2380
M. Xian et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2377–2380
9. Grant, S. K.; Green, B. G.; Stiffey-Wilusz, J.; Durette, P. L.;
Shah, S. K.; Kozarich, J. W. Biochemistry 1998, 37, 4174.
10. Renodon-Corniere, A.; Boucher, J. L.; Dijols, S.; Stuehr,
D. J.; Mansuy, D. Biochemistry 1999, 38, 4663.
11. Thomas, G.; Ramwell, P. W. J. Pharm. Exp. Ther. 1991,
259, 490.
12. Ishikawa, T.; Misonou, T.; Ikeno, M.; Sato, K.; Saka-
maki, T. Biochem. Biophys. Res. Commun. 1995, 214, 145.
13. Arima, T.; Kitamura, Y.; Nishiya, T.; Takagi, H.;
Nomura, Y. Neurosci. Lett. 1996, 212, 1.
14. Arima, T.; Kitamura, Y.; Nishiya, T.; Taniguchi, T.;
Takagi, H.; Nomura, Y. Neurochem. Int. 1997, 30, 605.
15. Davidoff, F. N. Engl. J. Med. 1973, 289, 141.
16. Durant, G. J.; Roe, A. M.; Green, A. L. Prog. Med.
Chem. 1970, 7, 124.
17. Berlinck, R. G. S. Nat. Prod. Rep. 1996, 13, 377.
18. Berlinck, R. G. S. Nat. Prod. Rep. 1999, 16, 339.
19. Hogikyan, R. V.; Supiano, M. A. Am. J. Physiol. 1994,
266, E717.
2H), 4.07 (dd, J=12, 6.8 Hz, 1H), 3.43–3.59 (m, 2H); 13C
NMR (100 MHz, CDCl3) d 143.1, 142.6, 122.2, 122.0, 117.7,
117.5, 72.2, 65.5, 50.9. HR-MS calcd for C9H9N3O2 191.0695,
1
found 191.0694. Compound 15: H NMR (400 MHz, CDCl3)
d 6.80–6.88 (m, 4H), 4.24 (dd, J=11.1, 2.1 Hz, 1H), 4.08–4.12
(m, 1H), 3.98 (dd, J=11.1, 7.5 Hz, 1H), 2.94–2.96, (m, 2H),
1.49 (s, 2H); 13C NMR (100 MHz, CDCl3) d 143.4, 121.8,
121.6, 117.5, 117.3, 74.7, 66.3, 42.8. HR-MS calcd for
C9H11NO2 165.0790, found 165.0789. Compound 18: 1H
NMR (400 MHz, CDCl3) d 2.64 (t, J=5.6 Hz, 2H), 2.42–2.47
(m, 4H), 1.79 (s, 2H), 1.49–1.58 (m, 10H); 13C NMR
(100 MHz, CDCl3) d 62.3, 54.8, 40.4, 28.4, 27.8, 26.3. Com-
pund 19: 1H NMR (400 MHz, CDCl3) d 3.11 (t, J=5.6 Hz,
2H), 2.58 (t, J=5.6 Hz, 2H), 2.51 (t, J=5.2 Hz, 4H), 1.54 (b,
10H); 13C NMR (100 MHz, CDCl3) d 117.0, 57.8, 54.6, 44.5,
28.1, 27.8, 26.2. CIMS 182 (M++1).
25. Baker, T. J.; Goodman, M. Synthesis 1999, 1423.
26. The enzymatic reaction was carried out in 50 mM HEPES
buffer (pH 7.4). Incubated mixtures containing 1 mM sub-
strate, 100 mM NADPH, 4 mM FAD, 4 mM FMN, 6 mM BH4,
400 mM DTT, 100 units SOD, 50 units Catalase and 0.5 unit of
NOS in a 500 mL final volume were shaken at 37 ꢁC for the
indicated time. For nNOS, 1 mM CaCl2 and 10 mg/mL CaM
were also added. For iNOS, 1 mM magnesium acetate was
added. Reactions were quenched with 100 mL of ethanol and
the mixtures allowed to stay at room temperature for at least
1 h before addition of the Griess reagent [150 mL of 1% sulfa-
nilamide in 1 N HCl and 150 mL of 0.1% N-(1-naphthyl)ethy-
lenediamine in 1 N HCl]. Absorbances were measured at
548 nm. Calibration curves were made from identical incu-
bated mixtures without NOS and containing various con-
centration of NaNO2 to properly determine the amounts of
20. Park, J. I.; Shin, C. Y.; Lee, Y. W.; Huh, I. H.; Sohn,
U. D. J. Pharm. Pharmcol. 2000, 52, 409.
21. Zheng, Z. L.; Shimamura, K.; Anthony, T. L.; Kreulen,
D. L. Neurosci. Lett. 2000, 288, 231.
22. Clement, B.; Boucher, J. L.; Mansuy, D.; Harsdorf, A.
Biochem. Pharm 1999, 58, 439.
23. Baker, T. J.; Goodman, M. Synthesis 1999, 1423.
1
24. Compound 4: H NMR (400 MHz, CD3OD) d 4.17–4.13
(m, 1H), 4.00 (dd, J=8.4, 6.4 Hz, 1H) 3.64 (dd, J=8.0, 6.4 Hz
1H), 3.31–3.27 (m, 1H), 3.22–3.18 (m, 1H), 1.61–1.40 (m,
10H); 13C NMR (100 MHz, CD3OD) d 161.0, 109.7, 75.0,
66.3, 42.2, 36.4, 34.7, 25.1, 23.9, 23.7; EI-MS 230 (M++1),
229 (M+). Compound 1: 1H NMR (500 MHz, CD3OD) d 4.64
(bs, 5H), 4.28–4.25 (m, 1H), 4.09 (dd, J=8.5, 6.5 Hz, 1H), 3.68
(dd, J=8.5, 6.0 Hz, 1H), 3.45 (dd, J=14.5, 3.5 Hz, 1H), 3.25
(dd, J=14.5, 6.0 Hz, 1H), 1.64–1.41 (m, 10H); HR-MS calcd
ꢀ
NO2 formed in the enzymatic reactions.
27. Jousserandot, A.; Boucher, J. J.; Henry, Y.; Niklaus, B.;
Clement, B.; Mansuy, D. Biochemistry 1998, 37, 17179.
28. George, M. V.; Kierstead, R. W.; Wright, G. F. Can. J.
Chem. 1959, 37, 679.
1
for C10H19N3O2 213.1477, found 213.1480. Compound 2: H
NMR (400 MHz, CD3OD) d 6.81–6.89 (m, 4H), 4.30–4.36 (m,
2H), 4.00 (dd, J=12.4, 7.2 Hz, 1H), 3.58 (dd, J=14.4, 4.0 Hz,
1H), 3.48 (dd, J=14.4, 7.2 Hz, 1H); 13C NMR (100 MHz,
CD3OD) d 157.9, 143.2, 142.6, 121.6, 121.5, 117.2, 117.0, 71.7,
65.2, 41.7. Compound 5: 1H NMR (400 MHz, CD3OD) d
6.81–6.88 (m, 4H), 4.31–4.35 (m, 2H), 3.99 (dd, J=12, 8 Hz,
1H), 3.50–3.59 (m, 2H); 13C NMR (100 MHz, CD3OD) d
159.6, 143.3, 142.7, 121.6, 121.5, 117.2, 117.0, 71.7, 65.3, 41.4.
EI-MS 224 (M++1). Compound 6: 1H NMR (400 MHz,
CD3OD) d 3.07 (t, J=6 Hz, 2H), 2.58–2.61 (m, 6H), 1.59–1.66
(m, 10H); 13C NMR (100 MHz, CD3OD) d 157.9, 57.1, 54.0,
39.4, 27.8, 27.3, 26.2. EI-MS 215 (M++1). Compound 11: 1H
NMR (500 MHz, CDCl3) d 4.17–4.12 (m, 1H), 3.96 (dd,
J=9.0, 6.5 Hz, 1H), 3.66–3.63 (m, 1H), 3.08 (dd, J=13.5,
4.5 Hz, 1H), 3.01 (dd, J=13.5, 6.0 Hz, 1H), 1.56–1.30 (m,
10H); 13C NMR (125 MHz, CDCl3) d 116.2, 110.8, 73.8, 66.1,
48.9, 36.7, 34.7, 25.2, 24.2, 23.9; HR-MS calcd for
C10H16N2O2 196.1212, found 196.1211. Compound 14: 1H
NMR (400 MHz, CDCl3) d 6.86–6.90 (m, 4H), 4.32–4.37 (m,
29. Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper,
P. L.; Wishnok, J. S.; Tannenbaum, S. R. Anal. Biochem.
1982, 126, 131.
30. This enzymatic reaction was carried out in PBS buffer (pH
7.4) with 1 mM EDTA. Incubated mixtures containing 0.6 mM
substrate, 0.6 mM H2O2, and 80 mg/mL horseradish perox-
idase in a 500 mL final volume were shaken at 25 ꢁC for 20 min.
ꢀ
The concentration of NO2 generated in the system was
determined by addition of the Griess reagent. Absorbances
were measured at 548 nm. Calibration curves were made
from identical incubated mixtures without the enzyme and
containing various concentratioꢀn of NaNO2 to properly
determine the amounts of NO2 formed in the enzymatic
reaction.
31. Marciniak, G.; Delgado, A.; Leclerc, G.; Velly, J.; Decker,
J.; Schwartz, J. J. Med. Chem. 1989, 32, 1402.
32. Galligan, J. J.; Hess, M. C.; Miller, S. B.; Fink, G. D. J.
Pharmacol. Exp. Ther. 2001, 296, 478.