Synthetic Strategies for the Modification of Diclofenac

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 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.

Key words

diclofenac - hapten synthesis - solid-phase synthesis - spacer introduction - amide linkage

• References

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• 21 Reagents All standard chemicals and reagents were purchased from Sigma-Aldrich (Steinheim, Germany). All solvents were chromatography grade. Diclofenac sodium salt, 1-N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), piperidine, trifluoroacetic acid (TFA), 1-hydroxybenzotriazol (HOBT), triisopropylsilane (TIS), chlorotrimethylsilane, N,N-dimethylformamide (DMF), dichloromethane (CH₂Cl₂), 6-aminohexanoic acid (Ahx), cyclohexane, and ethyl acetate (EtOAc) were also purchased from Sigma-Aldrich. Fmoc Ahx Wang resin was supplied by Iris Biotech (Marktredwitz, Germany). 2-Aminoethanol and sulfuric acid (98%) were purchased from J.T. Baker (Griesheim, Germany). Piperidine was supplied by Roth (Karls­ruhe, 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 production of ultrapure water a water purification system (Milli-Q Synthesis A10, Merck Millipore, Schwalbach, Germany) was employed. Instruments 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 X_B-C18, 2.6 μm, 150 mm × 3 mm (Phenomenex, 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 substances had been dissolved in CDCl₄. Trimethylsilane was used as reference. Synthesis of 6-{[2-(2,6-Dichlorophenylamino)phenyl]acetamido}-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. 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. 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 × CH_Ar), 124.43 (qC), 128.05, 128.87 (2 × CH_Ar), 130.19 (qC), 130.73 (CH_Ar), 137.38, 142.79 (2 × qC), 171.55 (CO), 178.50 (COOH) ppm. Synthesis Trial for 2-Aminoethyl-2-[2-(2,6-dichlorophenylamino)phenyl]acetate 100 mg DCF (sodium salt) was dissolved in 3 mL 2-aminoethanol. 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 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. Synthesis of Ahx Methyl Ester (AhxMe) 1 g Ahx was dissolved in 2 mL chlorotrimethylsilane. After addition 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