Journal of Medicinal Chemistry
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General Procedure for the Synthesis of Acyl Hydrazine
Derivatives. A solution of hydrazine hydrate 80% (NH2NH2; 5
mmol, 0.223 mL) in methanol (25 mL) was mixed with a solution of
4,5-dihydroisoxazol derivative (4e−f; 0.8 mmol) in methanol (20 mL).
The mixture remained at reflux for 24 h. The respective generated acyl
hydrazine derivatives (6e and 6f) were recrystallized from water and
filtered. The yields were 37 and 40%, respectively.
H), 1664 (CO), 1593 (CN). 1H NMR (DMSO-d6): 3.80 (d, H1,
2JH4a−H4b = 17.3 Hz, H-4a of isoxazoline ring), 3.40 (d, H1, 2JH4b−H4a
=
17,3 Hz, H-4b of isoxazoline ring), 8.9 (s, H1, NH of hydroxamic
acid), 10.9 (s, H1, OH of hydroxamic acid). Anal. Calcd for
C11H11BrN2O3: C, 44.2; H, 3.7; N, 9.4. Found: C, 44.9; H, 3.8; N, 9.6.
3-(3-Fluorophenyl)-N-hydroxy-5-methyl-4,5-dihydroisoxazole-5-
carboxamide (5j). Yield, 82%. mp 172−174 °C. IR (cm−1): 3244 (O−
H), 1670 (CO), 1610 (CN). 1H NMR (DMSO-d6): 3.76 (d, H1,
3-Phenyl-N-hydroxy-4,5-dihydroisoxazole-5-carboxamide (5a).
Yield, 50%. mp 148−150 °C. IR (cm−1): 3194 (O−H), 1639 (C
2JH4a−H4b = 17.6 Hz, H-4a of isoxazoline ring), 3.36 (d, H1, 2JH4b−H4a
=
1
2
O), 1601 (CN). H NMR (DMSO-d6): 3.67 (dd, H1, JH4a−H4b
∼
17.6 Hz, H-4b of isoxazoline ring), 8.8 (s, H1, NH of hydroxamic
acid), 10.9 (s, H1, OH of hydroxamic acid). Anal. Calcd for
C11H11FN2O3: C, 55.5; H, 4.6; N, 11.8. Found: C, 55.9; H, 4.7; N,
12.2.
3
17.0 Hz, JH4a−H5 ∼ 11.0 Hz, H4a of isoxazoline ring), 3.53 (dd, H1,
2JH4b−H4a ∼ 17.0 Hz, 3JH4b−H5 ∼ 7.3 Hz, H-4b of isoxazoline ring), 5.00
3
3
(dd, H1, JH5−H4a ∼ 11.0 Hz, JH5−H4b ∼ 7.3 Hz, H-5 of isoxazoline
ring), 9.0 (s, H1, NH of hydroxamic acid), 11.0 (s, H1, OH of
hydroxamic acid). Anal. Calcd for C10H10N2O3: C, 58.2; H, 4.9; N,
13.6. Found: C, 57.6; H, 4.8; N, 13.6.
3-(2-Fluorophenyl)-N-hydroxy-5-methyl-4,5-dihydroisoxazole-5-
carboxamide (5k). Yield, 41%. mp 100−102 °C. IR (cm−1): 3192
(O−H), 1667 (CO), 1601 (CN). 1H NMR (DMSO-d6): 3.78 (d,
2
H1, JH4a−H4b = 17.5 Hz, H-4a of isoxazoline ring), 3.39 (d, H1,
3-(2-Chlorophenyl)-N-hydroxy-4,5-dihydroisoxazole-5-carboxa-
mide (5b). Yield, 35%. mp 105−108 °C. IR (cm−1): 3130 (O−H),
1666 (CO), 1592 (CN). 1H NMR (DMSO-d6): 3.84 (m, H2, H-
4 of isoxazoline ring), 5.27 (dd, H1, 3JH5−H4a ∼ 9.8 Hz, 3JH5−H4b ∼ 7.2
Hz, H-5 of isoxazoline ring), 6.4 (s, H1, NH of hydroxamic acid), 9.4
(s, H1, OH of hydroxamic acid). Anal. Calcd for C10H9ClN2O3: C,
49.9; H, 3.8; N, 11.6. Found: C, 46.4; H, 3.7; N, 11.4.
2JH4b−H4a = 17.5 Hz, H-4b of isoxazoline ring), 8.8 (s, H1, NH of
hydroxamic acid), 10.9 (s, H1, OH of hydroxamic acid). Anal. Calcd
for C11H11FN2O3: C, 55.5; H, 4.6; N, 11.8. Found: C, 56.1; H, 4.9; N,
12.0.
3-(2-Ethoxyphenyl)-4,5-dihydroisoxazole-5-carbohydrazide (6e).
Yield, 37%. mp 130−131 °C. IR (cm−1): 3282 and 3223 (NH2), 1676
(CO), 1589 (CN). 1H NMR (DMSO-d6): 3.60 (dd, H1,
2JH4a−H4b = 17.7 Hz, 3JH4a−H5 = 11.0 Hz, H-4a of isoxazoline ring), 3.50
3-(3-Chlorophenyl)-N-hydroxy-4,5-dihydroisoxazole-5-carboxa-
mide (5c). Yield, 65%. mp 151−153 °C. IR (cm−1): 3256 (O−H),
1
2
3
1683 (CO), 1562 (CN). H NMR (DMSO-d6): 3.66 (dd, H1,
(dd, H1, JH4b−H4a = 17.7 Hz, JH4b−H5 = 7.8 Hz, H-4b of isoxazoline
3
2JH4a−H4b ∼ 17.2 Hz, JH4a−H5 ∼ 10.8 Hz, H-4a of isoxazoline ring),
3
3
ring), 4.98 (dd, H1, JH5−H4a = 11.0 Hz, JH5−H4b = 7.8 Hz, H-5 of
isoxazoline ring), 9.43 (s, H1, NH of hydrazine), 4.35 (s, H2, NH2 of
hydrazine).
2
3
3.56 (dd, H1, JH4b−H4a ∼ 17.2 Hz, JH4b−H5 ∼ 7.8 Hz, H-4b of
isoxazoline ring), 5.02 (dd, H1, 3JH5−H4a ∼ 10.8 Hz, 3JH5−H4b ∼ 7.8 Hz,
H-5 of isoxazoline ring), 9.1 (s, H1, NH of hydroxamic acid), 11.0 (s,
H1, OH of hydroxamic acid). Anal. Calcd for C10H9ClN2O3: C, 49.9;
H, 3.8; N, 11.6. Found: C, 48.1; H, 3.9; N, 11.6.
3-(3-Ethoxyphenyl)-4,5-dihydroisoxazole-5-carbohydrazide (6f).
Yield, 40%. mp 151−152 °C. IR (cm−1): 3300 and 3251 (NH2),
1
1683 (CO), 1563 (CN). H NMR (DMSO-d6): 3.55 (dd, H1,
2JH4a−H4b = 17.4 Hz, 3JH4a−H5 = 11.0 Hz, H-4a of isoxazoline ring), 3.45
3-(4-Chlorophenyl)-N-hydroxy-4,5-dihydroisoxazole-5-carboxa-
mide (5d). Yield, 40%. mp 140−143 °C. IR (cm−1): 3259 (O−H),
2
3
(dd, H1, JH4b−H4a = 17.4 Hz, JH4b−H5 = 7.3 Hz, H-4b of isoxazoline
1
3
3
1680 (CO), 1596 (CN). H NMR (DMSO-d6): 3.64 (dd, H1,
ring), 4.95 (dd, H1, JH5−H4a = 11.0 Hz, JH5−H4b = 7.3 Hz, H-5 of
isoxazoline ring), 9.40 (s, H1, NH of hydrazine), 4.30 (s, H2, NH2 of
hydrazine).
3
2JH4a−H4b ∼ 17.2 Hz, JH4a−H5 ∼ 11.2 Hz, H-4a of isoxazoline ring),
2
3
3.50 (dd, H1, JH4b−H4a ∼ 17.2 Hz, JH4b−H5 ∼ 7.7 Hz, H-4b of
3
3
isoxazoline ring), 5.0 (dd, H1, JH5−H4a ∼ 11.2 Hz, JH5−H4b ∼ 7.7 Hz,
H-5 of isoxazoline ring), 10.0 (s, H1, OH of hydroxamic acid).
3-(2-Ethoxyphenyl)-N-hydroxy-4,5-dihydroisoxazole-5-carboxa-
mide (5e). Yield, 50%. mp 171−174 °C. IR (cm−1): 3203 (O−H),
Biological Assays. Mice. The animals used for the in vivo assay
and to obtain macrophage cells were a susceptible mice BALB/c
lineage from 5 to 8 weeks of age from the UFRJ vivarium. All animals
were maintained at constant temperature (22 °C) and with light/dark
cycles of 12 h. The care and handling of the animals were in
accordance with the standards of the Ethics Committee of UFRJ.
Cells, Parasite, and Growth Conditions. T. cruzi epimastigote
forms (Y and Dm 28c strains) are part of our trypanosomatid culture
collection and were cultivated in LIT (liver infusion tryptose)
supplemented with yeast extract 0.5%, peptone 0.5%, KCl 2%, sucrose
2%, hemin 2 mg (w/v), and 10% fetal bovine serum at 28 °C. Cellular
viability was assessed by motility using trypan blue cell dye exclusion.43
Bloodstream trypomastigote forms of T. cruzi (Y and Dm 28c strains)
were maintained by blood passage in Balb/C mice every 7 days.44
Trypomastigotes were isolated from heparinized blood by low-speed
centrifugation and collection of the parasites that swam out of the
pellet. Experimental infection was performed by i.p. injection of 104
blood trypomastigotes. The total number of parasites/milliliter was
determined by quantification in a Neubauer chamber. Intracellular
amastigotes were obtained after infection with trypomastigote forms of
macrophage cells extracted from Balb/C mice and THP-1 cells. The
highly infected cells ruptured, releasing amastigote forms of T. cruzi
after 3−5 days of infection.45 Human acute monocytic leukemia cells
lines (THP-1) were maintained in DMEM supplemented with 10%
fetal bovine serum and antibiotics (100 μg/mL of streptomycin and
100 μg/mL of penicillin G). Macrophages were obtained by injecting
thioglycollate46 (4%) i.p. into Balb/C mice, and peritoneal cells were
collected by chilled RPMI washes 4 days after injection.
1
1672 (CO), 1600 (CN). H NMR (DMSO-d6): 3.68 (dd, H1,
2JH4a−H4b = 17.5 Hz, 3JH4a−H5 = 10.6 Hz, H-4a of isoxazoline ring), 3.6
2
3
(dd, H1, JH4b−H4a = 17.5 Hz, JH4b−H5 = 7.9 Hz, H-4b of isoxazoline
3
3
ring), 4.9 (dd, H1, JH5−H4a = 10.6 Hz, JH5−H4b = 7.9 Hz, H-5 of
isoxazoline ring); 4.3 (s, H1, NH of hydroxamic acid), 9.4 (s, H1, OH
of hydroxamic acid).
3-(4-Ethoxyphenyl)-N-hydroxy-4,5-dihydroisoxazole-5-carboxa-
mide (5f). Yield, 40%. mp 155−157 °C. IR (cm−1): 3205 (O−H),
1641 (CO), 1608 (CN). 1H NMR (DMSO-d6): 3.56 (m, H2, H-
4 of isoxazoline ring), 4.93 (m, H1, H-5 of isoxazoline ring), 9.0 (s,
H1, NH of hydroxamic acid), 11.0 (s, H1, OH of hydroxamic acid).
3-[4-(Benzyloxy)phenyl]-N-hydroxy-4,5-dihydroisoxazole-5-car-
boxamide (5g). Yield, 54%. mp 100−102 °C. IR (cm−1): 3211 (O−
1
H), 1639 (CO), 1597 (CN). H NMR (DMSO-d6): 3.60 (m,
3
H2, H-4 of isoxazoline ring), 4.95 (dd, H1, JH5−H4a = 10.6 Hz,
3JH5−H4b = 7.5 Hz, H-5 of isoxazoline ring), 9.0 (s, H1, NH of
hydroxamic acid), 11.0 (s, H1, OH of hydroxamic acid). Anal. Calcd
for C17H16N2O4: C, 65.4; H, 5.1; N, 8.9. Found: C, 64.6; H, 5.1; N,
8.7.
3-(3-Chlorophenyl)-N-hydroxy-5-methyl-4,5-dihydroisoxazole-5-
carboxamide (5h). Yield, 96%. mp 168−170 °C. IR (cm−1): 3188
(O−H), 1666 (CO), 1598 (CN). 1H NMR (DMSO-d6): 3.76 (d,
2
H1, JH4a−H4b = 17.4 Hz, H-4a of isoxazoline ring), 3.38 (d, H1,
2JH4b−H4a = 17,4 Hz, H-4b of isoxazoline ring), 8.8 (s, H1, NH of
Epimastigotes Extracts. Six-day-old cultured epimastigotes, 1 × 107
cells, at the log growth phase, were harvested by centrifugation (1500g,
15 min, 4 °C) and washed three times with phosphate-buffered saline
(PBS; 150 mM NaCl, 20 mM phosphate buffer, pH 7.2). The pellet
was resuspended in extraction lysis buffer (125 mM Tris, pH 6.8, 4%
hydroxamic acid), 10.9 (s, H1, OH of hydroxamic acid). Anal. Calcd
for C11H11Cl N2O3: C, 51.9; H, 4.5; N, 11.0. Found: C, 52.1; H, 4.4;
N, 11.3.
3-(3-Bromophenyl)-N-hydroxy-5-methyl-4,5-dihydroisoxazole-5-
carboxamide (5i). Yield, 99%. mp 176−178 °C. IR (cm−1): 3188 (O−
305
dx.doi.org/10.1021/jm400902y | J. Med. Chem. 2014, 57, 298−308