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V. Kumar et al. / European Journal of Pharmaceutical Sciences 92 (2016) 203–211
2.2.5. 4-(5,7-Dihydroxy-2-methyl-4-oxo-4H-chromen-8-yl)-1-
methylpiperidin-3-yl hexanoate (3d)
99.80, 72.69, 58.0, 55.97, 45.75, 36.20, 25.06, 20.36; IR (CHCl3) νmax
3385, 2923, 2852, 2280, 1724, 1660, 1526, 1418, 1387, 1351, 1271,
1186, 1115, 844, 757 cm−1; ESI-MS: m/z 454.80 [M + H]+; HRMS: m/
z 455.1461 calcd for C23H23N2O8 + H+ (455.1454).
White powder; HPLC purity: 97.14% (tR = 3.3 min); m.p. 203–205 °
C; 1H NMR (400 MHz, CDCl3): δ 12.80 (s, 1H), 6.10 (s, 1H), 6.03 (s, 1H),
5.16 (d, J = 4 Hz, 1H), 3.72–3.69 (m, 1H), 3.15–3.05 (m, 1H), 2.67 (s,
1H), 2.45–2.42 (m, 1H), 2.34 (s, 6H), 2.62–2.20 (m, 3H), 1.78 (s, 1H),
1.54–1.47 (m, 3H), 1.28–1.18 (m, 4H), 0.86 (t, J = 8, 16 Hz, 3H); 13C
NMR (125.76 MHz, CDCl3): δ 182.77, 172.9, 166.02, 162.44, 160.95,
108.64, 105.04, 104.9, 101.10, 71.77, 57.91, 54.76, 51.61, 45.66, 34.29,
33.43, 31.13, 24.87, 24.34, 22.28, 20.51, 13.89; IR (CHCl3) νmax 3357,
2927, 2855, 2351, 1731, 1659, 1589,1420, 1389, 1259, 1185, 1117,
1007, 845, 755 cm−1; ESI-MS: m/z 404.00 [M + H]+; HRMS: m/z
404.2102 calcd for C22H30NO6 + H+ (404.2073).
2.2.10. 4-(5,7-Dihydroxy-2-methyl-4-oxo-4H-chromen-8-yl)-1-
methylpiperidin-3-yl 4-methylbenzoate (3i)
Off white powder; HPLC purity: 96.27% (tR = 4.6 min); m.p. 236–
238 °C; 1H NMR (400 MHz, CDCl3): δ 12.68 (s, 1H), 7.61 (d, J = 8 Hz,
2H), 6.95 (d, J = 8 Hz, 2H), 6.07 (s, 1H), 5.79 (brs, 1H), 5.29 (s, 1H),
3.96 (s, 1H), 3.19–3.06 (m, 3H), 2.45 (d, J = 12 Hz, 1H), 2.27–2.17 (m,
10H), 1.87–1.77 (m, 1H); 13C NMR (100 MHz, CD3Cl): δ 182.62,
166.45, 166.06, 163.27, 160.60, 156.88, 143.66, 129.40, 128.81, 127.18,
108. 03, 105.02, 104.21, 100.25, 70.93, 57.54, 54.75, 45.45, 34.63,
24.90, 21.59, 20.29; IR (CHCl3) νmax 3584, 2923, 2852, 2786, 2326,
1711, 1660, 1612, 1588, 1389, 1271, 1180, 1080, 980, 753 cm−1; ESI-
2.2.6. 4-(5,7-Dihydroxy-2-methyl-4-oxo-4H-chromen-8-yl)-1-
methylpiperidin-3-yl 3-methylbutanoate (3e)
Cream white powder; HPLC purity: 94.5% (tR = 4.7 min); m.p. 187–
188 °C; 1H NMR (400 MHz, CDCl3): δ 12.81 (s, 1H), 6.19 (s, 1H), 5.94 (s,
1H), 5.12 (brs, 1H), 3.50–3.45 (m, 1H), 3.07 (d, J = 12 Hz, 2H), 2.93–2.89
(m, 1H), 2.37–2.33 (m, 1H), 2.29 (s, 3H), 2.27 (s, 3H), 2.17–2.2 (m, 1H),
2.05–1.92 (m, 2H), 1.82–1.70 (m, 2H), 0.67 (d, J = 8 Hz, 6H); 13C NMR
(125.76 MHz, CD3Cl): δ 182.79, 172.50, 166.19, 162.48, 160.85, 156.29,
108.45, 105.07, 104.77, 100.51, 71.27, 58.23, 55.42, 45.73, 43.36, 34.62,
25.50, 24.82, 22.13, 20.50; IR (CHCl3) νmax 3333, 2957, 2925, 2851,
2443, 1730, 1660, 1589, 1421, 1389, 1292, 1186, 1081, 754 cm−1, ESI-
MS: m/z 424.10 [M
C
+ ; HRMS: m/z 424.1765calcd for
H]+
24H26NO6 + H+ (424.1760).
2.2.11. 4-(5,7-Dihydroxy-2-methyl-4-oxo-4H-chromen-8-yl)-1-
methylpiperidin-3-yl acetate (3j)
Cream white powder; HPLC purity: 99.0% (tR = 17.7 min); m.p. 145–
147 °C; 1H NMR (400 MHz, CDCl3): δ 12.81 (s, 1H), 6.30 (s, 1H), 6.03 (s,
1H), 5.14 (brs, 1H), 3.71–3.66 (m, 1H), 3.20–3.08 (m, 2H), 2.82 (brs,
1H), 2.50–2.44 (m, 1H), 2.42 (s, 3H), 2.37 (s, 3H), 2.35 (brs, 1H), 2.03
(s, 3H), 1.83 (d, J = 16 Hz, 1H); 13C NMR (100 MHz, CD3OD): δ
182.40, 170.22, 166.97, 162.38, 159.95, 156.34, 106.73, 104.49, 103.20,
97.89, 69.98, 57.76, 55.64, 44.07, 35.60, 23.79, 18.94, 18.39; IR (CHCl3)
νmax 3584, 2924, 2852, 2792, 1737, 1660, 1588, 1389, 1258, 1116,
1081, 846, 754 cm−1; ESI-MS: m/z 348.00 [M + H]+; HRMS: m/z
348.1455 calcd for C18H22NO6 + H+ (348.1447).
MS: m/z 390.00 [M
C
+ ; HRMS: m/z 390.1882 calcd for
H]+
21H28NO6 + H+ (390.1917).
2.2.7. 4-(5,7-Dihydroxy-2-methyl-4-oxo-4H-chromen-8-yl)-1-
methylpiperidin-3-yl 3,5-dimethoxybenzoate (3f)
White powder; HPLC purity: 98.94% (tR = 4.542 min); m.p. 182–
183 °C; 1H NMR (400 MHz, CD3OD): δ 7.14 (s, 2H), 6.64 (s, 1H), 6.23
(s, 1H), 5.88 (s, 1H), 5.46 (brs, 1H), 3.79 (s, 6H), 3.61–3.58 (m, 1H),
3.40–3.36 (m, 1H), 3.28–3.21 (m, 2H), 2.69 (d, J = 16 Hz, 1H), 2.44 (s,
3H), 2.41–2.38 (m, 1H), 2.34 (s, 3H), 1.90 (d, J = 12 Hz, 1H); 13C NMR
(100 MHz, CD3Cl): δ 182.63, 166.16, 165.98, 163.52, 160.82, 160.56,
160.14, 156.70, 131.60, 108.24, 107.26, 106.87, 105.35, 104.38, 104.35,
100.94, 70.39, 55.99, 55.47, 55.30, 52.62, 44.52, 32.11, 24.29, 20.17; IR
(CHCl3) νmax 3584, 3332, 2923, 2849, 1715, 1660, 1594, 1426, 1389,
1233, 1205, 1156, 1050, 845, 763 cm−1; ESI-MS: m/z 470.00
[M + H]+; HRMS: m/z 470.1822 calcd for C25H28NO8 + H+ (470.1815).
2.3. Thermodynamic equilibrium solubility studies
Solubility of synthesized prodrugs was determined by miniaturized
shake˗flask method (Bharate and Vishwakarma, 2015). Briefly, an ex-
cess of the compound was added into 1.5 mL Eppendorf tube containing
200 μL of dissolution medium (water/PBS/SGF/SIF). Eppendorf tubes
were shaken at 300 rpm (IKA® MS 3 digital shaker) for 24 h at room
temperature in order to achieve equilibrium. After equilibration, the
tubes were centrifuged at 16 RCF (G force) for 10 min using Eppendorf
centrifuge 5430R. Supernatants were analyzed, to find out amount of
drug dissolved in respective dissolution medium. The concentration of
compound in dissolution media viz. water, PBS (pH 7.4), SGF (pH 1.2)
and SIF (pH 6.8) was determined as follow. Calibration curve of com-
pound in water, PBS, SGF and SIF was plotted in the concentration
range of 0˗160 μg/mL (0, 5, 10, 20, 40, 80 and 160 μg/mL). The optical ab-
sorbance of standard solutions and tests were measured at 254 nm,
using microplate reader (SpectraMax Plus384), against a blank. The
concentration of compound in the dissolution medium was calculated
using calibration equation. The analysis was performed in triplicate for
each compound.
2.2.8. 4-(5,7-Dihydroxy-2-methyl-4-oxo-4H-chromen-8-yl)-1-
methylpiperidin-3-yl 3,4,5-trimethoxybenzoate (3g)
White powder; HPLC purity: 98.25% (tR = 9.67 min); m.p. 228–230 °
C; 1H NMR (400 MHz, CDCl3 + CD3OD) δ: 7.23 (s, 2H), 6.10 (s, 1H), 5.74
(s, 1H), 5.44 (brs, 1H), 3.76 (s, 6H), 3.72 (s, 3H), 3.52–3.48 (m, 1H),
3.30–3.23 (m, 1H), 3.19–3.15 (m, 2H), 2.61 (d, J = 12 Hz, 1H), 2.38 (s,
3H), 2.33–2.28 (m, 1H), 2.15 (s, 3H), 1.77 (d, J = 12 Hz, 1H); 13C NMR
(100 MHz, CD3Cl + CD3OD) δ: 184.22, 168.74, 167.51, 164.41, 161.83,
158.12, 154.28, 143.79, 126.82, 108.99, 108.45, 106.48, 105.20, 99.98,
72.47, 61.24, 60.10, 57.91, 57.14, 46.43, 38.49, 30.74, 26.18, 20.366; IR
(CHCl3) νmax 3381.97, 2978.35, 2946.27, 2882.52, 2739.21, 2605.55,
2498.58, 2321.53, 1661.27, 1590.66, 1476.06, 1397.68, 1173.34,
2.4. Determination of log P and log D
1126.68, 1037.01, 851.08, 807.56 cm−1
; ESI-MS: m/z 500.00
[M + H]+; HRMS: m/z 500.1929 calcd for C26H30NO9 + H+ (500.1921).
Log P and log D was determined as per our recently published and
validated protocol (Bharate et al., 2016). Briefly, in 1.5 mL Eppendorf
tube, 200 μL of stock solution (1000 μg/mL prepared in n-octanol) was
added and volume was made up to 1 mL with 300 μL of presaturated
n-octanol and 500 μL of presaturated aqueous phase (water/PBS
pH 7.4). The Eppendorf tubes were shaken overnight at 500 rpm and
centrifuged at 16,000 RCF (G force) for 20 min so as to separate aqueous
and organic layer. The supernatants were removed using glass syringe
to avoid contamination of aqueous and organic aliquots with each
other. For the experiment, presaturated solutions were used. For log P
and log D experiments, organic phase consisted of n-octanol saturated
2.2.9. 4-(5,7-Dihydroxy-2-methyl-4-oxo-4H-chromen-8-yl)-1-
methylpiperidin-3-yl 4-nitrobenzoate (3h)
Yellow powder; HPLC purity: 97.23% (tR = 8.4 min); m.p. = 118–
120 °C; 1H NMR (400 MHz, CDCl3) δ: 12.79 (s, 1H), 8.19 (d, J = 8 Hz,
2H), 8.04 (d, J = 8 Hz, 2H), 6.23 (s, 1H), 5.90 (s, 1H), 5.55–5.49 (m,
1H), 4.02–3.93 (m, 1H), 3.25–3.05 (m, 2H), 2.98–2.81 (m, 2H), 2.73–
2.67 (m, 1H), 2.44 (s, 3H), 2.20 (s, 3H), 2.13–2.02 (m, 1H); 13C NMR
(125 MHz, CD3Cl) δ: 182.39, 166.12, 164.43, 162.89, 160.62, 156.64,
150.15, 135.43, 130.72, 130.43, 123.55, 122.97, 108.17, 104.75, 104.15,