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1H), 6.48 (d, J = 2.0 Hz, 1H), 7.67 (d, J = 16.0 Hz, 1H), 7.74 (d, J = 8.8 Hz, 2H),
7.78 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 16.0 Hz, 1H), 8.05 (d, J = 8.8 Hz, 2H), 10.3 (s,
1H); 13C NMR (100 MHz, DMSO-d6) 24.2, 55.5, 99.1, 108.3, 114.4, 117.8, 118.2,
129.5, 130.2, 132.7, 138.5, 143.3, 160.2, 161.9, 168.9, 187.5 ppm. Compound 10:
Dark brown solid (31.2%). Rf 0.78 (Methanol/Chloroform = 1:4); 1H NMR
(400 MHz, DMSO-d6) d 3.85 (s, 3H), 6.46 (dd, J = 2.0, 8.4 Hz, 1H), 6.54 (d,
J = 2.0 Hz, 1H), 6.72 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.4 Hz, 1H), 7.61 (d,
J = 15.6 Hz, 1H), 7.87 (d, J = 15.6 Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H); 13C NMR
(100 MHz, DMSO-d6) 55.7, 98.9, 108.6, 114.2, 114.7, 116.9, 118.3, 129.8, 130.0,
130.6, 137.0, 159.8, 163.7, 186.8 ppm. Compound 11: Weak yellow solid
(64.9%). Rf 0.23 (Ethyl acetate/n-hexane = 1:3); 1H NMR (400 MHz,
CDCl3 + CD3OD) d 6.87 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 8.4 Hz, 1H), 7.08 (d,
J = 8.8 Hz, 1H), 7.44 (d, J = 15.6 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.60 (d,
J = 8.8 Hz, 1H), 7.70 (d, J = 15.6 Hz, 1H), 7.92 (d, J = 8.8 Hz, 2H); 13C NMR
(100 MHz, CDCl3 + CD3OD) 115.6, 116.2 (d, J = 21.5 Hz), 121.8, 130.0, 130.4 (d,
J = 8.2 Hz), 131.3, 131.5, 142.8, 162.1, 164.8, 189.3 ppm. Compound 12: Orange
solid (73.9%). Rf 0.24 (Ethyl acetate/n-hexane = 1:3); 1H NMR (400 MHz,
CDCl3 + CD3OD) d 6.35 (d, J = 2.0 Hz, 1H), 6.40 (dd, J = 2.0, 8.4 Hz, 1H), 7.07
(d, J = 8.8 Hz, 1H), 7.09 (d, J = 8.4 Hz, 1H), 7.47 (d, J = 15.6 Hz, 1H), 7.61 (d,
J = 8.4 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 8.8 Hz, 1H), 7.78 (d,
J = 15.6 Hz, 1H); 13C NMR (100 MHz, CDCl3 + CD3OD) 103.3, 108.6, 113.6,
116.3 (d, J = 22.3 Hz), 120.4, 130.6 (d, J = 8.4 Hz), 131.3, 132.1, 142.9, 165.1,
165.5, 166.1, 191.6 ppm. Compound 13: Yellow solid (4.0%). Rf 0.60 (Ethyl
acetate/n-hexane = 1:3); 1H NMR (400 MHz, CDCl3) d 3.88 (s, 3H), 6.49 (d,
J = 2.8 Hz, 1H), 6.50 (dd, J = 2.8, 8.4 Hz, 1H), 7.13 (dd, J = 8.8, 8.8 Hz, 2H), 7.51 (d,
J = 15.6 Hz, 1H), 7.66 (dd, J = 8.8, 8.8 Hz, 2H), 7.83 (d, J = 8.4 Hz, 1H), 7.86 (d,
J = 15.6 Hz, 1H), 13.4 (s, 1H); 13C NMR (100 MHz, CDCl3) 55.9, 101.3, 108.1,
114.3, 116.4 (d, J = 21.5 Hz), 120.3, 120.4, 130.7 (d, J = 9.0 Hz), 131.3, 131.4,
165.6, 166.5, 167.0, 191.9 ppm. Compound 14: Yellow solid (76.5%). Rf 0.11
(Ethyl acetate/n-hexane = 1:3); 1H NMR (400 MHz, CDCl3) d 3.87 (s, 3H), 6.49
(d, J = 2.4 Hz, 1H), 6.50 (dd, J = 2.4, 8.0 Hz, 1H), 6.66 (s, 1H), 7.09 (dd, J = 8.4,
8.4 Hz, 2H), 7.46 (d, J = 15.6 Hz, 1H), 7.59 (dd, J = 8.4, 8.4 Hz, 2H), 7.67 (d,
J = 15.6 Hz, 1H), 7.70 (d, J = 8.0 Hz); 13C NMR (100 MHz, CDCl3) 56.0, 99.5,
108.3, 116.2 (d, J = 21.6 Hz), 121.7, 126.9, 127.0, 130.4 (d, J = 8.9 Hz), 131.8,
133.3, 161.1, 161.6, 165.3, 191.0 ppm. Compound 15: Red solid (3.8%). 1H NMR
(400 MHz, DMSO-d6) d 1.95–1.99 (m, 4H), 3.29–3.33 (m, 4H), 6.58 (d, J = 8.8 Hz,
2H), 6.86 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 16.0 Hz, 1H), 7.61 (d, J = 16.0 Hz, 1H),
7.66 (d, J = 8.8 Hz, 2H), 8.01 (d, J = 8.8 Hz, 2H); 13C NMR (100 MHz, DMSO-d6)
24.9, 47.3, 111.7, 115.2, 115.6, 121.7, 129.7, 130.6, 130.7, 114.1, 149.2, 161.7,
186.7 ppm. Compound 16: Red solid (17.7%). Rf 0.20 (Ethyl acetate/n-
hexane = 1:3); 1H NMR (400 MHz, CDCl3) d 2.05–2.07 (m, 4H), 3.37–3.41 (m,
4H), 6.43 (s, 1H), 6.57 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 14.8 Hz, 1H), 7.56 (d,
J = 8.4 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 8.4 Hz, 1H), 7.89 (d,
J = 14.8 Hz, 1H), 13.77 (s, 1H); 13C NMR (100 MHz, CDCl3) 25.7, 47.8, 103.9,
107.6, 112.0, 114.0, 122.2, 125.0, 131.1, 146.1, 150.0, 152.3, 162.5, 166.5,
190.6 ppm. Compound 17: Orange solid (38.4%). Rf 0.53 (Ethyl acetate/n-
hexane = 1:3); 1H NMR (400 MHz, CDCl3) d 2.03–2.07 (m, 4H), 3.35–3.40 (m,
4H), 6.57 (d, J = 8.8 Hz, 2H), 7.16 (dd, J = 8.6, 8.6 Hz, 2H), 7.29 (d, J = 15.6 Hz,
1H), 7.55 (d, J = 8.8 Hz, 2H), 7.81 (d, J = 15.6 Hz, 1H), 8.04 (dd, J = 8.6, 8.6 Hz,
2H); 13C NMR (100 MHz, CDCl3) 25.7, 47.8, 112.0, 115.7 (d, J = 21.5 Hz), 115.9,
122.2, 130.9, 131.0 (d, J = 9.0 Hz), 135.6, 135.7, 146.6, 149.9, 164.2, 166.7,
189.2 ppm. Compound 18: Red solid (31.8%). Rf 0.45 (Ethyl acetate:n-
hexane = 1:3); 1H NMR (400 MHz, CDCl3) d 2.08–2.12 (m, 4H), 3.41–3.45 (m,
4H), 6.61 (d, J = 8.8 Hz, 2H), 7.29 (d, J = 15.6 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H),
7.87 (d, J = 15.6 Hz, 1H), 8.16 (d, J = 8.8 Hz, 2H), 8.37 (d, J = 8.8 Hz, 2H); 13C NMR
(100 MHz, CDCl3) 25.7, 47.9, 112.1, 115.5, 121.7, 123.9, 129.4, 131.4, 144.7,
148.4, 150.4, 189.1 ppm.
14. Representative synthetic procedure: Method 1. A mixture of THP protected 4-
hydroxyacetophenone analogue, benzaldehyde analogue and NaOH or Ba(OH)2
base in ethanol was stirred (4–24 h) at room temperature. To the mixture was
added 4 M-HCl and the reaction mixture was stirred for more 20–40 min. After
adding water, the reaction mixture was extracted with ethyl acetate and
organic layer was washed with water. Organic layer was dried over anhydrous
MgSO4 and solvent was removed under reduced pressure. (1) The residue was
treated with EtOAc and hexane, filtered and collected to give product, or (2)
The residue was applied to silica gel column chromatography to give desired
compound.
Method 2.
A mixture of acetophenone analogue (or acetyl 5-membered
heteroaromatic analogue), benzaldehyde analogue and NaOH or Ba(OH)2 base
in ethanol was stirred at room temperature. (1) To the mixture was added
water and solid formed was filtered and dried on the air. Solid was triturated
with mixed solvent, filtered and dried to give desired compound. or (2) After
adding water, the reaction mixture was extracted with ethyl acetate and
organic layer was washed with water. Organic layer was dried over anhydrous
MgSO4 and solvent was removed under reduced pressure. The residue was
applied to silica gel column chromatography to give desired compound.
Compound 1: Yellow solid (39.7%). Rf 0.11 (Ethyl acetate/n-hexane = 1:3); 1H
NMR (400 MHz, CDCl3 + CD3OD) d 3.91 (s, 3H), 6.87 (d, J = 8.8 Hz, 2H), 6.90 (d,
J = 8.4 Hz, 1H), 7.39 (d, J = 15.6 Hz, 1H), 7.50 (dd, J = 2.0, 8.4 Hz, 1H), 7.63 (d,
J = 15.6 Hz, 1H), 7.84 (d, J = 2.0 Hz, 1H), 7.92 (d, J = 8.8 Hz, 2H); 13C NMR
(100 MHz, CDCl3 + CD3OD) 56.5, 112.0, 112.4, 115.6, 120.9, 129.3, 129.8, 130.1,
131.3, 132.7, 142.4, 157.6, 162.0, 189.2 ppm. Compound 2: Yellow solid (72.6%).
Rf 0.77 (Ethyl acetate/n-hexane = 1:3); 1H NMR (400 MHz, CDCl3 + CD3OD) d
3.89 (s, 3H), 6.32 (d, J = 2.0 Hz, 1H), 6.39 (dd, J = 2.0, 8.8 Hz, 1H), 6.89 (d,
J = 8.4 Hz, 1H), 7.40 (d, J = 15.6 Hz, 1H), 7.50 (dd, J = 1.2, 8.4 Hz, 1H), 7.68 (d,
J = 15.6 Hz, 1H), 7.76 (d, J = 8.8 Hz, 1H), 7.83 (d, J = 1.2 Hz, 1H); 13C NMR
(100 MHz, CDCl3 + CD3OD) 56.5, 103.2, 108.6, 112.0, 112.5, 113.6, 119.4, 129.0,
130.0, 132.0, 132.7, 142.4, 157.8, 165.0, 166.0, 191.6 ppm. Compound 3: Yellow
solid (11.7%). Rf 0.48 (Ethyl acetate:n-hexane = 1:3); 1H NMR (400 MHz, CDCl3)
d 3.88 (s, 3H), 3.97 (s, 3H), 6.49 (d, J = 2.0 Hz, 1H), 6.50 (dd, J = 2.0, 8.8 Hz, 1H),
6.95 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 15.2 Hz, 1H), 7.55 (dd, J = 2.0, 8.8 Hz, 1H),
7.79 (d, J = 15.2 Hz, 1H), 7.84 (d, J = 8.8 Hz, 1H), 7.91 (d, J = 2.0 Hz, 1H), 13.5 (s,
1H); 13C NMR (100 MHz, CDCl3) 55.8, 56.7, 101.3, 108.0, 112.1, 112.7, 114.3,
119.4, 129.1, 130.2, 131.4, 132.9, 142.8, 158.0, 166.5, 166.9, 191.8 ppm.
Compound 4: Off-white solid (89.0%). Rf 0.51 (Ethyl acetate/n-hexane = 1:3); 1
H
NMR (400 MHz, CDCl3) d 3.97 (s, 3H), 6.95 (d, J = 8.8 Hz, 1H), 7.18 (d, J = 8.4 Hz,
1H), 7.20 (d, J = 8.4 Hz, 1H), 7.40 (d, J = 15.2 Hz, 1H), 7.55 (dd, J = 2.0, 8.8 Hz,
1H), 7.73 (d, J = 15.2 Hz, 1H), 7.90 (d, J = 2.0 Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 8.07
(d, J = 8.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) 112.1, 112.8, 116.0 (d,
J = 21.5 Hz), 120.6, 129.1, 130.1, 131.2, 131.3 (d, J = 8.9 Hz), 132.9, 143.5,
153.0,158.0, 188.7 ppm. Compound 5: Weak yellow solid (91.4%). Rf 0.47 (Ethyl
acetate/n-hexane = 1:3); 1H NMR (400 MHz, CDCl3) d 3.96 (s, 3H), 6.94 (d,
J = 8.4 Hz, 1H), 7.20 (dd, J = 3.8, 4.8 Hz, 1H), 7.31 (d, J = 15.6 Hz, 1H), 7.55 (dd,
J = 2.0, 8.4 Hz, 1H), 7.69 (d, J = 4.8 Hz, 1H), 7.75 (d, J = 15.6 Hz, 1H), 7.88 (dd,
J = 1.2, 3.8 Hz, 1H), 7.90 (d, J = 2.0 Hz. 1H); 13C NMR (100 MHz, CDCl3) 112.1,
112.7, 120.7, 128.5, 129.0, 130.1, 132.0, 132.9, 134.1, 142.5, 145.8, 158.0,
182.3 ppm. Compound 6: Ivory solid (70.8%). Rf 0.37 (Ethyl acetate/n-
hexane = 1:3); 1H NMR (400 MHz, CDCl3) d 3.96 (s, 3H), 6.60–6.62 (m, 1H),
6.93 (d, J = 8.4 Hz, 1H), 7.33 (d, J = 3.2 Hz, 1H), 7.34 (d, J = 15.6 Hz, 1H), 7.54 (d,
J = 8.4 Hz, 1H), 7.66 (s, 1H), 7.77 (d, J = 15.6 Hz, 1H), 7.90 (s, 1H); 13C NMR
(100 MHz, CDCl3) 56.6, 112.1, 112.6, 112.8, 117.6, 120.3, 129.1, 130.2, 132.9,
142.4, 146.7, 153.9, 157.9, 178.0 ppm. Compound 8: Orange solid (70.8%). Rf
0.14 (Ethyl acetate/n-hexane = 1:3); 1H NMR (400 MHz, CDCl3) d 3.90 (s, 3H),
5.46 (s, 1H), 6.47–6.49 (m, 2H), 7.16 (d, J = 8.8 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H),
7.51 (d, J = 15.6 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 8.05 (d,
J = 8.8 Hz, 1H), 8.06 (d, J = 15.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) 55.8, 99.4,
108.1, 115.8 (d, J = 21.6 Hz), 117.2, 120.2, 131.2 (d, J = 8.9 Hz), 131.4, 135.0,
141.0, 146.8, 159.5, 160.9, 189.9 ppm. Compound 9: Brown solid (15.3%). 1H
NMR (400 MHz, DMSO-d6) d 2.08 (s, 3H), 3.84 (s, 3H), 6.45 (dd, J = 2.0, 8.4 Hz,
2009, 52, 3093; Cathepsin B and L inhibitory activities were assayed in the
reaction buffer (50 mM NaOAc–HCl, 2 mM dithiothreitol (DTT), 2 mM EDTA,
pH 5.5 for cathepsin
mercaptoethanol, pH 5.5 for cathepsin L) with 20
B
and 0.1 M NaOAc–HCl, 1 mM EDTA, 0.1% b-
M substrate and 1.5 nM
l
cathepsin B or 4 nM cathepsin L. Cathepsin B and L were obtained from
Calbiochem (Darmstadt, Germany). The substrates used were RR-AMC (Sigma,
USA) for cathepsin
B and Z-FR-AMC (Sigma, USA) for cathepsin L. The
cathepsins were reductively activated by preincubation in assay buffer at
37 °C for 30 min, prior to initiating the reaction by substrate and compounds.
Afterwards, the reaction mixture was incubated at room temperature for
30 min. Fluorescence intensities were determined by using 360 nm excitation
and 450 nm emission wavelengths. CA-074, Z-FF-FMK, and nimodipine were
used as cathepsin B inhibitor, cathepsin L inhibitor, calcium channel blocker,
respectively, and all were purchased from Sigma Chemical Co., (USA).