Synthetic Strategies for the Modification of Diclofenac

Article abstract of DOI:10.1055/s-0036-1588858

For many heterogeneous sensor applications as well as the synthesis of hapten antigens to produce antibodies, protein conjugates of the target substance are essential. A requirement is that the target substance already offers or is modified to contain a functionality that allows for coupling to a protein, that is, an amino acid residue. Ideally, to avoid shielding of the compound by the carrier protein, a sufficient distance to the protein surface should be provided. With its carboxyl function diclofenac (DCF) allows for direct binding to lysine residues after in situ synthesis of the NHS ester. One problem is that diclofenac as free acid tends to autocondensation, which results in low yields. Here we describe the 'insertion' of a C6 spacer via synthesis of the amide with 6-aminohexanoic acid. To carry out the reaction in solution, first the methyl ester of the amino acid had to be produced. Due to otherwise low yields and large cleaning efforts, solid-phase synthesis on Fmoc Ahx Wang resin is recommended. The crude product is mainly contaminated by cleavage products from the resin which were removed by chromatography. The structure of the highly pure hapten was completely determined by nuclear magnetic resonance (NMR) spectroscopy.

Full text of DOI:10.1055/s-0036-1588858

SYNLETT
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© Georg Thieme Verlag Stuttgart · New York
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S. Schmidt et al.
Letter
Synlett
Synthetic Strategies for the Modification of Diclofenac
Stephan Schmidta,b
Sven Haneltc
O
O
O
O
Fmoc deprotection
O
O
Carsten Canitzd
Holger Hoffmanna,e
Leif-Alexander Garbeb,d
NH2
O
O
Cl
N
N
H
H
Cl
OH
Fmoc Ahx Wang Resin
Rudolf J. Schneider*a,b
0
0
0
0
0-
0
0
3
2-
2
2
8
1-
2
4
8
O
Diclofenac coupling
O
O
a Bundesanstalt für Materialforschung und -prüfung (BAM),
Abteilung1: Analytische Chemie; Referenzmaterialien, Rich-
ard-Willstätter-Straße 11, 12489 Berlin, Germany
rudolf.schneider@bam.de
O
O
N
O
H
b Technische Universität Berlin, Institut für Biotechnologie,
Fachgebiet für Bioanalytik, Gustav-Meyer-Allee 25, 13355 Ber-
lin, Germany
Product cleavage
H
OH
N
Cl
O
H
N
H
c Versuchs- und Lehranstalt für Brauerei in Berlin (VLB) e.V., For-
schungsinstitut für instrumentelle Bier- und Getränkeanalytik
(FIBGA), Seestraße 13, 13353 Berlin, Germany
d Hochschule Neubrandenburg, Fachbereich Agrarwirtschaft
und Lebensmittelwissenschaften, Brodaer Straße 2, 17033
Neubrandenburg, Germany
N
Cl
Cl
Cl
e Humboldt-Universität zu Berlin, Institut für Chemie, Brook-
Taylor-Straße 2, 12489 Berlin, Germany
Received: 24.03.2017
Accepted after revision: 09.05.2017
Published online: 06.06.2017
ly used and accordingly also detected regularly in the
aquatic environment. In Germany alone, every year up to
80 t are sold.1 Around 65–70% of ingested DCF is excreted in
urine.2 Topical use leads to wash-off of the compound while
showering. Due to incomplete removal in wastewater treat-
ment plants (WWTP, depending on the study between 17–
75%3), residues in surface water and drinking water have
been detected. In India, the accumulation of DCF has led to a
mass extinction of vultures.4
DOI: 10.1055/s-0036-1588858; Art ID: st-2017-b0210-l
Abstract For many heterogeneous sensor applications as well as the
synthesis of hapten antigens to produce antibodies, protein conjugates
of the target substance are essential. A requirement is that the target
substance already offers or is modified to contain a functionality that
allows for coupling to a protein, that is, an amino acid residue. Ideally,
to avoid shielding of the compound by the carrier protein, a sufficient
distance to the protein surface should be provided. With its carboxyl
function diclofenac (DCF) allows for direct binding to lysine residues af-
ter in situ synthesis of the NHS ester. One problem is that diclofenac as
free acid tends to autocondensation, which results in low yields. Here
we describe the ‘insertion’ of a C6 spacer via synthesis of the amide
with 6-aminohexanoic acid. To carry out the reaction in solution, first
the methyl ester of the amino acid had to be produced. Due to other-
wise low yields and large cleaning efforts, solid-phase synthesis on
Fmoc Ahx Wang resin is recommended. The crude product is mainly
contaminated by cleavage products from the resin which were re-
moved by chromatography. The structure of the highly pure hapten
was completely determined by nuclear magnetic resonance (NMR)
spectroscopy.
Cl
H
N
Cl
OH
O
Figure 1 Diclofenac (DCF)
Due to these circumstances DCF was introduced to the
first Watch List of the EU Water Framework Directive.5 The
proposed limit of 100 ng/L specified for WWTP effluents is
clearly exceeded according to several studies.2,6 Current de-
tection in environmental samples is carried out mainly by
GC–MS and LC–MS methods. Another approach is the de-
tection by immunoassays. These are cost-effective meth-
ods, require less time, and enable for the high-throughput
analysis of many samples. Measurement at the point of
sampling is possible, too, as immunosensors7 and dipstick
assays8 are becoming widely spread. There are antibodies
Key words diclofenac, hapten synthesis, solid-phase synthesis, spacer
introduction, amide linkage
Diclofenac (DCF, Figure 1) is a pharmacologically active
compound which is used as a nonsteroidal anti-inflamma-
tory drug against fever, inflammation, rheumatic diseases,
and pain. Its mode of action is based on inhibition of cyclo-
oxygenase-2. Of all drugs, DCF is among the most common-
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

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S. Schmidt et al.
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against a variety of pharmacologically active substances, for
example, caffeine9, carbamazepine,10 isolithocholic acid,11
sulfonamides,12 2,4,6-trinitrotoluene (TNT),13 picoxystrob-
in,14 cocaine,15 and bisphenol A.16 A polyclonal anti-
diclofenac antibody is available, with which already mea-
surements were made in environmental samples.17 Recent-
ly, the development of a monoclonal antibody was pub-
lished.18 It is the first application of a monoclonal anti-
diclofenac antibody in environmental analysis. However, al-
ready in 2010, different monoclonal antibodies against di-
clofenac and a number of its metabolites were developed
for medical research. Yet, only the production of IgE-con-
taining polyclonal antisera was published.19 All these anti-
bodies are based on immunogens generated by the direct
coupling of diclofenac to the respective carrier protein via
its carboxyl function. However, through the introduction of
a spacer, more selective and possibly antibodies with high-
er affinity could be expected. An optimum of the length of
alkyl spacers is often achieved with six carbon atoms.20 As a
spacer of choice 6-aminohexanoic acid is selected. Via an
amide linkage between the spacer and DCF the hapten H1
DCF-Ahx (Figure 2) is created. Because of the carboxylic
acid group H1 can be bound to proteins under the same
conditions as DCF. An alternative is hapten H2, an ester of
DCF and 2-aminoethanol (Figure 3). H2 allows the coupling
via an amino function.
are very difficult. This paper is intended to provide an over-
view of successful syntheses21 resulting from some general
problem solving approaches and the application of the
products.
The goal of the experiments was the synthesis of an am-
ide of diclofenac and 6-aminohexanoic acid H1 as well as an
ester of diclofenac and 2-aminoethanol H2. The haptens
can be synthesized under homogeneous conditions. Alter-
natively, it is possible to produce H1 in a solid-phase syn-
thesis. A modification of diclofenac proved to be extremely
difficult because of the very rapidly occurring cyclization
under acidic conditions or after activation with several acti-
vation regents, like NHS, hydroxybenzotriazole (HOBT), or
oxalyl chloride.
Synthesis of H1 was performed in DMF. Ahx is not solu-
ble in DMF. This problem was circumvented by using an
Ahx methyl ester. Initial experiments aimed at activation of
DCF with NHS, 1-hydroxybenzotriazol (HOBT), or the syn-
thesis of various mixed anhydrides, followed by reaction
with AhxMe. Although a product formation was detected
by HPLC–MS, the reaction proceeded in low yield. More-
over, after two-stage purification by column chromatogra-
phy on silica gel and reversed-phase chromatography con-
taminants were still found in an LC–UV/Vis run (Figure 5).
The difference of 0.2 min in the UV trace and the MS trace is
caused by the transfer of sample from the HPLC to the MS
detector.
Cl
H
N
H
N
O
Cl
OH
O
Figure 2 Structure of hapten DCF-Ahx (H1)
Cl
H
N
Cl
O
NH2
O
Figure 3 Structure of hapten H2
Commercially, DCF is available as the sodium salt. If DCF
is converted into the free carboxylic acid or an activated
form, cyclisation is the result. The cyclisation product (DCF
lactam, Figure 4) was found in environmental samples, too.
Because of the cyclization side reaction chemical modi-
fications, such as the syntheses of novel diclofenac haptens,
O
N
Cl
Cl
Figure 5 HPLC chromatogram of DCF-AhxMe, UV/Vis trace (upper),
MS trace at m/z = 423 (lower)
Figure 4 Cyclisation product of DCF: DCF lactam
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Because DCF already forms a methyl ester, when it is
acidified in methanolic solution, it has been tried to make
this property employable for hapten synthesis of H2. For
this purpose, DCF was dissolved in 2-aminoethanol. The
solution was then acidified. HPLC–MS analysis confirmed
product formation, but in low yield (Figure 6). Higher
amounts in starting materials could provide higher yields.
However, the purification proved to be difficult, which is
why this synthesis must be assessed overall as ineffective.
why a glass column was used as a reactor. Reactants and
wash solutions can be easily rinsed from the resin. The du-
ration of the synthesis depends strongly on the type and
porosity of the resin, since the frit of the column may clog,
which results in very long rinsing times. As optimum resin
Fmoc Ahx Wang Resin was determined. Advantages are on
the one hand, that an appropriate C6 spacer is already
bound to the resin, so only the coupling of diclofenac is re-
quired and the consumption of solvents is drastically re-
duced. Moreover, all solutions can be rapidly withdrawn
from the resin. All resins in common is that together with
the product impurities are cleaved from the resin, which
cannot be completely removed under vacuum and act as
solvent for the product. For this reason, a preliminary puri-
fication on silica gel is required. The final purification was
carried out by HPLC. The yield was 0.4%, reproducibly. Us-
ing 5 g resin, 2.3 mg hapten DCF-Ahx could be obtained.
This amount is sufficient to synthesize protein conjugates
for immunization and development of an ELISA. In addition,
NMR spectra can be produced for structure confirmation.
As can be seen in the chromatogram with diode array de-
tection (DAD), the product is slightly contaminated (Figure
7).
Figure 6 HPLC chromatogram of H2, DAD trace (upper) and MS trace
at m/z = 339 (lower)
Another approach is the reaction with Grignard- or lith-
ium-activated spacers, where diclofenac does not require
activation. Diclofenac must be available as methyl ester as
starting material. The DCF methyl ester is easy to synthe-
size. However, the reaction with an organometallic com-
pound leads again only to the cyclic product DCF lactam. It
can be assumed that the ester is cleaved intramolecularly
under such basic conditions.
As described, a methyl ester of H1 could be synthesized
in homogeneous solution. Because of the impurities and a
required ester hydrolysis, this synthesis route was ineffec-
tive. For this reason, solid-phase synthesis was chosen, as
here impurities can be washed from the resin. Due to the
low loading, 5 g of resin were used for each batch to be able
to provide sufficient product. Various resins have been test-
ed in different reaction chambers. First, the reaction was
carried out in glass bottles, followed by filtration of the res-
in. However, these steps were too cumbersome, which is
Figure 7 HPLC chromatogram of DCF-Ahx, DAD (upper) and MS trace
at m/z = 409 (lower)
This contamination could not be removed and leads to
additional peaks in the NMR spectrum. Nevertheless, the
structure could be unambiguously confirmed by NMR spec-
troscopy. Furthermore, it had no effect on protein coupling.
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

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The main reason for the low yield is the aforementioned cy-
clization of DCF after activation. Also, synthesis in the mi-
crowave reactor did not improve the yield. A new approach
for synthesis of DCF-Ahx was the activation with NHS. This
activation is often used in peptide modifications. In classi-
cal solid-phase synthesis the activation and coupling is
done in situ in one hour. Activation with NHS for DCF-Ahx
needs to be conducted overnight. The long activation time
reduces side reactions, like cyclization, and leads to higher
yields. However, the coupling must be done under argon at-
mosphere. This approach gave 138 mg hapten DCF-Ahx. In
addition to the higher yield the product was cleaner. Only a
purification step by column chromatography on silica was
necessary. The HPLC chromatogram (Figure 8) and NMR
spectra (Figure 9 and Figure 10) show 100% purity. The high
amount of product allows to determine the structure. Par-
allel to our work another group synthesized an amide of
DCF and glycine (DCF-Gly) in similar ways by solid-phase
synthesis.22
Figure 9 1H NMR spectrum of DCF-Ahx (H1)
As described before, the reactivity of diclofenac allows
only very limited scope for syntheses. The added value to
the known synthesis of DCF-Gly22 is that our synthesis of
DCF-Ahx (aminohexanoic acid amide of diclofenac) pro-
vides via solid-phase synthesis a pure product in high yield
by avoiding cyclization. The product enables, due to the C6
Figure 10 13C NMR spectrum of DCF-Ahx (H1)
spacer, for protein conjugates with better exposure of the
diclofenac moiety, an important feature with antigens for
antibody production.
Funding Information
This study was supported by Bundesministerium für Wirtschaft und
Energie (BMWi) via the funding programme Zentrales Innovations-
programm Mittelstand (ZIM, KF 2201065AJ3 and KF 2132333AJ3)
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References
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Figure 8 HPLC chromatogram of DCF-Ahx (H1), DAD (upper) and MS
trace at m/z = 409 (lower)
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

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S. Schmidt et al.
Letter
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(4) Saini, M.; Taggart, M. A.; Knopp, D.; Upreti, S.; Swarup, D.; Das,
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employed.
Instruments
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Sci. Rep. 2017, 7, 42030.
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HPLC-MS studies were performed on an Agilent 1260 Infinity
LC system with binary pump, degasser, autosampler, column
heater, and UV detector. The chromatographic separation was
carried out on a Kinetex XB-C18, 2.6 μm, 150 mm × 3 mm (Phe-
nomenex, Aschaffenburg, Germany) analytical LC column with
UHPLC C18, 3 mm (Phenomenex) column guard. As mobile
phases Milli-Q water with 10 mM NH4Ac and 0.1% (v/v) acetic
acid (A) and MeOH with 10 mM NH4Ac and 0.1% (v:v) AcOH (B)
were used. The system was run with a flow rate of 350 μL/min
and a column heater temperature of 50 °C. An elution gradient
was applied, starting with 80% A for the first 3 min. Within 5
min A is reduced to 5% and maintained at this level for a further
4 min. Then A is again increased to 80% within 2 min and held
for 8 min to re-equilibrate the column. 10 μL sample was
injected. The mass determination was performed on an ABSciex
6500 triple quad mass spectrometer. For electrospray ionization
(ESI) the positive ionization mode was selected. For purification
of crude products an Agilent 1200 Series LC System was used
with the same method and an identical column. 30 μL sample
was injected, and fractions were collected from 12.25–12.45
min. Products were dried in a stream of nitrogen. Structure
determination was made by registering 1H NMR and 13C NMR
spectra under standard conditions with a Bruker Avance 600
MHz NMR spectrometer operating at 600.2 MHz. The sub-
stances had been dissolved in CDCl4. Trimethylsilane was used
as reference.
(10) Oberleitner, L.; Eremin, S. A.; Lehmann, A.; Garbe, L.-A.;
Schneider, R. J. Anal. Methods 2015, 7, 5854.
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Schneider, R. J. J. Immunoassay Immunochem. 2015, 36, 233.
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Rubio, F. R. J. Agric. Food Chem. 2008, 56, 6609. (c) Liu, N.; Han,
Z.; Lu, L.; Wang, L.; Ni, G.; Zhao, Z.; Wu, A.; Zheng, X. J. Sci. Food
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Pollut. Res. 2016, 23, 10714.
Synthesis of 6-{[2-(2,6-Dichlorophenylamino)phenyl]acet-
amido}-hexanoic Acid (DCF-Ahx)
5 g of Fmoc Ahx Wang resin (degree of loading 0.3 mmol/g)
were transferred to a glass column and swollen in DMF for 1 h.
For optimum mixing the glass column was shaken. For Fmoc
deprotection DMF/piperidine (4:1, v/v) was added and shaken
for 10 min. After this time the resin was washed twice with
DMF and twice with CH2Cl2. The resin was dried and transferred
in an argon atmosphere. The resin was loaded with 2 g DCF
(sodium salt), 806 mg of NHS, and 1.4 g DCC in 50 mL dry DMF.
The reaction mixture was shaken overnight. The next day, the
resin was washed four times with MeOH, twice with DMF and
twice with CH2Cl2. Cleavage of the product was done by adding
TFA/TIS/H2O (95:2.5:2.5, v/v/v). After 2 h reaction time, the
resin was extracted once with TFA. The dry resin was washed
with TFA again. The combined cleavage and extraction solutions
were concentrated at 75 °C on a rotary evaporator. The resulting
yellow oil was purified by column chromatography on silica gel
using the mobile phase cyclohexane/EtOAc (1:1, v/v). Every
fifth fraction was analyzed by HPLC–MS. The fractions which
contained the desired purified product were combined and the
solvent evaporated on a rotary evaporator. 138 mg (22.5%) of a
white solid were obtained.
(17) Deng, A.; Himmelsbach, M.; Zhu, Q.-Z.; Frey, S.; Sengl, M.;
Buchberger, W.; Niessner, R.; Knopp, D. Environ. Sci. Technol.
2003, 37, 3422.
(18) Huebner, M.; Weber, E.; Niessner, R.; Boujday, S.; Knopp, D.
Anal. Bioanal. Chem. 2015, 1.
(19) Harrer, A.; Lang, R.; Grims, R.; Braitsch, M.; Hawranek, T.;
Aberer, W.; Vogel, L.; Schmid, W.; Ferreira, F.; Himly, M. PLoS
ONE 2010, 5, 1.
(20) (a) Keuchel, C.; Weil, L.; Niessner, R. Fresenius' J. Anal. Chem.
1992, 343, 143. (b) Lopez-Moreno, R.; Mercader, J. V.; Agullo, C.;
Abad-Somovilla, A.; Abad-Fuentes, A. Org. Biomol. Chem. 2013,
11, 7361.
(21) Reagents
All standard chemicals and reagents were purchased from
Sigma-Aldrich (Steinheim, Germany). All solvents were chro-
matography grade. Diclofenac sodium salt, 1-N-hydroxysuccin-
imide (NHS), dicyclohexylcarbodiimide (DCC), piperidine, tri-
fluoroacetic acid (TFA), 1-hydroxybenzotriazol (HOBT),
triisopropylsilane (TIS), chlorotrimethylsilane, N,N-dimethyl-
formamide (DMF), dichloromethane (CH2Cl2), 6-aminohexanoic
acid (Ahx), cyclohexane, and ethyl acetate (EtOAc) were also
purchased from Sigma-Aldrich. Fmoc Ahx Wang resin was sup-
plied by Iris Biotech (Marktredwitz, Germany). 2-Aminoethanol
and sulfuric acid (98%) were purchased from J.T. Baker
(Griesheim, Germany). Piperidine was supplied by Roth (Karls-
ruhe, Germany). For HPLC the following solvent compounds
have been used: ammonium acetate (NH4Ac, analytical grade,
99.3%, Fischer Chemicals, Zurich, Switzerland), acetic acid
glacial (AcOH, analytical reagent grade, Fischer Chemicals) and
methanol (MeOH, HPLC gradient grade, J.T. Baker). For produc-
tion of ultrapure water a water purification system (Milli-Q
Analytical Data
1H NMR (600 MHz, CDCl3): δ = 1.33 (m, 2 H, CH2), 1.52 (m, J1 =
7.58 Hz, J2 = 7.58 Hz, J3 = 14.96 Hz, 2 H, CH2), 1.62 (m, J1 = 7.43
Hz, J2 = 7.43 Hz, J3 = 15.13 Hz, 2 H, CH2), 2.31 (t, J1 = 7.36 Hz, J2
= 7.36 Hz, 2 H, CH2), 3.25 (q, J1 = 7.11 Hz, J2 = 13.16 Hz, 2 H,
CH2), 3.67 (s, 2 H, CH2), 5.85 (m, J1 = 5.22 Hz, J2 = 5.22 Hz, 1 H,
ArH), 6.50 (dd, J1 = 1.01 Hz, J2 = 8.06 Hz, 1 H, ArH), 6.91 (t, J1 =
1.19 Hz, J2 = 7.42 Hz, J3 = 7.42 Hz, 1 H, ArH), 6.99 (m, J1 = 8.07
Hz, J2 = 8.07 Hz, 1 H, ArH), 7.11 (m, J1 = 1.38 Hz, J2 = 7.50 Hz, 1
H, ArH), 7.15 (m, J1 = 1.38 Hz, J2 = 7.50 Hz, 1 H, ArH), 7.33 (d, J =
8.06 Hz, 2 H, ArH) ppm.
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S. Schmidt et al.
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13C NMR (125 MHz, CDCl3): δ = 24.16, 26.17, 29.05, 33.64, 39.57,
40.99 (6 × CH2), 117.28, 121.48, 124.23 (3 × CHAr), 124.43 (qC),
128.05, 128.87 (2 × CHAr), 130.19 (qC), 130.73 (CHAr), 137.38,
142.79 (2 × qC), 171.55 (CO), 178.50 (COOH) ppm.
Synthesis Trial for 2-Aminoethyl-2-[2-(2,6-dichlorophenyl-
amino)phenyl]acetate
of 7 mL MeI, the reaction mixture was stirred overnight. On the
next day, the solvent was removed under vacuum. The solid was
suspended in EtOAc. The organic solution was washed with
water three times. The organic phase was dried with MgSO4.
After filtration, EtOAc was removed under vacuum. 893 mg
(90%) of a yellow solid was obtained.
100 mg DCF (sodium salt) was dissolved in 3 mL 2-aminoetha-
nol. Carefully 1 mL sulfuric acid was added at 0 °C. The reaction
mixture was stirred for 2 h. After this time the reaction mixture
was analyzed by HPLC–MS.
Synthesis of DCF Methyl Ester (DCFMe)
1 g DCF (sodium salt) was dissolved in 7 mL DMF. After addition
Synthesis of Ahx Methyl Ester (AhxMe)
1 g Ahx was dissolved in 2 mL chlorotrimethylsilane. After addi-
tion of 8 mL MeOH, the reaction mixture was stirred overnight.
On the next day, the solvent was removed under vacuum.
(22) Piccirillo, G.; Bochicchio, B.; Pepe, A.; Schenke-Layland, K.;
Hinderer, S. Acta Biomater. 2017, 52, 187.
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F