Y. Li et al. / Bioorg. Med. Chem. 17 (2009) 3536–3542
3541
Compound 5:
A
solution of 4-chlorobenzenethiol (8.7 g;
Compound 14: (oil, 42%) (CDCl3): d 0.88–0.96 (m, 3H), 1.00–2.00
(m, 4H), 2.43–2.52 (m, 4H), 2.79 (s, 3H), 3.58–3.65 (m, 1H), 3.63 (s,
3H), 5.39 (t, 1H), 7.18–7.50 (m, 8H).
Compound 15: (oil, 69%) (CDCl3): d 0.88–0.96 (m, 3H), 1.00–2.00
(m, 4H), 2.43–2.56 (m, 4H), 2.79 (s, 3H), 2.81 (t, 2H), 3.48 (t, 2H),
3.60–3.70 (m, 1H), 3.63 (s, 3H), 4.80 (s, 1H), 5.36–5.40 (m, 1H),
6.40–6.44 (d, 1H), 7.18–7.45 (m, 9H).
Compound 16: (oil, 78%) (CDCl3): d0.88–0.98 (m, 6H), 1.32–1.85
(m, 3H), 2.45–2.58 (m, 4H), 2.80 (d, 3H), 3.59–3.68 (m, 1H), 3.67 (s,
3H), 5.42–5.58 (m, 1H), 7.25 (dd, 2H), 7.45 (dd, 2H).
60 mmol) in anhydrous DMF (20 mL) was treated with triethyl-
amine (13.7 g; 135 mmol) and then stirred for 15 min. The reaction
mixture was cooled in an ice bath and ethyl 5-bromovalerate
(12.55 g; 60 mmol) was added. The reaction mixture was stirred
overnight and allowed to warm to room temperature. Ethyl acetate
(200 mL) was added and the resulting solution was washed with
5% aqueous HCl (50 mL), 5% aqueous sodium bicarbonate (50 mL)
and brine (50 mL). The organic layer was dried over anhydrous so-
dium sulfate, filtered, and the solvent was removed on a rotary
evaporator, leaving a crude product which was purified by flash
chromatography (silica gel/ethyl acetate/hexane) to give com-
pound 5 (11.57 g; 71% yield) as a white solid. 1H NMR (CDCl3): d
1.95 (t, 2H), 2.45 (t, 2H), 2.95 (t, 2H), 3.65 (s, 3H), 7.25 (s, 4H).
Compound 6: N-chlorosuccinimide (1.05 g; 7.85 mmol) was
added in small portions over 15 min to a solution of compound 5
(2.14 g; 7.85 mmol) in benzene (10 mL) and carbon tetrachloride
(10 mL) and the reaction mixture was stirred at room temperature
overnight. The mixture was filtered and the filtrate was concen-
trated using a rotary evaporator to give compound 6 (1.93 g; 80%
yield) as a yellow oil. The product was used in the next reaction
without further purification. 1H NMR (CDCl3): d 2.28 (m, 1H),
2.40 (m, 1H), 2.60 (t, 2H), 3.64 (s, 3H), 5.25 (t, 1H), 7.20 (m, 4H).
Compound 7: To a solution of compound 4 (19.6 g; 110 mmol)
and triethylamine (11.13 g; 110 mmol) in anhydrous DMF
(150 mL) was added compound 6 (30.60 g; 110 mmol) and the
reaction mixture was stirred at room temperature overnight. The
solvent was removed using a vacuum pump at 50 °C. The residue
was dissolved in ethyl acetate (800 mL) and washed with brine
(100 mL). The organic layer was dried over anhydrous sodium sul-
fate, filtered, and the solvent was removed on a rotary evaporator,
leaving a crude product which was purified by flash chromatogra-
phy (silica gel/ethyl acetate/hexane) to give compound 7 (1.35 g;
47%) as a yellow oil. 1H NMR (CDCl3): d 0.95 (m, 3H), 1.40-1.90
(m, 4H), 2.58 (m, 4H), 3.83 (m, 5H), 4.05 (m, 1H), 5.50 (m, 1H),
5.75 (s, 1H), 6.98-7.11 (m, 4H).
5.3. Enzyme assays and inhibition studies
5.3.1. Human neutrophil elastase
HNE was assayed by mixing 10
in 0.05 M sodium acetate/0.5 M NaCl buffer, pH 5.5, 10
sulfoxide and 980 L of 0.1 M HEPES buffer containing 0.5 M NaCl,
pH 7.25, in a thermostated cuvette. A 100 L aliquot was trans-
ferred to a thermostated cuvette containing 880 L 0.1 M HEPES/
0.5 M NaCl buffer, pH 7.25, and 20 L of a 70 M solution of MeO-
lL of a 70
l
M enzyme solution
l
L dimethyl
l
l
l
l
l
Suc-Ala-Ala-Pro-Val p-nitroanilide, and the change in absorbance
was monitored at 410 nm for 60 s. In a typical inhibition run,
10
10
l
l
L of inhibitor (3.5 mM) in dimethyl sulfoxide was mixed with
L of 70 M enzyme solution and 980 L 0.1 M HEPES/0.5 M
l
l
NaCl buffer, pH 7.25, and placed in a constant temperature bath.
Aliquots (100 L) were withdrawn at different time intervals and
transferred to a cuvette containing 20 L of MeOSuc-Ala-Ala-Pro-
Val p-nitroanilide (7 mM) and 880 L 0.1 M HEPES/0.5 M NaCl buf-
l
l
l
fer. The absorbance was monitored at 410 nm for 60 s.
5.3.2. Human neutrophil proteinase 3
Twenty microliters of 32.0 mM 5,50-dithio-bis(2-nitrobenzoic
acid) in dimethyl sulfoxide and 10 lL of a 3.45 lM solution of hu-
man proteinase 3 in 0.1 M phosphate buffer, pH 6.50 (final enzyme
concentration: 34.5 nM) were added to a cuvette containing a solu-
tion of 940
10 L 862.5
centration: 8.62
l
l
L 0.1 M HEPES buffer, pH 7.25, containing 0.5 M NaCl,
M inhibitor in dimethyl sulfoxide (final inhibitor con-
M) and 20 L 12.98 mM Boc-Ala-Ala-NVa-SBzl
Compound 8: A solution of compound 7 (22.98 g; 52.83 mmol)
in dry acetonitrile (100 mL) was treated with 60% w/w sodium hy-
dride (2.11 g; 52.83 mmol) at 0 °C with stirring. After the solution
was stirred for 15 min, methyl iodide (18.29 g; 128.85 mmol) was
added and the reaction mixture was stirred for 4 h at room temper-
ature. The solvent was removed and the residue was taken up in
methylene chloride (500 mL). The organic layer was washed with
water (100 mL) and then dried over anhydrous sodium sulfate. Re-
moval of the solvent left a crude product which was purified by
flash chromatography (silica gel/ethyl acetate/hexane) to give
compound 8 (7.87 g; 33%) as a yellow oil. 1H NMR (CDCl3): d
0.95 (m, 3H), 1.20–2.00 (m, 4H), 2.55 (m, 4H), 2.80 s, 3H), 3.62 (s,
3H), 3.65 (s, 3H), 5.57 (m, 1H), 7.20 (m, 4H).
l
l
l
and the change in absorbance was monitored at 410 nM for
2 min. A control (hydrolysis run) was also run under the same con-
ditions by adding 5,50-dithio-bis(2-nitrobenzoic acid) in dimethyl
sulfoxide and 10
in 0.1 M phosphate buffer, pH 6.50 (final enzyme concentration:
34.5 nM) to a cuvette containing a solution of 940 L 0.1 M HEPES
buffer, pH 7.25, containing 0.5 M NaCl, 10 L dimethyl sulfoxide
and 20 L 12.98 mM Boc-Ala-Ala-NVa-SBzl and the change in
lL of a 3.45 lM solution of human proteinase 3
l
l
l
absorbance was monitored at 410 nM for 2 min. PR 3 activity
remaining was determined using% remaining activity = (v/
vo) ꢄ 100 and is the average of duplicate or triplicate
determinations.
Compound 9: (oil, 38%) (CDCl3): d 0.88–0.96 (m, 3H), 1.30–1.80
(m, 4H), 2.40–2.60 (m, 4H), 2.80 (s, 3H), 3.62–3.72 (m, 1H), 3.65 (s,
3H), 5.38–5.53 (m, 1H), 7.00 (t, 2H), 7.52–7.62 (m, 2H).
5.3.3. Computational method
Compound 10: (oil, 71%) (CDCl3): d0.88–0.96 (m, 3H), 1.30–1.99
(m, 4H), 2.41–2.58 (m, 4H), 2.80 (s, 3H), 3.62–3.72 (m, 1H), 3.66 (s,
3H), 3.78 (s, 3H), 5.28–5.42 (m, 1H), 6.85 (d, 2H), 7.45–7.52 (m,
2H).
Compound 11: (oil, 59%) (CDCl3): d0.88–0.96 (m, 3H), 1.20–1.98
(m, 4H), 2.42–2.55 (m, 4H), 2.80 (s, 3H), 3.62–3.70 (m, 1H), 3.64 (s,
3H), 5.22–5.35 (m, 1H), 6.60 (d, 2H), 7.38 (d, 2H).
Compound 12: (oil, 97%) (CDCl3): d0.88–0.96 (m, 3H), 1.30–1.99
(m, 4H), 1.51 (s, 9H), 2.38–2.54 (m, 4H), 2.80 (s, 3H), 3.60–3.70 (m,
1H), 3.64 (s, 3H), 5.31–5.41 (m, 1H), 6.62 (s, 1H), 7.40 (m, 4H).
Compound 13: (oil, 74%) (CDCl3): d0.88–0.96 (m, 3H), 1.00–2.00
(m, 4H), 2.40–2.55 (m, 4H), 2.60–2.68 (t, 2H), 2.69–2.78 (t, 2H),
2.80 (s, 3H), 3.58–3.65 (m, 1H), 3.64 (s, 3H), 5.40 (t, 1H), 7.40–
7.50 (m, 4H), 8.05 (s, 1H).
Molecular docking simulations were performed via the SURFLEX
31
program.30 The structure of compound 8 was constructed in SYBYL
and was structurally optimized to default convergence thresholds
using the Tripos Force Field32 and Gasteiger–Marsili partial atomic
charges.33 Receptor models were prepared for HNE and PR 3 using
the 1HNE34 and 1FUJ25 crystal structures, respectively. These struc-
tures were protonated in SYBYL, stripped of all water molecules and
bound ligands, and electrostatically represented with Gasteiger–
Marsili charges.
Acknowledgments
This work was supported in part by a grant from the National
Institutes of Health (HL 57788).