Structure-activity relationship study of nitrosopyrimidines acting as antifungal agents

Article abstract of DOI:10.1016/j.bmc.2012.08.033

The design, synthesis, in vitro evaluation, and conformational study of nitrosopyrimidine derivatives acting as antifungal agents are reported. Different compounds structurally related with 4,6-bis(alkyl or arylamino)-5-nitrosopyrimidines were evaluated. Some of these nitrosopyrimidines have displayed a significant antifungal activity against human pathogenic strains. In this paper, we report a new group of nitrosopyrimidines acting as antifungal agents. Among them, compounds 2a, 2b and 15, the latter obtained from a molecular modeling study, exhibited antifungal activity against Candida albicans, Candida tropicalis and Cryptococcus neoformans. We have performed a conformational and electronic analysis on these compounds by using quantum mechanics calculations in conjunction with Molecular Electrostatic Potentials (MEP) obtained from B3LYP/6-31G(d) calculations. Our experimental and theoretical results have led us to identify a topographical template which may provide a guide for the design of new nitrosopyrimidines with antifungal effects.

Full text of DOI:10.1016/j.bmc.2012.08.033

Bioorganic & Medicinal Chemistry 20 (2012) 6109–6122
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationship study of nitrosopyrimidines acting
as antifungal agents
Monica Olivella a, Antonio Marchal b, Manuel Nogueras b, Adolfo Sánchez b, Manuel Melguizo b,
a,e,
Marcela Raimondi c,d, Susana Zacchino c, Fernando Giannini a, Justo Cobo b, , Ricardo D. Enriz
⇑
⇑
a Departamento de Química, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco 915, 5700 San Luis, Argentina
b Departamento de Química Inorgánica y Orgánica, Universidad de Jaén, 23071 Jaén, Spain
c Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina
d Microbiología, Facultad de Medicina, , Universidad Nacional de Rosario, Santa Fe 3100, 2000 Rosario, Argentina
e IMIBIO-SL (CONICET), Chacabuco 915, 5700 San Luis, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, synthesis, in vitro evaluation, and conformational study of nitrosopyrimidine derivatives act-
ing as antifungal agents are reported. Different compounds structurally related with 4,6-bis(alkyl or
arylamino)-5-nitrosopyrimidines were evaluated. Some of these nitrosopyrimidines have displayed a sig-
nificant antifungal activity against human pathogenic strains. In this paper, we report a new group of
nitrosopyrimidines acting as antifungal agents. Among them, compounds 2a, 2b and 15, the latter
obtained from a molecular modeling study, exhibited antifungal activity against Candida albicans, Candida
tropicalis and Cryptococcus neoformans. We have performed a conformational and electronic analysis on
these compounds by using quantum mechanics calculations in conjunction with Molecular Electrostatic
Potentials (MEP) obtained from B3LYP/6–31G(d) calculations. Our experimental and theoretical results
have led us to identify a topographical template which may provide a guide for the design of new nitr-
osopyrimidines with antifungal effects.
Received 6 June 2012
Revised 8 August 2012
Accepted 16 August 2012
Available online 31 August 2012
Keywords:
Nitrosopyrimidines
Antifungal compounds
Synthesis
Structure–activity relationships
Conformational study
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Among them, a series of 4-aryl- or 4-alkyl-N-arylamino-1-butenes
(homoallylamines) and related tetrahydroquinolines and quino-
Fungal infections have continued to be a major medical prob-
lem during the past two decades especially involving immunocom-
promised patients.1,3 Although it appears to be many drugs for the
treatment of systemic and superficial mycoses, there are in fact
only a limited number of efficacious antifungal drugs.2 Azoles that
inhibit ergosterol biosynthesis and polyenes that bind to mature
membrane sterols have been the mainstays of antifungal therapy
for more than two decades.4,5 However, the emergence of fluconaz-
ole resistance among different pathogenic strains and the high
toxicity of amphotericin B,6,7 have led to the search for new anti-
fungal agents.8 Although combination therapy has emerged as a
good alternative to overcome these disadvantages,9,10 there is a
real need for a next generation of safer and more potent antifungal
agents.2
lines displayed a range of antifungal properties against dermato-
phytes, which are responsible of most dermatomycoses in
humans.22 Afterwards, we extended our study by introducing a
new series of 4-N-arylamino-1-butenes containing the pyridinyl
or quinolinyl moieties at C4 and other structurally related
compounds.24 The structure–activity relationship (SAR) analysis
indicated that the presence of two aromatic rings and a particular
length for the connecting chain are operative for the homoallyl-
amine derivatives.28
Although the previously reported homoallylamines displayed
an interesting antifungal activity, unfortunately their effects were
limited to dermatophytes and they did not show any significant
activity against other opportunistic clinical relevant fungi. It is well
known that many fungal infections are caused by opportunistic
pathogens that may be endogenous or acquired from the environ-
ment. Patients with significant immunosuppression frequently
develop candidiasis or cryptococcosis.
In the course of our ongoing screening program for new
antifungal compounds, we have previously reported the antifungal
activity of peptide compounds11–17 as well as of different
molecules obtained from natural18–21 and synthetic22–31 sources.
Candidiasis has shown to be the fourth most common
nosocomial blood stream infection, Candida albicans representing
more than 60% of all isolates from clinical infections.32 Among the
non-albicans Candida species, Candida tropicalis showed to be
themost isolated species(20 %) in Latin Americancountriesbetween
⇑
Corresponding authors. Tel.: +34 953212695 (J.C.); tel.: +54 2664 424689x153
(R.D.E.).
E-mail addresses: jcobo@ujaen.es (J. Cobo), denriz@unsl.edu.ar (R.D. Enriz).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.033

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M. Olivella et al. / Bioorg. Med. Chem. 20 (2012) 6109–6122
2001 and 2004. Cryptococcosis, caused by the encapsulated yeast
Cryptococcus neoformans, has been the leading cause of fungal
mortality among immune-suppressed patients.6 This organism has
predilection for the central nervous system and leads to severe,
life-threatening meningitis.
Invasive aspergillosis is a rapidly progressive, often fatal infec-
tion that occurs in patients who are severely immunosuppressed,
including those who are profoundly neutropenic, those who have
received bone marrow or solid organ transplants, and patients with
advanced AIDS or chronic granulomatous disease.33 The majority
of human illness is caused by Aspergillus fumigatus and Aspergillus
niger and, less frequently, by Aspergillus flavus.
2. Results and discussion
2.1. Chemistry
We have already reported the synthesis of a family of N4-substi-
tuted 2,4-diamino-6-methoxy-5-nitrosopyrimidines 2 by selective
monoaminolysis of 1a,34a–c which was prepared through the intro-
duction of a 5-nitroso group into the commercial 2-amino-4,
6-dimethoxypyrimidine (see Scheme 1 and Table 1). A second ami-
nolysis with
a different amino compound permitted us the
preparation of a set of highly functionalized N4,N6-disubstituted
2,4,6-triamino-5-nitrosopyrimidines 3 (see Scheme 1 and Table
1). We have also isolated in some of these reactions the hydrolysis
products such as several N6-substituted 2,6-diamino-5-nitrosopyr-
imidin-4(3H)ones 4 (see Scheme 1).
Considering that homoalylamines previously reported dis-
played two aromatic rings, we start our study using compounds
possessing such structural characteristics. In addition taking
advantage of our previous experience in the synthesis of aminopyr-
imidines bearing the same structural characteristics,34 we decided
to look for new compounds structurally related to the homoallyl-
amines previously reported. In addition, we have recently reported
that the introduction of a nitroso group on C5 of a pyrimidine
nucleus highly activates it towards nucleophilic substitution.
This enables the easy and selective displacement of methoxy
groups in 2-amino-4,6-dimethoxy-5-nitrosopyrimidine by a large
series of amines under mild conditions, allowing efficient, time-
saving, one-pot preparations of symmetrical and non-symmetrical
2,4,6-tris(alkylamino)-5-nitrosopyrimidines, which are useful as
intermediates in common synthetic routes to heterobicyclic sys-
All of these ortho-aminonitrosopyrimidines, such as 2, 3 or 4, are
very useful as synthetic intermediates to purine or pteridine ana-
logues, which, by reduction of the nitroso to an amino group and
further cyclocondensation can afford easily this kind of compounds;
for example the cyclocondensation with formamidinium acetate or
with sodium nitrite in acid media, leads to the corresponding pur-
ine, or azapurine analogues, 5 or 6 respectively if starting from 2c
(Scheme 1). If starting from 2i, the reduction of the nitroso group
is followed by an intramolecular cyclization through an ester group
rendering the pteridine analogue 7 as showed in Scheme 1.
In order to increase the structural diversity and also to test the
influence of changing different groups, or the size of the fused ring
to pyrimidine, we have prepared the pteridine analogues 9a, b and
10 (Scheme 2). So, changing the methoxy group at 1a by a bulkier
group, such as cyclohexylmethylenoxy, by transalkoxylation we
have synthetized compound 1b, which, by further substitution
with different aminoesters yielded 8a,b, which were used, in a sim-
ilar procedure to that employed to get the pteridine 7, for the prep-
aration of analogues 9a,b.
34a
tems of great biological interest.
In this study we have determined the antifungal activity of
nitrosopyrimidine derivatives against a panel of clinically impor-
tant fungi. With these results we have performed a structure–
activity relationship (SAR) study in order to determine the
minimum structural requirements for these compounds to pro-
duce the antifungal effect. We also discuss here a possible phar-
macophoric model for these compounds and the stereoelectronic
requirements necessary to elicit the activity. In addition, a molec-
ular modeling study supported by quantum mechanic calculations
allowed us to design, synthesize and test new active compounds
of this series.
Compound 10 is a pteridine analogue, prepared from 2h by a
similar procedure, in which instead of a six membered ring it has
a seven membered ring fused to pyrimidine.
We have modified the free amino at C-2 of the pyrimidine ring
as well; thus, different derivatives related to 2 such as 11a,b,c,
Scheme 1. Synthetic sequence for the preparation of fused pyrimidines via nitrosation-aminolysis-reduction-cyclization from methoxypyrimidines.

M. Olivella et al. / Bioorg. Med. Chem. 20 (2012) 6109–6122
6111
Table 1
Structural features of compounds 2, 3 and 4 and 15
R2
N
R3
NH2
OCH3
N
NH2
NO
NO
NO
NO
N
N
N
R1
R1
R1
Ph
N
N
H
H3CO
H2
N
N
N
H
H2
N
N
N
H
N
N
H
H3CO
Type 4
Type 2
Type 3
Compound 15
Compd type 2
Compd type 3
Compd type 4
R1
R1
R1
R2
R3
a
b
c
d
e
f
g
h
i
(3-Cl)C6H4
Bu
Ad
(CH2)2O(CH2)2OH
(CH2)2-OH
CH2CO2Et
CHCH3CO2Et
(CH2)2CO2Et
CHBnCO2Et
(3-Cl)C6H4
Bu
Bn
Bn
iPr
Ph
iPr
CH3
Bn
H
H
H
H
H
H
CH(s-Bu)CO2H
CH(CH2OH)CO2H
Ad
Bn
(CH2)2CO2Et
Cyclohexyl
Ph
–(CH2)4–
–(CH2)4–
were obtained (Scheme 3). These compounds possess an ethylene
linker between amino and another potential hydrogen donor
and/or acceptor group, such hydroxyl 11a or acetoxy groups
11b,c. These molecules were prepared by a similar procedure to
that described above in Schemes 1 and 2; the modification of ami-
no group at C-2 was done in a first step from 2-chloro-4,6-dimeth-
oxypyrimidine I by nucleophilic substitution with ß-aminoethanol.
Finally, we have prepared several 5-nitroso derivatives, such 12,
13 and 14 with a 4-amino-2-methoxypirimidine moiety, in which
these two groups were exchanged respect to the previously
synthetized compounds. So, we have firstly prepared the nitroso-
pyrimidine 1c, isomer of 1a, from the commercial 4-amino-2,
6-dimethoxypyrimidine using the reported nitrosation proce-
dure,34b to be used as precursor for the preparation of compound
12, related to the set of compounds 2 (Scheme 4).
Compounds 2a, 2b, 5 and 14 displayed moderate to low
(although selective) antifungal activities (MICs between 31.2 to
>250 lg/mL) against the yeasts C.albicans, C. tropicalis, C. neoformans
and some of them against dermatophytes, being fungicide rather
than fungistatic. In contrast, Aspergillus spp. did not show suscepti-
bility to nitrosopyrimidines and related compounds (Table 2).
However, it is important to take into account that these MIC val-
ues refer to the minimum concentration necessary to inhibit 100% of
the fungal growth. If, as recommended by standardized docu-
ments,35,36 less stringent end-points such as MIC50 (the minimum
concentration of compounds that inhibit 50% of growth, respec-
tively) are determined, lower minimum inhibitory concentrations
for each compound would be obtained. In addition, the application
of less stringent end-points many times provide a better correlation
with other measurements of antifungal activity.22,23
As a last structural change, we included the lactamic form in the
pyrimidine ring instead of the heteroaromatic one, so we per-
formed a benzylation reaction on 4-amino-2-methoxypyrimidin-
4(3H)one III to get the corresponding N-benzylated derivative IV,
that is obtained along with its O-benzylated heteroaromatic iso-
mer V. Once those isomeric derivatives were separated, they were
independently subjected to our nitrosation procedure (see Scheme
Compounds 2a, 2b, 5 and 14 were tested again, against C. albi-
cans ATCC 10231 and C. neoformans ATCC 32264 by using a similar
microplate design than that used for determining MIC100. The aim
was determine the inhibition percentages displayed by each com-
pound at the different concentrations, which were calculated with
the aid of a microplate reader, as explained in material and meth-
ods. These results are presented in Table 3.
34a
5) to get their 5-nitroso derivatives, 13 and 14 respectively.
Compound 5 displayed just a moderated antifungal activity
Finally, after performing the antifungal assays of the above
compounds, and as a direct consequence of the results obtained
in our molecular modeling study (see Section 2.3), we decided to
synthesize compound 15 (Table 1) as described in Scheme 4 using
aniline in the selective monoaminolysis of 1c.
(MIC50 = 31.25–62.5
2a, 2b and 14 which displayed higher antifungal activities with
MIC50 values ranging from 1.95 to 7.8 g/mL.
Since the toxicity of any potential antifungal agent is a critical
aspect for its usefulness and limitations, the acute toxicity for com-
pounds 2a, 2b and 14 were evaluated using a toxicity test on fishes
which has been successfully used by our group with other anti-
fungal compounds.20–37 Our results indicated that compound 14
possessed a high acute toxicity. It displayed 100% of mortality at
lg/mL), which is in contrast with compounds
l
In summary compounds 9a–b, 10, 11a–c, 12, 13 and 15 are
reported here for the first time.
2.2. Antifungal activity and structure–activity relationships
1.6
Suplementary material). In contrast, acute toxicities were not ob-
served (0% mortality) with 38.4 g/mL of 2a or 35 g/mL of 2b,
concentrations clearly higher than the antifungal ones for both
compounds.
lg/mL and 0.8 lg/mL at 24 and 48 h respectively (Table 1S in
We tested, at first, the antifungal effects of the 23 previously
reported compounds: 1a, 2a–i, 3a–g, 4a–c, 5, 6, 7 and 14 against
a panel of clinically important fungi such as the yeasts C. albicans,
C. tropicalis, C. neoformans, the hialohyphomycetes Aspergillus fla-
vus, A. fumigatus and A. niger, and the dermatophytes Microsporum
gypseum, Trichophyton rubrum and Trichophyton mentagrophytes.
l
l
Nitrosopyrimidines and analogues reported in the previous sec-
tion provided a considerable diversity of chemical structures and

6112
M. Olivella et al. / Bioorg. Med. Chem. 20 (2012) 6109–6122
Scheme 2. Preparation of new pteridine analogues from alkoxynitrosopyrimidines.
Scheme 3. Preparation of some N2,N4-disubstituted 2,4 diamino-5-nitrosopyrimidines.
5 displayed only a moderate antifungal activity. The pres-
ence of a non-substituted amino group in type 2-compounds
appears to be necessary to produce the antifungal activity.
Type 3-compounds, possessing substituents of different size
3a, 3b, 3c, 3d, 3e and 3f were all inactive. These results sug-
gest that the presence of donor or acceptor groups such as
NH2 or OCH3 in the central ring might be necessary to pro-
duce the biological response. Compounds 3g and 7 were also
inactive giving an additional support to this hypothesis. In
order to corroborate this assumption, we synthesized com-
pounds 12 and 13 possessing different central rings. None
of them displayed any antifungal activity supporting, at least
in part, our assumption.
OCH3
N
NO
N
H
NO
N
N
CH2Cl2, rt
H3CO
N
NH2
H3CO
N
12
NH2
1c
Scheme 4. Synthesis of 4-amino-2-methoxy-5-nitroso-6-(pyrrolindin-1-yl)pyrim-
idine 12.
therefore, from the analysis of their structures and the antifungal
activities displayed, some Structure–Activity Relationships can be
extracted. This SAR analysis was performed considering the follow-
ing structural changes:
b) Regarding the results shown in Table 2 it is evident that the
presence of an aromatic central ring type 2 might be impor-
tant for the antifungal activity; however it is not by itself
enough to produce the biological response. The type of sub-
stituents on the central ring appears to play an important
role in the antifungal effect. Replacement of hydrophobic
groups by more polar systems at N4-substituent gives inac-
tive compounds. Compounds 2d–i, and 4a,b illustrate this
situation very well. In contrast, the replacement of m-ClPhe
(a) Different types of aromatic central rings
(b) Different substituents on the aromatic central ring
(c) Molecular flexibility
a) The active compounds 2a and 2b have the same aromatic
central ring; in contrast compounds 5 and 14 possess differ-
ent aromatic central rings. It should be noted that compound

M. Olivella et al. / Bioorg. Med. Chem. 20 (2012) 6109–6122
6113
O
O
PhH2C
NO
PhH2C
H3CO
isoamyl nitrite
N
N
N
DMSO, rt
NH2
H3CO
N
IV
N
13
NH2
O
BrCH2Ph
HN
+
K2CO3, DMSO
OCH2Ph
OCH2Ph
NO
H3CO
N
NH2
isoamyl nitrite
DMSO, rt
NH2
III
N
H3CO
N
H3CO
N
NH2
V
14
34a
Scheme 5. Synthesis of lactamic 4-amino-3-benzyl-2-methoxypyrimidin-4(3H)one 13 along with its 4-O-benzyl heteroaromatic isomer 14
.
Table 2
Minimum inhibitory concentrations and Minimum Fungicide Concentration (MIC and MFC in
lg/mL, showed as MIC/MFC). Ca (C. albicans), Ct (C.tropicalis), Cn
(C. neofromans), Afu A fumigatus, Ani (A. niger), Mg (M. gypseum), Tr (T. rubrum), Tm (T. mentagrophytes).
Compound 15 is highlighted in grey because it was synthesized and tested in a second instance as the result of a computational design. See Section 2.3.
Table 3
Antifungal activity (inhibition %, and MIC50 values) obtained for the most active compounds against C. albicans, C. tropicalis and C. neoformans.
Concentration in lg/ml
Candida albicans
Comp
2a
2b
5
14
250
100
100
100
100
100
125
100
100
86.8 1.8
100
62.5
92.7 0.3
100
43.4 0.1
100
100
31.25
15.6
7.8
3.9
MIC50
3.9
7.8
62.5
7.8
91.8 0.5
98.2 0.9
38.9 4.3
100
86,3 0.4
86.2 0.6
34.3 0.7
99.6 1.4
98.1 4.8
80.3 4.6
50.4 4.1
23.2 2.1
56.9 1.6
85.0 3.0
53.3 0.0
20.0 1.38
29.3 5.2
47.2 2.7
4.7 2.6
15
100
100
7.8
Amp
0.49
Candida tropicalis
2a
2b
5
14
15
Amp
100
100
100
100
100
100
100
81.2 0.8
100
100
87.9 1.5
100
53.3 6.6
100
85.2 1.2
93.2 5.8
47.3 0.5
100
75.2 4.8
85.2 1.8
41.5 3.9
96.7 2.6
88.2 8.9
64.5 2.9
57.7 6.1
34.5 3.8
56.7 2.6
87.2 2.2
48.8 6.4
16.3 5.9
36.4 6.4
19.7 1.3
4.1 2.9
3.9
7.8
62.5
7.8
7.8
100
100
0.49
Cryptococcus neoformans
2a
2b
5
14
15
Amp
100
100
100
100
100
100
100
88.0 2.9
100
100
83.7 2.0
85.5 1.4
51.1 0.2
100
81.3 4.8
75.0 8.6
56.9 4.9
88.1 2.9
100
80.6 6.2
63.1 8.6
39.2 1.2
80.2 3.2
100
74.9 8.9
52.4 1.3
9.5 1.9
78.1 1.5
91.2 1.6
53.2 3.9
11.3 1.1
0
49.2 4.1
76.2 1.9
3.9
7.8
31.25
3.9
1.95
0.98
100
of compound 2a by butyl in compound 2b was well toler-
that compound 2b possesses just one aromatic ring. This is a
ated, keeping the antifungal activity. It is interesting to note
striking difference with respect to the structural require-

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M. Olivella et al. / Bioorg. Med. Chem. 20 (2012) 6109–6122
ments previously reported for homoallylamines.22,24,28,29
(a)
However, it appears that not any hydrophobic substituent
is operative; note that the replacement of a butyl group of
2b by adamantine in 2c give an inactive compound. In order
to evaluate if a similar behavior might be also observed for
the N4-substituents if a 2-amino group is modified, we syn-
thesized compounds 11a,b,c. Our results indicated that the
presence of relativelly polar N4-substituents produces loss
of activity (compare 2a with 11c). Compounds 11a and
11b give additional support to this observation.
c) Compound 5, possessing a conformationally constrained
structure, displayed only a moderate antifungal activity;
whereas compound 7 possessing a comparable restricted
molecular flexibility was totally inactive. These results sug-
gest that a relative molecular flexibility could be necessary
in these compounds to produce the antifungal activity. To
better understand such situation we synthesized and tested
the conformationally restricted compounds 10, 9a and 9b.
None of these compounds displayed antifungal activity. It
should be noted that the only structural difference between
compounds 6 and 5 is the replacement of N by C. However,
this apparently minor structural change produces the com-
plete loss of activity for compound 6.
(b)
It must be pointed out that all the above structural changes
were carried out without taking into account the possible changes
in the physicochemical properties introduced with each structural
modification. None of the new nitrosopirimidines synthesized (9a,
9b, 10, 11a–c, 12 and 13) displayed antifungal activity; they do not
completely inhibit the growth of fungi even at high concentrations.
Therefore, these results prompted us to develop a new nitrosopyr-
imidine structure but now using a more rational design based on
theoretical calculations. Thus, in the next step of our study we
decided to perform an exhaustive conformational and electronic
study on the active compounds 2a, 2b and 5 in order to determine
the stereoelectronic characteristics which could be useful to design
new compounds possessing similar properties.
Figure 1. Spatial view of the lowest-energy conformations obtained for compounds
2a (a) and 2b (b). In these figures carbon atoms are marked with grey; chlorine with
green; hydrogens with white; nitrogens with blue and oxygen with red. The
different torsional angles are also shown in this figure.
method to answer the above questions is to find out how the local
energy minima and the transition states are linked together, and
this requires an exploration of the complete PESs. The PESs were
calculated using a 12 Â 12 grid generated by rotating through U2
and U3 in 30° increments from À180 to 180°. At each point a com-
plete geometry optimization was performed with U2 and U3 frozen
at their respective grid values. Thus, considering that U1 might
adopt two conformations, two different PESs were calculated for
compound 2a in which U1 is located near to 0° and near to 180°
(Fig. 2a and 2b, respectively).
The lowest-energy structures of compound 2a are planar forms
possessing a stabilizing hydrogen bond (see Fig. 1a). These con-
formers also can be seen in the contour and surface plots of fig.
2a as the minima centered at (U2 and U3) (0.0, 0.0, conformation
1) and its symmetry-related partner (0.0, 180.0, conformation 2).
The set of symmetry-related anti-coplanar structures (near to
180, 180 and 180, 0, conformations 5 and 6, respectively) lies
approximately 17 Kcal/mol above the preferred conformers. It is
interesting to note that the interconversion between the two pre-
ferred forms requires about 3 kcal/mol indicating that this mole-
cule possesses a high molecular flexibility in such spatial zone. In
contrast, the conformational interconversion betwen conformer 1
with 4 requires about 22 Kcal/mol.
2.3. Conformational and electronic study of nitrosopyrimidines
In an attempt to explain the above experimental results as well
as to find a new active compound, we conducted a computer-as-
sisted conformational and electronic study on compounds 2a, 2b
and 5. The purpose was to obtain more precise information about
how these compounds resemble each other in terms of the spatial
orientations of the essential components to produce the biological
response. Compound 2a looks like a relatively simple conforma-
tional problem with three torsional angles (U1, U2 and U3, Fig. 1).
In this compound the orientations of the ring systems are de-
scribed by the dihedral angles U2 and U3; whereas the orientations
of the nitroso group are described by the dihedral angle U1. Our
calculations predicts that the torsional angle U1 possesses only
two possible quasi-planar conformations: one of them near to 0°
which allows the formation of a hydrogen bond between the oxy-
gen atom of nitroso group and the NH of the connecting chain; in
contrast in the other conformer the oxygen atom of nitroso group
is located in the opposite position giving values near to 180°. Com-
pound 2a has a numerable number of conformers on its Potential
Energy Surface (PES) but, where does one form change to the other
and how far from an energy minimum can the molecule stray away
before ceasing to be in a conformation referred to as the energy
minimum? It is clear, that information about local and global min-
ima of a molecule such as compound 2a is not enough. We need to
have at least a good notion of the shape and also some indication
about the dynamic behavior of the internal degrees of freedom of
this molecule. Probably the most comprehensive computational
Figure 2b shows the PES obtained for compound 2a scanning U2
and U3 when U1 is located near to 180°. The four different low-en-
ergy conformers (conformations 3, 4, 7 and 8) might be well appre-
ciated in this surface. Note that the spatial positions obtained for
the different conformers as well as their respective energy gaps
displayed in Figure 2a and Table 4 are closely related but they
are not exactly equal. This is an expected result considering that
they were obtained from different levels of calculations.
It should be noted that these PESs were obtained from RHF cal-
culations using a modest basis set (3-21G); therefore, in order to
confirm that the molecular flexibility and the relative stability of

M. Olivella et al. / Bioorg. Med. Chem. 20 (2012) 6109–6122
6115
(a)
6
2
5
1
(b)
8
4
7
3
Figure 2. Contour graphic of the PES obtained for compound 2a from RHF/3-21G calculations. In figure 2a the torsional angle /1 adopts values near to cero and in Figure 2b
near to 180°. Full cycle of rotation (from 0° to 360°) is shown for variables /2 and /3. The iso-energy curves included in an energy window of 4 Kcal/mol are denoted in red.
Table 4
the various conformers are correct, we performed more accurate
Different conformations of compound 2a obtained from B3LYP/6-31G(d)
optimizations
DFT (B3LYP/6–31G(d)) calculations. With them, we evaluated the
molecular flexibility of compound 2a generating 1-D cross-sec-
Conf
Torsional Angles
Energy Gap D )
E (Kcal.molÀ1
tions, that is potential energy curves, along U2 and U3 (Fig. 3). To
understand the significance of the rotation barriers, it is important
to look not just at the magnitudes of the energy barriers, but at the
complete energy versus rotation angle behavior. By looking first
the torsional angle U2, we see that the energy minima occur at
U2 = 18, 150, 210 and 345°, the quasi-planar conformations, while
the energy maxima occur near to 90 and 270°, the perpendicular
forms. This profile can be understood by recognizing that the pla-
nar conformation is stabilized by resonance structures which per-
U1
U2
U3
1
2
3
4
5
6
7
8
180.0
179.97
0.0
0.0
0.0
0.0
0.0
146.31
145.84
156.52
203.49
0.0
180.1
0.0
180.0
169.71
344.5
152.8
207.17
0.00
0.65
4.53
5.12
13.54
13.73
14.19
14.19
0.0
172.33
172.63
352.8
7.17
mit the conjugated
p-electron system to delocalize the molecular
system. When the torsional angle U2 is rotated almost 90°, reso-
nance stabilization is eliminated, and there is an increase in en-
ergy. This increase due to the loss of resonance energy is
responsible, at least in part, for the approximately 7 Kcal/mol rota-
tion barriers seen in Figure 3. This barrier indicates that this rota-
tion is somewhat restricted. In contrast, the rotation barrier
displayed for torsional angle U3 is very low (less than 1 Kcal/mol,
Fig. 3). Although the minima for U3 are located near to 90 and
270°, DFT calculations predicts that this is practically a free rota-
tion and therefore different conformations might be adopted for
U3 without a significant energy requirement. In Table 4, the differ-

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M. Olivella et al. / Bioorg. Med. Chem. 20 (2012) 6109–6122
φ2
φ3
7
6
5
4
3
2
1
0
0
90
180
270
360
φ
Figure 3. Potential Energy Curves (PECs) obtained for the torsional angles /2 and /3 of compound 2a. Each PEC was calculated at B3LYP/6-31G (d) level of theory.
ent conformers obtained for compound 2a optimized from B3LYP/
6–31G(d) calculations, are summarized.
compound 2a, we evaluated the conformational properties of a
hypothetical compound 15. Note that in this compound the –O–
CH2– connecting chain was replaced by –NH– trying to mimic com-
pound 2a. As we expected, the conformational behavior obtained
for compound (named compound 15) was very similar to that at-
tained for 2a. The potential energy curves obtained for the torsional
angles U2 and U3 of compound 15 are shown in Figure 4.
Next, we performed a comparative conformational study of com-
pounds 2a, 2b and 5. In this comparative analysis, we also included
the hypothetical compounds 15.Figure 5 shows a spatial view of the
superimposed conformations of these compounds. From this figure,
it is clear that compound 15 can adopt spatial dispositions very sim-
ilar to those of the active compounds 2a, 2b and 5.
It is clear that not only the conformational aspects but the
electronic properties of these compounds are determinant for the
antifungal activities. Thus, the knowledge of the stereo-electronic
attributes and properties of these nitrosopyrimidines will contrib-
ute significantly to the elucidation of the structural requirements
to produce the antifungal activity. Molecular electrostatic potentials
(MEP) are of particular value because they allow the visualization
and assessment of the capacity of a molecule to interact electrostat-
ically with a putative-binding site.38–40 MEP can be interpreted in
The conformational behavior of compound 2b is mainly deter-
mined by six torsional angles (U1–U6, Fig. 1b). As we expected,
the conformational behaviors obtained for U1 and U2 are very sim-
ilar to those observed for the torsional angles U1 and U2 of com-
pound 2a. The nitroso group adopts only two forms (near to 0°
and near to 180°), whereas the potential energy curve obtained
for the dihedral angle U2 is closely related to that attained for U2
of compound 2a. The rest of the torsional angles displayed rotation
barriers lower than 3 kcal/mol suggesting that the hydrocarbon
side-chain of compound 2b is flexible enough to adopt different
conformations, from a folded form to an extended one. To catego-
rize the obtained structures, we introduced, intuitively rather than
by a precise definition, four forms: fully folded (FF), open folded
(OF), and fully extended (FE). By FF form we understand a close
structure with the flexible side chain (butyl group) completely
folded. By FE form we understand a form with a linearized con-
necting chain. By OF we denote a form intermediate between
folded and extended. Figure 1b shows a spatial view of the low-
est-energy conformation obtained for compound 2b; note that, in
this conformer, the flexible side chain displayed a partially folded
spatial ordering.
terms of
a stereo-electronic pharmacophore condensing all
Compound 5 possesses a very restricted molecular flexibility
being a spatial ordering with U2 near to 60° the highly preferred
conformer. In fact, this molecule possesses three equivalent con-
formations with U2 near to 60°, 180° and 300°. It is clear, however,
that the conformational interconversion is a restricted process. DFT
calculations predict an energy barrier of more than 7.8 Kcal/mol for
such process. As it was previously discussed, this restricted molec-
ular flexibility might be, at least in part, responsible for the lower
antifungal activity obtained for this compound in comparison to
2a and 2b.
It should be noted that compound 14 was the only active com-
pound possessing a central aromatic ring different to that of com-
pounds type 2 or 5. Unfortunately this compound displayed a high
acute toxicity in comparison with compounds 2a and 2b. Thus, we
hypothesize that one of the potentially responsible groups for such
effect could be the oxygen atom at the connecting chain in 14. With
the aim to design a new compound possessing a central ring similar
to 14 but on the other hand with similar structural feature to that of
available information on the electrostatic forces underlying affinity
and specificity. More positive potentials reflect nucleus predomi-
nance, while less positive values represent rearrangements of elec-
tronic charges and lone pairs of electrons. The fundamental
application of this study is the analysis of non-covalent interac-
tions,40 mainly by investigating the electronic distribution in the
molecule. Thus, this methodology was used to evaluate the elec-
tronic distribution around molecular surface for compounds re-
ported here.
Figure 6 gives the Molecular Electrostatic Potentials (MEPs) ob-
tained for compounds 2a, 2b, 5 and 15. The MEPs obtained for com-
pounds 2a, 2b and 15 account for the general characteristics of the
electronic behaviour for the active compounds. The general pattern
is very similar for all these systems. The MEPs exhibit four charac-
teristic regions: a clear minimum value (deep red zone with
V(r)min ꢀ À0.0874 el/au3) in the vecinity of the NO group; two po-
sitive regions (deep blue zones with V(r)max ꢀ 0.054 el/au3) located
near to the NH2 and OCH3 groups and an extended hydrophobic

M. Olivella et al. / Bioorg. Med. Chem. 20 (2012) 6109–6122
6117
8
7
6
5
4
3
2
1
0
φ2
φ3
0
90
180
270
360
φ
Figure 4. Potential Energy Curves (PECs) obtained for the torsional angles /2 and /3 of compound 15. Each PEC was calculated at B3LYP/6-31G (d) level of theory.
to synthesize (see Chemistry section) and to test the antifungal
activity for this compound in order to corroborate the results ob-
tained from our theoretical study. Interestingly enough, compound
15 displayed the strongest antifungal activity obtained for this ser-
ies. This compound displayed 76 % inhibition at a relatively low
molar concentration (3.9 lg/mL) possessing a MIC50 = 1.9 lg/mL
against C. Neoformans. Similar results were obtained against the
other two fungi tested here (Table 2). It is clear that these experi-
mental results are in a complete agreement with our theoretical re-
sults and give support to the molecular modeling study.
We evaluated the acute toxicity of this new compound. Our re-
sults indicated that compound 15 displayed just a moderate acute
toxicity; being non toxic (0% mortality) at 35
toxicity is markedly lower than that previously obtained for com-
pound 14 (non-toxic at 5 g/mL), the acute toxicity obtained for
lg/mL. Although this
Figure 5. Stereoview of overlapping of global minima for 2a (light blue), 2b
(yellow), 5 (fuchsia) and 15 (green).
l
compound 15 is still significant and could be a limitation for the
potential usefulness of this compound as antifungal agent.
Finally, it is important to highlight that the mechanism of action
of these compounds has not been determined yet; therefore it is
very difficult to perform a clear and definitive SAR study with this
series. Thus, it is prudent to remark that the results of our SAR
analysis might be considered like preliminary and exploratory
but not definitive ones; however in our opinion these results could
be useful to orientate new studies. As was pointed out, unfortunat-
elly we have not yet definitive results about the possible molecular
mechanism for these nitrosopyrimidines. Different bioassays are
being carried out in our laboratories in order to obtain sufficient
information regarding this matter. In addition new nitrosopyrimi-
dines are now being synthesized in our laboratory considering the
pharmacophoric pattern obtained.
zone (deep and light green zone with an almost a neutral potential
V(r)med ꢀ À0.0022 el/au3) throughout the second ring (or the flex-
ible side chain in the case of compound 2b). It should be noted that
the MEP obtained for compound 14 displayed the general charac-
teristics observed for all the active molecules in this series. This
MEP is shown in figure 1S as Supplementary material.
The MEP obtained for compound 5 displayed some differences
with respect to those of the compounds 2a, 2b. This MEP does
not show a deep red zone; displaying a generalized hydrophobic
potential. The different electronic behavior added to the restricted
molecular flexibility could explain the lower antifungal activity ob-
tained for this compound with respect to the active molecules 2a
and 2b.
FromFigure 6 it is clear that compounds 2a, 2b and 15 displayed
a closely related electronic distribution. Thus, our theoretical cal-
culations indicated that compound 15 possesses a similar stereo-
electronic behavior in comparison with the active compounds 2a
and 2b.
3. Conclusions
The synthesis, in vitro evaluation and SAR studies of 32 nitros-
opyrimidines and derivatives acting as antifungal agents are re-
ported. A molecular-modeling study using quantum mechanical
calculations allowed us to design a new compound possessing
the potential parent for this series. Thus, we designed, synthesized
and tested compound 15 which displayed the strongest antifungal
activity among the nitrospyrimidines reported here. From our
2.4. Experimental support for the theoretical calculations
Our molecular modeling study indicated that compound 15
possesses the minimal structural characteristics to produce the
antifungal effect. Thus, in the next step of our study we decided

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M. Olivella et al. / Bioorg. Med. Chem. 20 (2012) 6109–6122
Figure 6. Electrostatic potential-encoded electron density surfaces of compounds 2a (a), 2b (b), 5 (c) and 15 (d). The surfaces were generated with GAUSSIAN 03 using B3LYP/
6–311++G(d,p) single point calculations. The coloring represents electrostatic potential with red indicating the strongest attraction to a positive point charge and blue
indicating the strongest repulsion. The electrostatic potential is the energy of interaction of the positive point charge with the nuclei and electrons of a molecule. It provides a
representative measure of overall molecular charge distribution. The color-code is shown at the left.
results (antifungal activity and acute toxicity) it appears that com-
pounds 2a, 2b and 15 are interesting candidates for further studies.
On the other hand, our SAR study, supported by theoretical cal-
culations, helped us to identify and understand the minimum
structural requirements for the antifungal action of these com-
pounds. Thus, we believe that our results could provide a guide
in the design of nitrosopyrimidines with this biological activity.
4.1. Preparation of alkoxypyrimidines
2-Amino-4,6-bis-cyclohexylmethoxy-5-nitrosopyrimidine
1b: A suspension of 2-amino-4,6-dimethoxy-5-nitroso-pyrimidine
1a (368 mg, 2.00 mmol) in cyclohexylmethanol (10 mL) was
heated at 100 °C until no starting material was visualized by TLC
(Silica, eluent: CH2Cl2/MeOH 9:1, v/v). Then, solvent was evapo-
rated off by vacuum distillation and hexane was added. The precip-
itate was filtered, washed and finally, dried in
a vacuum
4. Experimental chemistry
dessiccator over potassium hydroxide pellets obtaining compound
1b as a green solid (557 mg, 80%). mp 165–167 °C . 1H NMR
(400 MHz, DMSO-d6, 25 °C) d (ppm): 8.18 (bs, 2H, NH2), 4.20 (d,
J = 8.3 Hz, 4H, 2OCH2), 1.80–1.63 (m, 12H, CH2), 1.31–1.13 (m,
10H, CH, CH2). 13C NMR (100 MHz, DMSO-d6) d: 163.0 (s), 141.5
Melting points were determined on a Barstead Electrothermal
9100 apparatus and are uncorrected. IR spectra were recorded in
KBr disks on Bruker TENSOR 27 spectrophotometer from ‘‘Centro
de Instrumentación Científico-Técnica (CICT) at Universidad de
Jaén’’. 1H and 13C NMR spectra were recorded on a Bruker Avance
400 spectrophotometer (CICT) operating at 400 MHz and
100 MHz respectively, using CDCl3 and [D6]DMSO as solvents and
tetramethylsilane as internal standard; Multiplicity of the carbons
was assigned with DEPT and gHSQC experiments, although usual
abbreviations according to off-resonance decoupling are used: (s)
singlet, (d) doublet, (t) triplet, (q) quartet. The same abbreviations
were used for the multiplicity of signals in 1H NMR and also: (m)
multiplet, (bs) broad singlet. Coupling constants (J) are reported
in Hertz. Mass spectra were run on a SHIMADZU-GCMS 2010-DI-
2010 spectrometer (equipped with a direct inlet probe) operating
at 70 eV. High resolution Mass spectra were run on a Waters Micro-
mass AutoSpec-Ultima spectrometer (equipped with a direct inlet
probe) operating at 70 eV. Silica gel aluminum plates (Merck 60
(s), 71.8 (t), 36.6 (d), 29.0 (t), 26.0 (t), 25.2 (t). IR (KBr):
3223, 2937, 1634, 1579, 1357, 1233 cmÀ1. UV–vis (MeOH): kmax
nm (log ) = 239 (4.08), 337 (4.44), 659 (1.80). MS (EI, 70 eV):
348 (M+Á,14), 252 (13), 156 (100), 139 (16), 55 (24). HR–MS (EI):
m = 3306,
e
C18H28N4O3 requires 348.2161, found 348.2164. Anal. Calcd for
C
18H28N4O3Á(348.44): C, 62.05; H, 8.10; N, 16.08. Found: C, 61.97;
H, 8.14; N, 15.73.
2-(2-Amino-6-cyclohexylmethoxy-5-nitroso-pyrimidin-4-
ylamino)-propionic acid ethyl ester 8a: To a suspension of 1b
(174 mg, 0.50 mmol) in t-BuOH (5 mL),
L-Alanine ethyl ester
hydrochloride (94 mg, 0.60 mmol) and Et3N (61.3 mg, 0.60 mmol)
were added. The mixture was stirred at 60 °C and monitored by
TLC (Silica, eluent: CH2Cl2/MeOH 9:1, v/v) until no starting mate-
rial was visualized (18 h). Then, solvent was put off by vacuum dis-
tillation and CH2Cl2 was added on the oil obtained. The organic
solution was washed with water (3 Â 15) and dried on anhydrous
sodium sulfate. Finally, solution was filtered and the solvent was
removed under vacuum evaporation to offer an oil which was puri-
fied by flash column chromatography on silica gel (eluent: CH2Cl2/
Me2CO 10:0.4, v/v) to render titled compound as a blue oil,
(124 mg, 71 %). 1H NMR (400 MHz, CDCl3, 25 °C) d (ppm): 11.52
(d, J = 7.1 Hz, 1H, NH), 5.63 (bs, 2H, NH2), 4.70 (q, J = 7.1 Hz, 1H,
NCH), 4.31 (d, J = 6.6 Hz, 2H, OCH2), 4.14 (q, J = 7.1 Hz, 2H, OCH2),
1.89–1.64 (m, 7H, CH, 3CH2), 1.44 (d, J = 7.7 Hz, 3H, CH3), 1.24 (t,
J = 7.1 Hz, 3H, CH3), 1.14–0.92 (m, 2H, 2CH2). 13C NMR (100 MHz,
F254) were used for analytical TLC. The microwaves assisted reac-
tions were carried out in a 10 mL glass sealed tube for focused
mono-mode microwaves oven (‘‘Discover’’ by CEM Corpotation)
working at standart mode by controlling the target temperature.
The amines, aminoesters, 2-amino-4,6-dimethoxypyrimidine,
4-amino-2,6-dimethoxypyrimidine were purchased from Aldrich,
Fluka and Acros (synthesis reagent grades) and were used without
further purification. Solvents were purchased from the same
vendors but purified according standard protocols. 6-Amino-2-
methoxypyrimidin-4(3H)-one and nitrosopyrimidines 1a, 2a–i,
3a–g, 4a–b were synthezed using reported procedures.34a

M. Olivella et al. / Bioorg. Med. Chem. 20 (2012) 6109–6122
6119
CDCl3) d: 172.1 (s), 171.8 (s, CO), 163.5 (s), 149.7 (s), 139.2 (s), 73.0
(t), 61.4 (t), 48.9 (d), 37.1 (d), 29.7 (t), 26.3 (t), 25.6 (t), 17.9 (q), 14.1
(q). MS (EI, 70 eV): 351 (M+Á,88), 182 (74), 166 (68), 140 (100).
2-(2-Amino-6-cyclohexylmethoxy-5-nitroso-pyrimidin-4-
ylamino)-3-phenyl-propionic acid ethyl ester 8b: To a suspen-
nylamino)-6-methoxy-5-nitroso-pyrimidin-2-ylamino]-ethyl ester
11c: To a suspension of II (712 mg, 4.00 mmol) in anhydride acetic
(2.5 mL), a drop of acetic acid was added. The mixture was stirred
at room temperature and monitored by TLC (Silica, eluent: CH2Cl2/
MeOH 9:1, v/v) until no starting material was visualized (18 h).
Then, cold water was slowly added and next, aqueous solution
was neutralised with solid sodium hydrogen carbonate and ex-
tracted with diethyl ether (3 Â 15). Organic phases were combined,
dried on anhydrous sodium sulfate and finally concentrated to give
an oily residue (683 mg, 2.83 mmol, 71%). This oil was nitrosated
sion of 1b (174 mg, 0.50 mmol) in t-BuOH (5 mL), L-phenylalanine
ethyl ester hydrochloride (139 mg, 0.60 mmol) and Et3N (61.3 mg,
0.60 mmol) were added. The mixture was stirred at 60 °C and
monotoried by TLC (Silica, eluent: CH2Cl2/MeOH 9:1, v/v) until
no starting material was visualized (12 h). Then, solvent was put
off by vaccum destillation and dichloromethane was added on
the oil obtained. The organic solution was washed with water
(3 Â 15) and dried over anhydrous sodium sulfate. Finally, solution
was filtrated and the solvent was removed under vacuum evapora-
tion to offer an oil which was purified by flash column chromatog-
raphy on silica gel (eluent: CH2Cl2/Me2CO 10:0.4, v/v) to render
titled compound as a blue oil (203 mg, 95 %). 1H NMR (400 MHz,
DMSO-d6, 25 °C) d (ppm): 11.55 (d, J = 7.1 Hz, 1H, NH), 7.39–7.32
(m, 5H, Ph), 5.54 (bs, 2H, NH2), 4.93 (dt, J = 7.7, 5.5 Hz, 1H, NCH),
4.31 (d, J = 6.6 Hz, 2H, OCH2), 4.11 (q, J = 7.1 Hz, 2H, OCH2), 3.21–
3.05 (dd, J = 7.7, 7.1 Hz, 2H, CH2), 1.90–1.67 (m, 7H, CH, 3CH2),
1.19 (t, J = 7.1 Hz, 3H, CH3), 1.30–0.96 (m, 2H, 2CH2). 13C NMR
(100 MHz, DMSO-d6) d: 172.1 (s), 170.5 (s, CO), 163.4 (s), 149.7
(s), 139.2 (s), 135.8 (s), 129.2 (t), 128.6 (t), 127.1 (t), 122.9 (d),
73.0 (t), 61.4 (t), 54.7 (d), 38.1 (t), 37.1 (d), 29.7 (t), 26.3 (t), 25.6
(t), 14.1 (q). MS (EI, 70 eV): 427 (M+Á,1), 352 (100), 256 (21), 182
(79), 166 (69), 149 (33), 140 (88), 55 (71).
2-(4-Methoxy-5-nitroso-6-phenylamino-pyrimidin-2-ylamino)
-ethanol 11a: To a solution of commercial 2-cloro-4,6-dimethoxy-
pyrimidine I (712 mg, 4.00 mmol) in t-BuOH (10 mL), 2-aminoeth-
anol (0.5 mL, 4.0 mmol) was added. The mixture was stirred at
85 °C and monotoried by TLC (Silica, eluent: CH2Cl2/MeOH 9:1, v/
v) until no starting material was visualized (18 h). Then, the sol-
vent was eliminated by vacuum distillation and diethylether was
added to the oil obtained. The organic solution was washed with
water (3 Â 15), dried on anhydrous sodium sulfate and finally con-
centrated to an oily residue which was purified by flash column
chromatography on silica gel (eluent: CH2Cl2/MeOH 10:0.4, v/v)
to render 2-(4,6-dimethoxy-pyrimidin-2-ylamino)-ethanol II as a
yellow oil (606 mg, 76%). 1H NMR (400 MHz, CDCl3, 25 °C) d
(ppm): 5.50 (bs, 2H, OH, NH), 5.39 (s, 1H, CH), 3.81 (s, 6H,
2OCH3), 3.82–3.76 (q, J = 4.9 Hz, CH2), 3.57–3.52 (q, J = 5.5 Hz,
CH2). 13C NMR (100 MHz, CDCl3) d: 172.1 (s), 162.2 (s), 79.0 (d),
63.2 (t), 53.7 (q), 44.3 (t). MS (EI, 70 eV): 199 (M+Á,20), 168 (100),
155 (23), 139 (18), 69 (63).
following the general procedure34a to afford
a green solid
(497 mg, 65%). mp 178–180 °C (d) . 1H NMR (400 MHz, DMSO-d6,
25 °C) d (ppm): 8.93 (t, J = 5.5 Hz, 1H, NH), 4.17 (t, J = 5.5 Hz, 2H,
OCH2), 3.98 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 3.62 (q, J = 5.5 Hz,
2H, NCH2), 1.99 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) d:
170.3 (s, CO), 161.0 (s), 141.5 (s), 62.0 (t), 54.3 (q), 40.4 (t), 20.7
(q). MS (EI, 70 eV): 270 (M+Á,12), 166 (5), 122 (7), 87 (42), 43 (100).
To obtain compound 11b, Aniline (93 mg, 1.0 mmol) was added
to a suspension of the above green solid (90 mg, 0.33 mmol) in
H2O/MeOH (6 mL, 2:1, v/v). The mixture was stirred at room tem-
perature and monitored by TLC (Silica, eluent: CH2Cl2/MeOH 9:1, v/
v) until no starting material was visualized (24 h). Then, the pre-
cipitate was filtered, washed and finally, dried in a vacuum dessic-
cator over potassium hydroxide pellets offering titled compound as
an orange solid (58 mg, 53%). mp 128–130 (d). 1H NMR (400 MHz,
DMSO-d6, 25 °C) d (ppm): 13.68, 13.30 (bs, 1H, NH), 8.99 (bs, 1H,
NH), 7.73 (m, 2H, Ph), 7.38 (m, 2H, Ph), 7.18 (m, 1H, Ph), 4.18 (m,
2H, OCH2), 4.12, 4.09 (s, 3H, OCH3), 3.71 (s, 1H, NCH2), 3.60 (s,
1H, NCH2), 2.00, 1.90 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6)
d: 171.0 (s, CO), 170.4, 170.3 (s), 162.0, 161.7 (s), 147.4, 147.0 (s),
138.4, 138.3 (s), 137.2, 136.9 (s), 129.0 (d), 125.1, 124.9 (d),
122.8 (d), 62.3, 62.0 (t), 54.5 (q), 41.1, 41.0 (t), 20.8, 20.7 (q). IR
(KBr):
m
= 3193, 2964, 1740, 1586, 1559, 1165 cmÀ1. MS (EI,
70 eV): 331 (M+Á,13), 272 (3), 254 (2), 227 (5), 87 (9), 43 (100).
HR–MS (EI): C15H17N5O4 requires 331.1281, found 331.1284.
To obtain compound 11c, 3-Chloroaniline (127 mg, 1.0 mmol)
was added to a suspension of the above green solid (90 mg,
0.33 mmol) obtained in H2O/MeOH (6 mL, 2:1, v/v). The mixture
was stirred at room temperature and monitored by TLC (Silica, elu-
ent: CH2Cl2/MeOH 9:1, v/v) until no starting material was visual-
ized (36 h). Then, the precipitate was filtered, washed and finally,
dried in a vacuum dessiccator over potassium hydroxide pellets
offering titled compound as an orange solid (70 mg, 58%), mp
153–155 °C (d). 1H NMR (400 MHz, DMSO-d6, 25 °C) d (ppm):
13.56, 13.27 (bs, 1H, NH), 9.09 (bs, 1H, NH), 8.09 (m, , 1H, C2’Ph),
7.57 (dd, J = 7.8, 7.4 Hz, 1H, C4’Ph), 7.36 (q, J = 8.3 Hz, 1H, C5’Ph),
7.21 (m, 1H, C6’Ph), 4.23, 4.20 (t, J = 5.6 Hz, 2H, OCH2), 4.15, 4.11
(s, 3H, OCH3), 3.72, 3.62 (q, J = 5.7 Hz, 2H, NCH2), 2.02, 1.97 (s,
3H, CH3). 13C NMR (100 MHz, DMSO-d6) d: 170.3, 170.2 (s), 162.0
, 161.7 (s),147.3 (s), 146.9 (s), 138.7 (s), 138.4 (s), 133.2 (s), 133.1
(s), 130.4, 130.7 (d), 124.7, 124.6 (d), 122.8, 122.5 (d), 121.3 (d),
62.3, 61.9 (t), 54.5 (q), 40.6, 40.4 (t), 20.7, 20.6 (q). IR (KBr):
Compound II (199 mg, 1.0 mmol) was dissolved in DMSO
(2.5 mL) and isoamyl nitrite was added (14 mg, 1.2 mmol). The
mixture was stirred at room temperature and monitored by TLC
(Silica, eluent: CH2Cl2/MeOH 9:1, v/v) until no starting material
was visualized (12 h). Then, H2O (25 mL) was added with continu-
ous stirring and 30 min later, aniline (3 mmol) was also added.
After 18 h, the precipitate was filtered, washed and finally, dried
in a vacuum dessiccator over potassium hydroxide pellets offering
titled compound as an orange oil (72 mg, 25 %). 1H NMR (400 MHz,
DMSO-d6, 25 °C) d (ppm): 13.70, 13.35 (bs, 1H, NH), 9.02 (bs, 1H,
NH), 7.70 (m, 2H, Ph), 7.39 (m, 2H, Ph), 7.16 (m, 1H, Ph), 4.74
(bs, 1H, OH), 4.11, 4.09 (s, 3H, OCH3), 3.56 (m, 2H, OCH2), 3.41
(m, 2H, NCH2). 13C NMR (100 MHz, DMSO-d6) d: 170.4, 170.3 (s),
162.0, 161.6 (s), 147.4, 147.1 (s), 138.4, 138.3 (s), 137.3, 137.0 (s),
129.1 (d), 125.2, 125.0 (d), 122.8 (d), 62.2, 62.0 (t), 54.5 (q), 41.1,
m
= 3255, 11741, 1580, 1241 cmÀ1. MS (EI, 70 eV): 365 (M+Á,66),
306 (12), 288 (9), 261 (16), 111 (14), 87 (47), 43 (100). HR–MS
(EI): C15H16ClN5O4 requires 365.0891, found 365.0898.
4-Amino-2-methoxy-5-nitroso-6-pyrrolidin-1-yl-pyrimidine
12: To a suspension of 4-amino-2,6-dimethoxy-5-nitroso-pyrimi-
dine 1c (184 mg, 1.00 mmol) in dichloromethane (10 mL), pyrroli-
dine (0.10 mL, 1.1 mmol) was added. The mixture was stirred at
room temperature and monitored by TLC (Silica, eluent: CH2Cl2/
MeOH 9:1, v/v) until no starting material was visualized (15 h).
Then, water (20 mL) was added and the organic phase was sepa-
rated and dried on anhydrous sodium sulfate. The solid was re-
moved and the solvent was eliminated, offering titled compound
as a blue solid (145 mg, 65%), mp 202–204 °C (d) .1H NMR
(400 MHz, DMSO-d6, 25 °C) d (ppm): 10.72 (bs, 1H, NH2), 8.34
41.0 (t). IR (KBr):
m
= 3366, 3263, 1586, 1562, 1484 cmÀ1. MS (EI,
70 eV): 289 (M+Á,50), 272 (18), 254 (13), 227 (20), 77 (100), 69
(34), 43 (57). HR–MS (EI): C13H15N5O3 requires 289.2899, found
289.2889.
Acetic acid 2-(4-methoxy-5-nitroso-6-phenylamino-pyrimidin-
2-ylamino)-ethyl ester 11b and Acetic acid 2-[4-(3-chloro-phe-

6120
M. Olivella et al. / Bioorg. Med. Chem. 20 (2012) 6109–6122
(bs, 1H, NH2), 3.85 (s, 7H, 2NCH2, OCH3), 1.98–1.91 (M, 4H, 2CH2).
13C NMR (100 MHz, DMSO-d6) d: 164.9 (s), 161.4 (s), 151.2 (s),
142.5 (s), 54.3 (q), 53.0 (t), 50.3 (t), 26.2 (t), 23.0 (t). IR (KBr):
4.2. Preparation of fused alkoxypyrimidines following a
reduction/cyclization process
m
= 3237, 3051, 1613, 1554, 1251 cmÀ1. MS (EI, 70 eV): 223
2-Amino-4-cyclohexylmethoxy-7-methyl-7,8-dihydro-5H-
pteridin-6-one 9a: To a suspension of nitrosopyrimidine 8a
(96 mg, 0.27 mmol) in MeCN/H2O (15 mL, 2:1, v/v) at 50 °C,
Na2S2O4 (235 mg, 1.35 mmol) was slowly added until decoloration.
Then, the mixture was evaporated to dryness, the residue was sus-
pended in water and left at 4 °C overnight. The precipitate was fil-
tered, washed with water and dried over blue silica offering finally
titled compound as a pale solid (51 mg, 65%), mp 193–195 °C. 1H
NMR (400 MHz, CDCl3, 25 °C) d (ppm): 7.32 (bs, 1H, NH), 4.87
(bs, 1H, NH), 4.55 (bs, 2H, NH2), 4.16 (q, J = 7.1, 6.6 Hz, 1H, CHCO),
4.05 (d, J = 6.6 Hz, 2H, OCH2), 1.78–1.67 (m, 5H, CH, 2CH2), 1.46 (d,
J = 7.1 Hz, 3H, CH3). 1.30–1.14 (m, 3H, CH, 3CH2), 1.03–0.87 (m, 3H,
CH, 3CH2). 13C NMR (100 MHz, CDCl3) d: 165.2 (s, CO), 157.0 (s),
156.2 (s), 151.4 (s), 94.8 (s), 71.6 (t), 51.9 (d), 37.4 (d), 29.7 (t),
(M+Á,9), 206 (100), 166 (36), 149 (20). HR–MS (EI): C9H13N5O2 re-
quires 223.1069, found 223.1079.
6-Amino-3-benzyl-2-methoxy-5-nitroso-pyrimidin-4(3H)-
one 13 and 4-Amino-6-benzyloxy-2-methoxy-5-nitroso-pyrimi-
dine 14: A suspension of 6-amino-2-methoxypyrimidin-4(3H)-one
III (10.0 g, 70.85 mmol) and K2CO3 (11.75 g, 85.02 mmol) in DMSO
(90 mL) was stirred at room temperature for 30 min. Benzyl chlo-
ride (9.9 mL, 85.02 mmol) was then added and the mixture was
stirred at room temperature for 18 h. Cold water (270 mL) was
added dropwise with continuous stirring and the solid in suspen-
sion was then collected by filtration, washed with water and dried
in vacuo. The dried solid was suspended in Et2O (300 mL) and stir-
red for 24 h. The solid in suspension was collected by filtration,
washed with diethyl ether and recrystallised from methanol to af-
ford the titled compound (5.47 g, 33%) as a white solid which was
characterized as 6-amino-3-benzyl-2-methoxypyrimidin-4(3H)-
one IV, on the other hand, from the ethereal filtrate the O-benzyl
isomer 6-amino-4-benzyloxy-2-methoxypyrimidine V was recov-
ered (5.67 g, 35%). Finally, compounds IV and V34a (2.31 g,
10.0 mmol) were separately subjected to the general nitrosation
procedure34a to give compounds 13 and 14,34a as blue and green
solids respectively (2,47 g, 95 %).
26.4 (t), 25.7 (t), 19.6 (q). IR (KBr):
m = 3376, 3220, 2926, 1651,
1599, 1472 cmÀ1. MS (EI, 70 eV): 291 (M+Á,32), 195 (100), 180
(48), 152 (41). HR-MS (EI): C14H21N5O2 requires 291.1695, found
291.1708.
2-Amino-7-benzyl-4-cyclohexylmethoxy-7,8-dihydro-5H-
pteridin-6-one 9b: To a suspension of nitrosopyrimidine 8b
(72 mg, 0.17 mmol) in MeCN/H2O (15 mL, 2:1, v/v) at 50 °C,
Na2S2O4 (148 mg, 0.85 mmol) was slowly added until decoloration.
Then, the mixture was evaporated to dryness, the residue was sus-
pended in water and left at 4 °C overnight. The precipitate was fil-
tered, washed with water and dried over blue silica offering finally
titled compound as a pale solid (47 mg, 75%) mp 213–215 °C. 1H
NMR (400 MHz, CDCl3, 25 °C) d (ppm): 7.34–7.20 (M, 6H, NH,
Ph), 4.89 (bs, 1H, NH), 4.57 (bs, 2H, NH2), 4.30–4.26 (ddd, J = 7.1,
6.6 Hz, 1H, CHCO), 4.04 (d, J = 6.6 Hz, 2H, OCH2), 3.38–3.32 (dd,
J = 13.7, 3.3 Hz, 1H, CH2), 2.94–2.86 (dd, J = 13.7, 9.9 Hz, 1H,
CH2),1.78–1.68 (m, 5H, CH, 2CH2), 1.34–1.16 (m, 4H, 2CH2), 1.05–
0.87 (m, 2H, CH2). 13C NMR (100 MHz, CDCl3) d: 164.1 (s, CO),
157.7 (s), 156.2 (s), 150.7 (s), 136.0 (s), 129.5 (d), 129.0 (d), 127.3
(d), 94.6 (s), 71.7 (t), 57.5 (d), 39.5 (t), 37.4 (d), 29.7 (t), 26.4 (t),
6-amino-3-benzyl-2-methoxypyrimidin-4(3H)-one IV: mp
200–202 °C. 1H NMR (300 MHz, DMSO-d6, 25 °C): d = 7.31–7.18
(m, 5H, Ph), 6.46 (bs, 2H, NH2), 4.95 (s, 2H, NCH2), 4.89 (s, 1H,
CH), 3.82 (s, 3H, OCH3) ppm. 13C NMR (100 MHz, DMSO-d6):
d = 162.1 (s), 156.4 (s), 137.6 (s), 128.3 (d), 127.1 (d), 78.9 (d),
55.24 (q), 42.37 (q) ppm. IR (KBr): 3403, 3337, 3197, 1643, 1545,
1395, 1299, 1095 cmÀ1. GC–MS (EI, 70 eV): m/z (%) = 231 (M+Á
100), 214 (16), 125 (36), 111 (8), 104 (30), 91 (93). HR–MS (EI):
,
C12H13N3O2 requires 231.1002, found 231.1008
6-Amino-3-benzyl-2-methoxy-5-nitroso-pyrimidin-4(3H)-
25.7 (t). IR (KBr):
m = 3354, 3226, 2924, 1677, 1624, 1600,
one 13: mp 148–150 °C (d). 1H NMR (400 MHz, DMSO-d6, 25 °C):
d = 11.15 (bs, 1H, NH2), 9.12 (bs, 1H, NH2), 7.33–7.28 (m, 5H, Ph),
5.13 (s, 2H, NCH2), 3.99 (s, 3H, OCH3) ppm. 13C NMR (100 MHz,
DMSO-d6): d = 161.8 (s), 158.1 (s), 148.3 (s), 142.9 (s), 136.5 (s),
128.5 (d), 127.4 (d), 127.3 (d), 56.8 (q), 43.7 (t) ppm. IR (KBr):
1468 cmÀ1. MS (EI, 70 eV): 367 (M+Á,25), 276 (43), 180 (100), 163
(15), 91 (17). HR-MS (EI): C20H25N5O2 requires 367.2008, found
367.2006.
2-Amino-4-methoxy-5,7,8,9-tetrahydro-pyrimido[4,5-
b][1,4]diazepin-6-one 10: To a suspension of 3-(2-amino-6-meth-
oxy-5-nitroso-pyrimidin-4-ylamino)-propionic acid ethyl ester
2h34a (269 mg, 1.00 mmol) in MeCN/H2O (20 mL, 3:1, v/v) at
50 °C, Na2S2O4 (871 mg, 5.00 mmol) was slowly added until decol-
oration. Then, the mixture was evaporated to dryness, the residue
was suspended in water and left at 4 °C overnight. The precipitate
was filtered, washed with water and dried over blue silica offering
finally titled compound as a pale solid (72 mg 34%), mp 178–180 °C
(d). 1H NMR (400 MHz, DMSO-d6, 25 °C) d (ppm): 8.01 (s, 1H, NH),
6.74 (t, 1H, NH), 5.85 (bs, 2H, NH2), 3.41 (m, 2H, NCH2). 2.47 (m,
2H, CH2), 3.77 (s, 3H, OCH3). 13C NMR (100 MHz, DMSO-d6) d:
171.8 (s, CO), 161.4 (s), 158.3 (s), 155.2 (s), 91.8 (s), 53.2 (q), 41.6
m
= 3255, 3034, 1693, 1575, 1504, 1272, 1250, 1058 cmÀ1. GC–MS
(EI, 70 eV): m/z (%) = 260 (M+Á , 20), 132 (13), 91 (100). UV–vis
(MeOH): kmax nm (log ) = 205 (4.28), 229 (4.27), 305 (sh), 335
e
(4.10), 617 (1.77). HR–MS (EI): C12H12N4O3 requires 260.0909,
found 260.0918.
4-Amino-6-phenylamino-2-methoxy-5-nitrosopyrimidine
15: To a suspension of 4-amino-2,6-dimethoxy-5-nitroso-pyrimi-
dine 1c (184 mg, 1.00 mmol) in water (10 mL), aniline (0.18 mL,
2.0 mmol) was added. The mixture was stirred at room tempera-
ture and monitored by TLC (Silica, eluent: CH2Cl2/MeOH 9:1, v/v)
until no starting material was visualized (24 h). Then, the precipi-
tate was filtered, washed and finally, dried in a vacuum dessiccator
over potassium hydroxide pellets offering titled compound as a
green solid (147 mg, 60%), mp 195–197 °C. 1H NMR (400 MHz,
DMSO-d6, 25 °C) d (ppm): 13.56, 9.15 (s, 1H, NH), 10.99, 8.69 (bs,
1H, NH2), 10.37, 8.34 (bs, 1H, NH2), 7.90–7.75 (dd, J = 8.2, 6.7 Hz,
2H, Ph), 7.42–7.35 (m, 2H, Ph), 7.21–7.12 (m, 1H, Ph), 3.91, 3.88
(s, 3H, OCH3). 13C NMR (100 MHz, DMSO-d6) d: 167.6, 167.2 (s),
163.3 (s), 149.7, 146.9 (s), 138.6, 138.1 (s), 137.6, 136.6 (s), 129.0,
128.4 (d), 125.3, 124.3 (d), 123.2, 123.1 (d), 55.1, 54.9 (q). IR
(t), 36.7 (t). IR (KBr):
m
= 3388, 3141, 1755, 1652, 1208 cmÀ1. MS
(EI, 70 eV): 209 (M+Á,100), 194 (49), 166 (65), 149 (16). HR–MS
(EI): C8H11N5O2 requires 209.0913, found 209.0921.
4.3. Antifungal evaluation
4.3.1. Microorganisms and media
For the antifungal evaluation, standardized strains from the
American Type Culture Collection (ATCC), Rockville, MD, USA,
and Colección de Culutivos de CEREMIC (CCC, Centro de Referencia
en Micología, Facultad de Ciencias Bioquímicas y Farmacéuticas,
Suipacha 531-(2000)-Rosario, Argentina were used in a first in-
(KBr):
m
= 3363, 3226, 1652, 1566, 1277 cmÀ1. MS (EI, 70 eV): 245
(M+Á,35), 228 (100), 211 (41), 157 (17), 77 (31). HR–MS (EI):
C11H11N5O2 requires 245.0913, found 245.0914.

M. Olivella et al. / Bioorg. Med. Chem. 20 (2012) 6109–6122
6121
stance of screening: C. albicans ATCC 10231, Cryptococcus neofor-
mans ATCC 32264, Aspergillus flavus ATCC 9170, A. fumigatus ATTC
26934, A. niger ATCC 9029, Trichophyton rubrum CCC 110,
T. mentagrophytes ATCC 9972 and M. gypseum CCC 115.
respectively and was determined from the results obtained in the
inhibition percentage determination.
4.4. Statistical analysis
Strains were grown on Sabouraud-chloramphenicol agar slants
for 48 h at 30 °C, maintained on slopes of Sabouraud-Dextrose agar
(SDA, Oxoid) and sub-cultured every 15 days to prevent pleomor-
phic transformations. Inocula of cell or spore suspensions were
obtained according to reported procedures and adjusted to
1–5 Â 103 cells/spores with colony forming units (CFU)/mL.36
Data of percentage of inhibition were statistically analyzed by
both, one-way analysis of variance and Student’s test. A p < 0.05
was considered significant.
4.5. Acute toxicity test
4.3.2. Antifungal susceptibility testing
Toxic effect of compounds was evaluated using a toxicity test on
fish. The static technique recommended by the US Fish and wildlife
Service Columbia National Fisheries Research Laboratory41 was
modified in order to use lower amounts of tested compounds.37
Fish of the specie Poecilia reticulata were born and grown in our
laboratory until they reached a size of 0.7–1 cm (15 days-old). In
the toxicity test, 10 specimens were exposed to each of the concen-
tration tested per drug in 2 L wide-mouthed jars containing the
test solutions. Aqueous stock solutions of pure compounds diluted
in DMSO were prepared and added to test chambers to get the final
concentrations. The test began upon initial exposure to the com-
pounds and continued for 96 h. The number of dead organisms in
each test chamber was recorded and the dead organisms were re-
moved every 24 h; general observations on the conditions of tested
organisms were also recorded at this time; however the percentage
of mortality was recorded at 96 h. Each experiment was performed
two times with three replicates each. We chose this technique be-
cause it is fast, economic, and easy to reproduce. This assay has
been previously used by our group testing the toxicity of synthetic
and natural compounds.20–37 The species P. reticulata has been
previously used in acute toxicity test.42
Minimum Inhibitory Concentration (MIC) of each extract or
compound was determined by using broth microdilution tech-
niques according to the guidelines of the National Committee for
Clinical Laboratory Standards for yeasts (M27-A3) and for filamen-
tous fungi (M 38-A2).36 MIC values were determined in RPMI-1640
(Sigma, St Louis, Mo, USA) buffered to pH 7.0 with MOPS. Microti-
ter trays were incubated at 30 °C for yeasts and species of Aspergil-
lus and at 28–30 °C for dermatophytes in a moist, dark chamber.
MICs were visually recorded at 48 h for yeasts, and at a time
according to the control fungus growth, for the rest of fungi.
For the assay, stock solutions of pure compounds were twofold
diluted with RPMI from 250–0.98
lg/mL (final volume = 100
ll)
l of inocu-
and a final DMSO concentration 61%. A volume of 100
l
lum suspension was added to each well with the exception of the
sterility control where sterile water was added to the well instead.
Amphotericin B, Terbinafine and Ketoconazole were used as posi-
tive controls.
Endpoints recorded in Table 2 were defined as the lowest con-
centration of drug resulting in total inhibition (MIC100) of visual
growth compared to the growth in the control wells containing
no antifungal.
4.6. Molecular Modeling
4.3.3. MFC determination
The minimum fungicidal concentration (MFC) of each com-
pound against each isolate was also determined as follows: After
determining the MIC, an aliquot of 5 lL sample was withdrawn
All calculations were carried out using the Gaussian 03
program.43
The search for low-energy conformations on the potential en-
ergy surface for compounds 2a, 2b, 5 and 15 was carried out by
using combined ab initio (RHF/3-21G) and DFT (B3LYP/6-31G(d))
calculations. Final DFT geometries were obtained from the geome-
try optimisation jobs. Minima were characterized through har-
monic frequency analysis calculated at DFT level. Correlations
effects were included using Density Functional Theory (DFT) using
the functional of Lee, Yang, and Parr44,45 as proposed and parame-
terized by Becke46,47 (RB3LYP), and the 6-31G(d) basis set.
Double scans were run at 30° intervals, from 0 to 360°, involv-
ing both U2 and U3 for compounds 2a and 15 using RHF 3-21G cal-
culations. The graphical presentations of the PESs were generated
using the program AXUM 5.0 plotting the total energy values as
a dependent variable generated by the double scans over the inde-
pendent variables U2 and U3. The optimum coordinates of U2 and
U3 for all possible minima were then visually estimated from the
level contours diagrams. Potential energy curves (PEC) for U2 and
U3 of compounds 2a and 15 have been obtained via one-dimen-
sional (1D)-scans using RHF/3-21G calculations. The energy has
been calculated at 30° intervals of the dihedral angles. The elec-
tronic study of compounds 2a, 2b, 5 and 15 was carried out using
molecular electrostatic potentials (MEPs). MEPs have been shown
to provide reliable information, both on the interaction sites of
the molecules with point charges and on the comparative reactiv-
ities of these sites.39–41 These MEPs were calculated using B3LYP/6-
311G (d,p) wave functions from the MOLEKEL program.48
Spatial views shown in Figures 2a, 2b, 5, 14 and 15 were con-
structed using the UCSF Chimera program49 as graphic interface.
from each clear well of the microtiter tray and plated onto a
150 mm RPMI-1640 agar plate buffered with MOPS (Remel, Le-
nexa, Kans.). Inoculated plates were incubated at 30 °C, and MFCs
were recorded after 48 h. The MFC was defined as the lowest con-
centration of each compound that resulted in total inhibition of
visible growth.
4.3.4. Inhibition percentage determination
The test was performed in 96-well microplates. Compound test
wells (CTWs) were prepared with stock solutions of each compound
in DMSO (maximumconcentration 61%), diluted with RPMI-1640 to
final concentrations 250–0.98
l
g/mL. Inoculum suspension (100
ll)
was added to each well (final volume in the well = 200
ll). A growth
control well (GCW) (containing medium, inoculum, the same
amount of DMSO used in CTW, but compound-free) and a sterility
control well (SCW) (sample, medium and sterile water instead of
inoculum) were included for each fungus tested. Microtiter trays
were incubated in a moist, dark chamber at 30 °C, 24 or 48 h for C.
albicans or C. neoformans, respectively. Microplates were read in a
VERSA Max microplate reader (Molecular Devices, Sunnyvale, CA,
USA). Amphotericin B was used as positive control.
Tests were performed in duplicate. Reduction of growth for
each compound concentration was calculated as follows: % of inhi-
bition = 100À(OD405 CTWÀOD405 SCW)/(OD405 GCWÀOD405 SCW).
4.3.5. MIC50 determination
MIC50 was defined as the lowest concentration of a compound
that showed 80% or 50% reduction of the growth control

6122
M. Olivella et al. / Bioorg. Med. Chem. 20 (2012) 6109–6122
25. Karolyhazy, L.; Freile, M. L.; Anwair, M.; Beke, G.; Giannini, F.; Sortino, M.;
Acknowledgments
Ribas, J. C.; Zacchino, S.; Matyus, P.; Enriz, R. D. Arzneim.-Forsch./-Drug Res.
2003, 53, 738.
RDE acknowledges ANPCyT, PICT 2010-1832. SAZ acknowledges
ANPCyT, PICT 2010-0608 and UNR BIO 258. Grants from Universi-
dad Nacional de San Luis (UNSL), partially supported this work.
Authors thank ‘‘Centro de Instrumentación Científico Técnica’’
and the staff for data collection. The financial support from the
Consejería de Economía, innovación y Ciencia (Junta de Andalucía,
Spain), the Universidad de Jaén and Ministerio de Ciencia e Innova-
cióon (project reference SAF2008-04685-C02-02) is thanked. RDE
is member of the Consejo Nacional de Investigaciones Científicas
y Técnicas (CONICET-Argentina) staff.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.08.033.
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