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A. Thalassitis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6433–6436
yield), mp 105–106 °C, 1H NMR (CDCl3) d 1.92–2.13 (m, 4H), 3.78 (br s, 2H),
4.18 (br s, 2H), 4.80 (d, 2H, J = 5.5 Hz), 5.17 (d, 1H, J = 16.4 Hz), 5.29 (d, 1H,
J = 10.9 Hz), 5.97–6.11 (m, 1H), 7.72 (s, 1H), 8.37 (s, 1H); 13C NMR (CDCl3) d
26.1, 45.5, 47.5, 118.4, 120.1, 132.1, 138.5, 150.0, 152.9, 153.1.
nyl-(5c) and morpholinyl-(5b) substituents. No inhibition was
found for compound 10 under the reported experimental condi-
tions. Compound 9 presents higher % inhibition values, followed
by 6 and 5a. For all the compounds the LO% inhibition values are
in agreement with these on lipid peroxidation.
We evaluated the ability of the compounds to inhibit throm-
bin.37,38 Compound 9 is the most potent inhibitor, followed by 5c
and 5a which are almost equipotent. The presence of a –COPh group
as substituent at position 6- is significant for the inhibition of throm-
bin (100% inhibition). (Table 1) The presence of a piperidinyl or of a
pyrrolidinyl ring leads to similar biological responses (67% and 69%).
Our studies confirm that the presence of substituent at position
6- is an important structural feature for the antioxidant/antiin-
flammatory and anti-thrombin activity. This provides an impetus
for designing new dual acting agents using the N-substituted 6-
amino purine scaffold as the starting point.
(d) General procedure. 1,3-Dipolar cycloaddition reaction with mesityl nitrile oxide
(4). A mixture of the allylpurine 3a (122 mg, 0.5 mmol) and mesityl nitrile
oxide (4) (81 mg, 0.5 mmol) in dry toluene (15 ml) was heated under reflux for
2 days. After cooling and evaporation of the solvent the residue was
chromatographed on a column (Silica Gel No. 60, ethyl acetate) to give from
the faster moving band 9-[(3-mesityl-4,5-dihydroisoxazol-5-yl)methyl]-6-
piperidin-1-yl-9H-purine (5a) (52% yield), mp 117–118 °C (DCM–hexane), IR
(KBr): 3023, 2930, 2851, 1586 cmꢀ1 1H NMR (CDCl3) d 1.64–1.80 (m, 6H), 1.99
;
(s, 6H), 2.25 (s, 3H), 2.99 (dd, 1H, J1 = 7.7 Hz, J2 = 18.0 Hz), 3.29 (dd, 1H,
J1 = 10.9 Hz, J2 = 18.0 Hz), 4.25 (br s, 4H), 4.50 (d, 2H, J = 4.5 Hz), 5.08–5.18 (m,
1H), 6.83 (s, 2H), 8.00 (s, 1H), 8.29 (s, 1H); 13C NMR (CDCl3) d 19.3, 21.0, 24.8,
26.2, 41.3, 45.7, 46.4, 78.2, 119.4, 125.4, 128.4, 136.3, 139.0, 151.0, 152.6,
153.9, 157.5; MS (ESI): 405 [M+H]+Å, 427 [M+Na]+; Anal. Calcd for C23H28N6O: C,
68.29; H, 6.98; N, 20.78. Found: C, 68.46; H, 6.81; N, 20.65.
(e) 9-[(3-Mesityl-4,5-dihydroisoxazol-5-yl)methyl]-6-morpholin-4-yl-9H-purine
(5b) (62% yield, after refluxing for 6 days), mp 179–180 °C (DCM–ethanol), IR
(KBr): 3015, 2947, 2859, 1584 cmꢀ1 1H NMR (CDCl3) d 2.00 (s, 6H), 2.26 (s,
;
3H), 2.98 (dd, 1H, J1 = 7.7 Hz, J2 = 18.0 Hz), 3.31 (dd, 1H, J1 = 10.9 Hz,
J2 = 18.0 Hz), 3.84 (t, 4H, J = 4.8 Hz), 4.32 (br s, 4H), 4.51 (d, 2H, J = 4.5 Hz),
5.08–5.18 (m, 1H), 6.84 (s, 2H), 8.02 (s, 1H), 8.33 (s, 1H); 13C NMR (CDCl3) d
19.3, 21.0, 41.4, 45.6, 45.8, 67.1, 78.1, 119.6, 125.3, 128.5, 136.3, 139.0, 139.7,
151.2, 152.4, 153.9, 157.5; MS (ESI): 407 [M+H]+Å, 429 [M+Na]+; Anal. Calcd for
C22H26N6O2: C, 65.01; H, 6.45; N, 20.68. Found: C, 65.16; H, 6.70; N, 20.86.
It is evident that, from all the tested compounds, 9 exhibits sat-
isfactory combined antioxidant-antiinflammatory activity and
thrombin inhibitory ability (100% inhibition at 100 lM), whereas
compound 5c presents high LO inhibitory activity in combination
to significant anti-thrombin activity. Therefore the design of this
type of dual acting molecules should be further explored.
(f)
(5c) (61% yield, after refluxing for 6 days), mp 131–132 °C (DCM–hexane), IR
(KBr): 3050, 2973, 2872, 1593 cmꢀ1 1H NMR (CDCl3) d 1.92–2.12 (m, 4H), 2.02
9-[(3-Mesityl-4,5-dihydroisoxazol-5-yl)methyl]-6-pyrrolidin-1-yl-9H-purine
;
(s, 6H), 2.26 (s, 3H), 2.98 (dd, 1H, J1 = 7.7 Hz, J2 = 18.0 Hz), 3.31 (dd, 1H,
J1 = 10.9 Hz, J2 = 18.0 Hz), 3.84 (br s, 2H), 4.19 (br s, 2H), 4.50 (d, 2H, J = 3.9 Hz),
5.09–5.19 (m, 1H), 6.84 (s, 2H), 8.01 (s, 1H), 8.33 (s, 1H); 13C NMR (CDCl3) d
19.4, 21.0, 24.1, 41.4, 43.2, 45.8, 78.3, 119.8, 125.4, 128.5, 136.3, 139.0, 139.8,
150.4, 152.9, 153.2, 157.5; MS (ESI): 391 [M+H]+Å, 413 [M+Na]+; Anal. Calcd for
C22H26N6O: C, 67.67; H, 6.71; N, 21.52. Found: C, 67.46; H, 6.79; N, 21.27.
(g) 6-Chloro-9-[(3-mesityl-4,5-dihydroisoxazol-5-yl)methyl]-9H-purine (6) (72%
yield, after refluxing for 5 days), mp 135–136 °C (DCM-ethanol), IR (KBr): 3040,
References and notes
1. Jacob, R. A.; Burri, B. J. Am. J. Clin. Nutr. 1996, 63, 985.
2. Garrido, G.; Gonzalez, D.; Delporte, C.; Backhouse, N.; Quintero, G.; Nunez-
Selles, A. J.; Morales, M. A. Phytother. Res. 2001, 15, 18.
3. Weber, V.; Coudert, P.; Rubat, C.; Duroux, E.; Vallee-Goyet, D.; Gardette, D.;
Bria, M.; Albuisson, E.; Leal, F.; Gramain, J.-C.; Couquelet, J.; Madesclaire, M.
Bioorg. Med. Chem. 2002, 10, 1647.
2949, 2860, 1596 cmꢀ1 1H NMR (CDCl3) d 2.01 (s, 6H), 2.26 (s, 3H), 2.95 (dd,
;
4. Muller, K. Arch. Pharm. 1994, 327, 3.
1H, J1 = 7.7 Hz, J2 = 18.0 Hz), 3.39 (dd, 1H, J1 = 10.9 Hz, J2 = 18.0 Hz), 4.57 (dd,
1H, J1 = 5.8 Hz, J2 = 14.8 Hz), 4.65 (dd, 1H, J1 = 3.5 Hz, J2 = 14.8 Hz), 5.13–5.25
(m, 1H), 6.85 (s, 2H), 8.45 (s, 1H), 8.76 (s, 1H); 13C NMR (CDCl3) d 19.4, 21.1,
41.7, 46.6, 77.7, 125.0, 128.5, 128.6, 130.1, 136.2, 139.3, 146.4, 151.4, 152.1,
157.6; MS (ESI): 378/380 [M+Na]+Å. Anal. Calcd for C18H18ClN5O: C, 60.76; H,
5.10; N, 19.68. Found: C, 60.53; H, 5.09; N, 19.39.
5. Merlini, P. A.; Cugno, M.; Rossi, M. L.; Agricola, P.; Repetto, A.; Fetiveau, R.;
Diotallevi, P.; Canosi, U.; Mannucci, P. M.;Ardissino, D. Am. J. Cardiol. 2004, 93, 822.
6. Viles-Gonzalez, J. F.; Badimon, J. J. Int. J. Biochem. Cell Biol. 2004, 36, 25.
7. Becker, R. C.; Spencer, F. A. J. Thromb. Thrombolysis 1998, 5, 73.
8. Maraganore, J. M. Thromb. Haemostasis. 1993, 70, 208.
9. Nyby, M. D.; Sasaki, M.; Ideguchi, Y.; Wynne, H. E.; Hori, M. T.; Berger, M. E.;
Golub, M. S.; Brickman, A. S.; Tuck, M. L. J. Pharm. Exp. Therap. 1996, 278, 503.
10. Vacca, J. P. Annu. Rep. Med. Chem. 1998, 33, 81.
(h) N-{9-[(3-Mesityl-4,5-dihydroisoxazol-5-yl)methyl]-9H-purin-6-yl}benzamide
(9) (37% yield, after refluxing for 7 days, eluting first from the column), mp 90–
92 °C (DCM), IR (KBr): 3291, 3098, 1694, 1615, 1579 cmꢀ1 1H NMR (CDCl3) d 2.04
;
11. De Clercq, E. J. Med. Chem. 1995, 38, 2491.
(s, 6H), 2.25(s, 3H), 3.00(dd, 1H, J1 = 7.4 Hz,J2 = 18.0 Hz),3.38(dd, 1H, J1 = 10.8 Hz,
J2 = 18.0 Hz), 4.58 (dd, 1H, J1 = 5.4 Hz, J2 = 14.3 Hz), 4.67 (dd, 1H, J1 = 3.4 Hz,
J2 = 14.3 Hz), 5.12–5.24 (m, 1H), 6.84 (s, 2H), 7.47 (t, 2H, J = 7.9 Hz), 7.61 (t, 1H,
J = 7.9 Hz), 8.11(d, 2H, J = 7.9 Hz), 8.40 (s, 1H), 8.82 (s, 1H), 9.94 (brs, 1H); 13C NMR
(CDCl3) d 19.4, 21.0, 41.7, 46.4, 77.9, 123.7, 128.3, 128.4, 128.6, 128.7, 129.5, 130.1,
132.7, 133.6, 143.6, 144.1, 147.2, 152.6, 155.4, 171.0; MS (EI): 440 (5%) [M]+Å, 412
(3%), 395 (5%), 253 (30%), 220 (53%), 149 (80%), 105 (100%). Anal. Calcd for
C25H24N6O2: C, 68.15; H, 5.49; N, 19.09. Found: C, 68.42; H, 5.67; N, 19.30.
(i) 9-[(3-Mesityl-4,5-dihydroisoxazol-5-yl)methyl]-9H-purin-6-amine (10) (6%
yield, after refluxing for 7 days, eluting at the end of the column), mp 256–
258 °C(dec.) (DCM), IR(KBr):3415, 3342, 3050, 1601, 1573 cmꢀ1; 1H NMR (CDCl3)
d 1.98 (s, 6H), 2.25 (s, 3H), 3.00 (dd, 1H, J1 = 7.6 Hz, J2 = 18.0 Hz), 3.32 (dd, 1H,
J1 = 11.2 Hz, J2 = 18.0 Hz), 4.52 (d, 2H, J = 3.9 Hz), 5.10–5.20 (m, 1H), 5.64 (br s, 2H,
exchanged by D2O), 6.83 (s, 2H), 8.10 (s, 1H), 8.34 (s, 1H); 13C NMR (CDCl3) d 19.4,
21.1, 41.0, 45.1, 77.9, 122.4, 126.4, 128.3, 130.6, 136.9, 138.0, 149.8, 151.2, 153.5,
157.3; MS (ESI): 359 [M+Na]+Å. Anal. Calcd for C18H20N6O: C, 64.25; H, 6.00; N,
24.99. Found: C, 64.46; H, 5.74; N, 25.30.
12. De Clercq, E. Curr. Med. Chem. 2001, 8, 1543.
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36. At pH 7.4 superoxide anion reduces the tetrazolium blue into formasan blue
(kmax = 560 nm). The production of superoxide was estimated by the nitroblue
tetrazolium method.
reflux.
A mixture of compound 1 (195 mg, 1 mmol) and piperidine (2a)
(170 mg, 0.198 ml, 2 mmol) in H2O (3 ml) was refluxed for 24 h. After cooling
the mixture was extracted with DCM (2 ꢂ 10 ml) and the organic layer was
washed with H2O (2 ꢂ 10 ml), dried with MgSO4 and evaporated to give 9-allyl-
6-piperidin-1-yl-9H-purine (3a) (230 mg, 95% yield), mp 53–55 °C (54–56 °C).28
(b) 9-Allyl-6-morpholin-4-yl-9H-purine (3b) (91% yield), mp 122–124 °C (122–
124 °C).28
(c) General procedure. Amination of 9-allyl-6-chloropurine under MW irradiation.
A mixture of purine 1 (195 mg, 1 mmol) and pyrrolidine (2c) (139 mg, 0.16 ml,
2 mmol) in H2O (3 ml) was irradiated at 100 °C in a Biotage (initiator 2.0)
scientific MW oven for 7 min. After cooling and evaporation the residue was
crystallized from Et2O to give 9-allyl-6-pyrrolidin-1-yl-9H-purine (3c) (90%
37. Michaelidou, A.; Hadjipavlou-Litina, D. J.; Matsini, I.; Tsitsogianni, E. Med.
Chem. 2007, 3, 439.
38. As a substrate tosyl-Gly-Pro-Arg-pNA was used at 1 mM final concentration.
Compounds were dissolved at a final concentration of 0.1 mV in a Tris-buffer
(0.05 M Tris, 0.154 M NaCl, ethanol 5%, pH 8.0). Three minutes after the
addition of bovine thrombin (2.5 unit/mg), the reaction was ended by adding
1 ml acetic acid 50%. The absorption of the released p-nitroaniline was
measured at 405 nm.