General Synthesis of 1-Aryl-6-azaisocytosines and Their Utilization for the Preparation of Related Condensed 1,2,4-Triazines

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.

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 [4][5][6]. Hence, it is desirable to design and develop 6-azaisocytosine based molecules [6,7], which, as isomeric structures of 6-azacytosine, should be more resistant to enzymatic deamination [6]. In addition, fused azaisocytosines, where the nitrogen atom is part of 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 β agonist, and is now in clinical trials for the treatment of dyslipidemia [11].
Unlike the 1-aryl-6-azauracils, which belong to the longest known and numerous derivatives of 1,2,4-triazine [12][13][14][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 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).
Molecules 2019, 24, x 2 of 13 substituted 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).

Results and Discussion
2-Cyanoacetylcyanamide sodium salt 2 [18] was treated with diazonium salts to afford the corresponding hydrazones 3. For the preparation of arylhydrazones 3a-3e we used a general method 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 2cyanobenzenediazonium 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-3g containing strong 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 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].

Results and Discussion
2-Cyanoacetylcyanamide sodium salt 2 [18] was treated with diazonium salts to afford the corresponding hydrazones 3. For the preparation of arylhydrazones 3a-3e we used a general method 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) (NO 2 , 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-3g containing strong 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 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]. 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). 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). 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 4 at room temperature. For this reason, the NMR data of arylhydrazones 3 were recorded immediately after sample preparation in DMSO-d6.
Compounds 3 (except 3e and 3g) were cyclized to the corresponding 1,2,4-triazines 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 3 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 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). 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 these forms, the phenyl derivative 4a was studied by 1 H-NMR spectroscopy in DMSO-d6 at two 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). 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 4 at room temperature. For this reason, the NMR data of arylhydrazones 3 were recorded immediately after sample preparation in DMSO-d 6 .
Compounds 3 (except 3e and 3g) were cyclized to the corresponding 1,2,4-triazines 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 3 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 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). 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 4 at room temperature. For this reason, the NMR data of arylhydrazones 3 were recorded immediately after sample preparation in DMSO-d6.
Compounds 3 (except 3e and 3g) were cyclized to the corresponding 1,2,4-triazines 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 3 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 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). 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 these forms, the phenyl derivative 4a was studied by 1 H-NMR spectroscopy in DMSO-d6 at two 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). 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 these forms, the phenyl derivative 4a was studied by 1 H-NMR spectroscopy in DMSO-d 6 at two 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).  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 1phenyl-6-azauracil-5-carboxylic acid 6.
The utility of the amino group of the 6-azaisocytosine skeleton for the synthesis of condensed 1,2,4-triazines was investigated. Formation of the fused ring between the amino group at position 3 and the nitrogen atom at position 4 of the 1,2,4-triazine cycle was demonstrated in the reaction of 1phenyl-6-azaisocytosine-5-carbonitrile 4a with phenacyl bromide, which resulted in the 2N-alkylated 6-azaisocytosine 7 which cyclized directly under the alkylation reaction conditions to the corresponding imidazo[2,1-c][1,2,4]triazine derivative 8. There are two main possible centers for the alkylation of 1-phenyl-6-azaisocytosine 4a: The amino group at position 3 and the nitrogen atom at position 4 of the 1,2,4-triazine cycle. The selective alkylation of compound 4a at the amino group was accomplished at a 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).  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 1phenyl-6-azauracil-5-carboxylic acid 6.
The utility of the amino group of the 6-azaisocytosine skeleton for the synthesis of condensed 1,2,4-triazines was investigated. Formation of the fused ring between the amino group at position 3 and the nitrogen atom at position 4 of the 1,2,4-triazine cycle was demonstrated in the reaction of 1phenyl-6-azaisocytosine-5-carbonitrile 4a with phenacyl bromide, which resulted in the 2N-alkylated 6-azaisocytosine 7 which cyclized directly under the alkylation reaction conditions to the corresponding imidazo[2,1-c][1,2,4]triazine derivative 8. There are two main possible centers for the alkylation of 1-phenyl-6-azaisocytosine 4a: The amino group at position 3 and the nitrogen atom at position 4 of the 1,2,4-triazine cycle. The selective alkylation of compound 4a at the amino group was accomplished at a 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). 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.
In addition to the given tautomer (A), compounds 10 and 11 may also be represented by two other tautomeric forms (B) and (C). In the varying tautomeric equilibrium between (A) and (B), two N-H groups are present; however, tautomer (C) has only an NH 2 group (Figure 4). From the NMR data, it was apparent that the compounds did not contain an NH 2 group (two sharp hydrogen signals were present in area of 9.0-9.3 ppm belonging to acidic N-H groups). 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 3,6dioxo-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 (A), compounds 10 and 11 may also be represented by two other tautomeric forms (B) and (C). In the varying tautomeric equilibrium between (A) and (B), two N-H groups are present; however, tautomer (C) has only an NH2 group (Figure 4). From the NMR data, it was apparent that the compounds did not contain an NH2 group (two sharp hydrogen signals were present in area of 9.0-9.3 ppm belonging to acidic N-H groups). 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][24][25][26], but also through forming intermolecular hydrogen bonds via the acidic NH groups.

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 μL/min. Mobile phases: 10 mM ammonium acetate in HPLC grade water and HPLC grade acetonitrile. 1 H-and 13 C-NMR spectra were measured on an ECA 400II ( 1 H: 399.78 MHz, 13 C: 100.53 MHz,) NMR spectrometer (JEOL Resonance, Tokyo, Japan). Chemical shifts (δ) are reported in ppm and referenced to the middle peak of the solvent signal (DMSO-d6: 2.49 ppm, 39.50 ppm; CDCl3: 7.27 ppm, 77.00 ppm. All recorded 1 H-and 13 C-NMR spectra are available as Supplementary material online. NMR data of arylhydrazones 3 were recorded immediately after sample preparation in DMSO-d6. The IR spectra were recorded in KBr wafers on an ATI Unicam 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.

Synthesis of Compounds 2-13
2-Cyanoacetylcyanamide sodium salt (2). To a solution of sodium ethoxide prepared from sodium (2.3 g, 0.1 mol) and anhydrous ethanol (50 mL) was added solution of cyanamide (4.2 g, 0.1 mol) in 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][24][25][26], but also through forming intermolecular hydrogen bonds via the acidic NH groups.

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 µL/min. Mobile phases: 10 mM ammonium acetate in HPLC grade water and HPLC grade acetonitrile. 1 H-and 13 C-NMR spectra were measured on an ECA 400II ( 1 H: 399.78 MHz, 13 C: 100.53 MHz,) NMR spectrometer (JEOL Resonance, Tokyo, Japan). Chemical shifts (δ) are reported in ppm and referenced to the middle peak of the solvent signal (DMSO-d 6 : 2.49 ppm, 39.50 ppm; CDCl 3 : 7.27 ppm, 77.00 ppm. All recorded 1 H-and 13 C-NMR spectra are available as Supplementary Materials online. NMR data of arylhydrazones 3 were recorded immediately after sample preparation in DMSO-d 6 . The IR spectra were recorded in KBr wafers on an ATI Unicam 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.

Synthesis of Compounds 2-13
2-Cyanoacetylcyanamide sodium salt (2). To a solution of sodium ethoxide prepared from sodium (2.3 g, 0.1 mol) and anhydrous ethanol (50 mL) was added solution of cyanamide (4.2 g, 0.1 mol) in 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 cold ethanol and dried at 60 • C for 90 min. Yield was 11.7 g (85%). 1  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.