5-Substituted 2-amino-4, 6-dihydroxypyrimidines and 2-amino-4, 6-dichloropyrimidines: Synthesis and inhibitory effects on immune-activated nitric oxide production

Article abstract of DOI:10.1007/s00044-014-1018-9

A series of 5-substituted 2-amino-4, 6-dihydr-oxypyrimidines were prepared by a modified condensation of the corresponding monosubstituted malonic acid diesters with guanidine in an excess of sodium ethoxide. The optimized procedure using Vilsmeier-Haack-Arnold reagent, followed by immediate deprotection of the (dimethylamino)methylene protecting groups, has been developed to convert the 2-amino-4, 6-dihydroxypyrimi-dine analogs to novel 5-substituted 2-amino-4, 6-dichloro-pyrimidines in high yields. Pilot screening for biological properties of the prepared compounds was done in mouse peritoneal cells using the in vitro nitric oxide (NO) assay. Irrespective of the substituent at the 5 position, 2-amino-4, 6-dichloropyrimidines inhibited immune-activated NO production. The most effective was 5-fluoro-2-amino-4, 6-dichloropyrimidine with an IC₅₀ of 2 lM (higher activity than the most potent reference compound) while the IC₅₀s of other derivatives were within the range of 9-36 μM. The 2-amino-4, 6-dihydroxypyrimidine counterparts were devoid of any NO-inhibitory activity. The compounds had no suppressive effects on the viability of cells. The Mechanism of action remains to be elucidated.

Full text of DOI:10.1007/s00044-014-1018-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-1018-9
ORIGINAL RESEARCH
5-Substituted 2-amino-4,6-dihydroxypyrimidines and 2-amino-4,
6-dichloropyrimidines: synthesis and inhibitory effects
on immune-activated nitric oxide production
•
Petr Jansa Antonın Holy Martin Dracınsky Viktor Kolman
•
•
•
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Zlatko Janeba Petra Kostecka Eva Kmonıckova Zdenek Zıdek
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•
•
•
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Received: 17 November 2013 / Accepted: 17 April 2014
Ó Springer Science+Business Media New York 2014
Abstract A series of 5-substituted 2-amino-4,6-dihydr-
oxypyrimidines were prepared by a modified condensation
of the corresponding monosubstituted malonic acid diesters
with guanidine in an excess of sodium ethoxide. The
optimized procedure using Vilsmeier–Haack–Arnold
reagent, followed by immediate deprotection of the
(dimethylamino)methylene protecting groups, has been
developed to convert the 2-amino-4,6-dihydroxypyrimi-
dine analogs to novel 5-substituted 2-amino-4,6-dichloro-
pyrimidines in high yields. Pilot screening for biological
properties of the prepared compounds was done in mouse
peritoneal cells using the in vitro nitric oxide (NO) assay.
Irrespective of the substituent at the 5 position, 2-amino-
4,6-dichloropyrimidines inhibited immune-activated NO
production. The most effective was 5-fluoro-2-amino-4,6-
dichloropyrimidine with an IC50 of 2 lM (higher activity
than the most potent reference compound) while the IC50s
of other derivatives were within the range of 9–36 lM. The
devoid of any NO-inhibitory activity. The compounds had
no suppressive effects on the viability of cells. The
Mechanism of action remains to be elucidated.
Keywords Pyrimidine ꢀ Nitric oxide ꢀ NO ꢀ
Anti-inflammatory
Introduction
This work was initially motivated by the finding that
2-amino-4,6-dichloropyrimidine inhibits replication of a
broad range of viruses such as members of the Herpes,
Picorna, and Pox groups (Marcialis et al., 1973, 1974; La
Colla et al., 1975). It has been reported that maturation of
viral particles was prevented as viral proteins synthesized
in the presence of this compound were not assembled into
new virions (Flore et al., 1977; La Colla et al., 1977;
Marcialis et al., 1979). This represents a unique and very
attractive mechanism of action for new antiviral drugs,
especially in combination with other antiviral agents with
the aim to suppress resistance development. The pyrimi-
dine heterocycle is an elemental structural motif of several
essential natural products (Undheim and Benneche, 1996;
Ralevic and Burnstock, 1998; Lagoja, 2005) and a number
of synthetic drugs. It was also reported that 2,4-diamino-6-
hydroxypyrimidine suppresses NO production in chicken
macrophages (Sung et al., 1994).
2-amino-4,6-dihydroxypyrimidine
counterparts
were
´
´
Antonın Holy: Deceased.
´
ˇ´
´
P. Jansa (&) ꢀ A. Holy ꢀ M. Dracınsky ꢀ V. Kolman ꢀ Z. Janeba
Institute of Organic Chemistry and Biochemistry, Academy of
´
Sciences of the Czech Republic, v.v.i., Flemingovo nam. 2,
166 10 Prague 6, Czech Republic
e-mail: jansa@uochb.cas.cz; jansap@email.cz
´
´ˇ
´
´
P. Kostecka ꢀ E. Kmonıckova ꢀ Z. Zıdek
Institute of Experimental Medicine, Academy of Sciences of the
´
There is a large class of pharmacologically important
pyrimidine derivatives that act as dihydrofolate reductase
(DHFR) inhibitors (Hitchings, 1989), as well as com-
´ ˇ
Czech Republic, Vıdenska 1083, 142 20 Prague 4,
Czech Republic
e-mail: zidekz@biomed.cas.cz
´
´
pounds with anti-HIV (Holy, 2003; De Clercq and Holy,
2005), anti-adenovirus (Naesens et al., 2005), and anti-
HBV activities (Ying et al., 2005), inhibitors of tetrahy-
drobiopterin synthesis (Bogdan et al., 1995; Kolinsky and
´ˇ
E. Kmonıckova
Institute of Pharmacology and Toxicology, Faculty of Medicine
´
in Pilsen, Charles University, Karlovarska 48, 30 166 Pilsen,
Czech Republic
´
123

Med Chem Res
Scheme 1 General synthesis of 5-substituted 2-amino-4,6-dihydroxypyrimidines (a) and 2-amino-4,6-dichloropyrimidines (b). For the source of
A1, B1, A12, and B12 see experimental
Gross, 2004), regulators of pain sensitivity and persistence
(Tegeder et al., 2006), antidepressants (Arvanitis et al.,
1999), compounds which suppress accumulation of cyto-
kine-induced NF-jB (Ikemoto et al., 2008), inhibitors of
EGFR and Her-2 tyrosine kinases (Suzuki et al., 2012) and
cyclin-dependent kinases as potential drug candidates for
cancer therapy (Beattie et al., 2003; Breault et al., 2003).
Substitution at position 5 of the pyrimidine moiety has been
used in the past to improve the biological property of several
pharmacologically interesting pyrimidine derivatives with
1963; Baraldi et al., 2003; Patel et al., 2007; Rostom et al.,
2009), optimization of the reaction conditions in our case
had a crucial impact on the isolation, yield, and purity of
the products especially in multi-gram scales (for details see
experimental). The following is our key findings: methanol
or ethanol is required as the solvent to achieve full con-
version and the clean reaction profile (alcohols are able to
at least partially dissolve the product which otherwise
precipitates in quantities that quickly precludes stirring);
addition of water after the reaction (which dissolves the
sodium salt of the product) and subsequent neutralization
by acetic acid is required to keep excess of guanidine in
solution while the product quantitatively precipitates from
the neutralized solution.
´
anti-HIV (Hockova et al., 2003, 2004) or anti-influenza
activity (Jansa et al., 2012), with inhibitory activity against
human thymidine phosphorylase (Nencka et al., 2007) or
´ ´
with antioxidative activity (Prochazkova et al., 2012a, b).
The aim of the present study was the preparation of
The second step in the synthesis of the targeted
5-substituted 2-amino-4,6-dichloropyrimidines B2–B11
(Scheme 1) consists of the transformation of the 4,6-dihy-
droxypyrimidine moiety into the 4,6-dichloropyrimidine
residue. For such reactions, chlorides of mineral acids such
as POCl3, PCl5, SOCl2, or COCl2 with diverse additives
like DMF, pyridine, 2-methylpyridine, diphenylamine, or
triethylamine are generally used (Altenbach et al., 2008;
Jang et al., 2011). Nevertheless, these classical procedures
turned out to be unsuitable for the preparation of the desired
5-substituted 2-amino-4,6-dichloropyrimidines B2–B11,
owing to their complicated isolations and very low isolated
yields (max. 30 %). This synthetic issue was resolved by
application of a modified synthetic procedure for the prep-
aration of 4,6-dichloro-2,5-bis{[(dimethylamino)methy-
lene]amino}pyrimidine (Daluge et al., 2000). The use of the
Vilsmeier–Haack–Arnold reagent (Reichardt, 1999), fol-
lowed by immediate deprotection of the (dimethyl-
amino)methylene protecting groups, gave final compounds
B2–B11 (Scheme 1). The final step was carried out
according to the method described in the literature (Daluge
et al., 2000), which was modified in order to obtain B2–B11
directly from the reaction mixture by simple precipitation
and filtration (for details see experimental).
5-substituted
2-amino-4,6-dihydroxypyrimidines
and
2-amino-4,6-dichloropyrimidines, and the pilot screening
of their intrinsic biological potential. Substituents at posi-
tion 5 of the pyrimidine ring were designed to probe this
position with a standard homologous series of alkyl sub-
stituents to study steric requirements in this position (e.g.,
length, branching, saturation, and desaturation). Possible
antiviral activity of compounds and their influence on the
immune-activated high output nitric oxide (NO) production
were analyzed. The NO assay has been employed for its
capacity to indicate both positive and negative interfer-
ences of compounds with cell immune responses including
´ˇ ´
secretion of cytokines and cytotoxicity (Kmonıckova et al.,
2007; Harmatha et al., 2013).
Chemistry
At first, 2-amino-4,6-dihydroxypyrimidines A2–A11
(Scheme 1) were prepared by the condensation of the
properly substituted malonic acid diesters with guanidine
under basic conditions. Despite the fact that this reaction is
well-known in the literature (Rembold and Schramm,
123

Med Chem Res
A
B
125
125
100
75
50
25
0
Group A
Group B
Group A
Group B
100
75
50
25
0
1
2
3
4
5
6
7
8
9
10 11 12
1
2
3
4
5
6
7
8
9
10 11 12
Substituent C5
Substituent C5
Fig. 1 Effects of 5-substituted 2-amino-4,6-dihydroxypyrimidines
A1–A12 and 2-amino-4,6-dichloropyrimidines B1–B12 on the pro-
duction of NO by mouse peritoneal cells (a) and their viability (b).
The compounds were applied at a concentration of 50 lM. The NO
data are means obtained from at least four experiments. The viability
data were averaged from two to four experiments. All columns are
mean SEM. Dotted lines denote 95 % confidential intervals of
control values. * p \ 0.05, ** p \ 0.01, *** p \ 0.001
100
75
Biological properties
Antiviral
None of the compounds A1–A12 or B1–B12 showed any
antiviral activity (EC50 [ 100 lg/mL) toward the follow-
ing viruses: HIV-1, HIV-2 in CEM cells, HCMV, HSV-1,
HSV-2, vaccinia virus and vesicular stomatitis virus (VSV)
in HeLa cells, Feline herpes virus and Feline corona virus
(FIPV) in CRFK cells, VSV, Coxsackie virus B4 and
respiratory syncytial virus (RSV) in HeLa cells, Parain-
fluenza 3-virus, Reovirus-1, Coxsackie B4 virus and Punta
Toro virus in MDCK cells. The parent 2-amino-4,6-
dichloropyrimidine (B1) was also tested and no antiviral
activity was observed (EC50 [ 100 lg/mL). It is important
to mention that antiviral activities described in the litera-
ture for this compound required relatively high concen-
trations in the range of the 100 lg/mL (Marcialis et al.,
1973, 1974, La Colla et al., 1975).
50
B3
B5
B6
25
B8
B10
B12
0
1
10
Concentration µM
Fig. 2 Dose-dependent inhibitory effects of 5-substituted 2-amino-
4,6-dichloropyrimidines B1–B12 on in vitro NO production. The
points are mean SEM. The data represent one of two identical
experiments. The concentration of nitrites following the LPS/IFN
stimulus (i.e. 100 % response) was 67.90 1.50 lM. It was
1.80 0.30 lM in untreated controls
NO production
compounds remained ineffective even at fourfold higher
(i.e., 200 lM) concentrations (data not shown).
The in vitro stimulation of mouse peritoneal cells with LPS
plus IFN-c substantially enhanced NO biosynthesis. The
concentration of nitrites varied from 53.30 0.90 lM to
74.00 3.30 lM (mean SEM) in various experiments.
The spontaneous production of NO by control cells was
negligible, ranging from 0.80 0.30 lM to 1.70 1.60 lM
(mean SEM) in individual experiments.
In contrast, all 5-substituted 2-amino-4,6-dichloropyr-
imidines B1–B12, applied at 50 lM concentrations, sup-
pressed NO production by at least 55 % (Fig. 1a). The
effects were highly statistically significant (F/12, 66/= 6.04,
p \ 0.0001). The most effective was 5-fluoro-2-amino-4,6-
dichloropyrimidine (B12).
The suppression of NO production by 5-substituted
2-amino-4,6-dichloropyrimidines B1–B12 was strictly
dose-dependent (Fig. 2). The effects of B6 and B12 were
already apparent at concentrations of 2.5 lM. The IC50s
estimates (Table 1) ranged between 2 lM and 36 lM (B12
and B8, respectively).
None of the 5-substituted 2-amino-4,6-dihydroxypyr-
imidines A1–A12 at 50 lM concentrations significantly
influenced (F/12, 39/= 0.85, p = 0.61) the IFN/LPS-acti-
vated NO biosynthesis (Fig. 1a). On average, the formation
of nitrites dropped to 91.6 % of control values. The
123

Med Chem Res
Conclusions
Table 1 The NO-inhibitory IC50
s
IC50 (lM)
95 % Limits of confidence
An optimized synthetic procedure for the preparation of
5-substituted 2-amino-4,6-dichloropyrimidines B1–B12
starting from the monosubstituted malonic acid diesters has
been developed. Final compounds B1–B12 inhibit NO
production in mouse peritoneal cells. Compounds B5, B6,
B10, and B12 are more potent than the most frequently
used inhibitor of the NO production (NG-monomethyl-L-
arginine). The most effective was compound B12 bearing
the fluorine atom at position 5 of the pyrimidine ring,
which nearly completely suppressed the production of NO
and which exhibited higher activity than the most potent
reference compound (1,400 W).
5-Substituted 2-amino-4,6-dichloropyrimidines
B3
35.7
19.64
9
26.78–47.59
15.76–24.48
6.26–12.93
22.83–57.49
18.18–32.31
1.81–2.19
B5
B6
B8
36.23
24.23
1.99
B10
B12
Reference NO-inhibitors
Aminoguanidine
L-NMMA
L-NIL
77.83
35.13
8.14
52.82–114.70
20.70–59.63
5.83–11.36
2.94–5.11
This interesting discovery of the inhibitory effect of the
5-substituted 2-amino-4,6-dichloropyrimidines on NO pro-
duction in vitro represents an initial hit in our development
of potent compounds with anti-inflammatory activity (patent
pending). Compounds of the next generations and further
in vitro and in vivo data will be published elsewhere.
1,400 W
3.87
100
75
Experimental
50
General
Aminoguanidine
L-NMMA
L-NIL
25
0
Unless stated otherwise, solvents were evaporated at 40 °C/
2 kPa and compounds were dried at 13 Pa. Analytical TLC
was performed on silica gel 60 F254 plates (Merck KGaA,
Darmstadt, Germany). The NMR spectra were measured on an
FT NMR spectrometer (Bruker Avance II 500) in DMSO-d6
(1H at 500 MHz and 13C at 125.7 MHz), referenced to the
residual solvent signal, chemical shift are expressed in parts
per million, ppm, and interaction constants J in Hz. GC/MS
analyses were measured using a 6,890 N gas chromatograph
(Agilent, Santa Clara, CA, USA) attached to a quadrupole
mass detector. A HP-5 ms capillary (30 m 9 0.25 mm;
0.25 lm; Agilent) was used for the analyses. The carrier gas
was helium with a flow rate of 1 mL/min. The EI mass spectra
were measured on a GCT Premier (Waters) OA-TOF GC mass
spectrometer. The elemental composition of the prepared
compounds was determined using a PE 2400 Series II CHNS/
O Elemental Analyzer (Perkin Elmer, USA, 1999). Melting
points were determined on a Stuart SMP3 Melting Point
Apparatus and are uncorrected. Compounds A12 and B12
were prepared according to the literature (Schostarez, 1992).
Compounds A1 and B1 were purchased from Sigma-Aldrich.
1400W
0.1
1
10
100
1000
Concentration µM
Fig. 3 Dose-dependent inhibitory effects of standard NO-inhibitors
on in vitro NO production. The points are mean SEM. The data
represent one of two identical experiments. The concentration of
nitrites following the LPS/IFN stimulus (i.e. 100 % response) was
64.10 1.50 lM. It was 0.30 0.20 lM in untreated controls.
L-NMMA, NG-monomethyl-L-arginine; L-NIL, L-N6-(1-iminoeth-
yl)lysine; 1,400 W, N-(3-aminomethyl)-benzylacetamide
The NO-inhibitory effects of tested pyrimidines were
compared to the effects of recognized NO-inhibitors
(Fig. 3). The most effective proved to be 1,400 W; the least
effective was aminoguanidine. The corresponding IC50s are
shown in Table 1. They are in good agreement with
activities reported previously (Stenger et al., 1995; Xu and
Krukoff, 2005).
Cell viability
General procedure for the preparation of 5-substituted
2-amino-4,6-dihydroxypyrimidines A2–A11
Applied at 50 lM concentrations, the compounds had no
statistically significant influence on the viability of cells
(Fig. 1b). No difference between the two groups of pyrim-
idine derivatives was observed (F/1, 35/= 0.25, p = 0.62).
Metallic sodium (12.9 g, 0.56 mol) was dissolved in abso-
lute ethanol (300 mL) under argon while being intensively
123

Med Chem Res
stirred with a mechanical stirrer. The reaction flask was
equipped with a reflux condenser with a chlorocalcium tube.
After all the sodium was dissolved and the reaction mixture
was cooled to room temperature; guanidine hydrochloride
(21.02 g, 0.22 mol) was added under intensive stirring,
followed by the corresponding monosubstituted malonic
acid diester (0.2 mol). The reaction mixture was further
intensively stirred due to the production of the solid product,
which is so massive that after 2 h it already practically
precludes stirring. After another 2 h, absolute ethanol
(200 mL) was added and the reaction mixture was refluxed
for 1 h while being stirred. Afterward, ethanol (ca
200–300 mL) was evaporated on a vacuum rotary evapo-
rator and water (500 mL) was added to the reaction mixture.
After stirring, the product (in the form of sodium salt) was
almost dissolved. The obtained mixture was subsequently
neutralized by dropwise addition of acetic acid, resulting in
immediate and quantitative precipitation of the desired
product in the form of a fine solid. This mixture was sub-
sequently heated under reflux for 10 min and then cooled to
laboratory temperature. This heating and cooling was repe-
ated twice to get a well-filterable solid product. The solid
product was filtered off, washed with water (2 9 50 mL),
ethanol (2 9 50 mL), and acetone (2 9 50 mL). The
product was dried under high vacuum at 60 °C for 2 days.
The obtained purity of the product prepared in this manner is
sufficient for the following reaction and based on analyses
contains only crystalline water.
OH), 6.31 (2H, bs, NH2), 2.10 (2H, t, J(10,20) = 7.5, H-10),
1.32 (2H, m, H-20), 0.80 (3H, t, J(3,20) = 7.4, H-30); 13C
NMR (DMSO-d6): d = 164.75 (C-4 and 6), 152.57 (C-2),
90.20 (C-5), 22.44 (C-10), 21.89 (C-20), 14.17 (C-30); Anal.
Calcd. for C7H11N3O2 ? 0.6 H2O: C, 46.71; H, 6.83; N,
23.35. Found: C, 46.80; H, 6.79; N, 23.32.
2-Amino-5-isopropylpyrimidine-4,6-diol (A5)
It was obtained as a white solid; yield: 32.36 g (93 %); m.p.
1
[250 °C; H NMR (DMSO-d6): d = 10.45 (2H, bs, 29
OH), 6.62 (2H, bs, NH2), 2.96 (1H, sept, J(CH,CH3) = 7.1,
H-CH), 1.08 (6H, d, J(CH3,CH) = 7.1, 29 CH3); 13C
NMR (DMSO-d6): d = 164.19 (C-4 and 6), 152.40 (C-2),
94.80 (C-5), 22.94 (CH), 20.96 (CH3); Anal. Calcd. for
C7H11N3O2 ? 0.3 H2O: C, 48.16; H, 6.70; N, 24.07.
Found: C, 48.11; H, 7.62; N, 23.98.
2-Amino-5-(prop-2-yn-1-yl)pyrimidine-4,6-diol (A6)
It was obtained as a white solid; yield: 33.47 g (96 %); m.p.
1
[250 °C; H NMR (DMSO-d6): d = 10.55 (2H, bs, 29
OH), 6.79 (2H, bs, NH2), 2.95 (2H, d, J(1,30) = 2.6, H-10),
2.43 (1H, t, J(30,10) = 2.6, H-30); 13C NMR (DMSO-d6):
d = 163.69 (C-4 and 6), 152.69 (C-2), 89.05 (C-20), 84.97
(C-5), 68.02 (C-30), 12.06 (C-10); Anal. Calcd. for
C7H7N3O2 ? 0.5 H2O: C, 48.28; H, 4.63; N, 24.13. Found:
C, 48.09; H, 4.33; N, 24.23.
2-Amino-5-methylpyrimidine-4,6-diol (A2)
5-Allyl-2-aminopyrimidine-4,6-diol (A7)
It was obtained as a white solid; yield: 30.86 g (91 %); m.p.
[250 °C; 1H NMR (DMSO-d6): d = 10.70 (2H, bs,
29 OH), 6.88 (2H, bs, NH2), 1.57 (3H, s, H-10); 13C NMR
(DMSO-d6): d = 164.97 (C-4 and 6), 152.53 (C-2), 84.06 (C-
5), 8.11 (C-10); Anal. Calcd. for C5H7N3O2 ? 1.6 H2O: C,
35.34; H, 6.05; N, 24.72. Found: C, 35.57; H, 6.15; N, 24.59.
It was obtained as a white solid; yield: 38.57 g (95 %); m.p.
1
[250 °C; H NMR (DMSO-d6): d = 10.35 (2H, bs, 29
OH), 6.40 (2H, bs, NH2), 5.73 (1H, ddt, J(20,10) = 6.1,
J(20,30cis) = 10.0, J(20,30trans) = 17.2, H-20), 4.87 (1H, ddt,
J(30trans,10) = 1.6, J(gem) = 2.3, J(30trans,20) = 17.2, H-30trans),
4.79 (1H, ddt, J(30cis,10) = 1.6, J(gem) = 2.3, J(30cis,20) =
10.0, H-30cis), 2.85 (2H, dt, J(10,30) = 1.6, J(10,20) = 6.1,
H-10); 13C NMR (DMSO-d6): d = 164.36 (C-4 and 6),
152.71 (C-2), 137.73 (C-20), 113.32 (C-30), 87.73 (C-5),
26.67 (C-10); Anal. Calcd. for C7H9N3O2 ? 2 H2O: C,
41.38; H, 6.45; N, 20.68. Found: C, 41.44; H, 6.17; N,
20.47.
2-Amino-5-ethylpyrimidine-4,6-diol (A3)
It was obtained as a white solid; yield: 32.54 g (88 %); m.p.
1
[250 °C; H NMR (DMSO-d6): d = 10.30 (2H, bs, 29
OH), 6.30 (2H, bs, NH2), 2.14 (2H, q, J(10,20) = 7.3, H-10),
0.88 (3H, t, J(20,10) = 7.3, H-20); 13C NMR (DMSO-d6):
d = 164.47 (C-4 and 6), 152.54 (C-2), 91.88 (C-5), 15.62
(C-10), 13.89 (C-20); Anal. Calcd. for C6H9N3O2 ? 1.7
H2O: C, 38.79; H, 6.73; N, 22.62. Found: C, 38.83; H, 7.90;
N, 22.41.
2-Amino-5-butylpyrimidine-4,6-diol (A8)
It was obtained as a white solid; yield: 39.61 g (97 %); m.p.
1
[250 °C; H NMR (DMSO-d6): d = 10.30 (2H, bs, 29
OH), 6.32 (2H, bs, NH2), 2.12 (2H, t, J(10,20) = 7.1, H-10),
1.28 and 1.23 (29 2H, 29 m, H-20 and H-30), 0.85 (3H, t,
J(40,30) = 7.2, H-40); 13C NMR (DMSO-d6): d = 164.67
(C-4 and 6), 152.54 (C-2), 90.36 (C-5), 31.04 (C-10), 22.32
(C-20), 21.98 (C-30), 14.22 (C-40); Anal. Calcd. for
2-Amino-5-propylpyrimidine-4,6-diol (A4)
It was obtained as a white solid; yield: 33.86 g (94 %); m.p.
1
[250 °C; H NMR (DMSO-d6): d = 10.30 (2H, bs, 29
123

Med Chem Res
C8H13N3O2 ? 1.2 H2O: C, 46.91; H, 7.58; N, 20.52.
The obtained mixture was quickly transferred into a se-
paratory funnel and immediately extracted with chloroform
(3 9 20 mL). The organic fractions were combined toge-
ther, dried over MgSO4, filtered through a 0.5 cm layer of
silica gel and concentrated down on a rotary evaporator.
This crude residue of 5-substituted 4,6-dichloro-2-
{[(dimethylamino)methylene]amino}pyrimidine was dis-
solved in the mixture of 99 % ethanol (20 mL) and 37 %
aqueous HCl (2 mL). The reaction mixture was heated at
50 °C for 2 h, during which a crystalline product began to
precipitate directly from the reaction mixture. After that,
water (30 mL) was added and the reaction mixture was
intensively stirred for 10 min. The precipitated product was
filtered off and washed with a water/ethanol mixture (1/1,
2 9 10 mL), 5 % aqueous solution of NaHCO3 (10 mL)
and a water/ethanol mixture (1/1, 10 mL). The product was
subsequently recrystallized from aqueous ethanol, filtered
off, washed with a water/ethanol mixture (1/1, 10 mL) and
dried under high vacuum.
Found: C, 47.15; H, 7.69; N, 20.36.
2-Amino-5-(sec-butyl)pyrimidine-4,6-diol (A9)
It was obtained as a white solid; yield: 36.89 g (93 %); m.p.
1
[250 °C; H NMR (DMSO-d6): d = 10.20 (2H, bs, 29
OH), 6.31 (2H, bs, NH2), 2.70 (1H, m, H-10), 1.65 and 1.40
(29 1H, 29 m, H-20), 1.06 (3H, d, J(100,10) = 7.0, H-100),
0.72 (3H, t, J(30,20) = 7.4, H-30); 13C NMR (DMSO-d6):
d = 164.46 (C-4 and 6), 152.47 (C-2), 93.61 (C-5), 30.08
(C-10), 27.09 (C-20), 19.00 (C-100), 13.03 (C-30); Anal.
Calcd. for C8H13N3O2 ? 0.8 H2O: C, 48.62; H, 7.45; N,
21.26. Found: C, 48.57; H, 7.47; N, 21.18.
2-Amino-5-phenylpyrimidine-4,6-diol (A10)
It was obtained as a white solid; yield: 41.43 g (94 %); m.p.
[250 °C; 1H NMR (DMSO-d6): d = 10.60 (2H, bs,
29 OH), 7.50 (2H, d, phenyl), 7.19 (2H, t, phenyl), 7.02
(1H, t, phenyl), 6.74 (2H, bs, NH2); 13C NMR (DMSO-d6):
d = 162.84 (C-4 and 6), 152.02 (C-2), 135.40, 130.26,
126.84 and 124.19 (C-phenyl), 106.11 (C-5); Anal. Calcd.
for C10H9N3O2 ? 1 H2O: C, 54.29; H, 5.01; N, 19.00.
Found: C, 54.17; H, 5.19; N, 18.82.
4,6-Dichloro-5-methylpyrimidin-2-amine (B2)
It was obtained as a white solid; yield: 1.26 g (71 %); m.p.
189–190 °C; 1H NMR (DMSO-d6): d = 7.26 (2H, bs,
NH2), 2.17 (3H, s, H-10); 13C NMR (DMSO-d6):
d = 161.01 (C-4 and 6), 160.78 (C-2), 113.60 (C-5), 14.93
(C-10); Anal. Calcd. for C5H5Cl2N3: C, 33.73; H, 2.83; Cl,
39.83; N, 23.60. Found: C, 33.53; H, 2.78, Cl, 40.02; N,
23.42; MS (EI), m/z (%): 177 and 179 [M?] (100); MS
(ESI ?), m/z (%): 178 and 180 [M?H?] (100).
2-Amino-5-benzylpyrimidine-4,6-diol (A11)
It was obtained as a white solid; yield: 43.21 g (91 %); m.p.
[250 °C; 1H NMR (DMSO-d6): d = 10.42 (2H, bs,
29 OH), 7.18 (4H, m, phenyl), 7.07 (1H, m, phenyl), 6.46
(2H, bs, NH2), 3.44 (2H, s, CH2); 13C NMR (DMSO-d6):
d = 164.47 (C-4 and 6), 152.68 (C-2), 143.10, 128.31,
127.90 and 125.20 (C-phenyl), 94.51 (C-5), 28.12 (CH2);
Anal. Calcd. for C11H11N3O2 ? 1.1 H2O: C, 55.74; H,
5.61; N, 17.73. Found: C, 55.71; H, 5.54; N, 17.60.
4,6-Dichloro-5-ethylpyrimidin-2-amine (B3)
It was obtained as a white solid; yield: 1.58 g (82 %); m.p.
183–185 °C; 1H NMR (DMSO-d6): d = 7.32 (2H, bs,
NH2), 2.61 (2H, q, J(10,20) = 7.4, H-10), 1.07 (3H, t,
J(20,10) = 7.4, H-20); 13C NMR (DMSO-d6): d = 160.83
(C-2), 160.76 (C-4 and 6), 118.90 (C-5), 22.41 (C-10), 12.92
(C-20); Anal. Calcd. for C6H7Cl2N3: C, 37.52; H, 3.67; Cl,
36.92; N, 21.88. Found: C, 37.59; H, 3.73; Cl, 36.76; N,
21.77; MS (EI), m/z (%): 191 and 193 [M?] (100).
General procedure for the preparation of 5-substituted
2-amino-4,6-dichloropyrimidines B2–B11
Prior to the reaction, the starting 5-substituted 2-amino-4,6-
dihydroxypyrimidine A2–A11 was dried in a vacuum drier
at 80 °C and under 0.1 mbar for 1 day, because crystalline
water increases the amount of the Vilsmeier–Haack–
Arnold reagent required for full conversion. Subsequently,
5-substituted 2-amino-4,6-dihydroxypyrimidine (10 mmol)
was suspended under inert atmosphere in a 2 M solution of
the Vilsmeier–Haack–Arnold reagent (80 mmol, 40 mL)
in chloroform. The reaction mixture was subsequently
heated at reflux for 4 h, during which the starting material
was completely dissolved. The reaction mixture was cooled
to the room temperature, poured onto ice and rapidly
neutralized with a saturated aqueous NaHCO3 solution.
4,6-Dichloro-5-propylpyrimidin-2-amine (B4)
It was obtained as a white solid; yield: 1.60 g (78 %); m.p.
182–183 °C; 1H NMR (DMSO-d6): d = 7.52 (2H, bs,
NH2), 2.69 (2H, t, J(10,20) = 7.6, H-10), 1.52 (2H, m, H-20),
0.91 (3H, t, J(300.20) = 7.2, H-30); 13C NMR (DMSO-d6):
d = 160.78 (C-2), 160.69 (C-4 and 6), 119.20 (C-5), 31.12
(C-10), 20.86 (C-20), 13.75 (C-30); Anal. Calcd. for
C7H9Cl2N3: C, 40.80; H, 4.40; Cl, 34.41; N, 20.39. Found:
C, 40.79; H, 4.32; Cl, 34.15; N, 20.19; MS (EI), m/z (%):
205 and 207 [M?] (100).
123

Med Chem Res
4,6-Dichloro-5-isopropylpyrimidin-2-amine (B5)
5-(Sec-butyl)-4,6-dichloropyrimidin-2-amine (B9)
It was obtained as a white solid; yield: 1.43 g (69 %); m.p.
175–176 °C; 1H NMR (DMSO-d6): d = 7.31 (2H, bs,
NH2), 3.46 (1H, sept, J(CH,CH3) = 7.2, CH), 1.28 (6H, d,
J(CH3,CH) = 7.2, 29 CH3); 13C NMR (DMSO-d6):
d = 160.62 (C-2), 160.32 (C-4 and 6), 121.65 (C-5), 28.57
(CH), 19.82 (CH3); Anal. Calcd. for C7H9Cl2N3: C, 40.80;
H, 4.40; Cl, 34.41; N, 20.39. Found: C, 40.57; H, 4.54; Cl,
34.69; N, 20.67; MS (EI), m/z (%): 205 and 207 [M?]
(100).
It was obtained as a white solid; yield: 1.66 g (75 %); m.p.
159–160 °C; 1H NMR (DMSO-d6): d = 7.33 (2H, s,
NH-2), 3.23 (1H, m, H-10), 1.83 and 1.64 (29 1H, 29 m,
H-20), 1.25 (3H, d, J(100,10) = 6.2, H-100), 0.77 (3H, t,
J(30,20) = 7.4, H-30); 13C NMR (DMSO-d6): d = 163.64
(C-2), C-4 and C-6 not found, 120.12 (C-5), 35.72 (C-10),
26.67 (C-20), 18.13 (C-100), 12.53 (C-30); Anal. Calcd. for
C8H11Cl2N3: C, 43.66; H, 5.04; Cl, 32.22; N, 19.09. Found:
C, 43.63; H, 4.82; Cl, 32.17; N, 18.86; MS (EI), m/z (%):
219 and 221 [M?] (100).
4,6-Dichloro-5-(prop-2-yn-1-yl)pyrimidin-2-amine
(B6)
4,6-Dichloro-5-phenylpyrimidin-2-amine (B10)
It was obtained as a white solid; yield: 1.63 g (81 %); m.p.
159–161 °C; 1H NMR (DMSO-d6): d = 7.50 (2H, bs,
NH2), 3.52 (2H, d, J(10,30) = 2.7, H-10), 2.96 (1H, t,
J(30,10) = 2.7, H-30); 13C NMR (DMSO-d6): d = 161.20
(C-2), 160.84 (C-4 and 6), 113.30 (C-5), 79.86 (C-20),
71.96 (C-30), 19.03 (C-10); Anal. Calcd. for C7H5Cl2N3: C,
41.61; H, 2.49; Cl, 35.09; N, 20.80. Found: C, 41.41; H,
2.48; Cl, 34.96; N, 20.55; MS (EI), m/z (%): 201 and 203
[M?] (100).
It was obtained as a white solid; yield: 1.71 g (71 %); m.p.
193–195 °C; 1H NMR (DMSO-d6): d = 7.60 (2H, bs,
NH2), 7.44 (2H, t, phenyl), 7.40 (1H, t, phenyl), 7.30 (2H,
d, phenyl); 13C NMR (DMSO-d6): d = 161.46 (C-2),
160.26 (C-4 and 6), 134.24, 130.47, 128.55 and 128.46
(phenyl), 119.71 (C-5); Anal. Calcd. for C10H7Cl2N3: C,
50.03; H, 2.94; Cl, 29.53; N, 17.50. Found: C, 49.86; H,
2.82; Cl, 38.44; N, 17.23; MS (EI), m/z (%): 239 and 241
[M?] (100).
5-Allyl-4,6-dichloropyrimidin-2-amine (B7)
5-Benzyl-4,6-dichloropyrimidin-2-amine (B11)
It was obtained as a white solid; yield: 1.50 g (74 %); m.p.
175–177 °C; 1H NMR (DMSO-d6): d = 7.40 (2H, bs,
NH-2), 5.83 (1H, ddt, J(20,10) = 5.8, J(20,30cis) = 10.1,
J(20,30trans) = 17.1, H-20), 5.06 (1H, dq, J(30cis,10) =
J(gem) = 1.6, J(30cis,20) = 10.1, H-30cis), 4.96 (1H, dq,
J(30trans,10) = J(gem) = 1.7, J(30trans,20) = 17.1, H-30trans),
3.36 (2H, dt, J(10,30) = 1.7, J(10,20) = 5.8, H-10); 13C
NMR (DMSO-d6): d = 161.33 (C-4 and 6), 161.08 (C-2),
133.57 (C-20), 116.28 (C-30), 115.01 (C-5), 32.70 (C-10);
Anal. Calcd. for C7H7Cl2N3: C, 41.20; H, 3.46; Cl, 34.75;
N, 20.59. Found: C, 41.12; H, 3.37; Cl, 34.54; N, 20.56;
MS (EI), m/z (%): 203 and 205 [M?] (100).
It was obtained as a white solid; yield: 2.02 g (80 %); m.p.
196–197 °C; 1H NMR (DMSO-d6): d = 7.46 (2H, bs,
NH2), 7.29 (2H, t, phenyl), 7.20 (1H, t, phenyl), 7.15 (2H,
d, phenyl), 4.01 (2H, s, CH2); 13C NMR (DMSO-d6):
d = 161.75 (C-4 and 6), 161.15 (C-2), 138.16, 128.72,
127.92 and 126.56 (C-phenyl), 116.29 (C-5), 34.15 (CH2);
Anal. Calcd. for C11H9Cl2N3: C, 51.99; H, 3.57; Cl, 27.90;
N, 16.54. Found: C, 51.92; H, 3.66; Cl, 28.02; N, 16.68;
MS (EI), m/z (%): 253 and 255 [M?] (100).
Preparation of the compound solutions for biological
assays
5-Butyl-4,6-dichloropyrimidin-2-amine (B8)
The 200 mM stock solutions of compounds were prepared
in DMSO. They were further diluted to working concen-
trations in complete RPMI-1640 culture medium (described
below). In order to exclude any possible interference of
DMSO with NO production and viability of cells, appro-
priately diluted DMSO was included in all experiments. It
was found ineffective in the assays (data not shown). Stan-
dard inhibitors of NO production, i.e., NG-monomethyl-L-
arginine (L-NMMA), aminoguanidine (AG), L-N6-(1-imi-
noethyl)lysine (L-NIL), and N-(3-aminomethyl)benzylaceta-
mide (1,400 W) were bought from Sigma-Aldrich. Stock
solutions (5 mM) were prepared in apyrogenic distilled water.
It was obtained as a white solid; yield: 1.92 g (87 %); m.p.
169–170 °C; 1H NMR (DMSO-d6): d = 7.29 (2H, bs,
NH2), 2.59 (2H, m, CH2), 1.45 (2H, m, CH2), 1.33 (2H, m,
CH2), 0.91 (3H, t, J(40,30) = 7.3, H-40); 13C NMR (DMSO-
d6): d = 160.78 (C-2), 160.76 (C-4 and 6), 117.71 (C-5),
30.37, 28.46 and 22.04 (C-10, 20 and 30), 13.81 (C-40); Anal.
Calcd. for C8H11Cl2N3: C, 43.66; H, 5.04; Cl, 32.22; N,
19.09. Found: C, 43.70; H, 4.93; Cl, 32.24; N, 18.87; GC/
MS-EI (RT 16.03 min), m/z (%): 219 and 221 [M?] (18),
176 and 178 [M?-Pr] (100), min. 99.5 % purity.
123

Med Chem Res
Animals; isolation and cultivation of cells
Statistical evaluation
Female mice of the inbred strain C57BL/6, 8- to 10-week-
old, were purchased from Charles River Deutschland
(Sulzfeld, Germany). They were kept in transparent plastic
cages in groups of ten, and maintained in an Independent
Environmental Air Flow Animal Cabinet (ESI Flufrance,
Wissous, France). Lighting was set on 06–18 h, tempera-
ture at 22 °C.
Animals, killed by cervical dislocation, were intraperi-
toneally injected with 8 mL of sterile saline. Pooled peri-
toneal cells collected from mice (n = 4–10 in individual
experiments) were washed in sterile saline, resuspended in
culture medium, and seeded into 96-well round-bottom
microplates (Costar). The final amount of cells was
2 9 106 cells/mL. The cultures were maintained at 37 °C,
5 % CO2 in humidified Heraeus incubator.
Analysis of variance (ANOVA) with subsequent Bonfer-
´
ronis multiple comparison test and graphical presentation
of data was done using the Prism program (GraphPad
Software, San Diego, CA). Several experiments were per-
formed. The concentration of nitrites as well as optical
density, the measure of viability of cells, varied among
experiments. In order to amalgamate the data from various
experiments, they were expressed as a percentage of con-
trol values.
Acknowledgments The research was supported by grant CZ: GA
ˇ
CR:303/12/0172 from the Grant Agency of the Czech Republic. The
antiviral testing provided by the group of prof. Jan Balzarini, Prof.
Lieve Naesens, Prof. Johan Neyts, Prof. Robert Snoeck and Prof.
Graciela Andrei (Rega Institute for Medical Research, Katholieke
Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Bel-
gium) is highly appreciated.
Complete RPMI-1640 culture medium (Sigma-Aldrich)
contained 10 % heat-inactivated fetal bovine serum,
2 mM L-glutamine, 50 lg/mL gentamicin, and 5 9 10-5
M 2-mercaptoethanol (all Sigma).
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