H)-one (1–30) have been synthesized by the cyclization of (E)-3-(4-(3-substitutedphenyl)acrylolyl)phenyl)-2-(substitutedphenyl)-7-substituted quinazolin-4-(3H)-one with hydroxylamine hydrochloride. The synthesized compounds were examined for their in vivo antihypertensive activity using albino rats. All the titled compounds exhibited good to moderate antihypertensive activity. Compounds 7-Chloro-3-(4-(3-(4-chlorophenyl)-4,5- dihydroisoxazol-5-yl)phenyl)-2-p-tolylquinazolin-4(3H)-one (23) and 7-Chloro-3-(4-(3-(4-chlorophenyl)-4,5-dihydroisoxazol-5-yl)phenyl)-2-(4-methoxyphenyl)quinazolin-4(3H)-one (24) exhibited potent antihypertensive activity through their anticipated α1-adrenergic receptor blocking property similar to its clinically used analogue, prazosin, without affecting heart rate with prolonged duration of action when tested in adrenaline induced hypertension in anaesthetized rats.A series of 7-substituted-3-(4-(3-(4-substitutedphenyl)-4,5-dihydroisoxazol-5-yl)phenyl)-2-substituted quinazolin-4(3 In 2prazosin , 6, teraerazosin  were foudiuretic –10, antidiuretic , antihisdiuretic , 13 analdiuretic , 15 antidiuretic  and antidiuretic  activitiH)-one derivatives possessing antihypertensive activity with minimum side effects  NMR spectrometers using dimethylsulfoxide (DMSO)-d6 solvent and tetramethylsilane (TMS) as an internal standard . 13C NMR spectra were recorded on a Bruker 400 Ultrashield (400 MHz) NMR spectrometers using dimethylsulfoxide (DMSO)-d6 solvent. Mass spectra recorded on UPLC-MS/MS  mass serial no. JAA-272 (Synapt MS-G1) were presented as m/z. Elemental analysis was carried out on CHN elemental  using sulphanilic acid as a standard and tungsten (VI) oxide as a combusting agent. All the results of elemental analyses corresponded to the calculated values within experimental error. The reaction was monitored by thin-layer chromatography (TLC) and spots were visualized by iodine vapors or irradiation with UV light (254 nm).All the chemicals used were of laboratory grade and procured from E. Merck  and S.D. Fine Chemicals . Melting points were determined by open capillary tubes in a Hicon melting point apparatus and are uncorrected. Purity of the compounds was checked by thin layer chromatography (TLC) plates (silica gel G). Iodine chamber and UV lamp were used for the visualization of TLC spots. The Fourier Transform Infrared (FT-IR) spectra were recorded on Bio-rad FTS-135 spectrophotometer using KBr pellets; To the substituted anthranilic acid (2 mmol) dissolved in 10% sodium hydroxide (10 mL), substituted benzoyl chloride (2.2 mmol) was added with stirring at room temperature for over 1 h. Upon completion, reaction mixture was quenched with cold water to obtain solid residue, which was washed with dilute HCl followed by water and recrystallized from ethanol. Yield: 90–96%.a1–a12, 2 mmol) in acetic anhydride (10 mL) was heated under reflux for 2 h and then poured into crushed ice. The solid residue thus obtained was filtered, dried, and recrystallized with ethanol; Yield is 85–90%.A solution of substituted 2-benzamidobenzoic acid  and p-aminoacetophenone (2 mmol) in methanol was heated under reflux for 3 h. The separated solid was collected by filtration, washed with water, dried, and recrystallized from ethanol.A mixture of substituted benzoxazine  in ethanol, substituted aldehyde (0.01 mmol) was added and the reaction mixture was stirred for 2 h. Upon completion of reaction, the mixture was poured on crushed ice and then acidified with dilute HCl. A precipitate formed which was filtered, washed with water, and recrystallized with ethanol.To a magnetically stirred solution of substituted quinazolinone (E)-3-(4-(3-substitutedphenyl)acrylolyl)phenyl)-2-(substitutedphenyl)-7-substituted quinazolin-4-(3H)-one  in absolute ethanol (25 mL) and water; hydroxylamine hydrochloride (0.01 mol) was added. The reaction mixture was heated under reflux for 5 h in the presence of 30% KOH. The mixture was poured in ice water and then acidified with dilute HCl. The resulting solid was filtered, washed with water, dried, and crystallized with ethanol. A mixture of ((1) 2-Phenyl-3-phenyl)quinazolin-4(3H)-one  (1). Yield: 78%; m.p. 138–140°C; TLC solvent ; Rf 0.63; IR (KBr) νmax (cm−1): 3052 (Ar-CH), 1695 (CO), 1630 (C=N), 1608, 1455 (C=C), 1220 (C–O–N), 1115 (C–N); 1H NMR (DMSO-d6) δ (ppm); 3.80 , 5.88 , 8.04 , 8.06 , 8.10 , 8.12 , 8.14 , 8.16 , 8.15–8.17 ; 13C NMR (DMSO-d6) δ; 128.5 (2C), 128.8 (2C), 131.8, 132.5 (phenyl), 43.7, 85.5, 155.7 , 128.5 (2C), 128.8 (2C), 130.2, 131.3 (Ar-C), 124.4 (2C), 128.5 (2C), 132.45, 137.66 (Ar-C), 120.6, 126.6, 128.2, 133.8, 135.2, 148.3, 156.9 (quinazoline), 160.4 ; (m/z): 443.49 (M+); % Anal. Cal. for C29H21N3O2: C: 78.54, H: 4.77, N: 9.47. Found: C: 78.50, H: 4.72, N: 9.46.(2) 2-(2-Chlorophenyl)-3-phenyl)quinazolin-4(3H)-one (2). Yield: 84%; m.p. 140–142°C; TLC solvent ; Rf 0.66; IR : 3022 (Ar-CH), 1685 (CO), 1610 (C=N), 1598, 1463 (C=C), 1230 (C–O–N), 1110 (C–N), 710 (C–Cl); 1H NMR (DMSO-d6) δ; 3.90 , 6.02 , 8.06 , 8.06–8.09 , 8.10 , 8.12 , 8.14 , 8.15 , 8.16–8.18 ; 13C NMR (DMSO-d6) δ; 128.2 (2C), 129.2 (2C), 130.8, 131.2 (phenyl), 42.2, 83.4, 156.5 , 123.5, 126.5, 128.8, 130.2, 131.3, 132.3 (Ar-C), 125.8 (2C), 128.2 (2C), 132.5, 138.3 (Ar-C), 120.4, 127.7, 128.8, 134.2, 135.6, 147.6, 155.4 (quinazoline), 162.3 ; % Anal. Cal. for C29H20ClN3O2; C, 72.88; N, 8.79; H, 4.22. Found; C, 72.86; N, 8.77; H, 4.20; Ms (m/z): 477.94 (M+).(3) 3-phenyl)-2-p-tolylquinazolin-4(3H)-one (3). Yield: 70%; m.p. 162–164°C; TLC solvent ; Rf 0.63; IR : 3012 (Ar-CH), 1690 (CO), 1634 (C=N), 1618, 1465 (C=C), 1225 (C–O–N), 1123 (C–N); 1H NMR (DMSO-d6) δ (ppm); 2.23 , 3.92 , 6.00 , 8.03–8.06 , 8.08 , 8.08-8.09 , 8.12 , 8.15 , 8.16-8.17 ; 13C NMR (DMSO-d6) δ; 20.5 , 128.5 (2C), 128.8 (2C), 131.0, 132.6 (phenyl), 40.8, 84.7, 157.2 , 125.2, 129.5 (2C), 130.8 (2C), 140.6 (Ar-C), 126.2 (2C), 128.8 (2C), 131.2, 137.5 (Ar-C), 120.8, 126.4, 128.8, 133.6, 136.3, 146.5, 156.4 (quinazoline), 160.6 ; % Anal. Cal. for C30H23N3O2; C, 78.75; N, 9.18; H, 5.07. Found; C, 78.76; N, 9.18; H, 5.05; Ms (m/z): 457.52 (M+).(4) 2-(4-Methoxyphenyl)-3-phenyl)quinazolin-4(3H)-one (4). Yield: 65%; m.p. 135–137°C; TLC solvent ; Rf 0.67; IR : 3022 (Ar-CH), 1675 (CO), 1625 (C=N), 1588, 1425 (C=C), 1310 (C–O), 1226 (C–O–N), 1110 (C–N); 1H NMR (DMSO-d6) δ (ppm); 2.70 , 3.80 , 5.78 , 8.02–8.04 , 8.07 , 8.09 , 8.11–8.20 ; 13C NMR (DMSO-d6) δ; 52.6 , 128.5 (2C), 129.8 (2C), 130.2, 131.5 (phenyl), 42.0, 84.2, 155.7 , 118.4 (2C), 120.5, 132.4 (2C), 158.4 (Ar-C), 124.4 (2C), 126.8 (2C), 131.4, 138.0 (Ar-C), 120.8, 127.6, 128.2, 133.6, 135.6, 148.3, 156.0 (quinazoline), 160.2 ; % Anal. Cal. for C30H23N3O3; C, 76.09; N, 8.87; H, 4.90. Found; C, 76.08; N, 8.87; H, 4.92; Ms (m/z): 473.52 (M+).(5) 7-Chloro-2-phenyl-3-phenyl)quinazolin-4(3H)-one (5). Yield: 80%; m.p. 166–168°C; TLC solvent ; Rf 0.54; IR : 3062 (Ar-CH), 1680 (CO), 1650 (C=N), 1618, 1465 (C=C), 1226 (C–O–N), 1122 (C–N), 720 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 3.83 , 5.88 , 7.98–8.00 , 8.04 , 8.06 , 8.08 , 8.12 , 8.14–8.16 ; 13C NMR (DMSO-d6) δ; 128.5 (2C), 129.0 (2C), 131.4, 132.5 (phenyl), 42.6, 84.6, 155.0 , 128.4 (2C), 128.6, 128.8 (2C), 132.5 (Ar-C), 125.2 (2C), 128.6 (2C), 131.0, 135.8 (Ar-C), 118.5, 122.5, 127.6, 130.8, 137.8, 152.4, 156.2 (quinazoline), 161.5 ; % Anal. Cal. for C29H20ClN3O2; C, 72.88; N, 8.79; H, 4.22. Found; C, 72.86; N, 8.77; H, 4.20; Ms (m/z): 478.94 (M+1).(6) 7-Chloro-2-(2-chlorophenyl)-3-phenyl)quinazolin-4(3H)-one (6). Yield: 85%; m.p. 130–132°C; TLC solvent ; Rf 0.65; IR : 3034 (Ar-CH), 1710 (CO), 1660 (C=N), 1598, 1480 (C=C), 1224 (C–O–N), 1176 (C–N), 750, 610 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 3.94 , 5.86 , 8.03–8.06 , 8.06–8.08 , 8.10 , 8.14 , 8.17 , 8.20–8.22 ; 13C NMR (DMSO-d6) δ; 128.2 (2C), 130.8 (2C), 131.4, 132.3 (phenyl), 45.7, 88.5, 154.7 , 120.6, 124.8, 130.2, 132.3 (2C), 134.5 (Ar-C), 125.8 (2C), 127.8 (2C), 132.4, 137.6 (Ar-C), 118.3, 120.6, 128.2, 130.8, 138.25, 151.3, 155.9 (quinazoline), 162.4 ; % Anal. Cal. for C29H19Cl2N3O2; C, 67.98; N, 8.20; H, 3.74. Found; C, 67.96; N, 8.22; H, 3.72; Ms (m/z): 512.38 (M+1). (7) 7-Chloro-3-phenyl)-2-p-tolylquinazolin-4(3H)-one (7). Yield: 70%; m.p. 164–166°C; TLC solvent ; Rf 0.67; IR : 3012 (Ar-CH), 1690 (CO), 1644 (C=N), 1602, 1445 (C=C), 1225 (C–O–N), 1154 (C–N), 720 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 2.32 , 3.96 , 5.80 , 8.00–8.02 , 8.04 , 8.07 , 8.10 , 8.12 , 8.11–8.18 ; 13C NMR (DMSO-d6) δ; 20.4 , 128.5 (2C), 129.8 (2C), 130.2, 131.8 (phenyl), 42.6, 84.2, 154.3 , 125.5, 128.5 (2C), 130.3 (2C), 138.9 (Ar-C), 125.2 (2C), 127.5 (2C), 131.8, 137.2 (Ar-C), 118.8, 122.6, 130.6, 134.8, 137.4, 153.4, 156.7 (quinazoline), 160.2 ; % Anal. Cal. for C30H22ClN3O2; C, 73.24; N, 8.54; H, 4.51. Found; C, 73.23; N, 8.52; H, 4.50; Ms (m/z): 491.96 (M+).(8) 7-Chloro-2-(4-methoxyphenyl)-3-phenyl)quinazolin-4(3H)-one (8). Yield: 85%; m.p. 170–172°C; TLC solvent ; Rf 0.70; IR : 3022 (Ar-CH), 1693 (CO), 1610 (C=N), 1588, 1463 (C=C), 1295 (C–O), 1233 (C–O–N), 1122 (C–N), 722 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 2.75 , 3.94 , 5.82 , 8.04–8.06 , 8.08 , 8.10 , 8.14 , 8.18 , 8.20–8.22 ; 13C NMR (DMSO-d6) δ; 52.5 , 128.2 (2C), 128.8 (2C), 130.6, 132.5 (phenyl), 42.0, 85.6, 155.6 , 125.8, 127.4 (2C), 132.3 (2C), 139.4 (Ar-C), 125.2 (2C), 127.5 (2C), 131.8, 137.2 (Ar-C), 118.2, 120.5, 131.4, 133.4, 136.8, 152.5, 157.3 (quinazoline), 162.5 ; % Anal. Cal. for C30H22ClN3O3; C, 70.93; N, 8.27; H, 4.37. Found; C, 70.92; N, 8.25; H, 4.35; Ms (m/z): 507.96 (M+1).(9) 7-Methyl-2-phenyl-3-phenyl)quinazolin-4(3H)-one (9). Yield: 55%; m.p. 133–135°C; TLC solvent ; Rf 0.80; IR : 3012 (Ar-CH), 1690 (CO), 1610 (C=N), 1588, 1420 (C=C), 1232 (C–O–N), 1100 (C–N); 1H NMR (DMSO-d6) δ (ppm); 2.30 , 3.97 , 5.86 , 7.94–8.00 , 8.02–8.04 , 8.07 , 8.10 , 8.13 , 8.17–8.20 ; 13C NMR (DMSO-d6) δ; 21.5 , 128.2 (2C), 128.8 (2C), 131.5, 132.5 (phenyl), 42.0, 83.2, 153.5 , 124.3, 128.8 (2C), 131.2 (2C), 132.3 (Ar-C), 125.8 (2C), 128.2 (2C), 130.8, 138.0 (Ar-C), 118.5, 120.6, 132.4, 133.8, 142.0, 151.2, 156.7 (quinazoline), 162.4 ; % Anal. Cal. for C30H23N3O2; C, 78.75; N, 9.18; H, 5.07. Found; C, 78.76; N, 9.16; H, 5.05; Ms (m/z): 457.52 (M+).(10) 2-(2-Chlorophenyl)-7-methyl-3-phenyl)quinazolin-4(3H)-one (10). Yield: 60%; m.p. 154–156°C; TLC solvent ; Rf 0.71; IR : 3002 (Ar-CH), 1710 (CO), 1624 (C=N), 1608, 1488 (C=C), 1220 (C–O–N), 1105 (C–N), 710 (C–Cl); 1H NMR (DMSO-d6) δ: 2.22 , 3.87 , 5.81 , 8.02–7.98 , 8.04–8.02 , 8.07–8.03 , 8.10–8.08 , 8.16–8.13 , 8.22–8.19 ; 13C NMR (DMSO-d6) δ; 13.46 , 106.16, 108.65, 110.54, 114.20, 122.62, 121.13 , 63.44, 150.12, 154.12 , 115.82, 122.13, 134.12, 134.84, 138.12, 154.25 , 108.16, 115.44, 118.65, 126.52, 127.06, 133.24 , 108.65, 110.58, 115.17, 126.52, 150.53, 147.62, 160.62 , 171.66 ; % Anal. Cal. for C30H22ClN3O2; C, 73.24; N, 8.54; H, 4.51. Found; C, 73.22; N, 8.52; H, 4.50; Ms (m/z): 492.96 (M+1).(11) 7-Methyl-3-phenyl)-2-p-tolylquinazolin-4(3H)-one (11). Yield: 66%; m.p. 178–180°C; TLC solvent ; Rf 0.58; IR : 3012 (Ar-CH), 1678 (CO), 1612 (C=N), 1572, 1453 (C=C), 1218 (C–O–N), 1112 (C–N); 1H NMR (DMSO-d6) δ (ppm); 2.21 , 2.36 , 3.80 , 5.74 , 8.04–8.08 , 8.10 , 8.12 , 8.15 , 8.22 , 8.30–8.32 ; 13C NMR (DMSO-d6) δ; 21.6 , 128.0 (2C), 128.6 (2C), 131.2, 132.5 (phenyl), 42.2, 85.5, 153.6 , 124.2, 127.6 (2C), 130.7 (2C), 139.2 (Ar-C), 125.8 (2C), 128.3 (2C), 130.8, 136.2 (Ar-C), 118.8, 120.3, 130.6, 133.2, 143.2, 152.4, 157.4 (quinazoline), 160.4 ; % Anal. Cal. for C31H25N3O2; C, 78.96; N, 8.91; H, 5.34. Found; C, 78.97; N, 8.90; H, 5.31; Ms (m/z): 471.54 (M+).(12) 2-(4-Methoxyphenyl)-7-methyl-3-phenyl)quinazolin-4(3H)-one (12). Yield: 76%; m.p. 172–174°C; TLC solvent ; Rf 0.58; IR : 3012 (Ar-CH), 1677 (CO), 1612 (C=N), 1596, 1453 (C=C), 1300 (C–O), 1226 (C–O–N), 1106 (C–N); 1H NMR (DMSO-d6) δ (ppm); 2.28 , 2.82 , 3.86 , 5.89 , 7.96–7.94 , 8.08 , 8.14 , 8.18 , 8.20 , 8.21–8.23 ; 13C NMR (DMSO-d6) δ; 20.4 , 50.5  128.2 (2C), 128.9 (2C), 131.6, 132.5 (phenyl), 42.0, 84.8, 153.5 , 125.2, 128.0 (2C), 131.4 (2C), 138.5 (Ar-C), 125.6 (2C), 128.0 (2C), 132.4, 138.2 (Ar-C), 120.8, 122.6, 130.6, 133.4, 138.0, 151.4, 155.7 (quinazoline), 161.5 ; % Anal. Cal. for C31H25N3O3; C, 76.37; N, 8.62; H, 5.17. Found; C, 76.32; N, 8.60; H, 5.15; Ms (m/z): 488.54 (M+1).(13) 3-(4-(3-(4-Chlorophenyl)-4, 5-dihydroisoxazol-5-yl)phenyl)-2-phenylquinazolin-4(3H)-one (13). Yield: 70%; m.p. 145–147°C; TLC solvent ; Rf 0.70; IR : 3012 (Ar-CH), 1684 (CO), 1618 (C=N), 1596, 1473 (C=C), 1222 (C–O–N), 1102 (C–N), 712 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 3.55 , 5.92 , 8.06–8.08 , 8.07–8.09 , 8.10 , 8.12 , 8.18–8.20 ; 13C NMR (DMSO-d6) δ; 128.0 (2C), 128.2, 129.2 (2C), 137.2 (phenyl), 42.3, 85.2, 155.6 , 127.2, 128.4 (2C), 128.8 (2C), 130.2 (Ar-C), 125.8 (2C), 128.2 (2C), 130.2, 138.0 (Ar-C), 120.4, 123.5, 130.3, 135.3, 138.0, 148.6, 155.7 (quinazoline), 160.5 ; % Anal. Cal. for C29H20ClN3O2; C, 72.88; N, 8.79; H, 4.22. Found; C, 72.87; N, 8.75; H, 4.20; Ms (m/z): 478.94 (M+1). (14) 2-(2-Chlorophenyl)-3-(4-(3-(4-chlorophenyl)-4,5-dihydroisoxazol-5-yl)phenyl)quinazolin-4(3H)-one (14). Yield: 76%; m.p. 150–152°C; TLC solvent ; Rf 0.66; IR : 3033 (Ar-CH), 1698 (CO), 1622 (C=N), 1590, 1473 (C=C), 1227 (C–O–N), 1132 (C–N), 752 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 3.74 , 5.92 , 8.03–8.05 , 8.06–8.08 , 8.12 , 8.13 , 8.38–8.40 ; 13C NMR (DMSO-d6) δ; 128.2 (2C), 129.8 (2C), 130.8, 137.4 (phenyl), 43.3, 84.6, 156.6 , 126.3, 128.2 (2C), 128.8 (2C), 138.0 (Ar-C), 122.2, 126.8, 130.2, 131.6, 132.0, 132.6 (Ar-C), 120.8, 124.6, 132.6, 133.8, 137.4, 148.4, 155.7 (quinazoline), 160.5 ; % Anal. Cal. for C29H19Cl2N3O2; C, 67.98; N, 8.20; H, 3.74. Found; C, 67.96; N, 8.18; H, 3.73; Ms (m/z): 515 (M+2). (15) 3-(4-(3-(4-Chlorophenyl)-4,5-dihydroisoxazol-5-yl)phenyl)-2-p-tolylquinazolin-4(3H)-one (15). Yield: 77%; m.p. 145–147°C; TLC solvent ; Rf 0.67; IR : 3038 (Ar-CH), 1710 (CO), 1605 (C=N), 1590, 1453 (C=C), 1226 (C–O–N), 1117 (C–N), 733 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 2.21 , 3.68 , 5.86 , 8.06–8.08 , 8.12–8.14 , 8.18 , 8.19 , 8.42–8.44 ; 13C NMR (DMSO-d6) δ; 21.8 , 128.2 (2C), 128.8 (2C), 131.2, 137.9 (phenyl), 42.2, 83.8, 155.6 , 125.6, 129.2 (2C), 130.5 (2C), 139.2 (Ar-C), 125.8 (2C), 128.5 (2C), 130.8, 138.2 (Ar-C), 120.8, 122.5, 130.3, 133.6, 134.4, 147.4, 157.7 (quinazoline), 162.4 ; % Anal. Cal. for C30H22ClN3O2; C, 73.24; N, 8.24; H, 4.51. Found; C, 73.22; N, 8.22; H, 4.52; Ms (m/z): 492.96 (M+1). (16) 3-(4-(3-(4-Chlorophenyl)-4,5-dihydroisoxazol-5-yl) phenyl)-2-(4-methoxyphenyl) quinazolin-4(3H)-one (16). Yield: 66%; m.p. 164–166°C; TLC solvent ; Rf 0.68; IR : 3010 (Ar-CH), 1696 (CO), 1622 (C=N), 1598, 1466 (C=C), 1285 (C–O), 1217 (C–O–N), 1120 (C–N), 717 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 2.78 , 3.96 , 5.92 , 8.04–8.02 , 8.04–8.06 , 8.08 , 8.10 , 8.18–8.20 ; 13C NMR (DMSO-d6) δ; 52.6 , 128.6 (2C), 129.2, 130.5 (2C), 137.9 (phenyl), 40.6, 84.3, 156.2 , 115.6 (2C), 121.6, 130.8 (2C), 162.8 (Ar-C), 124.2 (2C), 127.9 (2C), 130.2, 137.8 (Ar-C), 120.2, 126.4, 128.6, 129.2, 133.5, 147.8, 156.2 (quinazoline), 160.2 ; % Anal. Cal. for C30H22ClN3O3; C, 70.93; N, 8.27; H, 4.37. Found; C, 70.91; N, 8.25; H, 4.35; Ms (m/z): 508.96 (M+1). (17) 3-(4-(3-(4-Methoxyphenyl)-4,5-dihydroisoxazol-5-yl)phenyl)-2-phenylquinazolin-4(3H)-one (17). Yield: 60%; m.p. 190–192°C; TLC solvent ; Rf 0.70; IR : 3018 (Ar-CH), 1713 (CO), 1634 (C=N), 1586, 1453 (C=C), 1296 (C–O), 1234 (C–O–N), 1110 (C–N); 1H NMR (DMSO-d6) δ (ppm); 2.74 , 3.68 , 5.92 , 8.00–8.02 , 8.04–8.06 , 8.15 , 8.16 , 8.38–8.40 ; 13C NMR (DMSO-d6) δ; 54.6 , 115.8 (2C), 122.5, 127.3 (2C), 160.6 (phenyl), 40.8, 84.2, 156.4 , 127.2 (2C), 128.2 (2C), 128.8, 130.6 (Ar-C), 126.8 (2C), 127.8 (2C), 131.4, 137.5 (Ar-C), 120.4, 126.5, 127.3, 129.4, 134.8, 148.0, 155.4 (quinazoline), 161.2 ; % Anal. Cal. for C30H23N3O3; C, 76.09; N, 8.87; H, 4.90. Found; C, 76.07; N, 8.85; H, 4.92; Ms (m/z): 475.52 (M+2). (18) 2-(2-Chlorophenyl)-3-(4-(3-(4-methoxyphenyl)-4,5-dihydroisoxazol-5-yl)phenyl)quinazolin-4(3H)-one (18). Yield: 70%; m.p. 165–167°C; TLC solvent ; Rf 0.63; IR : 3018 (Ar-CH), 1713 (CO), 1634 (C=N), 1586, 1453 (C=C), 1308 (C–O), 1234 (C–O–N), 1110 (C–N); 1H NMR (DMSO-d6) δ (ppm); 2.68 , 3.88 , 5.80 , 8.06–8.08 , 8.10–8.12 , 8.15 , 8.30 , 8.38–8.40 ; 13C NMR (DMSO-d6) δ; 55.2 , 114.5 (2C), 123.5, 128.8 (2C), 161.2 (phenyl), 42.4, 83.8, 157.4 , 123.6, 126.8, 129.2, 130.4, 131.8, 132.5 (Ar-C), 125.6 (2C), 128.2 (2C), 130.4, 138.5 (Ar-C), 120.4, 126.2, 126.8, 127.9, 133.8, 147.5, 154.5 (quinazoline), 160.5 ; % Anal. Cal. for C30H22ClN3O3; C, 70.93; N, 8.27; H, 4.37. Found; C, 70.95; N, 8.25; H, 4.35; Ms (m/z): 508.96 (M+1). (19) 3-(4-(3-(4-Methoxyphenyl)-4,5-dihydroisoxazol-5-yl) phenyl)-2-p-tolylquinazolin-4(3H)-one (19). Yield: 73%; m.p. 176–178°C; TLC solvent ; Rf 0.60; IR : 3015 (Ar-CH), 1697 (CO), 1618 (C=N), 1588, 1468 (C=C), 1280 (C–O), 1230 (C–O–N), 1154 (C–N); 1H NMR (DMSO-d6) δ (ppm); 2.03 , 2.56 , 3.80 , 5.88 , 8.04 , 8.06 , 8.10 , 8.12 , 8.14 , 8.16 , 8.15–8.17 ; 13C NMR (DMSO-d6) δ; 20.4 , 55.3 , 114.4 (2C), 120.3, 128.8 (2C), 161.5 (phenyl), 42.2, 83.0, 155.6 , 125.2, 129.5 (2C), 130.3 (2C), 140.2 (Ar-C), 125.2 (2C), 128.2 (2C), 130.4, 138.2 (Ar-C), 120.4, 126.2, 127.8, 130.4, 133.8, 148.8, 156.2 (quinazoline), 160.8 ; % Anal. Cal. for C31H25N3O3; C, 76.37; N, 8.62; H, 5.17. Found; C, 76.35; N, 8.60; H, 5.19; Ms (m/z): 487.54 (M+).(20) 2-(4-Methoxyphenyl)-3-(4-(3-(4-methoxyphenyl)-4,5-dihydroisoxazol-5-yl)phenyl) quinazolin-4(3H)-one (20). Yield: 65%; m.p. 162–164°C; TLC solvent ; Rf 0.55; IR : 3024 (Ar-CH), 1688 (CO), 1612 (C=N), 1595, 1472 (C=C), 1298 (C–O), 1224 (C–O–N), 1160 (C–N); 1H NMR (DMSO-d6) δ (ppm); 2.80 , 2.86 , 3.84 , 5.92 , 8.08–8.10 , 8.12–8.14 , 8.15 , 8.17 , 8.20–8.22 ; 13C NMR (DMSO-d6) δ; 54.7, 116.2 (2C), 122.8, 127.2 (2C), 162.3 (phenyl), 42.5, 84.5, 156.2 , 115.6 (2C), 120.3, 130.3 (2C), 160.8 (Ar-C), 125.8 (2C), 129.5 (2C), 131.5, 137.5 (Ar-C), 120.3, 126.6, 128.3, 130.8, 132.5, 147.3, 157.5 (quinazoline), 162.3 ; % Anal. Cal. for C31H25N3O4; C, 73.94; N, 8.34; H, 5.00. Found; C, 73.92; N, 8.32; H, 5.02; Ms (m/z): 503.54 (M+).(21) 7-Chloro-3-(4-(3-(4-chlorophenyl)-4,5-dihydroisoxazol-5-yl)phenyl)-2-phenylquinazolin-4(3H)-one (21). Yield: 68%; m.p. 180–182°C; TLC solvent ; Rf 0.54; IR : 3032 (Ar-CH), 1684 (CO), 1608 (C=N), 1592, 1426 (C=C), 1228 (C–O–N), 1124 (C–N), 712 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 3.93 , 5.95 , 8.10–8.12 , 8.14–8.16 , 8.20 , 8.22 , 8.28–8.32 ; 13C NMR (DMSO-d6) δ; 128.2 (2C), 128.8, 129.2 (2C), 137.2 (phenyl), 41.5, 85.0, 156.2 , 128.2 (2C), 128.6, 129.5 (2C), 130.7 (Ar-C), 125.5 (2C), 127.8 (2C), 131.4, 137.6 (Ar-C), 118.4, 125.2, 126.8, 132.4, 137.5, 152.2, 157.5 (quinazoline), 160.8 ; % Anal. Cal. for C29H19Cl2N3O2; C, 67.98; N, 8.20; H, 3.74. Found; C, 67.96; N, 8.17; H, 3.72; Ms (m/z): 513.38 (M+1). (22) 7-Chloro-2-(2-chlorophenyl)-3-(4-(3-(4-chlorophenyl)-4,5-dihydroisoxazol-5-yl)phenyl)quinazolin-4(3H)-one (22). Yield: 75%; m.p. 157–159°C; TLC solvent ; Rf 0.67; IR : 3042 (Ar-CH), 1688 (CO), 1628 (C=N), 1587, 1420 (C=C), 1225 (C–O–N), 1113 (C–N), 734 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 3.84 , 5.90 , 8.10–8.12 , 8.12–8.14 , 8.16 , 8.18 , 8.18–8.20 ; 13C NMR (DMSO-d6) δ; 118.4 (2C), 122.6, 128.2 (2C), 136.2 (phenyl), 42.5, 84.0, 157.6 , 123.5, 126.5, 130.3, 131.2, 132.3, 132.8 (Ar-C), 124.8 (2C), 128.6 (2C), 130.2, 137.5 (Ar-C), 118.4, 122.2, 125.6, 129.4, 137.5, 150.8, 157.4 (quinazoline), 162.4 ; % Anal. Cal. for C29H18Cl3N3O2; C, 63.70; N, 7.68; H, 3.32. Found; C, 63.72; N, 7.66; H, 3.30; Ms (m/z): 547.83 (M+1). (23) 7-Chloro-3-(4-(3-(4-chlorophenyl)-4,5-dihydroisoxazol-5-yl)phenyl)-2-p-tolylquinazolin-4(3H)-one (23). Yield: 74%; m.p. 181–183°C; TLC solvent ; Rf 0.70; IR : 3038 (Ar-CH), 1694 (CO), 1622 (C=N), 1595, 1420 (C=C), 1305 (C–O), 1216 (C–O–N), 1114 (C–N), 710 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 2.23 , 3.76 , 5.86 , 7.96–7.98 , 8.02–8.04 , 8.06–8.08 , 8.16 , 8.20 ; 13C NMR (DMSO-d6) δ; 22.5 , 128.2 (2C), 128.6, 128.8 (2C), 135.8 (phenyl), 43.0, 83.5, 156.2 , 125.8, 129.0 (2C), 130.8 (2C), 138.8 (Ar-C), 125.8 (2C), 127.8 (2C), 131.2, 138.0 (Ar-C), 118.8, 122.7, 127.5, 130.6, 137.4, 152.5, 156.4 (quinazoline), 162.3 ; % Anal. Cal. for C30H21Cl2N3O2; C, 68.45; N, 7.98; H, 4.02. Found; C, 68.47; N, 7.96; H, 4.00; Ms (m/z): 528.41 (M+2). (24) 7-Chloro-3-(4-(3-(4-chlorophenyl)-4,5-dihydroisoxazol-5-yl)phenyl)-2-(4-methoxyphenyl)quinazolin-4(3H)-one (24). Yield: 80%; m.p. 186–188°C; TLC solvent ; Rf 0.69; IR : 3036 (Ar-CH), 1680 (CO), 1622 (C=N), 1596, 1420 (C=C), 1300 (C–O), 1233 (C–O–N), 1130 (C–N), 735 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 2.65 , 3.84 , 6.01 , 7.98–8.00 , 8.04–8.06 , 8.08–8.10 , 8.14 , 8.18 ; 13C NMR (DMSO-d6) δ; 52.4 , 128.3 (2C), 128.5, 128.9 (2C), 136.4 (phenyl), 43.8, 82.6, 158.6 , 115.5 (2C), 120.8, 131.5 (2C), 162.4 (Ar-C), 125.2 (2C), 128.5 (2C), 132.6, 138.5 (Ar-C), 118.2, 120.5, 128.3, 131.4, 137.8, 151.4, 157.3 (quinazoline), 160.2 ; % Anal. Cal. for C30H21Cl2N3O3; C, 66.43; N, 7.75; H, 3.90. Found; C, 66.41; N, 7.73; H, 3.88; Ms (m/z): 544.11 (M+2). (25) 3-(4-(3-(4-Chlorophenyl)-4,5-dihydroisoxazol-5-yl)phenyl)-7-methyl-2-phenylquinazolin-4(3H)-one (25). Yield: 64%; m.p. 150–152°C; TLC solvent ; Rf 0.62; IR : 3028 (Ar-CH), 1692 (CO), 1618 (C=N), 1590, 1422 (C=C), 1222 (C–O–N), 1120 (C–N); 1H NMR (DMSO-d6) δ (ppm); 2.12 , 3.80 , 5.88 , 7.92–7.94 , 8.08–8.10 , 8.12 , 8.14 , 8.33–8.35 ; 13C NMR (DMSO-d6) δ; 22.5 , 128.2 (2C), 128.6, 128.8 (2C), 135.8 (phenyl), 43.0, 83.5, 156.2 , 125.8, 129.0 (2C), 130.8 (2C), 138.8 (Ar-C), 125.8 (2C), 127.8 (2C), 131.2, 138.0 (Ar-C), 118.8, 122.7, 127.5, 130.6, 137.4, 152.5, 156.4 (quinazoline), 162.3 ; % Anal. Cal. for C30H22ClN3O2; C, 73.24; N, 8.54; H, 4.51. Found; C, 73.22; N, 8.52; H, 4.50; Ms (m/z): 491.96 (M+).(26) 2-(2-Chlorophenyl)-3-(4-(3-(4-chlorophenyl)-4,5-dihydroisoxazol-5-yl)phenyl)-7-methylquinazolin-4(3H)-one (26). Yield: 73%; m.p. 185–187°C; TLC solvent ; Rf 0.54; IR : 3040 (Ar-CH), 1682 (CO), 1622 (C=N), 1596, 1424 (C=C), 1232 (C–O–N), 1112 (C–N), 720 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 2.34 , 3.89 , 5.93, 7.94–7.96 , 8.10 , 8.13 , 8.14–8.16 , 8.20–8.24 ; 13C NMR (DMSO-d6) δ; 20.8 , 128.6 (2C), 129.6, 130.2 (2C), 136.2 (phenyl), 42.6, 84.2, 155.8 , 123.4, 126.6, 130.4, 131.8, 132.2, 132.8 (Ar-C), 124.5 (2C), 126.2 (2C), 130.6, 137.6 (Ar-C), 118.8, 124.7, 128.5, 132.6, 143.5, 151.0, 157.2 (quinazoline), 162.3 ; % Anal. Cal. for C30H21Cl2N3O2; C, 68.45; N, 7.98; H, 4.02. Found; C, 68.43; N, 7.96; H, 4.00; Ms (m/z): 528.41 (M+2). (27) 3-(4-(3-(4-Chlorophenyl)-4, 5-dihydroisoxazol-5-yl)phenyl)-7-methyl-2-p-tolylquinazolin-4(3H)-one (27). Yield: 64%; m.p. 176–178°C; TLC solvent ; Rf 0.74; IR : 3030 (Ar-CH), 1692 (CO), 1614 (C=N), 1588, 1420 (C=C), 1225 (C–O–N), 1130 (C–N), 708 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 2.20 , 2.24 , 3.84 , 5.90 , 7.88–7.90 , 7.97-7.98 , 8.10 , 8.12 , 8.16–8.18 ; 13C NMR (DMSO-d6) δ; 22.5 , 128.0 (2C), 128.8, 129.2 (2C), 136.0 (phenyl), 42.8, 84.2, 156.6 , 125.8, 129.2 (2C), 130.8 (2C), 140.2 (Ar-C), 125.2 (2C), 128.5 (2C), 130.4, 138.8 (Ar-C), 118.2, 120.7, 128.5, 130.2, 142.4, 151.2, 155.6 (quinazoline), 160.5 ; % Anal. Cal. for C31H24ClN3O2; C, 73.58; N, 8.30; H, 4.78. Found; C, 73.56; N, 8.28; H, 4.76; Ms (m/z): 506.99 (M+1). (28) 3-(4-(3-(4-Cchlorophenyl)-4, 5-dihydroisoxazol-5-yl) phenyl)-2-(4-methoxyphenyl)-7-methylquinazolin-4(3H)-one (28). Yield: 86%; m.p. 122–124°C; TLC solvent ; Rf 0.62; IR : 3036 (Ar-CH), 1687 (CO), 1624 (C=N), 1586, 1416 (C=C), 1288 (C–O), 1224 (C–O–N), 1104 (C–N), 732 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 2.19 , 2.96 , 3.96 , 5.78 , 8.00–8.02 , 8.04–8.06 , 8.08 , 8.12 , 8.13–8.15 ; 13C NMR (DMSO-d6) δ; 20.2 , 53.8 , 128.2 (2C), 128.7 129.8 (2C), 135.5 (phenyl), 42.4, 85.7, 155.8 , 115.2 (2C), 120.2, 131.2 (2C), 162.5 (Ar-C), 125.6 (2C), 128.8 (2C), 131.4, 137.4 (Ar-C), 117.0, 124.7, 127.5, 130.5, 143.5, 150.2, 156.2 (quinazoline), 161.8 ; % Anal. Cal. for C31H24ClN3O3; C, 71.33; N, 8.05; H, 4.63. Found; C, 71.31; N, 8.03; H, 4.65; Ms (m/z): 521.99 (M+).(29) 7-Chloro-3-(4-(3-(4-methoxyphenyl)-4,5-dihydroisoxazol-5-yl)phenyl)-2-phenylquinazolin-4(3H)-one (29). Yield: 65%; m.p. 190–192°C; TLC solvent ; Rf 0.75; IR : 3040 (Ar-CH), 1680 (CO), 1614 (C=N), 1595, 1417 (C=C), 1308 (C–O), 1230 (C–O–N), 1120 (C–N), 722 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 2.78 , 3.85 , 5.80 , 8.06–8.08 , 8.10 , 8.12 , 8.13–8.15 , 8.16–8.18 ; 13C NMR (DMSO-d6) δ; 52.0 , 115.5 (2C), 122.6 128.2 (2C), 160.8 (phenyl), 43.2, 84.7, 156.4 , 128.2 (2C), 128.6, 129.5 (2C), 130.2 (Ar-C), 124.5 (2C), 128.3 (2C), 130.4, 138.2 (Ar-C), 118.6, 122.6, 127.8, 130.5, 138.2, 152.8, 157.7 (quinazoline), 160.4 ; % Anal. Cal. for C30H22ClN3O3; C, 70.93; N, 8.27; H, 4.37. Found; C, 70.95; N, 8.25; H, 4.35; Ms (m/z): 507.96 (M+).(30) 7-Chloro-2-(2-chlorophenyl)-3-(4-(3-(4-methoxyphenyl)-4, 5-dihydroisoxazol-5-yl)phenyl)quinazolin-4(3H)-one (30). Yield: 86%; m.p. 179–181°C; TLC solvent ; Rf 0.64; IR : 3035 (Ar-CH), 1696 (CO), 1620 (C=N), 1598, 1430 (C=C), 1278 (C–O), 1234 (C–O–N), 1134 (C–N), 722 (C–Cl); 1H NMR (DMSO-d6) δ (ppm); 2.82 , 3.83 , 5.94 , 7.96–7.98 , 8.00–8.02 , 8.02–8.04 , 8.08 , 8.10 ; 13C NMR (DMSO-d6) δ; 55.9 , 116.4 (2C), 122.5 128.2 (2C), 160.4(phenyl), 43.4, 84.6, 156.2 , 122.5, 126.8, 130.5, 131.6, 132.2, 132.8 (Ar-C), 125.0 (2C), 127.6 (2C), 131.8, 138.9 (Ar-C), 118.6, 122.5, 128.2, 131.5, 137.8, 151.6, 155.8 (quinazoline), 160.2 ; % Anal. Cal. for C30H21Cl2N3O3; C, 66.43; N, 7.75; H, 3.90. Found; C, 66.41; N, 7.73; H, 3.92; Ms (m/z): 544.41 (M+2).All the experimental protocols were carried out with the permission from the Institutional Animal Ethics committee (IAEC), project proposal no. 781 and the guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments in Animal (CPCSEA). Animals were obtained from the Central Animal House Facility, Hamdard University, New Delhi. Registration number and date of registration are 173/CPCSEA, 28th of January, 2000. All rats were housed in a temperature and humidity controlled room at an ambient temperature of 25 ± 2°C with a 12 h light/dark cycle. Animals were provided with pellets diet  and water ad libitum.For conducting the BP measurement studies, the animals were kept in restrainers for 10 min every day for one week. This exercise was done to avoid the fluctuation in blood pressure due to aggressive behaviour of the animal.After recording the initial BP of rats, the animals were divided into groups of 6 animals each. One group was taken as control. Hypertension was induced in the remaining groups by subcutaneous injection of methyl prednisolone acetate (20 mg/kg body wt./wk) for 2 weeks as per method reported by Krakoff et al. .Systolic blood pressure was measured in conscious rats using CODA noninvasive blood pressure recorder by Tail Cuff method . The restrainers carrying the rat were placed in the BP instrument with the tail protruding out. The tail was gently placed in contact with a transducer membrane, which was connected to the digital BP display panel. The instrument was then turned on and allowed to stabilize until steady pulse rate was observed. Once the “pulse level ready” signal appeared, the BP recording button was pressed and the mean arterial BP was recorded. Albino rats (body weight 200–250 g) were used in the study. Rats were assigned to groups of six animals in each. Each compound  was administered orally after suspending in 1% carboxy methyl cellulose (CMC) solution. The blood pressure was recorded at various time intervals.α1-adrenergic receptor blocking effect of some synthesized compounds. In each experiment, the effect of adrenaline, at a dose of 3 μg/Kg intravenously, on the arterial blood pressure was recorded alone 30 min before i.p. injection  of the test compound and then its effect was determined again after 30, 60, and 120 min from the injection of the test compounds [Albino rats were classified into ten groups; each group comprises 6 animals each, to investigate the P < 0.05 was considered to be significant .The statistical analysis was performed using GRAPHPAD INSTAT 3 software . Data obtained from animal experiments were expressed as arithmetic mean ± SEM and ±S.E. The comparison between various groups was performed by one-way analysis of variance (ANOVA), and the effects in treatment groups were compared with toxic control or control group by Dunnett multiple comparison test. H)-one derivatives (1–30) were synthesized according to  (a1–a12). Some substituted benzoxazines (b1–b12) were obtained by heating (a1–a12) at reflux in acetic anhydride [H)-one (c1–c12) were yielded by the refluxing of substituted benzoxazines with p-aminoacetophenone [H)-one with aromatic aldehydes in equimolar amount were stirred in ethanol for 2 h to yield different chalcones [1–30) [ 24, The IR spectra of mentioned molecule showed characteristic absorption bands at 3036 cm−1 (Ar-CH), 1680 cm−1 (CO), 1622 cm−1 (C=N), 1596, 1420 cm−1 (C=C), 1300 cm−1, 1233 cm−1 (C–O–N), 1130 cm−1 (C–N), and 735 cm−1 (C–Cl). The 1H NMR spectra showed two doublet at δ 8.14  and 8.18  confirming N2-Ar ring, one doublet at δ 3.84  confirming CH2 isoxazole, and one multiplet at δ 6.01 confirming CH isoxazole. The multiplets at δ 8.00–7.98, 8.06–8.04, and 8.10–8.08 are indicatives of 4 protons of Ar2 ring, 3 protons of quinazoline moiety, and 4 protons of N3-Ar ring, respectively. The one singlet corresponding to OCH3 was obtained at δ 2.65. The mass spectrum shows the presence of molecular ion peak at m/z 544.11 (M+2) according to the molecular formula, C30H21Cl2N3O3. The structure was also supported by elemental analysis data which was ±0.4%. The other compounds are also characterized in a similar manner.7-Substituted-3-(4-(3-(4-substitutedphenyl)-4,5-dihydroisoxazol-5-yl)phenyl)-2-substituted quinazolin-4 . The pur 2, 18, 19, 23, and 27 were found to show substantial reduction in systolic blood pressure, whereas compound 24 showed significant antihypertensive activity as compared to standard drug prazosin , and when there was no further decrease, that is, ceiling effect in the blood pressure, observed at a certain dose, that dose was assumed as the minimum dose required to elicit the optimum antihypertensive activity of the synthesised compound. All titled compounds were screened to study their effect on the arterial blood pressure by using noninvasive Tail Cuff method. The results are compared with standard drug prazosin , as showprazosin . The ceiμg/Kg [P < 0.05) drop in arterial blood pressure due to selective blockade of α1-adrenergic receptor by prazosin  [ 1 showed the significant (P < 0.05) increase in SABP and DABP and without changing heart rate at 30 min prior to administration of adrenaline. Therefore this compound could serve as lead molecule for the synthesis of a series of compounds to treat hypotension. Compounds 23 and 24 showed reverse action of vasopressor effect of adrenaline with depressor reflex after 30 min, as shown in α1-adrenergic blocking property without causing reflex tachycardia. Compounds 16, 27, and 28 also exhibited moderate antagonist activity of adrenaline after 30 min. It indicates that these compounds also possess α1-adrenergic receptor blocking effect with shorter duration of action like prazosin.The injection of prazosin, 30 min prior to i.v injection of adrenaline at a dose of 3 μg/Kg , produceagonist) ; howeverα1-adrenergic blocking property was exhibited by the compound 24, having an SABP & DABP -11.6, −12.6 at 30 min. The replacement of piperazine moiety of prazosin at position-2 with substituted phenyl ring and incorporation of substituted isoxazole ring at position-3 of quinazoline moiety proved beneficial for antihypertensive activity. Our study shows that these synthesized compounds act as lead and may be useful in antihypertensive therapy.In conclusion, we have synthesized new quinazoline derivatives associated with isoxazole. These compounds showed significant antihypertensive activity with minimal side effects. The most potent


Nrp1 and Nrp2 knockout mice have disparate phenotypes, suggesting that these receptors may play a role in sorting NCCs biased towards sensory and sympathetic fates to appropriate locations.Neural crest cells (NCCs) are a transient embryonic cell type that give rise to a wide spectrum of derivatives, including neurons and glia of the sensory and autonomic nervous system, melanocytes and connective tissues in the head. Lineage-tracing and functional studies have shown that trunk NCCs migrate along two distinct paths that correlate with different developmental fates. Thus, NCCs migrating ventrally through the anterior somite form sympathetic and sensory ganglia, whereas NCCs migrating dorsolaterally form melanocytes. Although the mechanisms promoting migration along the dorsolateral path are well defined, the molecules providing positional identity to sympathetic and sensory-fated NCCs that migrate along the same ventral path are ill defined. Neuropilins (Nrp1 and Nrp2) are transmembrane glycoproteins that are essential for NCC migration. in situ hybridisation, immunohistochemistry and lineage-tracing analyses to demonstrate that neuropilins are expressed in a non-overlapping pattern within NCCs. Whereas Nrp1 is expressed in NCCs emigrating from hindbrain rhombomere 4 (r4) and within trunk NCCs giving rise to sympathetic and sensory ganglia, Nrp2 is preferentially expressed in NCCs emigrating from r2 and in trunk NCCs giving rise to sensory ganglia. By generating a tamoxifen-inducible lineage-tracing system, we further demonstrate that Nrp2-expressing NCCs specifically populate sensory ganglia including the trigeminal ganglia (V) in the head and the dorsal root ganglia in the trunk.Here we have combined Taken together, our results demonstrate that Nrp1 and Nrp2 are expressed in different populations of NCCs, and that Nrp2-expressing NCCs are strongly biased towards a sensory fate. In the trunk, Nrp2-expressing NCCs specifically give rise to sensory ganglia, whereas Nrp1-expressing NCCs likely give rise to both sensory and sympathetic ganglia. Our findings therefore suggest that neuropilins play an essential role in coordinating NCC migration with fate specification. Neural crest cells (NCCs) are transient embryonic cells that generate a wide spectrum of derivatives. Based on their origin along the anteroposterior axis, NCCs are broadly divided into different populations that give rise to a restricted set of derivatives. Cranial NCCs arise anterior to the fifth somite, and form derivates such as sensory, sympathetic, and parasympathetic ganglia, connective tissue, bone, cartilage, and muscle tendons of the head . Trunk NEven within these anatomically defined domains, NCCs can be further subdivided by additional properties, including their migration path and their developmental fate –7. For ein vivo optical imaging to analyse the behaviour of single cells, McKinney and colleagues recently found that pre-migratory NCCs in the chick lack positional identity or fate prior to delamination [There is mounting evidence from chick and mouse studies supporting the notion that NCCs migrating dorsolaterally are specified at the time of delamination , 11–13, mination , but ratmination –23.Nrp1 or Nrp2 knockout embryos identified distinct migration defects in different NCC lineages. In the head, NCCs emigrating from r4 are misguided in Nrp1 knockout embryos [Nrp2 knockout embryos [Nrp1 knockout embryos, while neurons from the trigeminal ganglia (V) are ectopically positioned in Nrp2 knockout embryos [Nrp1 knockout embryos have ectopically placed neurons in the sympathetic and sensory nervous systems, and Nrp2 knockout embryos have misplaced neurons in the sensory nervous system [The molecular cues that cause some ventrally migrating NCCs to remain near the neural tube to form sensory ganglia, while others continue migrating to adopt other fates (e.g. sympathetic ganglia) remain ill defined. In chick, the chemokine receptor CXCR4 is expressed in a specific subset of early migrating NCCs to control their migration towards a source of SDF1 around the anlagen of the sympathetic ganglia , 25. Neu embryos , while N embryos , 32. Acc embryos , 32. Thes system , 33, 34.s system . Howeverin situ hybridisation, immunohistochemistry and lineage-tracing analyses to directly compare the expression of Nrp1 and Nrp2 in migrating NCCs. Our results demonstrate that these receptors are expressed in a non-overlapping pattern within different populations of cranial and trunk NCCs. Whereas Nrp1 is expressed in cranial NCCs migrating out of hindbrain r4 and within trunk NCCs migrating towards the sympathetic and sensory ganglia, Nrp2 is preferentially expressed in cranial NCCs migrating out of r2 and in trunk NCCs migrating towards and positioned within the sensory ganglia. To determine whether Nrp2-expressing NCCs are fate-restricted towards the sensory lineage, we generated a tamoxifen-inducible Cre/LoxP tracing system to genetically label and lineage-trace this population of cells. In combination with our detailed expression analyses and previous phenotypic analyses [Here we have combined analyses , 34, ourNrp1 and Nrp2 knockout mice have non-overlapping phenotypes in different cranial NCC derivatives [in situ hybridisation on wild-type embryos between embryonic day (E) 8.5 to E9.5. By comparing the expression profiles of Nrp1 and Nrp2 with that of the pan-NCC marker Sox10, we found that neuropilins are expressed in different populations of cranial NCCs. At E8.5 and E9.0, the time at which NCCs have delaminated from the neural tube and begun to migrate into the branchial arch tissue, the expression of Nrp1 was restricted to the stream of NCCs emigrating out of r4, while Nrp2 was expressed reciprocally within NCCs emigrating out of r2  and Nrp2 has ectopically placed neurons of the trigeminal ganglia (V) [To compare expression of Nrp1 and Nrp2 in NCCs, we immunostained embryos generated by crossing Wnt1Cre mice witre[Z/EG reporterglia (V) , 32. FutNrp1 and Nrp2 knockout embryos also displayed disparate phenotypes in different trunk NCC derivatives [Sox10 and p75 over the forelimb region of E9.5 embryos. In situ hybridisation confirmed that Nrp1 was expressed within NCCs migrating in the anterior half of the somite and in non-NCC derivatives such as the dorsal aorta and intersomitic blood vessels . Consistent with our in vivo expression analysis, counterstaining of Nrp1 and Nrp2 identified three populations of trunk NCCs based on their neuropilin expression profiles: 1) Nrp1 high/Nrp2 high, 2) Nrp1 high/Nrp2 low, and 3) Nrp2 high/Nrp1 low  in cells and descendants of cells endogenously expressing Nrp2 at the time of injection. No lacZ labelling was detected without tamoxifen injection (data not shown).To permanently label and follow the fate of We next determined that intraperitoneal injection of tamoxifen at 2–4 mg per 40 g body weight to pregnant dams was sufficient to label Nrp2-expressing cells without resulting in embryonic lethality. As expected, higher doses of tamoxifen injected at E7.5 to E8.5 had detrimental effects on embryos.Having optimised the dosage of tamoxifen, we first validated that the transgene recapitulated the Nrp2 expression profile in NCCs. Tamoxifen was injected at E9.0 and E9.5, and embryos collected at E10.0. Analysis of X-gal staining in whole mount embryos demonstrated that the transgene is expressed in a highly overlapping pattern to endogenous Nrp2  ganglia that are derived from NCCs migrating out of r2  and glia (anti-Sox9 antibodies) Figure D-G. UndeOur expression analysis raised the hypothesis that ventrally migrating trunk NCCs expressing Nrp2 are biased towards neurons and glia of the sensory ganglia. To test this notion, we completed fate-mapping studies with the Nrp2-inducible lineage-tracing mouse model. Tamoxifen was administered to pregnant dams when embryos were E9.5 and E10.0, and these were later collected at E11.5, a stage at which the sensory and sympathetic ganglia have started to condense. Consistent with the staining of Nrp2 in NCCs and paraxial mesoderm at E9.5, X-gal staining identified derivatives of Nrp2-expressing cells within the DRG and skeletal muscle Figure A. Eosin Our fate-mapping results of Nrp2-expressing NCCs are strikingly similar to that presented for lineage tracing of Ngn2-expressing NCCs in mice . In the The work presented here identified molecularly distinct populations of cranial and trunk NCCs based on their expression profiles of the cell surface receptors Nrp1 and Nrp2. In combination with the recent analyses of Nrp1 and Nrp2 knockout mice –34, thisIn addition to their differential expression in restricted populations of NCCs along the anteroposterior axis, neuropilins also label divergent populations of NCCs along the dorsoventral axis Figure A. Trunk Our study identified three distinct NCC sub-types within and around the DRG that can be segregated based on their neuropilin expression profiles. While Nrp2-expressing NCCs (Nrp2-high/Nrp1-high and Nrp2-high/Nrp1-low) were traced to the DRG it will be important to complete the inverse experiment with lineage tracing of Nrp1-expressing NCCs to determine if this population is also fate-restricted.Our expression analysis also identified Nrp2 within presumptive NCC precursors in the dorsal neural tube Figure F. Given Taken together with previous reports, our studies demonstrate that neuropilins provide part of the molecular machinery to sort heterogeneous populations of ventrally migrating NCCs towards correct target regions.Wnt1Cre[Z/EG[All experimentation was approved by and conducted in accordance with the guidelines of the Animal Ethics Committee of the SA Pathology/Central Adelaide Local Health Network, and followed the Australian code of practice for the care and use of animals for scientific purposes. To obtain embryos of defined gestational ages, animals were mated in the evening, and the morning of vaginal plug formation was counted as embryonic day (E) 0.5. To lineage-trace NCCs and their derivatives throughout development, we crossed Wnt1Cre with Z/Ere[Z/EG mice.Nrp2 locus, was obtained from the BAC resource at Children’s Hospital Oakland Research Institute . A CreERT2 IRES FLAG-Kikume cassette was inserted at the initiation codon of the Nrp2 coding sequence, which is located within exon 1, by homologous recombination using a targeting vector designed for BAC recombineering [CreERT2 IRES FLAG-Kikume sequence in plasmid pBS-CreERT2 IRES FLAG-Kikume (Schwarz laboratory). The 5′ homology arm and CreERT2 IRES FLAG-Kikume fragment was then sub-cloned to the recombineering vector PL451 using XhoI restriction sites. A 3′ homology arm was amplified by PCR with a forward primer 5′-taggatccgtaagcccttcaaagtttttc-3′ and reverse primer 5′-tagcggccgcaaagaatccacacatgtgaaaag-3′, and inserted downstream of CreERT2 IRES FLAG-Kikume Neo/Kan in the PL451 vector using unique BamHI/NotI sites. This created the Nrp2- CreERT2/Kikume PL451 recombineering vector.The BAC clone RP24-250G22, containing 62 kb of DNA upstream of the first exon to 4 kb past the final coding exon of the ineering , 40. A 5Escherichia coli strain EL250 and maintained with chloramphenicol resistance. EL250 cells carrying RP24-250G22 BAC were electroporated with Nrp2-CreERT2/Kikume PL451, and homologous recombined positive clones were selected with kanamycin and chloramphenicol. The Neo/Kan cassette was removed by L-arabinose induction of flp recombinase, resulting in a Nrp2-CreERT2/Kikume BAC. Homologous recombination was confirmed by PCR and sequencing of the BAC using primers that flanked upstream of the Nrp2 5′ homology arm and downstream of the 3′ homology arm. High-quality BAC DNA was prepared using the Large Construct Kit  and analysed by pulsed field gel electrophoresis, restriction analysis, and BAC end sequencing. Prior to microinjection, the DNA elution buffer was replaced with fresh microinjection buffer  by microdialysis. The Nrp2-CreERT2/Kikume BAC was microinjected into fertilized embryos by standard pronuclear injection techniques at the Transgenic Animal Services Queensland . Genomic DNA was isolated from tail samples [CreERT2: iCre forward 5′-gagagatggatctctgtgtc-3′ and iCre reverse 5′-gacttcatcagaggtggcatc-3′, yielding a 580 bp product. Founders and offspring were subsequently genotyped with the same primer pair. One founder line was produced that transmitted the transgene in a normal mendelian inheritance pattern, with all offspring appearing grossly normal.The RP24-250G22 BAC was transformed into the recombinogenic  samples , and fouin situ hybridisation was performed as described previously [Sox10, Nrp1, and Nrp2[Whole mount and section eviously . Riboproand Nrp2, 34.Embryos were fixed in 4% paraformaldehyde in PBS. All sections were cut at a thickness of 14 μm on a CM1850 cryostat  and air-dried for 60 min before staining. For immunolabelling, cryosections or whole mount embryos were blocked in PBS containing 0.2% BSA and 0.5% Triton X-100, and stained with the indicated primary antibodies: rabbit anti-Sox9 1:1000; rabbit tyrosine hydoxylase 1:300 ; goat anti-Nrp1 1:500; rabbit anti-Nrp2 1:500 ; rabbit anti-p75-NTR 1:200 ; mouse anti β-tubulin isotype III  1:750; and chicken anti-GFP 1:1000 . Cyrosections were mounted in Prolong Gold Antifade Reagent containing DAPI . Confocal images were acquired under a confocal microscope . All images were prepared with Photoshop software .2, 5 mM EGTA, 0.01% sodium deoxycholate, 0.02% NP-40, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/ml X-gal substrate, at 37°C until blue staining was sufficient. Sections were counterstained with eosin.Embryos were fixed in PBS containing 4% paraformaldehyde. Whole embryos or cryosections were incubated in staining solution: 19 mM sodium dihydrogen phosphate, 81 mM disodium hydrogen phosphate, 2 mM MgClTamoxifen 1 g  was suspended in 5 ml of ethanol, and then dissolved in 45 ml of sunflower oil to produce a final stock at 20 mg/ml concentration. To dissolve the tamoxifen completely, the stock was sonicated in an ice bath for 5 min . Pregnant dams were injected intraperitoneally with 2 mg tamoxifen per 40 g body weight at the indicated time points.Wnt1Cre; Z/EG embryos as previously described [4 cells/well. After 4 hours, cells were fixed with 4% PFA for 10 minutes, and immunolabelled with anti-Nrp1 and anti-Nrp2 antibodies. Images were acquired on an Olympus IX81 microscope using Cell X-cellence software .Primary NCCs were isolated from E9.5 escribed . Trunk r


Rehabilitation care is fundamental to health and human dignity and a human right enshrined in the United Nations Convention on the Rights of Persons with Disabilities. The provision of rehabilitation is important for reducing the need for formal support and enabling persons with disabilities to lead an independent life. Increasingly scholars and advocacy groups voice concerns over the significant barriers facing people with disabilities in accessing appropriate and quality rehabilitation. A growing body of research highlights a “respond-need” gap in the provision of rehabilitation and assistive technologies and underscore the lack of indicators for assessing performance of rehabilitation systems and monitoring States compliance with human rights standards in rehabilitation service planning and programming. While research on human rights and health monitoring has increased exponentially over the last decade far too little attention has been paid to rehabilitation services. The proposed research aims to reduce this knowledge gap by developing a human rights based monitoring framework with indicators to support human rights accountability and performance assessment in rehabilitation.Concept mapping, a stakeholder-driven approach will be used as the core method to identify rights based indicators and develop the rehabilitation services monitoring framework. Concept mapping requires participants from various stakeholders groups to generate a list of the potential indicators through on line brainstorming, sort the indicators for conceptual similarity into clusters and rate them against predefined criteria. Multidimensional scaling and hierarchical cluster data analysis will be performed to develop the monitoring framework while bridging analysis will provide useful insights about patterns of agreement or disagreement among participants views on indicators.This study has the potential to influence future practices on data collection and measurement of compliance with human rights standards in rehabilitation service delivery and organization. The development of a valid and universally applicable set of indicators will have a profound impact on the design and implementation of evidence informed disability policies and programs as it can support countries in strengthening performance measurement through documentation of comparative information on rehabilitation care systems. Most importantly, the resulting indicators can be used by disabled people’s organizations as well as national and international institutions to define a minimal standard for monitoring and reporting progress on the implementation of the Convention on the Rights of Persons with Disabilities in the area of rehabilitation. Disability is a major issue facing global health policy. It is estimated that one billion people around the world experience some significant degree of disability with 80 % of those living in low and middle income countries . FiguresRehabilitation – defined by the World Health Organization (WHO) as “a set of measures that assist individuals who experience or are likely to experience disability to achieve and maintain optimal functioning in interaction with their environment”  p.96] – –6]. AltDemographic and epidemiological trends suggest that the number of people who could benefit from interventions aiming to reduce dependency, optimize functions and promote social participation is set to increase due to, among other factors, population growth and ageing , thus puex-novo to drive the collection of data in consistency with the legal requirements set by the CRPD in areas where data are not available. Data collection on disability (and rehabilitation) is a key obligation of States stipulated in Article 31(1) of the CRPD, under which “States Parties undertake to collect appropriate information, including statistical and research data, to enable them to formulate and implement policies to give effect to the present Convention.” [Health systems performance measurement and human rights monitoring require meaningful, objective and reliable indicators to quantify progress achieved. While it is true that the use of indicators depends critically on the availability and quality of data, in disability rights monitoring some indicators must be constructed ention.” . At presention.” –17. The ention.”  which maDriven by this absence of a unified framework for estimating disability prevalence and measuring environmental barriers to social participation, WHO is undertaking research to harmonize and advance data collection practices. This research, which is mostly guided by the WHO Disability Action Plan 2014–2021 “Better health for all people with disabilities”  aims at These aspects are sidelined in international initiatives to strengthen disability data and left out of States’ monitoring priorities. The narrowed approach to disability data collection that dictates current technocratic agendas  is inappThus far, international efforts to strengthen data collection practices have limited their focus on survey based data and screening for disability through interview questions in population surveys ignoring other types of data from population based disease registries, hospital discharge records, and facility based reviews, all of which are essential for both evidence-based service planning and human rights reporting , 29. ThiGlobal health leaders, agencies and independent expert groups have increasingly been calling upon States to intensify their efforts to collect appropriate data suitable to monitor the human rights status and situation of vulnerable groups and enhance their collaboration with specialized agencies to build the technical capacity required for health monitoring and human rights assessment. These calls have led WHO and the World Bank recently to propose a global framework for monitoring universal health coverage across the entire spectrum of essential health services . The pilCurrently we lack a validated international set of indicators, especially process indicators , that coWhich indicators are appropriate for monitoring the implementation of the rehabilitation aspects of the CRPD?Which indicators are relatively more important and feasible and how the views of researchers, rehabilitation professionals and people with disabilities differ on the subject?What does a human rights monitoring framework for rehabilitation consists of, and how can it be put in practice?Seeking to cover this knowledge gap, this paper proposes a study to develop a monitoring framework with indicators to support countries to assess progress in the implementation of the CRPD in the area of rehabilitation. Specifically the study aims to provide answers to the following research questions:The general approach to answering these questions and the methodological details of this study are explained in the Methods section. But before describing the research process, it is essential to define what a human rights based indicator is and understand some of the challenges associated with their development.In general terms indicators can be defined as numerical or textual values that provide information about a phenomenon. Indicators are essential elements of monitoring and evaluation systems that allow for assessment of progress towards the achievement of goals, enabling comparisons of different units or comparisons of the same unit at different time intervals. Unlike development and performance indicators, which follow programming principles, human rights indicators are explicitly anchored in human rights values and standards . “Such iMethodologically, the development of human rights indicators is a challenging undertaking. In academic research, scholars have proposed and used many ways to accomplish the difficult task of indicators development, ranging from traditional research methods to more sophisticated approaches . CurrentRegardless of the method being used to develop human rights indicators, one of the major challenges lies in the conceptualization of the treaty provisions (what to measure) and the operationalization of qualitative or textual information into measurable entities (how to measure). A thorough legal analysis is therefore essential to delineate the attributes of the right under examination and identify the key obligations of signatories to a specific treaty. These obligations must be then mapped onto indicators for which data are available and reliable. But as Fukuda Par notes, the criterion of measurability cannot dictate the selection of indicators since not all aspects relevant to human rights can be quantifiable or captured with measurement tools such as, people’s participation in the conduct of health affairs , 48. ThuFor developing the rehabilitation monitoring framework and identifying appropriate human rights-based indicators we propose to carry out a Group Concept Mapping study (GCM) that is underpinned by the methodological principles of structured conceptualization . GCM is Conceptually, the study will be guided by key international legal instruments pertaining to disability and rehabilitation, specifically the CRPD and particularly Article 25 , Article 26 (Rehabilitation) and Article 31 (Data collection and Statistics). An analysis of the obligations of States under the CRPD with respect to rehabilitation and its resulting framework will serve as a conceptual platform for identifying potential indicators in key human rights commitment areas . This pln = 150 participants through purposive and self-selection from the following stakeholder groups: (i) n = 50 rehabilitation professionals and service managers, (ii) n = 50 researchers with expertise in various disciplines such as global health law, health services research and rehabilitation medicine, and (iii) n = 50 people with disabilities and representatives of disabled people’s organizations. This will result in n = 27 participants sorting and rating the indicators which is above the minimum number recommended for ensuring the validity and reliability of the study results (n = 15) [There is no limit on the number of people who can participate in GCM. Based on a pooled analysis of 69 concept mapping studies Kane et al. found a range of 20–649 participants with an average of 75 participants completing the rating phase . AssuminFor recruiting experts in the study the investigators will contact international professional organizations and societies, disabled people’s organizations and individual academics and peers asking them to participate in the study and nominate additional experts for participation. A pool of participants who fulfil the criteria for participation  will then be stratified by stakeholder group. A random sample will be drawn from the experts pool for each stakeholder group and sent an email invitation to participate in the survey. Invitees who decline the invitation are replaced by other randomly drawn experts from the same expert pool.In GCM data can be collected either in paper form during pre-scheduled workshops or remotely through dedicated web based platforms. In this study data collection will be facilitated by the Concept Systems Global MAX™, a web based software specifically designed to facilitate concept mapping activities . Data peBased on the principles of GCM  a sequen“An appropriate indicator for monitoring the implementation of the CRPD in the area of rehabilitation is…” Using this question, participants will brainstorm a wide array of measures which will result in a long unfiltered list of potential indicators. After the conclusion of the brainstorming session, the research investigators will review and content analyze the brainstormed indicators to remove duplicate or other irrelevant statements and agree with stakeholder representatives on a final set of measures for further elaboration.At this stage, a focus prompt – an incomplete sentence designed to invite people to complete it – will be used to elicit participants’ best ideas and expertise on the topic. The focus prompt is: In the first data collection activity of this phase all participants will be invited by email to organize the indicators into categories in a way that makes sense to them and thus identify the conceptual building blocks for the rehabilitation monitoring framework. Participants will also rate the indicators for importance and feasibility, thus allowing patterns of agreement and/disagreement among different stakeholder groups to be identified.Data analysis will be performed using special functions of the Concept Systems Global MAX™ software . MultidiThese analyses will finally lead to a general discussion of the reasonability of the resulting monitoring framework and its implications for human rights compliance and rehabilitation systems performance assessment. Specifically, in the final phase of the GCM, stakeholder groups' representatives and experts in public health monitoring will be invited to review the clusters of indicators and the underlying relationships between them and to collectively interpret the results in a way that can drive evidence based action.Ethics review and clearance has been obtained for this project by the Ethics Commission for Northwest and Central Switzerland.This study aims to reduce the information gap in the assessment of performance of rehabilitation services and programmes for people with disabilities. It also has the potential to influence future practices on data collection and measurement of compliance with human rights standards in the delivery and organization of rehabilitation care. The development of a valid and universally applicable set of indicators will have a profound impact on decision making and implementation of evidence informed policies for rehabilitation at national and global level. Furthermore, the resulting monitoring framework can support the documentation of comparative information on national rehabilitation care systems and could be used as a minimal standard for monitoring and reporting progress on the implementation of the CRPD in the area of rehabilitation.A unique feature and major strength of the GCM is that it employs a truly participatory process for developing the indicators. Unlike other research methods the approach to data collection and elaboration is highly structured and under the control of the participants. Specifically and significantly, GCM allows stakeholders to contribute their expert knowledge by identify relevant metrics, participate in the interpretation of their perceptions and elaborate on their potential use of the resulting evaluation framework. An additional strength lies in the use of advanced statistical techniques to provide useful insights about the relationships of the ideas of different stakeholder groups, which provides rigor and enhances the credibility of information obtained during qualitative research processes. This results in visual representations of the indicators in the form of maps that are relatively easy to understand and interpret in the subsequent phases of the research process.Alternative approaches that have been used for the development or selection of indicators in health research either lack scientific credibility  or adoptAs with conducting on line surveys, GCM presents a number of challenges for the investigators to address when collecting, organizing and interpreting the data. Remote data collection in concept mapping may allow researchers to access large and geographically distributed samples of participants, but they carry with them specific limitations when compared to the traditional paper based collection of data. These include the risk of a low response rate and a resource intensive process for collecting and analyzing the data . In ordeThe results of this study are relevant to key stakeholders at both the national and international level in a variety of ways. First, the resulting indicators set will equip national public health and statistical authorities and human rights institutions with a valuable tool to monitor States’ compliance with international human rights law standards in the organization and delivery of rehabilitation services. In so doing, competent authorities may choose to either adjust their own data collection and monitoring systems to include human rights based indicators or use the new metrics separately on an ad hoc basis during periodic country performance reviews. Second, the results will be relevant to researchers, policy analysts, advocates and global health stakeholders, providing them with an evaluation framework and menu of newly created, evidence based measures that can be used to standardize the assessment of rehabilitation services and programs and facilitate comparative evaluation research.In addition to satisfying the need for human rights monitoring and performance assessment, the rehabilitation indicators will be extremely valuable for UN agencies, Member States and other international stakeholders. The metrics proposed by this study will augment existing quantitative and qualitative measures thus offering a more comprehensive evaluation toolbox that WHO and other organizations can use in their programmatic activities, as for example to assess progress in the implementation of the WHO Global Disability Action Plan. Finally, the indicators set will be based on the legal requirements of the CRPD that represents a nearly universal consensus as it has been ratified by nearly 160 countries in the world. This makes the set of indicators applicable in all contexts and thus might be an appropriate start for the development of specific human rights monitoring guidelines for Article 25 and 26 by the UN committee responsible for the oversight of the CRPD.Lastly, from a consumer’s perspective, the long standing misapprehensions surrounding the goals of rehabilitation and the contested role of health professionals in disability management make the results of this research even more important. Since people with disabilities have been traditionally excluded from decision making in rehabilitation care planning and programming, rights based indicators will prompt rehabilitation professionals and service designers to reconsider oppressing attitudes and approaches in their everyday clinical practice and pay more attention to human rights issues in patients care . More imThe results of this study will provide the global health and disability community with a preliminary set of cross-cutting indicators for assessing performance of and monitoring compliance with human rights standards in rehabilitation service planning and delivery. It will help identify gaps in quantitative data and other qualitative information that are essential for the comparative evaluation of national rehabilitation care systems. This will stimulate action toward the expansion of information gathering on rehabilitation services and further research on the development, testing and validation of indicators for various tracer conditions. The study described in this paper is a major step toward advancing the current practice of rehabilitation systems performance comparison and promoting evidence-based human rights monitoring and reporting.


The 14-3-3 (YWHA) proteins are highly conserved in higher eukaryotes, participate in various cellular signaling pathways including cell cycle regulation, development and growth. Our previous studies demonstrated that 14-3-3ε (YWHAE) is responsible for maintaining prophase | arrest in mouse oocyte. However, roles of 14-3-3ε in the mitosis of fertilized mouse eggs have remained unclear. Here, we showed that 14-3-3ε interacts and cooperates with CDC25B phosphorylated at Ser321 regulating G2/M transition of mitotic progress of fertilized mouse eggs.Disruption of 14-3-3ε expression by RNAi prevented normal G2/M transition by inhibition of MPF activity and leaded to the translocation of CDC25B into the nucleus from the cytoplasm. Overexpression of 14-3-3ε-WT and unphosphorylatable CDC25B mutant (CDC25B-S321A) induced mitotic resumption in dbcAMP-arrested eggs. In addition, we examined endogenous and exogenous distribution of 14-3-3ε and CDC25B. Endogenous 14-3-3ε and CDC25B were co-localized primarily in the cytoplasm at the G1, S, early G2 and M phases whereas CDC25B was found to accumulate in the nucleus at the late G2 phase. Upon coexpression with RFP–14-3-3ε, GFP–CDC25B–WT and GFP–CDC25B–S321A were predominantly cytoplasmic at early G2 phase and then GFP–CDC25B–S321A moved to the nucleus whereas CDC25B-WT signals were observed in the cytoplasm without nucleus accumulation at late G2 phase at presence of dbcAMP.Our data indicate that 14-3-3ε is required for the mitotic entry in the fertilized mouse eggs. 14-3-3ε is primarily responsible for sequestering the CDC25B in cytoplasm and 14-3-3ε binding to CDC25B-S321 phosphorylated by PKA induces mitotic arrest at one-cell stage by inactivation of MPF in fertilized mouse eggs. Progression through the cell-division cycle requires phosphorylation events carried out by cyclin-dependent protein kinases (CDKs) and the activation of the CDKs is a central issue of cell cycle regulation. The WEE1/MYT1 protein kinases mediate inhibitory phosphorylation of CDK1 (CDC2) on tyrosine 15 (Tyr15) and threonine 14 (Thr14) , whereasThe 14-3-3 proteins are highly conserved in eukaryotes binding to their phospho-serine and phospho-threonine-containing ligands to regulate a wide range of cellular phenomena involved in development and growth including cell cycle control, apoptosis and signal transduction –13]. Se. Se13]. Studies in HeLa cells have demonstrated that Ser323 is a primary 14-3-3 binding site in CDC25B, and this binding blocks the access of the catalytic site, thereby directly inhibiting the activity of CDC25B . Ser323 14-3-3ε existed in G1 phase of fertilized mouse eggs   at concentrations of 20 μmol (10 pl) was microinjected into the cytoplasm of fertilized mouse eggs at G1 stage (12 h after the hCG injection) to knock down endogenous 14-3-3ε, which resulted in the strongest suppression without embryo lethality caused by over-microinjection. The fertilized eggs were then cultured in M16 medium at 37°C for 15 h to allow time for RNAi-mediated targeting of mRNA, which was assessed by RT-PCR . The morphology change and cleavage rate in each group were calculated after counting and observed under a phase-contrast microscope 19 h after the injection of siRNA (31 h after the hCG injection). In the two control groups, 60.9% (no injection) and 61.7% (injection of control siRNA) of embryos had reached the two-cell stage at 31 h after the hCG injection, and there was no significant difference between the two control groups (P > 0.05). A high number of embryos microinjected with 14-3-3ε siRNA arrested at one-cell stage, and only 20% of embryos reached two-cell stage 19 h after the injection of siRNA (31 h after the hCG injection) (P <0.01 vs. no injection or control siRNA group). In addition, abnormal cleavage rate was significantly increased in the 14-3-3ε siRNA eggs (P <0.05 vs. no injection or control siRNA group). Fewer than 5% of eggs were dead after the various injection (P > 0.05) , increased initially at 16 h (28 h after the hCG injection), and reached its maximal level at 16.5 h (28.5 h after the hCG injection) and began to decrease at 17 h (29 h after the hCG injection). In contrast, the MPF activity remained at low levels at 15-17 h after 14-3-3ε siRNA injection (27-29 h after the hCG injection) (P <0.05 vs. no injection group or control siRNA injection group) , none of the mouse eggs was able to enter the M phase of mitosis because of inhibition of G2/M transition induced by dbcAMP which was similar to our previous results . Ou. Ou29]. 14-3-3ε and Cdc25b-S321A mRNAs or injected with Cdc25b-Ser321A mRNA solely increasing initially at 8 h (30 h after hCG injection), and peaking at 9 h after microinjection . In contrast, MPF activity remained at a relatively low level in the 14-3-3ε mRNA or Cdc25b-WT mRNA solely injected, co-injection of 14-3-3ε and Cdc25b-WT mRNA or two negative controls at 7-10 h after microinjection (29-32 h after hCG injection)  after It has been shown that endogenous CDC25B is mainly nuclear, but a fraction resides in the cytoplasm during the G2 phase of the cell cycle in HeLa cells . ContrarTo confirm the subcellular localization of exogenous 14-3-3ε and CDC25B, the pEGFP-CDC25B-WT and pEGFP-CDC25B-S321A plasmids were co-injected with pRFP-HA-14-3-3ε into fertilized mouse eggs, respectively, at the G1 phase (19 h after hCG injection), and then the microinjected eggs were transferred into M16 medium containing 2 mmol/l dbcAMP. Thirty eggs from early G2 and late G2 phases for each group were analyzed, respectively. As shown in Figure It has been reported that 14-3-3β and 14-3-3ε specifically bind to Ser309 of CDC25B and that mutation of CDC25B Ser309 to Ala impairs 14-3-3 binding and completely abolished the cytoplasmic localization of CDC25B . In contAn important observation made by Kornbluth and colleagues is that 14-3-3 binding to Xenopus CDC25 phosphorylated on S287 protects this mitotic phosphatase from premature dephosphorylation and activation . RemovalKip1 and downregulation of Cyclin E1, respectively, which in turn is likely to depress progression into G2/M . In. In10]. nto G2/M . Proteomnto G2/M ,44]. Th. ThKip1 In this study, we did not give the evidence that which protein phosphates dephosphorylate and activate CDC25B at early G2 phase. In the future, it is of great importance to probe the molecular mechanisms how CDC25B is dephosphorylated and activated at early G2 phase under appropriate conditions in fertilized mouse eggs. Moreover, additional functional experiments will be needed to determine the timing of 14-3-3ε absence from CDC25B in the fertilized mouse eggs.In this study, for the first time, we provide the experimental evidence for an important role of 14-3-3ε regulating G2/M transition and CDC25B distribution in the fertilized mouse eggs. The results of the study indicate that 14-3-3ε is required for the mitotic entry in the fertilized mouse eggs. The activation of MPF by CDC25B in the cytoplasm at early G2 phase is responsible for initiation of G2/M transition in the fertilized mouse eggs. These results suggest that 14-3-3ε binding to CDC25B-S321 phosphorylated by PKA blocks the mitotic division by inactivation of MPF at G1 and S phases in fertilized mouse eggs. 14-3-3ε knockdown also resulting in the mitotic arrest may due to the lost of cytoplasmic localization of CDC25B, thereby inactivating MPF initially in the cytoplasm.Kunming strain mice  were obtained from the Department of Laboratory Animals, China Medical University (CMU). All experiments were performed at CMU in accordance with the National Institutes of Health guidelines for the Care and Use of Laboratory Animals. The protocol for animal handling and the treatment procedures were reviewed and approved by the CMU Animal Care and Use Committee. Reagents, unless otherwise specified, were from Sigma.2 in air after injection with various siRNA or mRNA or plasmids.One-cell stage mouse embryos were collected and cultured according to the method described by our previous report  on the b14-3-3ε or Cdc25b mRNAs were microinjected into the nucleus or cytoplasm of one-cell embryos at G1 or S phases according to our previous report . Ty. Ty14-3-Mitotic stages  were defined as previously described. G1 phase: 10-19 h after hCG injection. S phase: 19-25 h after hCG injection. G2 phase: 25-28 h after hCG injection. M phase: 28-31 h after hCG injection . The rat14-3-3ε, the siRNA for mouse 14-3-3ε was microinjected into the cytoplasm of one-cell embryos at G1 phase. The G1 phase embryos were transferred to M2 medium, and the cytoplasm was injected with 10 pl of siRNA (20 μmol) solution. Following microinjection, embryos were transferred to M16 medium and incubated at 37°C in 5% CO2 for 15 h, and the rate of cleavage was scored with a DMI4000B inverted microscope fitted with a differential interference contrast (DIC) lens. The sequence of 14-3-3ε siRNA was 5′-UGUACAUCCAGAAUGUCACAACAGA-3′. A 25 bp duplex oligoribonucleotide of RNAi Neg Ctl LO GC (Invitrogen) was used as the control siRNA. All siRNA duplexes were resuspended in 1 ml DEPC-treated water according to the manufacturer’s instructions and stored in single use aliquots at -20°C.For targeted knockdown of 14-3-3ε primer was designed according to published mouse cDNA. 14-3-3ε (NM_009536.4), forward primer: 5′-TATCTCGAGGCCGCAATGGATGATCGGGAG -3′ and reverse primer: 5′-CGCGGATCCTACGTCTCACTGATTCTCATC-3′. β-actin (NM_007393.3), forward primer: 5′-GTGGCATCCATGAAACTACAT-3′ and reverse primer: 5′-AACGCAGCTCAGTAACAGTC-3′. The primers of 14-3-3β, 14-3-3γ, 14-3-3ζ, 14-3-3η, 14-3-3σ, and 14-3-3τ were designed as previously described ..3VO4, anProtein extracts of mouse fertilized eggs were prepared by adding approximately 200 or 300 eggs in a minimal volume of collection medium to 20 μl of protein extraction buffer  containing 1 mM phenylmethylsulfonyl fluoride and 1 μg/ml leupeptin and pepstatin. Laemmli sample buffer was added to the protein extracts, and the mixture was boiled for 5 min and subjected to a 10% SDS-PAGE gel. For immunoblotting, the fractionated proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 3% BSA in Tris-buffered saline containing 0.05% Tween 20 and probed with primary antibody against 14-3-3ε (1:200), CDC25B(1:200), β-actin (1:400) (Santa Cruz Biotechnology) and phospho-WEE1B-Ser15 antibody (1:100)  overnight at 4°C. The membrane was then incubated with a horseradish peroxidase-conjugated anti-mouse, anti-goat or anti-rabbit secondary antibody at 1:3000 (Beijing Zhongshan Biotechnology). The proteins were detected by using an enhanced chemiluminescence detection system (Piece Biotechnology).The proteins expression of 14-3-3ε and β-actin were detected by Western blot. Densitometry of bands was performed with Quantity One Software, densitometry of 14-3-3ε bands/densitometry of β-actin bands was used as quantitation of endogenous 14-3-3ε expression.Embryos were washed in PBS with 0.1% BSA, and fixed in 4% paraformaldehyde in PBS (pH7.4) for 1 h at room temperature. After being permeablilzed with 0.1% TritonX-100 in PBS at room temperature for 30 min, embryos were blocked in 5% BSA in PBS for 1 h and incubated overnight at 4°C with monoclonal mouse anti-14-3-3ε antibody  and polyclonal goat anti-CDC25B antibody  at 4°C . After being washed three times in PBS with 0.1% BSA, the embryos were incubated with FITC-conjugated goat anti-mouse secondary antibody  and TRITC-conjugated rabbit anti-goat secondary antibody  at 37°C for 1 h. Then, The DNA was stained with 25 μg/ml Hoechst33258 for 10 min at room temperature. The signals of subcellular localization of endogenous 14-3-3ε, CDC25B and DNA stained were detected by a Laser Confocal Scanning Microscope at 488 nm, 530 nm and 260 nm, respectively.x2 test or one-way analysis of variance followed by a Least Significant Difference (LSD) test was used to evaluate the differences between multiple experimental groups with SSPS software (version 13.0). A probability level of 0.05 was considered significant.All experiments were performed independently at least three times. A The authors have no competing interests.CC, BY and EW designed the project and gave final approval of the version to be published. XR, XD and XQ performed the experiments. CC and DL analyzed the data and combined to draft the manuscript. XZ contributed reagents/materials/analysis tools. All authors read and approved the final manuscript.


Androgen deprivation therapy (ADT) is the first-line treatment to metastatic prostate cancer (PCa). However, sustained expression and function of the androgen receptor (AR) gene contribute to the progression of castration resistant prostate cancers (CRPC). Additionally, tumors can adapt the PI3K/AKT survival pathway to escape ADT. Co-targeting AR and PI3K/AKT signaling has been proposed to be a more effective therapeutic means for CRPC patients. Many clinical trials are ongoing to test whether PI3K/AKT inhibitors are beneficial to PCa patients. However whether these inhibitors have any impacts on the expressions of full length AR (AR-FL) and its splice variant (AR-V7) remains unclear.Four human prostate cancer cell lines  with different genetic backgrounds were treated with five PI3K/AKT inhibitors  and or AKT siRNA. AR and AR-V7 protein and mRNA levels were measured by immunoblotting and real-time PCR assays. AR gene transcription initiation, alternative RNA splicing and AR mRNA degradation rates were also determined.PI3K/AKT inhibitors had various impacts on AR protein expressions primarily through alterations of AR gene transcription initiation and RNA splicing. However, these effects remained unchanged in the presence RNA silencing of the AKT genes.PI3K/AKT inhibitors have off-target effects on AR gene expression in prostate cancer cells, which shall be considered when applying these inhibitors to PCa patients, particularly patients under ADT treatment. Androgen deprivation therapy (ADT) is the standard treatment for metastatic prostate cancer (PCa). However, progression to castration resistant prostate cancer (CRPC) occurs to majority of patients Additionally, the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling is frequently activated in PCa and has been demonstrated to play important roles for CRPC progression and resistance to therapy-induced cell death There are several inhibitors targeting different key components of the PI3K/AKT pathway including PI3K, AKT and mTOR. However, PI3K inhibitors such as LY294002 have also been demonstrated to bind and inhibit other kinases that do not belong to the PI3K/AKT signaling In this study, four PC cell lines were treated with five PI3K/AKT inhibitors. We measured both AR mRNA and protein levels and determined AR gene transcription initiation, RNA splicing and AR mRNA degradation rates. We reported there existed complex impacts of PI3K/AKT inhibitors to AR gene expression that are independent to AKT knockdown. These off-target effects on AR gene expression need to be considered when applying PI3K/AKT inhibitors to PCa patients.LNCaP, VCaP and 22Rv1 human prostate cancer cell lines were obtained from the American Type Culture Collection . LNCaP cells were between 42–50 passages. LNCaP95 cell line was provided by Dr. Plymate (University of Washington) and was reported in previous studies Protein lysates were collected with lysis buffer  supplemented with protease and phosphatase inhibitors from Roche  as we described Real-time PCR assays were performed as we described 6 cells were washed with cold PBS, harvested and lysed on ice in 0.5% Nonidet P-40 lysis buffer  and centrifuged at 500×g for 10 min. Supernatants were removed and nuclei were incubated in reaction buffer  containing 2.5 mM NTP plus Biotin-16-UTP mix from Roche  for 45 min at 30°C. Biotinylated nascent RNA transcripts were precipitated by streptavidin beads from Gold Biotechnology  and washed with Saline Sodium Citrate buffer plus 15% formaldehyde. Precipitated RNA was eluted from streptavidin beads and used as templates for real-time PCR analyses with primers amplifying mRNAs of total AR or 18S rRNA (18rS) as described in Nuclear run-on assays were performed as described Total RNA was extracted using Purelink RNA mini kit from Invitrogen . Real-time PCR was conducted to measure AR gene RNA splicing. The relative AR-FL and AR-V7 splicing efficiency was determined by normalization with an internal PCR product within AR exons 2 and 3. Primer information was listed in t-test was carried out using GraphPad Prism (version 4) with the level of significance set at P<0.05 as *, P<0.01 as ** and P<0.001 as ***.Results are expressed as the mean ± SEM. To determine the differences among experimental groups, one-way ANOVA followed by student LY294002 is a strong competitive inhibitor to PI3K and prevents PI3K from phosphorylating and activating downstream effectors of AKT AR mRNA levels were also measured in PCa cell lines under treatments of PI3K/AKT inhibitors. The changes of AR-FL and AR-V7 mRNA levels  were conTo further investigate whether the impacts of LY294002 and AZD5363 on AR protein and mRNA levels were mediated through AKT, we performed AKT knockdown assays using pooled siRNA against all three AKT1–3 isoforms. Efficiency of AKT siRNA was shown by Western blotting . We obseThe impacts of LY294002 and AZD5363 on AR mRNA levels could be at multiple possible levels including AR gene transcription initiation, pre-mRNA splicing and AR mRNA degradation. Using nuclear run-on assay, we measured both AR and 18rS gene transcription initiation rates in presence of vehicle or PI3K/AKT inhibitors . The 18rbona fide effects of LY294002 and AZD5363 on AR-FL and AR-V7 RNA splicing, but exclude their impacts on AR gene transcription initiation, we designed three sets of primers to measure the relative splicing rates for AR-FL and AR-V7. Since AR-V7 RNA splicing is a mutually exclusive RNA splicing process of exon 3b and exon 4 of the AR pre-mRNA, one set of primers cover the exon 3 and 4 junction of the AR gene for measuring AR-FL mRNA levels, one set of primers cover exon 3 and 3b for AR-V7 mRNA levels and the other set of primers are within exons 2 and 3 for total AR mRNA levels. We showed that AR-FL RNA splicing was not affected by LY294002, AZD5363 or AKT knockdown. However, LY294002 dramatically inhibited, while AZD5363 increased AR-V7 splicing in LNCaP95 cells  and one AR-V7 regulated gene (UGT2b17). In both LNCaP and VCaP cells, AR-FL agonist R1881 stimulated PSA expression, which actions were suppressed by LY294002 . By contAberrant activation of the PI3K/AKT pathway through either genetic or epigenetic mechanisms frequently occurs in tumors, rendering this pathway an attractive therapeutic target for a wide spectrum of cancers. Many inhibitors are available to block PI3K/AKT signaling and some are under early clinical trials. However, heterogeneity of prostate cancers is a special feature, which makes it difficult to anticipate whether inhibition of PI3K/AKT signaling would benefit PCa patients. Additionally, many possible biological sequelae of PI3K/AKT inhibition may further undermine the potential therapeutic gain. This study aims to investigate whether PI3K/AKT inhibitors have any off-target effects to AR gene expression in a panel of PCa cell lines that represent prostate tumor cells with a broad range of genetic variations. It is important to document the expression profile of AR-FL and AR splice variants under treatments of PI3K/AKT inhibitors, since sustained AR expression and function are one of the critical molecular mechanisms by which prostate tumors progress into CRPC. We reported that PI3K/AKT inhibitors had complex impacts on AR gene expression that were independent to AKT, suggesting that these PI3K/AKT inhibitors could affect signaling beyond the PI3K/AKT pathway in PCa cells pending upon their genetic backgrounds. These off-target effects cautioned potential application of PI3K/AKT inhibitors to certain, if not all, PCa patient populations.Off-target effects of PI3K/AKT inhibitors had been well documented and repeatedly observed in this study. LY294002 and Wortmannin were demonstrated to bind many other kinases that were not related to PI3K/AKT signaling Both AR and PI3K/AKT signaling had been demonstrated to be important for PCa cells to develop therapy resistance. Development of inhibitors to these signaling provides new opportunities for PCa patients. The counterbalance of these opportunities is the challenge of intrapatient PCa cell heterogeneity In summary, our studies demonstrated PI3K/AKT inhibitors have various off-target effects on AR gene expressions in PCa cells with different genetic backgrounds. This information should be considered when treating PCa patients with these inhibitors.Table S1Information of antibodies and primers used in this study.(DOCX)Click here for additional data file.