Synthesis and biological activity of fluorinated 7-aryl-2-pyridyl-6,7-dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amines [1]

Article abstract of DOI:10.1016/j.jfluchem.2008.02.007

In our lead finding program, 12 new fluorinated 7-aryl-2-pyridyl-6,7-dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amines were prepared via a practical three-step procedure starting from (iso)nicotinic hydrazides. The fluorine substituted aryl fragment was introduced in the last step through cyclocondensation of N-(3-pyridyl-1,2,4-triazol-5-yl)guanidines and fluoro/trifluoromethyl substituted benzaldehydes. The structures of the compounds were confirmed by ¹H and ¹³C NMR spectral data. The tautomeric preferences for the compounds were established using 2D NOESY experiments. The antiproliferative activity of the synthesized 1,2,4-triazolo[1,5-a][1,3,5]triazines was evaluated against breast, colon and lung cancer cell lines. The highest antiproliferative activity in the series was found for 2-(pyridine-3-yl)-7-(4-trifluoromethylphenyl)-6,7-dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amine. The lack of inhibitory activity against bovine dihydrofolate reductase (DHFR) indicated that the antiproliferative activity was realized via other mechanisms.

Full text of DOI:10.1016/j.jfluchem.2008.02.007

Journal of Fluorine Chemistry 129 (2008) 429–434
Contents lists available at ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
Synthesis and biological activity of fluorinated 7-aryl-2-pyridyl-
6,7-dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amines [1]§
a
a,b
Anton V. Dolzhenko a, , Bee Jen Tan , Anna V. Dolzhenko
,
*
Gigi Ngar Chee Chiu a, Wai Keung Chui a
a Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore
b Perm State Pharmaceutical Academy, 48 Lenin Street, Perm 614990, Russian Federation
A R T I C L E I N F O
A B S T R A C T
Article history:
In our lead finding program, 12 new fluorinated 7-aryl-2-pyridyl-6,7-dihydro[1,2,4]triazolo[1,5-
a][1,3,5]triazin-5-amines were prepared via a practical three-step procedure starting from (iso)nicotinic
hydrazides. The fluorine substituted aryl fragment was introduced in the last step through
cyclocondensation of N-(3-pyridyl-1,2,4-triazol-5-yl)guanidines and fluoro/trifluoromethyl substituted
benzaldehydes. The structures of the compounds were confirmed by 1H and 13C NMR spectral data. The
tautomeric preferences for the compounds were established using 2D NOESY experiments. The
antiproliferative activity of the synthesized 1,2,4-triazolo[1,5-a][1,3,5]triazines was evaluated against
breast, colon and lung cancer cell lines. The highest antiproliferative activity in the series was found for
2-(pyridine-3-yl)-7-(4-trifluoromethylphenyl)-6,7-dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amine.
The lack of inhibitory activity against bovine dihydrofolate reductase (DHFR) indicated that the
antiproliferative activity was realized via other mechanisms.
Received 7 February 2008
Received in revised form 26 February 2008
Accepted 26 February 2008
Available online 4 March 2008
Keywords:
Hydrazides
1,3,5-Triazines
1,2,4-Triazoles
Guanidines
5-Azapurines
Tautomerism
Anticancer activity
ß 2008 Elsevier B.V. All rights reserved.
1. Introduction
N-(3-phenyl-1,2,4-triazol-5-yl)guanidine using a number of
aldehydes and ketones. In continuation of our lead finding
program in the area of fused 1,3,5-triazines, particularly fluorine
The unique character of fluorinated molecules is well known
in medicinal chemistry [2–8]. Fluorine substitution has been
extensively practiced in the design of new biologically active
compounds, including those with potential anticancer properties
substituted derivatives [13], we applied herein
a similar
approach for the preparation of new fluorinated 7-aryl-2-
pyridyl-6,7-dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amines
(5). We also aimed to investigate antiproliferative effect of the
compounds towards three cancer cell lines. The monocyclic
dihydro-1,3,5-triazines (e.g. cycloguanil) and some fused dihy-
dro-1,3,5-triazines [14,15] were reported to inhibit the enzyme
DHFR, which is a well recognized target in anticancer therapy
[16]. Therefore, the potential DHFR inhibitory activity of 5 as one
of the possible mechanism of the anticancer effect was evaluated
in the present study [1].
We designed the synthesis of 1,2,4-triazolo[1,5-a][1,3,5]tria-
zines 5 starting from hydrazides of nicotinic and isonicotinic acids
(1) which comprised three steps and involved subsequent
formation of the 1,2,4-triazole and the 1,3,5-triazine rings (Scheme
1). In order to generate a library of fluorinated 1,2,4-triazolo[1,5-
a][1,3,5]triazine derivatives for biological screening, fluorine/
trifluoromethyl substituted benzaldehydes were used in the step
of 1,3,5-triazine ring closure.
[8].
A variety of attractive pharmacological effects were
attributed to the fluorinated mono- and polycyclic 1,3,5-
triazines [9]. A bicyclic system comprising 1,3,5-triazine and
1,2,4-triazole rings, namely 1,2,4-triazolo[1,5-a][1,3,5]triazine
nucleus, which is 5-aza-bioisoster of purine, has been shown
to be
a favorable scaffold for the design of biologically
active compounds [10]. However, data concerning anticancer
properties of these compounds are limited [11]. Recently, we
have developed a new method [12] for the synthesis of 5-
azapurine derivatives, viz. 2-phenyl-6,7-dihydro[1,2,4]tria-
zolo[1,5-a][1,3,5]triazin-5-amines via ring closure reaction of
§
Part 10 in the series ‘‘Fused heterocyclic systems with s-triazine ring’’.
* Corresponding author. Tel.: +65 6516 2657; fax: +65 6779 1554.
E-mail address: phada@nus.edu.sg (A.V. Dolzhenko).
0022-1139/$ – see front matter ß 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jfluchem.2008.02.007

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A.V. Dolzhenko et al. / Journal of Fluorine Chemistry 129 (2008) 429–434
Scheme 1. Synthesis of fluorinated 7-aryl-2-pyridyl-6,7-dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amines (5a–l).
2. Results and discussion
and proton of endocyclic NH in 1H NMR spectra was observed for
several compounds (J = 0–1.1 Hz). Despite J value was small and
not always detectable, cross-peaks between these signals in 2D
NOESY experiments clearly indicated that the NH proton was
located in the vicinity of sp3-hybridized carbon.
2.1. Chemistry
The reaction of cyanoguanidine with hydrazides of nicotinic
and isonicotinic acids (1) in the presence of hydrochloric acid
provided N-(iso)nicotinamidobiguanides (2) (Scheme 1). The
biguanides 2 were further cyclized under alkaline conditions to
afford N-(3-pyridyl-1,2,4-triazol-5-yl)guanidines (3).
Heating of guanidines 3 with fluorinated benzaldehydes in
ethanol in the presence of piperidine gave 7-aryl-2-pyridyl-6,7-
dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amines (5) (Scheme
1). The characteristic signal at 64.2–64.8 ppm for the compounds
with ortho-substituted phenyl group and 67.6–67.8 ppm for the
compounds with meta- and para-substituted phenyl group was
found in 13C NMR spectra. This indication of the presence of
sp3-hybridized carbon atom in the structure supported the triazine
ring closure and ruled out the structure of conceivable Schiff
base-like intermediates 4. The heterocyclization was found to
proceed regioselectively resulting in the formation of fused
triazolotriazine system 5 with the [1,5-a] ring junction, exclu-
sively. The theoretically possible regioisomeric 1,2,4-triazolo[4,3-
a][1,3,5]triazines 6 were not isolated from the reaction mixture.
The 2D NOESY experiments were used to differentiate between
triazolotriazine structures 5 and 6. The pyridine ring protons gave
no cross-peaks neither with proton at sp3-hybridized carbon nor
with protons of fluorinated aryl group. The results are in
agreement with those obtained in our previous studies [12] for
structurally related compounds.
The annular tautomerism, possible for 5, might result in the
existence of four different forms, viz. 6,7- (A), 1,7- (B), 4,7- (C), and
3,7-dihydro- (D) tautomers (Scheme 2). However, form A was
found to be predominant in Me2SO solution as indicated by the
NMR data. The coupling of proton at sp3-hybridized carbon (H-7)
Scheme 2. Tautomeric forms of fluorinated 7-aryl-2-pyridyl-dihydro[1,2,
4]triazolo[1,5-a][1,3,5]triazin-5-amines (5a–l).

A.V. Dolzhenko et al. / Journal of Fluorine Chemistry 129 (2008) 429–434
431
Table 1
Antiproliferative activity of the fluorinated 7-aryl-2-pyridyl-6,7-dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amines (5a–l)
Compound
Py
RF
Concentration (mM)
Cell growth inhibition (%)*
MDA-MB-231
HT29
A549
5a
3-Py
2-F
62.5
125
62.5
62.5
125
62.5
125
62.5
125
62.5
125
62.5
125
62.5
125
62.5
125
50
12 ꢀ 5.9
17 ꢀ 3.8
21 ꢀ 6.3
17 ꢀ 9.4
29 ꢀ 9.3
28 ꢀ 9.7
13 ꢀ 9.3
29 ꢀ 7.0
13 ꢀ 6.7
26 ꢀ 9.6
12 ꢀ 7.2
5b
5c
3-Py
3-Py
3-F
4-F
8
ꢀ 7.9
2
6
1
6
ꢀ 5.5
ꢀ 6.7
ꢀ 9.6
ꢀ 6.7
18 ꢀ 6.1
ꢀ 7.2
5d
5e
5f
3-Py
3-Py
3-Py
4-Py
4-Py
4-Py
2-CF3
3-CF3
4-CF3
2-F
3
1
7
ꢀ 13.0
ꢀ 10.0
12 ꢀ 6.5
17 ꢀ 8.0
41 ꢀ 7.7
65 ꢀ 12.5
75 ꢀ 6.4
29 ꢀ 6.1
51 ꢀ 5.1
40 ꢀ 16.1
48 ꢀ 17.2
16 ꢀ 6.4
25 ꢀ 7.4
25 ꢀ 16.1
54 ꢀ 16.9
13 ꢀ 6.4
34 ꢀ 5.2
14 ꢀ 6.9
14 ꢀ 11.7
42 ꢀ 15.5
20 ꢀ 17.1
29 ꢀ 11.1
12 ꢀ 8.3
20 ꢀ 5.5
32 ꢀ 11.1
41 ꢀ 9.3
ꢁ3 ꢀ 13.7
ꢁ1 ꢀ 10.2
5g
5h
5i
3
ꢀ 10.4
11 ꢀ 9.8
19 ꢀ 18.8
44 ꢀ 12.7
21 ꢀ 5.1
30 ꢀ 6.2
22 ꢀ 6.4
3-F
7
ꢀ 16.4
30 ꢀ 12.5
ꢁ2 ꢀ 5.5
10 ꢀ 6.3
14 ꢀ 6.5
4-F
5j
4-Py
4-Py
2-CF3
3-CF3
5k
62.5
125
62.5
125
2
ꢀ 6.7
0
ꢀ 9.0
19 ꢀ 5.1
30 ꢀ 12.5
44 ꢀ 13.3
15 ꢀ 8.8
27 ꢀ 15.1
44 ꢀ 10.2
5l
4-Py
4-CF3
*
ꢀ, S.D., n = 5–6.
2.2. Biological activity
However, the most active compound, namely 2-(pyridine-3-yl)-
7-(4-trifluoromethylphenyl)-6,7-dihydro[1,2,4]triazolo[1,5-a][1,
3,5]triazin-5-amine (5f) was more effective against breast cancer
sell line MDA-MB-231 (IC50 = 28 mM). The position of fluoro/
trifluoromethyl group seemed to play more important role in the
anticancer effect of the compounds than position of nitrogen in
the pyridine ring. Thus, the active compounds were almost
equally distributed within 3- and 4-pyridyl substituted groups
(5a–f and 5g–l). At the same time, the pairs of the compounds with
pronounced anticancer effect were identified, those included 5f
and 5l (R = 4-CF3) as well as 5b and 5h (R = 3-F). The pairs showed
similar activity and selectivity towards the cell lines used for the
evaluation.
The antiproliferative and DHFR inhibitory activities were
evaluated for the prepared fluorinated 7-aryl-2-pyridyl-6,7-
dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amines (5a–l). For
both the antiproliferative and DHFR inhibition bioassays the
compounds were dissolved in Me2SO. In order to ensure that the
solvent per se had no effect on the cell growth and enzymatic
activity, negative control tests were performed using Me2SO at the
same concentrations. The antiproliferative activity was performed
using MTT assay [17]. The following cancer cell lines were used for
the screening: breast cancer MDA-MB-231, colon cancer HT-29
and lung cancer A549. The DHFR inhibition bioassay was carried
out using bovine DHFR (Fluka Chemie) according to the previously
described method [13]. The concentrations of the compounds for
the biological tests were varied according to their solubility in the
media. The effect of the compounds was expressed as percentage
of cell growth inhibition (antiproliferative assay) and percentage of
the enzymatic activity inhibition (DHFR assay). IC50 value was
determined for the most active compound.
None of 1,2,4-triazolo[1,5-a][1,3,5]triazines 5 demonstrated
significant inhibition of the bovine DHFR activity in the screening
(Table 2). Therefore, the anticancer effect of the compounds
appeared to be realized via mechanisms other than DHFR inhibition.
3. Conclusions
Twelve new fluorinated 7-aryl-2-pyridyl-6,7-dihydro[1,2,4]tria-
zolo[1,5-a][1,3,5]triazin-5-amines (5) were prepared using simple
and effective three-step procedure. The biological evaluation
showed that compound 5 possessed the anticancer properties not
associated with DHFR inhibition. The most active anticancer agents
identified in this study was 2-(pyridine-3-yl)-7-(4-trifluoromethyl-
phenyl)-6,7-dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amine
(5f). Therefore, it may be used as a lead for further development.
The results of the antiproliferative tests of compounds 5 are
shown in Table 1. In general, the lung cancer cells were more
resistant to the treatment with 1,2,4-triazolo[1,5-a][1,3,5]tria-
zines 5, while the colon cancer cells were more susceptible.
Table 2
Bovine DHFR inhibitory activity of the fluorinated 7-aryl-2-pyridyl-6,7-dihy-
dro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amines (5a–l)
4. Experimental
Compound
Py
RF
Concentration (mM)
Inhibition (%)
5a
5b
5c
5d
5e
5f
3-Py
3-Py
3-Py
3-Py
3-Py
3-Py
4-Py
4-Py
4-Py
4-Py
4-Py
4-Py
2-F
500
500
500
500
250
100
500
500
500
250
500
100
14
12
na
32
na
na
11
20
9
Melting points (uncorrected) were determined on a Gallen-
kamp melting point apparatus. Analytical TLC were carried out on
aluminum plates coated with Silica gel 60 F254 (Merck) with
detection by UV light. 1H and 13C NMR spectra were recorded on a
Bruker DPX-300 spectrometer, using Me2SO-d6 as a solvent and
TMS as an internal reference.
3-F
4-F
2-CF3
3-CF3
4-CF3
2-F
5g
5h
5i
3-F
4-F
4.1. N-Nicotinamidobiguanide hydrochloride (2a)
5j
2-CF3
3-CF3
4-CF3
22
7
5k
5l
na
To the solution of nicotinic hydrazide (1a, 6.80 g, 50 mmol) in
ethanol (50 mL), hydrochloric acid (37%, 5 ml, 50 mmol) and
na, inactive.

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A.V. Dolzhenko et al. / Journal of Fluorine Chemistry 129 (2008) 429–434
cyanoguanidine (4.62 g, 55 mmol) were added. The reaction
mixture was heated under reflux with stirring for 8 h. After
cooling, the product was filtered, washed with cold ethanol and
dried. Yield 80%; mp 176–177 8C (aq. EtOH) [lit. [18] mp 178–
189 8C]. 1H NMR (300 MHz, DMSO-d6): d 7.58 (7H, br s, NH–
C( NH)NH–C( NH)NH2 HCl), 7.74 (1H, dd, 3J = 7.5 Hz, 3J = 4.9 Hz,
H-5), 8.52 (1H, d, 3J = 7.5 Hz, H-4), 8.86 (1H, d, 3J = 4.9 Hz, H-6), 9.21
(1H, s, H-2), 11.16 (1H, s, CONH). 13C NMR (75 MHz, DMSO-d6): d
124.3 (C-5), 128.8 (C-3), 138.0 (C-4), 146.8 (C-6), 150.0 (C-2), 157.3,
158.5 (NH–C( NH)NH–C( NH)NH2), 163.6 (C O).
H-7), 7.22–7.34 (3H, m, H-300, H-500 and H-600), 7.42 (1H, dd,
3
3J = 7.7 Hz, J = 4.3 Hz, H-50), 7.44–7.52 (1H, m, H-400), 8.03 (1H, s,
4
NH), 8.17 (1H, dt, 3J = 7.9 Hz, J = 1.9 Hz, H-40), 8.55 (1H, dd,
3J = 4.7 Hz, 4J = 1.7 Hz, H-60), 9.02 (1H, d, 4J = 1.5 Hz, H-20). 13C NMR
3
2
(75 MHz, Me2SO-d6): d 64.2 (d, JC–F = 3.5 Hz, C-7), 116.0 (d, JC–
F = 20.6 Hz, C-300), 123.4 (C-50), 124.8 (d, 4JC–F = 3.5 Hz, C-500), 127.0
(d, 2JC–F = 11.8 Hz, C-100), 127.4 (C-30), 128.4 (d, 3JC–F = 3.5 Hz, C-600),
3
131.4 (d, JC–F = 8.2 Hz, C-400), 132.6 (C-40), 146.5 (C-60), 149.5 (C-
1
20), 155.6, 156.7, 157.3 (C-2, C-3a and C-5), 159.7 (d, JC–
F = 248.1 Hz, C-200). Anal. Calcd. for C15H12FN7: C, 58.25; H, 3.91;
N, 31.70. Found: C, 57.96; H, 4.11; N, 31.58.
4.2. N-Isonicotinamidobiguanide dihydrochloride (2b)
4.4.2. 7-(3-Fluorophenyl)-2-(pyridin-3-yl)-6,7-
To the solution of isonicotinic hydrazide (1b, 6.80 g, 50 mmol)
in methanol (70 mL), hydrochloric acid (37%, 10 mL, 100 mmol)
and cyanoguanidine (4.62 g, 55 mmol) were added. The reaction
mixture was heated under reflux with stirring for 8 h. After cooling,
the product was filtered, washed with cold ethanol and dried. Yield
82%, mp 198 8C (aq EtOH) [lit. [19] mp 200 8C].
dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amine (5b)
Yield 87%; mp 288 8C (EtOH–DMF); TLC (silica gel, EtOH): Rf
0.43. 1H NMR (300 MHz, Me2SO-d6): d 6.60 (2H, s, NH2), 6.81 (1H, s,
H-7), 7.16–7.31 (3H, m, H-200, H-400 and H-600), 7.44 (1H, dd,
3J = 7.9 Hz, 3J = 4.9 Hz, H-50), 7.51 (1H, td, 3J = 7.6 Hz, 4JH–F = 6.4 Hz,
4
H-500), 8.12 (1H, s, NH), 8.20 (1H, dt, 3J = 7.9 Hz, J = 1.9 Hz, H-40),
1H NMR (300 MHz, DMSO-d6): d 7.59 (8H, br s, NH–C( NH)NH–
C( NH)NH2 2HCl), 8.04 (2H, d, 3J = 5.9 Hz, 4J = 1.5 Hz, H-3 and H-
5), 8.85 (2H, dd, 3J = 5.9 Hz, 4J = 1.5 Hz, H-2 and H-6), 11.18 (1H, s,
CONH). 13C NMR (75 MHz, DMSO-d6): d 122.6 (C-3 and C-5), 141.4
(C-4), 148.0 (C-2 and C-6), 156.7, 158.4 (NH–C( NH)NH–
C( NH)NH2), 163.3 (C O).
8.57 (1H, dd, 3J = 4.5 Hz, J = 1.5 Hz, H-60), 9.06 (1H, d, 4J = 1.5 Hz,
4
H-20). 13C NMR (75 MHz, Me2SO-d6): d 67.6 (d, 4JC–F = 1.8 Hz, C-7),
2
2
113.1 (d, JC–F = 21.8 Hz, C-200), 116.0 (d, JC–F = 21.2 Hz, C-400),
4
122.3 (d, JC–F = 2.9 Hz, C-600), 123.6 (C-50), 127.4 (C-30), 131.1 (d,
3JC–F = 8.2 Hz, C-500), 132.7 (C-40), 142.9 (d, JC–F = 6.5 Hz, C-100),
3
146.6 (C-60), 149.5 (C-20), 155.6, 156.6, 157.4 (C-2, C-3a and C-5),
1
162.1 (d, JC–F = 245.2 Hz, C-300). Anal. Calcd. for C15H12FN7: C,
4.3. General method for the syntheses of N-(3-pyridyl-1H-1,2,4-
triazol-5-yl)guanidine (3a,b)
58.25; H, 3.91; N, 31.70. Found: C, 57.92; H, 4.07; N, 31.62.
4.4.3. 7-(4-Fluorophenyl)-2-(pyridin-3-yl)-6,7-
dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amine (5c)
Yield 91%; mp 281 8C (EtOH–DMF); TLC (silica gel, EtOH): Rf
0.40. 1H NMR (300 MHz, Me2SO-d6): d 6.55 (2H, s, NH2), 6.79 (1H, d,
N-(Iso)nicotinamidobiguanide (di)hydrochloride (2, 20 mmol)
was heated at 80 8C in 10% aqueous sodium hydroxide solution
(10 mL) for 4–6 h. After cooling, the product was filtered, washed
with cold water and dried.
3
3J = 1.1 Hz, H-7), 7.28 (2H, dd, 3J = 8.7 Hz, JH–F = 9.0 Hz, H-300 and
H-500), 7.39–7.48 (3H, m, H-50, H-200 and H-600), 8.03 (1H, d,
3J = 1.1 Hz, NH), 8.18 (1H, dt, 3J = 7.9 Hz, 4J = 1.9 Hz, H-40), 8.56 (1H,
4.3.1. N-[3-(Pyridin-3-yl)-1H-1,2,4-triazol-5-yl]guanidine (3a)
Yield 86%, mp 269 8C (EtOH) [lit. [18] mp 275–276 8C]; TLC
(silica gel, EtOH): Rf 0.15. 1H NMR (300 MHz, DMSO-d6): d 6.70 (4H,
br s, NH–C( NH)NH2), 7.45 (1H, dd, 3J = 7.9 Hz, 3J = 4.5 Hz, H-5),
8.28 (1H, dt, 3J = 7.9 Hz, 4J = 1.7 Hz, H-4), 8.56 (1H, dd, 3J = 4.5 Hz,
4J = 1.5 Hz, H-6), 9.16 (1H, d, 4J = 1.9 Hz, H-2), 12.58 (1H, br s, NH).
13C NMR (75 MHz, DMSO-d6): d 123.5 (C-50), 127.9 (C-30), 132.4 (C-
40), 146.5 (C-60), 149.0 (C-20), 154.9 (C-3), 157.9 (NH–C( NH)NH2),
160.8 (C-5).
4
4
dd, 3J = 4.9 Hz, J = 1.5 Hz, H-60), 9.05 (1H, d, J = 1.9 Hz, H-20). 13C
NMR (75 MHz, Me2SO-d6): d 67.7 (C-7), 115.7 (d, 2JC–F = 21.8 Hz, C-
300 and C-500), 123.6 (C-50), 127.4 (C-30), 128.6 (d, 3JC–F = 8.2 Hz, C-200
and C-600), 132.6 (C-40), 136.5 (d, 4JC–F = 2.9 Hz, C-100), 146.6 (C-60),
149.5 (C-20), 155.6, 156.6, 157.3 (C-2, C-3a and C-5), 162.4 (d, 1JC–
F = 245.2 Hz, C-400). Anal. Calcd. for C15H12FN7: C, 58.25; H, 3.91; N,
31.70. Found: C, 58.03; H, 3.97; N, 31.62.
4.4.4. 2-(Pyridin-3-yl)-7-(2-trifluoromethylphenyl)-6,7-
dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amine (5d)
4.3.2. N-[3-(Pyridin-4-yl)-1H-1,2,4-triazol-5-yl]guanidine (3b)
Yield 92%, mp 289–290 8C (EtOH) [lit. [18] mp 292 8C]; TLC
(silica gel, EtOH): Rf 0.20. 1H NMR (300 MHz, DMSO-d6): d 6.73 (4H,
Yield 87%; mp 285 8C (EtOH–DMF); TLC (silica gel, EtOH): Rf
0.49. 1H NMR (300 MHz, Me2SO-d6): d 6.50 (2H, s, NH2), 6.99 (1H, s,
4
br s, NH–C( NH)NH2), 7.88 (2H, dd, 3J = 4.5 Hz, J = 1.5 Hz, H-30
3
H-7), 7.34 (1H, d, J = 7.9 Hz, H-600), 7.41 (1H, dd, 3J = 7.9 Hz,
and H-50), 8.62 (2H, dd, 3J = 4.5 Hz, 4J = 1.5 Hz, H-20 and H-60), 12.70
(1H, br s, NH). 13C NMR (75 MHz, DMSO-d6): d 119.5 (C-30 and C-50),
139.2 (C-10), 149.9 (C-20 and C-60), 155.2 (C-3), 157.9 (NH–
C( NH)NH2), 160.9 (C-5).
3
3J = 4.7 Hz, H-50), 7.64 (1H, t, J = 7.5 Hz, H-500), 7.76 (1H, t,
3
3J = 7.5 Hz, H-400), 7.85 (1H, d, J = 7.5 Hz, H-300), 8.00 (1H, s, NH),
8.16 (1H, dt, 3J = 7.9 Hz, 4J = 1.9 Hz, H-40), 8.55 (1H, dd, 3J = 4.9 Hz,
4J = 1.5 Hz, H-60), 9.02 (1H, d, 4J = 1.5 Hz, H-20). 13C NMR (75 MHz,
Me2SO-d6): d 64.7 (q, 4JC–F = 2.9 Hz, C-7), 123.6 (C-50), 127.2 (C-30),
123.9 (q, 1JC–F = 274.4 Hz, CF3), 125.8 (q, 3JC–F = 5.7 Hz, C-300), 126.0
4.4. General method for the syntheses of 7-aryl-2-pyridyl-6,7-
2
(q, JC–F = 30.6 Hz, C-200), 128.4 (C-600), 129.9 (C-400), 132.7 (C-40),
dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amine (5a–l)
3
133.8 (C-500), 138.6 (q, JC–F = 1.2 Hz, C-100), 146.6 (C-60), 149.6 (C-
20), 155.3, 157.1, 157.7 (C-2, C-3a and C-5). Anal. Calcd. for
The solution of N-(3-pyridyl-1,2,4-triazol-5-yl)guanidine (3,
0.50 g, 2.5 mmol), fluorine/trifluoromethyl substituted benzalde-
hyde (2.5 mmol) and piperidine (0.10 mL, 1.0 mmol) in ethanol (7–
10 mL) was heated under reflux for 5–18 h. After cooling, the
product was filtered, washed with cold ethanol, dried and
recrystallized from ethanol or ethanol–DMF mixture.
C16H12F3N7: C, 53.48; H, 3.37; N, 27.29. Found: C, 53.35; H, 3.56; N,
27.06.
4.4.5. 2-(Pyridin-3-yl)-7-(3-trifluoromethylphenyl)-6,7-
dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amine (5e)
Yield 86%; mp 260 8C (EtOH–DMF); TLC (silica gel, EtOH): Rf
0.45. 1H NMR (300 MHz, Me2SO-d6): d 6.63 (2H, s, NH2), 6.92 (1H, s,
H-7), 7.43 (1H, dd, 3J = 7.7 Hz, 3J = 4.7, H-50), 7.63 (1H, d, 3J = 7.5 Hz,
H-400), 7.71 (1H, t, 3J = 7.9 Hz, C-500), 7.80 (1H, s, H-200), 7.81 (1H, d,
3J = 7.9 Hz, H-600), 8.15 (1H, s, NH), 8.19 (1H, dt, 3J = 7.9 Hz,
4.4.1. 7-(2-Fluorophenyl)-2-(pyridin-3-yl)-6,7-
dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amine (5a)
Yield 85%; mp 280 8C (EtOH–DMF); TLC (silica gel, EtOH): Rf
0.45. 1H NMR (300 MHz, Me2SO-d6): d 6.48 (2H, s, NH2), 6.97 (1H, s,

A.V. Dolzhenko et al. / Journal of Fluorine Chemistry 129 (2008) 429–434
433
4
4J = 1.9 Hz, H-40), 8.57 (1H, dd, 3J = 4.7 Hz, J = 1.7 Hz, H-60), 9.06
Calcd. for C15H12FN7: C, 58.25; H, 3.91; N, 31.70. Found: C, 58.33; H,
3.95; N, 31.54.
(1H, d, 4J = 1.9 Hz, H-20). 13C NMR (75 MHz, Me2SO-d6): d 67.7 (C-7),
3
1
123.0 (q, JC–F = 3.7 Hz, H-400), 123.6 (C-50), 123.9 (q, JC–
3
F = 272.4 Hz, CF3), 126.0 (q, JC–F = 3.3 Hz, C-200), 127.4 (C-30),
4.4.10. 2-(Pyridin-4-yl)-7-(2-trifluoromethylphenyl)-6,7-
dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amine (5j)
Yield 86%; mp 278 8C (EtOH–DMF); TLC (silica gel, EtOH): Rf
0.51. 1H NMR (300 MHz, Me2SO-d6): d 6.52 (2H, s, NH2), 7.01 (1H, s,
129.6 (q, 2JC–F = 31.9 Hz, C-300), 130.2 (C-600), 130.4 (q, 4JC–F = 1.2 Hz,
H-500), 132.7 (C-40), 141.5 (C-100), 146.6 (C-60), 149.6 (C-20), 155.6,
156.7, 157.5 (C-2, C-3a and C-5). Anal. Calcd. for C16H12F3N7: C,
53.48; H, 3.37; N, 27.29. Found: C, 53.16; H, 3.55; N, 26.98.
3
3
H-7), 7.36 (1H, d, J = 7.5 Hz, H-600), 7.64 (1H, t, J = 7.5 Hz, H-500),
7.76 (2H, dd, 3J = 4.5 Hz, J = 1.5 Hz, H-30 and H-50), 7.77 (1H, t,
4
3
4.4.6. 2-(Pyridin-3-yl)-7-(4-trifluoromethylphenyl)-6,7-
dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amine (5f)
Yield 93%; mp 274 8C (EtOH); TLC (silica gel, EtOH): Rf 0.41. 1H
NMR (300 MHz, Me2SO-d6): d 6.58 (2H, s, NH2), 6.88 (1H, s, H-7),
3J = 7.5 Hz, H-400), 7.85 (1H, d, J = 7.5 Hz, H-300), 8.03 (1H, s, NH),
8.58 (2H, dd, 3J = 4.5 Hz, J = 1.5 Hz, H-20 and H-60). 13C NMR
4
(75 MHz, Me2SO-d6): d 64.8 (q, 4JC–F = 2.9 Hz, C-7), 119.7 (C-30 and
C-50), 123.9 (q, 1JC–F = 274.6 Hz, CF3), 125.8 (q, 3JC–F = 5.3 Hz, C-300),
126.0 (q, 2JC–F = 30.6 Hz, C-200), 128.5 (C-600), 129.9 (C-400), 133.8 (C-
500), 138.5 (C-10 and C-100), 150.0 (C-20 and C-60), 155.4, 157.2, 157.8
(C-2, C-3a and C-5). Anal. Calcd. for C16H12F3N7: C, 53.48; H, 3.37;
N, 27.29. Found: C, 53.60; H, 3.42; N, 26.02.
7.42 (1H, dd, J = 7.9 Hz, J = 4.5 Hz, H-50), 7.58 (2H, d, 3J = 8.3 Hz,
H-200 and H-600), 7.84 (2H, d, 3J = 8.3 Hz, H-300 and H-500), 8.09 (1H, s,
NH), 8.17 (1H, dt, 3J = 8.3 Hz, 4J = 1.8 Hz, H-40), 8.55 (1H, dd, 3J = 4.9,
4J = 1.5 Hz, H-60), 9.03 (1H, d, 4J = 1.9 Hz, H-20). 13C NMR (75 MHz,
3
3
1
Me2SO-d6): d 67.7 (C-7), 123.6 (C-50), 123.9 (q, JC–F = 272.2 Hz,
CF3), 125.8 (q, 3JC–F = 3.7 Hz, C-300 and C-500), 127.2 (C-200 and C-600),
127.3 (C-30), 129.5 (q, 2JC–F = 31.8 Hz, C-400), 132.7 (C-40), 144.4 (C-
100), 146.5 (C-60), 149.5 (C-20), 155.5, 156.7, 157.5 (C-2, C-3a and C-
5). Anal. Calcd. for C16H12F3N7: C, 53.48; H, 3.37; N, 27.29. Found: C,
53.34; H, 3.62; N, 27.01.
4.4.11. 2-(Pyridin-4-yl)-7-(3-trifluoromethylphenyl)-6,7-
dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amine (5k)
Yield 90%; mp 258 8C (EtOH–DMF); TLC (silica gel, EtOH): Rf
0.46. 1H NMR (300 MHz, Me2SO-d6): d 6.62 (2H, s, NH2), 6.92 (1H, s,
3
3
H-7), 7.62 (1H, d, J = 7.9 Hz, H-600), 7.70 (1H, t, J = 7.7 Hz, H-500),
7.77 (2H, dd, 3J = 4.5 Hz, 4J = 1.5 Hz, H-30 and H-50), 7.79 (1H, s, H-
3
4.4.7. 7-(2-Fluorophenyl)-2-(pyridin-4-yl)-6,7-
200), 7.81 (1H, d, J = 7.5 Hz, H-400), 8.13 (1H, s, NH), 8.60 (2H, dd,
3J = 4.5 Hz, J = 1.5 Hz, H-20 and H-60). 13C NMR (75 MHz, Me2SO-
4
dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amine (5g)
Yield 85%; mp 285 8C (EtOH–DMF); TLC (silica gel, EtOH): Rf
0.46. 1H NMR (300 MHz, Me2SO-d6): d 6.57 (2H, s, NH2), 7.01 (1H, s,
H-7), 7.22–7.39 (3H, m, H-300, H-500 and H-600), 7.44–7.52 (1H, m, H-
400), 7.78 (2H, dd, 3J = 4.5 Hz, 4J = 1.5 Hz, H-30 and H-50), 8.11 (1H, s,
d6): d 67.7 (C-7), 119.6 (C-30 and C-50), 123.1 (q, 3JC–F = 3.9 Hz, C-200),
123.9 (q, 1JC–F = 272.2 Hz, CF3), 126.0 (q, 3JC–F = 3.5 Hz, C-400), 129.4
(q, 2JC–F = 31.8 Hz, C-300), 130.2 (C-600), 130.4 (q, 4JC–F = 1.2 Hz, C-500),
138.6 (C-10), 141.3 (C-100), 150.0 (C-20 and C-60), 155.6, 156.8, 157.7
(C-2, C-3a and C-5). Anal. Calcd. for C16H12F3N7: C, 53.48; H, 3.37;
N, 27.29. Found: C, 53.36; H, 3.32; N, 27.40.
4
NH), 8.60 (2H, dd, 3J = 4.5 Hz, J = 1.5 Hz, H-20 and H-60). 13C NMR
3
2
(75 MHz, Me2SO-d6): d 64.4 (d, JC–F = 2.9 Hz, C-7), 116.1 (d, JC–
4
F = 20.6 Hz, C-300), 119.6 (C-30 and C-50), 124.8 (d, JC–F = 3.5 Hz, C-
2
3
500), 126.9 (d, JC–F = 11.8 Hz, C-100), 128.5 (d, JC–F = 3.5 Hz, C-600),
4.4.12. 2-(Pyridin-4-yl)-7-(4-trifluoromethylphenyl)-6,7-
3
131.5 (d, JC–F = 8.2 Hz, C-400), 138.6 (C-10), 150.0 (C-20 and C-60),
dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amine (5l)
1
Yield 88%; mp 297–298 8C (EtOH–DMF); TLC (silica gel, EtOH):
Rf 0.46. 1H NMR (300 MHz, Me2SO-d6): d 6.61 (2H, s, NH2), 6.90 (1H,
d, 3J = 1.1 Hz, H-7), 7.59 (2H, d, 3J = 8.3 Hz, H-200 and H-600), 7.77 (2H,
dd, 3J = 4.5 Hz, 4J = 1.5 Hz, H-30 and H-50), 7.84 (2H, d, 3J = 8.3 Hz, H-
300 and H-500), 8.11 (1H, d, 3J = 1.1 Hz, NH), 8.59 (2H, dd, 3J = 4.5 Hz,
4J = 1.5 Hz, H-20 and H-60). 13C NMR (75 MHz, Me2SO-d6): d 67.8 (C-
155.6, 156.8, 157.5 (C-2, C-3a and C-5), 159.7 (d, JC–F = 248.7 Hz,
C-200). Anal. Calcd. for C15H12FN7: C, 58.25; H, 3.91; N, 31.70. Found:
C, 58.04; H, 4.08; N, 31.52.
4.4.8. 7-(3-Fluorophenyl)-2-(pyridin-4-yl)-6,7-
dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amine (5h)
Yield 87%; mp 288 8C (EtOH–DMF); TLC (silica gel, EtOH): Rf
0.44. 1H NMR (300 MHz, Me2SO-d6): d 6.61 (2H, s, NH2), 6.81 (1H, d,
3J = 1.1 Hz, H-7), 7.16–7.31 (3H, m, H-200, H-400 and H-600), 7.51 (1H,
1
7), 119.7 (C-30 and C-50), 123.9 (q, JC–F = 272.8 Hz, CF3), 125.9 (q,
2
3JC–F = 3.5 Hz, C-300 and C-500), 127.3 (C-200 and C-600), 129.7 (q, JC–
F = 31.8 Hz, C-400), 138.6 (C-10), 144.3 (C-100), 150.0 (C-20 and C-60),
155.7, 156.8, 157.7 (C-2, C-3a and C-5). Anal. Calcd. for C16H12F3N7:
C, 53.48; H, 3.37; N, 27.29. Found: C, 53.23; H, 3.28; N, 27.52.
4
td, 3J = 8.1 Hz, JH–F = 6.0 Hz, H-500), 7.79 (2H, dd, 3J = 4.5 Hz,
4J = 1.5 Hz, H-30 and H-50), 8.11 (1H, d, 3J = 1.1 Hz, NH), 8.60 (2H,
4
dd, 3J = 4.5 Hz, J = 1.5 Hz, H-20 and H-60). 13C NMR (75 MHz,
4
2
Me2SO-d6): d 67.6 (d, JC–F = 1.2 Hz, C-7), 113.2 (d, JC–F = 22.3 Hz,
C-200), 116.0 (d, 2JC–F = 20.6 Hz, C-400), 119.6 (C-30 and C-50), 122.3 (d,
Acknowledgment
4JC–F = 2.4 Hz, C-600), 131.1 (d, JC–F = 7.6 Hz, C-500), 138.6 (C-10),
3
This work is supported by the Academic Research Fund from the
National University of Singapore (WBS R-148-000-069-112).
3
142.7 (d, JC–F = 6.5 Hz, C-100), 150.0 (C-20 and C-60), 155.6, 156.7,
1
157.5 (C-2, C-3a and C-5), 162.1 (d, JC–F = 245.2 Hz, C-300). Anal.
Calcd. for C15H12FN7: C, 58.25; H, 3.91; N, 31.70. Found: C, 58.14; H,
4.02; N, 31.43.
References
4.4.9. 7-(4-Fluorophenyl)-2-(pyridin-4-yl)-6,7-
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Rf 0.46. 1H NMR (300 MHz, Me2SO-d6): d 6.54 (2H, s, NH2), 6.78 (1H,
s, H-7), 7.28 (2H, dd, 3J = 8.7 Hz, 3JH–F = 9.0 Hz, H-300 and H-500), 7.42
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4
(2H, dd, 3J = 8.7 Hz, JH–F = 5.7 Hz, H-200 and H-600), 7.77 (2H, dd,
3J = 4.5 Hz, 4J = 1.5 Hz, H-30 and H-50), 8.02 (1H, s, NH), 8.59 (2H, dd,
4
3J = 4.5 Hz, J = 1.5 Hz, H-20 and H-60). 13C NMR (75 MHz, Me2SO-
d6): d 67.8 (C-7), 115.7 (d, 2JC–F = 21.8 Hz, C-300 and C-500), 119.6 (C-
3
4
30 and C-50), 128.6 (d, JC–F = 8.8 Hz, C-200 and C-600), 136.4 (d, JC–
F = 2.9 Hz, C-100), 138.7 (C-10), 150.0 (C-20 and C-60), 155.7, 156.7,
157.5 (C-2, C-3a and C-5), 162.4 (d, JC–F = 245.2 Hz, C-400). Anal.
1

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