6
K. R. A. Abdellatif et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
7.95 (d, J ¼ 8.7 Hz, 1H, fluorophenyl H-6), 13.25 (s, 1H, OH, 152–154 ꢀC; IR (KBr disk) 3408–3324 (OH, NH), 3066 (C–H
D2O exchangeable); 13C NMR (CDCl3) ꢀ 15.1, 105.3, 107.2, aromatic), 2917 (C–H aliphatic), 1602 (C ¼ N); 1H NMR
117.1, 118.6, 125.9, 129.1, 130.8, 131.9, 143.3, 145.2, 166.1, (DMSO-d6) ꢀ 2.47 (s, 3H, SCH3), 3.29 (dd, J ¼ 16.5, 8.7 Hz,
168.7, 192.4; MS (m/z): 288 (M+, 97%) 137 (100%); Anal Calcd 1H, pyrazole H-4), 3.65 (dd, J ¼ 16.5, 10.2 Hz, 1H, pyrazole H’-
for C16H13FO2S: C, 66.65; H, 4.54; found: C, 66.84; H, 4.92.
4), 5.01 (dd, J ¼ 10.2, 8.7 Hz, 1H, pyrazole H-5), 6.64–6.80 (m,
1-(4-Fluoro-2-hydroxyphenyl)-3-(4-methoxylphenyl)prop-2-
3H, fluorophenyl H-3, H-5, H-6), 7.13–7.33 (m, 4H, methysulfa-
en-1-one (5d). Yield (82%); yellow crystals; mp 122–124 ꢀC; IR nylphenyl H-2, H-3, H-5, H-6), 10.52 (s, 1H, NH, D2O
(KBr disk) 3430 (OH), 3073 (C–H aromatic), 2966 (C–H exchangeable), 13.25 (s, 1H, OH, D2O exchangeable); 13C
1
aliphatic), 1641 (CO); H NMR (CDCl3) ꢀ 3.88 (s, 3H, OCH3), NMR (DMSO-d6) ꢀ13C NMR (DMSO-d6) ꢀ15.7, 40.4, 60.3,
6.67–6.74 (m, 2H, fluorophenyl H-3, H-5), 6.99 (d, J ¼ 8.7 Hz, 103.5, 106.9, 113.9, 126.3, 127.2, 132.1, 134.6, 138.1, 155.7,
2H, methoxyphenyl H-3, H-5), 7.37 (d, J ¼ 8.7 Hz, 2H, methox- 161.3, 165.1; MS (m/z): 302 (M+, 0.12%) 80 (100%); Anal Calcd
yphenyl H-2, H-6), 7.56 (d, J ¼ 15.6 Hz, 1H, COCH ¼ CH), 7.91 for C16H15FN2OS: C, 63.56; H, 5.00; N, 9.26; found: C, 63.19; H,
(d, J ¼ 15.6 Hz, 1H, COCH ¼ CH), 7.94 (d, J ¼ 8.7 Hz, 1H, 5.03; N, 9.39.
fluorophenyl H-6), 13.18 (s, 1H, OH, D2O exchangeable); 13C
3-(4-Fluoro-2-hydroxyphenyl)-5-(4-methoxylphenyl)-4,5-dihy-
NMR (CDCl3) ꢀ 55.4, 105.3, 107.3, 113.8, 116.7, 120.2, 121.3, dro-1H-pyrazole (6d). Yield (70%); yellow crystals; m.p. 115–
130.1, 135.8, 145.7, 160.0, 166.2, 168.8, 192.5; MS (m/z): 272 117 ꢀC; IR (KBr disk) 3409–3321 (OH, NH), 3056 (C–H
(M+, 5%) 80 (100%); Anal. Calcd for C16H13FO3: C, 70.58; H, aromatic), 2932 (C–H aliphatic), 1609 (C ¼ N); 1H NMR
4.81; found: C, 70.89; H, 4.49.
(DMSO-d6) ꢀ 3.30 (dd, J ¼ 16.5, 8.7 Hz, 1H, pyrazole H-4),
3.67 (dd, J ¼ 16.5, 10.2 Hz, 1H, pyrazole H’-4), 3.93 (s, 3H,
OCH3), 5.13 (dd, J ¼ 10.2, 8.7 Hz, 1H, pyrazole H-5), 6.67–6.86
(m, 3H, fluorophenyl H-3, H-5, H-6), 7.18–7.48 (m, 4H,
methysulfanylphenyl H-2, H-3, H-5, H-6), 10.55 (s, 1H, NH,
General method for preparation of 3,5-diaryl-4,5-dihydro-1H-
pyrazoles(6a–d)
To a solution of the appropriate chalcone 5a, 5b, 5c or 5d D2O exchangeable), 13.31 (s, 1H, OH, D2O exchangeable); 13C
(1.0 mmol) in ethanol (10 mL), hydrazine hydrate (1.2 mmol, NMR (DMSO-d6) ꢀ40.3, 55.8, 60.9, 103.8, 106.6, 111.5, 113.4,
0.06 g) was added, and the reaction mixture was refluxed for 2– 117.3, 128.2, 135.1, 152.7, 159.6, 162.6, 165.0; MS (m/z): 286
4 h. The reaction was monitored every 60 min interval on TLC (M+., 0.03%) 80 (100%); Anal Calcd for C16H15FN2O2: C, 67.12;
plates using chloroform:methanol (9:1 V/V). The excess solvent H, 5.28; N, 9.78; found: C, 66.87; H, 5.17; N, 9.95.
was removed in vacuum, and the residue was washed with water,
crystallized from absolute ethanol/methanol mixture (1:1) to give Biological evaluation
the respective dihydropyrazoles 6a–d for which physical and
Animals
spectral data are listed below.
5-(2,3-Dimethoxyphenyl)-3-(4-fluoro-2-hydroxyphenyl)-4,5-
Swiss albino mice 20–25 g and Wistar rats (150–175 g) were
dihydro-1H-pyrazole (6a). Yield (78%); white crystals; m.p. 96– obtained from the National Research Center, Cairo, Egypt) were
98 ꢀC; IR (KBr disk) 3427–3373 (OH, NH), 3079 (C–H used throughout the study and were kept at controlled conditions
aromatic), 2938 (C–H aliphatic), 1609 (C ¼ N); 1H NMR (temperature 23 2 ꢀC, humidity 60 10%) and a 12/12 h light/
(CDCl3) ꢀ 3.09 (dd, J ¼ 16.5, 8.7 Hz, 1H, pyrazole H-4), 3.56 dark cycle. All procedures relating to animal care and treatments
(dd, J ¼ 16.5, 10.2 Hz, 1H, pyrazole H’-4), 3.89 (s, 3H, OCH3), were conducted in accordance with protocols approved by the
3.90 (s, 3H, OCH3), 5.23 (ddd, J ¼ 10.2, 8.7, 0.9 Hz, 1H, pyrazole Research Ethical Committee of Faculty of Pharmacy Beni-Suef
H-5), 6.60 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H, dimethoxyphenyl H-6), University (2014-Beni-Suef, Egypt).
6.72 (dd, J ¼ 7.8, 1.8 Hz, 1H, dimethoxyphenyl H-4), 6.91
(dd, J ¼ 7.8, 7.8 Hz, 1H, dimethoxyphenyl H-5), 7.04–7.27 (m, Material
3H, fluorophenyl H-3, H-5, H-6), 10.55 (s, 1H, NH, D2O
DPPH radical was obtained from Sigma-Aldrich (St. Louis, MO).
exchangeable), 13.27 (s, 1H, OH, D2O exchangeable); 13C NMR
Ascorbic was purchased from Merck (Rahway, NJ). All the used
(DMSO-d6) ꢀ 40.9, 56.2, 56.7, 60.8, 103.5, 106.8, 112.5, 114.4,
chemicals and solvents were of analytical grade.
118.0, 124.5, 129.7, 136.2, 146.7, 152.8, 159.0, 161.9, 164.3; MS
(m/z): 316 (M+, 0.2%) 271 (100%); Anal Calcd for C17H17FN2O3:
C, 64.55; H, 5.42; N, 8.86; found: C, 64.69; H, 5.57; N, 8.86.
DPPH radical scavenging activity
5-(3,4-Dimethoxyphenyl)-3-(4-fluoro-2-hydroxyphenyl)-4,5-
dihydro-1H-pyrazole (6b). Yield (69%); white crystals; m.p. 144–
146 ꢀC; IR (KBr disk) 3393–3331 (OH, NH), 3071 (C–H
aromatic), 2934 (C–H aliphatic), 1605 (C ¼ N); 1H NMR
(DMSO-d6) ꢀ 3.22 (dd, J ¼ 6.5, 8.7 Hz, 1H, pyrazole H-4), 3.61
(dd, J ¼ 16.5, 10.2 Hz, 1H, pyrazole H’-4), 3.87 (s, 3H, OCH3),
3.88 (s, 3H, OCH3), 4.97 (dd, J ¼ 10.2, 8.7 Hz, 1H, pyrazole H-5),
6.61 (dd, J ¼ 8.4, 2.4 Hz, 1H, dimethoxyphenyl H-2), 6.72 (dd,
J ¼ 8.4, 2.4 Hz, 1H, dimethoxyphenyl H-6), 6.78 (d, J ¼ 8.4 Hz,
1H, dimethoxyphenyl H-5), 6.91–6.97 (m, 2H, fluorophenyl H-3,
H-5), 7.15–7.27 (m, 1H, fluorophenyl H-6), 10.53 (s, 1H, NH,
D2O exchangeable), 13.31 (s, 1H, OH, D2O exchangeable);13C
NMR (DMSO-d6) ꢀ41.4, 55.9, 56.0, 62.5, 103.6, 106.8, 111.0,
112.2, 114.4, 119.3, 129.8, 134.8, 149.2, 152.7, 159.1, 161.9,
164.3; MS (m/z): 316 (M+., 35%) 151 (100%); Anal Calcd for
C17H17FN2O3: C, 64.55; H, 5.42; N, 8.86; found: C, 64.82; H,
5.61; N, 9.11.
Antioxidant activity was evaluated as the scavenging activity of
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical essentially as men-
tioned before16–18. In a brief, 2 mL of DPPH solution (0.2 mmol/L
in ethanol) was incubated with different concentrations of the test
compounds. The reaction mixtures were shaken and wrapped in
aluminum foil and kept at room temperature for 30 min in dark.
The spectrophotometric measurements at 517 nm were done
under dim light. All data are presented as mean standard
deviation (SD) (n ¼ 3). The IC50 (the concentration of sample
necessary to cause 50% inhibition of DPPH radical scavenging
activity) was calculated for each compound. Ascorbate (Vitamin
C) was used as a positive antioxidant control.
Yeast-based antioxidant screening assay
The yeast-based biological assay detects antioxidant activities of
test compounds against physiologically relevant oxidants. DPPH
radical scavenging activity method provides only an indication of
the ability of a compound to scavenge oxidants19. For this reason,
3-(4-Fluoro-2-hydroxyphenyl)-5-(4-methylsulfanylphenyl)-4,5-
dihydro-1H-pyrazole (6c). Yield (65%); pale yellow crystals; m.p.