Fused heterocyclic systems with an s-triazine ring. 34. Development of a Practical Approach for the Synthesis of 5-Aza-isoguanines

Article abstract of DOI:10.3390/molecules24081453

Purine isosteres present excellent opportunities in drug design and development. Using isosteres of natural purines as scaffolds for the construction of new therapeutic agents has been a valid strategy of medicinal chemistry. Inspired by the similarity to isoguanine, we attempted to develop a practical method for the preparation of 5-aza-isoguanines. Several synthetic approaches were explored to establish a robust general protocol for the preparation of these compounds. The significant difference in the reactivity of the C-5 and C-7 electrophilic centers of 1,2,4-triazolo[1,5-a][1,3,5]triazines (5-azapurines) towards nucleophiles was demonstrated. The most practical and general method for the preparation of 5-aza-isoguanines involved a regioselective reaction of ethoxycarbonyl isothiocyanate with a 5-aminotriazole. The intramolecular ring closure of the resulted product followed by the S-methylation afforded 7-methylthio-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one, which could be effectively aminated with various amines. The resulted 5-aza-isoguanines resemble a known purine nucleoside phosphorylase inhibitor and could be interesting for further investigations as potential anticancer agents.

Full text of DOI:10.3390/molecules24081453

molecules
Article
Fused Heterocyclic Systems with an s-Triazine Ring.
34. Development of a Practical Approach for the
Synthesis of 5-Aza-isoguanines
Ahmad Junaid 1, Felicia Phei Lin Lim 1 , Yvonne Peijun Zhou 2, Wai Keung Chui 2 and
Anton V. Dolzhenko 1,3,
*
1
School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway,
Selangor Darul Ehsan 47500, Malaysia; ahmad.junaid@monash.edu (A.J.); felicia.lim@monash.edu (F.P.L.L.)
Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4,
Singapore 117543, Singapore; marz_vonne@yahoo.com.sg (Y.P.Z.); phacwk@nus.edu.sg (W.K.C.)
School of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research Institute,
Faculty of Health Sciences, Curtin University, GPO Box U1987, Perth, WA 6845, Australia
Correspondence: anton.dolzhenko@monash.edu; Tel.: +60-3-5514-5867
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Panayiotis A. Koutentis and Andreas S. Kalogirou
Received: 12 March 2019; Accepted: 10 April 2019; Published: 12 April 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Purine isosteres present excellent opportunities in drug design and development. Using
isosteres of natural purines as scaffolds for the construction of new therapeutic agents has been a valid
strategy of medicinal chemistry. Inspired by the similarity to isoguanine, we attempted to develop
a practical method for the preparation of 5-aza-isoguanines. Several synthetic approaches were
explored to establish a robust general protocol for the preparation of these compounds. The significant
difference in the reactivity of the C-5 and C-7 electrophilic centers of 1,2,4-triazolo[1,5-a][1,3,5]triazines
(5-azapurines) towards nucleophiles was demonstrated. The most practical and general method for the
preparation of 5-aza-isoguanines involved a regioselective reaction of ethoxycarbonyl isothiocyanate
with a 5-aminotriazole. The intramolecular ring closure of the resulted product followed by the
S-methylation afforded 7-methylthio-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one, which could be
effectively aminated with various amines. The resulted 5-aza-isoguanines resemble a known purine
nucleoside phosphorylase inhibitor and could be interesting for further investigations as potential
anticancer agents.
Keywords: triazine; triazole; azapurine; purine isostere
1. Introduction
Purine isosteres attract significant attention from medicinal chemists due to their potential for the
development of new therapeutic agents. These compounds are known to modulate a complex network
of processes, which involves regulatory biogenic purines. The purine-like scaffolds incorporating
a 1,3,5-triazine ring became privileged in the construction of bioactive molecules [
that pyrazolo[1,5-a][1,3,5]triazine (5-aza-9-deazapurine) [ ] and 1,2,4-triazolo[1,5-a][1,3,5]triazine
(5-azapurine) [ ] were the most promising skeletons in this group of purine isosteres. We developed
1]. It was found
2
3,4
efficient methods for the synthesis of adenine and xanthine analogues built on these scaffolds [5–15].
The synthesis of 5-aza-guanines has also been reported [16,17].
Isoguanine has been reported as
a skeleton for several biologically important
alkaloids [18 20]. Some interesting results were obtained on a compound with the
–
4-aminopyrazolo[1,5-a][1,3,5]triazin-2-one skeleton (a triazine-based 5-aza-9-deaza-isostere of
isoguanine) [21]. This compound was an efficient inhibitor of purine nucleoside phosphorylase,
Molecules 2019, 24, 1453; doi:10.3390/molecules24081453
www.mdpi.com/journal/molecules

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which is a valid target for anticancer and antiparasitic therapy [22
–
25]. This brought our attention
to a similar purine-like heterocyclic system, i.e., 5-aza-isoguanine. Herein, we report our attempts to
develop an efficient synthesis of 5-aza-isoguanines bearing various substituents on the amino group.
2. Results and Discussion
Earlier we developed convenient syntheses of amino-substituted pyrazolo[1,5-a][1,3,5]triazines
and 1,2,4-triazolo[1,5-a][1,3,5]triazines exploiting good reactivity of highly electrophilic 1,3,5-triazine
ring substituted with the trichloromethyl group [26
was the ring closure reaction of azolylguanidine
,
27]. The key step in this synthetic approach
using trichloroacetonitrile (Scheme 1). The
1
subsequent nucleophilic substitution of the introduced trichloromethyl leaving group of
2 with
variety of amines resulted in the formation of the corresponding pyrazolo[1,5-a][1,3,5]triazines and
1,2,4-triazolo[1,5-a][1,3,5]triazines 3.
Scheme 1. Synthesis of diamino-substituted azolo[1,5-a][1,3,5]triazines 3.
With our interest leaning towards the synthesis of 5-aza-isoguanines, which are 5-oxo-analogues
of compounds
trichloromethyl group was based on the previously developed reaction using triazolylurea
of guanidine (Scheme 2). However, despite a number of attempts to achieve the triazine ring
3, an initial attempt to prepare 1,2,4-triazolo[1,5-a][1,3,5]triazine 5 bearing reactive
4
in place
1
closure using trichloroacetonitrile under various reaction conditions, we were able to isolate starting
urea 4 only.
Scheme 2. An attempt to prepare 5-aza-isoguanines via trichloromethyl-substituted intermediate 5.
To further explore the trichloromethyl chemistry in the synthesis of 7-amino-substituted
1,2,4-triazolo[1,5-a][1,3,5]triazin-5-ones
6, we subjected 1-guanyl-1,2,4-triazole 7, prepared as reported
previously [28], to the reaction with trichloroacetonitrile (Scheme 3). Similar to triazolylguanidine
1
(X = N), the reaction pathway of
7
was solvent dependent. In ethanol, diamine
8
9
, identical to
was isolated
that reported in reference [29], was formed, whereas 1,2,4-triazolo[1,5-a][1,3,5]triazine
exclusively when the reaction was carried out in toluene.
Scheme 3. Synthesis of 5-aza-isoguanine 6a.

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1
In the H-NMR spectrum of
9, two protons of the amino group gave independent signals
appearing at rather low field: 9.22 and 9.65 ppm. These observations can be attributed to the
prominent delocalisation of an electron pair on the amino group nitrogen directly attached to the
highly electron-deficient 1,3,5-triazine ring. The electron-withdrawing trichloromethyl group further
decreased electron density on the triazine ring, enhancing the partial double bond character of the
C-NH2 bond and restricting the rotation around it. The activation energy (∆
G‡) for the hindered
rotation around this bond was estimated using dynamic 1H-NMR spectroscopy and was equal 68.8 kJ
at the coalescence temperature 343 K.
The trichloromethyl group of
carbonate to afford 6a. The substitution of trichloromethyl group of 9 with nucleophiles (particularly
amines) was more problematic in comparison to the aminolysis of the isomeric structure . No
9 was hydrolytically removed using aqueous solution of sodium
2
reaction was observed when similar reaction conditions were applied. Moreover, unexpected
results were obtained after prolonged heating with excess of amine depending on the reaction
conditions and amine structure (Scheme 4). Replacement of the amino group in position 7 of
the 1,2,4-triazolo[1,5-a][1,3,5]triazine ring was observed instead of the proposed substitution of the
trichloromethyl group of 9 (e.g., in the synthesis of 10) or both the processes took place together
(e.g., in the preparation of 11). It was demonstrated earlier that compounds with identical leaving
groups in positions 5 and 7 of the 1,2,4-triazolo[1,5-a][1,3,5]triazine ring were suitable for the sequential
nucleophilic substitution with amines: first in position 7, then in position 5 [30]. This strategy
was successfully applied for the synthesis of bioactive 1,2,4-triazolo[1,5-a][1,3,5]triazines [31,32].
However, to the best of our knowledge, no examples on transaminations at the position 7 of
1,2,4-triazolo[1,5-a][1,3,5]triazines have been reported. For similar pyrazolo[1,5-a][1,3,5]triazines,
the replacement of a N-methylanilino substituent in the corresponding position with other amines was
effectively employed in drug-discovery programs to prepare a variety of compounds with a diverse
substitution pattern [33–40].
Scheme 4. Reaction of 9 with 4-methoxybenzylamine and morpholine.
The hydrolysis of 7-amino substituted 10 can be applied to generate 6k. Nevertheless, difficulties
with controlling reaction outcome in the synthesis of analogues of 10 and a long reaction time led us to
design an intermediate with a leaving group more accessible for the nucleophilic substitution by amines
than 9. This strategy was realized by the preparation of 1,2,4-triazolo[1,5-a][1,3,5]triazine (15) (Scheme 5).
Aminotriazole 12 reacted with carbon disulfide in DMF in the presence of potassium hydroxide followed
by the selective S-methylation affording 13. Treatment of 13 with trichloroacetonitrile resulted in the
formation of 15. Trichloroacetonitrile provided a C–N fragment for the construction of the triazine
ring. We propose that the reaction involved an initial addition to the triple bond of trichloroacetonitrile
followed by the intramolecular ring closure of intermediate 14 via nucleophilic substitution at the
methyl dithiocarboxylate moiety. Surprisingly, hydrogen sulfide instead of methylthiol played the role

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of a leaving group in this reaction. The methylthio group of 15 can be readily replaced by stoichiometric
quantity of amines, e.g., 4-methoxybenzylamine, providing 10. However, a long reaction time at the
hydrolysis step arising from the low aqueous solubility of 10 and the necessity of chromatographic
purification limited the practicality of this approach.
Scheme 5. Synthesis of 5-trichloromethyl-7-methylthio-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazine (15).
Considering the smooth replacement of the methylthio group at position 7 of the
1,2,4-triazolo[1,5-a][1,3,5]triazine with amines, we implemented an alternative synthetic approach
for the preparation of
6
.
The key intermediate in this protocol was 16, prepared from
5-amino-3-phenyl-1,2,4-triazole (12) (Scheme 6). The selective addition of 12 to ethoxycarbonyl
isothiocyanate was challenging due to the presence of several competing nucleophilic centers on
12. It was found that regioselective addition occurs only at the endocyclic N-1 atom of 16 when the
reaction was carried out under kinetic control in cold acetone for not more than 15 min. Extension of
the reaction time or increasing temperature led to the formation of more thermodynamically stable
N-carbethoxy-N0-(1,2,4-triazol-5-yl)thiourea, which existed in solution as a mixture of two tautomers
17 and 170 (KT = 0.74) crystalizing in the form of the predominant tautomer 17 (Figure 1) [41].
Scheme 6. Synthesis of 7-methylthio-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (18).
Figure 1. X-ray structure of 17.
In solutions, 16 readily underwent rearrangement to 17 even at room temperature. Therefore,
instant preparation of sufficiently pure 16 was critical, as changes in the product structure during
purification were unavoidable. The alkali-induced cyclocondensation of 16 followed by S-methylation
afforded 7-methylthio-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (18).
The nucleophilic replacement of the methylthio group of 18 proceeded smoothly, with a variety
of amines affording
6 with reasonable yields and high purity (Scheme 7). The reaction remained

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chemoselective even when aqueous solutions of fairly basic amines (synthesis of 6a
–
c) were used. The
structure of the prepared compounds
6 was confirmed by NMR spectroscopy, clearly showing that the
signal of methylthio group of 18 was replaced by signals of the corresponding amines in spectra of
6
.
The X-ray data for a representative example 6l also confirmed the proposed structure (Figure 2) [42].
Scheme 7. Synthesis of 5-aza-isoguanines 6.
Figure 2. X-ray structure of 6l.
The synthetic route described in Schemes 6 and 7 was not placed on the top priority at the initial
design of the synthetic routes due to the relatively low yield of the intermediate 16 and the uncertainty
involved in the preparation of a pure regioisomer 16 as a result of the addition reaction between

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ethoxycarbonyl isothiocyanate to 5-amino-3-phenyl-1,2,4-triazole (12). However, this synthetic route
unexpectedly became the most practical and versatile method that could be applied to the synthesis of
a library of 7-amino substituted 2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-ones (6a–p).
3. Conclusions
In conclusion, several synthetic approaches to the synthesis of 5-aza-isoguanines
6 were
explored to develop a practical method for their preparation. The significant difference in the
reactivity of the C-5 and C-7 electrophilic centers of 1,2,4-triazolo[1,5-a][1,3,5]triazines towards
nucleophiles was demonstrated. The most practical and general method for the preparation of
5-aza-isoguanines
ethoxycarbonyl isothiocyanate followed by an intramolecular ring closure and S-methylation to afford
18, which could be effectively aminated with various amines. The prepared compound resemble a
6 involved a regioselective addition of 5-amino-3-phenyl-1,2,4-triazole (12) to
6
known purine nucleoside phosphorylase inhibitor and could be interesting for further investigations
as potential anticancer agents.
4. Materials and Methods
4.1. General
The melting points (uncorrected) were determined via the use of the Gallenkamp melting point
apparatus. The 1H NMR and 13C NMR spectra were recorded on a Bruker DPX-300 spectrometer
(Fällanden, Switzerland), using DMSO-d6 as a solvent and TMS as the internal reference. All the
reactions were monitored with the use of the analytical TLC carried out on aluminum plates coated on
Silica gel 60 F254 (Merck, Darmstadt, Germany) with detection by UV light.
4.2. Synthesis
4.2.1. Synthesis of 2-Phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5,7-diamine 8
1-Guanyl-3-phenyl-1,2,4-triazol-5-amine (7, 0.3 g, 1.5 mmol) and trichloroacetonitrile (0.45 mL,
4.5 mmol) were heated in EtOH (10 mL) under reflux for 6 h. After cooling, the precipitated colorless
product was filtered and washed with cold EtOH to give the product, which was found to be identical
to compound 8 prepared earlier via an alternative method [29]. Yield 210 mg, 62%.
4.2.2. Synthesis of 7-Amino-2-phenyl-5-trichloromethyl-1,2,4-triazolo[1,5-a][1,3,5]triazine 9
1-Guanyl-3-phenyl-1,2,4-triazol-5-amine (7, 0.3 g, 1.5 mmol) and trichloroacetonitrile (0.45 mL,
4.5 mmol) were refluxed in toluene (10 mL) for 6 h. After cooling, the colorless product was filtered
and recrystalized from EtOH. Yield 310 mg, 63%; mp > 300 ◦C (EtOH). 1H-NMR (300 MHz, DMSO-d6):
δ
7.53–7.65 (3H, m, H-30, H-40 and H-50), 8.19–8.28 (2H, m, H-20 and H-60), 9.22 (1H, s, NH), 9.65 (1H,
s, NH). 13C-NMR (75 MHz, DMSO-d6):
δ 96.2, 126.9 (2C), 128.9 (2C), 129.7, 130.9, 151.6, 157.2, 164.1,
164.6. Combustion elemental analysis calculated for C11H7Cl3N6: C, 40.09; H, 2.14; N, 25.50. Found:
C, 39.96; H, 2.35; N, 25.32.
4.2.3. Synthesis of 7-Amino-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-ones 6a from 9
To 10% aqueous Na2CO3 (10 mL), 7-amino-2-phenyl-5-trichloromethyl-1,2,4-triazolo[1,5-a][1,3,5]
triazine (9, 0.33 g, 1 mmol) was added, and the mixture was heated under reflux for 72 h. The reaction
mixture was allowed to cool and HCl (1 N) was added dropwise to pH 4. The precipitate formed was
filtered and washed with water to give colorless crystalline powder. Yield 36 mg, 16%; mp > 300 ◦C
(MeOCH2CH2OH). 1H-NMR (300 MHz, DMSO-d6):
δ
7.48–7.58 (3H, m, H-30, H-40 and H-50), 8.01–8.11
(2H, m, H-20 and H-60), 8.26 (1H, s, NH), 8.64 (1H, brs, NH), 12.17 (1H, brs, N(4)H). 13C-NMR (75 MHz,
DMSO-d6): 126.5 (2C), 128.8 (2C), 129.6, 130.4, 149.8, 153.1, 153.6 (2C), 161.4. Combustion elemental
analysis for C10H8N6O: C, 52.63; H, 3.53; N, 36.83. Found: C, 52.42; H, 3.71; N, 36.66.
δ

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4.2.4. Synthesis of N-(4-Methoxybenzyl)-2-phenyl-5-trichloromethyl-1,2,4-triazolo[1,5-a][1,3,5]
triazin-7-amine 10
To the suspension of 9 (0.33 g, 1 mmol) in EtOH (5 mL), 4-methoxybenzylamine (0.36 mL, 3 mmol)
was added and the mixture was heated under reflux for 24 h. After cooling, the resulting precipitate
was filtered, washed with cold EtOH, dried and recrystallized from EtOH. Yield 212 mg, 47%; mp
174–175 ◦C (EtOH). 1H-NMR (300 MHz, DMSO-d6):
δ 3.73 (3H, s, OMe), 4.73 (2H, d, J = 5.8 Hz, CH2),
6.91 (2H, d, J = 8.7 Hz, C-300 and C-500), 7.47 (2H, d, J = 8.7 Hz, C-200 and C-600), 7.52–7.61 (3H, m, H-30,
H-40 and H-50), 8.16–8.28 (2H, m, H-20 and H-60), 10.20 (1H, t, J = 5.8 Hz, NH). 13C-NMR (75 MHz,
DMSO-d6):
δ
43.7, 55.0, 96.1, 113.7 (2C0), 126.9 (2C), 128.9 (2C), 129.3, 129.7, 129.8 (2C), 130.9), 149.6,
157.1, 158.7, 163.9, 164.5. Combustion elemental analysis calculated for C19H15Cl3N6O: C, 50.74; H, 3.36;
N, 18.69. Found: C, 50.55; H, 3.52; N, 18.42.
4.2.5. Synthesis of 5,7-Bis(morpholino)-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazine 11
To 5 mL of morpholine, 9 (0.33 g, 1 mmol) was added, and the mixture was heated under reflux
for 5 h. After cooling, the resulting precipitate was filtered, washed with cold EtOH, dried and
recrystallized from EtOH. Yield 180 mg, 49%; mp 278–279 ◦C (EtOH). 1H-NMR (300 MHz, DMSO-d6):
δ
3.61–3.85 (12H, m), 4.27 (4H, br. s, W1/2 = 24.9 Hz), 7.46–7.55 (3H, m, H-30, H-40 and H-50), 8.05–8.15
(2H, m, H-20 and H-60). 13C-NMR (75 MHz, DMSO-d6):
δ 44.0 (br. s), 46.7, 65.7, 65.8, 126.6 (2C), 128.6
(2C), 130.2 (2C), 148.5, 158.8, 161.1, 162.1. Combustion elemental analysis calculated for C18H21N7O2:
C, 58.84; H, 5.76; N, 26.69. Found: C, 58.61; H, 6.02; N, 26.41.
4.2.6. Synthesis of Methyl 5-Amino-3-phenyl-1,2,4-triazoyl-1-dithiocarbonate 13
3-Phenyl-1,2,4-triazol-5-amine (12, 3.20 g, 20 mmol) was dissolved in cold DMF (8 mL). Carbon
disulphide (1.2 mL, 20 mmol) was added drop-wise, followed by 10 N KOH solution (2 mL, 20 mmol).
The reaction mixture was stirred for 1 h on an ice-bath. Iodomethane (1.25 mL, 20 mmol) was added,
and stirring was continued for 10 min. Subsequently, the ice-bath was removed and the reaction
mixture was stirred for 2 h. Cold water (15 mL) was added to the mixture, the yellow precipitate was
filtered, washed with water, dried and recrystalized from EtOH. Yield 80 mg, 32%; mp 207–209 ◦C
(EtOH). 1H-NMR (300 MHz, DMSO-d6):
δ
2.63 (3H, s, SMe), 7.47–7.57 (3H, m, H-30, H-40 and H-50),
8.03 (2H, dd, J = 6.8, 3.0 Hz, H-20 and H-60), 8.64 (2H, s, NH2). 13C-NMR (75 MHz, DMSO-d6): δ 18.8,
126.6 (2C), 128.8 (2C), 129.1, 130.6, 157.5, 158.8, 199.5. Combustion elemental analysis calculated for
C10H10N4S2: C, 47.98; H, 4.03; N, 22.38. Found: C, 48.06; H, 4.12; N, 22.14.
4.2.7. Synthesis of 7-Methylthio-2-phenyl-5-trichloromethyl-1,2,4-triazolo[1,5-a][1,3,5]triazine 15
Methyl 5-amino-3-phenyl-1,2,4-triazoyl-1-dithiocarbonate
(
13
,
0.75 g,
3 mmol) and
trichloroacetonitrile (0.75 mL, 7.5 mmol) were heated under reflux in toluene (10 mL) for 12 h.
Solvent was evaporated under reduced pressure, the residue was triturated with diethyl ether and
1
solid was collected by filtration. Yield 245 mg, 68%. H-NMR (300 MHz, DMSO-d6):
δ 2.85 (3H, s, SMe),
7.56–7.68 (3H, m, H-30, H-40 and H-50), 8.26 (2H, dd, J = 6.6, 2.8 Hz, H-20 and H-60). 13C-NMR (75 MHz,
DMSO-d6): 13.1, 95.2, 127.2 (2C), 129.0, 129.1 (2C), 131.6, 155.2, 161.2, 164.4, 166.0. Combustion
δ
elemental analysis calculated for C12H8Cl3N5S: C, 39.97; H, 2.24; N, 19.42. Found: C, 40.12; H, 2.33;
N, 19.11.
4.2.8. Reactions of 3-Phenyl-1,2,4-triazol-5-amine (12) with Ethoxycarbonyl Isothiocyanate
Synthesis of 5-amino-1-carbethoxythiocarbamoyl-1,2,4-triazole (16). To a solution of acetone
(30 mL), 3-phenyl-1,2,4-triazol-5-amine (12, 3.20 g, 20 mmol) and ethoxycarbonyl isothiocyanate
(2.62 mL, 20 mmol) were added. The reaction mixture was stirred on an ice-bath for 15 min. The yellow
precipitate formed was filtered immediately. Yield 2.16 g, 37%; mp 207–209 ◦C. 1H-NMR (300 MHz,
DMSO-d6):
δ
2.63 (3H, s, SMe), 7.47–7.57 (3H, m, H-30, H-40 and H-50), 8.03 (2H, dd, J = 6.8, 3.0 Hz,

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H-20 and H-60), 8.64 (2H, s, NH2). 13C-NMR (75 MHz, DMSO-d6):
δ
18.8, 126.6 (2C), 128.8 (2C), 129.1,
130.6, 157.5, 158.8, 199.5. Combustion elemental analysis calculated for C12H13N5O2S: C, 49.47; H, 4.50;
N, 24.04. Found: C, 49.20; H, 4.67; N, 23.88.
Synthesis of N-carbethoxy-N0-(3-phenyl-1H-1,2,4-triazol-5-yl)thiourea (17). To the solution of
3-phenyl-1,2,4-triazol-5-amine (12, 0.48 g, 3 mmol) in anhydrous DMF (4 mL), ethoxycarbonyl
isothiocyanate (0.43 mL, 3.3 mmol) was added. After stirring the mixture for 5 h at room temperature,
cold water (60 mL) was added. The precipitated product was filtered, washed with cold water and
recrystalized from EtOH. Yield 0.74 g, 85%; mp 180–181 ◦C (EtOH). 1H-NMR (300 MHz, DMSO-d6):
δ
1.28 (3H, t, J = 7.1 Hz, CH3), 4.25 (2H, q, J = 7.1 Hz, CH2), 7.37–7.66 (3H, m, H-30, H-40 and H-50), 8.01
(2H, d, J = 7.2 Hz, H-20 and H-60), 11.56* (12H, brs, 2NH), 11.87 (1H, brs, NH), 12.16 (1H, brs, NH), 13.93
(1H, brs, N(1)H), 14.47 (1H, brs, N(1)H)*. *—signals of the minor tautomer. Combustion elemental
analysis calculated for C12H13N5O2S: C, 49.47; H, 4.50; N, 24.04. Found: C, 49.33; H, 4.62; N, 23.95.
4.2.9. Synthesis of 7-Methylthio-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one 18
5-Amino-1-carbethoxythiocarbamoyl-1,2,4-triazole (16, 2.9 g, 10 mmol) was heated under reflux
in aqueous EtOH (80%, 20 mL) for 20 min and then cooled. The creamy product was filtered, dissolved
in 5 mL of water and then stirred with iodomethane (0.62 mL, 10 mmol) for 30 min at 10 ◦C with a slow
warming to room temperature. The colorless precipitate formed was filtered and dissolved in water
(30 mL). A solution of HCl (1 N) was then added drop-wise to pH 3. The colorless precipitate formed
was filtered, washed with water and dried. Yield 1.68 g, 65%; mp 275–276 ◦C. 1H-NMR (300 MHz,
DMSO-d6):
δ
2.62 (3H, s, SMe), 7.49–7.59 (3H, m, H-30, H-40 and H-50), 8.00–8.11 (2H, m, H-20 and
12.8, 126.7 (2C), 128.9 (2C), 130.0, 130.8,
H-60), 13.07 (1H, s, N(4)H). 13C-NMR (75 MHz, DMSO-d6):
δ
151.2, 152.1 (2C), 162.2, 162.6. Combustion elemental analysis calculated for C11H9N5OS: C, 50.96;
H, 3.50; N, 27.01. Found: C, 50.77; H, 3.62; N, 26.90.
4.2.10. Synthesis of 7-Amino-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one 6a from 18
7-Methylthio-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (18, 0.52 g, 2 mmol) was added to a
solution of aqueous NH3 (28%, 20 mL), and the mixture was stirred on a water bath at 55 ◦C for 18 h.
The colorless precipitate formed was filtered and recrystalized from MeOCH2CH2OH. Yield 238 mg,
52%. Analytical data obtained were identical to those of compound 6a obtained from the method
described under Section 4.2.3.
4.2.11. Synthesis of 7-Amino-substituted-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-ones 6b and 6c
7-Methylthio-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (18, 0.52 g, 2 mmol) was added to
an aqueous solution of methylamine (40%) or dimethylamine (40%) (10 mL) and the mixture was
stirred at room temperature for 12 h. Solvent was evaporated under reduced pressure and recrystalized
from a suitable solvent.
7-Methylamino-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazine-5-one (6b). Colorless crystalline powder; yield
474 mg, 98%; mp 298–299 ◦C (EtOH); 1H-NMR (300 MHz, DMSO-d6):
δ
2.94 (3H, d, J = 3.8 Hz, NHMe),
7.48–7.58 (3H, m, H-30, H-40 and H-50), 8.02–8.11 (2H, m, H-20 and H-60), 8.71 (1H, q, J = 3.8 Hz, NH),
12.18 (1H, s, N(4)H). 13C-NMR (75 MHz, DMSO-d6):
27.3, 126.5 (2C), 128.8 (2C), 129.6, 130.4, 148.3,
δ
152.8, 153.4 (brs), 161.3. Combustion elemental analysis calculated for C11H10N6O: C, 54.54; H, 4.16; N,
34.69. Found: C, 54.46; H, 4.21; N, 34.60.
7-Dimethylamino-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazine-5-one (6c). Colorless crystalline powder;
yield 500 mg, 98%; mp > 300 ◦C (MeOCH2CH2OH); 1H-NMR (300 MHz, DMSO-d6):
δ 3.47 (6H, brs,
NMe2), 7.49–7.57 (3H, m, H-30, H-40 and H-50), 8.00–8.10 (2H, m, H-20 and H-60), 12.24 (1H, s, N(4)H).
Combustion elemental analysis calculated for C12H12N6O: C, 56.24; H, 4.72; N, 32.79. Found: C, 56.11;
H, 4.89; N, 32.62.

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4.2.12. Synthesis of 7-Amino-substituted-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-ones 6d–6p.
7-Methylthio-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (18, 0.52 g, 2 mmol) was added to a
solution of appropriate amine (3 mmol) in DMF (5 mL), and the mixture was heated at 70–80 ◦C with
stirring for 4–14 h. After cooling, ice cold water (40 mL) was added and the product was filtered and
recrystalized from a suitable solvent.
7-Cyclohexylamino-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (6d). Colorless crystalline powder;
yield 245 mg, 36%; mp > 300 ◦C (EtOH). 1H-NMR (300 MHz, DMSO-d6):
δ 1.03–1.21 (1H, m, Hc-Hex),
1.23-1.42 (2H, m, Hc-Hex), 1.44–1.69 (3H, m, Hc-Hex), 1.71–1.92 (4H, m, Hc-Hex), 3.85–4.01 (1H, m, H-100),
7.47–7.59 (3H, m, H-30, H-40 and H-50), 8.02–8.16 (2H, m, H-20 and H-60), 8.56 (1H, d, J = 8.3 Hz, NH),
12.17 (1H, s, N(4)H). 13C-NMR (75 MHz, DMSO-d6):
δ 24.85 (2C), 24.89, 31.5 (2C), 50.0, 126.6 (2C),
128.7 (2C), 129.6, 130.4, 147.1, 152.9, 153.5 (2C), 161.3. Combustion elemental analysis calculated for
C16H18N6O: C, 61.92; H, 5.85; N, 27.08. Found: C, 61.86; H, 6.02; N, 26.89.
2-Phenyl-7-pyrrolidino-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (6e). Colorless crystalline powder; yield
508 mg, 90%; mp > 300 ◦C (DMSO); 1H-NMR (300 MHz, DMSO-d6):
δ 1.82–2.08 (4H, m,
C(300)H2C(400)H2), 3.64 (1H, t, J = 6.0 Hz, C(500)H2), 4.25 (1H, t, J = 6.0 Hz, C(200)H2), 7.48–7.57
(3H, m, H-30, H-40 and H-50), 8.00–8.09 (2H, m, H-20 and H-60), 12.15 (1H, s, N(4)H). Combustion
elemental analysis calculated for C14H14N6O: C, 59.56; H, 5.00; N, 29.77. Found: C, 59.43; H, 5.24;
N, 29.51.
7-Piperidino-2-phenyl-1,◦2,4-triazolo[1,5-a][1,3,5]triazin-5-one (6f). Colorless crystalline powder; yield
1
492 mg, 83%; mp 286 C (MeOCH2CH2OH). H-NMR (300 MHz, DMSO-d6):
δ
1.57–1.78 (6H, m,
C(300)H2, C(400)H2, C(500)H2, 3.80–4.50 (4H, m, C(200)H2, C(600)H2, 7.47–7.58 (3H, m, H-30, H-40 and
H-50), 7.98–8.10 (2H, m, H-20 and H-60), 12.29 (1H, s, N(4)H). 13C-NMR (75 MHz, DMSO-d6):
δ
23.7,
25.5, 47.9, 126.5 (2C), 128.8 (2C), 129.3, 130.5, 147.4, 152.8, 154.9 (2C), 160.4. Combustion elemental
analysis calculated for C15H16N6O: C, 60.80; H, 5.44; N, 28.36. Found: C, 60.68; H, 5.63; N, 28.21.
7-Morpholino-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (6g). Colorless crystalline powder; yield
573 mg, 96%; mp > 300 ◦C (MeOCH2CH2OH); 1H-NMR (300 MHz, DMSO-d6):
δ
3.77 (4H, t, J = 4.5 Hz,
CH2OCH2), 4.23 (4H, brs, CH2NCH2), 7.47–7.57 (3H, m, H-30, H-40 and H-50), 8.00–8.10 (2H, m, H-20
and H-60), 12.39 (1H, s, N(4)H). 13C-NMR (75 MHz, DMSO-d6):
47.2 (brs), 65.7, 126.6 (2C), 128.8 (2C),
δ
129.1, 130.6, 147.7, 152.7, 154.9 (2C), 160.5. Combustion elemental analysis calculated for C14H14N6O2:
C, 56.37; H, 4.73; N, 28.17. Found: C, 56.22; H, 4.87; N, 28.02.
7-N-methylpiperazino-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (6h). Colorless crystalline powder;
yield 510 mg, 82%; mp 266–268 ◦C (MeOCH2CH2OH); 1H-NMR (300 MHz, DMSO-d6):
δ
2.24 (3H, s,
NMe), 2.44–2.55 (4H, m, CH2N(Me)CH2), 4.24 (4H, brs, CH2NCH2), 7.47–7.59 (3H, m, H-30, H-40 and
H-50), 7.99–8.10 (2H, m, H-20 and H-60), 12.11 (1H, brs, N(4)H). 13C-NMR (75 MHz, DMSO-d6):
45.3,
δ
46.6 (brs), 54.1, 126.5 (2C), 128.8 (2C), 129.2, 130.6, 147.6, 152.8, 154.9 (2C), 160.5. Combustion elemental
analysis calculated for C15H17N7O: C, 57.87; H, 5.50; N, 31.49. Found: C, 57.64; H, 5.62; N, 31.35.
7-Benzylamino-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (6i). Colorless crystalline powder; yield
572 mg, 90%; mp > 300 ◦C (DMF); 1H-NMR (300 MHz, DMSO-d6):
δ 4.63 (2H, d, J = 6.0 Hz, CH2), 7.27
(1H, t, J = 7.0 Hz, H-400), 7.35 (2H, t, J = 7.3 Hz, H-300 and H-500), 7.41 (2H, d, J = 7.9 Hz, H-200 and H-600),
7.48–7.59 (3H, m, H-30, H-40 and H-50), 8.03–8.13 (2H, m, H-20 and H-60), 9.31 (1H, t, J = 6.0 Hz, NH),
12.24 (1H, s, N(4)H). 13C-NMR (75 MHz, DMSO-d6):
δ 43.4, 126.5 (2C), 127.0, 127.4 (2C), 128.2 (2C),
128.8 (2C), 129.6, 130.5, 138.0, 148.2, 152.9, 153.5 (2C), 161.4. Combustion elemental analysis calculated
for C17H14N6O: C, 64.14; H, 4.43; N, 26.40. Found: C, 63.89; H, 4.64; N, 26.19.

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7-(4-Chlorobenzylamino)-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (6j). Colorless crystalline powder;
yield 614 mg, 87%; mp > 300 ◦C (DMF); 1H-NMR (300 MHz, DMSO-d6):
δ 4.61 (2H, d, J = 5.7 Hz, CH2),
7.40 (2H, d, J = 8.7 Hz, H-300 and H-500), 7.44 (2H, d, J = 8.7 Hz, H-200 and H-600), 7.48–7.59 (3H, m, H-30,
H-40 and H-50), 8.03–8.13 (2H, m, H-20 and H-60), 9.32 (1H, t, J = 5.7 Hz, NH), 12.25 (1H, s, N(4)H).
13C-NMR (75 MHz, DMSO-d6):
δ 42.8, 126.5 (2C), 128.1 (2C), 128.8 (2C), 129.3 (2C), 129.5, 130.5, 131.6,
137.0, 148.2, 152.9, 153.4 (2C), 161.5. Combustion elemental analysis calculated for C17H13ClN6O: C,
57.88; H, 3.71; N, 23.82. Found: C, 57.71; H, 4.00; N, 23.69.
7-(4-Methoxybenzylamino)-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (6k). Colorless crystalline
powder; yield 670 mg, 96%; mp > 300 ◦C (MeOCH2CH2OH); 1H-NMR (300 MHz, DMSO-d6):
δ 3.73
(3H, s, OMe), 4.56 (2H, d, J = 6.0 Hz, CH2), 6.90 (2H, d, J = 8.7 Hz, H-300 and H-500), 7.35 (2H, d,
J = 8.7 Hz, H-200 and H-600), 7.48–7.58 (3H, m, H-30, H-40 and H-50), 8.03–8.12 (2H, m, H-20 and H-60),
9.25 (1H, t, J = 6.0 Hz, NH), 12.25 (1H, s, N(4)H). 13C-NMR (75 MHz, DMSO-d6):
δ 42.9, 55.0, 113.6 (2C),
126.5 (2C), 128.8 (2C), 128.9 (2C), 129.5, 129.9, 130.4, 148.0, 152.9, 153.5 (brs), 158.4, 161.4. Combustion
elemental analysis calculated for C18H16N6O2: C, 62.06; H, 4.63; N, 24.12. Found: C, 61.96; H, 4.88;
N, 23.93.
2-Phenyl-7-[(pyridin-4-ylmethyl)amino]-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (6l). Colorless crystalline
powder; yield 556 mg, 87%; mp 293–294 ◦C (EtOH); 1H-NMR (300 MHz, DMSO-d6):
δ 4.66 (2H, d,
J = 5.7 Hz, CH2), 7.40 (2H, d, J = 4.9 Hz, H-300 and H-500), 7.48–7.61 (3H, m, H-30, H-40 and H-50),
8.02-8.15 (2H, m, H-20 and H-60), 8.52 (2H, d, J = 4.9 Hz, H-200 and H-600), 9.36 (1H, t, J = 5.7 Hz, NH),
12.29 (1H, s, N(4)H). 13C-NMR (75 MHz, DMSO-d6):
δ 42.5, 122.1 (2C), 126.5 (2C), 128.8 (2C), 129.5,
130.5, 146.9, 148.4, 149.4 (2C), 153.0, 153.4 (2C), 161.5. Combustion elemental analysis calculated for
C16H13N7O: C, 60.18; H, 4.10; N, 30.70. Found: C, 59.97; H, 4.34; N, 30.51.
2-Phenyl-7-phenylamino-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (6m). Colorless crystalline powder; yield
◦
1
545 mg, 90%; mp 298–299 C (MeOCH2CH2OH); H-NMR (300 MHz, DMSO-d6):
δ 7.23 (1H, t,
J = 7.9 Hz, H-400), 7.43 (2H, t, J = 7.9 Hz, H-300 and H-500), 7.49–7.61 (3H, m, H-30, H-40 and H-50), 7.80
(2H, d, J = 7.9 Hz, H-200 and H-400), 8.09–8.21 (2H, m, H-20 and H-60), 10.44 (1H, s, NH), 12.45 (1H, s,
N(4)H). 13C-NMR (75 MHz, DMSO-d6):
δ 123.3 (2C), 125.2, 126.7 (2C), 128.4 (2C), 128.8 (2C), 129.5,
130.5, 136.4, 146.5, 153.1, 153.3 (2C), 161.4. Combustion elemental analysis calculated for C16H12N6O:
C, 63.15; H, 3.97; N, 27.62. Found: C, 62.99; H, 4.24; N, 27.41.
7-(4-Chlorophenylamino)-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (6n). Colorless crystalline
◦
1
powder; yield 562 mg, 83%; mp > 300 C (MeOCH2CH2OH); H-NMR (300 MHz, DMSO-d6):
7.48 (2H, d, J = 8.7 Hz, H-300 and H-500), 7.52–7.61 (3H, m, H-30, H-40 and H-50), 7.87 (2H, d, J = 8.7 Hz,
H-200 and H-400), 8.08–8.20 (2H, m, H-20 and H-60), 10.55 (1H, s, NH), 12.49 (1H, s, N(4)H). 13C-NMR
(75 MHz, DMSO-d6): 124.7 (2C), 126.7 (2C) 128.4 (2C), 128.8 (2C), 129.0, 129.5, 130.6, 135.5, 146.4, 153.1
δ
δ
(2C), 161.5. Combustion elemental analysis calculated for C16H11ClN6O: C, 56.73; H, 3.27; N, 24.81.
Found: C, 56.62; H, 3.36; N, 24.70.
7-(4-Methoxyphenylamino)-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (6o). Colorless crystalline
powder; yield 650 mg, 96%; mp 296–297 ◦C (MeOCH2CH2OH); 1H-NMR (300 MHz, DMSO-d6):
δ
3.78 (3H, s, OMe), 6.99 (2H, d, J = 8.7 Hz, H-300 and H-500), 7.49–7.60 (3H, m, H-30, H-40 and H-50), 7.65
(2H, d, J = 8.7 Hz, H-200 and H-400), 8.08–8.19 (2H, m, H-20 and H-60), 10.35 (1H, s, NH), 12.37 (1H, s,
N(4)H). 13C-NMR (75 MHz, DMSO-d6):
δ 55.2, 113.6 (2C), 125.0 (2C), 126.7 (2C), 128.8 (2C), 129.1, 129.5,
130.6, 146.6, 153.1, 153.3 (2C), 156.8, 161.4. Combustion elemental analysis calculated for C17H14N6O2:
C, 61.07; H, 4.22; N, 25.14. Found: C, 60.87; H, 4.49; N, 24.98.
7-Indolino-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one (6p). Colorless crystalline powder; yield
588 mg, 89%; mp 297 ◦C (MeOCH2CH2OH); 1H-NMR (300 MHz, DMSO-d6):
δ 3.27 (2H, t, J = 8.3 Hz,

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C(300)H2), 4.91 (2H, d, J = 8.3 Hz, C(200)H2), 7.13 (1H, t, J = 7.3 Hz, H-500), 7.27 (1H, t, J = 7.7 Hz, H-600),
7.34 (1H, d, J = 7.2 Hz, H-500), 7.44–7.60 (3H, m, H-30, H-40 and H-50), 8.00–8.15 (2H, m, H-20 and
H-60), 8.40 (1H, d, J = 7.9 Hz, H-700), 12.48 (1H, s, N(4)H). 13C-NMR (75 MHz, DMSO-d6):
δ 27.9, 52.3,
118.9, 124.7, 124.8, 126.5 (2C), 126.7, 128.8 (2C), 129.3, 130.5, 133.1, 141.7, 146.1, 153.0, 154.6 (2C), 160.5.
Combustion elemental analysis calculated for C18H14N6O: C, 65.44; H, 4.27; N, 25.44. Found: C, 65.23;
H, 4.54; N, 25.19.
Author Contributions: Conceptualization, A.V.D.; Methodology, F.P.L.L. and A.V.D.; Investigation, A.J., Y.P.Z.
and A.V.D.; Resources, W.K.C. and A.V.D.; Data Curation, F.P.L.L., Y.P.Z. and A.V.D.; Writing—Original Draft
Preparation, A.J. and A.V.D.; Writing—Review and Editing, F.P.L.L., and A.V.D.; Supervision, W.K.C. and A.V.D.;
Project Administration, W.K.C. and A.V.D.
Funding: This research was funded by the Ministry of Higher Education, Malaysia under Fundamental Research
Grant Scheme, grant number FRGS/1/2015/SG01/MUSM/03/1.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of compounds 6a–p are available from the authors.
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