General synthesis of 1-Aryl-6-azaisocytosines and their utilization for the preparation of related condensed 1,2,4-Triazines

Article abstract of DOI:10.3390/molecules24193558

A simple general synthesis of 1-aryl-6-azaisocytosine-5-carbonitriles 4 is described. This method is based on coupling diazonium salts with cyanoacetylcyanamide 2 and then cyclization of the formed 2-arylhydrazono-2-cyanoacetylcyanamides 3. The 6-azaisocytosines 4 were studied with respect to tautomeric equilibrium and the transformation of functional groups, and used in the synthesis of the condensed heterocyclic compounds: Purine isosteric imidazo[2,1-c]-[1,2,4]triazine 8 and the 1,2,4-triazino[2,3-a]quinazolines 9-12.

Full text of DOI:10.3390/molecules24193558

molecules
Article
General Synthesis of 1-Aryl-6-azaisocytosines and
Their Utilization for the Preparation of Related
Condensed 1,2,4-Triazines
František Zálešák , Jan Slouka and Jakub Stýskala *
Department of Organic Chemistry, Faculty of Science, Palacký University, 17. listopadu 1192/12, CZ-771 46
Olomouc, Czech Republic; frantisek.zalesak@upol.cz (F.Z.); katka.sloukova@email.cz (J.S.)
*
Correspondence: jakub.styskala@upol.cz; Tel.: +42-058-563-4466
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Andrea Penoni
Received: 18 September 2019; Accepted: 30 September 2019; Published: 1 October 2019
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: A simple general synthesis of 1-aryl-6-azaisocytosine-5-carbonitriles
4
is described.
and then cyclization
. The 6-azaisocytosines were studied
with respect to tautomeric equilibrium and the transformation of functional groups, and used in the
This method is based on coupling diazonium salts with cyanoacetylcyanamide
of the formed 2-arylhydrazono-2-cyanoacetylcyanamides
2
3
4
synthesis of the condensed heterocyclic compounds: Purine isosteric imidazo[2,1-c]-[1,2,4]triazine
and the 1,2,4-triazino[2,3-a]quinazolines 9–12.
8
Keywords: 1-aryl-6-azaisocytosine; 1,2,4-triazine; condensed 1,2,4-triazine; fused azaisocytosines;
cyclization of arylhydrazones
1
. Introduction
The 1,2,4-triazine ring is a prominent core structure that is found in numerous biologically
active compounds, which have a wide range of potent pharmacological activities [1–3]. Certain aza
analogs of pyrimidine nucleobases (6-azacytosine and 6-azauracil), which are structurally based on
the 1,2,4-triazine heterocyclic system, have been shown to display antitumor, antiviral, antifungal,
tuberculostatic, and anti-HIV activities.
The nucleosides of 6-azacytosine have shown antiproliferative activity against a wide variety of
cancer cells. However, these azacytidines are deactivated in serum by the enzyme cytidine deaminase,
resulting in inactive azauridines [
based molecules [ ], which, as isomeric structures of 6-azacytosine, should be more resistant to
enzymatic deamination [ ]. In addition, fused azaisocytosines, where the nitrogen atom is part of
4–6]. Hence, it is desirable to design and develop 6-azaisocytosine
6,7
6
a condensed ring, have promising medical applications as antiviral, antitumor, and antihemolytic
agents [8].
The 6-aza analogs of pyrimidine nucleobases substituted with an aryl group at position 1 are
of interest as 6-azacytosine analogs. For example, some 1-aryl-6-azauracils are used in veterinary
medicine as antiprotozoal drugs (clazuril, diclazuril, nitromezuril) to combat the detrimental effects
of protozoan parasites [9,10]. A substituted 1-aryl-6-azauracil-5-carbonitrile (MGL 3196) has been
determined to be a highly selective thyroid hormone receptor
the treatment of dyslipidemia [11].
β agonist, and is now in clinical trials for
Unlike the 1-aryl-6-azauracils, which belong to the longest known and numerous derivatives
of 1,2,4-triazine [12 15], there are only a few known derivatives of 1-aryl-6-azaisocytosines. These
–
compounds can be prepared only with substitutions at the amino group at position 3 [16] or the nitrogen
atom at position 4 [17] of the 1,2,4-triazine ring. Derivatives that are unsubstituted at these positions
are not known and it is not possible to prepare them by established methods. To date, substituted
Molecules 2019, 24, 3558; doi:10.3390/molecules24193558
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1-aryl-6-azaisocytosines have not been investigated in detail. These substances are interesting, not
only in terms of potential biological activity, but also as useful starting materials for the synthesis of
various condensed 1,2,4-triazines. In the preparation of potential new pharmaceutics, emphasis is
placed on the use of readily available reactants using convenient and highly scalable reactions. Herein,
we describe the development of a novel general method for the synthesis of 1-aryl-6-azaisocytosines
(2-aryl-3-amino-1,2,4-triazin-5(2H)-ones).
For the preparation of 1-aryl-6-azaisocytosines, a previously known method [12–15] for the
synthesis of 1-aryl-6-azauracils was modified using a cyanamide group instead of a carbamate
Figure 1). For subsequent modification of the newly prepared 1,2,4-triazines, we reacted the amino
(
group in position 3 of the 1,2,4-triazine ring with functional groups located at position 2 of the attached
aryl group, or with the nitrogen atom at position 4 of the 1,2,4-triazine ring. For example, using this
method 1,2,4-triazino[2,3-a]quinazoline and purine isosteric imidazo[2,1-c][1,2,4]triazine derivatives
could be obtained. These heterocycles are related to 6-azaisocytosines in which the nitrogen atom of
amino group is incorporated into a fused ring.
Figure 1. Schematic diagram of this work and atom numbering for the 6-azaisocytosine and
1
,2,4-triazine skeletons.
2
. Results and Discussion
-Cyanoacetylcyanamide sodium salt
corresponding hydrazones . For the preparation of arylhydrazones 3a
2
2
[
18] was treated with diazonium salts to afford the
3e we used a general method
3
–
for azo-coupling reactions using sodium acetate as a base (Method A). Unfortunately, this method
failed with the benzenediazonium salts containing strong electron withdrawing groups (EWG) (NO2,
CN). In this case, the formazans 13f–13g were predominantly observed, as a mixture with the desired
hydrazones 3f 3g. The known cyano formazan 13g
–
[
19] was also prepared for structure confirmation
by different methods in this work based on the azo-coupling reaction of the 2-cyanobenzenediazonium
salt with cyanoacetic acid. To avoid formazan 13 formation, we modified the azo-coupling process
and carried out the reaction under strong acidic conditions without sodium acetate (Method B).
This modified procedure enabled the preparation of the desired hydrazones 3f
electron withdrawing groups in high yield. It is important to note that this method is not suitable
for the preparation of the arylhydrazones 3a 3e because of the insufficient electrophilicity of the
–3g containing strong
–
corresponding diazonium salts (see Table 1 and Scheme 1). A similar coupling reaction in a strongly
acidic environment has previously been performed using the reactive pyridine-1-oxide-2-diazonium
salt [20].

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Table 1. Comparison of Methods A and B for the reaction of diazonium salts with compound 2.
Arylhydrazone 3 [Isolated Yield %]
Diazonium Salt Ar-N +
2
Method A
Method B
Ar: phenyl
3a [80%]
3b [87%]
3c [83%]
3d [79%]
3e [72%]
3f [15%] *
3g [10%] *
3a [5%] *
3b [0%]
3c [0%]
3d [5%] *
3e [50%]
3f [93%]
3g [80%]
Ar: 4-methylphenyl
Ar: 4-methoxyphenyl
Ar: 4-chlorophenyl
Ar: 2-carboxyphenyl
Ar: 4-nitrophenyl
Ar: 2-cyanophenyl
*
yield based on HPLC analysis.
Scheme 1. Preparation of arylhydrazones
3 showing the formation of undesired formazans 13 for
very reactive diazonium salts f, g. Reagents and conditions: (i) NH CN, EtONa, EtOH, reflux, 15 min;
2
◦
Method A): Diazonium salt: ArNH , HCl, H O or AcOH (for g), NaNO , 0–5 C, 15–60 min, then
2 2 2
(
◦
addition to compound
2
in AcONa/H O, 0–5 C, 18 h; (Method B): Diazonium salt: Similar as above,
2
◦
then addition to compound 2 in H O, 0–5 C, 18 h.
2
The different behaviors of the diazonium salts can be explained on the basis of their electrophilicity
in the reaction with the twice-activated methylene group in cyanoacetylcyanamide 2. It is well known
that electrophilic agents can react with an activated methylene group. In the case of strong electrophiles,
usually one group is sufficient for activation. For weaker electrophilic agents, such as diazonium
salts, two activators are required. Such coupling reactions most often take place in basic medium
forming azo compounds that are tautomeric with the corresponding hydrazones. However, when
using reactive diazonium salts containing strong EWG in the reaction with cyanoacetylcyanamide
2 in
basic medium, the azo compounds formed initially, deprotonated, and then a further azo-coupling
reaction proceeded to give a double-coupled cyanoacetylcyanamide 14, which formed a formazan
after hydrolysis. In the case of reactive diazonium salts, this second azo coupling reaction leading to a
formazan could be suppressed in a strongly acidic environment in which the mentioned subsequent
azo-coupling reaction cannot take place (Scheme 2).

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Scheme 2. Plausible mechanisms for formazan 13 formation in the reaction of reactive diazonium salts
with 2-cyanoacetylcyanamide 2 under basic conditions.
The prepared arylhydrazono-cyanoacetylcyanamides
3 are very poorly soluble in non-polar
solvents. In polar solvents, particularly with traces of water, compounds 3 are transformed slowly to
the less polar cyclized products at room temperature. For this reason, the NMR data of arylhydrazones
were recorded immediately after sample preparation in DMSO-d6.
Compounds (except 3e and 3g) were cyclized to the corresponding 1,2,4-triazines
4
3
3
4 by heating
in a mixture of ethanol–water in good yields. It is interesting to note that in anhydrous ethanol this
cyclization does not proceed (also not by adding of TsOH as an acid catalyst). The fact that hydrazones
3
do not cyclize on heating in anhydrous ethanol, but only on heating in aqueous ethanol, can be
explained by the fact that these compounds under normal conditions (or in anhydrous solvents) are
present in the form 3-A, which is stabilized by an intramolecular hydrogen bond and is unfavorable
3
for the cyclization. This stable conformation prevents part of the molecule from spinning to form 3-B
which is the conformation required to enable the addition of the NH group to the CN group. This
intramolecular hydrogen bond is likely to be disrupted by the presence of water in polar solvents, or
,
by heating the hydrazones
3 to a higher temperature, above the melting point, at which temperature
the hydrogen bond also ceases to exist (see Scheme 3).
Scheme 3. Cyclization of hydrazones
3 leading to 1-aryl-6-azaisocytosine-5-carbonitriles 4. Reagents
and conditions: (i) EtOH:H O (3:1), reflux 60 min.
2
The prepared 1,2,4-triazines
4 (1-aryl-6-azaisocytosin-5-carbonitriles) can exist in two possible
tautomeric forms 4-A and 4-B, which may influence further chemical modifications. To investigate
1
these forms, the phenyl derivative 4a was studied by H-NMR spectroscopy in DMSO-d at two
6
different temperatures. We found that compound 4a at laboratory temperature was present in the
imino form 4-A (two broad hydrogen atom signals were apparent at 8.2 and 7.0 ppm) (Figure 2), at a
◦
higher temperature of 80 C, the amino tautomer 4-B was observed (one hydrogen signal at 7.25 ppm
with double the integral intensity) (Figure 3).

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1
◦
Figure 2. H-NMR spectra of compound 4a in DMSO-d recorded at 23 C.
6
1
◦
Figure 3. H-NMR spectra of compound 4a in DMSO-d recorded at 80 C.
6
The reactivity of the prepared 1-aryl-6-azaisocytosines 4, with respect to the amino (imino) group
in position 3 of the 1,2,4-triazine ring, was studied using the phenyl derivative 4a. We found that
the amino (imino) group underwent relatively easy hydrolysis under mild conditions to give the
corresponding 1-phenyl-6-azauracil-5-carbonitrile 5 [12,15] without hydrolysis of the nitrile group
(Scheme 4). Under more drastic conditions [21], this hydrolysis resulted in the corresponding
1
-phenyl-6-azauracil-5-carboxylic acid 6.
The utility of the amino group of the 6-azaisocytosine skeleton for the synthesis of condensed
,2,4-triazines was investigated. Formation of the fused ring between the amino group at position
1
3
1
6
and the nitrogen atom at position 4 of the 1,2,4-triazine cycle was demonstrated in the reaction of
-phenyl-6-azaisocytosine-5-carbonitrile 4a with phenacyl bromide, which resulted in the 2N-alkylated
-azaisocytosine which cyclized directly under the alkylation reaction conditions to the corresponding
imidazo[2,1-c][1,2,4]triazine derivative . There are two main possible centers for the alkylation of
-phenyl-6-azaisocytosine 4a: The amino group at position 3 and the nitrogen atom at position 4 of the
,2,4-triazine cycle. The selective alkylation of compound 4a at the amino group was accomplished at a
7
8
1
1
high temperature and without a base, when only the amino form is present (proofed by NMR). It can
◦
be assumed that at 130 C a thermodynamic equilibrium exists, in which the alkylation takes place
at the more nucleophilic center—the amino group. In contrast, the nucleophilicity of the NH center
at position 4 of the 1,2,4-triazine ring close to the C=O group is very low. Thus, the most probable
cyclized product would be isomer 8 (Scheme 4).

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O
O
N
H
O
CN
O
CN
N
N
N
N
N
O
N
N
H
H
CN
O
N
N
(i)
CN
N
N
HN
(iii)
6
7%
5
N
N
65%
O
O
(
ii)
H
H
COOH
O
CN
N
N
8
8%
8
4a
N
N
O
N
N
N
7
6
Scheme 4. The hydrolytic reactions of 6-azaisocytosine 4a and use in the synthesis of the condensed
system . Reagents and conditions. (i) 2% HCl in EtOH:H O (1:1), reflux, 24 h; (ii) aq. 20% HCl, reflux, 2
8
2
◦
h; (iii) PhCOCH Br, DMF, 130 C, 24 h.
2
The reactivity of the amino (imino) group of compound 4a with substituents located at position
2
of the attached aryl group was exploited for the preparation of 1,2,4-triazino[2,3-a]quinazoline
derivatives. First, the cyclization reaction of (2-carboxyphenyl)hydrazone 3e, formed the corresponding
-(2-carboxyphenyl)-6-azaisocytosin-5-carbonitrile 4e, which was used without isolation to smoothly
provide 3,6-dioxo-3,4-dihydro-6H-triazino[2,3-a]quinazolin-2-carbonitrile (Scheme 5). Thus, closure
1
9
of the quinazoline ring by this lactamization was much more advantageous than closure of this ring
by cyclocondensation of the amide group of 1-(o-carbamoylphenyl)-6-azauracil-5-carbonitrile [22].
A triazino-quinazoline skeleton was also prepared via a similar cyclocondensation of (2-cyanophenyl)
hydrazone 3g. In this reaction, the intermediate 1-(2-cyanophenyl)-6-azaisocytosin-5-carbonitrile 4g
was not observed and 6-imino-3-oxo-4,6-dihydro-3H-[1,2,4]triazino[2,3-a]quinazoline-2-carbonitrile 10
was prepared directly. The structure of compound 10 was unambiguously confirmed by IR spectroscopy
in which only one CN group was apparent.
Scheme 5. The preparation of 1,2,4-triazino[2,3-a]quinazoline derivatives
9–12. Reagents and conditions.
(
i) EtOH:H O (3:1), reflux, 24 h; (ii) EtOH:H O (3:1), reflux, 60 min; (iii) aq. HCl (1:1), reflux, 20 min,
2
2
then NH , pH = 3–4; (iv) aq. 35% HCl, reflux, 2 h.
3

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We were interested in the stability of the functional groups of compound 10 under acid
hydrolysis. Surprisingly, we found the imino group to be more stable than the nitrile group. Under
mild conditions, 6-imino-3-oxo-4,6-dihydro-3H-[1,2,4]triazino[2,3-a]quinazoline-2-carboxylic acid 11
was formed, whereas under more drastic conditions the hydrolysis resulted in formation of the
3,6-dioxo-4,6-dihydro-3H-[1,2,4]triazino[2,3-a]quinazoline-2-carboxylic acid 12. We can conclude that
the imino group in the quinazoline ring of compound 10 is much more stable to hydrolysis than the
amino (imino) group connected to the 1,2,4-triazine ring of model compound 4a.
In addition to the given tautomer (
other tautomeric forms ( ) and ( ). In the varying tautomeric equilibrium between (
N-H groups are present; however, tautomer ( ) has only an NH group (Figure 4). From the NMR
A
), compounds 10 and 11 may also be represented by two
B
C
A
) and ( ), two
B
C
2
data, it was apparent that the compounds did not contain an NH group (two sharp hydrogen signals
2
were present in area of 9.0–9.3 ppm belonging to acidic N-H groups).
Figure 4. Possible tautomeric forms of 1,2,4-triazino[2,3-a]quinazolines 10 and 11.
Many of the prepared compounds (especially condensed derivatives 9–12) can serve as model
compounds that have the potential for intercalation with DNA. These compounds may exhibit
biological activity, not only through intercalation, as is the case with a number of isocyclic condensed
aromatics [23
–
26], but also through forming intermolecular hydrogen bonds via the acidic NH groups.
3
. Materials and Methods
3
.1. General Informations
All commercially available reagents were used without further purification and purchased from
standard chemical suppliers. Reactions were monitored by LC/MS analyses on a UHPLC-MS system
(
Thermo Scientific, Waltham, MA, USA) consisting of a UHPLC chromatograph equipped with a
◦
photodiode array detector and a triple quadrupole mass spectrometer using a C18 column at 30 C
and flow rate of 800
HPLC grade acetonitrile. H- and C-NMR spectra were measured on an ECA 400II ( H: 399.78
MHz, 13C: 100.53 MHz,) NMR spectrometer (JEOL Resonance, Tokyo, Japan). Chemical shifts (
are reported in ppm and referenced to the middle peak of the solvent signal (DMSO-d : 2.49 ppm,
µL/min. Mobile phases: 10 mM ammonium acetate in HPLC grade water and
1
13
1
δ
)
6
1
13
3
9.50 ppm; CDCl : 7.27 ppm, 77.00 ppm. All recorded H- and C-NMR spectra are available as
3
Supplementary Materials online. NMR data of arylhydrazones
3 were recorded immediately after
sample preparation in DMSO-d . The IR spectra were recorded in KBr wafers on an ATI Unicam
6
Genesis FTIR instrument. High resolution mass spectra (HRMS) measurements were performed on an
Orbitrap Elite mass analyzer Thermo Exactive Plus equipped with Heated Electrospray Ionization
(HESI) and Dionex Ultimate 3000 system (Thermo Scientific, MA, USA). Thin layer chromatography
(TLC) was performed on pre-coated silica gel 60 F254 plates and visualized by exposure to UV light
(254 or 366 nm). Melting points were measured on Boetius stage apparatus and are uncorrected.
3
.2. Synthesis of Compounds 2–13
-Cyanoacetylcyanamide sodium salt (
2.3 g, 0.1 mol) and anhydrous ethanol (50 mL) was added solution of cyanamide (4.2 g, 0.1 mol) in
2
2). To a solution of sodium ethoxide prepared from sodium
(

Molecules 2019, 24, 3558
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◦
anhydrous ethanol (100 mL). After stirring at 50 C for 5 min, to the formed suspension of sodium salt
was added ethyl cyanoacetate (11.7 g, 0.1 mol). The resulting mixture was refluxed for 15 min and after
cooling it was concentrated under reduced pressure to the volume of about 60 mL. This mixture was
◦
allowed to stand at 0–5 C for 2 h. The precipitated white crystalline solid was filtered off, washed with
◦
1
cold ethanol and dried at 60 C for 90 min. Yield was 11.7 g (85%). H-NMR (400 MHz, DMSO-d ):
δ
6
3
.27 (s, 2H); 13C-NMR (101 MHz, DMSO-d ): δ 27.3, 117.8, 120.7, 171.6.
6
3
.2.1. General Procedure for the Preparations of 2-Arylhydrazono-2-cyanoacetylcyanamides (3)
Method A
A solution of corresponding aniline (8 mmol) in a mixture of water (20 mL) and 35% hydrochloric
◦
acid (2 mL) was diazotized at 0–5 C with a solution of sodium nitrite (0.55 g, 8 mmol) in ice water
(10 mL). The mixture was stirred in ice bath for 15 min and then added portionwise to a solution of
2
-cyanoacetylcyanamide sodium salt
2
(1.05 g, 8 mmol), sodium acetate (6 g) in water (50 mL), which
◦
◦
was pre-cooled to 0–5 C. After 20 min of stirring the reaction mixture was let to stand at 5 C for 18
h. Then, aqueous solution of hydrochloric acid (1:2) was added to adjust pH to 3. The precipitated
yellow-orange solid was filtered off, washed with water and dried.
Method B
Diazonium salt prepared in Method A was added with stirring to a solution of
2
-cyanoacetylcyanamide sodium salt
2
(1.05 g, 8 mmol) in water (50 mL), which was pre-cooled
◦
◦
to 0–5 C. After 20 min of stirring the reaction mixture was let to stand at 5 C for 18 h. Precipitated
solid was filtered off, washed with water and dried.
2
-Phenylhydrazono-2-cyanoacetylcyanamide (3a). This compound was prepared according to Method A
◦
1
using aniline hydrochloride (1.04 g). Yield 1.3 g (80%) orange solid, m.p. 166–170 C. H-NMR (400
MHz, DMSO-d ):
δ
7.17–7.21 (m, 1H, Ar), 7.39–7.44 (m, 2H, Ar), 7.72–7.75 (m, 2H, Ar), 12.06–12.78 (bs,
6
13
−1
1
H); C-NMR (101 MHz, DMSO-d6):
δ
104.7, 108.5, 110.5, 116.8, 125.5, 129.3, 141.5, 161.9; IR (cm ):
+
759, 1285, 1428, 1493, 1558, 1710, 2213 (CN), 2259 (CN), 3228; HRMS (HESI m/z): [M + H] calcd for
C H N O 214.0723; found 214.0724.
10
8
5
2
-(4-Methylphenylhydrazono)-2-cyanoacetylcyanamide (3b). This compound was prepared according to
◦
1
Method A using 4-methylaniline (0.86 g). Yield 1.60 g (87%) yellow solid, m.p. 162–166 C. H-NMR
(
1
1
400 MHz, DMSO-d ): δ 2.30 (s, 3H, CH ), 7.22 (d, J = 8.3 Hz, 2H, Ar) 7.63 (d, J = 8.4 Hz, 2H, Ar),
6 3
13
2.04–12.63 (bs, 1H); C-NMR (101 MHz, DMSO-d6): δ 20.5, 103.9, 108.5, 110.6, 116.8, 129.7, 134.9,
−
1
39.3, 161.9; IR (cm ): 815, 1299, 1447, 1608, 1678, 2217 (CN), 2254 (CN), 3237; HRMS (HESI m/z): [M +
+
H] calcd for C H N O 228.0880; found 228.0881.
11
10
5
2
-(4-Methoxyphenylhydrazono)-2-cyanoacetylcyanamide (3c). This compound was prepared according to
◦
Method A using 4-methoxyaniline (0.98 g). Yield 1.62 g (83%) yellow-orange solid, m.p. 166–170 C
1
(
with observed cyclization). H-NMR (400 MHz, DMSO-d ):
δ
3.77 (s, 3H, OCH ), 6.96–7.00 (m, 2H,
6
3
13
Ar), 7.67–7.71 (m, 2H, Ar), 12.32–12.56 (bs, 1H); C-NMR (101 MHz, DMSO-d6):
δ
55.4, 103.1, 108.6,
−1
110.8, 114.5, 118.4, 135.1, 157.4, 162.0; IR (cm ): 1033, 1253, 1447, 1562, 1691, 2217 (CN), 2254 (CN),
+
3
205; HRMS (HESI m/z): [M + H] calcd for C H N O 244.0829; found 244.029.
11 10 5 2
2
-(4-Chlorophenylhydrazono)-2-cyanoacetylcyanamide (3d). This compound was prepared according to
◦
Method A using 4-chloroaniline (1.02 g). Yield 1.47 g (79%) yellow-orange solid, m.p. 166–168 C (with
1
cyclization). H-NMR (400 MHz, DMSO-d ):
δ 7.45–7.49 (m, 2H, Ar), 7.72–7.76 (m, 2H,Ar), 12.06–12.87
6
bs, 1H); 13C-NMR (101 MHz, DMSO-d6):
δ
105.5, 108.6, 110.4, 118.4, 129.2, 129.3, 140.5, 161.9; IR (cm ):
−1
(
+
8
38, 1290, 1456, 1562, 1682,2218 (CN), 2264 (CN), 3089, 3242; HRMS (HESI m/z): [M + H] calcd for
C H ClN O 248.0334; found 248.0333.
10
7
5

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2
-(2-Carboxyphenylhydrazono)-2-cyanoacetylcyanamide (3e). This compound was prepared according
◦
to Method A using 2-aminobenzoic acid (1.1 g). Yield 1.55 g (72%) yellow solid, m.p. 198–202 C.
1
H-NMR (400 MHz, DMSO-d6):
δ 7.09–7.13 (m, 1H, Ar), 7.60–7.64 (m, 1H, Ar), 7.73 (d, J = 8.3 Hz, 1H,
1
3
Ar), 7.95 (dd, J = 7.9, 1.2 Hz, 1H, Ar), 12.41–12.86 (bs, 1H); C-NMR (101 MHz, DMSO-d6):
δ 112.2,
−
1
113.9, 115.1, 122.5, 131.3, 134.8, 144.4, 166.4, 169.5; IR (cm ): 755, 1253, 1530, 1604, 1673, 2176 (CN),
−
2
250 (CN), 3491; HRMS (HESI m/z): [M − H] calcd for C H N O 256.0476; found 256.0476.
11
6
5
3
2
-(4-Nitrophenylhydrazono)-2-cyanoacetylcyanamide (3f). This compound was prepared according to
◦
1
Method B using 4-nitroaniline (1.1 g). Yield 1.91 g (93%) yellow-orange solid m.p. 185–186 C; H-NMR
13
(
400 MHz, DMSO-d6):
δ 7.87–7.91 (m, 2H, Ar), 8.26-8.29 (m, 2H, Ar); C-NMR (101 MHz, DMSO-d6): δ
−1
110.1, 112.4, 116.7, 125.3, 126.4, 143.5, 161.9; IR (cm ): 847, 1258, 1341, 1530, 1595, 2167 (CN), 2365
+
(CN), 3228, 3412; HRMS (HESI m/z): [M + H] calcd for C H N O 259.0574; found 259.0572.
10 7 6 3
2-(2-Cyanophenylhydrazono)-2-cyanoacetylcyanamide (3g). This compound was prepared according to
Method B with different diazotation process. A solution of 2-aminobenzencabonitrile (0.472 g, 4 mmol)
◦
in a mixture of acetic acid (10.5 mL) and 35% hydrochloric acid (3 mL) was diazotized at 0–5 C with a
solution of sodium nitrite (0.275 g, 4 mmol) in ice water (3 mL). The mixture was stirred in ice bath
for 1 h and then added portionwise to a solution of 2-cyanoacetylcyanamide sodium salt
mmol) in water (25 mL), which was pre-cooled to 0–5 C. After 20 min of stirring the reaction mixture
2 (0.525 g, 4
◦
◦
◦
1
was let to stand at 5 C for 6 h. Yield 0.81 g (85%) yellow solid m.p. 185–186 C; H-NMR (400 MHz,
DMSO-d ): 7.36 (td, J = 7.5, 1.1 Hz, 1H, Ar), 7.72–7.77 (m, 1H, Ar), 7.84–7.88 (m, 2H,Ar), 11.22–11.93
δ
6
1
3
(
1
bs, 2H); C-NMR (101 MHz, DMSO-d ): δ 101.9, 108.0, 108.5, 110.2, 116.9, 119.8, 126.0, 134.1, 134.3,
6
44.0, 161.7; IR (cm 1): 1291, 1432, 1445, 1490, 1607, 1703, 2216 (CN), 2255 (CN), 2298 (CN), 3200; HRMS
−
+
(
HESI m/z): [M + H] calcd for C H N O 239.0676; found 239.0675.
11
7
6
3
.2.2. General Procedure for the Preparation of 2-Aryl-3-imino-5-oxo-2,3,4,5-tetrahydro-1,2,4-
triazine-6-carbonitriles 4
Corresponding arylhydrazone 3 (2 mmol) was suspended in mixture of ethanol-water (3:1; 25 mL)
and resulting mixture was refluxed with stirring for 60 min. After this time, water was added to the
hot mixture (15 mL) and allowed to cool down. The precipitated crystalline compound was filtered off,
◦
washed with water and dried at 80 C. For the highest purity it is possible to crystalize the prepared
compounds 4 from ethanol.
3
-Imino-5-oxo-2-phenyl-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (4a). Yield 88% as beige crystalline
◦
1
solid, m.p. 238–242 C. H-NMR (400 MHz, DMSO-d ):
7
δ
6.47–7.47 (bs, 1H,), 7.55–7.62 (m, 5H, Ar),
6
13
.74–8.62 (bs, 1H); C-NMR (101 MHz, DMSO-d6): δ 113.5, 125.7, 126.9, 130.1, 130.4, 139.0, 154.7,
−
1
+
159.2; IR (cm ): 769, 1202, 1415, 1567, 1664, 2231 (CN), 3085; HRMS (HESI m/z): [M + H] calcd for
C H N O 214.0723; found 214.0724.
10
8
5
3
-Imino-2-(4-methylphenyl)-5-oxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (4b). Yield 92% as light
◦
1
yellow crystalline solid, m.p. 246–250 C. H-NMR (400 MHz, DMSO-d ):
δ 2.39 (s, 3H, CH ), 6.70–7.19
3
6
1
3
(
bs, 1H), 7.38–7.40 (m, 2H, Ar), 7.43–7.45 (m, 2H, Ar), 7.74–8.59 (bs, 1H); C-NMR (101 MHz, DMSO-d6):
−
1
δ
20.9, 113.6, 125.7, 126.7, 130.6, 136.6, 140.1, 154.8, 159.2; IR (cm ): 787, 1428, 1516, 1562,1659, 2236
+
(CN), 3099; HRMS (HESI m/z): [M + H] calcd for C H N O 228.0880; found 228.0879.
11 10 5
3
-Imino-2-(4-methoxyphenyl)-5-oxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (4c). Yield 93% as light
◦
1
yellow crystalline solid, m.p. 285–289 C. H-NMR (400 MHz, DMSO-d ):
δ 3.82 (s, 3H, CH ), 6.58–7.09
3
6
1
3
(
bs, 1H), 7.10–7.16 (m, 2H, Ar), 7.47–7.51 (m, 2H, Ar), 7.75–8.45 (bs, 1H); C-NMR (101 MHz, DMSO-d6):
−
1
δ
55.6, 113.6, 115.2, 125.6, 128.4, 131.7, 155.1, 159.2, 160.3; IR (cm ): 999, 1175, 1244, 1511, 1636, 2236
+
(CN), 3131; HRMS (HESI m/z): [M + H] calcd for C H N O 244.0829; found 244.028.
11 10 5 2

Molecules 2019, 24, 3558
10 of 13
2
-(4-Chlorophenyl)-3-imino-5-oxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (4d). Yield 89% as light
◦
1
yellow crystalline solid, m.p. 249–253 C. H-NMR (400 MHz, DMSO-d ):
δ
6.87–7.52 (bs, 1H), 7.60–7.63
6
13
(
m, 2H, Ar), 7.65–7.68 (m, 2H, Ar) 7.68–7.79 (bs, 1H); C-NMR (101 MHz, DMSO-d6):
δ
113.4, 125.8,
−
1
128.9, 130.2, 135.0, 137.8, 154.7, 159.1; IR (cm ): 856, 1018, 1294, 1405, 1511, 1637, 2236 (CN), 3140;
+
HRMS (HESI m/z): [M + H] calcd for C H ClN O 248.0334; found 248.0335.
10
7
5
3
-Imino-2-(4-nitrophenyl)-5-oxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (4f). Yield 89% as light
◦
1
yellow crystalline solid, m.p. 238–242 C. H-NMR (400 MHz, DMSO-d ):
δ
6.85-7.75 (bs, 1H), 7.87–7.91
6
13
(m, 2H, Ar), 8.43–8.46 (m, 2H, Ar) C-NMR (101 MHz, DMSO-d6): δ 113.4, 125.6, 126.2, 128.7, 144.0,
−1
+
148.2, 154.4, 159.0; IR (cm ): 856, 1189, 1350, 1535, 1682, 2227 (CN), 3171; HRMS (HESI m/z): [M + H]
calcd for C H N O 259.0574; found 259.0572.
10
7
6
3
3,5-Dioxo-2-phenyl-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (5). Nitrile 4a (200 mg; 0.94 mmol) was
refluxed in a mixture of ethanol (10 mL), water (10 mL) and aqueous 10% hydrochloric acid (3 mL)
for 24 h. After this time the reaction mixture was concentrated under reduced pressure to one half
of its original volume, when the product started to crystalize. The product was filtered off, washed
◦
with water and dried. Yield 134 mg (67%) as a colorless crystalline solid, m.p. 244–246 C (243–245
◦
1
13
C [12
DMSO-d6):
,15]). H-NMR (400 MHz, DMSO-d ): δ 7.45–7.55 (m, 5H, Ar), 13.03 (s, 1H); C-NMR (101 MHz,
6
−1
δ
112.6, 122.4, 125.8, 129.00, 129.02, 139.3, 147.2, 154.8; IR (cm ): 575, 769, 1139, 1535, 1714,
240 (CN), 2826, 3057; HRMS (HESI m/z): [M − H] calcd for C H N O 213.0418; found 213.0405.
−
2
10
5
4
2
3
,5-Dioxo-2-phenyl-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxylic acid (6). Nitrile 4a (100 mg; 0.47 mmol)
was refluxed in aqueous 20% hydrochloric acid (4 mL) for 2 h. After this time, the reaction mixture was
concentrated under reduced pressure to one third of its original volume, when the product started to
crystalize. The product was filtered off, washed with water and dried. Yield 96 mg (88%) as a colorless
◦
◦
1
crystalline solid, m.p. 222–226 C (224–226 C [12
5
,
21]). H-NMR (400 MHz, DMSO-d ):
δ
7.49–7.53 (m,
6
H, Ar), 12.46 (s, 1H), 13.23–13.86 (bs, 1H); 13C-NMR (101 MHz, DMSO-d ):
δ 125.9, 128.4, 128.7, 135.8,
6
−
1
39.7, 147.8, 154.6, 161.8. HRMS (HESI m/z): [M
−
H] calcd for C H N O 232.0364; found 232.0354.
10
6
3
4
4
-Oxo-1,6-diphenyl-1,4-dihydroimidazo[2,1-c][1,2,4]triazine-3-carbonitrile (
8
). A mixture of nitrile 4a (48
◦
mg, 0.23 mmol) and phenacyl bromide (46 mg, 0.23 mmol) in DMF (1.5 mL) was heated at 130 C for
2
4 h. Cooled mixture was poured into water (20 mL) and precipitated solid was filtered off, washed
with water and dried. Yield 46 mg (65%). The crude product was crystalized from DMF (0.4 mL) to get
◦
1
an orange crystalline compound (30 mg). m.p 244–246 C. H-NMR (400 MHz, DMSO-d ):
δ
7.37–7.40
6
(
7
1
m, 1H, Ar), 7.44–7.47 (m, 2H, Ar), 7.60–7.64 (m, 1H, Ar), 7.67–7.71 (m, 2H, Ar), 7.92–7.94 (m, 2H, Ar),
13
.98–8.01 (m, 2H, Ar), 8.69 (s, 1H, Ar); C-NMR (101 MHz, DMSO-d6): δ 106.8, 113.5, 115.0, 124.6,
+
26.0, 128.9, 129.0, 129.4, 129.8, 131.4, 139.9, 142.2, 143.0, 149.2. HRMS (HESI m/z): [M + H] calcd for
C H N O 314.1036; found 314.1035.
18
12
5
3
,6-Dioxo-4,6-dihydro-3H-[1,2,4]triazino[2,3-a]quinazoline-2-carbonitrile (9). Hydrazone 3e (400 mg, 1.56
mmol) was suspended in mixture of ethanol-water (3:1; 25 mL) and resulting mixture was refluxed
with stirring for 24 h. After this time, water was added to the hot mixture (15 mL) and allowed to cool
◦
down. The precipitated crystalline compound was filtered off, washed with water and dried at 80 C.
◦
◦
1
Yield 250 mg (67%), m.p. over 360 C (367–370 C [22]). H-NMR (400 MHz, DMSO-d ): 7.49–7.53 (m,
6
1
1
1
H, Ar), 7.78 (ddd, J = 8.7, 7.2, 1.6 Hz, 1H, Ar), 7.98 (d, J = 8.0 Hz, 1H, Ar), 8.03 (dd, J = 7.8, 1.5 Hz,
H, Ar); 13C-NMR (101 MHz, DMSO-d ):
δ
114.1; 114.8; 119.5; 126.7; 126.8; 127.1; 133.4; 139.1; 151.7;
6
−
1
+
59.6; 166.3. IR (cm ): 759, 122, 152, 1571, 1638, 2245 (CN); HRMS (HESI m/z): [M + H] calcd for
C H N O 240.0516; found C H N O .
11
6
5
2
11
6
5
2
6
-Imino-3-oxo-4,6-dihydro-3H-[1,2,4]triazino[2,3-a]quinazoline-2-carbonitrile (10). Cyanohydrazone 3g (255
mg, 1.07 mmol) was suspended in a mixture of ethanol-water (3:1; 20 mL) and the resulting mixture

Molecules 2019, 24, 3558
11 of 13
was refluxed with stirring for 60 min. After cooling, the precipitated crystalline compound was filtered
◦
◦
off, washed with water and dried at 80 C. Yield 222 mg (87%) beige crystalline solid, m.p. 350–360 C
1
(
decomp). H-NMR (400 MHz, DMSO-d ):
δ
7.70 (ddd, J = 8.2, 7.4, 1.0 Hz, 1H, Ar), 7.99 (ddd, J = 8.6,
6
7
.3, 1.3 Hz, 1H, Ar), 8.21 (dd, J = 8.6, 0.8 Hz, 1H, Ar), 8.33 (dd, J = 8.2, 1.0 Hz, 1H, Ar) 9.13 (s, 1H), 9.23
(
s, 1H); 13C-NMR (101 MHz, DMSO-d ):
δ
112.2, 113.5, 115.8, 125.0, 127.5, 128.8, 135.6, 138.7, 150.8,
6
60.0, 161.2; IR (cm 1): 759, 1175, 1354, 1444, 1492, 1567, 2241 (CN), 3087; HRMS (HESI m/z): [M + H]
−
+
1
calcd C H N O 239.0676; found 239.0676.
11
7
6
6
-Imino-3-oxo-4,6-dihydro-3H-[1,2,4]triazino[2,3-a]quinazoline-2-carboxylic acid (11). Nitrile 10 (50 mg, 0.21
mmol) was suspended in a mixture of aqueous hydrochloric acid (1:1, 4 mL) and refluxed for 20 min.
Formed solution was cooled and neutralized with diluted ammonium hydroxide (1:2) to pH = 3–4.
Precipitated white microcrystalline solid was filtered off, washed with water and dried. Yield 44 mg
◦
82%), m.p. 252–254 C (decomp). H-NMR (400 MHz, DMSO-d ): δ 7.65–7.69 (m, 1H, Ar), 7.96–8.00
6
1
(
(
(
m, 1H, Ar), 8.24 (d, J = 8.4 Hz, 1H, Ar), 8.31 (d, J = 7.9 Hz, 1H, Ar), 9.12 (s, 1H), 9.21 (s, 1H); 15.09–15.38
bs, 1H, COOH); 13C-NMR (101 MHz, DMSO-d ):
δ 112.4, 116.0, 125.0, 127.4, 135.6, 139.0, 139.2, 151.5,
6
−
1
61.4, 162.4, 163.7. HRMS (HESI m/z): [M − H] calcd for C H N O 256.0476; found 256.0475.
11
6
5
3
3,6-Dioxo-4,6-dihydro-3H-[1,2,4]triazino[2,3-a]quinazoline-2-carboxylic acid (12). Nitrile 10 (166 mg, 0.7
mmol) was suspended in concentrated hydrochloric acid (4.5 mL) and refluxed for 24 h. After this
time, the precipitated white crystalline solid was filtered off, washed with water and dried. Yield 126
◦
◦
1
mg (70%), m.p. 256–258 C (decomp) (255–257 C [22]). H-NMR (400 MHz, DMSO-d ):
δ
7.60–7.64
6
(
1
1
2
m, 1H, Ar), 7.96 (ddd, J = 8.6, 7.3, 1.5 Hz, 1H, Ar), 8.07 (d, J = 8.3 Hz, 1H, Ar), 8.14 (dd, J = 7.9, 1.3 Hz,
13
H, Ar), 12.66–13.28 (bs, 1H), 13.81–14.90 (bs, 1H); C-NMR (101 MHz, DMSO-d ):
δ
115.4, 117.7, 127.3,
6
−
27.4, 135.9, 138.8, 143.2, 149.0, 159.1, 159.4, 162.8. HRMS (HESI m/z): [M
−
H] calcd for C H N O
11
5
4
4
57.0305; found 257.0309.
3-Cyano-1,5-bis(2-cyanophenyl)formazan (13g). a) This compound was prepared according to Method
A with different diazotation process. A solution of 2-aminobenzencabonitrile (0.472 g, 4 mmol) in a
◦
mixture of acetic acid (10.5 mL) and 35% hydrochloric acid (3 mL) was diazotized at 0–5 C with a
solution of sodium nitrite (0.275 g, 4 mmol) in ice water (3 mL). The mixture was stirred in ice bath
for 1 h and then added portionwise to a solution of 2-cyanoacetylcyanamide sodium salt
2 (0.525 g, 4
◦
mmol), sodium acetate (3 g) in water (25 mL), which was pre-cooled to 0–5 C. After 5 h of stirring
the precipitated red solid was filtered off, washed with water and dried. A sample for analysis was
crystallized from ethanol (0.1 g/10 mL). Yield 510 mg (85%, based on 2-aminobenzencabonitrile), m.p.
◦ ◦
87–191 C (190 C [19]) as red crystals. H-NMR (400 MHz, CDCl ): δ 7.44–7.48 (td, J = 7.6, 1.0 Hz,
3
1
1
13
2
H, Ar), 7.71–7.77 (m, 2H, Ar), 7.94 (d, J = 8.3 Hz, 2H, Ar), 12.33 (s, 1H); C-NMR (101 MHz, CDCl3): δ
1
1
04.5, 112.0, 116.6, 116.8, 118.3, 120.8, 125.2, 127.6, 129.4, 132.5, 133.1, 133.5, 134.2, 134.3, 134.4, 134.9,
−
47.9. HRMS (HESI m/z): [M − H] calcd for C H N 298.0836; found 298.0838.
16
8
7
b) Product with the same spectral and physical properties was obtained using cyanoacetic acid in
a similar manner as described above. Cyanoacetic acid (42.5 mg, 0.5 mmol), 2-aminobenzencabonitrile
118 mg, 1 mmol), were used for the reaction. Yield 80%.
(
4
. Conclusions
In this work, we extended and modified a previously developed method for the preparation of novel
-aryl-3-imino-5-oxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitriles (4-A), which at high temperature
2
occur as their 3-amino tautomers (4-B). These compounds are forms of 1-aryl-6-azaisocytosine
derivatives, which to date have not been well studied. The method was based on the coupling of
diazonium salts with cyanoacetylcyanamide
the corresponding 2-arylhydrazono-2-cyanoacetylcyanamides
polar solvents containing water to form the 1-aryl-6-azaisocytosines
2
carried out in slightly basic or acidic conditions to obtain
, which were cyclized by heating in
. The amino (imino) group at
3
4

Molecules 2019, 24, 3558
12 of 13
position 3 of the 1,2,4-triazine ring of the model phenyl derivative 4a was used for the preparation of the
novel imidazo[2,1-c][1,2,4]triazine and 1,2,4-triazino[2,3-a]quinazoline derivatives ( 12), potentially
8
9–
interesting as model compounds for intercalation testing with DNA.
Supplementary Materials: The copies of H- and 13C-NMR spectra are available online.
1
Author Contributions: F.Z. synthesized compounds; J.S. (Jan Slouka) conceived and designed the experiments;
J.S. (Jakub Stýskala) synthesized compounds and wrote the paper.
Funding: This research was funded by the Palacky University grant numbers IGA_PrF_2019_027 and
ˇ
IGA_LF_2019_019 and TACR grant number TE02000058.
Acknowledgments: Victoria Muir, from Edanz Group (www.edanzediting.com/ac) edited a draft of
this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
Arshad, M.; Khan, T.A.; Khan, M.A. 1,2,4-Triazine Derivatives: Synthesis and Biological Applications. Int. J.
Pharm. Sci. Res. 2015, 5, 149–162. [CrossRef]
2
.
Cascioferro, S.; Parrino, B.; Spanò, V.; Carbone, A.; Montalbano, A.; Barraja, P.; Diana, P.; Cirrincione, G. An
overview on the recent developments of 1,2,4-triazine derivatives as anticancer compounds. Eur. J. Med.
Chem. 2017, 142, 328–375. [CrossRef] [PubMed]
3.
4.
5.
Kumar, R.; Sirohi, T.S.; Singh, H.; Yadav, R.; Roy, R.K.; Chaudhary, A.; Pandeya, S.N. 1,2,4-Triazine Analogs
as Novel Class of Therapeutic Agents. Mini-Reviews Med. Chem. 2014, 14, 168–207. [CrossRef] [PubMed]
Farràs, J.; del Mar Lleó, M.; Vilarrasa, J.; Castillón, S.; Matheu, M.; Solans, X.; Font-Bardia, M. New bicyclic
nucleosides related to 6-azaisocytidine. Tetrahedron Lett. 1996, 37, 901–904. [CrossRef]
Mahfouz, R.Z.; Jankowska, A.; Ebrahem, Q.; Gu, X.; Visconte, V.; Tabarroki, A.; Terse, P.; Covey, J.; Chan, K.;
Ling, Y.; et al. Increased CDA Expression/Activity in Males Contributes to Decreased Cytidine Analog
Half-Life and Likely Contributes to Worse Outcomes with 5-Azacytidine or Decitabine Therapy. Clin. Cancer
Res. 2013, 19, 938–948. [CrossRef]
6
.
Tzeng, C.-C.; Hwang, L.-C.; Wang, C.-J.; Lee, G.-H.; Wang, Y. Synthesis and Structure Assignment of
1
4
-[(2-Hydroxyethoxy)methyl]- and 1-[(1,3-Dihydroxy-2-propoxy)methyl]-6-azaisocytosine. Heterocycles 1995
1, 293. [CrossRef]
Pit’ha, J.; Fiedler, P.; Gut, J. Nucleic acids components and their analogues. LXXXII. The fine structure of
-azaisocytosine and its derivatives. Collect. Czechoslov. Chem. Commun. 1966, 31, 1864–1871. [CrossRef]
,
7
.
.
6
´
8
Sztanke, M.; Sztanke, K.; Rajtar, B.; Swia˛tek, Ł.; Boguszewska, A.; Polz-Dacewicz, M. The influence of some
promising fused azaisocytosine-containing congeners on zebrafish (Danio rerio) embryos/larvae and their
antihaemolytic, antitumour and antiviral activities. Eur. J. Pharm. Sci. 2019, 132, 34–43. [CrossRef]
Stock, M.L.; Elazab, S.T.; Hsu, W.H. Review of triazine antiprotozoal drugs used in veterinary medicine. J.
9
1
.
Vet. Pharmacol. Ther. 2018, 41, 184–194. [CrossRef]
®
0. Diaferia, M.; Veronesi, F.; Morganti, G.; Nisoli, L.; Fioretti, D.P. Efficacy of Toltrazuril 5% Suspension (Baycox ,
®
Bayer) and Diclazuril (Vecoxan , Janssen-Cilag) in the Control of Eimeria spp. in Lambs. Parasitol. Res.
2
013, 112, 163–168. [CrossRef]
1. Kelly, M.J.; Pietranico-Cole, S.; Larigan, J.D.; Haynes, N.-E.; Reynolds, C.H.; Scott, N.;
Vermeulen, J.; Dvorozniak, M.; Conde-Knape, K.; Huang, K.-S.; et al. Discovery
of 2-[3,5-Dichloro-4-(5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yloxy)phenyl]-3,5-dioxo-2,3,4,5-tetrahydro-
1,2,4]triazine-6-carbonitrile (MGL-3196), a Highly Selective Thyroid Hormone Receptor Agonist in Clinical
Trials for the Treatment of Dys. J. Med. Chem. 2014, 57, 3912–3923. [CrossRef] [PubMed]
1
[
β
1
1
1
2. Slouka, J. 1-Aryl-6-azauracile: Die Synthese von 1-Phenyl-6-azauracil-5-carbonsäure und einiger ihrer
Derivate. Monatshefte für Chemie 1963, 94, 258–262. [CrossRef]
3. Neunhoeffer, H. Chemistry of 1,2,3-Triazines and 1,2,4-triazines, tetrazines and pentazines; J. Wiley and sons: New
York, NY, USA, 1978.
4. Frysova, I.; Mutina, V.; Slouka, J.; Hlavac, J. Chemistry of 1-Aryl-6-azauracil-5-carbonitriles. Acta Upol. Fac.
Rer. Nat. Chem. 2004, 43, 15–58. [CrossRef]

Molecules 2019, 24, 3558
13 of 13
0 0 0 0
5. Slouka, J.; Pe cˇ , P. 1-Aryl-6-azauracile, 4. Mitt.: Die Synthese des 1-Phenyl-5-[5 -methyl-1 ,2 ,4 -
1
0
oxdiazolyl-(3 )]-6-azauracils und einiger seiner Derivate. Monatshefte für Chemie 1965, 96, 1874–1877.
CrossRef]
[
1
1
1
1
6. Wardakhan, W.W.; Ouf, S.A. Reaction of cyanoacetamide with benzoyl isothiocyanate: synthesis of pyridine,
pyrido[2,1-a]triazine, pyrazolo[3,4-d]triazine and thiazole derivatives. Egypt. J. Chem. 2005, 48, 393–406.
7. El-Bannany, A.A.A.; Ghozlan, S.A.S.; Ibraheim, L.I. Synthesis of some new 1,2,4-triazine, 1,2,3-triazole and,
pyridazine derivatives. Pharmazie 1987, 42, 695–696.
8. Hirayama, T.; Kamada, M.; Tsurumi, H.; Mimura, M. A novel synthesis of pyrimidines. I. Cyclization of
N-cyano-cyanoaceto derivatives. Chem. Pharm. Bull. 1976, 24, 26–35. [CrossRef]
9. Khattab, T.A.; Haggag, K.M.; Elnagdi, M.H.; Abdelrahman, A.A.; Abdelmoez Aly, S. Microwave-Assisted
Synthesis of Arylazoaminopyrazoles as Disperse Dyes for Textile Printing. Zeitschrift fur Anorg. und Allg.
Chemie 2016, 642, 766–772. [CrossRef]
2
2
2
2
2
2
2
0. Slouka, J.; Bekárek, V. Synthesis and cyclization of some N-oxides of 2-pyridylhydrazones of mesoxalic acid
derivatives. Collect. Czechoslov. Chem. Commun. 1988, 53, 626–632. [CrossRef]
1. Slouka, J. 1-Aryl-6-azauracile, 3. Mitt.: Die Synthese von 1-Phenyl-6-azauracil und einiger seiner Derivate.
Monatshefte für Chemie 1965, 96, 134–137. [CrossRef]
2. Slouka, J.; Bekárek, V. 1-Aryl-6-azauracile. XVII. Synthese einiger 1,2,4-Triazino[2,3-a]chinazolin-Derivate. J.
für Prakt. Chemie 1974, 316, 943–951. [CrossRef]
3. Li, S.; Cooper, V.R.; Thonhauser, T.; Lundqvist, B.I.; Langreth, D.C. Stacking interactions and DNA
intercalation. J. Phys. Chem. B 2009, 113, 11166–11172. [CrossRef] [PubMed]
4. Almaqwashi, A.A.; Paramanathan, T.; Rouzina, I.; Williams, M.C. Mechanisms of small molecule-DNA
interactions probed by single-molecule force spectroscopy. Nucleic Acids Res. 2016, 44, 3971–3988. [CrossRef]
5. Almaqwashi, A.A.; Andersson, J.; Lincoln, P.; Rouzina, I.; Westerlund, F.; Williams, M.C. DNA intercalation
optimized by two-step molecular lock mechanism. Sci. Rep. 2016, 6, 1–11. [CrossRef] [PubMed]
6. Biebricher, A.S.; Heller, I.; Roijmans, R.F.H.; Hoekstra, T.P.; Peterman, E.J.G.; Wuite, G.J.L. The impact of
DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding
kinetics. Nat. Commun. 2015, 6, 1–12. [CrossRef]
Sample Availability: Samples of the compounds are not available.
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