3700
O. Bruno et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3696–3701
(m, 2H, H2 + NH, 1H disappears with D2O), 7.28–7.40
disappears with D2O), 3.98–4.27 (m, 2H, CH2N), 4.80 (br
s, 2H, NH2, disappears with D2O), 5.08–5.18 (m, 1H,
CHOH), 7.26–7.45 (m, 5H, Ar), 7.46 (s, 1H, H3). Anal.
calcd for C12H12N4O: C, 63.15; H, 5.30; N, 24.55. Found:
C, 62.99; H, 5.25; N, 24.60.
(m, 5H, Ar), 7.45 (s, 1H, H6). Anal calcd for C17H20N4O:
C, 68.89; H, 6.80; N, 18.90. Found: C, 69.02; H, 6.90; N,
19.11.
Morpholin-4-yl-(2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]-
pyrazol-7-yl)methanone (4e): Yield: 65%. mp 202–204 ꢁC.
IR (KBr) cmꢀ1: 3230 (NH), 1610 (CO). 1H NMR (CDCl3):
d 3.64–3.77 (m, 8H, 4CH2-morph.), 3.96 (near t, 1H, H3),
4.54 (near t, 1H, H3), 5.40–5.52 (m, 1H, H2) 5.58 (br s, 1H,
NH, disappears with D2O), 7.28–7.40 (m, 5H, Ar), 7.44 (s,
1H, H6). Anal calcd for C16H18N4O2: C, 64.41; H, 6.08; N,
18.78. Found: C, 64.10; H, 6.09; N, 18.89.
15. 5-Amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-car-
boxamide (9). Preparation: To a solution of compound 8
(2.28 g, 10 mmol) in absolute ethanol (10 mL) 2M NaOH
(10 mL) was added, and the mixture was refluxed for 2 h.
After cooling, the white solid obtained was filtered,
washed with water and recrystallized from ethanol. Yield:
65%. mp 228 ꢁC. IR (KBr) cmꢀ1: 3389 (OH), 3299, 3169
(NH2), 1643 (CO) 1H NMR (CDCl3): d 3.80–3.90 (m, 2H,
CH2N), 4.80–4.90 (m, 1H, CHOH), 5.61 (d, J = 4, 1H,
OH, disappears with D2O), 5.98 (s, 2H, NH2, disappears
with D2O), 6.60 (br s, 2H, CONH2, disappears with D2O),
7.10–7.35 (m, 5H, Ar), 7.56 (s, 1H, H3). Anal. calcd for
C12H14N4O2: C, 58.53; H, 5.73; N, 22.75 Found: C, 58.55;
H, 5.66; N, 22.94.
Azepan-1-yl-(2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyra-
zol-7-yl)methanone (4f): Yield: 74%. mp 142–143 ꢁC. IR
1
(KBr) cmꢀ1: 3260 (NH), 1600 (CO). H NMR (CDCl3): d
1.6–1.85 (m, 8H, 4CH2-hex), 3.50–3.80 (m, 4H, 2CH2N-
hex), 3.98 (near t, 1H, H3), 4.55 (near t, 1H, H3), 5.40–5.55
(m, 2H, H2 + NH, 1H disappears with D2O), 7.26–7.41 (m,
5H, Ar), 7.49 (s, 1H, H6). Anal calcd for C18H22N4O: C,
69.65; H, 7.14; N, 18.05. Found: C, 69.45; H, 7.06; N,
17.89.
16. 2-Phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole
7-car-
boxamide (10). Preparation: Starting from compound 9
(2.46 g, 10 mmol), the same procedure for compound 2 was
used. Yield: 41%. mp 248–249 ꢁC. IR (KBr) cmꢀ1: 3450,
2-Phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carbox-
ylic acid benzylamide (4g): Yield: 48%. mp 232–234 ꢁC. IR
1
1
(KBr) cmꢀ1: 3300 (NH), 1630 (CO). H NMR (CDCl3): d
3156 (NH, NH2), 1665 (CO). H NMR (CDCl3) d: 3.67
3.98 (near t, 1H, H3), 4.50–4.65 (m, 3H, H3 + CH2Ar), 5.35
(br s, 1H, NH, disappears with D2O), 5.40–5.54 (m, 1H,
H2), 5.90 (br s, 1H, NHCO, disappears with D2O), 7.27–
7.40 (m, 10H, Ar), 7.48 (s, 1H, H6). Anal calcd for
C19H18N4O: C, 71.68; H, 5.70; N, 17.60. Found: C, 71.55;
H, 5.76; N, 17.69.
(4-Benzyl-piperazin-1-yl)-(2-phenyl-2,3-dihydro-1H-imi-
dazo[1,2-b]pyrazol-7-yl)methanone (4h): Yield: 75%. mp
161–162 ꢁC (dec). IR (KBr) cmꢀ1: 3270 (NH), 1590 (CO).
1H NMR (CDCl3): d 2.49–2.51 (m, 4H, 2CH2-pip), 3.55 (s,
2H, CH2Ar), 3.68–3.80 (m, 4H, 2CH2N-pip), 3.97 (near t,
1H, H3), 4.55 (near t, 1H, H3), 5.34–5.50 (m, 2H, H2 + NH,
1H disappears with D2O), 7.30–7.40 (m, 10H, Ar), 7.45 (s,
1H, H6). Anal calcd for C23H25N5O: C, 71.29; H, 6.50; N,
18.02. Found: C, 70.97; H, 6.57; N, 18.01.
(near t, 1H, H3), 4.46 (near t, 1H, H3), 5.31 (near t, 1H, H2),
6.70 (br s, 1H, NH, disappears with D2O), 6.95 (s, 2H,
CONH2, disappears with D2O), 7.21-7.40 (m, 5H, Ar), 7.56
(s, 1H, H6). Anal. calcd for C12H12N4O. H2O: C, 58.53; H
5.70; N, 22.75. Found: C, 58.63; H, 5.40; N, 22.67.
17. Giordano, C.; Lucente, G.; Masi, A.; Paglialunga Paradisi, M.;
Sansone, A.; Spisani, S. Bioorg. Med. Chem. 2006, 14, 2642.
8. (a) Neutrophil preparation: Neutrophils were obtain from
the blood of healthy subjects and cells were purified
employing standard techniques, and were resuspended in
Krebs-Ringer-phosphate containing 0.1% w/v glucose
(KRPG), pH 7.4.; (b) Random locomotion and chemotaxis
were evaluated using a 48-well microchemotaxis chamber
(BioProbe, Milan Italy), estimating the distance in micro-
metres which the leading edge of the cell migrated.; (c) O2ꢀ
production was measured by superoxide dismutase-inhibit-
able reduction of ferricytochrome c, modified for micro-
plated-based assays.; (d) Release of neutrophil granule
enzymes was evaluated by determining lysozyme activity,
modified for microplate-based assays. Lysozyme was quan-
tified nephelometrically by the rate of lysis of a cell wall
suspension of Micrococcus lysodeikticus.; (e) Receptor
binding assays: In competition experiments, carried out to
determine Ki values, 10 nM of [3H]fMLP was incubated
with 100 ll of human neutrophils (5 · 106) and at least 6–8
different concentrations of the examined compounds at
37 ꢁC for 15 min. Non-specific binding was measured in the
presence of 10 lM fMLP and was about 20% of total
binding. Bound and free radioactivity was separated by
rapid filtration through Whatman GF/C glass-filters, which
were washed with ice-cold buffer. The filter-bound radio-
activity was measured by scintillation spectrometer with an
efficiency of 57%. (f) Preparation of the tested compounds:
10ꢀ2 M in dimethylsulfoxide (DMSO) of fMLP-OMe and
the tested compounds were diluted in buffer before use. At
the concentrations used, DMSO did not interfere with any
of the biological assays performed.
11. Shioiri, T.; Ninomiya, K.; Yamada, S. J. Am. Chem. Soc.
1972, 94, 6203.
12. Bondavalli, F.; Botta, M.; Bruno, O.; Ciacci, A.; Corelli,
F.; Fossa, P.; Lucacchini, A.; Manetti, F.; Martini, C.;
Menozzi, G.; Mosti, L.; Ranise, A.; Schenone, S.; Tafi, A.;
Trincavelli, M. L. J. Med. Chem. 2002, 45, 4875.
13. 2-Phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole
(7).
Method b: Starting from compound 6 (2.03 g, 10 mmol),
the same procedure for compound 2 was used. Yield: 65%.
Method i: Compound 3 was heated at 190 ꢁC until
complete development of CO2. The crude was dissolved
in CHCl3, washed twice with saturated NaHCO3 solution
and dried (MgSO4). After solvent evaporation, the pale
solid obtained was recrystallized from absolute ethanol.
Yield: 74%. mp 136 ꢁC. IR (KBr) cmꢀ1: 3170 (NH). 1H
NMR (CDCl3) : d 3.89 (near t, 1H, H3), 4.12 (br s, 1H,
NH, decreases with D2O), 4.45 (near t, 1H, H3), 5.25 (near
t, 1H, H2), 5.33 (d, J = 1.8, 1H, H7), 7.16–7.40 (m, 6H,
5Ar + H6). Anal. calcd for C11H11N3: C, 71.33; H, 5.99; N,
22.69. Found: C, 71.49; H, 5.87; N, 22.73.
14. 5-Amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-car-
bonitrile (8). Preparation: To a solution of 2-hydrazino-1-
phenylethanol (6.08 g, 40 mmol) in absolute ethanol
(50 mL), ethoxymethylenemalononitrile (4.88 g, 40 mmol)
was added and the reaction mixture was refluxed for 6 h.
The solvent was concentrated until 50% of the initial
volume and cooled. The yellow solid obtained was filtered
and recrystallized from absolute ethanol. Yield: 63%. mp
180–181 ꢁC. IR (KBr) cmꢀ1: 3438, 3339, 3199 (OH, NH2),
2223 (CN). 1H NMR (CDCl3): d 3.15 (br s, 1H, OH,
19. Spisani, S.; Fabbri, E.; Muccinelli, M.; Cariani, A.; Barbin,
L.; Trotta, F.; Dovigo, L. Rheumatology 2001, 40, 794.
20. Tiberghien, F.; Wenandy, T.; Loor, F. J. Antibiotic
(Tokyo) 2000, 53, 509.
21. Spisani, S.; Turchetti, M.; Varani, K.; Falzarano, S.;
Cavicchioni, G. Eur. J. Pharmacol. 2003, 469, 13.
22. Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmacol. 1973,
22, 3099.