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(3· 20 ml). The organic layer was washed with water and
concentrated to get 12 as white crystalline solid. (b)
Synthesis of 2-[3,4,5-trimethoxybenzoyl naphthalene-2-
yloxy]-ethanoic acid amide 14: In a round-bottomed flask
2-[1-(3,4,5-trimethoxy-benzoyl) naphthalene-2-yloxy]-eth-
yl ethanoate 10 (100 mg, 0.25 mmol) was taken in 5 ml
aqueous ammonia (30%). The reaction mixture was kept
as such for overnight at room temperature. Later the
reaction mixture was diluted with water and extracted
with ethyl acetate (3· 25 ml). The organic layer was
washed with water, dried over anhydrous sodium sulfate
and distilled off to get a creamy white solid compound 14.
(c) General procedure for the synthesis of amide deriva-
tives 15–23. Synthesis of 4-[1-(3,4,5-trimethoxybenzoyl)
d 3.75 (6H, s, 3,5-OCH3), 3.92 (3H, s, 4-OCH3), 4.77
(2H, s, OCH2), 6.2 (1H, br s, exchangeable, COOH),
7.16–7.96 (8H, m, all aromatic protons); 13C NMR
(75 MHz, CDCl3): d 56.73, 56.73, 60.86, 67.17, 108.94,
108.94, 114.66, 124.94, 124.94, 125.11, 126.06, 127.70,
128.30, 129.97, 131.77, 132.15, 133.33, 150.67, 152.60,
153.68, 170.39, 196.11. Electrospray mass (CH3CN): 397
[M+H]+, 419 [M+Na]+, 435 [M+K]+. Elemental Anal.
Calcd for C22H20O7: C, 66.66; H, 5.09. Obsd: C, 67.14;
H, 4.87.
Compound 13: Yield: 69%; mp 132–35 ꢁC; 1H NMR
(300 MHz, CDCl3): d 3.87 (6H, s, 3,5-OCH3), 4.08 (s,
4-OCH3), 6.63 (1H, d, @CHCOOH, J = 15.6 Hz), 6.72
(2H, s, OCH2), 7.53–7.58 (1H, d, CH@, J = 15.6 Hz),
7.40–7.96 (8H, m, all aromatic protons); 13C NMR
(75 MHz, CDCl3): d 14.14, 56.55, 56.55, 60.38, 60.81,
67.02, 108.0, 108.0, 114.56, 122.45, 124.58, 124.67,
127.57, 128.14, 129.54, 131.16, 131.30, 132.16, 148.13,
152.83, 153.52, 165.73, 194.61. Electrospray mass: 445
[M+Na]+. Elemental Anal. Calcd for C24H22O7: C,
68.24; H, 5.25. Obsd: C, 68.46; H, 5.48.
naphthalene-2-yloxy]-but-2-enoic
acid-3,4,5-trimethoxy
phenyl amide 23: In a 25 ml round-bottomed flask
4-[1-(3,4,5-trimethoxy-benzoyl)-naphthalen-2-yloxy]-but-2-
enoic acid 10 (100 mg, 0.171 mmol) was taken in dry
dichloromethane (10 ml). To this, EDC (41 mg,
0.21 mmol), HOBt (29 mg, 0.21 mmol) and DMAP
(5 mg) were added and stirred at room temperature for
20 min. Later 3,4,5-trimethoxy aniline (46 mg, 0.25 mmol)
was added to the reaction mixture and refluxed for 3 h. On
completion, water (10 ml) was added to it and extracted
with chloroform (3· 20 ml). The organic layer was washed
with water, dried over anhydrous sodium sulfate and
evaporated. The residue thus obtained was purified
through a silica gel column by eluting with hexane–ethyl
acetate. The desired amide was obtained at 5% ethyl
acetate–hexane fraction, which on concentration yielded a
creamy white solid compound.
Compound 14: Yield: 48%, mp 107–109 ꢁC, 1H NMR
(300 MHz, CDCl3): d 3.75 (6H, s, 3 and 5 OCH3), 3.92
(3H, s, 4-OCH3), 5.44 and 6.60 (2H, br s, NH2), 4.63
(2H, s, OCH2), 7.11–8.01 (8H, m, all the aromatic
protons). Electrospray mass (CH3CN): 418[M+Na]+.
Elemental Anal. Calcd for C22H21NO6: C, 66.83; H,
5.35; N, 3.54. Obsd: C, 67.28; H, 5.07; N, 3.22.
1
Compound 15: Yield: 62%, mp oil, H NMR (300 MHz,
CDCl3): d 0.85 (3H, br s, CH3), 1.25–1.28 (12H, br s, 6·
CH2), 3.11–3.16 (2H, br s, NCH2), 3.70 (6H, s, 3,5-
OCH3), 3.93 (3H, s, 4-OCH3), 6.43 (1H, bs, NH), 4.58
(2H, s, OCH2), 7.13–7.72 (8H, m, all the aromatic
protons). Electrospray mass (CH3CN): 508 [M+H]+.
Elemental Anal. Calcd for C30H37NO6: C, 70.98; H,
7.35; N, 2.76. Obsd: C, 70.85; H, 7.42; N, 3.04.
Compound 17: Yield: 62%, mp 143–145 ꢁC, 1H NMR
(300 MHz, CDCl3): d 3.79 (6H, s, 3,5-OCH3),3.83 (3H,
s, OCH3 of p-anisidine ring), 3.91 (3H, s, 4-OCH3),
4.81 (2H, s, OCH2), 6.83–7.94 (12H, m, all the
aromatic protons). Electrospray mass (CH3CN): 502
[M+H]+. Elemental Anal. Calcd for C29H27NO7: C,
69.45; H, 5.43; N, 2.79. Obsd: C, 69.22; H,5.83; N,
3.16.
16. Biological evaluation. Cathepsin
D inhibition assay:
The enzyme inhibition assay was performed using
spectrophotometric stop rate determination method. A
reaction cocktail containing haemoglobin solution
(10 ml, 2.5% w/v), citrate buffer (400 mM, pH 2.8,
2.5 ml) and deionised water (10 ml) was freshly pre-
pared. The pH of the contents was adjusted to 3.0 by
using dil HCl. To the 2.24 ml of reaction cocktail,
0.01 ml of different concentrations (1000, 100, 10 and
1 lg/ml, in DMSO) of test samples (pepstatin and
compounds 10–23) was added, mixed well and left for
30 min.
A blank sample was prepared by adding
0.01 ml DMSO into 2.24 ml of reaction cocktail. To
the reaction mixture (except blank where enzyme was
added after adding TCA), 0.01 ml enzyme solution
(0.1 lg/ml) was added and incubated for 10 min at
37 ꢁC. After this, 2.25 ml (5%, v/v) trichloroacetic acid
was added to stop the enzymatic reaction and the
contents were shaken well and filtered through 0.45 lM
syringe filter. The supernatant was taken and read
spectrophotometrically at 280 nm. The enzyme activity
was calculated by the formula
Compound 18: Yield: 38%, mp 152–155 ꢁC, 1H NMR
(300 MHz, CDCl3): d 3.74 (3H, s, OCH3), 3.78 (6H, s,
2· OCH3), 3.81 (6H, s, 2· OCH3), 3.90 (3H, s, 4-
OCH3), 4.89 (2H, s, OCH2), 6.68–7.89 (10H, m, all the
aromatic protons). Electrospray mass (CH3CN): 562
[M+H]+. Elemental Anal. Calcd for C31H31NO9: C,
66.33; H, 5.56; N, 2.49. Obsd: C, 65.92; H, 5.23; N,
2.94.
Compound 19: Yield: 57%, mp oil, 1H NMR
(300 MHz, CDCl3): d 0.85 (3H, t, CH3, J = 7.22 Hz),
1.32 (4H, m, CH2-CH2), 3.30 (2H, br d, N–CH2), 3.77
(3H, s, OCH3), 3.78 (6H, s, 3,5-OCH3), 3.91 (3H, s,
OCH3), 6.52 (1H, d, @CH, J = 15.2 Hz), 6.62 (2H, s,
OCH2), 6.63–7.75 (9H, m, all the aromatic protons and
CH@). Electrospray mass (CH3CN): 478 [M+H]+.
Elemental Anal. Calcd for C28H31NO6: C, 70.42; H,
6.54; N, 2.93. Obsd: C, 69.92; H, 6.23; N, 3.34.
Compound 22: Yield: 46%, mp 127–129 ꢁC, 1H NMR
(300 MHz, CDCl3): d 3.78 (3H, s, OCH3), 3.84 (6H, s,
3,5-OCH3), 3.97 (3H, s, 4-OCH3), 6.69 (2H, s, OCH2),
6.72 (1H, d, @CH, J = 15 Hz), 6.85–7.84 (13H, m, all
the aromatic protons and CH@). Electrospray mass
(CH3CN): 528 [M+H]+. Elemental Anal. Calcd for
C31H29NO7: C, 70.58; H, 5.54; N, 2.65. Obsd: C, 70.66;
H, 5.92; N, 3.09.
Units=ml enzyme activity
ðA280nmTest ꢀ A280nmBlankÞð4:51ÞðdfÞ
¼
;
ð10Þð0:01Þð1Þ
where 4.51 = total volume of assay (in ml), df = dilution
factor, 10 = time of assay (in minutes), 0.01 = volume of
enzyme (in ml), 1 = increase in 280 nm per minute as per
the unit definition. The IC50 values were calculated from
this enzyme activity value for each test compound by using
Michaelis–Menten equation in ‘Graph pad prism’
software.
17. Selected physical data: Compound 12: Yield: 78%; mp
132–135 ꢁC; IR (KBr, cmꢀ1): 3451 (OH, acid), 1713
(acid carbonyl), 1659 (ketone), 1585 and 1506 (aromat-
1
ic), 1249 and 1127 (ether); H NMR (300 MHz, CDCl3):