides by enhancing detoxification process. In
these studies, the activities of two enzymes implicated in insect detoxification, namely 7-
ethoxycoumarine-O-deethylase (ECOD) and glutathione-S-transferase (GST) were assessed.
While ECOD activity remained unchanged, GST activity was enhanced in the midgut and fat
body, in contrast with the enhancement of infected honeybee susceptibility to fipronil. This
means that GST would not be involved in detoxification process of fipronil. In 2013 the
metabolic fate and the elimination of fipronil in rats were investigated using radiolabeled
fipronil.3 This study revealed that fipronil was slowly metabolized and excreted in these
animals. Indeed, except in feces, no traces of parent compound were found in the analyzed
samples. However, the highest level of radioactivity was found in adipose tissue and adrenals.
To investigate further the toxic effect of fipronil and allow mechanistic studies, we wished to
label fipronil with a fluorescent probe. For this purpose, we covalently functionalized fipronil
with fluorescein using different chemical ligations and we investigated the stability of
fluorescent fipronil (thermal and light sensitivity). For in vivo imaging, the conjugate has to
display high stability to guarantee the colocalization of fipronil with the probe. We found that
the strategy based on copper(I) catalyzed azide-alkyne cycloaddition (CuAAC) reaction was
the most efficient to obtain a stably labeled fipronil derivative that will be used for cellular
imaging.
Derivatization of fipronil with fluorescein through a thiourea bond
As the 3-cyano and 4-trifluoromethylsulfinyl groups of phenyl pyrazole of fipronil are key
pharmacophores, the main approaches reported for the derivatization of fipronil rely on the
modification of the amino group on the pyrazole ring.8 In this context, we initially relied on a
previous work that investigated the derivatization of the free amine of fipronil 1 with
fluorescein isothiocyanate (FITC) 2 through a thiourea bond (Scheme 1).9 Fipronil reacted
2

with FITC in the presence of triethylamine at 40°C, giving the expected fluorescent conjugate
3 with 49% yield.
Scheme 1. Synthesis of fipronil functionalized with fluorescein through a thiourea bond.
The desired compound 3 was characterized by 1H and 13C NMR in different deuterated
solvents and by LC-MS. First, NMR in DMSO-D6 displayed unexpected signals in the
aromatic area. This may be due to cis-trans equilibrium around the thiourea bond10 and to the
possible formation of the lactone on the fluorescein moiety. To avoid this equilibrium, NMR
analysis was performed by increasing the temperature to 50°C (Figure S1). This analysis
confirmed our hypothesis as less peaks were observed, but there were still undefined signals.
However, when NMR was performed in deuterated methanol at 20°C, the peaks were better
defined and their integration perfectly corresponded to the proton number of compound 3
(Figure S2). This result shows that the solvent certainly plays a crucial role in the
conformation of the molecule. LC-MS analysis confirmed the synthesis of the desired
compound (m/z = 827.8 [M+H]+).
For in vivo experiments, the compound has to be stable at physiological temperature
for many hours or days. We performed HPLC analysis in order to study the stability of the
conjugate (Figure 1). We observed that an aqueous solution of compound 3 stored at room
temperature for 10 hours was unstable as four news peaks appeared. Compound 3 is probably
photo- and temperature-sensitive. We also analyzed a solution stored at 4°C and we observed
that it was slightly more stable, but still some degradation peaks appeared. Even when stored
as a powder at 4°C, compound 3 displayed a certain level of degradation.
3

Figure 1. HPLC chromatograms of compound 3 (top) just after the synthesis and (bottom)
stored in different conditions: in solution at rt (blue) and at 4°C (black), and as powder at 4°C
(red).
The main degradation product at 5.8 min was isolated by preparative HPLC and
analyzed by LC-MS. This product corresponded to a mass of m/z = 348.2 identified as 5-
aminofluorescein ([M+H]+), confirming the low stability of the thiourea bond of compound 3
especially in solution. These results highlight the water sensitivity of thiourea bonds. Some
studies have reported the instability of thiourea bonds in water, leading to ammonium and
cyanate ions as hydrolysis products.11-12
Although this fluorescent compound was previously used in biological studies, we
would like to underline that fipronil functionalized with FITC is not stable, even at low
temperature and in powder form. Therefore, we can clearly conclude that this derivative is not
suitable for biological experiments as only the fluorophore (not fipronil) would be detected.
We recommend that careful stability studies are performed with fipronil derivatives before
any in vitro and/or in vivo assay.
Synthesis of fipronil-fluorescein via the formation of an amide bond
Due to the low stability of the thiourea bond, we designed a new fipronil derivative based on
the formation of an amide bond between the amino group of fipronil and the carboxylic
function of 5(6)-carboxyfluorescein 4 (Scheme 2). The amide should be more stable
compared to the thiourea bond. The reaction was performed by pre-activation of 5(6)-
4

carboxyfluorescein 4 with N,N’-diisopropylcarbodiimide (DIC) and N-hydroxysuccinimide
(NHS). The activated ester reacted then with the amino group of fipronil in the presence of
triethylamine to form desired compound 5.
Scheme 2. Synthesis of fipronil functionalized with fluorescein through an amide bond.
The amidation reaction was monitored by HPLC, which confirmed the activation of
both isomers of 5(6)-carboxyfluorescein as NHS esters (Figure 2). Indeed, after 2 h a peak
shift of 2 min (from 6.9 and 7.1 to 8.9 and 9.1 min, respectively) was observed, confirming
the formation of the activated 5(6)-carboxyfluorescein. However, after addition of fipronil
and triethylamine, several peaks appeared at ~13.5 and 16 min but with very low intensity.
After 3 h we could identify by LC-MS one peak with m/z = 794.8 corresponding to the
desired product ([M+H]+). However, even after heating at 40°C for 5 h, the reaction did not
proceed further and the main peaks observed were still the activated fluorescein at 8.9 and 9.1
min and starting fipronil at 15.0 min. The crude mixture was purified by preparative HPLC.
Nevertheless, we were not able to isolate a reasonable amount of compound 5. This difficulty
can be attributed to two factors: i) the low reactivity of the amino group of fipronil as the
electron lone pair of the nitrogen is not highly nucleophilic due to the conjugation on the
pyrazole ring; ii) steric hindrance due to the fact that all the aromatic groups considerably
decrease the degree of freedom of the intermediates formed. As a consequence, we decided to
try another approach to overcome these issues.
5

Figure 2. HPLC chromatograms at 254 nm of 5(6)-carboxyfluorescein (A), the reaction
mixture just after the addition of fipronil t=0 (B), and the reaction mixture after stirring for
3 h at room temperature (C).
Synthesis of fipronil-fluorescein conjugate via click chemistry
In view of the difficulties to synthesize a stable fluorescently labeled fipronil derivative, we
explored a third strategy using click chemistry, in particular the CuAAC reaction,13,14 between
fipronil and fluorescein derivatized with an acetylene and an azide linker, respectively. The
concept of click chemistry was introduced by K. B. Sharpless in 2001 and it refers to
reactions that are orthogonal, high yielding, and fast.15 Moreover, in our case the presence of
linkers allowed decreasing steric hindrance. For this purpose, fipronil initially reacted with a
slight excess of propargyl bromide in the presence of potassium hydroxide (Scheme 3).16
However, following a procedure reported in literature we isolated only the di-alkylated
1
derivative S1 (80% yield), as confirmed by H and 13C NMR and LC-MS (m/z = 513.0
[M+H]+) (Figure S3). To solve this problem, we improved the published protocol. We
increased the dilution to reach lower concentrations, reduced the amount of propargyl
bromide to a sub-stoichiometric amount (0.9 eq.), and decreased temperature from rt to 0°C
during the addition of propargyl bromide in order to avoid the formation of the di-alkylated
6

compound. Moreover, propargyl bromide was added dropwise as a solution in acetone for 15
min. Using these optimized conditions, we were able to obtain the desired mono-alkylated
compound 7 in a reasonable yield (50%).
Scheme 3. Synthesis of fluorescein-fipronil derivative 9 through CuAAC.
In parallel, fluorescein was derivatized with an azide group. For this purpose, 5-
carboxyfluorescein
6
was
activated
with
2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethylaminium tetrafluoroborate (TBTU) and then reacted with 3-azidopropylamine to
give the desired fluorescein-azide derivative 8 (Scheme 3).17 Surprisingly, we discovered that
the work-up reported in literature did not work in our case (addition of 10% aqueous NaOH
followed by diethyl ether extraction). Therefore, we modified the work-up by acidification of
the reaction mixture to reach pH = 2 using HCl 6N and extraction of the compound with ethyl
acetate. The crude residue was purified by preparative HPLC to give the desired compound 8
in 15% yield. The low amount of isolated product can be explained by the difficulty to dilute
the compound in a water miscible solvent that forced us to add DMF, resulting in an
important loss of the compound in the HPLC injection peak.
The last step consisted in the CuAAC reaction between the two precursors previously
synthesized. The reaction was performed in the presence of a catalytic amount of copper
sulfate and ascorbic acid in 1:1 THF/water, leading to the formation of compound 9 in mild
conditions and with 85% yield after purification by silica gel column (Scheme 3). Overall, the
synthetic strategy based on CuAAC was longer (3 steps) compared to the other approaches (1
7

step) investigated in this work, but it resulted much more efficient. The stability of compound
9 was investigated by HPLC and we observed no degradation peak both in powder form and
in solution over a period of 24 h. In addition, we stored the compound as a powder and in
DMSO (1 mg/mL) at -20°C for more than six months without observing any degradation.
In conclusion we have investigated different chemical ligations to derivatize fipronil
with fluorescein. The approaches relying on the formation of thiourea or amide bond between
the two entities were not satisfying due to instability and low yield, respectively. The most
efficient strategy was based on CuAAC reaction allowing to obtain a compound with high
stability and not photosensitive. These properties ensure the relevance of in vitro and in vivo
biological experiments. The fipronil-fluorescein conjugate will be used for cellular imaging
for mechanistic investigation of fipronil activity. As we encountered many problems while
trying to reproduce protocols reported in literature, we optimized conditions to solve these
issues. Hence, we believe this work will be very helpful for the design of fipronil-based
derivatives with high stability in biological assays.
Acknowledgments
This work was supported by the Centre National de la Recherche Scientifique (CNRS) and by
the International Center for Frontier Research in Chemistry (icFRC). The authors gratefully
acknowledge financial support from the Agence Nationale de la Recherche (ANR) through
the LabEx project Chemistry of Complex Systems (ANR-10-LABX-0026_CSC). Preliminary
experiments were performed by Jean-Baptiste Madinier.
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Graphical Abstract
Unstable
Stable
fluorescent fipronil
fluorescent fipronil
10