3.1. Materials
Ketoprofen was isolated from commercially available medicines in pills (Ketonal forte® (100 mg) tablet manufactured by SANDOZ, Holzkirchen, Germany) by extraction with ethanol, then purification of the extract by filtration and then precipitation from extract with water. Identification of isolated compounds was performed by NMR analysis and purity was determined by acid-base (acidimetric) titration, according to the Pharmacopoeia and elementary analysis. l-valine and l-leucine with high purity (>99.0%) were purchased from Carl Roth (Karlsruhe, Germany). Trimethylsilyl chloride with high purity (≥99.0%) (TMSCl) was obtained from Sigma-Aldrich (Steinheim am Albuch, Germany). Ethanol (EtOH), propan-2-ol (i-PrOH), propan-1-ol (PrOH), butan-1-ol (BuOH), dimethyl sulfoxide, chloroform, ethyl acetate, diethyl ether, toluene and n-hexane and ammonia solution were high purity provided by Chempur (Gliwice, Poland). All reagents, solvents, and other materials were of analytical grade and were used without any further purification. Deuterated chloroform (CDCl3) (99.8%) (+0.03% TMSCl) was obtained from Eurisotop (Cheshire, England).
Bovine serum albumin (BSA), heat shock fraction, protease-free, fatty acid-free, essentially globulin free (pH 7, ≥98%), Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4 mM L-glutamine, 4500 mg L−1 glucose, 1 mM sodium pyruvate, and 1500 mg L−1 sodium bicarbonate, trypsin-EDTA solution, and penicillin–streptomycin–neomycin stabilized solution were purchased from Merck (Darmstadt, Germany).
Murine macrophages RAW 264.7 was obtained from the American Tissue Culture Collection (ATCC) (Manassas, VA, USA). Alamar Blue cell viability reagent was purchased from Thermo Scientific (Waltham, MA, USA).
3.3. Characterization of the Ketoprofen Amino Acid Alkyl-Esters [AAOR][KETO]
1H-NMR (400.13 MHz) and 13C-NMR (100.62 MHz) spectra were recorded in CDCl3 on a BRUKER DPX-400 Avance III HD spectrometer (Billerica, MA, USA). TMS was used as an internal standard. ATR–FTIR spectra were collected in a Thermo Fisher Scientific Nicolet FTIR 380 FTIR Spectrometer (Waltham, MA, USA) equipped with attenuated total reflectance (ATR) sampling accessory (diamond plate). Spectra were recorded in transmittance mode from 400 to 4000 cm–1, co–adding 16 interferograms at a resolution of 4 cm–1. The content of elements, i.e., hydrogen, nitrogen, carbon, and oxygen were determined by CHNS/O elemental analysis. The elemental analysis was performed using a Thermo Scientific™ FLASH 2000 CHNS/O Elemental Analyzer (Waltham, MA, USA). Thermogravimetric analysis was carried out on thermomicrobalance TG 209 F1 Libra® from NETZSCH (Selb, Germany), in Al2O3 crucible. Samples between 5–10 mg were heated from 25 °C to 1000 °C with a heating rate of 10 °C min−1, under air atmosphere (flow rate: air—25 cm3 min−1, nitrogen (as protective gas)—10 cm3 min−1). Phase transformation temperatures were measured using a TA Instruments DSC analyzer model Q-100 (New Castle, DE, USA). Measurements were performed within the temperature range of 0 °C to 100–200 °C (depends on thermal stability), in a nitrogen atmosphere. The heating rate was 10 °C min−1. The sample was loaded on an aluminum pan sealed with a pinhole cap. The specific rotation [α]D20 measurements were determined with an AUTOPOL IV Polarimeter from Rudolph Research Analytical (Hackettstown, NJ, USA) for aqueous solutions of AAILS (c = 0.01 g cm−3). The measurements were performed at 20 °C.
3.3.1. [l-LeuOEt][KETO]—l-Leucine Ethyl Ester Ketoprofenate
The compound was obtained according general procedure in 94.0% yield as white solid. 1H-NMR (400 MHz, CDCl3) δ in ppm: 7.71–7.81 (m, 3H, H7, H12, H16); 7.62 (d, J9,8 = 7.6 Hz, 1H, H9); 7.54–7.60 (m, 2H, H13, H14); 7.44–7.49 (m, 2H, H5, H15); 7.38 (t, J6,5 = 7.6 Hz, 1H, H6); 6.67 (s, 3H, H22); 4.12–4.18 (m, 2H, H24); 3.68–3.72 (m, 1H, H2); 3.59 (dd, J20,19 = 4.6 Hz, 1H, H21); 1.69–1.73 (m, 1H, H20); 1.58–1.62 (m, 1H, H19); 1.44–1.51 (m, 4H, H3, H20); 1.24 (t, J25,24 =7.2 Hz, 3H, H25); 0.85–0.89 (dd, J17(18),19 = 7.6 Hz, 6H, H17, H18); 13C-NMR (100 MHz, CDCl3) δ in ppm: 196.72 (C1); 178.79 (C23); 173.68 (C10); 142.37 (C8); 137.56 (C11); 132.43 (C4); 131.84 (C5); 130.09 (C14); 129.29 (C12); 128.57 (C16); 128.26 (C9); 128.23 (C6); 61.46 (C24); 51.79 (C21); 46.55 (C2); 42.09 (C20); 24.52 (C19); 22.58 (C18); 21.85 (C17); 18.72 (C3); 14.10 (C25); FTIR: ν (ATR): 2957; 2933; 2872; 1742; 1653; 1597; 1539; 1479; 1456; 1446; 1391; 1362; 1317; 1281; 1252; 1231; 1196; 1172; 1132; 1093; 1074; 1026; 995; 968; 932; 921; 880; 824; 780; 728; 706; 644; 607; 574; 523; 444 cm−1; Elemental analysis: Calc. (%) for C24H31NO5 (413.51 g mol−1) C (69.71), H (7.56), N (3.39), O (19.35), Found C (69.82), H (7.53), N (3.37), O (19.33).
3.3.2. [l-ValOEt][KETO]—l-Valine Ethyl Ester Ketoprofenate
The compound was obtained according general procedure in 94.0% yield as white solid. 1H-NMR (400 MHz, CDCl3) δ in ppm: 7.75–7.81 (m, 3H, H7, H12, H16); 7.64 (d, J9,8 = 7.6 Hz, 1H, H9); 7.53–7.60 (m, 2H, H13, H14); 7.48 (t, J6,5 = 7.3 Hz, 2H, H5, H15); 7.40 (t, J6,5 = 7.7 Hz, 1H, H6); 5.75 (s, 3H, H21); 4.02–4.26 (m, 2H, H23); 3.68–3.72 (m, 1H, H2); 3.44 (d, J20,19 = 4.6 Hz, 1H, H20); 1.98–2.16 (m, 1H, H19); 1.49 (d, J3,2 = 7.1 Hz, 3H, H3); 1.25 (t, J24,23 = 7.2 Hz, 3H, H24); 0.88–0.95 (m, 6H, H17, H18); 13C-NMR (100 MHz, CDCl3) δ in ppm: 196.65 (C1); 178.49 (22); 173.58 (C10); 141.64 (C8); 137.72 (C11); 137.54 (C4); 132.45 (C5); 131.76 (C14); 130.10 (C12/C16); 129.31 (C9); 128.79 (C6); 128.37 (C7); 128.28 (C13); 61.17 (C23); 58.95 (C20); 46.00 (C2); 31.39 (C19); 18.72 (C18), 18.52 (C17); 17.29 (C3); 14.21 (C24); FTIR: ν (ATR): 2968; 2929; 2876; 2610; 1741; 1651; 1596; 1574; 1517; 1481; 1463; 1446; 1387; 1361; 1316; 1280; 1257; 1218; 1204; 1180; 1173; 1135; 1107; 1074; 1065; 1053; 1015; 996; 966; 952; 878; 859; 778; 718; 705; 642 cm−1; Elemental analysis: Calc. (%) for C23H29NO5 (399.48 g mol−1) C (69.15), H (7.32), N (3.51), O (20.03), Found C (69.20), H (7.30), N (3.55), O (20.10).
3.3.3. [l-ValOiPr][KETO]—l-Valine Isopropyl Ester Ketoprofenate
The compound was obtained according general procedure in 90.0% yield as white solid. 1H-NMR (400 MHz, CDCl3) δ in ppm: 7.73–7.83 (m, 3H, H7, H12, H16); 7.61–7.67 (dt, 1H, H9); 7.52–7.60 (m, 2H, H13, H14); 7.47 (t, J6,5 = 7.6 Hz, 2H, H5, H15); 7.39 (t, J6,5 = 7.7 Hz, 1H, H6); 5.63 (s, 3H, H21); 4.95–5.12 (m, 2H, H23); 3.66–3.79 (m, 1H, H2); 3.40 (d, J20,19 = 4.6 Hz, 1H, H20); 2.03–2.12 (m, 1H, H19); 1.49 (d, J3,2 = 7.2 Hz, 3H, H3); 1.24 (d, J24,23 = 7.1 Hz, 6H, H24, 25); 0.87–0.95 (dd, 6H, H17, H18); 13C-NMR (100 MHz, CDCl3) δ in ppm: 196.70 (C1); 178.48 (C22); 141.95 (C10); 137.65 (C8); 137.54 (C11); 132.43 (C4); 131.79 (C5); 130.10 (C14); 129.33 (C12/C16); 129.31 (C9); 128.69 (C6); 128.32 (C7); 128.27(C13); 68.90 (C23); 58.90 (C20); 46.21 (C2); 31.30 (C19); 21.76 (C18); 18.65 (C17); 18.60 (C3); 17.26 (C24/C25); FTIR: ν (ATR): 2976; 2933; 2877; 1738; 1660; 1597; 1577; 1558; 1448; 1387; 1358; 1318; 1283; 1233; 1180; 1144; 1105; 1075; 999; 954; 915; 880; 822; 775; 721; 705; 643 cm−1; Elemental analysis: Calc. (%) for C24H31NO5 (413.51 g mol−1) C (69.71), H (7.57), N (3.39), O (19.35), Found C (69.75), H (7.56), N (3.38), O (19.34).
3.3.4. [l-ValOPr][KETO]—l-Valine Propyl Ester Ketoprofenate
The compound was obtained according general procedure in 93.0% yield as white solid. 1H-NMR (400 MHz, CDCl3) δ in ppm: 7.74–7.82 (m, 3H, H7, H12, H16); 7.63–7.68 (dt, 1H, H9); 7.52–7.60 (m, 2H, H13, H14); 7.47 (t, J6,5 = 7.6 Hz, 2H, H5, H15); 7.41 (t, J6,5 = 7.7 Hz, 1H, H6); 4.85 (s, 3H, H21); 4.03–4.12 (m, 2H, H23); 3.72–3.79 (m, 1H, H2); 3.42 (d, J20,19 = 4.7 Hz, 1H, H20); 2.03–2.13 (m, 1H, H19); 1.61–1.69 (m, 2H, H24); 1.51 (d, J3,2 = 7.1 Hz, 3H, H3); 0.89–0.97 (m, 6H, H17, H18, H25); 13C-NMR (100 MHz, CDCl3) δ in ppm: 196.64 (C1); 178.35 (C10); 174.20 (C22); 141.41 (C8); 137.75 (C11); 137.52 (C4); 132.47 (C5); 131.73 (C14); 130.10 (C12); 129.32 (C9); 128.88 (C6); 128.42 (C7); 128.29 (C13/C15); 66.71 (C23); 59.14 (C20); 45.81 (C2); 31.59 (C19); 21.95 (C24); 18.88 (C18); 18.46 (C17); 17.23 (C3); 10.40 (C25); FTIR: ν (ATR): 2969; 2935; 2879; 1742; 1659; 1597; 1578; 1462; 1448; 1389; 1358; 1318; 1283; 1222; 1179; 1140; 1059; 999; 955; 929; 910; 881; 777; 721; 705; 643; cm−1; Elemental analysis: Calc. (%) for C24H31NO5 (413.51 g mol−1) C (69.71), H (7.57), N (3.39), O (19.35), Found C (69.74), H (7.55), N (3.37), O (19.33).
3.3.5. [l-ValOBu][KETO]—l-Valine Butyl Ester Ketoprofenate
Compound was obtained according general procedure in 95.0% yield as white solid. 1H-NMR (400 MHz, CDCl3) δ in ppm: 7.75–7.82 (m, 3H, H7,H12, H16); 7.61–7.66 (dt, 1H, H9); 7.53–7.60 (m, 2H, H13, H14); 7.47 (t, J6,5 = 7.6 Hz, 2H, H5, H15); 7.39 (t, J6,5 = 7.7 Hz, 1H, H6); 5.62 (s, 3H, H21); 4.05–4.16 (m, 2H, H23); 3.68–3.76 (m, 1H, H2); 3.44 (d, J20,19 = 4.5 Hz, 1H, H20); 2.04–2.12 (m, 1H, H19); 1.56–1.64 (m, 2H, H24); 1.48 (d, J3,2 = 7.2 Hz, 3H, H3); 0.87–0.95 (m, 9H, H17, H18, H25, H26); 13C-NMR (100 MHz, CDCl3) δ in ppm: 196.70 (C1); 178.52 (C10); 173.51 (C22); 141.94 (C8); 137.65 (C11); 137.53 (C4); 132.45 (C5); 131.80 (C14); 130.10 (C12); 129.31 (C9); 128.70 (C6); 128.32 (C7); 128.27 (C13/C15); 65.10 (C23); 58.91 (C20); 46.21 (C2); 31.31 (C19); 30.56 (C24); 19.09 (C25); 18.60 (C18); 18.60 (C17); 17.32 (C3); 13.65 (C26); FTIR: ν (ATR): 2964; 2934; 2875; 1742; 1660; 1597; 1578; 1462; 1448; 1389; 1358; 1318; 1283; 1242; 1219; 1179; 1140; 1062; 1021; 999; 954; 881; 779; 721; 705; 643 cm−1; Elemental analysis: Calc. (%) for C25H33NO5 (427.53 g mol−1) C (70.23), H (7.78), N (3.27), O (18.71), Found C (70.25), H (7.80), N (3.25), O (19.69).
3.3.6. Ketoprofen
1H-NMR (400 MHz, CDCl3) δ in ppm: 7.67–7.74 (m, 3H, H9, H16, H12); 7.59–7.61 (dt, 1H, H7); 7.44–7.54 (m, 2H, H4, H5); 7.30–7.42; (m, 3H, H); 3.69–3.79 (m, 1H, H); 1.46 (d, J2,1 = 7.1 Hz, 3H, H1); 13C-NMR (100 MHz, CDCl3) δ in ppm: 196.56 (C10); 180.24 (1); 140.12 (C8); 137.93 (C11); 137.41 (C4); 132.60 (C5); 131.71 (C14); 130.14 (C12/C16); 129.39 (C9); 129.31 (C6); 128.63 (C7); 128,34 (C13/C15); 45.25 (C2); 18.13 (C3); FTIR: ν (ATR): 2978; 2926; 1697; 1655; 1598; 1576; 1457; 1419; 1370; 1319; 1308; 1285; 1227; 1195; 1175; 1134; 1178; 1063; 1061; 968; 928; 916; 866; 717; 703; 691; 643; 614 cm−1; Elemental analysis: Calc. (%) for C16H14O3 (254.28 g mol−1) C (75.58), H (5.55), N (0.00), O (18.88), Found C (75.56), H (5.56), N (0.00), O (18.88).