Synthesis and antimicrobial activity of new 3-(1-R-3(5)-methyl-4-nitroso-1H-5(3)-pyrazolyl)-5-methylisoxazoles

Article abstract of DOI:10.1016/S0968-0896(00)00211-X

A number of new 3-(1-R-3(5)-methyl-4-nitroso-1H-5(3)-pyrazolyl)-5-methylisoxazoles 6a-g (7b-f) were synthesized and tested for antibacterial and antifungal activity. Some of these compounds displayed antifungal activity at non-cytotoxic concentrations. Derivative 6c was 9 times more potent in vitro than miconazole and 20 times more selective against C. neoformans. 6c was also 8- and 125-fold more potent than amphotericin B and fluconazole, respectively. None of the compounds was active against bacteria. Preliminary structure-activity relationship (SAR) studies showed that the NO group at position 4 of the pyrazole ring is essential for the activity. Lipophilicity of the pyrazole moiety, N-alkyl chain length and planarity of the two heterocyclic rings appear to play a decisive role in modulating cytotoxicity and antifungal activity. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00211-X

Bioorganic & Medicinal Chemistry 8 (2000) 2719±2728
Synthesis and Antimicrobial Activity of New 3-(1-R-3(5)-Methyl-
4-nitroso-1H-5(3)-pyrazolyl)-5-methylisoxazoles
Enrico Aiello, a,* Stefania Aiello, a Francesco Mingoia, b Alessia Bacchi, c
Giancarlo Pelizzi, c Chiara Musiu, d Maria Giovanna Setzu, d Alessandra Pani, d
Paolo La Colla d and Maria Elena Marongiu d
a
Á
Dipartimento Farmacochimico Tossicologico e Biologico, Universita degli Studi, Via Archira® 32, 90123 Palermo, Italy
bIstituto di Chimica e Tecnologia dei Prodotti Naturali-CNR, Via Ugo La Malfa 153, 90146 Palermo, Italy
Á
Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Universita degli Studi di Parma,
c
Parco Area delle Scienze 17A, I-43100 Parma, Italy
Á
d
Dipartimento Biologia sperimentale, Sezione Microbiologia, Universita degli Studi di Cagliari, Cittadella Universitaria,
S.S.554, Km 4.5, 09042 Monserrato, Cagliari, Italy
Received 12 January 2000; accepted 20 July 2000
AbstractÐA number of new 3-(1-R-3(5)-methyl-4-nitroso-1H-5(3)-pyrazolyl)-5-methylisoxazoles 6a±g (7b±f) were synthesized and
tested for antibacterial and antifungal activity. Some of these compounds displayed antifungal activity at non-cytotoxic con-
centrations. Derivative 6c was 9 times more potent in vitro than miconazole and 20 times more selective against C. neoformans. 6c
was also 8- and 125-fold more potent than amphotericin B and ¯uconazole, respectively. None of the compounds was active against
bacteria. Preliminary structure±activity relationship (SAR) studies showed that the NO group at position 4 of the pyrazole ring is
essential for the activity. Lipophilicity of the pyrazole moiety, N-alkyl chain length and planarity of the two heterocyclic rings appear
to play a decisive role in modulating cytotoxicity and antifungal activity. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
M. canis at 6.25 mg/mL.2 Moreover, the occurrence of
4-nitrosopyrazoles of biological interest is indicated by
the large number of reports dealing with their numerous
biological activities,3 10 but despite the existing interest on
this class of compounds, virtually nothing is known about
the possible structure±activity relationships (SARs).
Cryptococcosis is a disseminated infection caused by the
ubiquitous yeast Cryptococcus neoformans.1 Since this
fungus is primarily a pathogen for immunocompro-
mised patients, individuals with AIDS are at high risk of
cryptococcosis. In the setting of AIDS, cryptococcal
infections are particularly dicult to treat because anti-
fungal therapy does not usually eradicate the infection.
Given the high incidence of relapse after initial anti-
fungal therapy, current management of C. neoformans
infections in patients with AIDS includes lifelong suppres-
sive therapy with antifungal drugs. Therefore, the need for
novel antifungal agents for cryptococcosis and other
opportunistic infections is apparent in light of the sig-
ni®cant problems associated with current drugs and makes
the development of new drug entities all the more urgent.
These ®ndings and the increased interest on new anti-
fungal agents led us to synthesize, to de®ne the struc-
tures of title compounds and to test in vitro their
antifungal properties in order to identify the structural
features essential for the activity of 4-nitrosopyrazoles.
Chemistry
Derivatives 4b±d, 5b±d, 6a±g and 7b±f were prepared by
reaction of b-diketones 1 and 2 with suitable commer-
cial hydrazino derivatives 3a±g according to modi®ed
methods previously reported by us.2 The synthetic
pathway (Scheme 1) allows the formation of a mixture
of two isomers that were easily separated by ordinary
silica gel column chromatography. Yields, mp, selected
reaction conditions to obtain the two isomeric series in
In our previous papers, we reported that some nitroso-
pyrazolyl-isoxazoles inhibited the growth of the fungus
*Corresponding author. Tel.: +39-91-616-3454; fax: +39-91-617-
7955; e-mail: eaiello@unipa.it
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00211-X

2720
E. Aiello et al. / Bioorg. Med. Chem. 8 (2000) 2719±2728
Scheme 1. (i) NaNO2/HCl in water at rt; (ii) method A: water at rt; B: dichloromethane at rt for 10 min; C ethanol at 0 ꢀC to rt; D: ethanol at re¯ux
for 1 h; E: water/OH at rt; F: acetic acid at re¯ux for 1 h; (iii) H2SO4/HNO3 at rt for 3 h; (iv) HNO3 on ice bath in 5 h; (v) SnCl2/HCl concd at 0 ꢀC.
1
good yields, m/z, IR and H NMR spectral data are
given in Tables 1 and 2.
a carbon adjacent to an alkylated nitrogen resonates
up®eld of the signal of the same carbon in the other
isomer.11 In fact the 13C NMR signals of C-5 4b±d
derivatives were up®eld with respect to the signals of the
corresponding C-3 5b±d isomers. In the same manner
the C-3 and C-5 signals relative to the compounds of the
series I and II were assigned according to literature
data.12
The reaction of 2 with 3d (R=CH2CH2OH) in water
produced 7d (80%) and 6d (10%) after 3 days. Similar
yields were obtained in dichloromethane, but in this
case the reaction was completed in just 10 min. When
the reaction was initially performed in absolute ethanol
at 0 ꢀC and then kept at room temperature for 3 days
both isomers in comparable yields were obtained.
Reaction of 2 with phenylhydrazine, carried out in sev-
eral di€erent experimental conditions, gave only the
isomer 6g in low yields (30% in re¯uxing acetic acid and
20% in re¯uxing ethanol). Compound 9c was easily
obtained by oxidation of 6c with nitric acid at 5 ꢀC.
Finally, reduction of 6c carried out in concentrated
hydrochloric acid and stannous choride a€orded in
good yields compound 10c.
With regard to the series of compounds III and IV, the
presence of a nitroso substituent at position 40 involves
a high degree of chemical shielding anisotropy.13,14 In
the case of 6e and 7e, the chemical shifts were 159.21
and 158.78 ppm, respectively.
The singlet which appeared as the most deshielded one
was assigned to C-5 and the less shielded one to C-3.
The only doublet observed at low ®eld for nitroso deri-
vatives 6b±g and 7b±f was assigned by DEPT to C-4.
Owing to the availability of both III and IV pure iso-
meric series, the chemical structure of all derivatives was
elucidated by elemental analysis, IR, 1H NMR, 13C
NMR and X-ray di€ractometric data.
In all derivatives, signals observed between 11.62 and
11.80 ppm, not showing signi®cant chemical shift varia-
tions, were assigned to the isolated 5-methyl carbon.
The experimental 13C NMR spectral data are listed in
Table 3. The interpretation of 13C NMR spectra of iso-
meric N-alkylated azoles was made by considering that
The signals attributed to 5-methyl carbon of 7b±f
derivatives consistantly resonate up®eld with respect to
the 30-methyl carbon of compounds 6b±g: the observed

E. Aiello et al. / Bioorg. Med. Chem. 8 (2000) 2719±2728
Table 1. Yields, physical and spectroscopic data of series I and II
2721
Compound
Mp (ꢀC)d
Eluante
Yield (%)f
m/z
Molecular
formula
IR
(cm
1H NMR (d, ppm), (DMSO-d6)
1
)
4ba
4cb
35
(c)
57
(c)
(1)
(1)
67
40
177
191
C9H11N3O
C10H13N3O
2.19 (3H, s, CH3), 2.47 (3H, s, CH3), 4.00
(3H, s, CH3), 6.55 (1H, s, CH), 6.63 (1H, s, CH)
1.32 (3H, t, J=6.8 Hz, CH3), 2.21 (3H, s, CH3),
2.47 (3H, s, CH3), 4.40 (2H, q, J=6.8 Hz,CH2),
6.55 (1H, s, CH), 6.62 (1H, s, CH)
2.22 (3H, s, CH3), 2.47 (3H, s, CH3), 3.78
(2H, q, J=5.8 Hz, CH2), 4.46 (2H, t,
4dc
87
(b)
(3)
90
207
C10H13N3O2
3320
(OH, broad)
J=5.1 Hz, CH2), 4.91 (1H, t, J=5.1 Hz, OH),
6.51 (1H, s, CH), 6.60 (1H, s, CH)
5ba
5cb
125±126
(c)
72
(1)
(2)
11
40
177
191
C9H11N3O
C10H13N3O
2.28 (3H, s, CH3), 2.42 (3H, s, CH3), 3.78
(3H, s, CH3), 6.44 (1H, s, CH), 6.46 (1H, s, CH)
1.33 (3H, t, J=7.3 Hz, CH3), 2.30 (3H, s, CH3),
2.42 (3H, s, CH3), 4.10 (2H, q, J=7.3 Hz, CH2),
6.44 (1H, s, CH), 6.48 (1H, s, CH)
(c)
5dc
88
(b)
(3)
6
207
C10H13N3O2
3395
(OH, broad)
2.31 (3H, s, CH3), 2.42 (3H, s, CH3), 3.72
(2H, q, J=5.8 Hz, CH2), 4.11 (2H, t,
J=5.8 Hz,CH2), 4.90 (1H, t, J=5.8 Hz, OH),
6.41 (1H, s, CH) 6.47 (1H, s, CH)
aR=Me.
bR=Et.
cR=CH2CH2OH.
dCrystallization solvent: (c)=cyclohexane, (b)=benzene.
eChromatographic eluant: (1)=petroleum ether 40±60ꢀ: ethyl acetate 1:1; (2)=petroleum ether 40±60ꢀ: ethyl acetate 7:3; (3)=dichloromethane:ethyl
acetate 7:3.
fRe¯uxed in ethanol for 1 h.
signal pattern is justi®ed, as mentioned above, by the
e€ects of the alkyl group on the nearest nitrogen
atom.11,15
re®nement for these two compounds. The results pro-
vided useful data for preliminary SAR studies.
Because of the nitroso group rotation,16 the C-30 and
C-50 carbon signals, registered at room temperature at
50.3 MHz, appeared as two broad and weak peaks at
the shift values listed in Table 3. These assignments were
supported by structural X-ray analysis assuming a good
structure correspondence between the solution and the
solid state,14 in which the NO group assumes a stable
conformation.
Biology
Compounds were tested for antimicrobial activity against
representative human pathogenic fungi (C. albicans, C.
parapsilosis, C. paratropicalis, C. kruzei, C. glabrata, C.
laurentii, C. neoformans, A. fumigatus, T. mentagrophytes,
T. capitatum), against Gram-negative (Shigella spp., Sal-
monella spp.) and Gram-positive bacteria (Staphylo-
coccus aureus, group D Streptococcus). Test compounds
were also evaluated for antiretroviral activity in MT-4
cells infected with HIV-1. Cytotoxicity against MT-4
cells, carried out in parallel with anti-HIV-1 activity, was
evaluated to determine whether the compounds were
endowed with selective antimicrobial/antiviral activity.
Due to the availability of only one isomer, the carbon
signals of 6g were assigned on the basis of an analogous
signal pattern of compounds belonging to series III.
Additional evidence for the above assignments came from
1
the H NMR spectra. The 3-CH3 protons of compounds
6b±g appeared as a singlet down®eld with respect to the
5-CH3 protons of compounds 7b±f, according to the
electronic and steric e€ects of substituents situated on
the vicinal nitrogen atom.11
Miconazole, ¯uconazole, and amphotericin B were used
as reference compounds in antimycotical assays. Strep-
tomycin and AZT were used in antibacterial and anti-
retroviral assays, respectively.
With regard to IR spectra, all 6a±g and 7b±f nitroso
derivatives showed a characteristic and strong NO
absorption band in the range 1327±1372 cm 1. In the
series 7b±f, the frequency value of the absorption band
was slightly higher than the value found for the 6a±g
series having the same N-alkyl group (see Table 2).
Results and Discussion
Test compounds showed inhibitory activity against
fungi at concentrations ranging between 0.1 and 200 mM
(MIC/MFC in Table 4). Derivatives 6b±e were active
against C. neoformans at concentrations equal to or
lower than those of miconazole (MIC and MFC,
0.9 mM). Due to a lower cytotoxicity for MT-4 cells
(CC50=42 mM), compound 6c was 9 times more potent
and 20-fold more selective than miconazole, 6c was also
8- and 125-fold more potent than amphotericin B and
X-ray di€raction
Since it was possible to produce well formed crystals of
6e and 7e by recrystallization from dilute ethanolic solu-
tion we were able to obtain crystal data and structure

2722
E. Aiello et al. / Bioorg. Med. Chem. 8 (2000) 2719±2728
Table 2. Yields, physical and spectroscopic data of series III and IV
Mp (ꢀC)h
Eluanti
Yield (%)/
methodk
m/z
Molecular
formula
IR (cm )
1H NMR (d ppm), (DMSO-d6)
1
Compound
6aa
6bb
6cc
188
(b) (1)
57
(e) (1)
107
(e) (2)
(4)
(1)
(5)
50/A
192
206
220
C8H8N4O2
C9H10N4O2
C10H12N4O2
1327 (NO)
3170 (NH)
1334 (NO, strong)
2.37 (3H, s, CH3), 2.55 (3H, s, CH3),
6.80 (1H, s, CH), 12.98 (1H, broad NH)
2.12 (3H, s, CH3), 2.59 (3H, s, CH3),
4.09 (3H, s, CH3), 7.03 (1H, s, CH)
1.45 (3H, t, J=7.3 Hz, CH3), 2.15
(3H, s, CH3), 2.60 (3H, s, CH3), 4.46
(2H, q, J=7.3 Hz, CH2), 7.02 (1H, s, CH)
2.16 (3H, s, CH3), 2.60 (3H, s, CH3),
3.84 (2H, t, J=5.8 Hz, CH2), 4.51 (2H,
t, J=5.8 Hz, CH2), 5.00 (1H, t, J=5.8 Hz,
OH), 7.00 (1H, s, CH)
0.90 (3H, t, J=7.3 Hz, CH3), 1.89 (2H, m,
J=7.3 Hz, CH2), 2.15 (3H, s, CH3), 2.60
(3H, s, CH3), 4.40 (2H, t, J=7.3 Hz, CH2),
7.02 (1H, s, CH)
0.90 (3H, t, J=7.3 Hz, CH3), 1.33 (2H, m,
J=7.3 Hz, CH2), 1.85 (2H, m, J=7.3 Hz,
CH2), 2.14 (3H, s, CH3), 2.60 (3H, s, CH3),
4.45 (2H, t, J=7.3 Hz, CH2), 7.02 (1H, s, CH)
2.26 (3H, s, CH3), 2.51 (3H, s, CH3), 6.76
(1H, s, CH), 7.55 (1H, s, C6H5)
30/C
j/E
35/A
30/E
1337 (NO, strong)
6dd
6ee
6ff
148
(e) (2)
(6)
(7)
(5)
10/A
10/B
30/C
236
234
248
C10H12N4O3
C11H14N4O2
C12H16N4O2
1342 (NO, strong)
3440 (OH, broad)
67±68
(e) (3)
50/A
1341 (NO, strong)
1341 (NO, strong)
48±49
(e±w) (2)
45/A
30/E
6gg
7bb
7cc
120±121
(e) (2)
72
(e) (3)
60
(e) (3)
(8)
(2)
(5)
20/D
30/F
40/C
j/E
50/A
45/E
268
206
220
C14H12N4O2
C9H10N4O2
C10H12N4O2
1346 (NO, strong)
1348 (NO, strong)
1345 (NO, strong)
2.52 (3H, s, CH3Â2), 3.88 (3H, s, CH3),
6.69 (1H, s, CH)
1.4 (3H, t, J=7.3 Hz, CH3), 2.53 (3H, s,
CH3), 2.56 (3H, s, CH3), 4.23 (2H, q,
J=7.3 Hz, CH2), 6.69 (1H, s, CH)
2.53 (3H, s, CH3), 2.59 (3H, s, CH3), 3.84
(2H, t, J=5.1 Hz, CH2), 4.27 (2H, t,
J=5.1 Hz, CH2), 5.07 (1H, t, J=5.1 Hz,
OH), 6.69 (1H, s, CH)
0.91 (3H, t, J=7.3 Hz, CH3), (3H, s, CH3),
1.84 (2H, m, J=7.3 Hz, CH2), 2.54 (3H, s,
CH3), 2.57 (3H, s, CH3), 4.17 (2H, t,
J=7.3 Hz, CH2), 6.70 (1H, s, CH)
0.92 (3H, t, J=7.3 Hz, CH3), 1.33 (2H, m,
J=7.3 Hz, CH2), 1.80 (2H, m, J=7.3 Hz,
CH2), 2.53 (3H, s, CH3), 2.56 (3H, s, CH3),
4.20 (2H, t, J=7.3 Hz, CH2), 6.69
(1H, s, CH)
1.32 (3H, t, J=6.8 Hz, CH3), 2.50 (3H, s,
CH3), 2.56 (3H, s, CH3), 4.07 (2H, q,
J=6.8 Hz, CH2), 6.68 (1H, s, CH)
1.21 (3H, t, J=6.8 Hz, CH3), 2.08 (3H, s,
CH3), 2.46 (3H, s, CH3), 4.15 (2H, bs,
NH2), 4.19 (2H, q, J=6.8 Hz, CH2), 6.68
(1H, s, CH)
1.28 (3H, t, J=7.3 Hz, CH3), 2.16 (3H, s,
CH3), 2.42 (3H, s, CH3), 4.01 (2H, q,
J=7.3 Hz, CH2), 4.20 (2H, bs, NH2),
6.43 (1H, s, CH)
7dd
7ee
7ff
146±147
(e) (3)
(5)
(7)
(5)
80/A
80/B
40/C
236
234
248
C10H12N4O3
C11H14N4O2
C12H16N4O2
1362 (NO, strong)
3468 (OH, broad)
58±59
(e) (3)
30/A
1345 (NO, strong)
1348 (NO, strong)
52±53
(e±w)(3)
45/A
35/E
9cc
98±100
(e) (4)
(2)
(9)
75
80
236
206
C10H12N4O3
C10H14N4O
1516,1344 (NO2)
3412, 3342 (NH2)
10cc
Oil
11cc
118±119
(c) (5)
(3)
80
206
C10H14N4O
3412, 3328 (NH2)
aa R=H.
bb R=Me.
cc R=Et.
dd R=CH2CH2OH.
ee R=n-Pr.
ff R=n-Bu.
gg R=Ph.
hCrystallization solvent: (b) benzene, (e) ethanol, (e±w) ethanol±water, (c) petroleum ether. Crystal appearance: (1) blue-green prism, (2) emerald-
green prism, (3) blue-green needle, (4) white needle, (5) yellow needle.
iChromatographic eluant: (1)=petroleum ether 40±60ꢀ:ethyl acetate 1:1; (2)=petroleum ether 40±60ꢀ:ethyl acetate 7:3; (3)=dichloromethane:ethyl
acetate 7:3; (4)=dichloromethane:ethyl acetate 9:1; (5)=dichloromethane; (6)=diethyl ether:methanol 96:4; (7)=dichloromethane:petroleum ether
40±60ꢀ 6:4; (8)=petroleum ether 40±60ꢀ:diethyl ether 9:1; (9)=diethyl ether.
jDark intractable rubber.
kMethod: A water, rt; B dichloromethane, rt for 10 min; C ethanol at 0 ꢀC to rt; D ethanol at re¯ux for 1 h; E water/OH , rt; F acetic acid at re¯ux
for 1 h.
¯uconazole, respectively, against C. neoformans. Com-
pounds 6b±e were also active against Candida spp., A.
fumigatus and T. mentagrophytes at concentrations
slightly higher than those of miconazole.
Derivative 6e had a wider range of activity, with a
potency comparable to that of miconazole against C.
albicans, C. paratropicalis, C. glabrata, C. neoformans
and A. fumigatus.

E. Aiello et al. / Bioorg. Med. Chem. 8 (2000) 2719±2728
2723
Table 3. 13C NMR Chemical shifts registered at 50.3 MHz, in DMSO-d6
a
Compound
C-5
C-4
C-3
C-5
C-4
C-3
3-CH3
5-CH3
5-CH3
N-C1
N-C2
N-C3
N-C4
4b
4c
4d
5b
5c
5d
6a
6b
6c
6d
6e
6f
169.83
169.81
169.78
169.36
169.34
169.34
170.38
171.23
171.35
171.11
171.27
171.20
171.02
101.51
101.61
101.82
99.78
99.74
99.76
102.49
104.28
104.50
104.78
104.50
104.42
104.53
154.11
154.01
154.21
157.11
157.54
157.51
155.23
152.52
152.43
152.56
152.42
152.42
152.20
131.92
131.06
132.27
139.38
139.61
139.82
133.23
133.20
133.69
133.80
133.56
133.39
135.24
107.94
107.50
107.35
103.43
103.54
103.31
158.84
159.37
159.32
159.46
159.21
159.16
159.59
146.61
146.84
147.13
140.24
139.40
140.83
139.67
138.69
137.97
139.48
138.31
138.24
138.03
12.97
13.10
13.10
11.64
11.62
11.61
11.74
11.73
11.75
11.80
11.72
11.74
11.71
11.68
11.62
11.68
38.77
45.06
52.81
36.19
43.59
51.26
15.46
60.19
10.63
10.44
10.88
15.11
60.28
11.15
12.89
14.82
13.04
12.96
13.27
12.86
39.50
46.78
59.34
52.73
51.03
13.04
53.56
22.57
31.15
10.67
19.08
12.91
6g
139.06 (C-1-Ph); 129.54 (C-3,5-Ph);
125.54 (C-2-6-Ph); 129.24 (C-4-Ph)
36.33
43.95
59.47
50.12
48.46
46.05
45.41
43.91
7b
7c
7d
7e
7f
9c
10c
11c
170.13
170.10
170.16
170.09
170.08
170.85
169.36
168.24
102.40
102.46
102.53
102.46
102.45
103.98
100.35
99.32
155.36
155.43
155.49
155.42
155.40
152.27
153.94
158.58
135.11
134.74
136.77
134.98
134.75
130.59
129.54
123.47
158.88
158.89
159.01
158.78
158.79
145.49
116.33
127.19
136.78
136.77
138.81
137.03
137.00
131.49
135.86
127.84
10.06
9.81
10.31
9.98
9.94
11.73
11.71
11.75
11.70
11.69
11.79
11.74
11.56
14.11
51.60
22.10
30.67
15.09
15.44
15.07
10.70
19.14
13.37
13.49
10.58
8.03
aa R=H, b R=Me, c R=Et, d R=CH2CH2OH, e R=n-Pr, f R=n-Bu, g R=Ph.
Compounds of the series I and II and the derivative 8b
were noncytotoxic for MT-4 cells at doses as high as
200 mM, whereas compounds of the series III and IV
showed a cytotoxicity comparable to that of miconazole
(CC50=18 mM) with the exception made for derivative
7d, which was 6-fold more toxic.
length of the alkylic carbon chain, the activity increases
passing through a maximum with the ethyl group,
decreasing progressively with the propyl and butyl groups.
Lipophilicity measurements (RM) pointed out constantly
higher RM values for derivatives 6b±f (series III) than for
corresponding isomers 7b±f (series IV). Compounds
belonging to the series III were more active and less
cytotoxic than isomeric 1H-3-pyrazolyl derivatives of
series IV.
All derivatives were neither signi®cantly active against
Gram-negative and Gram-positive bacteria (Table 5),
nor capable of protecting MT-4 cells from the cyto-
pathic e€ect induced by HIV-1, at least at noncytotoxic
concentrations. Under the same experimental condi-
tions AZT was active at 0.01 mM.
The trend of the observed activity can be correlated
with data obtained in the determination of crystal
structures of two isomeric compounds 6e and 7e (Figs 1
and 2). The nitroso group of 6e adopts trans con®gura-
tion with respect to the alkyl chain on the pyrazole
nitrogen and extended perpendicularly to the average
molecular plane, as already observed for related com-
pounds both in solution and in the solid state.14 On the
contrary, the nitroso group of 7e adopts cis con®gura-
tion. The geometry of 6e can be compared with the
other two nitrosopyrazole compounds whose structures
are known, 1 H-3,5-di-tert-butyl-4-nitrosopyrazole and
1,3,5-trimethyl-4-nitrosopyrazole.14 These compounds
present partial cis/trans disorder in the solid state, while
6e is found in the pure trans form. This is probably due
to the di€erent steric hindrance of the 3-methyl and
4-isoxazole substituents, which favor the orientation of
the NO group away from the latter. Moreover, in 6e the
The absence of the NO group (series I and II) or its
replacement with the NO2 group (9c) or with the amino
group (10c) gave compounds devoid of antimycotical
activity. The dinitro derivative 8b was also inactive.
Only compounds belonging to the two series III and IV
proved active as antifungal agents. Compounds 6a±c,
6e±f and 7b±f displayed selective activity against some
fungi (Table 4).
The potency of NO-carrying derivatives appeared to be
modulated by the type of substituent and by the length
of the carbon chain of the alkyl group bound to the
nitrogen of the pyrazole ring: the N±H (6a) and N-phenyl
(6g) derivatives were poorly active. With the increasing

Table 4. Antifungal activity and RM value
MICb/MFCc
a
Compound
CC50
MT-4
C. albicans
C. parapsilosis C. paratropicalis
C. kruzei
C. glabrata
C. laurenti
C. neoformans A. fumigatus T. mentagrophytes
T. capitum
RM
4b
5b
4c
5c
4d
5d
6a
6b
7b
6c
7c
6d
7d
6e
7e
6f
>200
>200
>200
>200
>200
>200
26
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
25/25
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
66/>200
>200/>200
9/9
>200/>200
>200/>200
>200/>200
66/66
66/66
66/200
>200/>200
200/200
>200/>200
>200
>200
0.9/0.9
200/200
0.8/3.1
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
25/>25
200/200
19/19
66/200
66/66
>200/>200 >200/>200 >200/>200
>200/>200 >200/>200 >200/>200
>200/>200 >200/>200 >200/>200
>200/>200 >200/>200 >200/>200
>200/>200 >200/>200 >200/>200
>200/>200 >200/>200 >200/>200
>200/>200 >200/>200 >200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
30/60
0.8/1.6
3.1/3.1
0.1/0.1
3.1/3.1
0.8/0.8
22/22
0.8/0.8
2.4/2.4
2.4/2.4
9/9
66/66
>200/>200
>200
>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
12/22
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
25/25
66/66
19/19
200/200
2.4/7.4
>200/>200
2.4/7.4
2.4/7.4
200/200
75/75
66/200
>200/>200
ND
ND
0.9/0.9
ND
ND
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
12/12
0.158
0.286
0.084
0.203
0.357
0.434
0.277
0.008
0.248
0.089
0.149
0.194
0.360
0.163
0.059
0.038
0.278
0.142
0.078
ND
11.6
9.1
42
9.5
13.5
6/6
25/50
30/60
100/100
66/66
25/25
100/100
19/19
50/100
66/66
12/25
50/50
9/19
25/50
ND
>200/>200
19/19
66/66
22/66
9/9
66/66
22/22
25/100
75/>75
100/100
66/200
>200/>200
66/66
66/66
66/66
3.35 >200/>200
20
14
15
92
14
>200/>200
7.4/7.4
22/22
>200/>200 >200/>200 >200/>200
>200/>200
7.4/7.4
7.4/7.4
7.4/200
37/37
66/200
>200/>200
ND
ND
7.4/7.4
22/22
22/22
>200/>200
>200/>200
>200/>200
>200
>200
7/7
200/>200
3.1/3.1
22/22
66/66
66/66
7.4/7.4
22/22
22/200
ND
ND
ND
22/22
66/66
7f
>200/>200
>200/>200
>200/>200
>200
>200
7/7
12.5/>200
0.2/3.1
>200/>200
200/>200
>200/>200 >200/>200 >200/>200
150/150
>200/>200
75/150
ND
>200/>200
200/>200
>200/>200
ND
6g
8b
9c
10c
>200
>200
>200
18
>200
>200
>200
>200
2/4
200/200
12.5/12.5
>200
>200
6/6
200/>200
3.1/3.1
ND
ND
0.5/0.9
ND
ND
ND
4/4
ND
ND
ND
ND
ND
ND
d
-
0.9/0.9
12.5/12.5
0.8/0.8
4/4
ND
ND
e
-
f
-
aCompound dose (mM) required to reduce the viability of mock-infected cells by 50%.
bMinimum inhibitory concentration.
cMinimum fungicidal concentration.
dMiconazole.
eFluconazole.
fAmphotericin B.

E. Aiello et al. / Bioorg. Med. Chem. 8 (2000) 2719±2728
2725
Table 5. Antibacterial activity
MICb/MBCc (mM)
a
Compound
CC50
d
MT-4
Shigella
Salmonella
Staphylococcus
Streptococcus
RM
4b
5b
4c
5c
4d
5d
6a
6b
7b
6c
7c
6d
7d
6e
7e
6f
>200
>200
>200
>200
>200
>200
26
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
200/>200
66/66
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
200/>200
200/>200
>200/>200
200/>200
100/100
22/66
200/200
200/200
>200/>200
100/100
50/100
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
200/>200
200/>200
>200/>200
200/>200
100/100
22/66
200/200
200/200
>200/>200
100/100
50/50
0.158
0.286
0.084
0.203
0.357
0.434
0.277
0.008
0.248
0.089
0.149
0.194
0.360
0.163
0.059
0.278
0.038
0.142
0.078
ND
11.6
9.1
42
9.5
13.5
3.35
20
14
92
15
66/66
>200/>200
66/66
100/100
>200/>200
>200/>200
100/200
22/66
66/66
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
3.1/3.1
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
>200/>200
6.2/6.2
7f
6g
8b
9c
14
>200
>200
>200
>200
>200/>200
>200/>200
>200/>200
3.1/3.1
>200/>200
>200/>200
>200/>200
3.1/3.1
10c
Streptomycin
ND
aCompound dose (mM) required to reduce the viability of mock-infected cells by 50%.
bMinimum inhibitory concentration.
cMinimum bactericidal concentration.
dHigher RM value correspond to higher lipophilicity.
isoxazole and pyrazole rings, both planar within 0.03 A,
de®ne a dihedral angle of 13.3ꢀ due to the intramolecular
interaction between the propyl chain and the isoxazole
ring. In the isomer 7e the two rings, both again planar
within 0.03 A, de®ne a dihedral angle of 2.7ꢀ, as the
entire molecule is planar within 0.06 A. In 7e the nitroso
group is cis with respect to the N3±C9 bond, that is, it
prefers pointing towards the methyl than towards the
isoxazole, con®rming that the cis/trans preference is
driven by the steric hindrance of the 3,5 substituents of
the ring. Therefore, also the con®guration of the NO
group and the planarity of the entire molecule seem to
play a crucial role in modulating the activity of the
described compounds.
Figure 1. Perspective view of the molecular structure of 6e. Thermal
ellipsoids are drawn at the 50% probability level.
Conclusion
In conclusion, we have synthesized and evaluated
in vitro the antimicrobial activity of a new class of
4-nitrosopyrazoles. Compound 6c exhibits an antifungal
activity 9-fold more potent than miconazole and is 20-
fold more selective against C. neoformans. 6c was also 8-
and 125-fold more potent than amphotericin B and ¯u-
conazole, respectively. Compounds 6b±e were also
active against Candida spp., A. fumigatus and T. menta-
grophytes at concentrations comparable with those of
miconazole. We should point out that all compounds
belonging both to the series III and IV, except 6a, were
active against C. neoformans at concentrations com-
parable with or smaller than ¯uconazole and ampho-
tericin B. According to these in vitro results,
compound 6c could be a promising candidate for future
development.
Figure 2. Perspective view of the molecular structure of 7e. Thermal
ellipsoids are drawn at the 50% probability level.

2726
E. Aiello et al. / Bioorg. Med. Chem. 8 (2000) 2719±2728
Experimental
in ice±water. After neutralization with sodium hydrogen
carbonate, extraction with dichloromethane and
removal of the solvent left a residue which was chro-
matographed.
Chemistry
All melting points were taken on a Buchi-Tottoli capil-
lary apparatus and are uncorrected. IR spectra were
determined in bromoform with a Jasco FT/IR 5300
spectrophotometer. 1H and 13C NMR spectra were
measured at 200 and 50.3 MHz, respectively, in
(CD3)2SO solution unless otherwise speci®ed, using a
Bruker AC series 200 MHz spectrometer (TMS as
internal reference). Mass spectra were obtained with an
HP 5889A-GC/MS apparatus. Column chromato-
graphy was perfomed with Merck silica gel 230±400
Mesh ASTM. Analyses indicated by the symbols of the
elements or functions were within Æ0.4% of theoretical
values.
Yields, melting points, recrystallization solvent, eluants
of column chromatography, molecular ion (m/z), molec-
1
ular formula, IR and H NMR spectral data are repor-
ted in Tables 1 and 2, and 13C NMR in Table 3.
3-(1,3-Dimethyl-4-nitro-1H-3-pyrazolyl)-5-methyl-4-ni-
troisoxazole (8b).17 Eluted with petroleum ether (bp 40±
60 ꢀC):ethyl acetate 1:1, yield 90%, and recrystallized
from ethanol, mp 124 ꢀC. H NMR (DMSO-d6) d 2.55
1
(3H, s, CH3), 2.98 (3H, s, CH3), 3.87 (3H, s, CH3); 13C
NMR (DMSO-d6) d 13.14 (q), 13.64 (q), 38.42 (q),
127.80 (s), 130.08 (s), 132.49 (s), 145.62 (s), 147.51 (s),
174.12 (s); m/z 267. Anal. C9H9N5O5 (C,H,N).
1-(5-Methyl-3-isoxazolyl)-1,3-butanedione 1 and 1-(5-
methyl-3-isoxazolyl)-2-hydroxyimino-1,3-butanedione 2
were prepared according to the procedures previously
reported.2 Derivatives 4 and 5b,c were prepared accord-
ing to ref 17.
3-(1-Ethyl-3-methyl-4-nitro-1H-5-pyrazolyl)-5-methyl-
isoxazole (9c). To a cooled solution ( 5 ꢀC) of nitric acid
65% (2 mL), 2 mmol of 6c were added in small portions.
The reaction mixture was stirred for 5 h maintaining the
temperature at 5 ꢀC. After disappearance of the starting
material (TLC), the solution was poured in ice±water
and neutralized with sodium carbonate. The white solid
formed was ®ltered, air dried, and puri®ed by crystal-
lization.
General methods. For the preparation of 3-(3-methyl-4-
nitroso-1-R-1H-5-pyrazolyl)-5-methylisoxazoles 6a±g, 3-
(5-methyl-4-nitroso-1-R-1H-3-pyrazolyl)-5-methylisox-
azoles 7a±f, 3-(3-methyl-1-R-1H-5-pyrazolyl)-5-methyl-
isoxazole 4d, and 3-(5-methyl-1-R-1H-3-pyrazolyl)-5-
methylisoxazole 5d.
3-(4-Amino-1-ethyl-3-methyl-1H-5-pyrazolyl)-5-methyl-
isoxazole (10c) and 3-(4-amino-1-ethyl-5-methyl-1H-3-
pyrazolyl)-5-methylisoxazole (11c). Compound 6c or 7c
(2 mmol) was slowly added into a stirred and cooled
(0 ꢀC) solution of SnCl2 (2 g) in 2 mL of hydrochloric
acid (37%). Within 10 min, the reaction was complete
(TLC). Neutralization with sodium carbonate and
extraction with dichloromethane gave an oil, which was
puri®ed by column chromatography.
Method A. To an aqueous suspension of ®nely powdered
hydroxyimine 2 0.97 g (5 mmol), a stoichiometric amount
of suitable hydrazine salt dissolved in distilled water
(20 mL) was added dropwise at room temperature. The
reaction mixture was stirred until no starting material was
detected (TLC). After completion of reaction (1±24 h), the
mixture was extracted with dichloromethane (3Â100 mL)
and dried (anhydrous sodium sulphate). Removal of the
solvent under reduced pressure gave a blue-green resi-
due, which was puri®ed by column chromatography.
Lipophilicity measurements. The relative lipophilicity of
the compounds was measured by reversed-phase TLC
according to the method already described.22 RM values
were calculated from the experimental Rf values (calcu-
lated as mean values for ®ve determinations) according
to the equation RM=log[(1/Rf) 1]. Higher RM values
correspond to higher lipophilicity.
Method B. A mixture of 1,3-diketone 2, 0.97 g (5 mmol),
and suitable equimolar hydrazine salt, in 20 mL of dry di-
chloromethane, was stirred at room temperature until
starting material disappeared. After completion of the
reaction thesolvent wasevaporatedunderreduced pressure
and the residue chromatographed by silica gel column.
Crystal X-ray di€raction analysis. Single crystal X-ray
di€raction analyses were carried out at room tempera-
ture by a Siemens AED di€ractometer for 6e and an
Enraf±Nonius CAD4 di€ractometer for 7e. In both
cases graphite-monochromated CuKa radiation was
used (l=1.54138 A). Relevant details concerning data
collection and structure re®nements were provided to
the editor. For 6e, crystal decay was observed, and the
data were rescaled accordingly. The intensity data were
processed with a peak-pro®le analysis procedure and
corrected for Lorentz and polarization e€ects. The
phase problem was solved by direct methods using
SIR97.18 Full matrix least-squares re®nements were
carried out by SHELXL9719 on F2, using all measured
unique data. Anisotropic thermal displacement param-
eters were re®ned for all non-hydrogen atoms. H atoms
Method C. The equimolar mixture of diketone 2 and
suitable hydrazine salt (5 mmol) in dry ethanol (30 mL)
was stirred at 0 ꢀC to room temperature for 72 h. At the
end of the reaction, the solvent was removed and the
residue chromatographed as above.
Method D. As method C but the reactants were re¯uxed
for 1 h. Removal of the solvent a€orded a dark residue
which was puri®ed by silica gel column.
Method E. As method A, except for the 1,3-diketone 2 that
was dissolved in water with pellets of sodium hydroxide.
Method F. The equimolar mixture of 2 and 3g (5 mmol)
in AcOH (30 mL) was re¯uxed for 1 h and then poured

E. Aiello et al. / Bioorg. Med. Chem. 8 (2000) 2719±2728
2727
were introduced in idealized positions, riding on their
carrier atoms, for 6e and for the methyl groups of 7e.
The remaining H atoms of 7e were located on ÁF maps
and re®ned isotropically. Programs PARST9720 and
ZORTEP21 were used for analyzing and drawing the
molecular structures. Use was made of the packages of
the Cambridge Structural Database (release October
1998). All the calculations were performed on a Digital
Alpha 255 workstation at the Centro di Studio per la
Strutturistica Di€rattometrica del C.N.R. in Parma.
homogenizer and then diluting to 0.05 OD590/mL. Then,
20 mL of the above suspensions were added to each well
of ¯at-bottomed microtiter trays containing 80 mL of
medium with serial dilutions of test compounds, and
were incubated at 37 ꢀC. Growth controls were visually
determined after 2 days (yeast) or 3 days (dermato-
phytes). MIC was de®ned as the compound concentra-
tion at which no macroscopic sign of fungal growth was
detected. The minimal germicidal concentrations (MBC
or MFC) were determined by subcultivating in Sabour-
aud dextrose agar samples from cultures with no
apparent growth.
Biological assays
Compounds. Test compounds were dissolved in DMSO
at an initial concentration of 200 mM and then were
serially diluted in culture medium.
Linear regression analysis. Viral and cell growth at each
drug concentration was expressed as percentage of
untreated controls and the concentrations resulting in
50% (EC50, CC50) growth inhibition were determined
by linear regression analysis.
Cells. Cell lines were from American Type Culture Col-
lection (ATCC); bacterial and fungal strains were either
clinical isolates (obtained from Clinica Dermosililopa-
tica, University of Cagliari) or collection strains from
ATCC. H9/IIIB, MT-4 and C8166 cells [grown in RPMI
1640 containing 10% fetal calf serum (FCS), 100 UI/mL
penicillin G and 100 mg/mL streptomycin] were used for
anti-HIV-1 assays. Cell cultures were checked periodi-
cally for the absence of mycoplasma contamination
with a MycoTect Kit (Gibco).
Acknowledgements
This work was funded by Consiglio Nazionale delle
Ricerche, Ministero dell'Universita e della Ricerca Sci-
enti®ca and Istituto Superiore di Sanita, XI Progetto
AIDS 1997 (Grant 40A.0.55).
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cultures grown at 37 ꢀC for 5 days in Sabouraud dex-
trose broth by ®nely dispersing clumps with a glass

2728
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