N. Puttaswamy et al. / European Journal of Medicinal Chemistry 114 (2016) 153e161
159
1609 (C]N). 1H NMR (400 MHz, DMSO-d6):
d
5.8 (d, J ¼ 8.3 Hz, 1H,
66.46. LCMS: (Mþ) (438), (440). Anal. Calcd. for: C23H16ClNO6: C,
63.09; H, 3.68; N, 3.20; Found: C, 63.19; H, 3.64; N, 3.23%.
CeCHeAr), 6.6 (d, J ¼ 8.3 Hz, 1H, HeCeC]O), 7.0e7.8 (m, 11H, ArH)
10.6 (s, 1H, OH), 11.7 (s, 1H, COOH). 13C NMR: 188.41, 169.21, 163.20,
144.24, 136.38, 135.12, 134.17, 133.21, 131.34, 129.38, 129.21, 128.32,
122.23,119.32 118.02,114.31,113.46, 80.44, 60.61. LCMS (Mþ): (456),
(458), (460) Anal. Calcd. for: C23H15Cl2NO5: C, 60.54; H, 3.31; N,
3.07. Found: C, 60.52; H, 3.32; N, 3.07%.
4.1.5.7. 3(3-Chlorophenyl)-4(3-hydroxy-4-carboxybenzoyl)-5(3-
chlorophenyl)4,5-dihydrodihydroisoxazoline (8g). Yield 82%. m.p.
185e188 ꢀC; IR (KBr, /cmꢁ1): 3690e3620 (OH), 3430e3522 (OH of
g
COOH), 1700 (C]O of COOH), 1687 (C]O), 1239(CeO) 1616(C]N).
1H NMR (400 MHz, DMSO-d6):
d
5.2 (d, J ¼ 8.3 Hz, 1H CeCH-Ar), 6.5
4.1.5.2. 3(4-Chlorophenyl)-4(3-hydroxy-4-carboxybenzoyl)-5(4-
(d, J ¼ 8.3 Hz, 1H, HeCeC]O), 7.0e8.0 (m, 11H, ArH), 10.6 (s, 1H,
OH), 11.1 (s, 1H, COOH). 13C NMR: 187.36, 167.43, 163.41, 145.23,
139.12,138.33,136.82,135.39,134.04,132.23,130.22,128.48,126.32,
124.21, 124.52, 123.22, 122.02, 121.03, 118.32, 115.25, 83.03, 67.46.
LCMS: (Mþ) (455), (457), (459). Anal. Calcd. for: C23H15Cl2NO5: C,
60.54; H, 3.31; N, 3.07; Found: C, 60.52; H, 3.33; N, 3.04%.
bromophenyl)4,5-dihydrodihydroisoxazoline (8b). Yield 72%, m.p.
172e175 ꢀC: IR (KBr, /cmꢁ1): 3710e3630 (OH), 3425e3534 (OH of
g
COOH), 1710 (C]O of COOH), 1670 (C]O), 1238 (CeO) 1612 (C]N).
1H NMR (400 MHz, DMSO-d6):
d
5.6 (d, J ¼ 8.2 Hz, 1H, CeCHeAr),
6.8 (d, J ¼ 8.2 Hz, 1H, HeCeC]O), 7.0e7.8 (m, 11H, ArH) 10.7 (s, 1H,
OH), 12.3 (s, 1H, COOH). 13C NMR 186.91, 177.02, 165.10, 143.04,
137.08, 136.34, 132.79, 131.94, 129.34, 128.83, 126.21, 122.34, 120.23,
118.34, 117.32, 116.02, 113.04, 80.53, 59.62. LCMS (Mþ): (500), (502),
(504) Anal. Calcd. for: C23H15BrClNO5: C, 55.17; H, 3.02; N, 2.80.
Found:C, 55.11; H, 3.03; N, 2.82%.
4.1.5.8. 3(4-Chlorophenyl)-4(3-hydroxy-4-carboxybenzoyl)-5(4-
methoxyphenyl)4,5-dihydrodihydroisoxazoline (8h). Yield 73%. m.p.
154e157 ꢀC; IR (KBr, /cmꢁ1): 3720e3620 (OH), 3425e3520 (OH of
g
COOH), 1707 (C]O of COOH), 1682 (C]O), 1245 (CeO) 1626 (C]N).
1H NMR (400 MHz, DMSO-d6):
d
3.1 (s, 3H, OCH3), 5.2 (d, J ¼ 8.3 Hz,
4.1.5.3. 3(3-Chlorophenyl)-4(3-hydroxy-4-carboxybenzoyl)-5(4-
1H, CeCHeAr), 6.0 (d, J ¼ 8.3 Hz, 1H, HeCeC]O), 7.1e7.9 (m, 11H,
ArH), 10.7 (s, 1H, OH), 12.2 (s, 1H, COOH). 13C NMR: 184.36, 168.53,
164.13, 146.21, 139.61, 137.32, 138.21, 134.49, 133.54, 132.43, 126.31,
127.83, 124.36, 123.42, 122.34, 121.22, 118.52, 84.53, 62.43, 65.23.
LCMS: (Mþ) (452), (454). Anal. Calcd. for: C24H18ClNO6: C, 63.79; H,
4.02; N, 3.10. Found: C, 63.63; H, 4.10; N, 3.12%.
bromophenyl)4,5-dihydrodihydroisoxazoline (8c). Yield 82%, m.p.
158e171 ꢀC; IR (KBr, /cmꢁ1): 3700e3640 (OH), 3412e3528 (OH of
g
COOH), 1708 (C]O of COOH), 1686 (C]O), 1236 (CeO) 1614 (C]N).
1H NMR (400 MHz, DMSO-d6):
d
5.5 (d, J ¼ 8.3 Hz, 1H, CeCHeAr),
6.1 (d, J ¼ 8.3 Hz, 1H, HeCeC]O), 7.0e8.0 (m, 11H, ArH) 10.9 (s, 1H,
OH), 11.7 (s, 1H, COOH). 13C NMR 187.14, 174.52, 162.30, 145.42,
137.83, 136.12, 134.32, 133.09, 132.34, 131.35, 126.38, 125.21, 124.23,
123.13, 122.61, 122.10, 120.13, 119.12, 115.34, 81.54, 61.5. LCMS: (Mþ)
(500), (502), (504) Anal. Calcd. for:C23H15BrClNO5: C, 55.17; H, 3.02;
N, 2.80. Found: C, 55.13; H, 3.02; N, 2.88%.
4.1.5.9. 3(3-Chlorophenyl)-4(3-hydroxy-4-carboxybenzoyl)-5(4-
methoxyphenyl)4,5-dihydrodihydroisoxazoline (8i). Yield 73%, m.p.
154e157 ꢀC; IR (KBr, /cmꢁ1): 3700e3620 (OH), 3400e3585 (OH of
g
COOH), 1703 (C]O of COOH), 1687 (C]O),1239 (CeO) 1620 (C]N).
1H NMR (400 MHz, DMSO-d6):
d
3.0 (s, 3H, OCH3), 4.9 (d, J ¼ 8.2 Hz,
4.1.5.4. 3(3-Methoxyphenyl)-4(3-hydroxy-4-carboxybenzoyl)-5(4-
1H, CeCHeAr), 6.3 (d, J ¼ 8.2 Hz, 1H, HeCeC]O), 7.1e8.0 (m, 11H,
ArH), 10.5 (s, 1H, OH), 11.9 (s, 1H, COOH). 13C NMR, 185.346, 165.33,
165.23, 147.31, 140.61, 137.21, 133.29, 132.54, 128.83, 128.10, 127.34,
126.41, 125.26, 124.32, 123.48, 123.05, 122.32, 120.23, 119.42, 85.43,
67.27, 63.53. LCMS: (Mþ) (452), (454). Anal. Calcd. for: C24H18ClNO6:
C, 63.79; H, 4.02; N, 3.10. Found: C, 63.76; H, 4.08; N, 3.19%.
hydroxyphenyl)4,5-dihydrodihydroisoxazoline (8d). Yield 82%, m.p.
158e171 ꢀC; IR (KBr, /cmꢁ1): 3690e3640 (OH), 3422e3530 (OH of
g
COOH), 1706 (C]O of COOH), 1680 (C]O), 1249 (CeO) 1616 (C]N).
1H NMR (400 MHz, DMSO-d6):
d
2.9 (s, 3H), 5.3 (d, J ¼ 8.3 Hz, 1H,
CeCHeAr), 6.9 (d, J ¼ 8.3 Hz, 1H, HeCeC]O), 7.0e8.0 (m, 11H,
ArH), 10.4 (s, 1H, OH), 10.8 (s, 1H, OH), 11.7 (s, 1H, COOH). 13C NMR:
186.24, 168.52, 162.30, 146.22, 138.38, 137.12, 136.12, 134.29, 132.32,
131.94, 127.38, 126.26, 125.23, 124.53, 121.36, 120.42, 116.42, 82.54,
63.56, 62.23. LCMS: (Mþ) (433) Anal. Calcd. for: C24H19NO7: C,
66.51; H, 4.42; N, 3.23. Found: C, 66.51; H, 4.42; N, 3.32%.
4.1.5.10. 3-Phenyl-4(3-hydroxy-4-carboxybenzoyl)-5(2,4-
dimethoxyphenyl)4,5-dihydrodihydroisoxazoline (8j). Yield 73%,
m.p. 154e157 ꢀC; IR (KBr, /cmꢁ1): 3700e3620 (OH), 3410e3588
g
(OH of COOH), 1704 (C]O of COOH), 1675 (C]O), 1239 (CeO) 1620
(C]N). 1H NMR (400 MHz, DMSO-d6):
d
3.8 (s, 6H, OCH3), 5.8 (d,
4.1.5.5. 3(4-Methoxyphenyl)-4(3-hydroxy-4-carboxybenzoyl)-5(3-
J ¼ 8.2 Hz,1H, CeCHeAr), 6.7 (d, J ¼ 8.2 Hz,1H, HeCeC]O), 7.1e8.0
(m, 11H, ArH), 10.5 (s, 1H, OH), 11.9 (s, 1H, COOH). 13CNMR, 185.34,
165.33, 165.23, 147.31, 140.61, 137.21, 135.62, 134.43, 133.29, 133.09,
132.54, 128.83, 127.45, 125.26, 123.48, 122.32, 120.45, 118.42, 117.32,
85.43, 67.27, 63.53. LCMS: (Mþ) (448), Anal. Calcd. for: C25H21NO7:
C, 67.11; H, 4.73; N, 3.13. Found: C, 67.21; H, 4.70; N, 3.11%.
chlorophenyl)4,5-dihydrodihydroisoxazoline (8e). Yield 73%, m.p.
163e166 ꢀC; IR (KBr, /cmꢁ1): 3710e3640 (OH), 3421e3526 (OH of
g
COOH), 1706 (C]O of COOH), 1683 (C]O), 1240 (CeO) 1624 (C]N).
1H NMR (400 MHz, DMSO-d6):
d
2.9 (s, 3H, OCH3), 5.4 (d, J ¼ 8.2 Hz,
1H, CeCHeAr), 6.2 (d, J ¼ 8.2 Hz, 1H, HeCeC]O), 7.0e8.0 (m, 11H,
ArH), 10.6 (s, 1H, OH), 12.1 (s, 1H, COOH). 13C NMR, 185.26, 167.53,
163.31, 145.22, 138.21, 137.11, 136.42, 135.39, 134.12, 132.94, 128.38,
127.23, 125.26, 123.32, 122.12 121.23, 120.42, 117.52, 116.31, 83.53,
65.46, 63.23. LCMS: (Mþ) (452), (454) Anal. Calcd. for: C24H18ClNO6
C, 63.79; H, 4.02; N, 3.10. Found: C, 63.73; H, 4.01; N, 3.10%.
4.2. Biology
4.2.1. Chemicals and reagents
[Concanavalin A (Con A)] Ficoll-hypaque, tissue culture grade
sodium bicarbonate and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide A.R.] were procured from Sigma-
eAldrich Co., St. Louis, MO, USA. RPMI-1640 medium, Penstrip and
Fetal bovine serum (FBS) were purchased from Gibco-Invitrogen,
USA. Cell culture plates (96 well) were products of eppendorf,
Germany. Anti-Interleukin antibodies for IL-2, IL-12 and IL-23 were
procured from Merck Millipore. The COX assay kit was from
Cayman chemicals. All other chemicals and reagents used in the
experiments were of analytical grade.
4.1.5.6. 3(4-Hydroxyphenyl)-4(3-hydroxy-4-carboxybenzoyl)-5(3-
chlorophenyl)4,5-dihydrodihydroisoxazoline (8f). Yield 77%, m.p.
184e186 ꢀC: IR (KBr, /cmꢁ1), 3710e3640 (OH): 3465e3525 (OH of
g
COOH), 1702 (C]O of COOH), 1684 (C]O), 1241 (CeO), 1615(C]N).
1H NMR (400 MHz, DMSO-d6):
d
5.3 (d, J ¼ 8.3 Hz, 1H, CeCHeAr),
6.4 (d, J ¼ 8.3 Hz, 1H, HeCeC]O), 7.0e8.0 (m, 11H, ArH) 10.3 (s, 1H,
OH), 10.6 (s, 1H, OH), 12.1 (s, 1H, COOH). 13CNMR, 186.26, 166.53,
163.12, 162.41, 144.32, 137.24, 139.12, 137.12, 135.18, 134.93, 133.04,
129.62, 128.24, 127.38, 124.52, 123.43, 121.02, 120.54, 116.25, 82.03,