Synthesis and antileukemic activity of new fluorinated 5-aza-9-deazapurines1

Article abstract of DOI:10.3987/COM-16-13464

Novel fluorinated 4-benzylamino-7-phenylpyrazolo[1,5-a]-[1,3,5]triazin-2-amines were prepared using an efficient and practical approach. The chemoselectivity of condensation of pyrazolylguanidine and trichloroacetonitrile was found to be solvent-dependent

Full text of DOI:10.3987/COM-16-13464

HETEROCYCLES, Vol. 92, No. 6, 2016, pp. -. © 2016 The Japan Institute of Heterocyclic Chemistry
Received, 16th March, 2016, Accepted, 11th April, 2016, Published online, 28th April, 2016
DOI: 10.3987/COM-16-13464
SYNTHESIS
AND
ANTILEUKEMIC
ACTIVITY
OF
NEW
FLUORINATED 5-AZA-9-DEAZAPURINES1
Felicia Phei Lin Lim,a Koon Kee Kow,a Eng Hwa Yeo,b Sek Chuen Chow,b
and Anton V. Dolzhenkoa,c,d
*
a School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, Bandar
Sunway, Selangor Darul Ehsan 47500, Malaysia
b
School of Science, Monash University Malaysia, Jalan Lagoon Selatan, Bandar
Sunway, Selangor Darul Ehsan 47500, Malaysia
c
School of Pharmacy, Curtin Health Innovation Research Institute, Faculty of
Health Sciences, Curtin University, GPO Box U1987 Perth, Western Australia
6845, Australia
d Faculty of Pharmacy, South Ural State Medical University, 64 Vorovskogo Street,
Chelyabinsk, 454092, Russian Federation
 Corresponding author. Tel.: +60-3-5514-5867; fax: +60-3-5514-6364; e-mails:
anton.dolzhenko@monash.edu, dolzhenkoav@gmail.com
Abstract
–
Novel fluorinated 4-benzylamino-7-phenylpyrazolo[1,5-a]-
[1,3,5]triazin-2-amines were prepared using an efficient and practical approach.
The chemoselectivity of condensation of pyrazolylguanidine and
trichloroacetonitrile was found to be solvent-dependent and, when conducted in
toluene, this reaction afforded 7-phenyl-4-trichloromethylpyrazolo[1,5-a]-
[1,3,5]triazin-2-amine as the main product. This key intermediate underwent
nucleophilic replacement of the trichloromethyl group with fluorinated
benzylamines providing a series of the target compounds. Antiproliferative activity
of the prepared compounds against Jurkat T cells was explored using MTS assay.
Morphological changes observed in cells treated by the most potent compounds of
this series, suggested that these compounds induced apoptosis in cells.
The unique character of fluorinated molecules have been well exploited in drug discovery programs.2-4
Utilizing fluorine substitution, new biologically active compounds have been discovered including those
with anticancer properties.5 Notable examples include fluorinated purines, which displayed significant

antiproliferative activity against several cancer cell lines. Particularly, all three anticancer lead compounds,
recently identified in the biological evaluation of an extensive library of purine derivatives, possessed a
trifluoromethyl group on the aminosubstituent in position 6 of the heterocyclic system.6 In another focused
library of purines with the substituted 6-benzylamino fragment, compounds with fluorinated benzene ring
of this moiety were found to be active in cytokinin, antitumor and kinase bioassays.7 On the basis of the
purine motif, advancements in drug design and development have inspired extensive research into isosteric
heterocyclic systems. Purine isosteres with a 1,3,5-triazine ring have been demonstrated to be versatile
scaffolds for the discovery and development of new bioactive compounds.8 Among this group of
heterocyclic systems, 1,2,4-triazolo[1,5-a][1,3,5]triazines (5-azapurines) and pyrazolo[1,5-a]-
[1,3,5]triazines (5-aza-9-deazapurines) were identified as particularly promising scaffolds for the
development of new bioactive compounds.8 Previously, our studies on fluorinated
7-benzylamino-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-amines (1) lead to the development of
practical synthesis and identification of compounds with anticancer properties (Figure 1).9 In our
continuous search for new antiproliferative agents utilizing purine analogues, we attempted to alter scaffold
1 by removing a nitrogen atom to obtain 5-aza-9-deazapurine isostere 2 with identical substitution pattern.
The choice of this scaffold was also based on our previous promising results on 5-aza-9-deazapurines
demonstrating anticancer properties.10,11 Herein, we report synthesis of a small library of new fluorinated
4-benzylamino-7-phenylpyrazolo[1,5-a][1,3,5]triazin-2-amines and antiproliferative activity of these
compounds against leukemic Jurkat T cells.
Figure 1. Design of fluorinated 4-benzylamino-7-phenylpyrazolo[1,5-a][1,3,5]triazin-2-amine with
potential anticancer properties
Previously, we synthesized bioactive 1,2,4-triazolo[1,5-a][1,3,5]triazines 19 using a practical method12,13
based on the construction of
a
trichloromethyl-substituted 1,3,5-triazine ring onto
N-(3-phenyl-1,2,4-triazol-5-yl)guanidine followed by successful nucleophilic replacement of the
trichloromethyl group by amines. Following the previous general approach for the preparation of 1, we now
designed a synthesis of structurally related pyrazolo[1,5-a][1,3,5]triazines 2, albeit utilizing pyrazole-based
intermediates to obtained the desired products.

The conversion of 5(3)-amino-3(5)-phenylpyrazole (3), obtained from the reaction of benzoylacetonitrile
and hydrazine14 to N-(3-phenylpyrazol-5-yl)guanidine (4) was successfully achieved by the treatment of 3
with cyanamide in the presence of acid (Scheme 1). In the next step, chemoselectivity of the ring closure
reaction of guanidine 4 with trichloroacetonitrile was found to be solvent-dependent. This reaction
proceeded in ethanol media with elimination of chloroform, but in toluene, a mixture of two products 5
(major 77%) and 6 (minor 8%) was obtained. The formation of compound 5 involved elimination of
ammonia, which was probably present in the reaction mixture for sufficient time to displace the
trichloromethyl group of 5, therefore leading to the formation of side product 6. We manage to isolate the
desired product 5 in pure form via fractional recrystallization exploiting its significantly higher solubility in
hot toluene compared to 6. The structure of 5 was supported by NMR spectra, particularly by the
1
characteristic singlet at 7.66 ppm in the H NMR spectra attributed to the amino group on C-2 of the
13
heterocyclic system and the signal at 90 ppm in the C NMR spectra indicative of the trichloromethyl
group.
Scheme 1. Synthesis of fluorinated 4-benzylamino-7-phenylpyrazolo[1,5-a][1,3,5]triazin-2-amines (2a-g)
A small library of fluorinated 4-benzylamino-7-phenylpyrazolo[1,5-a][1,3,5]triazin-2-amines 2 was
generated by nucleophilic substitution on C-4 of the heterocyclic system using fluorine / trifluoromethyl
substituted benzylamines, which replaced the trichloromethyl group. One of the obvious advantages of
employing trichloromethyl group as a leaving group in this reaction was volatility and relatively inert
characteristics of the byproduct chloroform.
The structure of the synthesized compounds 2 was confirmed by NMR spectral data, which were similar to
1
their structural analogues 1 with 1,2,4-triazolo[1,5-a][1,3,5]triazine skeleton. However, in the H NMR
spectra of compounds 2, the NH proton signal of the benzylamino moiety appeared at 8.66-8.81 ppm, while

the signal of the same NH proton of analogues 1 was downfield shifted to 9.01-9.12 ppm due to the higher
deshielding effect of the more electron-deficient 1,2,4-triazolo[1,5-a][1,3,5]triazine ring system.9
The prepared compounds 2 were subjected to biological screening for antiproliferative activity against
Jurkat T cells. The antiproliferative activity of the prepared compounds 2 against Jurkat T cell lines was
evaluated in vitro using MTS assay and activity was expressed as IC50 values measured at two time
intervals: 24 h and 48 h (Table 1). In general, the trifluoromethyl-substituted 4-benzylamino-7-phenyl-
pyrazolo[1,5-a][1,3,5]triazin-2-amines (2d, 2e, and 2f) were found to be almost twice more potent in
inhibiting growth of Jurkat T cells as compared to fluoro-substituted compounds 2a, 2b, and 2c. However,
adding another trifluoromethyl group to the benzyl moiety (compound 2g) dramatically decreased
antiproliferative activity with no observable effect on viability of Jurkat T cells at the highest tested
concentration 100 μM. The most active compounds were the 2-trifluoromethyl- and 4-trifluoromethyl-
substituted derivatives 2d and 2f, which demonstrated similar IC50 values of 38 μM and 41 μM against
Jurkat T cells 48 hours after the treatment. Both 2d and 2f affected the viability of Jurkat T cells in a
concentration- and time-dependent manner reducing cell numbers at concentration 100 μM to less than 5%
of the control values.
Table 1. Antiproliferative activity of fluorinated 4-benzylamino-7-phenylpyrazolo[1,5-a][1,3,5]-
triazin-2-amines (2a-g) against Jurkat T cells (MTS assay)
Compound
ArF
IC50, μM
24 h
48 h
2-FC6H4
86 ± 39.6
85 ± 30.1
83 ± 41.9
50 ± 5.1
73 ± 17.2
57 ± 15.7
>100
70 ± 16.9
83 ± 26.9
61 ± 12.3
37 ± 6.9
53 ± 4.9
43 ± 9.4
>100
2a
2b
2c
2d
2e
2f
3-FC6H4
4-FC6H4
2-CF3C6H4
3-CF3C6H4
4-CF3C6H4
3,5-(CF3)2C6H3
2g
The morphological changes of Jurkat T cells induced by compounds 2d and 2f were further examined using
fluorescence microscopy after fixing the cells with 4% paraformaldehyde and staining with Hoechst
33358 dye. Normal nuclear morphology, represented by a dark blue color, with no prominent apoptotic
and necrotic cells was observed in the untreated control Jurkat T cells (Figure 2a). Jurkat T cells, 24 hours
after treatment with 50 μM of compound 2d and 2f, showed typical hallmarks of apoptosis, such as

fragmentation of the nuclei into apoptotic bodies (Figure 2b,c).15 Another observed characteristic of
apoptosis was chromatin condensation as represented by the bright fluorescent blue colour owing to the
condensed nuclear material. The Jurkat T cells also appeared as multinucleated cell and displayed nuclear
margination. Hence, we proposed that compounds 2d and 2f manifest their antiproliferative effect by
induction of apoptosis in Jurkat T cells.
(a)
(b)
(c)
Figure 2. Morphological changes in intact Jurkat T cells and cells treated with compounds 2d and 2f. (a)
Jurkat T cells – untreated control; (b) Jurkat T cells 24 h after treatment with 2d at 50 μM; (c) Jurkat T cells
24 h after treatment with 2f at 50 μM. Typical hallmarks of apoptosis include: formation of apoptotic bodies
(white arrow); chromatin condensation (red arrow); multinucleated cell (yellow arrow); nuclear
margination (green arrow). Bar: 100 μM
We
successfully
designed
and
prepared
new
fluorinated
4-benzylamino-7-phenyl-
pyrazolo[1,5-a][1,3,5]triazin-2-amines possessing antileukemic properties. It was found that the most
active compounds 2d and 2f induced apoptosis in Jurkat T cells resulting in their significant
antiproliferative effect, which make them promising leads for further development of new therapeutic
agents targeting T-cell leukemia.
EXPERIMENTAL
1
General Methods. Melting points were determined on a Gallenkamp melting point apparatus. H and
13C NMR spectra were recorded on a Bruker Avance III spectrometer (400 MHz), using DMSO-d6 as a
solvent and TMS as an internal reference. TLC analysis was performed on aluminum plates coated with
Silica gel F (Analtech) with detection by UV light.

Synthesis of N-(3-phenylpyrazol-5-yl)guanidine (4).
5(3)-Amino-3(5)-phenylpyrazole (3, 3.4 g, 21.4 mmol), cyanamide (2.6 g, 60 mmol) and conc. HCl
(3.75 mL, 38.5 mmol) were heated in EtOH (17 mL) under reflux for 7 h. After cooling, aqueous solution of
K2CO3 (5%, 250 mL) was added and the product formed was filtered and dried under vacuum. An
analytical sample was recrystallized from MeOH.
Yield 56%; mp 137-138 °C.
1H NMR (400 MHz, Me2SO-d6): δ 6.18 (1H, br s, H-8), 6.59 (4H, br s, NH(=NH)NH2), 7.29 (1H, t, 3J =
7.3 Hz, H-4′), 7.39 (2H, t, 3J = 7.6 Hz, H-3′ and H-5′), 8.03 (2H, d, 3J = 8.0 Hz, H-2′ and H-6′).
13C NMR (100 MHz, Me2SO-d6): δ 92.1 (br s, C-4), 125.0 (C-3′ and C-5′), 127.5 (C-4′), 128.7 (C-2′ and
C-6′), 131.6 (br s, C-1′), 145.3 (br s, C-3), 152.4 (br s, C-5), 154.8 (br s, NHC(=NH)NH2).
Anal. Calcd for C10H11N5: C, 59.69; H, 5.51; N, 34.80%. Found: C, 59.55; H, 5.64; N, 34.68%.
Reaction of N-(3-phenylpyrazol-5-yl)guanidine (4) with trichloroacetonitrile.
N-(3-Phenylpyrazol-5-yl)guanidine (4, 1.6 g, 7.85 mmol) and trichloroacetonitrile (1.6 mL, 16 mmol) were
heated in toluene (15 mL) under reflux for 30 min. After cooling, the precipitate was filtered and underwent
fractional recrystallization from toluene to furnish pure 2-amino-7-phenyl-4-trichloromethyl-
pyrazolo[1,5-a][1,3,5]triazine (5)
Yield 77%; mp 240-242 °C; Rf 0.74 (AcOEt : hexane – 1:1).
1H NMR (400 MHz, Me2SO-d6): δ 6.68 (1H, s, H-8), 7.45-7.55 (3H, m, H-3′, H-4′ and H-5′), 7.66 (2H, s,
NH2), 8.03 (2H, dd, 3J = 8.2 Hz, 4J = 1.5 Hz, H-2′ and H-6′).
13C NMR (100 MHz, Me2SO-d6): δ 87.9 (C-8), 90.0 (CCl3), 126.5 (C-3′ and C-5′), 128.8 (C-2′ and C-6′),
129.7 (C-4′), 131.8 (C-1′), 149.6 (C-4), 153.9 (C-8a), 156.3 (C-7), 157.3 (C-2).
Anal. Calcd for C12H8Cl3N5: C, 43.86; H, 2.45; N, 21.31. Found: C, 43.75; H, 2.52; N, 21.22.
An insoluble in toluene fraction was recrystallized from EtOH to give 2,4-diamino-7-phenyl-
pyrazolo[1,5-a][1,3,5]triazine (6)
Yield 8%; mp 338-340 °C; Rf 0.40 (AcOEt).
1H NMR (400 MHz, Me2SO-d6): δ 6.21 (1H, s, H-8), 6.40 (2H, s, NH2), 7.39 (1H, t, 3J = 7.3 Hz, H-4′), 7.46
(2H, t, 3J = 7.3 Hz, H-3′ and H-5′), 7.82 (2H, brs, NH2), 7.96 (2H, d, 3J = 7.3 Hz, H-2′ and H-6′).
13C NMR (100 MHz, Me2SO-d6): δ 86.9 (C-8), 126.1 (C-3′ and C-5′), 128.6 (C-2′ and C-6′), 128.7 (C-4′),
132.9 (C-1′), 150.3 (C-4), 152.3 (C-8a), 154.9 (C-7), 159.2 (C-2).
Anal. Calcd for C11H10N6: C, 58.40; H, 4.46; N, 37.15. Found: C, 58.28; H, 4.50; N, 36.97.
General method for the synthesis of fluorinated 4-benzylamino-7-phenylpyrazolo[1,5-a][1,3,5]triazin-
2-amines (2a-g). 2-Amino-7-phenyl-4-trichloromethylpyrazolo[1,5-a][1,3,5]triazine (6, 200 mg, 0.61
mmol) was added to the solution of the appropriate fluorinated benzylamine (0.78 mmol) in MeOH (7 mL),

the mixture was stirred at ambient temperature for 24 h and the reaction progress was monitored by TLC.
After evaporation of the solvent, the residue was recrystallized from MeOH (2a-2e, 2g) or toluene (2f) to
give pure compounds 2a-g.
4-(2-Fluorobenzylamino)-7-phenylpyrazolo[1,5-a][1,3,5]triazin-2-amine (2a).
Yield 90%; mp 177-179 °C; Rf 0.47 (AcOEt : hexane – 1:1).
3
1H NMR (400 MHz, Me2SO-d6): δ 4.75 (2H, d, J = 6.2 Hz, CH2), 6.26 (1H, s, H-8), 6.53 (2H, s, NH2),
7.15-7.24 (2H, m, H-3′′ and H-5′′), 7.29-7.49 (5H, m, H-3′, H-4′, H-5′, H-4′′ and H-6′′), 7.98 (2H, d, 3J =
7.3 Hz, H-2′ and H-6′), 8.66 (1H, t, 3J = 6.2 Hz, NH).
13C NMR (100 MHz, Me2SO-d6): δ 36.9 (d,3JCF = 4.9 Hz, CH2), 87.1 (C-8), 115.0 (d, 2JCF = 20.9 Hz, C-3′′),
124.9 (d, 4JCF = 3.5 Hz, C-5′′), 125.5 (d, 2JCF = 14.5 Hz, C-1′′), 126.0 (C-3′ and C-5′), 128.5 (C-2′ and C-6′),
128.7 (C-4′), 128.9 (d, 3JCF = 8.1 Hz, C-4′′), 129.0 (d, 3JCF = 4.3 Hz, C-6′′), 132.8 (C-1′), 148.9 (C-4), 152.1
(C-8a), 155.0 (C-7), 159.8 (d, 1JCF = 244.3 Hz, C-2′′),158.8 (C-2).
Anal. Calcd for C18H15FN6: C, 64.66; H, 4.52; N, 25.14. Found: C, 64.60; H, 4.70; N, 24.95.
4-(3-Fluorobenzylamino)-7-phenylpyrazolo[1,5-a][1,3,5]triazin-2-amine (2b).
Yield 75%; mp 176-177 °C; Rf 0.38 (AcOEt : hexane – 1:1).
1H NMR (400 MHz, Me2SO-d6): δ 4.69 (2H, d, 3J = 6.4 Hz, CH2), 6.27 (1H, s, H-8), 6.57 (2H, s, NH2), 7.09
(1H, ddd, 3J = 8.6 Hz, 3JHF = 8.6 Hz, 4J = 2.6 Hz, H-4′′), 7.23-7.28 (2H, m, H-2′′ and H-6′′), 7.36-7.43 (2H,
m, H-4′ and H-5′′), 7.47 (2H, t, 3J = 7.3 Hz, H-3′ and H-5′), 7.98 (2H, d, 3J = 7.3 Hz, H-2′ and H-6′), 8.75
(1H, t, 3J = 6.4 Hz, NH).
13C NMR (100 MHz, Me2SO-d6): δ 42.5 (d, 4JCF = 1.7 Hz, CH2), 113.7 (d, 2JCF = 20.9 Hz, C-4′′), 114.2 (d,
4
2JCF = 21.6 Hz, C-2′′), 123.4 (d, JCF = 2.8 Hz, C-6′′), 126.1 (C-3 and C-5), 128.5 (C-2 and C-6), 128.7
(C-4′), 130.2 (d, 3JCF = 8.3 Hz, C-5′′), 132.8 (C-1′), 141.8 (d, 3JCF = 7.1 Hz, C-1′′), 148.8 (C-4), 152.1 (C-8a),
155.0 (C-7), 158.8 (C-2), 162.1 (d, 1JCF = 243.4 Hz, C-3′′).
Anal. Calcd for C18H15FN6: C, 64.66; H, 4.52; N, 25.14. Found: C, 64.56; H, 4.68; N, 25.02.
4-(4-Fluorobenzylamino)-7-phenylpyrazolo[1,5-a][1,3,5]triazin-2-amine (2c).
Yield 48%; mp 178-179 °C; Rf 0.44 (AcOEt : hexane – 1:1).
1H NMR (400 MHz, Me2SO-d6): δ 4.65 (2H, d, 3J = 6.2 Hz, CH2), 6.25 (1H, s, H-8), 6.55 (2H, s, NH2), 7.16
(2H, dd, 3J = 8.7 Hz, 3JHF = 9.1 Hz, H-3′′ and H-5′′), 7.40 (1H, t, 3J = 7.4 Hz, H-4′), 7.44-7.49 (4H, m, H-3′,
H-5′, H-2′′ and H-6′′), 7.97 (2H, d, 3J = 7.9 Hz, H-2′ and H-6′), 8.71 (1H, t, 3J = 6.3 Hz, NH).
13C NMR (100 MHz, Me2SO-d6): δ 42.3 (CH2), 87.2 (C-8), 115.0 (d, 2JCF = 21.3 Hz, C-3′′ and C-5′′) 126.1
(C-3′ and C-5′),128.6 (C-2′ and C-6′), 128.8 (C-4′), 129.6 (d, 3JCF = 8.1 Hz, C-2′′ and C-6′′), 132.9 (C-1′),

4
1
135.1 (d, JCF = 3.0 Hz, C-1′′), 148.8 (C-4), 152.2 (C-8a), 155.0 (C-7), 158.9 (C-2), 161.3 (d, JCF
242.5 Hz, C-4′′).
=
Anal. Calcd for C18H15FN6: C, 64.66; H, 4.52; N, 25.14. Found: C, 64.60; H, 4.58; N, 25.09.
7-Phenyl-4-(2-trifluoromethylbenzylamino)pyrazolo[1,5-a][1,3,5]triazin-2-amine (2d).
Yield 56%; mp 138-140 °C; Rf 0.44 (AcOEt : hexane – 1:1).
1H NMR (400 MHz, Me2SO-d6): δ 4.92 (2H, d, 3J = 6.3 Hz, CH2), 6.30 (1H, s, H-8), 6.54 (2H, s, NH2), 7.41
(1H, t, 3J = 7.3 Hz, H-4′), 7.48 (2H, t, 3J = 7.3 Hz, H-3′ and H-5′), 7.49 (1H, t, 3J = 7.7 Hz, H-5′′), 7.56 (1H,
d, 3J = 7.8 Hz, H-6′′), 7.65 (1H, t, 3J = 7.6 Hz, H-4′′), 7.76 (1H, d, 3J = 7.7 Hz, H-3′′), 8.00 (2H, d, 3J =
7.4 Hz, H-2′ and H-6′), 8.76 (1H, t, 3J = 6.4 Hz, NH).
13C NMR (100 MHz, Me2SO-d6): δ 39.7 (CH2), 87.3 (C-8), 124.5 (q, 1JCF = 274.3 Hz, CF3), 125.8 (q, 3JCF =
2
5.6 Hz, C-3′′), 125.9 (q, JCF = 30.2 Hz, C-2′′), 126.1 (C-3′ and C-5′), 127.4 (C-4′′), 131.7 (C-6′′), 128.6
3
(C-2′ and C-6′), 128.8 (C-4′), 132.78 (C-5′′), 132.82 (C-1′), 137.0 (q, JCF = 1.5 Hz, C-1′′), 149.1 (C-4),
152.1 (C-8a), 155.2 (C-7), 158.8 (C-2).
Anal. Calcd for C19H15F3N6: C, 59.37; H, 3.93; N, 21.87. Found: C, 59.28; H, 4.12; N, 21.63.
7-Phenyl-4-(3-trifluoromethylbenzylamino)pyrazolo[1,5-a][1,3,5]triazin-2-amine (2e).
Yield 76%; mp 162-164 °C; Rf 0.44 (AcOEt : hexane – 1:1).
1H NMR (400 MHz, Me2SO-d6): δ 4.76 (2H, d, 3J = 6.3 Hz, CH2), 6.26 (1H, s, H-8), 6.56 (2H, s, NH2), 7.40
(1H, t, 3J = 7.3 Hz, H-4′), 7.47 (2H, t, 3J = 7.3 Hz, H-3′ and H-5′), 7.59 (1H, t, 3J = 7.6 Hz, H-5′′), 7.64 (1H,
d, 3J = 7.8 Hz, H-6′′), 7.75 (1H, d, 3J = 7.6 Hz, H-4′′), 7.78 (1H, s, H-2′′), 7.98 (2H, d, 3J = 7.4 Hz, H-2′ and
H-6′), 8.81 (1H, t, 3J = 6.4 Hz, NH).
13C NMR (100 MHz, Me2SO-d6): δ 42.6 (CH2), 87.2 (C-8), 123.8 (q, 3JCF = 3.7 Hz, C-4′′), 124.1 (q, 3JCF
=
3.8 Hz, C-2′′), 124.2 (q, 1JCF = 272.2 Hz, CF3), 126.1 (C-3′ and C-5′), 128.6 (C-2′ and C-6′), 128.8 (C-4′),
129.0 (q, 2JCF = 31.5 Hz, C-3′′), 129.4 (C-5′′), 131.7 (C-6′′), 132.8 (C-1′), 140.4 (C-1′′), 148.8 (C-4), 152.2
(C-8a), 155.1 (C-7), 158.9 (C-2).
Anal. Calcd for C19H15F3N6: C, 59.37; H, 3.93; N, 21.87. Found: C, 59.30; H, 4.08; N, 21.73.
7-Phenyl-4-(4-trifluoromethylbenzylamino)pyrazolo[1,5-a][1,3,5]triazin-2-amine (2f).
Yield 72%; mp 188-189 °C; Rf 0.34 (AcOEt : hexane – 1:1).
1H NMR (400 MHz, Me2SO-d6): δ 4.76 (2H, d, 3J = 6.4 Hz, CH2), 6.26 (1H, s, H-8), 6.54 (2H, s, NH2), 7.40
(1H, t, 3J = 7.3 Hz, H-4′), 7.47 (2H, t, 3J = 7.3 Hz, H-3′ and H-5′), 7.62 (2H, d, 3J = 8.2 Hz, H-2′′ and H-6′′),
7.71 (2H, d, 3J = 8.2 Hz, H-3′′ and H-5′′), 7.98 (2H, d, 3J = 7.4 Hz, H-2′ and H-6′), 8.81 (1H, t, 3J = 6.4 Hz,
NH).

13C NMR (100 MHz, Me2SO-d6): δ 42.6 (CH2), 87.2 (C-8), 124.3 (q, 1JCF = 271.8 Hz, CF3), 125.1 (q, 3JCF =
3.8 Hz, C-3′′ and C-5′′), 126.0 (C-3′ and C-5′), 127.6 .3 (q, 2JCF = 31.7 Hz, C-4′′), 128.0 (C-2′′ and C-6′′),
128.5 (C-2′ and C-6′), 128.7 (C-4′), 132.8 (C-1′), 143.7 (C-1′′), 148.8 (C-4), 152.1 (C-8a), 155.0 (C-7),
158.8 (C-2).
Anal. Calcd for C19H15F3N6: C, 59.37; H, 3.93; N, 21.87. Found: C, 59.18; H, 4.15; N, 21.67.
4-[3,5-bis-(Trifluoromethyl)benzylamino]-7-phenylpyrazolo[1,5-a][1,3,5]triazin-2-amine (2g).
Yield 68%; mp 238-240 °C; Rf 0.56 (AcOEt : hexane – 1:1).
1H NMR (400 MHz, Me2SO-d6): δ 4.85 (2H, d, 3J = 6.5 Hz, CH2), 6.27 (1H, s, H-8), 6.59 (2H, s, NH2), 7.41
(1H, t, 3J = 7.2 Hz, H-4′), 7.47 (2H, t, 3J = 7.3 Hz, H-3′ and H-5′), 7.98 (2H, d, 3J = 7.3 Hz, H-2′ and H-6′),
8.03 (1H, s, H-4′′), 8.15 (2H, s, H-2′′ and H-6′′), 8.86 (1H, t, 3J = 6.5 Hz, NH).
3
13C NMR (100 MHz, Me2SO-d6): δ 42.4 (CH2), 87.3 (C-8), 120.9 (sept, JCF = 3.8 Hz, C-4′′), 123.3 (q,
1JCF = 272.8 Hz, CF3  2), 126.1 (C-3′ and C-5′), 128.5 (q, 3JCF = 3.7 Hz, C-2′′ and C-6′′), 128.6 (C-2′ and
C-6′), 128.8 (C-4′), 130.1 (q, 2JCF = 32.8 Hz, C-3′′ and C-5′′), 132.8 (C-1′), 142.4 (C-1′′), 148.8 (C-4), 152.2
(C-8a), 155.2 (C-7), 158.8 (C-2).
Anal. Calcd for C20H14F6N6: C, 53.10; H, 3.12; N, 18.58. Found: C, 52.92; H, 3.22; N, 18.44.
Cell viability assay
Cell viability was assessed colourimetrically using the Cell Titer 96TM Aqueous cell proliferation assay16
based on the reduction of 5-(3-carboxymethoxyphenyl)2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl)-
tetrazolium, inner salt (MTS) by dehydrogenase enzymes in living cells to coloured water-soluble formazan,
concentration of which is directly proportional to the number of living cells in the culture.17 With the
appropriate blank (medium alone), aliquots of 100 μL Jurkat T cell suspension (~25,000 cells) were
dispensed into the wells of a 96-well flat-bottomed plate followed by treatment with compounds 2a-2g at
final concentrations 12.5, 25, 50, and 100 μM. In corresponding control wells of the same plates, Jurkat T
cells were grown without treatment. Two 96-well flat-bottomed plates were incubated at 37 °C in an
incubator for 24 and 48 h respectively. After incubations, 20 μL of freshly prepared MTS solution
(333 μg/mL) in phosphate-buffered saline (PBS) was added to each well and the plate was incubated at
37 °C for 2 h. Absorbance was measured at 490 nm using a Tecan i-control plate reader. Assays were
conducted in duplicates in 3 independent experiments for each of 24 and 48 h time intervals. The IC50
values were calculated using Graphpad Prism (version 6) biostatistical software. Quantitative experimental
data were presented as mean ± standard deviation.
Fluoroscence microscopy

Aliquots of 1000 μL Jurkat T cell suspension (~250,000 cells) were dispensed into wells of a 24-well flat
bottomed plate, followed by treatment with compounds 2d and 2f (final concentration 50 μM). A series of
wells with Jurkat T cell on the same plate was remained untreated and was used as a control. The plate was
incubated at 37 °C in an incubator for 24 h; cells were harvested and centrifuged down. The supernatant
was removed and cell pellet was suspended in 50 μL of paraformaldehyde solution (4% in PBS) at room
temperature for 30 min. After removal of supernatant, the cells were re-suspended in 50 μL of PBS
containing 50 μg/mL Hoechst 33342 DNA dye and incubated for 30 min at room temperature. After
removal of supernatant, the stained cells were re-suspended in 20 μL glycerol / PBS (50:50, v/v) and 2-5 μL
of the mixture with stained cells was placed on slides and viewed using an Olympus BX51 fluorescence
microscope. Cells were judged to be apoptotic if they displayed the characteristic hallmarks of apoptosis.18
ACKNOWLEDGEMENTS
This work is supported by the Ministry of Higher Education, Malaysia under Fundamental Research Grant
Scheme (FRGS) and by a seed grant from the Infection and Immunity Cluster of the Tropical Medicine,
Biology and Genomics Platform (Monash University Malaysia).
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