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13. Wood, W. W.; Kremp, G.; Petry, T.; Simon, W. E. J. Synth. Commun. 1999, 29,
619. and references therein.
14. Gray, A. C. G.; Kremp, G.; Naisby, T. W.; Petry, T.; Simon, W.; Wilkin, J.; Wood,
W. W. In Synthesis and Chemistry of Agrochemicals VI, ACS Symposium Series,
2001; Vol. 800, pp 292 302.
15. Fruttero, R.; Mulatero, G.; Calvino, R.; Gasco, A. J. Chem. Soc., Chem. Commun.
1984, 323.
16. Vaglio, G. A.; Mortarini, V.; Frattini, C.; Gasco, A. Ann. Chim. Rome 1976, 66, 521.
17. Yamada, Y.; Yamamoto, T.; Okawara, M. Chem. Lett. 1975, 4, 361.
which erythromycin and ciprofloxacin are also inactive, as deter-
mined following the established clinical breakpoint P1 g/mL.
l
From this standpoint, the lowest MIC values were displayed by
derivatives 4a, 5a, and 7a, respectively bearing the 3-methyl-5-
oxazolyl, 5-methyl-3-oxazolyl, and thiophen-2-yl substituent at
the 3-position of the pyrazolylazoxycyanide scaffold R3. As far as
antifungal activity is concerned, the most active product was 7a:
this is a potent antifungal product active against almost all the spe-
cies tested. Candida parapsilosis and Candida tropicalis isolates had
higher MIC values than those of the other yeasts tested. The activ-
ity against Candida krusei, which displays intrinsic fluconazole
resistance, and against Candida glabrata, which has acquired resis-
tance to azoles, are of note (Table 2).24 When the thienyl substitu-
ent was moved to position 5 of the pyrazole ring, to give compound
8a, the level of activity remained high, MIC values being within
2 log2 dilutions. Compound 8a was particularly active against
the strains of Cryptococcus neoformans, which was susceptible to
all the pyrazolylazoxycyanide derivatives tested. When in 7a the
furan moiety was substituted for the thiophene, giving compound
9a, activity against all the species under study remained high,
declining only for the strains of C. tropicalis. The presence of the
azoxycyano function in these products appeared to be essential
to their activity: the cyano group was substituted in 7a with two
other electron-withdrawing moieties, the carbamoyl and the tosyl
moiety respectively, giving products 10 and 11, which were
inactive.
18. 4-(Cyano-NNO-azoxy)-1,5-dimethyl-3-(thiophen-2-yl)-1H-pyrazole
(7a): FC
(petroleum ether/EtOAc 7:3) gives 7a (85%) as a brown solid. Mp 131–132 °C
(EtOAc/hexane). IR (KBr DRIFT/cmÀ1): 2195 (C„N), 1460, 1326 (N(O)@N). 1H
NMR (CDCl3, 300 MHz) d (ppm): 7.72 (dd, 3J = 3.7 Hz, 4J = 1.2 Hz 1H, Th), 7.43
(dd, 3J = 5.1 Hz, 4J = 1.2 Hz, 1H, Th), 7.11 (dd, 3J = 5.1 Hz and 3.7 Hz, 1H, Th),
3.90 (s, 3H, 1-CH3-Pz), 2.66 (s, 3H, 5-CH3-Pz). 13C NMR (CDCl3, 75 MHz) d
(ppm): 141.7, 140.7, 131.3, 129.8, 128.1, 127.5, 126.4, 110.8, 37.6, 12.6. ES-MS
(70 eV, m/z): 247 (M+, 100%) 207 (MÀ40). Anal. Calcd for C10H9N5OS: C, 48.57;
H, 3.67; N, 28.32. Found: C, 48.54; H, 3.71; N, 28.14.
19. 4-(Cyano-NNO-azoxy)-1,3-dimethyl-5-(thiophen-2-yl)-1H-pyrazole (8a): general
procedure was modified as follows: acetonitrile as reaction solvent, 50 °C
reaction temperature, 1:1 nitroso/IBA molar ratio. FC (hexane/EtOAc 75:25)
gives 8a (30%) as a brown solid. Mp 92–93 °C dec. (EtOAc/hexane). IR (KBr
DRIFT/cmÀ1): 2185 (C„N), 1458, 1367 (N(O)@N). 1H NMR (CDCl3, 300 MHz) d
(ppm): 7.63 (d, J = 4.8 Hz, 1H, Th), 7.21–7.18 (m, 2H, 2 Th), 3.75 (s, 3H, 1-CH3-
Pz), 2.55 (s, 3H, 3-CH3-Pz). 13C NMR (CDCl3, 75 MHz) d (ppm): 146.0, 136.1,
131.5, 130.3, 128.5, 127.6, 125.4, 110.8, 38.1, 14.7. ES-MS (70 eV, m/z): 247
(M+) 207 (MÀ40, 100%). Anal. Calcd for C10H9N5OS: C, 48.57; H, 3.67; N, 28.32.
Found: C, 48.48; H, 3.59; N, 28.28.
20. 4-(Cyano-NNO-azoxy)-3-(furan-2-yl)-1,5-dimethyl-1H-pyrazole (9a): A mixture
of the nitroso-derivative 9 (0.57 g, 3 mmol) and cyanamide (0.15 g, 3.6 mmol)
in methylene chloride (5 mL) was treated at 0 °C with (diacetoxyiodo)benzene
(0.97 g, 3 mmol) in portions over 15 min. The reaction mixture was directly
deposited into the column and purified by FC (CH2Cl2/acetone 99.75:0.25) to
obtain 9a (0.10 g, 14%) as a yellow solid. Mp 152–154 °C dec. (EtOH). IR (KBr
DRIFT/cmÀ1): 2188 (C„N), 1453, 1344 (N(O)@N). 1H NMR (CDCl3, 300 MHz) d
(ppm): 7.56 (d, 3J = 1.5 Hz, 1H, Fu), 7.22 (d, 3J = 3.6 Hz, 1H, Fu), 6.54 (dd,
In conclusion, interesting pyrazole derivatives displaying anti-
fungal activity were developed. In particular, compounds 7a, 8a,
9a deserve further structural modulation, owing to their potent ac-
tion against C. krusei and C. glabrata, two fungal species resistant to
azoles.
3J = 3.6 Hz and 1.5 Hz, 1H, Fu), 3.95 (s, 3H, 1-CH3-Pz), 2.68 (s, 3H, 5-CH3-Pz). 13
C
NMR (CDCl3, 75 MHz) d (ppm): 144.05, 143.98, 140.5, 138.4, 126.1, 114.2,
111.7, 110.8, 37.9, 12.7. ES-MS (70 eV, m/z): 231 (M+,100%), 191 (MÀ40,). Anal.
Calcd for C10H9N5O2: C, 51.95; H, 3.93; N, 30.29. Found: C, 51.93; H, 3.75; N,
30.18.
21. Clinical and laboratory Standards Institute. 2008. M07-A7.
22. Clinical and laboratory Standards Institute. 2008. M27-A3
Supplementary data
23. In vitro activity. The yeast isolates used in this study were collected from
human sterile clinical specimens (blood and cerebrospinal fluid) to be sure of
their pathogenic role. After overnight growth on Sabouraud dextrose agar at
35 °C, each yeast isolate was suspended in 5 mL of sterile distilled water and
thoroughly vortexed to achieve a smooth suspension. Turbidity (read at a
wavelength of 530 nm) was adjusted to a McFarland standard of 0.5 with
water. This suspension (approximately 1–5 Â 106 CFU/mL) was used as
inoculum for susceptibility testing. Antifungal susceptibility testing was
Supplementary data (synthesis of intermediates 7, 8, full exper-
imental procedures, physicochemical characterization, and ele-
mental analyses for the compounds described, is available free of
performed with
a microdilution broth method using 96-well microtiter
plates. The wells of each row contained a single compound dissolved in
DMSO and diluted in RPMI 1640 medium buffered with MOPS 0.165 M and
supplemented with 2% glucose. Ten wells in the row contained ten different
scalar concentrations of the compound, ranging from 0.25 mg/L to 256 mg/L.
For each isolate, the inoculum suspension was diluted twice with RPMI 1640
medium (1:100 and then 1:20). Aliquots (0.1 mL) of the latter dilution were
then placed in 11 wells of a single row (10 wells contained the drug, the 11th
as growth control, the 12th as blank). The plates were incubated at 35 °C. An
initial visual examination was made after 24 h of incubation, and the lowest
concentration that had inhibited visible growth was recorded as the MIC. After
48 h of incubation, the panels were analyzed spectrophotometrically (after
shaking), and the MIC was recorded as the concentration that produced a 50%
reduction in turbidity compared with that of the growth-control well. The 48 h
readings were used to analyze results. Three quality control strains were
included: C. krusei ATCCÒ 6258, C. parapsilosis ATCCÒ 22019, Candida albicans
ATCCÒ 90028. Antibacterial susceptibility was tested as for yeasts, with a
microdilution broth method using 96-well microtiter plates; Mueller Hinton
broth was used instead of RPMI 1640 as medium. After overnight growth on
Mueller Hinton broth at 35 °C, each bacterial isolate was diluted to achieve a
suspension of approximately 1–5 Â 108 CFU/mL. Aliquots (0.1 mL) of the latter
dilution was then placed in 11 wells of a single row and incubated at 35 °C.
Reading was after 24 h of incubation, and the lowest concentration that had
inhibited visible growth was recorded as the MIC.
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