A new series of antibacterial nitrosopyrimidines: Synthesis and structure-activity relationship

Article abstract of DOI:10.1002/ardp.201400271

New nitrosopyrimidines were synthesized and evaluated as potential antibacterial agents. Different compounds structurally related with 4,6-bis(alkyl or arylamino)-5-nitrosopyrimidines were evaluated. Some of these nitrosopyrimidines displayed significant antibacterial activity against human pathogenic bacteria. Among them compounds 1c, 2a-c, and 9a-c exhibited remarkable activity against methicillin-sensitive and -resistant Staphylococcus aureus, Escherichia coli, Yersinia enterocolitica, and Salmonella enteritidis. A detailed structure-activity relationship study, supported by theoretical calculations, aided us to identify and understand the minimal structural requirements for the antibacterial action of the nitrosopyrimidines reported here. Thus, our results have led us to identify a topographical template that provides a guide for the design of new nitrosopyrimidines with antibacterial effects.

Full text of DOI:10.1002/ardp.201400271

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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–13
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Full Paper
A New Series of Antibacterial Nitrosopyrimidines: Synthesis and
Structure–Activity Relationship
Monica Olivella1, Antonio Marchal2, Manuel Nogueras2, Manuel Melguizo2, Beatriz Lima3,
3
3
4
1
1,5
´
Alejandro Tapia , Gabriela E. Feresin , Oscar Parravicini , Fernando Giannini , Sebastian A. Andujar
,
Justo Cobo2*, and Ricardo D. Enriz1,5
1
´
´
Facultad de Quımica, Bioquımica y Farmacia, Universidad Nacional de San Luis, San Luis, Argentina
2
3
´
´
´
´
´
Departamento de Quımica Inorganica y Organica, Universidad de Jaen, Jaen, Spain
Facultad de Ingenierıa, Instituto de Biotecnologıa, Universidad Nacional de San Juan, San Juan,
´
´
Argentina
4
5
´
´
´
´
Facultad de Bioquımica Quımica y Farmacia, Instituto de Quımica Organica, Universidad Nacional de
´
´
Tucuman, S. M. de Tucuman, Argentina
IMIBIO-SL (CONICET), San Luis, Argentina
New nitrosopyrimidines were synthesized and evaluated as potential antibacterial agents. Different
compounds structurally related with 4,6-bis(alkyl or arylamino)-5-nitrosopyrimidines were evaluated.
Some of these nitrosopyrimidines displayed significant antibacterial activity against human pathogenic
bacteria. Among them compounds 1c, 2a–c, and 9a–c exhibited remarkable activity against methicillin-
sensitive and -resistant Staphylococcus aureus, Escherichia coli, Yersinia enterocolitica, and Salmonella
enteritidis. A detailed structure–activity relationship study, supported by theoretical calculations, aided
us to identify and understand the minimal structural requirements for the antibacterial action of the
nitrosopyrimidines reported here. Thus, our results have led us to identify a topographical template that
provides a guide for the design of new nitrosopyrimidines with antibacterial effects.
Keywords: Antibacterial compounds / 4,6-Bis(alkyl or arylamino)-5-nitrosopyrimidines / Conformational
and electronic analysis / SAR
Received: July 7, 2014; Revised: September 2, 2014; Accepted: October 1, 2014
DOI 10.1002/ardp.201400271
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
and improved antibacterial agents to combat the worrying
Introduction
ability of bacteria to acquire resistance to current drugs.
Although combination therapy has emerged as a good
alternative to bypass these disadvantages, there is a real
need for a next generation of antibacterial agents [4–7].
In the course of our ongoing screening program for new
antimicrobial compounds, we have previously reported
antifungal [8–12] and antibacterial [13] activities of peptide
compounds as well as of different molecules obtained from
natural [14–17] and synthetic [18–23] sources. Among them,
recently we reported a new series of compounds structurally
related with 4,6-bis(alkyl or arylamino)-5-nitrosopyrimidines
possessing a moderate antifungal effect against human
pathogenic strains [24]. Compounds 2b, 9b, and 13 (see
Section 2.1) exhibited antifungal activity against Candida
albicans, Candida tropicalis, and Cryptococcus neoformans.
From these results, we were intrigued to know if these
compounds could also display antibacterial effect. Thus, in the
The continuous use of chemical antibiotics has led to the
appearance of multi-resistant bacterial strains all over the
world, which resulted in higher mortality on many infectious
diseases [1–3]. Thus, the increasing resistance of pathogenic
bacteria to conventional antibiotics has become a serious
problem in health care, which requires alternatives to be
developed. Medicinal chemists are still actively seeking new
´
Correspondence: Dr. Ricardo D. Enriz, Facultad de Quımica,
´
Bioquımica y Farmacia, Universidad Nacional de San Luis,
Chacabuco 915, 5700 San Luis, Argentina.
E-mail: denriz@unsl.edu.ar
Fax: þ54 02664 426367
ꢀAdditional correspondence: Dr. Justo Cobo,
E-mail: jcobo@ujaen.es
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series of nitrosopyrimidine derivatives, most of them not
described to date in the literature.
Results and discussion
Chemistry
In our aim of developing new bioactive pyrimidine-based
compounds, we have optimized a three-step strategy con-
sisting of a nitrosation, selective aminolysis of alkoxy groups,
and a tandem reduction/cyclization [25–28]. In this fashion,
we have already reported the synthesis of a family of N4-
substituted 2,4-diamino-6-methoxy-5-nitrosopyrimidines 2 by
selective monoaminolysis of 1a [25–27], which was prepared
through the introduction of
a 5-nitroso group to the
commercial 2-amino-4,6-dimethoxypyrimidine (see Scheme 1
and Table 1).
An excess of amine on 1a offered the N4,N6-disubstituted
2,4,6-triamino-5-nitrosopyrimidine 3. We have also per-
formed the hydrolysis of the methoxy group via acid catalysis
of compound 2d rendering the 5-nitrosopyrimidin-4(3H)one
4.
On the other hand, if starting from those derivatives
with an aminoester at C4 such as 2e–h, the reduction of the
nitroso group was followed by a spontaneous intramolecular
cyclization through the ester group to render the fused
pyrimidine derivatives either with pyrimidodiazepine nucleus
such as 5 or with tetrahydropteridine structure such as 6, as
shown in Scheme 1.
Scheme 1. Synthetic sequence for the preparation of 5-nitro-
sopyrimidines and fused pyrimidines via nitrosation-aminolysis
from methoxypyrimidines.
In order to increase the structural diversity and also to test
the influence of the alkoxy group, we have prepared the
tetrahydropteridine analogs 8a–c (Scheme 2), in a similar way
to that described above, but changing the methoxy group at
1a by a bulkier group, such as cyclohexylmethylenoxy by
transalkoxylation.
present study, we use as starting structures those previously
reported as the strongest antifungal compounds [24]. We
tested these compounds against different pathogenic bacte-
ria and some of them have displayed a strong antibacterial
effect; therefore, we performed different structural modifi-
cations in this series in order to obtain a structure–activity
relationship (SAR) as well as to determine the minimal
structural requirements (a possible pharmacophoric pattern)
to produce the antibacterial activity. In short, we here report
the synthesis, antibacterial activities, and a SAR study, for a
Next, we exchanged the alkoxy and amino group position
in the starting material and so from 1c several N-substituted
diamino and triamino-5-nitrosopyrimidine derivatives, such
as 9 and 10 (Scheme 3) (isomers to previously synthesized
5-nitrospyrimidines), were prepared in a similar procedure [26].
Finally, several pyrimidine derivatives with different alkoxy
or thioalkyl groups, namely 11–15 (Fig. 1), were prepared in
order to test their activity in comparison with the active
nitrosopyrimidine 1c [25, 29].
Table 1. Structural features of compounds 9 and 10.
Compounds type 9
Compounds type 10
Entry
R1
R2
Entry
R1
R2
9a
9b
9c
9d
9e
9f
Bu
C6H5
(3-Cl)C6H4
C6H5CH2
(3,4-OMe)C6H4CH2
–(CH2)4–
H
H
H
H
H
10a
10b
10c
10d
10e
Bu
C6H5
(3,4-OMe)C6H4CH2
(CH2)2–OH
(CH2)2–OH
H
H
H
H
¼ R1
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–13
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Figure 1. Structures of compounds 11–15.
ATCC27853 (Table 2). The three compounds showed similar
antibacterial activities with MIC values ranging from 0.1 to
2.5 mg/mL (Table 2). It should be noted that the antibacterial
activities obtained for these compounds are stronger than
those attained acting as antifungal agents [24].
Scheme 2. Preparation of pteridine analogs including cyclo-
hexylmethylenoxy group.
In the next step of our study, we performed different
chemical modifications on these three compounds in order to
obtain an SAR, which could help us to understand a possible
pharmacophoric patron for this series. Thus, we synthesized
and tested 35 compounds structurally related with com-
pounds 2b, 9b, and 13; such compounds have been described
in the previous section (compounds 1–15).
Antibacterial activity and SARs
For this study, we chose as starting structures compounds 2b,
9b, and 13, which were reported as the most active
compounds against C. albicans, C. tropicalis, and C. neofor-
mans in our previous paper [24]. As expected, these
compounds displayed a strong antibacterial activity against
all the species tested except Pseudomonas aeruginosa
To evaluate the SARs, the effects of structural changes on
the general structure shown in Fig. 2 were considered; thus,
we performed different substitutions at R1, R2, and R3. Several
compounds showed strong antibacterial activities in this
series, of which the most prominent are 2a–c, 9a–e, and 13.
It should be noted that the active compounds displayed
significant antibacterial effects against all the bacteria tested
here except against P. aeruginosa. Unfortunately, none of
them show any activity against this specie, but many
compounds were active against methicillin-resistant Staphy-
lococcus aureus.
Thirty-five compounds were evaluated against Gram-(þ)
and Gram-(ꢁ) bacteria. The compounds type 9 (a–e) and
compounds 11–13 exhibited remarkable antibacterial activity
against methicillin-sensitive and -resistant S. aureus, Escher-
ichia coli ATCC, and clinical isolate, Yersinia enterocolitica,
Salmonella enteritidis and LM-S. sp., with MIC values between
0.5 and 15 mg/mL, while the compounds type 2 (a–c) were
stronger active against the same strains except against
Staphylococcus aureus (mr) and Yersinia enterocolitica
(MIC ¼ 40 and MIC ¼ 50 mg/mL).
Regarding the influence of the substituents on R1, R2, and R3,
it appears that the presence of a group possessing properties to
produce hydrogen bonds (either acting like donor or acceptor
of protons) is necessary to produce the antibacterial effect. It
should be noted that all the active compounds possess NH2
or OCH3 groups at R1 or R3 indistinctly. In fact in these
Scheme 3. Preparation of some N2,N4-disubstituted 2,4-diami-
no-5-nitrosopyrimidines 9 and 10.
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Table 2. Antibacterial activity of the nitrosopyrimidine compounds.
MICa) (mg/mL)
Comp.
Sa (ms)
Sa (mr)
Ec
LM1-Ec
LM2-Ec
Pa
PI-Ye
MI-Se
Ssp (LM)
1b
1c
2a
2b
2c
2d
2e
2f
2g
2h
3
>50
5
>50
40
40
>50
25
>50
2.5
0.5
0.5
0.5
>50
50
50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
2.5
5
1
2.5
2.5
>50
>50
>50
>50
>50
>50
2.5
1
1
>50
>50
0.5
>50
2.5
5
>50
12.5
>50
>50
12.5
>50
10
12.5
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
12.5
1
>50
2.5
5
5
5
>50
10
40
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>40
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
7.5
>50
30
>50
40
50
>50
25
>50
2.5
0.5
1
5
>50
25
>50
2.5
2.5
2.5
5
>50
25
20
5
6.25
>50
10
20
40
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
6.25
1
1
1
15
>50
>50
>50
40
20
40
30
40
40
40
40
40
>50
>50
>50
>50
>50
>50
25
>50
>50
>50
6.25
1.25
0,5
>50
>50
>50
>50
>50
>50
30
>50
>50
>50
5
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
5
0.5
2.5
5
0.5
>50
50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
5
1
5
5
1
>50
>50
>50
50
4
5
6a
6b
7b
8a
8b
8c
9a
9b
9c
1
1
1
0.5
1
2.5
6.25
25
>50
>50
>50
50
9d
9e
9f
15
2.5
2.5
5
>50
>50
>50
50
25
>50
>50
>50
>50
>50
>50
2.5
12.5
12.5
>50
>50
0.5
>50
>50
>50
50
10a
10b
10c
10d
10e
11
12
13
14
15
Cefb)
>50
>50
>50
>50
2.5
1
40
50
50
50
50
>50
2.5
2.5
2.5
>50
>50
0.5
>50
12.5
6.25
6.25
>50
>50
5
>50
3.25
2.5
2.5
>50
>50
0.5
>50
6.25
5
2.5
>50
>50
0.5
>50
2.5
2.5
2.5
>50
>50
0.5
1
>50
>50
12.5
a)The minimal inhibitory concentration (MIC) of the peptides were determined (n ¼ 3) for Sa (ms): Staphylococcus aureus
methicillin-sensitive ATCC 29213, Sa (mr): Staphylococcus aureus methicillin-resistant ATCC 43300, Ec: Escherichia coli ATCC
25922, LM1-Ec: LM1-Escherichia coli, LM2-Ec: LM2-Escherichia coli, Pa: Pseudomonas aeruginosa ATCC 27853, PI-Ye: PI-Yersinia
enterocolitica, MI-Se: MI-Salmonella enteritidis, Ssp (LM): Salmonella sp. (LM).
b)Cefotaxime.
compounds when NH2 is located at R1, then OCH3 group is at
R3 and vice versa. On the other hand, noting the structures
taken as a starting point (compounds 2b, 9b, and 13) it would
be reasonable to think that the presence of a second aromatic
ring is necessary to produce the antibacterial effect. Such idea
is reinforced when we observe the strong activities obtained
for compounds 2a and 9c–e (Table 2). However, the
replacement of phenyl group by butyl was well tolerated,
keeping the antibacterial activity (observe activities of
compounds 2a and 2c). A similar result was observed for
compounds 9a and 9b. These results clearly indicate that in the
presence of a second aromatic ring, it is not a structural
requirement to produce the biological response. However, it
Figure 2. General structure of the nitrosopyrimidine com-
pounds analyzed here.
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is also important to note that not any group is operative in
this position since if this group becomes more polar, the
antibacterial activity is lost. This can be seen comparing the
activities obtained for compounds 2e–h with 9b. On the other
hand, the replacement of the aromatic ring in compound 9b
by an adamantyl group (compound 2d) gives a compound
devoid of any antibacterial effect indicating that not any
hydrophobic substituent at R2 is operative for these com-
pounds. In addition, when we performed significant structur-
al modifications simultaneously on R1 and R2, we observed a
near-total loss of the activity (compounds 1b, 3, 7a, 8a–c, 9f,
and 10a,d).
acting as antifungal agents [24]. On the other hand, although
1c and 9a showed potent antibacterial activity, at the same
time these compounds displayed a relatively significant acute
toxicity (7.5 and 10 mg/mL, respectively), which does not
allow to have a wide margin between the concentration
required to produce antibacterial activity and the concentra-
tion at which it begins to be toxic. In contrast to these results,
the compounds that showed a good relationship between the
concentrations in which they display antibacterial activity
with respect to those having toxicity were 2a, 2b, 9b, 9c, and
9e. In fact, these compounds showed no acute toxic effects at
least at concentrations above 50 mg/mL. It is important to note
that we did not measure toxic effects at higher concentrations
than those reported here and therefore, it might be expected
that these compounds display such effects at substantially
higher concentrations.
Using the concept of homology, compound 9d was
synthesized, which is the higher homolog of 9b. This
compound showed a very similar antibacterial activity to
that of 9b. Similar activities were also obtained for 9c, which is
a structural analog of 9b. Thus, these results indicate that an
extra methylene group is well tolerated in the connecting
chain of the aromatic rings for these compounds.
To confirm the results obtained from the acute toxicity
study, we performed
a study of cytotoxicity of three
representative compounds of this series (compounds 2a, 2b,
and 13) using the well-known MTT bioassay. We have
previously used this technique successfully to the study of
other compounds of biological interest [30, 31].
At this point of our study, we were interested to determine
the pharmacophoric patron of this series and to achieve this
we conducted a molecular simplification. By applying the
concept of molecular disjunction, we obtained compound 1c.
This compound showed a strong antibacterial activity being
the simplest structure that retains this effect. It should be
noted that compound 1c is a molecular simplification of 13 in
which the O-benzyl group has been replaced by OCH3. Once
the simplest structure with antibacterial activity of this series
was obtained, we applied the concept of isosterism by
replacing SCH3 by OCH3. Thus, we obtained compound 11,
which showed an antibacterial effect similar to 1c. The same
molecular modification was also applied on compound 13 and
in this case, we obtained compound 12, which also presented
a strong antibacterial activity.
Figure 3 shows the cytotoxic effects obtained for com-
pounds 2a, 9b, and 13 using the HepG2 cell culture.
Compound 13 displays a high cytotoxicity against hepatocytes
even at relatively low concentrations. In contrast, compounds
2a and 9b showed a moderate cytotoxicity at least until
32 mg/mL. These results are in agreement with those obtained
by the acute toxicity test.
From the results of these exploratory and preliminary
studies, we cannot discard the putative inconvenience of
these compounds as lead structures. However, it should be
noted that compounds reported here constitute a set of
candidate structures, which has been evaluated considering
In the next stage of our study, we wanted to find out if the
presence of the NO group was a necessary requisite for the
antibacterial activity of this series. Thus, other modification
that was done was to remove this group replacing it by
H. From compounds 12 and 13, we obtained 14 and 15,
respectively; both compounds were completely inactive
indicating that the presence of NO group is a structural
requirement necessary to produce antibacterial activity. Cyclic
compounds 5, 6a, 6b, 8a, 8b, and 8c give an additional support
to this observation. It is clear however that the mere presence
of the NO group is not sufficient to produce the biological
effect because many compounds possessing this group were
inactive.
Having in mind the potential limitations that could present
these compounds because of possible toxicities they may
display, we performed an acute toxicity study for those
derivatives that showed major antibacterial activities. We
observed that compound 13 and its structurally related
derivative 12 exhibited high acute toxicity (2.5 mg/mL both
compounds), which severely limits their applicability as
antibacterial agents. These results are in agreement with
those previously reported for nitrosopyrimidine derivatives
Figure 3. Viability of rat hepatocytes (HepG2) after treatment
with compounds 2a, 9b, and 13. Results are expressed as the
percentage of controls (untreated hepatocytes).
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their antimicrobial effect in order to obtain initial structures,
which probably need further optimization to be considered a
lead compound.
Conformational and electronic study
To better understand the above experimental results, we have
carried out a conformational and electronic study on the most
active compounds of this series to reveal the stereo-electronic
characteristics of these molecules. We performed calculations
for many compounds of this series but in order to keep the
size of this paper, we only show here the results obtained for
2a, 2c, 9c, and 13 because they are representatives of the
whole series. The details of how this study was conducted are
given as molecular modeling in Section 4.3.
Previously, we reported an exhaustive conformational and
electronic study for compounds 2b, 9a, and 9b [24]; therefore,
taking advantage of this situation, we compared those results
with the obtained here. As we expected, the conformational
behavior obtained for compounds 2a and 9c is almost the
same as that previously reported for 9b, while the results
obtained for 2c are very similar to those reported for
compound 9a.
Figure 5. Structures of the rotamers a2, a1, b1, and b2 of
compound 10a.
1H and 13C NMR spectra of non-symmetrically substituted
compounds type 9 show two sets of signals, indicating that,
in solution (DMSO-d6), these compounds exist as two
structures of 3-amino-2-nitrosocyclohex-2-en-1-one [33], 5-
amino-4-nitrosopyrazole [33], and 6-amino-5-nitrosopyrimi-
dines [34–38]. Our results are in agreement with those of
Prochazkova et al. reporting that a mixture of two forms with
distinct hydrogen-bond patterns were found in the solutions
of compounds with two hydrogen bond donors neighboring
the nitroso group [32]. It is interesting to note that these
authors also reported that the ratio of the two forms was
significantly substituent-dependent, and the concentrations
of the two forms can be found in a broad range.
–
Ar–N O rotamers a, b in equilibrium (Fig. 4). For compound
–
9b, B3LYP/6-31G(d,p) calculation predicts that rotamer b
possesses 0.04 kcal/mol above rotamer a and the energy
barrier for the conformational interconversion is 27.6 kcal/mol.
These results are in
a complete agreement with our
experimental results (see the spectrum shown in Supporting
Information Fig. S1), with a ratio a/b around 57:43, as well as
with the theoretical results recently reported by Prochazkova
et al. [32]. Similar results were obtained for the rest of
compounds type 9. In the case of compounds type 10,
rotamers showed four sets of signals (see Supporting
Information Fig. S2). DFT calculations predict the following
energy gaps in kcal/mol for such rotamers of compound 10a:
a2 (0.0), a1 (0.06), b1 (0.26), and b2 (0.40), which is also in
agreement with the 1H and 13C NMR data obtained (Fig. 5).
Conjugated nitrosamines belong to the same family as the
enols of b-diketones and other conjugated systems with
intramolecular hydrogen bonds. Therefore, the formation of
intramolecular hydrogen bonds may be expected, and
indeed, such interactions were found in the solid-state
Figure
6 shows a spatial view of the superimposed
energetically preferred conformations obtained for the most
active compounds (2a–c, 9a,b,e, and 13). The conformations
of compounds 2b, 9b, and 13 previously reported [24] have
been included here for comparison. From this figure, it is
evident that all these compounds can adopt very similar
conformations displaying the reactive groups in a similar
spatial ordering. However, it is clear that not only the
conformational aspects but also the electronic properties of
these compounds are determinant for the antibacterial
activities. Thus, the knowledge of the stereo-electronic
attributes and properties of these nitrosopyrimidines will
contribute significantly to the elucidation of the structural
requirements to produce the antibacterial activity. Molecular
electrostatic potentials (MEP) are of particular value because
they allow the visualization and assessment of the capacity
of a molecule to interact electrostatically with a putative
binding site [39–41]. Thus, this methodology was used to
evaluate the electronic distribution around molecular surface
for compounds reported here.
Figure 7 gives the MEPs obtained for compounds 2a, 9a, 9e,
and 13. The MEPs obtained for these compounds account
Figure 4. Structures of the rotamers a and b in equilibrium of
compound 9.
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Table 3. log S and log P obtained for active compounds.
Comp.
log S
log P
2a
2b
2c
9a
9b
9c
9d
9e
13
ꢁ4.12
ꢁ4.85
ꢁ3.44
ꢁ3.44
ꢁ4.12
ꢁ3.71
ꢁ3.92
ꢁ4.85
ꢁ3.28
2.53
3.09
1.81
1.81
2.53
2.04
2.29
3.09
2.67
V(r)min ꢂ ꢁ0.0874 el/au3) in the vicinity of the NO group; two
positive regions (deep blue zones with V(r)max ꢂ 0.054 el/au3)
located near to the NH2 and OCH3 groups and an extended
hydrophobic zone (deep and light green zone with an
almost neutral potential V(r)med ꢂ ꢁ0.0022 el/au3) throughout
the second ring (or the flexible side chain in the case of
compound 9a).
Figure 6. Spatial view of overlapping of the energetically
preferred conformations obtained for compounds 2a (yellow),
2b (green), 2c (white) 9a (pink), 9b (red), 9e (violet), and 13
(blue).
Finally, let us make some comments regarding pharmaco-
kinetic properties of these compounds. Predictions of ADME,
absorption, and distribution parameters and the calculated
physicochemical properties (log S and log P in Table 3) for the
active compounds, are within the typical ranges desired for a
drug, as well as the fulfillment of Lipinski’s rule, which permit
us to consider these compounds as interesting starting
structures for antibacterial activity. All these aspects serve
to justify future research on new series of nitrosopyrimidines
focused to the structural optimization that would lead to a
substantial improvement of potency and spectrum in the
antibacterial activity. Such research must be complemented
with in vivo toxicity and efficacy evaluations and the
establishing of the mechanism of action at molecular level.
Conclusions
The synthesis, in vitro evaluation and SAR studies of 35
nitrosopyrimidines and structurally related derivatives acting
as antibacterial agents are reported. Several compounds
showed a strong antibacterial activity, some of them with
activities comparable to the reference compound used
(cefotaxime). Unfortunately, some of these compounds also
showed a significant acute toxicity, which severely limits
its potential therapeutic use. However, other compounds
showed strong antibacterial activity and relatively low acute
toxicity like for example compounds 2a, 2b, 2c, and 9b. Thus,
special attention might be given to these compounds since
they not only showed the strongest antibacterial effects but at
the same time they showed a relatively low acute toxicity,
which converts these compounds like appropriate structures
for the search of new antibacterial compounds. Our SAR
study helped us to identify and understand the minimal
structural requirements for the antibacterial action of these
Figure 7. Electrostatic potential-encoded electron density
surfaces of compounds 9c (a), 13 (b), 2a (c), and 9a (d). The
surfaces were generated with GAUSSIAN 03 using B3LYP/6-
311þþG(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.
for the general characteristics of the electronic behavior
for the active compounds. The general pattern is very
similar for all these systems. The MEPs exhibit four character-
istic regions: a clear minimum value (deep red zone with
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Archiv der Pharmazie
compounds. Thus, we believe that our results may be helpful
in the structural identification and understanding of the
minimum structural requirements for the activity of these
molecules and could provide a guide in the design of
compounds with this biological activity.
170.8 (s), 169.0 (s, CO), 163.3 (s), 150.0 (s), 138.8 (s), 71.7 (t),
60.7 (t), 41.2 (t), 36.7 (d), 29.1 (t), 25.9 (t), 25.1 (t), 14.0 (q). MS
(EI, 70 eV): 338 (100), 337 (Mþ, 63), 242 (22), 152 (40), 140 (30),
55 (68). HR-MS (EI): C15H23N5O4 requires 337.1750, found
337.1749.
2-Amino-4-cyclohexylmethoxy-7,8-dihydro-5H-pteridin-6-
one (8a)
Experimental
To a suspension of nitrosopyrimidine 7a (168 mg, 0.50 mmol)
in MeCN/H2O (15 mL, 2:1, v/v) at 50˚C, Na2S2O4 (435 mg,
2.5 mmol) was slowly added until decoloration. 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 to give compound
8a as a yellow solid (116 mg, 84%), m.p. 214–216˚C (d). 1H NMR
(400 MHz, CDCl3, 25˚C) d (ppm): 7.41 (bs, 1H, NH), 4.98 (bs, 1H,
NH), 4.58 (bs, 2H, NH2), 4.12 (s, 2H, NCH2), 4.05 (d, J ¼ 6.6 Hz,
2H, OCH2), 1.78–1.67 (m, 6H, 2CH, 2CH2), 1.30–0.87 (m, 5H, CH,
2CH2). 13C NMR (100 MHz, CDCl3) d: 162.1 (s, CO), 157.9 (s),
156.3 (s), 151.5 (s), 94.45 (s), 71.6 (t), 46.0 (t), 37.4 (d), 29.8 (t),
29.7 (t), 26.4 (t), 25.7 (t). IR (KBr): n ¼ 3344, 3220, 2925, 2851,
1682, 1626, 1605, 1482 cmꢁ1. MS (EI, 70 eV): 277 (Mþ, 22), 181
(100), 152 (50), 55 (25). HR-MS (EI): C13H19N5O2 requires
277.1539, found 277.1533.
Chemistry
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 Instrumentacion Cientıfico-Tecnica (CICT) at Uni-
versidad de Jaen. H and 13C NMR spectra were recorded on a
1
´
Bruker Avance 400 spectrophotometer (CICT) operating at
400 and 100 MHz, respectively, using CDCl3 and DMSO-d6 as
solvents and tetramethylsilane as internal standard. Multi-
plicity 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, and (q) quartet. The same abbreviations were used for
the multiplicity of signals in 1H NMR and also: (m) multiplet
and (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
4-Amino-6-butylamino-2-methoxy-5-nitrosopyrimidine
(9a)
on
a
Waters Micromass AutoSpec-Ultima spectrometer
To a suspension of 4-amino-2,6-dimethoxy-5-nitrosopyrimi-
dine 1c (184 mg, 1.00 mmol) in dichloromethane (10 mL),
n-butylamine (0.11 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 (20 h). Then, water (20 mL) was added and the
organic phase was separated and dried on anhydrous sodium
sulfate. The solid was removed and the solvent was
eliminated, offering compound 9a as a blue solid (183 mg,
81%), m.p. 139–141˚C. 1H NMR (400 MHz, CDCl3, 25˚C) d (ppm):
11.51/7.95 (bs, 1H, NH), 10.62/7.68 (bs, 1H, NH2), 6.51/6.31 (bs,
1H, NH2), 3.99/3.97 (s, 3H, OCH3), 3.81/3.53 (q, J ¼ 7.1 Hz, 2H,
NCH2), 1.62 (m, 2H, CH2); 1.42 (m, 2H, CH2), 1.91 (m, 3H, CH3).
13C NMR (100 MHz, CDCl3) d: 168.2 (s), 168.1 (s), 150.2 (s), 137.8
(s), 55.3 (q), 39.6 (t), 31.0 (t), 20.1 (t), 13.7 (q). IR (KBr): n ¼ 3379,
3191, 2957, 1643, 1593, 1342, 1275 cmꢁ1. MS (EI, 70 eV): 225
(Mþ, 49), 208 (100), 182 (30), 154 (44). HR-MS (EI): C9H15N5O2
requires 225.1226, found 225.1223.
(equipped with a direct inlet probe) operating at 70 eV. Silica
gel aluminum plates (Merck 60 F254) were used for analytical
TLC. The amines, aminoesters, 2-amino-4,6-dimethoxypyrimi-
dine, and 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 to
the standard protocols. Compounds 1a–c, 2–6, 7b–c, 8b–c, 9b,
9f, 10e, and 11–15 were synthesized using reported proce-
dures [25–27].
2-(2-Amino-6-cyclohexylmethoxy-5-nitrosopyrimidin-4-
ylamino)acetic acid ethyl ester (7a)
To a suspension of 1b (174 mg, 0.50 mmol) in t-BuOH (5 mL),
glycine ethyl ester hydrochloride (84 mg, 0.60 mmol) and Et3N
(61.3 mg, 0.60 mmol) were added. The mixture was stirred at
60˚C and monotorized by TLC (silica, eluent: CH2Cl2/MeOH 9:1,
v/v) until no starting material was visualized (4 h). Then,
solvent was removed under reduced pressure and CH2Cl2 was
added until solution. The organic solution was washed with
water (3 ꢃ 15 mL) and dried on anhydrous sodium sulfate, and
the solvent was removed under vacuum to offer compound 7a
as a blue solid (110 mg, 65%); m.p. 182–184˚C (d), 1H NMR
(400 MHz, DMSO-d6, 25˚C) d (ppm): 11.22 (t, J ¼ 6.0 Hz, 1H, NH),
7.98 (bs, 1H, NH2), 7.94 (bs, 1H, NH2), 4.29 (d, J ¼ 6.6 Hz, 2H,
OCH2), 4.16 (d, J ¼ 6.0 Hz, 2H, NCH2), 4.11 (q, J ¼ 7.1 Hz, 2H,
OCH2), 1.82–1.64 (m, 7H, CH, 3CH2), 1.14–0.92 (m, 4H, 2CH2),
1.18 (t, J ¼ 7.1 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) d:
4-Amino-6-(3-chlorophenyl)amino-2-methoxy-5-
nitrosopyrimidine (9c)
To a suspension of 4-amino-2,6-dimethoxy-5-nitrosopyrimi-
dine 1c (184 mg, 1.00 mmol) in water (10 mL), 3-chloroaniline
(0.21 mL, 2.0 mmol) was added. The mixture was stirred at 80˚C
and monitored by TLC (silica, eluent: CH2Cl2/MeOH 9:1, v/v)
until no starting material was visualized (5 h). Then, the
precipitate was filtered, washed, and finally, dried in a
vacuum desiccator over potassium hydroxide pellets offering
compound 9c as a green solid (182 mg, 65%), m.p. 203–205˚C
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(d). 1H NMR (400 MHz, DMSO-d6, 25˚C) d (ppm): 13.41/10.32 (s,
1H, NH), 11.09/9.19 (bs, 1H, NH2), 8.71/8.39 (bs, 1H, NH2), 8.13–
8.07 (m, 1H, Ph), 7.89–7.60 (m, 1H, Ph), 7.63–7.37 (m, 1H, Ph),
7.26–7.19 (m, 1H, Ph), 3.92/3.91 (s, 3H, OCH3). 13C NMR
(100 MHz, DMSO-d6) d: 167.6/167.0 (s), 163.5 (s), 149.5/146.8
(s), 139.8/138.6 (s), 138.2/137.5 (s), 133.1/132.6 (s), 130.5/130.0
(d), 124.9/123.8 (d), 123.0/122.4 (d), 121.6/121.3 (d), 55.0 (q). IR
(KBr): n ¼ 3245, 3076, 2958, 1612, 1554, 1473, 1329, 1267,
1046 cmꢁ1. MS (EI, 70 eV): 279 (Mþ, 56), 262 (100), 245 (39), 192
(19), 111 (37), 58 (62). HR-MS (EI): C11H10N5O2 requires
279.0523, found 279.0518.
4-Amino-6-butylamino-2-(2-hydroxyethylamino)-5-
nitrosopyrimidine (10a)
To suspension of 5-nitrosopyrimidine 9a (225 mg,
a
1.00 mmol) in methanol (10 mL), 2-aminoethanol (0.12 mL,
2.0 mmol) was added. The mixture was stirred, refluxed, and
monitored by TLC (silica, eluent: CH2Cl2/MeOH 9:1, v/v) until
no starting material was visualized (12 h). After cooling, the
precipitate was filtered, washed, and finally, dried in a
vacuum desiccator over potassium hydroxide pellets offering
compound 10a as a pink solid (182 mg, 72%), m.p. 184–186˚C.
1H NMR (400 MHz, DMSO-d6, 25˚C) d (ppm): 11.65/11.39/10.61/
10.28 (t, J ¼ 5.5, 5.6 Hz, 1H, NH), 8.82/8.56/7.95/7.66 (t, J ¼ 6.0,
5.8 Hz, 1H, NH), 8.23/7.66/7.51 (bs, 1H, NH2), 8.02/7.66/7.26 (bs,
1H, NH2), 4.72 (bs, 1H, OH), 3.86 (m, 2H, OCH2), 3.52–3.37 (m,
4H, NCH2), 1.64–1.44 (m, 2H, CH2); 1.39–1.26 (m, 2H, CH2), 1.91
(m, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) d: 165.8/165.2 (s),
163.0/162.8/162.3 (s), 150.9/150.8/150.5/150.2 (s), 137.3/136.7
(s), 59.9/59.8 (t), 43.7/43.5 (t), 39.6/38.2 (t), 31.0/30.6 (t), 19.8/
19.7 (t), 13.8/13.6 (q). IR (KBr): n ¼ 3308, 2964, 1665, 1592, 1360,
1198, 1048 cmꢁ1. MS (EI, 70 eV): 255 (MHþ, 100), 237 (58), 183
(31), 43 (27). HR-MS (EI): C10H8N6O2 requires 254.1491, found
254.1494.
4-Amino-6-benzylamino-2-methoxy-5-nitrosopyrimidine
(9d)
To a suspension of 4-amino-2,6-dimethoxy-5-nitrosopyrimi-
dine 1c (184 mg, 1.00 mmol) in acetonitrile (10 mL), benzyl-
amine (0.13 mL, 1.2 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
(20 h). Then, water (20 mL) was added and the solid in
suspension was filtered, washed, and finally, dried in a
vacuum desiccator over potassium hydroxide pellets offering
compound 9d as a blue solid (233 mg, 90%), m.p. 218–220˚C.
1H NMR (400 MHz, DMSO-d6, 25˚C) d (ppm): 11.72/10.00
(t, J ¼ 6.0, 6.6 Hz; 1H, NH), 10.34/8.94 (bs, 1H, NH2), 8.52/8.11
(bs, 1H, NH2), 7.42–7.24 (m, 5H, Ph), 4.71/4.62 (d, J ¼ 6.6,
6.0 Hz; 2H, NCH2), 3.84/3.83 (s, 3H, OCH3). 13C NMR (100 MHz,
DMSO-d6) d: 167.8/167.5 (s), 164.7 (s), 149.9/149.2 (s), 139.1 (s),
138.1, 138.0 (s), 128.5/128.3 (d), 127.7/127.5 (d), 127.2/126.9
(d), 54.8/54.7 (q), 43.7/42.7 (t). IR (KBr): n ¼ 3356, 3203, 2952,
1642, 1593, 1528, 1456, 1339, 1288 cmꢁ1. MS (EI, 70 eV):
259 (Mþ, 12), 242 (100), 210 (19), 154 (32), 105 (16), 91 (60),
65 (27). HR-MS (EI): C12H13N5O2 requires 259.1069, found
259.1067.
4-Amino-6-phenylamino-2-(2-hydroxyethylamino)-5-
nitrosopyrimidine (10b)
To
a
suspension of 5-nitrosopyrimidine 9d (123 mg,
0.50 mmol) in methanol (5 mL), 2-aminoethanol (0.06 mL,
1.0 mmol) was added. The mixture was stirred, refluxed, and
monitored by TLC (silica, eluent: CH2Cl2/MeOH 9:1, v/v) until
no starting material was visualized (12 h). After cooling, the
precipitate was filtered, washed, and finally, dried in a
vacuum dessiccator over potassium hydroxide pellets offering
compound 10b as a pink solid (82 mg, 60%), m.p. 208–210˚C.
1H NMR (400 MHz, DMSO-d6, 25˚C) d (ppm): 14.07/13.72/10.42/
10.19 (bs, 1H, NH), 10.57/10.23/8.46/8.25 (bs, 1H, NH2), 8.18–
8.04 (bs, 1H, NH), 8.01/7.98/7.70/7.51 (bs, 1H, NH2), 7.95–7.77
(m, 2H, Ph), 7.39–7.29 (m, 2H, Ph), 7.16–7.05 (m, 1H, Ph), 4.78–
4.72 (m, 1H, OH), 3.58–3.50 (m, 2H, OCH2), 3.48–3.39 (m, 2H,
NCH2). 13C NMR (100 MHz, DMSO-d6) d: 165.7/165.0 (s), 163.0/
162.9/162.5/160.7 (s), 150.7/150.6/147.8/147.5 (s), 139.0/138.7/
138.0/137.2 (s), 137.6/136.2 (s), 129.0/128.9/128.4 (d), 124.5/
124.2/123.55/123.31 (d), 122.4/122.3/122.1/122.0 (d), 59.7/59.6
(t), 44.0/43.7 (t). IR (KBr): n ¼ 3242, 2953, 1613, 1587, 1558,
1340, 1053 cmꢁ1. MS (EI, 70 eV): 274 (Mþ, 100), 257 (63), 239
(16), 212 (47), 144 (16), 77 (40). HR-MS (EI): C12H14N6O2
requires 274.1178, found 274.1177.
4-Amino-6-(3,4-dimethoxybenzyl)methylamino-2-
methoxy-5-nitrosopyrimidine (9e)
To a suspension of 4-amino-2,6-dimethoxy-5-nitrosopyrimi-
dine 1c (184 mg, 1.00 mmol) in acetonitrile (10 mL), 3,4-
dimethoxybenzylamine (0.19 mL, 1.2 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 (20 h). Then, water (20 mL) was added
and the solid in suspension was filtered, washed, and finally,
dried in a vacuum desiccator over potassium hydroxide pellets
offering compound 9e as a blue solid (232 mg, 73%), m.p.
195–197˚C. 1H NMR (400 MHz, CDCl3, 25˚C) d (ppm): 11.58/8.12
(bs, 1H, NH), 10.44/7.58 (bs, 1H, NH2), 6.95–6.81 (m, 3H, Ph),
5.90/5.74 (bs, 1H, NH2), 4.78/4.63 (d, J ¼ 5.8 Hz; 2H, NCH2), 4.00/
3.99 (s, 3H, OCH3), 3.87/3.86 (s, 6H, 2OCH3). 13C NMR (100 MHz,
CDCl3) d: 168.2 (s), 165.2 (s), 149.7 (s), 149.3 (s), 148.9/148.8 (s),
138.4/137.9 (s), 129.6/129.1 (d), 120.5/120.3 (d), 111.5/111.3 (d),
56.0 (q), 55.4 (q), 45.0, 43.7 (t). IR (KBr): n ¼ 3405, 3305, 2954,
1632, 1598, 1520, 1465, 1370, 1274 cmꢁ1. MS (EI, 70 eV): 319
(Mþ, 8), 302 (100), 286 (37), 151 (30). HR-MS (EI): C14H17N5O4
requires 319.1281, found 319.1279.
4-Amino-6-(3,4-dimethoxyphenyl)methylamino-2-(2-
hydroxyethylamino)-5-nitrosopyrimidine (10c)
To
a
suspension of 5-nitroso-pyrimidine 9c (159 mg,
0.50 mmol) in methanol (5 mL), 2-aminoethanol (0.06 mL,
1.0 mmol) was added. The mixture was stirred, refluxed, and
monitored by TLC (silica, eluent: CH2Cl2/MeOH 9:1, v/v) until
no starting material was visualized (12 h). After cooling, the
precipitate was filtered, washed, and finally, dried in a
vacuum dessiccator over potassium hydroxide pellets offering
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Archiv der Pharmazie
title compound as a pink solid (104 mg, 60%), m.p. 200–202˚C
(s), 162.8, 162.6 (s), 150.7, 150.5, 150.2, 149.9 (s), 137.3, 136.7
(s), 72.7 (t), 72.6 (t), 69.4, 69.3 (t), 68.9, 68.9 (t), 60.7 (t), 41.2,
41.1 (t), 39.1, 39.0 (t). IR (KBr): n ¼ 3368, 3323, 2938, 1580, 1555,
1384, 1175, 1121 cmꢁ1. MS (EI, 70 eV): 330 (Mþ, 4), 313 (4), 227
(7), 177 (10), 84 (13), 66 (21), 45 (100). HR-MS (EI): C12H22N6O5
requires 330.1652, found 330.1653.
1
(d). H NMR (400 MHz, DMSO-d6, 25˚C) d (ppm): 11.84, 11.52,
9.31, 9.00 (t, J ¼ 5.5, 5.6 Hz, 1H, NH), 10.54, 10.22, 8.25, 8.02 (bs,
1H, NH2), 8.01, 7.55, 7.53, 7.27 (bs, 1H, NH2), 7.70 (bs, 1H, NH),
7.04–6.84 (m, 3H, Ph); 4.72–4.65 (m, 1H, OH), 4.65–4.45 (m,
NCH2), 3.70 (s, 6H, 2OCH3), 3.55–3.44 (m, 2H, OCH2), 3.41–3.34
(m, 2H, NCH2). 13C NMR (100 MHz, DMSO-d6) d: 165.8, 165.2,
163.0, 162.8, 162.7, 162.6, 150.8, 150.0, 149.7, 148.7, 148.6,
148.0, 147.8, 137.4, 136.8, 136.7, 132.1, 132.0, 130.8, 130.6 (s),
120.1 119.9 (d), 112.0, 111.9 (d), 111.8 (d), 59.7 (t), 55.4 (q), 43.6
(t), 42.1 (t). IR (KBr): n ¼ 3510, 3341, 3168, 2938, 1648, 1602,
1544, 1321, 1255, 1168, 1032 cmꢁ1. MS (EI, 70 eV): 348 (Mþ, 3),
330 (63), 299 (98), 283 (39), 183 (41), 151 (100), 120 (42), 107
(47), 43 (84). HR-MS (EI): C15H20N6O4 requires 348.1546, found
348.1546.
Biological screenings
Microorganisms
The following strains were used: S. aureus methicillin-sensitive
ATCC 29213, S. aureus methicillin-resistant ATCC 43300, E. coli
´
ATCC 25922, E. coli LM1 (LM: Laboratorio de Microbiologıa,
´
Facultad de Ciencias Medicas, Universidad Nacional de Cuyo,
Mendoza, Argentina), E. coli LM2, P. aeruginosa ATCC 27853,
Y. enterocolitica PI (PI: Pasteur Institute), S. enteritidis MI (MI:
´
Malbran Institute), and Salmonella sp. LM.
4-Amino-2,6-bis(2-hydroxyethylamino)-5-
nitrosopyrimidine (10d)
Antibacterial activity
To a suspension of 4-amino-2,6-dimethoxy-5-nitrosopyrimi-
dine 1c (184 mg, 1.00 mmol) in methanol (10 mL), 2-amino-
ethanol (0.14 mL, 2.4 mmol) was added. The mixture was
stirred, refluxed, and monitored by TLC (silica, eluent: CH2Cl2/
MeOH 9:1, v/v) until no starting material was visualized (12 h).
After cooling, the precipitate was filtered, washed, and
finally, dried in a vacuum desiccator over potassium hydroxide
pellets offering title compound as a pink solid (187 mg,
77%), m.p. 210–212˚C (d). 1H NMR (400 MHz, DMSO-d6, 25˚C) d
(ppm): 11.64/11.37/8.63/8.42 (t, J ¼ 5.5, 5.6 Hz, 1H, NH), 10.57/
10.24/8.24/8.02 (bs, 1H, NH2), 7.64 (bs, 1H, NH), 8.01/7.71/7.51/
7.25 (bs, 1H, NH2), 4.88 (bs, 1H, OH), 4.72 (bs, 1H, OH), 3.62–
3.17 (m, 8H, 2OCH2 þ 2NCH2). 13C NMR (100 MHz, DMSO-d6) d:
165.9, 165.3 (s), 163.0, 162.8 (s), 150.9, 150.8, 150.4, 150.2 (s),
137.3, 136.8 (s), 59.9, 59.8, 59.6 (t), 59.5, 59.2, 59.1 (t), 43.7,
43.5, 42.8, 42.6 (t), 41.3, 41.4 (t). IR (KBr): n ¼ 3370, 3324, 2940,
1651, 1602, 1560, 1382, 1192, 1058 cmꢁ1. MS (EI, 70 eV): 242
(Mþ, 100), 225 (53), 211 (52), 195 (79), 183 (73), 177 (39), 68
(57), 43 (98). HR-MS (EI): C8H14N6O3 requires 242.1127, found
242.1123.
The minimal inhibitory concentration (MIC) values were
determined using the microbroth dilution method according
to the protocols of the Clinical and Laboratory Standards
Institute (CLSI, 20012) [42].
All tests were performed in Mueller-Hinton broth (MHB),
and cultures of each strain were prepared overnight. Micro-
organisms suspensions were adjusted in a spectrophotometer
with sterile physiological solution to give a final organism
density of 0.5 McFarland scale (1–5 ꢃ 105 CFU/mL). Stock
solutions of peptides in DMSO were diluted to give serial
twofold dilutions that were added to each medium to obtain
final concentrations ranging from 0.25 to 50 mg/mL. The final
concentration of DMSO in the assay did not exceed 1%. The
Cefotaxime1 Argentia Pharmaceutica antimicrobial agent
was included in the assays as a positive control. The plates
were incubated for 24 h at 37˚C. The MIC was defined as the
lowest peptide concentration showing no visible bacterial
growth after incubation time. Tests were done in triplicate.
Acute toxicity test
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 Labo-
ratory [43] was modified in order to use lower amounts of
tested compounds [44]. 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 concentration 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
peptides and continued for 96 h. The number of dead
organisms in each test chamber was recorded and the dead
organisms were removed 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 experience was performed two times with three
2-{2-[(4-Amino-6-{[2-(2-hydroxyethoxy)ethyl]amino}-5-
nitrosopyrimidin-2-yl)amino]ethoxy}ethanol (10e)
To a suspension of 4-amino-2,6-dimethoxy-5-nitrosopyrimi-
dine 1c (184 mg, 1.00 mmol) in methanol (10 mL), 2-(2-
aminoethoxy)ethanol (0.25 mL, 2.4 mmol) was added. The
mixture was stirred, refluxed, and monitored by TLC (silica,
eluent: CH2Cl2/MeOH 9:1, v/v) until no starting material was
visualized (24 h). After cooling, solution was concentrated in
vacuum to remove methanol and then, acetonitrile was
added. The precipitate was filtered, washed, and finally, dried
in a vacuum dessiccator over potassium hydroxide pellets
offering title compound as a pink solid (230 mg, 70%), m.p.
144–146˚C (d). 1H NMR (400 MHz, DMSO-d6, 25˚C) d (ppm):
11.59, 11.33, 8.71, 8.47 (bs, 1H, NH), 10.53, 10.21, 8.26, 8.06 (bs,
1H, NH2), 7.73 (bs, 1H, NH), 8.04, 7.751, 7.54, 7.26 (bs, 1H, NH2),
4.61 (bs, 1H, OH), 4.59 (bs, 1H, OH), 4.79–3.38 (m, 16H,
4OCH2 þ 4NCH2). 13C NMR (100 MHz, DMSO-d6) d: 165.7, 165.2
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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replicates each. We chose this technique because it is fast,
economic, and easy to reproduce. This assay has been
previously used by our group to test the toxicity of synthetic
and natural compounds [16, 44]. The species P. reticulata has
been previously used in acute toxicity test [45].
eigenvalue, and the transition states were also confirmed
by animating the negative eigenvectors coordinate with a
visualization program and internal reaction coordinate (IRC)
calculations [52, 53]. HF/6-31G(d) IRC calculations were
performed on the transition state structures to check that
the TSs structures lead to the initial conformer and to the final
conformation (forward and reverse directions of the confor-
mational interconversion path). IRC calculations steps 6 points
in Cartesian coordinates in the forward direction and 6 points
in the reverse direction, in step of 03 amu1/2 Bohr along the
path were carried out.
Culture of HepG2 cells
HepG2 cells were cultured in Ham’s F-12/Leibovitz L-15 (1:1 v/v)
supplemented with 7% newborn calf serum, 50 U penicillin/
mL, and 50 mg streptomycin/mL. For sub-culturing purposes,
cells were detached by treatment with 0.25% trypsin/0.02%
EDTA at 37˚C. Cultures were used at 75% confluency.
The electronic study of nitrosopyrimidines was carried out
using MEPs [54]. The low energy conformations were
obtained for these compounds from our conformational
search. Subsequently, single point calculations were carried
out. Thus, these MEPs were calculated using B3LYP/6-
311þþG(d,p) wave functions and MEPs graphical presenta-
tions were created using the MOLEKEL program [55]. The
overlapping graphics images were produced using the UCSF
Chimera package [56].
MTT assay
Cytotoxicity effects were measured by using the MTT
(tetrazoliun salt) reduction assay after 24 h of exposure to
different concentrations of the compound (g/mL). The stock
solution of the extract or compounds was prepared at a
concentration of 1 mg/mL in culture medium and convenient-
ly diluted in culture medium to obtain the final desired
concentrations. Control cells (only medium-treated cells) were
included in all experiments.
R.D.E. acknowledges ANPCyT, PICT 2010-1832. Grants from
Universidad Nacional de San Luis (UNSL), partially supported
The MTT assay was performed according to the method of
Jover et al. [46] Briefly, the medium was removed after
exposure and cultures were washed with PBS. Then, 100 mL/
well MTT reagent (5 mg/mL in medium) was added to each
well and plates were placed in the incubator for a further 3 h.
The corresponding supernatants were discarded and cells
were washed again. The dye was extracted with DMSO and
optical density was read at 490 nm on a microplate reader. The
percentage of inhibition (%) of the succinic dehydrogenase
reduction of MTT was calculated in relation to control cells in
each experimental series (control cells were assumed to have
100% viability).
´
this work. Authors thank “Centro de Instrumentacion
´
´
Cientıfico Tecnica” and the staff for data collection. The
´
´
financial support from the Consejerıa de Economıa, Innova-
´
´
cion y Ciencia (Junta de Andalucıa, Spain), the Universidad de
´
´
Jaen and Ministerio de Ciencia e Innovacion (project reference
SAF2008-04685-C02-02) is thanked. R.D.E., G.E.F., and S.A.A.
are members of the Consejo Nacional de Investigaciones
´
´
Cientıficas y Tecnicas (CONICET-Argentina) staff. B.L. is a
fellowship of CONICET and thanks CICITCA-UNSJ for a grant.
The authors have declared no conflict of interest.
Molecular modeling
All the calculations reported here were performed using the
GAUSSIAN 09 program [47]. Critical points (low-energy
conformations and transition state structures) were optimized
at DFT level of theory. Density functional theory (DFT) with
the Becke-3-Lee-Yang-Parr (RB3LYP) functional [48–50] and 6-
31G(d) basis set calculations were used in the optimization
jobs of minimum obtained from potential energy surface
(PES). An extensive search to localize the critical points on the
PEHSs was carried out first by using starting geometries
suggested by GASCOS algorithm [51]. These input files were
used to obtain the low-energy conformations and TS
structures using RHF/6-31G(d) calculations. Vibrational fre-
quencies for the optimized structures were computed to
evaluate the zero-point energies (ZPE) as well as to confirm
the nature of the singular points along the PES. The stationary
points have been identified as a minimum with no imaginary
frequencies, or as a first-order transition state characterized
by the existence of only one imaginary frequency in the
normal mode coordinate analysis. Transition state structures
were located until the Hessian matrix had only one imaginary
References
[1] B. A. Rogers, Z. Aminzadeh, Y. Hayashi, D. L. Paterson,
Clin. Infect. Dis. 2011, 53, 49–56.
´
[2] M. Delgado-Valverde, J. Sojo-Dorado, A. Pascual,
´
˜
J. Rodrıguez-Bano, Ther. Adv. Infect. Dis. 2013, 1, 49–69.
[3] D. Talon, J. Hosp. Infect. 1999, 43, 13–17.
[4] A. Persidis, Nat. Biotechnol. 1999, 17, 1141–1142.
[5] A. Polak, Mycoses 1999, 42, 355–370.
´
[6] a) J. Bartroli, E. Turmo, M. Alguero, E. Boncompte,
´
M. Vericat, L. Conte, J. Ramis, M. Merlos, J. Garcıa-
Rafanell, J. Forn, J. Med. Chem. 1998, 41, 1855–1868;
´
b) J. Bartroli, E. Turmo, M. Alguero, E. Boncompte,
´
M. Vericat, L. Conte, J. Ramis, M. Merlos, J. Garcıa-
Rafanell, J. Med. Chem. 1998, 41, 1869–1882.
[7] T. J. Walsh, A. Groll, J. Hiemenz, R. Fleming, E. Roilides,
E. Anaissie, Clin. Microbiol. Infect. 2004, 10, 48–66.
[8] M. F. Masman, A. M. Rodriguez, L. Svetaz, S. A. Zacchino,
C. Somlai, I. G. Csizmadia, B. Penke, R. D. Enriz, Bioorg.
Med. Chem. 2006, 14, 7604–7614.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
11

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 1–13
M. Olivella et al.
Archiv der Pharmazie
´
[9] M. F. Masman, A. M. Rodriguez, M. Raimondi, S. A.
Zacchino, P. G. Luiten, C. Somlai, T. Kortvelyesi, B. Penke,
R. D. Enriz, Eur. J. Med. Chem. 2009, 44, 212–228.
[10] F. Garibotto, A. D. Garro, C. Somlai, M. F. Masman,
P. Lutien, S. A. Zacchino, A. M. Rodriguez, B. Penke,
R. D. Enriz, Bioorg. Med. Chem. 2010, 18, 158–170.
[11] M. S. Olivella, A. M. Rodriguez, C. Somlai, B. Penke,
V. Farkas, A. Perczel, R. D. Enriz, Bioorg. Med. Chem. Lett.
2010, 20, 4808–4811.
[27] A. Marchal, M. Melguizo, M. Nogueras, A. Sanchez,
J. N. Low, Synlett 2002, 20, 255–258.
´
[28] J. Quiroga, J. Trilleras, B. Insuasty, R. Abonıa,
M. Nogueras, A. Marchal, J. Cobo, Tetrahedron Lett.
2008, 49, 3257–3259.
[29] C. Glidewell, J. N. Low, A. Marchal, A. Quesada, Acta
Crystallogr. Sect. C 2003, 59, 454–460.
´
´
[30] E. R. Correche, S. A. Andujar, R. R. Kurdelas, M. J. Gomez
´
Lechon, M. L. Freile, R. D. Enriz, Bioorg. Med. Chem.
´
[12] F. M. Garibotto, A. D. Garro, A. M. Rodrıguez, M.
2008, 16, 3641–3651.
Raimondi, S. A. Zacchino, A. Perczel, C. Somlai, B. Penke,
R. D. Enriz, Eur. J. Med. Chem. 2011, 46, 370–377.
[31] C. Somlai, E. Correche, M. Olivella, L. Tolos, M. J. Gomez
Lechon, G. Dombi, G. K. To’th, B. Penke, R. D. Enriz,
Bioorg. Med. Chem. Lett. 2012, 22, 4233–4237.
[13] A. D. Garro, M. S. Olivella, J. A. Bombasaro, B. Lima,
A. Tapia, G. Feresin, A. Perczel, C. Somlai, B. Penke,
´
[32] E. Prochazkova, L. Cechova, Z. Janeba, M. Dracˇınsky, J.
´
´
J. Lopez Cascales, A. M. Rodrıguez, R. D. Enriz, Chem.
Org. Chem. 2013, 78, 10121–10133.
[33] P. Gilli, V. Bertolasi, V. Ferretti, G. J. Gilli, Am. Chem. Soc.
2000, 122, 10405–10417.
Biol. Drug Des. 2013, 82, 167–177.
[14] S. Zacchino, S. A. Lopez, G. Pezzenati, R. Furlan,
˜
C. Santecchia, L. Munoz, F. Giannini, A. M. Rodriguez,
[34] M. H. Holschbach, D. Sanz, R. M. Claramunt, L. Infantes,
S. Motherwell, P. R. Raithby, M. L. Jimeno, D. Herrero,
I. Alkorta, N. Jagerovic, J. Elguero, J. Org. Chem. 2003, 68,
8831–8837.
R. D. Enriz, J. Nat. Prod. 1999, 63, 1353–1357.
[15] R. R. Kurdelas, B. Lima, A. Tapia, G. Feresin, M. Gonzalez
´
Sierra, M. V. Rodrıguez, S. Zacchino, R. D. Enriz,
M. L. Freile, Molecules 2010, 15, 4898–4907.
[16] M. L. Freile, F. Giannini, G. Pucci, A. Sturniollo,
L. R. Dodero, O. Pucci, V. Balzareti, R. D. Enriz, Fitoter-
apia 2003, 74, 702–705.
[35] A. Marchal, M. Nogueras, A. Sanchez, J. N. Low, L. Naesens,
E. De Clercq, M. Melguizo, Eur. J. Org. Chem. 2010, 20,
3823–3830.
[36] G. Urbelis, I. Susvilo, S. Tumkevicius, J. Mol. Model. 2007,
13, 219–224.
[17] M. Derita, I. Montenegro, F. Garibotto, R. D. Enriz, M. C.
Fritis, S. A. Zacchino, Molecules 2013, 18, 2029–2050.
[37] I. Susvilo, A. Brukstus, S. Tumkevicius, Tetrahedron Lett.
2005, 46, 1841–1844.
´
[18] M. Derita, E. Del Olmo, B. Barboza, A. E. Garcıa-Cadenas,
´
´
´
J. L. Lopez-Perez, S. Andujar, R. D. Enriz, S. A. Zacchino,
A. S. Feliciano, Molecules 2013, 18, 3479–3501.
[19] L. J. Gutierrez, M. L. Mascotti, M. B. Kurina-Sanz,
C. Pungitore, R. D. Enriz, F. A. Giannini, Nat. Prod.
Commun. 2012, 7, 1639–1644.
[38] M. Melguizo, A. Quesada, J. N. Low, C. Glidewell, Acta
Crystallogr, Sect. B 2003, 59, 263–276.
[39] P. Politzer, D. G. Truhlar, Chemical Applications of
Atomic and Molecular Electrostatic Potentials, Plenum
Press, New York 1981, p. 1.
´
[20] M. Sortino, F. Garibotto, V. Cechinel Filho, M. Gupta,
R. Enriz, S. Zacchino, Bioorg. Med. Chem. 2011, 19, 2823–
2833.
[40] P. A. Carrupt, N. El Tayar, A. Karlen, B. Festa, Methods
Enzymol. 1991, 202, 638–650.
[41] P. Greeling, W. Langenaeker, F. De Proft, A. Baeten, in
Molecular Electrostatic Potentials: Concepts and Appli-
cations. Theoretical and Computational Chemistry, Vol. 3,
Elsevier Science B.V., Amsterdam 1996, pp. 587–617.
[42] CLSI, Performance standards for Antimicrobial Suscepti-
bility Testing, Twenty-Second Informational Supplement.
CLSI document M100-S22, Clinical and Laboratory Stand-
ards Institute, Wayne, PA 2012.
[21] V. V. Kouznetsov, L. Y. Vargas Mendes, M. Sortino,
Y. Vasquez, M. P. Gupta, M. Freile, R. D. Enriz,
S. Zacchino, Bioorg. Med. Chem. 2008, 16, 794–809.
[22] M. A. Sortino, P. Delgado, S. F. Juarez, J. Quiroga,
´
R. Abonıa, B. Insuasty, M. Nogueras, L. Rodero,
F. Garibotto, R. D. Enriz, S. A. Zacchino, Bioorg. Med.
Chem. 2007, 15, 484–494.
[23] L. Karolyhazy, M. L. Freile, M. Anwair Gy Beke,
F. Giannini, M. Sortino, J. C. Ribas, S. Zacchino,
P. Matyus, R. D. Enriz, Arzneim.- Forsch./-Drug Res.
2003, 53, 738–743.
[43] W. W. Johnsosn, M. T. Finley, Handbook of Acute
Toxicity of Chemicals to Fish and Aquatic Invertebrates,
United States Department of the Interior Fish and
Wildlife Service, Washington, DC 1980, p. 1.
´
[24] M. Olivella, A. Marchal, M. Nogueras, A. Sanchez,
[44] F. Bisogno, L. Mascotti, C. Sanchez, F. Garibotto,
F. Giannini, M. Kurina, R. D. Enriz, J. Agric. Food Chem.
2007, 55, 10635–10640.
M. Melguizo, M. Raimondi, S. Zacchino, F. Giannin,
J. Cobo, R. D. Enriz, Bioorg. Med. Chem. 2012, 20, 6109–
6122.
[45] W. Slooff, D. De Zwart, J. Van de Kerkhoff, Aquat.
Toxicol. 1983, 4, 189–198.
´
[25] A. Marchal, M. Nogueras, A. Sanchez, J. N. Low,
´
´
L. Naesens, E. De Clercq, M. Melguizo, Eur. J. Org.
Chem. 2010, 2, 3823–3830.
[46] R. Jover, X. Ponsoda, J. V. Castell, M. J. Gomez-Lechon,
Toxicol. In Vitro 1992, 6, 47–52.
´
[26] J. Cobo, M. Nogueras, J. N. Low, R. Rodrıguez, Tetrahe-
[47] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone,
dron Lett. 2008, 49, 7271–7273.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
12

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 1–13
New Antibacterial Nitrosopyrimidines
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B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato,
X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng,
J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda,
J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta,
F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers,
K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand,
K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar,
J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene,
J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo,
R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin,
R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin,
K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador,
[50] C. Lee, W. Yang, R. Parr, Phys. Rev. B 1988, 37, 785–789.
[51] a) L. N. Santagata, F. D. Suvire, R. D. Enriz, J. L. Torday,
I. G. Csizmadia, J. Mol. Struct. (Theochem) 1999, 465, 33–
67; b) L. N. Santagata, F. D. Suvire, R. D. Enriz, J. Mol.
Struct. (Theochem) 2000, 507, 89–95; c) L. N. Santagata,
F. D. Suvire, R. D. Enriz, J. Mol. Struct. (Theochem) 2001,
536, 173–188.
[52] C. Gonzalez, H. B. Schlegel, J. Chem. Phys. 1989, 90, 2154–
2161.
[53] C. Gonzalez, H. B. Schlegel, J. Chem. Phys. 1990, 94, 5523–
5527.
[54] P. Politzer, D. G. Truhlar, in Chemical Applications of
Atomic and Molecular Electrostatic Potentials, Plenum
Publishing, New York 1991, p. 749.
¨
J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas,
J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox, Gaussian
09 (Revision D.01), Gaussian, Inc., Wallingford, CT 2009.
[48] A. Becke, Phys. Rev. A 1988, 38, 3098–3100.
[55] S. Portmann, H. Lu¨ thi, Chimia 2000, 54, 766–770.
[56] E. Pettersen, T. Goddard, C. Huang, G. Couch, D. Greenblatt,
E. Meng, T. Ferrin, J. Comput. Chem. 2004, 25, 1605–
1612.
[49] A. Becke, J. Chem. Phys. 1993, 98, 5648–5652.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
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