Comparative study between the anti-P. falciparum activity of triazolopyrimidine, pyrazolopyrimidine and quinoline derivatives and the identification of new PfDHODH inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112941

In this work, we designed and synthesized 35 new triazolopyrimidine, pyrazolopyrimidine and quinoline derivatives as P. falciparum inhibitors (3D7 strain). Thirty compounds exhibited anti-P. falciparum activity, with IC₅₀ values ranging from 0.030 to 9.1 μM. The [1,2,4]triazolo[1,5-a]pyrimidine derivatives were more potent than the pyrazolo[1,5-a]pyrimidine and quinoline analogues. Compounds 20, 21, 23 and 24 were the most potent inhibitors, with IC₅₀ values in the range of 0.030–0.086 μM and were equipotent to chloroquine. In addition, the compounds were selective, showing no cytotoxic activity against the human hepatoma cell line HepG2. All [1,2,4]triazolo[1,5-a]pyrimidine derivatives inhibited PfDHODH activity in the low micromolar to low nanomolar range (IC₅₀ values of 0.08–1.3 μM) and did not show significant inhibition against the HsDHODH homologue (0–30% at 50 μM). Molecular docking studies indicated the binding mode of [1,2,4]triazolo[1,5-a]pyrimidine derivatives to PfDHODH, and the highest interaction affinities for the PfDHODH enzyme were in agreement with the in vitro experimental evaluation. Thus, the most active compounds against P. falciparum parasites 20 (R = CF₃, R₁ = F; IC₅₀ = 0.086 μM), 21 (R = CF₃; R₁ = CH₃; IC₅₀ = 0.032 μM), 23, (R = CF₃, R₁ = CF₃; IC₅₀ = 0.030 μM) and 24 (R = CF₃, 2-naphthyl; IC₅₀ = 0.050 μM) and the most active inhibitor against PfDHODH 19 (R = CF₃, R₁ = Cl; IC₅₀ = 0.08 μM - PfDHODH) stood out as new lead compounds for antimalarial drug discovery. Their potent in vitro activity against P. falciparum and the selective inhibition of the PfDHODH enzyme strongly suggest that this is the mechanism of action underlying this series of new [1,2,4]triazolo[1,5-a]pyrimidine derivatives.

Full text of DOI:10.1016/j.ejmech.2020.112941

Journal Pre-proof
Comparative study between the anti-P. falciparum activity of triazolopyrimidine,
pyrazolopyrimidine and quinoline derivatives and the identification of new PfDHODH
inhibitors
Flávia F. Silveira, Juliana O. de Souza, Lucas V.B. Hoelz, Vinícius R. Campos,
Valquíria A.P. Jabor, Anna C.C. Aguiar, M. Cristina Nonato, Magaly G. Albuquerque,
Rafael V.C. Guido, Nubia Boechat, Luiz C.S. Pinheiro
PII:
S0223-5234(20)30913-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112941
EJMECH 112941
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 August 2020
Revised Date: 9 October 2020
Accepted Date: 11 October 2020
Please cite this article as: F.F. Silveira, J.O. de Souza, L.V.B. Hoelz, V.R. Campos, V.A.P. Jabor, A.C.C.
Aguiar, M.C. Nonato, M.G. Albuquerque, R.V.C. Guido, N. Boechat, L.C.S. Pinheiro, Comparative study
between the anti-P. falciparum activity of triazolopyrimidine, pyrazolopyrimidine and quinoline derivatives
and the identification of new PfDHODH inhibitors, European Journal of Medicinal Chemistry, https://
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Comparative study between the anti-P. falciparum activity of triazolopyrimidine,
pyrazolopyrimidine and quinoline derivatives and the identification of new
PfDHODH inhibitors
Flávia F. Silveira, Juliana O. de Souza, Lucas V. B. Hoelz, Vinícius R. Campos,
Valquíria A. P. Jabor, Anna C. C. Aguiar, M. Cristina Nonato, Magaly G. Albuquerque,
Rafael V. C. Guido, Nubia Boechat and Luiz C. S. Pinheiro

Comparative study between the anti-P. falciparum activity of triazolopyrimidine,
pyrazolopyrimidine and quinoline derivatives and the identification of new PfDHODH inhibitors
Flávia F. Silveira1,2#, Juliana O. de Souza3# Lucas V. B. Hoelz1, Vinícius R. Campos4,
,
Valquíria A. P. Jabor5, Anna C. C. Aguiar3, M. Cristina Nonato5, Magaly G. Albuquerque2, Rafael V. C.
Guido3*, Nubia Boechat1,2,* and Luiz C. S. Pinheiro1*
1 Laboratorio de Sintese de Farmacos, Instituto de Tecnologia em Farmacos, Farmanguinhos - FIOCRUZ, Fundacao Oswaldo Cruz. Rua
Sizenando Nabuco 100, Manguinhos, Rio de Janeiro, RJ, 21041-250, Brazil.
2
Programa de Pós-Graduação em Química, PGQu Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ,
Brazil.
3 Instituto de Física de São Carlos, Universidade de São Paulo, Av. João Dagnone, 1.100, Jd. Santa Angelina São Carlos-SP, Brazil.
4
Departamento de Química Orgânica, Programa de Pós-Graduação em Química, Instituto de Química, Universidade Federal
Fluminense, Niterói, RJ, Brazil.
5
Laboratório de Cristalografia de Proteínas, Departamento de Ciências BioMoleculares, Faculdade de Ciências Farmacêuticas de
Ribeirão Preto, Universidade de São Paulo, Avenida do Café s/n Monte Alegre, 14040-903 Ribeirão Preto, SP, Brazil.
#These authors contributed equally to the work
*Correspondence: nubia.boechat@far.fiocruz.br; luiz.pinheiro@far.fiocruz.br; rvcguido@ifsc.usp.br Tel.: + 55-21-3977-2464 (N.B.;
L.C.S.P.); +55-16-3373-8673 (R.V.C.G.)
ABSTRACT
In this work, we designed and synthesized 35 new triazolopyrimidine, pyrazolopyrimidine and
quinoline derivatives as P. falciparum inhibitors (3D7 strain). Thirty compounds exhibited anti-P.
falciparum activity, with IC50 values ranging from 0.030 to 9.1 µM. The [1,2,4]triazolo[1,5-
a]pyrimidine derivatives were more potent than the pyrazolo[1,5-a]pyrimidine and quinoline analogues.
Compounds 20, 21, 23 and 24 were the most potent inhibitors, with IC50 values in the range of 0.030 -
0.086 µM and were equipotent to chloroquine. In addition, the compounds were selective, showing no
cytotoxic activity against the human hepatoma cell line HepG2. All [1,2,4]triazolo[1,5-a]pyrimidine
derivatives inhibited PfDHODH activity in the low micromolar to low nanomolar range (IC50 values of
0.08 - 1.3 μM) and did not show significant inhibition against the HsDHODH homologue (0 - 30% at 50
μM). Molecular docking studies indicated the binding mode of [1,2,4]triazolo[1,5-a]pyrimidine
derivatives to PfDHODH, and the highest interaction affinities for the PfDHODH enzyme were in
agreement with the in vitro experimental evaluation. Thus, the most active compounds against P.
falciparum parasites 20 (R = CF3, R1 = F; IC50 = 0.086 μM), 21 (R = CF3; R1 = CH3; IC50 = 0.032 μM),
23, (R = CF3, R1 = CF3; IC50 = 0.030 μM) and 24 (R = CF3, 2-naphthyl; IC50 = 0.050 μM) and the most
active inhibitor against PfDHODH 19 (R = CF3, R1 = Cl; IC50 = 0.08 μM - PfDHODH) stood out as new
lead compounds for antimalarial drug discovery. Their potent in vitro activity against P. falciparum and
the selective inhibition of the PfDHODH enzyme strongly suggest that this is the mechanism of action
underlying this series of new [1,2,4]triazolo[1,5-a]pyrimidine derivatives.
Keywords: triazolopyrimidine, pyrazolopyrimidine, quinoline, malaria, P. falciparum, PfDHODH

1.
Introduction
The World Health Organization (WHO) has reported a reduction of approximately 23 million
cases of malaria since 2010, when 251 million cases were reported with 585 000 deaths. However, the
number of cases remain alarming. In 2018, there were approximately 405 000 deaths, mainly children
under 5 years of age, representing 67% of all malaria deaths in the world and the death of one child
every 2 minutes [1]. The development of new and effective antimalarials is considered necessary due to
the resistance of the malaria parasite to existing drugs. Thus, the identification of drugs with new
mechanisms of action is imperative for malaria chemotherapy [2].
The WHO recommends artemisinin-based combination therapy (ACT) as the gold-standard
treatment for this disease. This therapy combines a fast-acting artemisinin derivative with a long-acting
antimalarial with a different mode of action. However, the future of this treatment is at risk due to the
development of resistance to both drugs, and this has already led to some treatment failures [3,4].
There are still many challenges to overcome, regardless of the advancement in the control and
elimination of malaria in recent years. With the emergence of Plasmodium falciparum drug resistance
and insecticide-resistant mosquitoes in addition to the difficulty of formulating a potent malaria vaccine
and the existence of many areas where there are populations that are not appropriately cared for by the
health system, it is not difficult to understand why malaria is still considered a global public health
emergency [1,5,6]. Moreover, there is a need to develop antimalarials to decrease the high number of
deaths caused by severe malaria [5]. Therefore, new strategies to achieve malaria eradication are needed
[7,8].
Currently, researchers have increased interest in exploiting dihydroorotate dehydrogenase enzyme
(DHODH) inhibition as a strategy to treat malaria [9]. Pyrimidines are important metabolites that are
vital for DNA and RNA biosynthesis. The malaria parasite cannot save preformed pyrimidine bases or
nucleosides, and pyrimidines must be developed through a de novo biosynthetic pathway. This
biosynthesis is catalysed by DHODH and shows the importance of DHODH to parasite survival [10].
An example of a P. falciparum dihydroorotate dehydrogenase enzyme (PfDHODH) inhibitor is the
[1,2,4]-triazolo[1,5-a]pyrimidine derivative DSM265, which was discovered by Phillips and co-workers
and has advanced to clinical development [11,12].
Molecular hybridization has been used in current medicinal chemistry and is based on hybrid
derivatives that combine two or more different pharmacophoric fragments in a single molecule with
possible dual or multiple activities [13,14]. Our research group has been synthesizing several hybrids
that have shown anti-P. falciparum activity [15–17]. One example is a series of [1,2,4]triazolo[1,5-
a]pyrimidine derivatives, which are hybrid derivatives of mefloquine and amodiaquine (Fig. 1). In vitro
test results against chloroquine-resistant (CQR) P. falciparum W2 strains showed that compound 1,

which contains the bulky 2-naphthylamine group has an IC50 of 0.023 µM but showed no toxicity to the
human hepatoma cell line HepG2 and was 10-fold more potent than chloroquine (IC50 = 0.22 µM) (Fig.
1). Enzymatic assays confirmed that compound 1 is a PfDHODH inhibitor (IC50 = 0.70 µM) [18].
Another example is the pyrazolo[1,5-a]pyrimidine series system synthesized using ring isosteric
replacement and molecular hybridization based on compound 1. These derivatives were active against
the P. falciparum W2 CQR strain and showed no toxicity to the kidney epithelial cell line BGM.
Compound 2 displayed the highest and most selective inhibition of the PfDHODH enzyme with an IC50
value of 0.16 µM (Fig. 1) [19].
A quinoline-sulfadoxine hybrid series was active in vitro against the P. falciparum W2 CQR clone
and not toxic to mammalian kidney BGM cells. Compound 3 (Fig. 1), with an IC50 value of 0.09 µM,
was more potent than chloroquine (IC50 = 0.22 µM). An in vivo activity test (P. berghei model) showed
that compound 3 reduced parasitaemia by 49% 5 days postinfection, contributing to the discovery of a
new prototype with antimalarial activity [17]. Inspired by the promising results obtained by our group
with prototypes 1–3, the [1,2,4]triazolo[1,5-a]pyrimidine scaffold was chosen for the design of new
compounds as antimalarial candidates.
In this work, new [1,2,4]triazolo[1,5-a]pyrimidine derivatives 4-24 were designed based on the
concepts of molecular hybridization. The study focused on maintaining the pharmacophoric groups:
arylamines present in prototype 1 (blue) and the benzenesulfonamide moiety present in prototype 3
(green) (Fig. 1). At the 7 position of the [1,2,4]triazolo[1,5-a]pyrimidine ring, the 4-amino-N-
arylbenzenesulfonamide moiety was added in order to insert the benzenesulfonamide moiety between
the heterocyclic system and arylamines, continuing the study of the anti-P. falciparum activity of this
system [18]. In previous works [17,19], we observed that the pyrazolo[1,5-a]pyrimidine and quinoline
rings play important roles in antiplasmodial activity. The 4-amino-N-arylbenzenesulfonamide moiety
was also added to this system in compounds 25-31 and 32-38 to investigate their anti-P. falciparum
activity.

Fig. 1: Rational approach to the design of compounds 4-38.
2.
Results and discussion
2.1.
Chemistry
The synthetic route used to prepare the 35 new compounds 4-38 was developed in 3 steps
according to Scheme 1. In the first step, the condensation reaction of aminoguanidine bicarbonate with
the appropriate carboxylic acid in refluxing toluene (110 °C) for 24 h afforded the respective 1H-1,2,4-
triazoles-5-amines 39a-c in 70-97% yield [20]. 5-Methyl-1H-pyrazol-3-amine (39d) was obtained
commercially. The reaction of 39a-d with ethyl acetoacetate in toluene at 110 °C for 20 h in the
presence of catalytic p-toluenesulfonic acid gave 5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-ones
40a-c in 71-95% yields and the 2,5-dimethylpyrazolo[1,5-a]pyrimidin-7(4H)-one (40d) in 98% yield
[18]. Compounds 40a-d were treated with phosphorus oxychloride (POCl3) at 105 °C for 4 h to produce
key intermediaries 41a-d in 51-97% yield [18,19].
In the second step, the chlorosulfonation reaction of acetanilide was carried out without solvent at
0 °C for 1 h and then heated to 60 °C for another hour to obtain 4-acetamidobenzene-1-sulfonyl chloride
(42) in 77% yield without purification [21]. The addition-elimination reaction between 42 and the
respective aniline using chloroform, triethylamine (TEA) under reflux for 5 h gave protected
sulfonamides 43a-g in 42-87% yield. The hydrolysis reactions of 43a-g with 6N HCl at 100 °C for 6 h
followed by neutralization with 20% NaOH, produced 4-amino-N-arylbenzenesulfonamides 44a-g in
63-89% yield without purification [22].
The third step is a convergent synthesis was a nucleophilic substitution reaction between 44a-g
and the appropriate intermediary 41a-d was performed in ethanol at 78 °C for 2 h to obtain target
compounds 4-31 [18,19]. To obtain quinolinic derivatives 32-38, the reaction between 4,7-
dichloroquinoline (45) and the appropriate counterpart 44a-g was carried out under the same conditions.
Purification was performed by recrystallization or thin layer chromatography (TLC). After purification,
target compounds 4-38 were obtained in yields of 24 to 98%.
The derivatives 4-38 were isolated as hydrochloride and their structures were confirmed by IR,
1
NMR (1H, 13C and 19F) and HRMS. The H and 13C NMR assignments were confirmed by COSY
(homonuclear COrrelation SpectroscopYy), HSQC (Heteronuclear Single Quantum Coherence –
Editing) and HMBC (Heteronuclear MultipleBond Coherence) experiments.
For [1,2,4]triazolo[1,5-a]pyrimidine derivatives 4-10, compound 5 was used to exemplify the
1
NMR assignments. In the H NMR spectrum, singlets at 8.79 ppm, 6.78 ppm and 2.50 ppm was
identified as H2, H6, and methyl hydrogens (CH3), respectively. The phenyl hydrogens showed signals

at 7.17 - 7.86 ppm. For the hydrogens of the sulfonamide group (SO2NH) a singlet around 10 ppm was
assigned. In the 13C NMR spectrum, C2, methyl carbon (CH3) and C6 were assigned as signals at a
signal at 152.4 ppm, 23.5 ppm and 91.7 ppm, respectively.
For derivatives 11-17, two singlets around 2.43 ppm and 2.48 ppm referring to the hydrogen of the
CH3 group can be observed in the spectrum of 1H NMR.
In the 13C NMR spectrum of derivatives 18-24, quartets with J = 269 Hz referring to the C-F
coupling of the CF3 group, and a quartet with J = 38 Hz by the coupling of the fluorine atoms with C2
were observed. In the 19F NMR spectrum, the signal can be observed at -64.30 ppm for the CF3 group.
The data of compound 18 will be used as an example for assignments of the pyrazolo[1,5-
a]pyrimidine derivatives. In the 1H NMR spectrum, two singlets can be observed at 2.36 ppm and 2.42
ppm referring to the hydrogen of CH3 group. For H3 and H6, singlets were assigned at 6.22 ppm and
6.40 ppm, respectively. Hydrogens of phenyl rings are in the range of 7.01-7.78 ppm. The simplets at
10.02 ppm and 10.30 ppm were attributed to the hydrogens linked to the NH and SO2NH groups. In the
13C NMR spectra, the two carbons of CH3 group presented the signals at 14.1 ppm and 24.4 ppm. The
C3 and C6 carbons of the pyrazolo[1,5-a]pyrimidine ring showed a signal at 87.6 and 93.7 ppm,
respectively.
The data of compound 36 will be used as an example for the assignments of the quinoline
derivatives. H2 couple with H3 (J = 6.2 Hz) and these hydrogens were assigned as a doublet at 8.61
ppm and 7.09 ppm, respectively. H6 was assigned as a double doublet at 7.79 ppm by coupling with H5
and H8 (J = 9.1 Hz and J = 2.0 Hz). H5 and H8 were assigned as a doublet at 8.64 ppm and 8.08 ppm,
respectively. For hydrogen of NH and the sulfonamide group (SO2NH), singlets were assigned at 10.52
ppm and 9.97 ppm, respectively. For hydrogens of the OCH3 group, a singlet was assigned at 3.67 ppm.
In the NMR 13C spectra, the assignments were as follow: C2 at 146,9ppm, C3 at 102,5 ppm, C5 at 125,4
ppm, C6 at 126,8 ppm and C8 122,5 ppm. It was further confirmed by HSQC. Quaternary carbons were
identified with HSQC. The spectra are in the supplementary material.
Based on X-ray crystallographic analysis, the molecular structure of the derivative 17 was
confirmed and allowed us to determine that the 4-amino-N-arylbenzenesulfonamide moiety was
introduced at the 7 position of the [1,2,4]triazolo[1,5-a]pyrimidine ring. Fig. 2 shows the Oak Ridge
Thermal Ellipsoid Plot (ORTEP) diagram of this compound, and the crystal data and refinements are
provided in Table S1 (Supplementary Information).

Reagents and conditions: (i) toluene, 110 °C, 24 h, 70-97%; (ii) toluene, cat. p-TsOH, 110 °C, 20 h, 71-98%; (iii)
POCl3, 105 °C, 4 h, 51-97%; (iv) HSO3Cl, 1) 0 °C, 1 h; 2) 60 °C, 1 h, 77%; (v) CHCl3, TEA, 61 °C, 5 h, 42-87%;
(vi) 1) 6N HCl, 100 °C, 6 h; 2) 20% NaOH, 63-89%; (vii) EtOH, 78 °C, 2 h, 24-98%; (viii) HCl/H2O 1:1, 15
minutes.
Scheme 1: Synthetic route used to prepare compounds 4-38.

(17)
(7)
Fig. 2: Asymmetric unit representation of derivatives 7 and 17 (ellipsoids at 50% probability).
In addition, it was possible to identify those molecules that did not need a purification process,
such as derivative 7, which was found in the form of a hydrochloride salt, since the X-ray diffraction
study revealed protonation of the nitrogen atom N(4)–H(4)…Cl(1) of derivative 7, as shown Fig. 2. The
same did not apply to the purified molecules that have a neutral form, such as derivative 17.
It was not possible to observe that some compounds were present as hydrochlorides in the
analyses previously carried out. Therefore, based on the results of the X-ray analyses, it was decided to
standardize all compounds in the form of a hydrochloride. Initially, the molecules were dissolved in a
water/methanol (1:1) solution, and the respective pH was measured. The fact that there were molecules
in both the hydrochloride and free base forms was confirmed after analysis of the melting points (m.p.
values), when it was determined that the molecules had different patterns, some in the range of 174-195
°C and others in the range of 200-288 °C. The molecules with the lower m.p. values (174-195 °C) had a
neutral pH, and the molecules with higher m.p. values (200-288 °C) had a more acidic pH.
It was observed that the molecules that did not need to be purified were in the form of a
hydrochloride and presented a lower pH range (2.6-5.4), which was the case for derivative 7. The
molecules that were purified, either by recrystallization or by preparative TLC, lost their hydrochloride
form and presented a higher pH (6.5-7.8), which was the case for derivative 17. Thus, molecules with
pH values in the range of 6.5-7.8 were treated with a HCl/H2O (1:1) solution for 15 minutes and then
concentrated under vacuum. The respective hydrochlorides were used in biological tests.
2.2.
Biological evaluation
New compounds 4-38 were assayed against the P. falciparum 3D7 chloroquine-sensitive (CQS)
strain and human hepatoma cell line HepG2. Chloroquine and artesunate were used as positive controls
for inhibition (Table 1).

Comparing the [1,2,4]triazolo[1,5-a]pyrimidine derivatives 4-24, it can be seen that a substituent
at the 2 position of the [1,2,4]triazolo[1,5-a]pyrimidine ring influences activity. All compounds
containing the 2-trifluoromethyl group 18-24 were more potent than the corresponding unsubstituted 4-
10 and methylated 11-17 derivatives. This result reinforced the importance of the trifluoromethyl group
at the 2 position for anti-P. falciparum activity [18].
Analyses of the importance of the substituents at the 4 position of the 4-amino-N-
phenylbenzenesulfonamide moiety indicated that they play a significant role in the anti-P. falciparum
activity. 4-Trifluoromethylated compounds 9 (R = H, R1 = CF3; IC50 = 0.15 μM), 16 (R = CH3, R1 =
CF3; IC50 = 0.39 μM) and 23 (R = CF3, R1 = CF3; IC50 = 0.03 μM) showed enhanced inhibitory activity
(up to 61-fold) compared with unsubstituted analogues 4 (R = H, R1 = H; IC50 = 9.1 μM), 11 (R = CH3,
R1 = H; IC50 = 6.9 μM) and 18 (R = CF3, R1 = H; IC50 = 1.3 μM), which were the least potent analogues
among the triazolopyrimidine derivatives. The 4-trifluoromethylated compounds were also more potent
than analogues containing methoxy groups, such as 8 (R = H, R1 = R = OCH3; IC50 = 3.5 μM), 15 (R =
CH3, R1 = OCH3; IC50 = 4 μM) and 22 (R = CF3, R1 = OCH3; IC50 = 0.8 μM). The greater activity of the
trifluoromethylated derivatives was also seen after comparison with other substituents in the series. This
leads us to believe that electron withdrawing groups at the 4 position is favourable for inhibitory
activity. Another factor to be considered is the greater lipophilicity that these compounds have related to
the others, which is another factor attributed to the CF3 group [23].
Compounds containing a methoxy substituent (R1 = OCH3; 8, 15 and 22) did not show relevant
inhibitory activity when compared to the other compounds in the series; however, introduction of this
substituent increased the potency of the compounds 1.6- to 2.6-fold when compared with their
unsubstituted analogues. The introduction of a methyl at the 4 position significantly increased the
activity of the compounds (up to 40-fold), as seen when comparing the IC50 values of compounds 18 (R
= CF3, R1 = H; IC50 = 1.3 μM) and 21 (R = CF3, R1 = CH3; IC50 = 0.032 μM). The introduction of a
halogen, such as in 19 (R = CF3, R1 = Cl; IC50 = 0.4 μM) and 20 (R = CF3, R1 = F; IC50 = 0.086 μM),
was favourable for inhibitory activity when compared to the unsubstituted analogues. In this sense,
fluorinated derivative 20 was 15-fold more potent than 18.
The bulky N-(naphthalen-2-yl)benzenesulfonamide group present in compounds 10 (R = H; IC50 =
0.23 μM), 17 (R = CH3; IC50 = 0.64 μM) and 24 (R = CF3; IC50 = 0.05 μM) proved to be very important
for anti-P. falciparum activity, significantly increasing the activity of these compounds up to 39-fold
when compared to compounds 4 (R = H, R1 = H; IC50 = 9.1 μM), 11 (R = CH3, R1 = H; IC50 = 6.9 μM)
and 18 (R = CF3, R1 = H; IC50 = 1.3 μM). This finding reinforces the importance of the presence of a
bulky group with the [1,2,4]triazolo[1,5-a]pyrimidine ring for anti-P. falciparum activity.

Derivatives containing the pyrazolo[1,5-a]pyrimidine ring 25-31 were the least potent inhibitors.
Compounds 25 (R1 = H) and 30 (R1 = CF3), the only active compounds in this series, had IC50 values of
2.8 and 8.0 μM, respectively; however, they were less potent than the [1,2,4]triazolo[1,5-a]pyrimidine
and quinoline analogues. It is interesting to note that among the active compounds, derivative 30 is
trifluoromethylated. What is surprising is the activity of 25 and the loss of activity of the other
compounds in the series containing compounds 26-29, including derivative 31 that has the bulky N-
(naphthalen-2-yl)benzenesulfonamide group. This indicates that the [1,2,4]triazolo[1,5-a]pyrimidine
ring is important for activity in molecules containing a pattern of bulky substituents. Thus, we can
hypothesize that compounds containing a pyrazolo[1,5-a]pyrimidine nucleus are not effective with this
substitution pattern.
Quinoline derivatives 32-38 showed activity with IC50 values ranging from 0.89 to 4.6 μM.
Compounds 35 (R1 = CH3) and 38 (2-naphthyl) were the most potent, with IC50 values of 0.97 and 0.89
μM, respectively. Quinoline derivatives 32 (R1 = H; IC50 = 2.0 µM) and 36 (R1 = OCH3; IC50 = 2.8 µM)
were more potent than their respective unsubstituted or 2-methylated [1,2,4]triazolo[1,5-a]pyrimidine
derivatives; however, all of these derivatives showed decreased inhibitory activity compared to the 2-
trifluoromethylated [1,2,4]triazolo[1,5-a]pyrimidine derivatives 18-24.
Compound 23 (IC50 = 0.03 µM) was selected as a representative compound from this series for
cross-resistance evaluation. In this assay, a panel of drug-resistant strains of P. falciparum, including K1
(resistant to chloroquine), DD2 (resistant to chloroquine and mefloquine), IPC4912 (partial resistance to
artemisinin) and a laboratory-generated strain resistant to phosphatidylinositol 4-kinases PI4K inhibitors
(PI4KRES), was used. The evaluated ratios of IC50 values (IC50resistant/IC503D7) were equal to or less than
one (Fig. S1 and Table S3, Supplementary Information). These ratios suggest that the mechanism of
action of compound 23 is distinct from the standard antimalarials, thereby indicating no cross-resistance
against genetically diverse strains of the parasite.
In summary, [1,2,4]triazolo[1,5-a]pyrimidine hydrochlorides 4-24 showed in vitro inhibitory
activity against P. falciparum with IC50 values ranging from 0.030 to 9.1 μM and were not toxic in the
HepG2 cell assay. Compounds 20, 21, 23 and 24 were the most potent inhibitors, with IC50 values of
0.086, 0.032, 0.03 and 0.05 µM, respectively, being equipotent to chloroquine (IC50 = 0.03 µM).
Compound 23 stood out as the most potent and selective in the series with a selectivity index (SI) of
3000 and no signs of cross-resistance in a panel of drug-resistant strains of P. falciparum.
The [1,2,4]triazolo[1,5-a]pyrimidine scaffold is a privileged scaffold active against DHODH
enzymes [10]. Therefore, the P. falciparum and human DHODH (PfDHODH and HsDHODH,
respectively) inhibitory activities of compounds 4-31 were assessed (quinoline derivatives 32-38 were
not evaluated against PfDHODH because it is known that the quinoline moiety is not recognized by this

enzyme). The enzymatic inhibitory activity evaluation was carried out in two stages. The first stage
involved the evaluation of each compound’s inhibitory activity against both HsDHODH and PfDHODH
enzymes at a single concentration (50 µM), Table 2S (Supplementary Information). The compounds
that considerably inhibited the enzymatic activity (≥ 80% inhibition) were evaluated in a second step
(Table 2S, Supplementary Information). In this stage, IC50 values against PfDHODH were determined.
Compounds DSM265 [24] and ML390 [25] were used as position controls for PfDHODH and
HsDHODH inhibition, respectively. The results of the enzyme inhibition evaluation are shown in Table
1.
Compound 18 showed PfDHODH inhibition below 80% at 50 µM; for this reason, this compound
was not subjected to IC50 evaluation. The IC50 values of compounds 5, 8, 9, 11 and 23 were not
determined because it was not possible to measure consumption of the substrates at a certain
concentration on the IC50 curve.
The IC50 values of the [1,2,4]triazolo[1,5-a]pyrimidine derivatives 4-24 spanned from 0.08 to 1.3
μM. On the other hand, pyrazolo[1,5-a]pyrimidine derivatives 25-31 did not show significant
PfDHODH inhibition (25 to 66% at 50 µM), which is in line with the in vitro inhibitory activity against
P. falciparum. It is interesting to note that compounds 4-31 showed no significant inhibition against the
HsDHODH enzyme (0-30% at 50 µM), thereby indicating that they are selective inhibitors of the
PfDHODH enzyme.
The [1,2,4]triazolo[1,5-a]pyrimidines 20 (R1 = F, IC50 = 0.5 µM) and 21 (R1 = CH3; IC50 = 0.8
μM) had very close IC50 values to prototype 1 (IC50 = 0.7 µM) [19], and can be considered equipotent
bioactive compounds, whereas 22 (R1 = OCH3; IC50 = 0.21 μM), 24 (R1 = 2-naphthyl; IC50 = 0.22 μM),
and 19 (R1 = Cl; IC50 = 0.08 μM) were 3.5, 3.5 and 8.75-fold more potent than prototype 1, respectively.
It is worth mentioning that a reasonable correlation between enzymatic and whole parasite
inhibition was verified for the low micromolar and submicromolar inhibitors 4-18, suggesting that
PfDHODH is the main target underlying the inhibitory activity of this series. However, a difference in
the IC50 values of 5- to 25-fold against PfDHODH and the P. falciparum 3D7 strain was observed for
the most potent parasite inhibitors (20, 21 and 24, Table 2). These inconsistencies may be due to the
lower limit of detection of the enzymatic assay. In the inhibition assay, the PfDHODH enzyme was used
at a final concentration of 50 nM. Therefore, the enzyme concentration might have a significant effect
on the IC50 value assessment of the most potent inhibitors as their IC50 values approach the enzyme
concentration [26].

Table 1: In vitro inhibitory activity against P. falciparum parasites (3D7 strain, chloroquine-
sensitive), human hepatocarcinoma cells (HepG2), and PfDHODH, and the selectivity index (SI) values
for compounds 4-38. Chloroquine and artesunate were used as positive controls for whole parasite
inhibition.
Substituents IC503D7 (μM)
IC50 (μM)
PfDHODH
0.8 ± 0.1
>50
0.3 ± 0.1
0.41 ± 0.04
>50
>50
0.3 ± 0.1
>50
0.6 ± 0.1
1.0 ± 0.2
0.3 ± 0.2
1.3 ± 0.1
0.6 ± 0.3
0.4 ± 0.1
>50
0.08 ± 0.01
0.5 ± 0.1
0.8 ± 0.1
0.21 ± 0.06
>50
Compounds
IC50HepG2 (μM)
SI*
R1
P. falciparum
9.1 ± 0.9
3.0 ± 0.5
2.0 ± 0.4
0.50 ± 0.05
4 ± 1
0.15 ± 0.01
0.23 ± 0.01
6.9 ± 0.2
0.8 ± 0.1
2.7 ± 0.8
0.91 ± 0.09
4 ± 1
0.39 ± 0.06
0.64 ± 0.01
1.3 ± 0.4
0.4 ± 0.2
0.086 ± 0.004
0.032 ± 0.005
0.8 ± 0.1
0.030 ± 0.003
0.050 ± 0.002
2.8 ± 0.1
>10
R1 = H
R1 = Cl
R1 = F
R1 = CH3
R1 = OCH3
R1 = CF3
-
R1 = H
R1 = Cl
R1 = F
R1 = CH3
R1 = OCH3
R1 = CF3
-
R1 = H
R1 = Cl
R1 = F
R1 = CH3
R1 = OCH3
R1 = CF3
-
R1 = H
R1 = Cl
R1 = F
365 ± 10
92 ± 2
367 ± 12
367 ± 10
46 ± 2
>100
56 ± 2
193 ± 9
>50
150 ± 20
110 ± 10
170 ± 10
125 ± 12
56 ± 2
92 ± 4
47 ± 2
101 ± 7
54 ± 4
93 ± 6
90 ± 10
>100
> 40
>31
>187
>728
>13
>667
243
28
>62.5
55.6
121
42.5
321
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
87.4
>70
>123
1174
1688
>114
3000
>2000
9.6
0.22 ± 0.07
>50
27 ± 4
ND
ND
ND
ND
>50
>50
>10
R1 = CH3
R1 = OCH3
R1 = CF3
-
>10
>10
8.0 ± 1
>10
ND
ND
42 ± 4
ND
ND
ND
5.25
ND
>50
>50
>50
>50
28
29
30
31
R = H
R = Cl
R = F
R = CH3
R = OCH3
R = CF3
-
2.0 ± 0.2
1.7 ± 0.2
4.6 ± 0.6
0.97 ± 0.08
2.8 ± 0.4
2.1 ± 0.2
0.89 ± 0.06
0.03 ± 0.01
0.009 ± 0.002
92 ± 4
93 ± 5
92 ± 2
92 ± 6
92 ± 4
23 ± 1
13 ± 1
384 ± 62
267 ± 21
>46
>56
>21
>95
>33
11
15.1
12,800
29,666
-
-
-
-
32
33
34
35
36
37
38
-
-
-
-
chloroquine
artesunate
3D7
*SI = IC50HepG2/IC50
2.3.
Molecular docking
Molecular docking simulations were performed considering only the triazolo[1,5-a]pyrimidine
derivatives, which were active against P. falciparum and inhibited the PfDHODH enzyme (Table 1).

Validation of the docking protocol was carried out using the inhibitor DSM265 complexed to
PfDHODH (PDB code: 5BOO, Fig. S2A-B, Supplementary Information) [12]. Thus, redocking of this
inhibitor showed a root-mean-square deviation (RMSD) value of 1.16 Å, predicting the co-crystallized
binding pose correctly (Fig. S2A) with a MolDock score value of -169.45 arbitrary units (a.u.). The
inhibitor DSM265 interacts via H-bonds with Arg265 and His185 (H-bond energy = -5.56 a.u.) and
presents steric interactions with Arg265, His185 and Leu240 (steric interaction energy = -187.48 a.u.)
(Table S4 and Fig. S3A, Supplementary Information).
Consequently, the predicted complexes between the selected triazolo[1,5-a]pyrimidine inhibitors
and PfDHODH showed their lowest energy poses docked into the same binding pocket as the inhibitor
DSM265 (Fig. S2B). Hydrogen bonds (H-bonds) and steric interactions were also evaluated (Table S4
and Fig. S3A-P). Among the active compounds, only 19 (Fig. 3A-C), the most potent in the enzymatic
assay (IC50 = 0.08 μM), and 21 (Fig. S4A-D) (Supplementary Information) presented similar
interactions with the enzyme compared to those of DSM265. Thus, here, we described only the
interactions concerning these two inhibitors, with the others reported in the supplementary material (Fig.
S3A-P).
Interestingly, the best superposition of the triazolo[1,5-a]pyrimidine and the phenylamine groups
occurs between the predicted pose of 19 and the co-crystallized structure of DSM265. Thus, this
inhibitor interacts via H-bonds with the same residues as described for the inhibitor DSM265, Arg265
and His185 (H-bond energy = -3.85 a.u.), and shows steric interactions with Arg265, Cys233, His185,
Gly192, Leu240, Leu531, Leu197, Met536, and Phe188 (steric interaction energy = -196.20 a.u.) (Fig.
3B-C).
Inhibitor 21 also interacts via the same H-bonds as the inhibitor DSM265 (H-bond energy = -3.85
a.u.), and shows steric interactions with Arg265, Cys233, Gly192, His185, Leu197, Leu240, Leu531,
Met536, and Phe188 (steric interaction energy = -194.90 a.u.) (Table S4 and Fig. S3N). However, its
predicted pose shows superposition only with the triazolo[1,5-a]pyrimidine ring when compared to
DSM265.
Overall, the molecular docking results suggest that the active triazolo[1,5-a]pyrimidine derivatives
interact within at same binding site as DSM265. However, among the newly synthesized derivatives,
compound 19 presents the most similar binding mode as that described for DSM265, with a higher
interaction affinity for the PfDHODH enzyme and a negative interaction energy value (Table S4).
Therefore, the molecular docking simulations corroborate the tests carried out on the PfDHODH
enzyme since the most active triazolo[1,5-a]pyrimidine (19), which shows similar interactions and a
similar pose to that of DSM265, inhibited PfDHODH activity at low nanomolar concentrations (IC50 of
0.08 μM; Table 1).

Fig. 3: (A) Representation of the superposition between DSM265 (grey) and 19 (green)
complexed to PfDHODH. (B) Hydrogen-bonding interactions (B) and steric interactions (C) between 19
and the amino acid residues of PfDHODH. The structures are represented as sticks and coloured by
atom: nitrogen atoms in blue, sulfur atoms in yellow, fluorine atoms in pink, oxygen atoms in red, and
carbon atoms in grey, green or white.
3.
Conclusions
Among the 35 synthesized compounds, 30 exhibited in vitro inhibitory activity against P.
falciparum (3D7 strain) with IC50 values ranging from 0.030 to 9.1 µM. Of the three series synthesized,
the [1,2,4]triazolo[1,5-a]pyrimidine derivatives 4-24 were the most potent and showed no cytotoxicity.
Compounds 20 (R = CF3, R1 = F; IC50 = 0.086 μM), 21 (R = CF3, R1 = CH3; IC50 = 0.032 μM), 23 (R =

CF3, R1 = CF3; IC50 = 0.03 μM), and 24 (R = CF3, 2-naphthyl; IC50 = 0.05 μM) were equipotent to
chloroquine (IC50 = 0.03 μM), and compound 23 stood out as the most active in the series. Compounds
with a trifluoromethyl group at the 2 position of the [1,2,4]triazolo[1,5-a]pyrimidine ring were the most
potent among the four series. This finding is in good agreement with a previous structure-activity
relationship study [18].
Compounds 9 (R = H, R1 = CF3; IC50 = 0.150 μM), 16 (R = CH3, R1 = CF3; IC50 = 0.39 μM) and
23 (R = CF3, R1 = CF3; IC50 = 0.030 μM) containing a CF3 group at the 4 position of the 4-amino-N-
phenylbenzenesulfonamide moiety showed substantially increased activity (up to 61-fold) compared to
the unsubstituted analogues. These results indicated that CF3 groups, with greater lipophilicity at the 2
position of the [1,2,4]triazolo[1,5-a]pyrimidine ring and the 4 position of the 4-amino-N-
phenylbenzenesulfonamide moiety are attractive substituents to increase potency. Compound 20 (R =
CF3, R1 = F; IC50 = 0.086 μM) and compound 21 (R = CF3, R1 = CH3; IC50 = 0.032 μM) showed an
increase in activity by 15- and 40-fold, respectively, when compared with their unsubstituted (R1 = H)
analogues. In addition, the bulky 4-amino-N-(naphthalen-2-yl)benzenesulfonamide moiety proved to be
important for the inhibitory activity (up to 40-fold more potent when compared to the unsubstituted
analogue). Compound 23 (IC50 = 0.03 µM), a representative compound of this series, showed no cross-
resistance against genetically diverse strains of the parasite.
The series of pyrazolo[1,5-a]pyrimidine derivatives 25-31 were less potent than the
[1,2,4]triazolo[1,5-a]pyrimidine and quinoline analogues. These results suggest that the pyrazolo[1,5-
a]pyrimidine ring was not relevant for anti-P. falciparum activity among molecules that have this
pattern of substituents. A similar trend was observed for quinoline derivatives 32-38, which showed
decreased inhibitory activity related to the [1,2,4]triazolo[1,5-a]pyrimidine series. The most potent
quinoline derivatives were low micromolar inhibitors (35, R1 = CH3; IC50 = 0.97 μM and 38, 2-
naphthyl; IC50 = 0.89 μM).
The [1,2,4]triazolo[1,5-a]pyrimidine derivatives 4-24 selectively inhibited the PfDHODH enzyme,
confirming that this system is active against this target. These derivatives showed potent inhibitory
activity of the PfDHODH enzyme with IC50 values in the range of 1.3-0.08 μM. These derivatives did
not show significant inhibition against the HsDHODH enzyme (0-30% at 50 µM), thereby indicating
that they are selective for the parasite homologue. The potent in vitro activity against P. falciparum and
inhibition of the PfDHODH enzyme strongly suggest that this is the mechanism of action underlying
this series of new [1,2,4]triazolo[1,5-a]pyrimidine derivatives. Pyrazolo[1,5-a]pyrimidines 25-31 did not
significantly inhibit the PfDHODH enzyme or the parasite in the in vitro tests.
Molecular docking studies suggest that compounds 4-24 interact at the same site as DSM265, a
PfDHODH inhibitor, which is in phase II of clinical trials. Among the pyrazolo[1,5-a]pyrimidine

inhibitors, compound 19 presented the most similar binding mode when compared to DSM265 but
showed a higher interaction energy for the PfDHODH enzyme.
Thus, the most active inhibitor against PfDHODH 19 (R = CF3, R1 = Cl; IC50 = 0.08 μM -
PfDHODH) and the most active compounds against P. falciparum parasites 20, (R = CF3, R1 = F; IC50 =
0.086 μM; 21, R = CF3, R1 = CH3; IC50 = 0.032 μM; 23, R = CF3, R1 = CF3; IC50 = 0.030 μM; and 24, R
= CF3, 2-naphthyl; IC50 = 0.050 μM) stand out as the new prototypes of the group.
Overall, these results demonstrate the potential of [1,2,4]triazolo[1,5-a]pyrimidine derivatives as
inhibitors of the PfDHODH enzyme and represent new lead compounds for antimalarial drug discovery.
4.
Materials and methods
4.1.
Chemistry
All reagents and solvents used were of analytical grade. TLC (thin layer chromatography) was
performed using silica gel Merck TLC F-254 PTLC (Preparative TLC) Glass Plates (20×20 cm). The
melting points (m.p. values) were determined using a Buchi model B-545 apparatus. Electron-ionization
mass spectrometry (EI-MS, scan ES+capillary (3.0 kV)/cone (30 V)/extractor (1 V)/RF lens (1.0
V)/source temperature (150 °C)/desolvation temperature (300 °C)) spectra were recorded using a
Micromass/Waters spectrometer (model: ZQ-4000). High-resolution mass spectrometry (HRMS) data
were obtained using an LC-MS Bruker Daltonics MicroTOF (time of flight) analyser. Fourier transform
infrared (FTIR) absorption spectra were recorded on a Shimadzu mode IR Prestige-21
1
spectrophotometer using the attenuated total reflection (ATR) technique. H, 13C and 19F nuclear
magnetic resonance (NMR) spectra were obtained at 400.00, 100.00 and 376.00 MHz, respectively,
using a Bruker Avance instrument equipped with a 5 mm probe. Tetramethylsilane was used as the
internal standard. The chemical shifts (δ) are reported in ppm, and the coupling constants (J) are
reported in Hertz. Analysis by high-performance liquid chromatography (HPLC) was performed on an
LC-20AD Shimadzu liquid chromatograph using a Hypersil BDS C18 column (5 μm, 250 × 4.6 mm).
4.2.
Synthesis
4.2.1. General procedure for the preparation of 1H-1,2,4-triazole-5-amines 39a-c
A mixture of 6.8 g (0.05 mol) of aminoguanidine bicarbonate and 5 mL (0.066 mol) of the
appropriate acid (formic, acetic or trifluoroacetic acid) was kept under magnetic stirring until the release
of carbon dioxide was complete. Then, toluene (100 mL) was added, and the reaction mixture was
heated to reflux (110 °C) with a Dean-Stark apparatus under stirring for 24 h. A white precipitate
formed and was cooled, vacuum filtered and washed with cold toluene [20].

1H-1,2,4-triazol-5-amine (39a) Yield: 97%. m.p. 152-154 °C. IR (cm-1): 3399-3324; 1590-1533; 1631;
1
1266. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 5.77 (s; 2H; NH2); 7.44 (s; 1H; H3); 11.99 (s;
1H; NH). 13C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 147.7 (C3); 157.9 (C5). ESI [M + 1]+
85.47.
3-methyl-1H-1,2,4-triazol-5-amine (39b) Yield: 70%. m.p. 145-146 °C. IR (cm-1): 3419-3189; 1708;
1
1599-1544; 1255. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.05 (s; 3H; CH3); 5.49 (s; 2H;
NH2); 11.68 (s; 1H; NH).13C NMR (100 MHz, DMSO-d6,TMS, δ in ppm): 13.2 (CH3); 168.5 (C3);
172.7 (C5). ESI [M+1]+ 99.38.
3-(trifluoromethyl)-1H-1,2,4-triazol-5-amine (39c) Yield: 93%. m.p. 197 °C. IR (cm -1): 3487-3360;
1634; 1580-1490; 1361, 1177, 758. 1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 6.46 (s; 2H; NH2);
13
12.71 (s; 1H; NH). C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 119.6 (q; J = 267.1 Hz; CF3);
19
149.9 (q; J = 36.8 Hz; C3); 157.8 (C5). F NMR (376 MHz, DMSO-d6, TMS, δ in ppm): -64.78. ESI
[M + 1]+ 153.10.
4.2.2. General procedure for the preparation of 5-methyl[1,2,4]triazolo[1,5-a]pyrimidine-7(4H)-
ones 40a-c and 2,5-dimethylpyrazolo[1,5-a]pyrimidin-7(4H)-one (40d)
To 0.01 mol of the appropriate 1H-1,2,4-triazole-5-amines 39a-c or 5-methyl-1H-pyrazol-3-amine
(39d) was added 13 mL of ethyl acetoacetate (0.1 mol), 15 mL of toluene and a catalytic amount of p-
toluenesulfonic acid. The reaction mixture stirred under reflux (110 °C) for 24 h. The mixture was
cooled to room temperature, and the white precipitate formed was vacuum filtered and washed with cold
toluene [18,19].
5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (40a) Yield: 71%. m.p. 278-279 °C. IR (cm-1):
1
2807; 1697; 1620; 1335. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.32 (s; 3H; CH3); 5.83 (s;
13
1H; H6); 8.18 (s; 1H; H2). C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 18.6 (CH3); 98.1 (C6);
150.6 (C5); 151.7 (C3a); 151.8 (C2); 155.8 (C7). ESI [M-1]- 149.17.
2,5-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (40b) Yield: 95%. m.p. 313-314°C. IR (cm-1):
1
2703; 1692; 1641; 1323. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.29 (s; 3H; CH3’); 2.33 (s;
3H; CH3); 5.76 (d; 1H; J = 0.56 Hz; H6); 13.00 (s; 1H; NH). 13C NMR (100 MHz, DMSO-d6, TMS, δ in
ppm): 14.2 (CH3’); 18.6 (CH3); 98.1 (C6); 150.8 (C5); 151.0 (C3a); 155.5 (C2); 160.7 (C7). ESI [M-1]-
163.18.
5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (40c) Yield: 84%. m.p. 262-263
1
°C. IR (cm-1): 2887; 1694; 1605; 1399; 751. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.35 (d;
3H; J = 0.60 Hz; CH3); 5.99 (d; 1H; J = 0.72 Hz; H6); 13.58 (s; 1H; NH). 13C NMR (100 MHz, DMSO-
d6, TMS, δ in ppm): 18.6 (CH3); 99.1 (C6); 119.1 (q; J = 269.0 Hz; CF3); 151.4 (C5); 151.8 (q; J = 38.5
Hz; C2); 152.6 (C3a); 155.1 (C7).19F NMR (376 MHz, DMSO-d6, TMS, δ in ppm): -64.96. ESI [M-1]-
217.06.
2,5-dimethylpyrazolo[1,5-a]pyrimidin-7(4H)-one (40d) Yield: 98%. m.p. 253-255 °C. IR (cm-1): 1664;
1
1612; 1330. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.26 (d; 3H; J = 0.36 Hz; CH3); 2.27 (s;
13
3H; CH3’); 5.50 (d; 1H; J = 0.56 Hz; H6); 5.91 (s; 1H; H3); 12.10 (s; 1H; NH). C NMR (100 MHz,

DMSO-d6, TMS, δ in ppm): 14.0 (CH3’); 18.5 (CH3); 87.9 (C3); 94.8 (C6); 142.0 (C3a); 149.5 (C5);
151.6 (C2); 156.0 (C7). ESI [M+23]+ 186.0687.
4.2.3. General procedure for the preparation of 7-chloro-5-methyl-[1,2,4]triazolo[1,5-
a]pyrimidines 41a-c and 7-chloro-2,5-dimethylpyrazolo[1,5-a]pyrimidine (41d)
A mixture of 0.006 mol of the appropriate carbonylated intermediate 40a-d and 10 mL of POCl3
was stirred under reflux (105 °C) for 4 h. The reaction mixture was carefully poured into ice water and
made alkaline with 20% NaOH to reach pH 9-10. The mixture was diluted with water (50 mL) and
extracted with ethyl acetate (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate
and filtered, and the solvent was evaporated under vacuum [18,19].
7-chloro-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (41a) Yield: 51%. m.p. 139-140 °C. IR (cm-1):
1619; 1525; 868. 1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.63 (s; 3H; CH3); 7.66 (s; 1H; H6).
8.67 (s; 1H; H2). 13C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 4.4 (CH3); 111.8 (C6); 138.0 (C5);
155.2 (C3a); 155.5 (C2); 165.4 (C7). ESI [M+1]+ 169.10.
7-chloro-2,5-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine (41b) Yield: 67%. m.p. 143-145 ºC. IR (cm-1):
3065; 1610; 1519; 862. 1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.50 (s; 3H; CH3); 3.60 (s; 3H;
13
CH3’); 7.55 (s; 1H; H6). C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 14.6 (CH3’); 24.3 (CH3);
110.9 (C6); 137.2 (C5); 155.5 (C3a); 164.7 (C2); 165.0 (C7). ESI [M+1]+ 182.97.
7-chloro-5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidine (41c) Yield: 79%. m.p. 107-108
1
ºC. IR (cm-1): 3061; 1613; 1522; 877; 748. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.68 (s;
3H; CH3); 7.88 (s; 1H; H6). 13C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 24.6 (CH3); 113.9 (C6);
119.1 (q; J = 269.0 Hz; CF3); 138.8 (C5); 154.5 (q; J = 38.6 Hz; C2); 155.3 (C3a); 167.8 (C7). 19F NMR
(376 MHz, DMSO-d6, TMS, δ in ppm): -64.60. HRMS (ESI): [M+1]+ 237.0142.
7-chloro-2,5-dimethylpyrazolo[1,5-a]pyrimidine (41d) Yield: 97%. m.p. 51-53 °C. IR (cm-1): 1603;
1543; 831. 1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.43 (s; 3H; CH3’); 2.50 (s; 3H; CH3); 6.51
(s; 1H; H3); 7.22 (s; 1H; H6). 13C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 14.1 (CH3’); 23.8
(CH3); 95.9 (C3); 108.2 (C6); 136.3 (C3a); 149.4 (C5); 154.5 (C2); 158.4 (C7). ESI [M+1]+ 182.0451.
4.2.4. General procedure for the preparation of 4-acetamidobenzene-1-sulfonyl chloride (42)
Ten grams (0.073 mol) of N-phenylacetamide was slowly added to 24,6 mL of chlorosulfonic acid
(0.37 mol) with magnetic stirring. During the addition, an ice-water bath was used to keep the
temperature at approximately 0 °C; then, the bath was removed, and the temperature was raised to 60
°C. The reaction mixture was stirred and heated for 1 h. The mixture was then slowly poured into ice,
and the precipitate formed was vacuum filtered and washed with water. The product was used without
purification [21].
4-acetamidobenzene-1-sulfonyl chloride (42) Yield: 77%. HRMS (ESI): calc. for C8H7ClNO3S
231.9841; found [M-1]- 231.9861.

4.2.5. General procedure for the preparation of N-(4-(N-arylsulfamoyl)phenyl)acetamides 43a-g
A mixture of 2.3 g (0.01 mol) of 4-acetamidobenzenesulfonyl chloride (42), 1 equivalent of the
respective aniline, 5 mL of TEA (2.5 equivalents), and 25 mL of chloroform was kept with stirring
under reflux for 5 h. Then, the solvent was evaporated, and ice water was added to the mixture. The
precipitate formed was vacuum filtered, washed with distilled water and recrystallized from
ethanol/water (1:1).
N-(4-(N-phenylsulfamoyl)phenyl)acetamide (43a) Yield: 67%. m.p. 197-199 °C. IR (cm-1): 3445-3249;
1
1669; 1592; 1314-1150. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.04 (s; 3H; CH3); 6.93 (t;
1H; J = 7.4 Hz; H4’); 7.02 (dd; 2H; J = 1.2 Hz; J = 7.4 Hz; H2’, H6’); 7.16 (t; 2H; J = 7.4 Hz; H3’,
H5’); 7.66 (s; 4H; H2, H3, H5, H6); 10.26 (s; 1H; SO2NH). 13C NMR (100 MHz, DMSO-d6, TMS, δ in
ppm): 23.9 (CH3); 118.3 (C3, C5); 119.9 (C2’, C6’); 122.8 (C4’); 127.6 (C2, C6); 128.8 (C3’, C5’);
134.0 (C1); 139.2 (C1’); 142.5 (C4); 168.8 (C=O). HRMS (ESI): [M+23]+ 313.0612.
N-(4-(N-(4-chlorophenyl)sulfamoyl)phenyl)acetamide (43b) Yield: 42%. m.p. 194-195 °C. IR (cm-1):
3358-3192; 1682; 1601; 1326-1150; 845. 1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.05 (s; 3H;
CH3); 7.07 (d; 2H; J = 8.8 Hz; H2’, H6’); 7.28 (d; 2H; J = 8.8 Hz; H3’, H5’); 7.67 (d; 4H; J = 9.1 Hz;
13
H2, H6); 7.70 (d; 4H; J = 9.1 Hz; H3, H5); 10.30 (s; 1H; NH); 10.31 (s; 1H; SO2NH). C NMR (100
MHz, DMSO-d6, TMS, δ in p.p.m): 24.0 (CH3); 118.4 (C3, C5); 121.4 (C2’, C6’); 127.84 (C2, C6);
127.88 (C4’); 128.9 (C3’, C5’); 132.4 (C1); 136.7 (C1’); 143.1 (C4); 168.9 (C=O). HRMS (ESI): [M-1]-
323.0143.
N-(4-(N-(4-fluorophenyl)sulfamoyl)phenyl)acetamide (43c) Yield: 59%. m.p. 177-179 °C. IR (cm-1):
1
3356-3229; 1688; 1592; 1324-1147; 1091. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.05 (s;
3H; CH3); 7.07-7.06 (m; 4H; H2’, H3’, H5’, H6’); 7.63 (d; 2H; J = 7.0 Hz; H2, H6); 7.68 (d; 2H; J = 7.0
13
Hz; H3, H5); 10.09 (s; 1H; SO2NH); 10.29 (s; 1H; NH). C NMR (100 MHz, DMSO-d6, TMS, δ in
ppm): 24.1 (CH3); 115.8 (d; J = 22.3 Hz; C3’, C5’); 118.4 (C3, C5); 122.7 (d; J = 8.1 Hz; C2’, C6’);
127.9 (C2, C6); 132.6 (C1); 134.0 (d; J = 2.3 Hz; C1’); 143.1 (C4); 158.9 (d; J = 239.2 Hz; C4’); 169.0
(C=O). 19F NMR (376 MHz, DMSO-d6, TMS, δ in ppm): -118.57. HRMS (ESI): [M+23]+ 331.0462.
N-(4-(N-(p-tolyl)sulfamoyl)phenyl)acetamide (43d) Yield: 74%. m.p. 190-192 °C. IR (cm-1): 3352-
1
3199; 1683; 1594; 1397; 1327-1145. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.05 (s; 3H;
CH3); 2.17 (s; 3H; CH3’); 6.94 (d; 2H; J = 8.4 Hz; H2’, H6’); 7.01 (d; 2H; J = 8.4 Hz; H3’, H5’); 7.63
(d; 4H; J = 9.0 Hz; H2, H6); 7.67 (d; 4H; J = 9.0 Hz; H3, H5); 9.97 (s; 1H; SO2NH); 10.27 (s; 1H; NH).
13C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 20.2 (CH3’); 24.0 (CH3); 118.4 (C3, C5); 120.5 (C2’,
C6’); 127.8 (C2, C6); 129.4 (C3’, C5’); 133.0 (C4’); 133.2 (C1); 135.1 (C1’); 142.9 (C4); 168.9 (C=O).
HRMS (ESI): [M+23]+ 327.0778.
N-(4-(N-(4-methoxyphenyl)sulfamoyl)phenyl)acetamide (43e) Yield: 87%. m.p. 195-197 °C. IR (cm-1):
1
3302-3242; 1668; 1586; 1315-1156; 1032. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.05 (s;
3H; CH3); 3.66 (s; 3H; OCH3); 6.78 (d; 2H; J = 9.0 Hz; H2’, H6’); 6.95 (d; 2H; J = 9.0 Hz; H3’, H5’);
7.59 (d; 2H; J = 7.0 Hz; H2, H6); 7.67 (d; 2H; J = 7.0 Hz; H3, H5); 9.76 (s; 1H; SO2NH); 10.27 (s; 1H;
13
NH). C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 24.1 (CH3); 55.1 (OCH3); 114.2 (C3’, C5’);

118.4 (C3, C5); 123.4 (C2’, C6’); 127.9 (C2, C6); 130.2 (C1); 133.0 (C1’); 142.9 (C4); 156.4 (C4’);
168.9 (C=O). HRMS (ESI): [M+23]+ 343.0702.
N-(4-(N-(4-(trifluoromethyl)phenyl)sulfamoyl)phenyl) acetamide (43f) Yield: 44%. m.p. 185-188 °C.
IR (cm-1): 1673; 1591; 1320-1152. 1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.05 (s; 3H; CH3);
7.27 (d; 2H; J = 8.4 Hz; H2’, H6’); 7.59 (d; 2H; J = 8.6 Hz; H3’, H5’); 7.72 (d; 4H; J = 9.2 Hz; H2,
H6); 7.76 (d; 4H; J = 9.2 Hz; H3, H5); 10.33 (s; 1H; SO2NH); 10.77 (s; 1H; NH). 13C NMR (100 MHz,
DMSO-d6, TMS, δ in ppm): 24.0 (CH3); 118.52 (C3, C5); 118.58 (C2', C6'); 123.4 (q; J = 31.8 Hz;
C4’); 124.1 (q; J = 269.7 Hz; CF3); 126.4 (q; J = 3.7 Hz; C3', C5'); 127.9 (C2, C6) 132.4 (C1); 141.5
(C1’); 143.3 (C4); 168.9 (C=O). 19F NMR (376 MHz, DMSO-d6, TMS, δ in ppm): -60.42. HRMS (ESI):
[M-1]- 357.0459.
N-(4-(N-(naphthalen-2-yl)sulfamoyl)phenyl)acetamide (43g) Gross Yield: 61%. HRMS (ESI): calc. for
[M-1]- C18H15N2O3S 339,0803; found [M-1]- 339,0812 (used without purification).
4.2.6. General procedure for the preparation of 4-amino-N-arylbenzenesulfonamides (44a-g)
A mixture of 0.006 mol of the respective N-(4-(N-arylsulfamoyl)phenyl)acetamide 43a-g in 30 mL
of 6N HCl was kept with stirring under reflux (100 °C) for 6 h. Then, the solution was cooled and
neutralized with 20% NaOH. The precipitate formed was vacuum filtered, washed with water and
recrystallized from ethanol/water (1:1).
4-amino-N-phenylbenzenesulfonamide (44a) Yield: 81%. m.p. 189 °C. IR (cm-1): 3250-3219; 1593;
1
1300-1147. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 5.95 (s; 2H; NH2); 6.51 (d; 2H; J = 8.7
Hz; H3, H5); 6.96 (t; 1H; J = 7.5 Hz; H4’); 7.05 (dd; 2H; J = 0.9 Hz; J = 7.5 Hz; H2’, H6’) 7.19 (t; 2H;
13
J = 7.5 Hz; H3’, H5’); 7.37 (d; 2H; J = 8.7 Hz; H2, H6); 9.83 (s; 1H; SO2NH). C NMR (100 MHz,
DMSO-d6, TMS, δ in ppm): 112.5 (C3, C5); 119.3 (C2’, C6’); 123.2 (C4’); 124.3 (C1); 128.6 (C3’,
C5’); 128.9 (C2, C6); 138.4 (C1’); 152.7 (C4). HRMS (ESI): [M+23]+ 271.0531.
4-amino-N-(4-chlorophenyl)benzenesulfonamide (44b) Yield: 75%. m.p. 191-193 °C. IR (cm-1): 3413-
3341; 1594; 1312-1147; 817. 1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 5.99 (s; 2H; NH2); 6.53
(d; 2H; J = 8.1 Hz; H3, H5); 7.06 (d; 2H; J = 8.2 Hz; H2’, H6’); 7.24 (d; 2H; J = 8.2 Hz; H3’, H5’); 7.37
(d; 2H; J = 8.1 Hz; H2, H6); 10.00 (s; 1H; SO2NH). 13C NMR (100 MHz, DMSO-d6, TMS, δ in ppm):
112.4 (C3, C5); 120.8 (C2’, C6’); 123.8 (C1); 127.1 (C4’); 128.6 (C2, C6); 128.8 (C3’, C5’); 137.3
(C1’); 152.8 (C4). HRMS (ESI): [M+23]+ 305.0214.
4-amino-N-(4-fluorophenyl)benzenesulfonamide (44c) Yield: 84%. m.p. 163-164 °C. IR (cm-1): 3391-
3345; 1593; 1308-1146; 1088. 1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 5.96 (s; 2H; NH2); 6.51
(d; 2H; J = 8.7 Hz; H3, H5); 7.05 (d; 4H; J = 6.8 Hz; H2’, H3’, H5’, H6’); 7.33 (d; 2H; J = 8.7 Hz; H2,
H6); 9.77 (s; 1H; SO2NH). 13C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 112.4 (C3, C5); 115.5 (d;
J = 22.4 Hz; C3’, C5’); 121.9 (d; J = 8.1 Hz; C2’, C6’); 123.9 (C1); 128.5 (C2, C6); 134.6 (d; J = 2.2
Hz; C1’); 152.7 (C4); 158.5 (d; J = 238.5 Hz; C4’). 19F NMR (376 MHz, DMSO-d6, TMS, δ in ppm): -
119.43. HRMS (ESI): [M+23]+ 289.0442.
4-amino-N-(p-tolyl)benzenesulfonamide (44d) Yield: 89%. m.p. 187-188 °C. IR (cm-1): 3411-3343;
1
1594; 1397; 1317-1147. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.17 (s; 3H; CH3); 5.92 (s;
2H; NH2); 6.50 (d; 2H; J = 8.7 Hz; H3, H5); 6.93 (d; 2H; J = 8.4 Hz; H2’, H6’); 6.99 (d; 2H; J = 8.4 Hz;

13
H3’, H5’); 7.34 (d; 2H; J = 8.7 Hz; H2, H6); 9.65 (s; 1H; SO2NH). C NMR (100 MHz, DMSO-d6,
TMS, δ in ppm): 20.2 (CH3); 112.4 (C3, C5); 119.9 (C2’, C6’); 124.4 (C1); 128.6 (C3’, C5’); 129.3 (C2,
C6); 132.4 (C4’); 135.8 (C1’); 152.6 (C4). HRMS (ESI): [M+23]+ 285.0681.
4-amino-N-(4-methoxyphenyl)benzenesulfonamide (44e) Yield: 66%. m.p. 194-195 °C. IR (cm-1):
1
3401-3258; 1589; 1317-1149; 1031. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 3.65 (s; 3H;
OCH3); 5.91 (s; 2H; NH2); 6.50 (d; 2H; J = 8.7 Hz; H3, H5); 6.76 (d; 2H; J = 8.9 Hz; H2’, H6’); 6.94
13
(d; 2H; J = 8.9 Hz; H3’, H5’); 7.2 (d; 2H; J = 8.7 Hz; H2, H6); 9.44 (s; 1H; SO2NH). C NMR (100
MHz, DMSO-d6, TMS, δ in ppm): 55.0 (OCH3); 112.4 (C3, C5); 114.0 (C3’, C5’); 122.8 (C2’, C6’);
124.4 (C1); 128.6 (C2, C6); 131.0 (C1’); 152.6 (C4); 156.0 (C4’). HRMS (ESI): [M+23]+ 301.0631.
4-amino-N-(4-(trifluoromethyl)phenyl)benzenesulfonamide (44f) Yield: 63%. m.p. 165-167 °C. IR (cm-
1
1): 3348; 1592; 1316-1144; 731. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 6.04 (s; 2H; NH2);
6.54 (d; 2H; J = 8.7 Hz; H3, H5); 7.23 (d; 2H; J = 8.5 Hz; H2’, H6’); 7.44 (d; 2H; J = 8.7 Hz; H2, H6);
7.57 (d; 2H; J = 8.6 Hz; H3’, H5’); 10.45 (s; 1H; SO2NH). 13C NMR (100 MHz, DMSO-d6, TMS, δ in
ppm): 112.5 (C3, C5); 117.9 (C2', C6'); 122.7 (q; J = 31.8 Hz; C4’); 123.5 (C1); 124.2 (q; J = 269.6 Hz;
19
CF3); 126.2 (q; J = 3.8 Hz; C3', C5'); 128.6 (C2, C6); 142.1 (C1’); 153.0 (C4). F NMR (376 MHz,
DMSO-d6, TMS, δ in ppm): -60.29.HRMS (ESI): [M-1]- 315.0314.
4-amino-N-(naphthalen-2-yl)benzenesulfonamide (44g) Yield: 67%. m.p. 207-208 °C. IR (cm-1): 3412-
1
3341; 1594; 1307-1142. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 5.93 (s; 2H; NH2); 6.49 (d;
2H; J = 8.7 Hz; H3, H5); 7.27 (dd; 1H; J = 2.1 Hz; J = 8,8 Hz; H4’); 7.38-7.34 (m; 1H; H5’); 7.43 (d;
3H; J = 8.7 Hz; H2, H6); 7.45-7.40 (m; 3H; H8’); 7.51 (d; 1H; J = 1.76 Hz; H1’); 7.78-7.72 (m; 3H;
13
H3’, H6’, H7’); 10.10 (s; 1H; SO2NH). C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 112.5 (C3,
C5); 114.9 (C1'); 120.0 (C4’); 124.2 (C1); 124.6 (C5’); 126.5 (C8'); 127.0 (C6’ or C7’); 127.4 (C6’ or
C7’); 128.7 (C2, C6, C3’); 129.6 (C4a’); 133.2 (C8a’); 136.1 (C2’); 152.8 (C4). EM (ESI): [M-1]-
296.97.
4.2.7. General procedure for the preparation of 4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-
yl)amino)-N-arylbenzenesulfonamide hydrochlorides (4-24), 4-((2,5-dimethylpyrazolo[1,5-a]pyrimidin-
7-yl)amino)-N-arylbenzenesulfonamide hydrochlorides (25-31) and 4-((7-chloroquinolin-4-yl)amino)-
N-arylbenzenesulfonamide hydrochlorides (32-38)
A mixture of 0.001 mol of compound (41a-d or 45), 1 equivalent of the respective 4-amino-N-
arylbenzenesulfonamides (44a-g) and 3 mL of ethanol was stirred and refluxed (78 °C) for 2 h. The
reaction mixture was poured into ice water, and the precipitate formed was vacuum filtered and washed
with water. The precipitate was purified by recrystallization from methanol/water (2:1) or
chromatography TLC Glass Plates (Merck TLC Silica gel 60 RP-18 F254s) in chloroform/methanol
(9.5:0.5). After purification, the respective hydrochloride was prepared, and the molecules were
solubilized and stirred for 15 minutes in a 1:1 solution of HCl/H2O and concentrated under vacuum [17–
19].

4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)-N-phenylbenzenesulfonamide hydrochloride
1
(4) Yield: 48%. m.p. 250-252 °C. IR (cm-1): 3036; 1655; 1608; 1332-1153. H NMR (400 MHz,
DMSO-d6, TMS, δ in ppm): 2.49 (s; 3H; CH3); 6.75 (s; 1H; H6); 7.04 (t; 1H; J = 7.3 Hz; H4’’); 7.15 (d;
2H; J = 7.5 Hz; H2’’, H6’’); 7.25 (t; 2H; J = 7.4 Hz; H3’’, H5’’); 7.63 (d; 2H; J = 8.7 Hz; H2’, H6’);
7.86 (d; 2H; J = 8.7 Hz; H3’, H5’); 8.76 (s; 1H; H2) 10.45 (s; 1H; SO2NH). 13C NMR (100 MHz,
DMSO-d6, TMS, δ in ppm): 23.5 (CH3); 91.6 (C6); 119.8 (C2’’, C6’’); 123.6 (C2’, C6’); 123.9 (C4’’);
128.2 (C3’, C5’); 129.1 (C3’’, C5’’); 136.3 (C1’); 137.5 (C1’’); 140.5 (C4’); 145.5 (C5); 152.5 (C3a);
152.6 (C2); 164.1 (C7). HRMS (ESI): calc. for C18H17N6O2S 381.1128; found [M+1]+ 381.1115. HPLC:
98%.
N-(4-chlorophenyl)-4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)benzenesulfonamide
hydrochloride (5) Yield: 98%. m.p. 286-288 °C. IR (cm-1): 3100-3034; 1655; 1605; 1327-1156; 826. 1H
NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.50 (s; 3H; CH3); 6.78 (s; 1H; H6); 7.17 (d; 2H; J = 8.9
Hz; H2’’, H6’’); 7.32 (d; 2H; J = 8.9 Hz; H3’’, H5’’); 7.64 (d; 2H; J = 8.7 Hz; H2’, H6’); 7.86 (d; 2H; J
= 8.7 Hz; H3’, H5’); 8.79 (s; 1H; H2) 10.65 (s; 1H; SO2NH). 13C NMR (100 MHz, DMSO-d6, TMS, δ
in ppm): 23.5 (CH3); 91.7 (C6); 121.4 (C2’’, C6’’); 123.6 (C2’, C6’); 128.0 (C4’’); 128.2 (C3’, C5’);
129.0 (C3’’, C5’’); 135.9 (C1’); 136.5 (C1’’); 140.6 (C4’); 145.5 (C5); 152.41 (C3a); 152.48 (C2);
164.1 (C7). HRMS (ESI): calc. for C18H16ClN6O2S 415.0738; found [M+1]+ 415.0729. HPLC: 98.5%.
N-(4-fluorophenyl)-4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)benzenesulfonamide
1
hydrochloride (6) Yield: 79%. m.p. 274-276 °C. IR (cm-1): 3293; 1624; 1587; 1333-1154; 1090. H
NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.46 (s; 3H; CH3); 6.68 (s; 1H; H6); 7.14-7.08 (m; 4H;
H2’’, H3’’, H5’’; H6’’); 7.63 (d; 2H; J = 8.5 Hz; H2’, H6’); 7.77 (d; 2H; J = 8.5 Hz; H3’, H5’); 8.53 (s;
13
1H; H2) 10.29 (s; 1H; SO2NH); 10.47 (s; 1H; NH). C NMR (100 MHz, DMSO-d6, TMS, δ in ppm):
24.6 (CH3); 90.6 (C6); 115.8 (d; J = 22.4 Hz; C3’’, C5’’) 122.5 (d; J = 8.2 Hz; C2’’, C6’’); 122.7 (C2’,
C6’); 128.1 (C3’, C5’); 133.7 (d; J = 2.5 Hz; C1’’); 134.9 (C1’); 141.3 (C4’); 144.3 (C5); 154.3 (C2);
155.3 (C3a); 158.9 (d; J = 239.5 Hz; C4’’); 164.6 (C7). 19F NMR (376 MHz, DMSO-d6, TMS, δ in
ppm): -118.41. HRMS (ESI): calc. for C18H16FN6O2S 399.1033; found [M+1]+ 399.1035. HPLC: 99.7%.
4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)-N-(p-tolyl)benzenesulfonamide hydrochloride
1
(7) Yield: 83%. m.p. 266-267 °C. IR (cm-1): 3103; 1659; 1614; 1385; 1339-1190. H NMR (400 MHz,
DMSO-d6, TMS, δ in ppm): 2.19 (s; 3H; CH3’); 2.50 (s; 3H; CH3); 6.76 (s; 1H; H6); 7.04 (d; 4H; J =
9.0 Hz; H2’’, H3’’, H5’’, H6’’); 7.62 (d; 2H; J = 8.7 Hz; H2’, H6’); 7.83 (d; 2H; J = 8.7 Hz; H3’, H5’);
8.79 (s; 1H; H2) 10.28 (s; 1H; SO2NH). 13C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 20.3 (CH3’)
23.5 (CH3); 91.8 (C6); 120.5 (C2’’, C6’’); 123.7 (C2’, C6’); 128.3 (C3’, C5’); 129.6 (C3’’, C5’’) 133.4
(C4’’); 134.9 (C1’); 136.5 (C1’’); 140.4 (C4’); 145.7 (C5); 152.4 (C3a); 152.5 (C2); 164.2 (C7). HRMS
(ESI): calc. for C19H19N6O2S 395.1284; found [M+1]+ 395.1283. HPLC: 98.5%.
N-(4-methoxyphenyl)-4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)benzenesulfonamide
hydrochloride (8) Yield: 53%. m.p. 261-262 °C. IR (cm-1): 3100-3034; 1655; 1605; 1327-1156; 826. 1H
NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.50 (s; 3H; CH3); 3.67 (s; 3H; OCH3); 6.75 (s; 1H; H6);
6.82 (d; 2H; J = 9.0 Hz; H2’’, H6’’); 7.03 (d; 2H; J = 9.0 Hz; H3’’, H5’’); 7.62 (d; 2H; J = 8.7 Hz; H2’,
H6’); 7.79 (d; 2H; J = 8.7 Hz; H3’, H5’); 8.78 (s; 1H; H2) 10.07 (s; 1H; SO2NH). 13C NMR (100 MHz,
DMSO-d6, TMS, δ in ppm): 23.6 (CH3); 55.1 (OCH3); 91.7 (C6); 114.3 (C3’’, C5’’); 123.4 (C2’, C6’);
123.6 (C2’’, C6’’); 128.3 (C3’, C5’); 130.0 (C1’); 136.4 (C1’’); 140.3 (C4’); 145.7 (C5); 152.5 (C3a);

152.6 (C2); 156.5 (C4’’); 164.2 (C7). HRMS (ESI): calc. for C19H19N6O3S 411.1233; found [M+1]+
411.1236. HPLC: 99.2%.
4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)-N-(4-
(trifluoromethyl)phenyl)benzenesulfonamide hydrochloride (9) Yield: 25%. m.p. 265-268 °C. IR (cm-1):
3266; 1621; 1589; 1325-1150; 773. 1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.45 (s; 3H; CH3);
6.68 (s; 1H; H6); 7.27 (d; 2H; J = 8.5 Hz; H2’’, H6’’); 7.58 (d; 2H; J = 8.6 Hz; H3’’, H5’’); 7.63 (d; 2H;
J = 8.7 Hz; H2’, H6’); 7.87 (d; 2H; J = 8.7 Hz; H3’, H5’); 8.52 (s; 1H; H2) 10.45 (s; 1H; NH); 10.60 (s;
13
1H; SO2NH). C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 24.6 (CH3); 90.5 (C6); 118.6 (C2’’,
C6’’); 122.4 (q; J = 32.8 Hz; C4’’); 122.8 (C2’, C6’); 124.2 (q; J = 269.5; CF3); 126.3 (q; J = 3.7 Hz;
C3’’, C5’’); 128.0 (C3’, C5’); 135.9 (C1’); 141.4 (C4’); 143.1 (C1’’); 144.4 (C5); 154.3 (C2); 155.3
(C3a); 164.5 (C7). 19F NMR (376 MHz, DMSO-d6, TMS, δ in ppm): -60.21. HRMS (ESI): calc.
C19H16F3N6O2S 449.1002; found [M+1]+ 449.1010. HPLC: 99.3%.
4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)-N-(naphthalen-2-yl)benzenesulfonamide
1
hydrochloride (10) Yield: 32%. m.p. 264-265 °C. IR (cm-1): 3287; 1621; 1569; 1320-1153. H NMR
(400 MHz, DMSO-d6, TMS, δ in ppm): 2.43 (s; 3H; CH3); 6.65 (s; 1H; H6); 7.34 (dd; 1H; J = 2.16 Hz;
J = 8.8 Hz; H4’’); 7.41-7.37 (m; 1H; H5’’); 7.47-7.43 (m; 1H; H8’’); 7.62-7.61 (m; 3H; H2’, H6’,
H1’’); 7.80-7.78 (m; 3H; H3’’, H6’’, H7’’); 7.87 (d; 2H; J = 8.7 Hz; H3’, H5’); 8.50 (s; 1H; H2); 10.43
(s; 1H; NH); 10.58 (s; 1H; SO2NH). 13C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 24.6 (CH3); 90.5
(C6); 115.7 (C1’’); 120.1 (C4’’); 122.7 (C2’, C6’); 124.9 (C5’’); 126.5 (C8’’); 127.0 (C6’’ ou C7’’);
127.4 (C6’’ ou C7’’); 128.1 (C3’, C5’); 129.0 (C3’’); 129.8 (C4a’’); 133.1 (C8a’’); 135.20 (C1’);
135.25 (C2’’); 141.3 (C4’); 144.2 (C5); 154.3 (C2); 155.3 (C3a); 164.6 (C7). HRMS (ESI): calc. for
C22H18N6O2S 431.1285; found [M+1]+ 431.1286. HPLC: 98.3%
4-((2,5-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)-N-phenylbenzenesulfonamide
1
hydrochloride (11) Yield: 27%. m.p. 256-258 °C. IR (cm-1): 3380; 1626; 1586; 1339-1162. H NMR
(400 MHz, DMSO-d6, TMS, δ in ppm): 2.42 (s; 3H; CH3’) 2.47 (s; 3H; CH3); 6.62 (s; 1H; H6); 7.03 (t;
1H; J = 7.4 Hz; H4’’); 7.12 (d; 2H; J = 7.6 Hz; H2’’, H6’’); 7.24 (t; 2H; J = 7.5 Hz; H3’’, H5’’); 7.60
(d; 2H; J = 8.7 Hz; H2’, H6’); 7.80 (d; 2H; J = 8.7 Hz; H3’, H5’); 10.33 (s; 1H; SO2NH); 10.40 (s; 1H;
NH). 13C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 14.7 (CH3’); 24.7 (CH3); 90.3 (C6); 119.8
(C2’’, C6’’); 122.6 (C2’, C6’); 124.0 (C4’’); 128.2 (C3’, C5’); 129.2 (C3’’, C5’’); 135.2 (C1’); 137.7
(C1’’); 141.5 (C4’); 143.8 (C5); 155.8 (C3a); 163.6 (C2); 164.1 (C7). HRMS (ESI): calc. for
C19H19N6O2S 395.1284; found [M+1]+ 395.1269. HPLC: 99.9%
N-(4-chlorophenyl)-4-((2,5-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)benzenesulfonamide
hydrochloride (12) Yield: 44%. m.p. 270-271 °C. IR (cm-1): 3371-3099; 1620; 1586; 1338-1160; 833.
1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.43 (s; 3H; CH3’) 2.48 (s; 3H; CH3); 6.64 (s; 1H;
H6); 7.14 (d; 2H; J = 8.8 Hz; H2’’, H6’’); 7.32 (d; 2H; J = 8.8 Hz; H3’’, H5’’); 7.62 (d; 2H; J = 8.6 Hz;
H2’, H6’); 7.80 (d; 2H; J = 8.7 Hz; H3’, H5’); 10.43 (s; 1H; NH); 10.50 (s; 1H; SO2NH). 13C NMR (100
MHz, DMSO-d6, TMS, δ in ppm): 14.5 (CH3’); 24.6 (CH3); 90.3 (C6); 121.2 (C2’’, C6’’); 122.5 (C2’,
C6’); 127.9 (C4’’); 128.1 (C3’, C5’); 129.1 (C3’’, C5’’); 134.7 (C1’); 136.6 (C1’’); 141.6 (C4’); 143.7
(C5); 155.7 (C3a); 163.5 (C2); 164.0 (C7). HRMS (ESI): calc. for C19H18ClN6O2S 429.0894; found
[M+1]+ 429.0901. HPLC: 100%.
4-((2,5-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)-N-(4-fluorophenyl)benzenesulfonamide
1
hydrochloride (13) Yield: 25%. m.p. 252-254 °C. IR (cm-1): 1625; 1559; 1328-1157; 1095. H NMR

(400 MHz, DMSO-d6, TMS, δ in ppm): 2.43 (s; 3H; CH3’); 2.48 (s; 3H; CH3); 6.62 (s; 1H; H6); 7.15-
7.08 (m; 4H; H2’’, H3’’, H5’’; H6’’); 7.60 (d; 2H; J = 8.7 Hz; H2’, H6’); 7.76 (d; 2H; J = 8.7 Hz; H3’,
H5’); 10.29 (s; 1H; SO2NH); 10.43 (s; 1H; NH). 13C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 14.5
(CH3’); 24.5 (CH3); 90.2 (C6); 115.8 (d; J = 22.5 Hz; C3’’, C5’’) 122.4 (d; J = 7.1 Hz; C2’’, C6’’);
122.5 (C2’, C6’); 128.1 (C3’, C5’); 133.7 (d; J = 2.5 Hz; C1’’); 134.8 (C1’); 141.4 (C4’); 143.7 (C5);
19
155.6 (C3a); 158.9 (d; J = 239.5 Hz; C4’’); 163.4 (C2); 164.0 (C7). F NMR (376 MHz, DMSO-d6,
TMS, δ in ppm): -118.45. HRMS (ESI): calc. for C19H18FN6O2S 413.1190; found [M+1]+ 413.1184.
HPLC: 100%.
4-((2,5-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)-N-(p-tolyl)benzenesulfonamide
hydrochloride (14) Yield: 29%. m.p. 257-260 °C. IR (cm-1): 3372-3128; 1621; 1562; 1387; 1337-1158.
1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.18 (s; 3H; CH3’’); 2.42 (s; 3H; CH3’); 2.47 (s; 3H;
CH3); 6.61 (s; 1H; H6); 7.05-6.99 (m; 4H; H2’’, H3’’, H5’’, H6’’); 7.59 (d; 2H; J = 8.6 Hz; H2’, H6’);
13
7.76 (d; 2H; J = 8.6 Hz; H3’, H5’); 10.16 (s; 1H; SO2NH); 10.40 (s; 1H; NH). C NMR (100 MHz,
DMSO-d6, TMS, δ in ppm): 14.6 (CH3’); 20.2 (CH3’’) 24.7 (CH3); 90.2 (C6); 120.3 (C2’’, C6’’); 122.6
(C2’, C6’); 128.1 (C3’, C5’); 129.6 (C3’’, C5’’) 133.3 (C4’’); 135.0 (C1’); 135.2 (C1’’); 141.4 (C4’);
143.8 (C5); 155.9 (C3a); 163.6 (C2); 164.1 (C7). HRMS (ESI): calc. for C20H21N6O2S 409.1441; found
[M+1]+ 409.1441. HPLC: 98%.
4-((2,5-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)-N-(4-methoxyphenyl)benzenesulfonamide
1
hydrochloride (15) Yield: 82%. m.p. 281-284 °C. IR (cm-1): 1656; 1578; 1332-1159; 1025. H NMR
(400 MHz, DMSO-d6, TMS, δ in ppm): 2.49 (s; 3H; CH3’); 2.56 (s; 3H; CH3); 3.67 (s; 3H; OCH3); 6.76
(s; 1H; H6); 6.82 (d; 2H; J = 9.0 Hz; H2’’, H6’’); 7.03 (d; 2H; J = 9.0 Hz; H3’’, H5’’); 7.59 (d; 2H; J =
13
8.7 Hz; H2’, H6’); 7.79 (d; 2H; J = 8.7 Hz; H3’, H5’); 10.09 (s; 1H; SO2NH); 11.09 (s;1H; NH). C
NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 13.4 (CH3’); 23.5 (CH3); 55.0 (OCH3); 92.2 (C6); 114.2
(C3’’, C5’’); 123.2 (C2’, C6’); 123.5 (C2’’, C6’’); 128.2 (C3’, C5’); 129.9 (C1’); 136.4 (C1’’); 140.0
(C4’); 145.2 (C5); 151.5 (C4’’); 156.4 (C3a); 160.3 (C2); 164.4 (C7). HRMS (ESI): calc. for
C20H21N6O3S 425.1390; found [M+1]+ 425.1387. HPLC: 99.2%.
4-((2,5-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)-N-(4-
(trifluoromethyl)phenyl)benzenesulfonamide hydrochloride (16) Yield: 30%. m.p. 250-252 °C. IR (cm-
1
1): 3369; 1621; 1562; 1321-1157; 846. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.42 (s; 3H;
CH3’); 2.47 (s; 3H; CH3); 6.63 (s; 1H; H6); 7.30 (d; 2H; J = 8.4 Hz; H2’’, H6’’); 7.61 (d; 2H; J = 8.4
Hz; H3’’, H5’’); 7.62 (d; 2H; J = 8.6 Hz; H2’, H6’); 7.87 (d; 2H; J = 8.8 Hz; H3’, H5’); 10.43 (s; 1H;
SO2NH); 10.91 (s; 1H; NH). 13C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 14.7 (CH3’); 24.6 (CH3);
90.4 (C6); 118.6 (C2’’, C6’’); 122.7 (C2’, C6’); 123.2 (q; J = 32.0 Hz; C4’’); 124.2 (q; J = 269.6; CF3);
126.5 (q; J = 3.7 Hz; C3’’, C5’’); 128.2 (C3’, C5’); 135.1 (C1’); 141.8 (C4’); 142.2 (C1’’); 143.8 (C5);
19
155.8 (C3a); 163.6 (C2); 164.1 (C7). F NMR (376 MHz, DMSO-d6, TMS, δ in ppm): -60.28. HRMS
(ESI): calc. for C20H18F3N6O2S 463.1158; found [M+1]+ 463.1147. HPLC: 98%.
4-((2,5-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)-N-(naphthalen-2-yl)benzenesulfonamide
1
hydrochloride (17) Yield: 27%. m.p. 246-248 °C. IR (cm-1): 3335; 1620; 1563; 1308-1151. H NMR
(400 MHz, DMSO-d6, TMS, δ in ppm): 2.40 (s; 3H; CH3’); 2.45 (s; 3H; CH3); 6.58 (s; 1H; H6); 7.33
(dd; 1H; J = 2.16 Hz; J = 8.8 Hz; H4’’); 7.41-7.37 (m; 1H; H5’’); 7.47-7.43 (m; 1H; H8’’); 7.61-7.58
(m; 3H; H2’, H6’, H1’’); 7.83-7.78 (m; 3H; H3’’, H6’’, H7’’); 7.86 (d; 2H; J = 8.7 Hz; H3’, H5’); 10.38
(s; 1H; NH); 10.57 (s; 1H; SO2NH). 13C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 14.6 (CH3); 24.7

(CH3); 90.3 (C6); 115.7 (C1’’); 120.2 (C4’’); 122.6 (C2’, C6’); 125.0 (C5’’); 126.6 (C8’’); 127.2 (C6’’
or C7’’); 127.5 (C6’’ or C7’’); 128.2 (C3’, C5’); 129.1 (C3’’); 129.9 (C4a’’); 133.2 (C8a’’); 135.1
(C1’); 135.3 (C2’’); 141.5 (C4’); 143.7 (C5); 155.8 (C3a); 163.6 (C2); 164.1 (C7). HRMS (ESI): calc.
for C23H20N6O2S 445.1437; found [M+1]+ 445.1438. HPLC: 99.9%.
4-((5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)-N-
phenylbenzenesulfonamide hydrochloride (18) Yield: 27%. m.p. 201-203 °C. IR (cm-1): 3350; 1629;
1562; 1296-1163. 1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.49 (s; 3H; CH3); 6.81 (s; 1H; H6);
7.06-7.02 (m; 1H; H4’’); 7.14-7.12 (m; 2H; H2’’, H6’’); 7.27-7.23 (m; 2H; H3’’, H5’’); 7.63 (d; 2H; J =
13
8.6 Hz; H2’, H6’); 7.83 (d; 2H; J = 8.6 Hz; H3’, H5’); 10.35 (s; 1H; SO2NH); 10.72 (s; 1H; NH). C
NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 24.7 (CH3); 92.5 (C6); 119.4 (q; J = 269.1; CF3); 119.8
(C2’’, C6’’); 123.2 (C2’, C6’); 123.9 (C4’’); 128.1 (C3’, C5’); 129.1 (C3’’, C5’’); 135.8 (C1’); 137.5
(C1’’); 140.8 (C4’); 145.2 (C5); 154.1 (q; J = 38.3; C2); 155.6 (C3a); 166.7 (C7). 19F NMR (376 MHz,
DMSO-d6, TMS, δ in ppm): -64.30. HRMS (ESI): calc. for C19H15F3N6NaO2S 471.0821; found [M+23]+
471.0811. HPLC: 97.1%.
N-(4-chlorophenyl)-4-((5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-
yl)amino)benzenesulfonamide hydrochloride (19) Yield: 31%. m.p. 216-218 °C. IR (cm-1): 1628; 1566;
1290-1155; 828. 1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.49 (s; 3H; CH3); 6.82 (s; 1H; H6);
7.13 (d; 2H; J = 8.8 Hz; H2’’, H6’’); 7.32 (d; 2H; J = 8.8 Hz; H3’’, H5’’); 7.64 (d; 2H; J = 8.7 Hz; H2’,
13
H6’); 7.82 (d; 2H; J = 8.7 Hz; H3’, H5’); 10.51 (s; 1H; SO2NH); 10.72 (s; 1H; NH). C NMR (100
MHz, DMSO-d6, TMS, δ in ppm): 14.5 (CH3’); 24.9 (CH3); 92.7 (C6); 119.5 (q; J = 269.1; CF3); 121.4
(C2’’, C6’’); 123.3 (C2’, C6’); 128.1 (C4’’); 128.2 (C3’, C5’); 129.2 (C3’’, C5’’); 135.5 (C1’); 136.6
(C1’’); 141.1 (C4’); 145.3 (C5); 154.2 (q; J = 38.1; C2); 155.7 (C3a); 166.8 (C7). 19F NMR (376 MHz,
DMSO-d6, TMS, δ in ppm): -64.31. HRMS (ESI): calc. for C19H14ClF3N6NaO2S 505.0431; found
[M+23]+ 505.0409. HPLC: 95.4%.
N-(4-fluorophenyl)-4-((5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-
yl)amino)benzenesulfonamide hydrochloride (20) Yield: 36%. m.p. 206-209 °C. IR (cm-1): 3356; 1629;
1
1562; 1297-1151; 1132. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.49 (s; 3H; CH3); 6.80 (s;
1H; H6); 7.12-7.10 (m; 4H; H2’’, H3’’, H5’’, H6’’); 7.62 (d; 2H; J = 8.6 Hz; H2’, H6’); 7.78 (d; 2H; J =
8.6 Hz; H3’, H5’); 10.30 (s; 1H; SO2NH); 10.72 (s; 1H; NH). 13C NMR (100 MHz, DMSO-d6, TMS, δ
in ppm): 24.9 (CH3); 92.6 (C6); 115.9 (d; J = 22.5 Hz; C3’’, C5’’) 119.5 (q; J = 269.0; CF3); 122.7 (d; J
= 8.1 Hz; C2’’, C6’’); 123.3 (C2’, C6’); 128.2 (C3’, C5’); 133.8 (d; J = 2.4 Hz; C1’’); 135.6 (C1’);
141.0 (C4’); 145.3 (C5); 154.2 (q; J = 38.3; C2); 155.7 (C3a); 159.0 (d; J = 239.6 Hz; C4’’); 166.8 (C7).
19F NMR (376 MHz, DMSO-d6, TMS, δ in ppm): -64.31 (CF3); -118.37 (F). HRMS (ESI): calc. for
C19H14F4N6NaO2S 489.0727; found [M+23]+ 489.0706. HPLC: 97.1%.
4-((5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)-N-(p-
tolyl)benzenesulfonamide hydrochloride (21) Yield: 25%. m.p. 210-212 °C. IR (cm-1): 1627; 1565;
1
1328; 1288-1150. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.19 (s; 3H; CH3’); 2.38 (s; 3H;
CH3); 6.47 (s; 1H; H6); 7.05-6.99 (m; 4H; H2’’, H3’’, H5’’, H6’’); 7.42 (d; 2H; J = 8.7 Hz; H2’, H6’);
13
7.72 (d; 2H; J = 8.7 Hz; H3’, H5’); 10.10 (s; 1H; SO2NH); 10.70 (s; 1H; NH). C NMR (100 MHz,
DMSO-d6, TMS, δ in ppm): 20.1 (CH3’) 24.5 (CH3); 91.5 (C6); 119.6 (q; J = 268.9; CF3); 120.3 (C2’’,
C6’’); 122.8 (C2’, C6’); 128.1 (C3’, C5’); 129.4 (C3’’, C5’’) 133.1 (C4’’); 134.1 (C1’); 135.0 (C1’’);
145.0 (C4’); 146.2 (C5); 156.1 (C3a); 153.5 (q; J = 37.9; C2); 164.9 (C7). 19F NMR (376 MHz, DMSO-

d6, TMS, δ in ppm): δ -64.35. HRMS (ESI): calc. for C20H17F3N6NaO2S 485.0970; found [M+23]+
485.0970. HPLC: 98.5%.
N-(4-methoxyphenyl)-4-((5-methyl-2-(trifluoromethyl)-[1,2,4triazolo[1,5-a]pyrimidin-7-
yl)amino)benzenesulfonamide hydrochloride (22) Yield: 67%. m.p. 201-202 °C. IR (cm-1): 3322-3263;
1628; 1569; 1312-1144; 1092. 1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.49 (s; 3H; CH3); 3.67
(s; 3H; OCH3); 6.79 (s; 1H; H6); 6.82 (d; 2H; J = 9.0 Hz; H2’’, H6’’); 7.02 (d; 2H; J = 9.0 Hz; H3’’,
H5’’); 7.61 (d; 2H; J = 8.7 Hz; H2’, H6’); 7.75 (d; 2H; J = 8.7 Hz; H3’, H5’); 9.97 (s; 1H; SO2NH);
13
10.71 (s;1H; NH). C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 24.8 (CH3); 55.0 (OCH3); 92.4
(C6); 114.2 (C3’’, C5’’); 119.4 (q; J = 269.0; CF3); 123.2 (C2’, C6’); 123.4 (C2’’, C6’’); 128.1 (C3’,
C5’); 129.9 (C1’); 135.8 (C1’’); 140.6 (C4’); 145.2 (C5); 154.1 (q; J = 38.2; C2); 155.6 (C3a); 156.4
19
(C4’’); 166.7 (C7). F NMR (376 MHz, DMSO-d6, TMS, δ in ppm): -64.31. HRMS (ESI): calc. for
C20H17F3N6NaO3S 501.0927; found [M+23]+ 501.0932. HPLC: 99.1%.
4-((5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)-N-(4-
(trifluoromethyl)phenyl)benzenesulfonamide hydrochloride (23) Yield: 28%. m.p. 230-231 °C. IR (cm-
1
1): 1628; 1566; 1323-1150; 749. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.41 (s; 3H; CH3);
6.60 (s; 1H; H6); 7.30 (d; 2H; J = 8.5 Hz; H2’’, H6’’); 7.52 (d; 2H; J = 8.5 Hz; H3’’, H5’’); 7.59 (d; 2H;
13
J = 8.6 Hz; H2’, H6’); 7.85 (d; 2H; J = 8.6 Hz; H3’, H5’); 10.83 (s; 1H; NH). C NMR (100 MHz,
DMSO-d6, TMS, δ in ppm): 24.6 (CH3); 91.9 (C6); 118.6 (C2’’, C6’’); 119.5 (q; J = 268.9; CF3); 123.1
(C2’, C6’); 122.7 (q; J = 32.3 Hz; C4’’); 124.2 (q; J = 269.4; CF3); 126.3 (q; J = 3.6 Hz; C3’’, C5’’);
128.1 (C3’, C5’); 135.0 (C1’); 142.6 (C4’); 143.8 (C1’’); 145.8 (C5); 155.9(C3a); 153.7 (q; J = 37.9 Hz;
C2); 165.5 (C7). 19F NMR (376 MHz, DMSO-d6, TMS, δ in ppm): -60.28 (CF3’); -64.34 (CF3); HRMS
(ESI): calc. for C20H14F6N6O2S 516.0803; found [M+23]+ 539.0686. HPLC: 98.8%.
4-((5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)-N-(naphthalen-2-
yl)benzenesulfonamide hydrochloride (24) Yield: 26%. m.p. 225-226 °C. IR (cm-1): 3272; 1629; 1560;
1354-1151. 1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.19 (s; 3H; CH3); 6.02 (s; 1H; H6); 7.17
(d; 2H; J = 8.1 Hz; H2’, H6’); 7.32 (dd; 1H; J = 2.1 Hz; J = 8.8 Hz; H4’’); 7.40-7.36 (m; 1H; H5’’);
7.46-7.42 (m; 1H; H8’’); 7.56 (d; 1H; J = 1.8 Hz; H1’’); 7.81-7.76 (m; 3H; H3’’, H6’’, H7’’); 7.72 (d;
13
2H; J = 8.6 Hz; H3’, H5’); 10.45 (s; 1H; NH). C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 24.3
(CH3); 90.3 (C6); 115.6 (C1’’); 119.9 (q; J = 268.5 Hz; CF3); 120.3 (C4’’); 122.3 (C2’, C6’); 125.7
(C5’’); 126.4 (C8’’); 126.9 (C6’’ or C7’’); 127.3 (C6’’ or C7’’); 128.1 (C3’, C5’); 128.8 (C3’’); 129.7
(C4a’’); 131.8 (C8a’’); 133.1 (C1’); 135.7 (C2’’); 147.4 (C4’); 152.8 (q; J = 37.4 Hz; C2); 156.9 (C5);
19
162.7 (C3a); 171.9 (C7). F NMR (376 MHz, DMSO-d6, TMS, δ in ppm): -64.42 (CF3).HRMS (ESI):
calc. for C23H17F3N6NaO2S 521.0966; found [M+23]+ 521.0967. HPLC: 100%.
4-((2,5-dimethylpyrazolo[1,5-a]pyrimidin-7-yl)amino)-N-phenylbenzenesulfonamide
hydrochloride
1
(25) Yield: 27%. m.p. 255-257 °C. IR (cm-1): 1617; 1551; 1340-1153. H NMR (400 MHz, DMSO-d6,
TMS, δ in ppm): 2.36 (s; 3H; CH3’) 2.42 (s; 3H; CH3); 6.22 (s; 1H; H3); 6.40 (s; 1H; H6); 7.05-7.01 (m;
1H; H4’’); 7.14-7.11 (m; 2H; H2’’, H6’’); 7.27-7.22 (m; 2H; H3’’, H5’’); 7.61 (d; 2H; J = 8.8 Hz; H2’,
13
H6’); 7.78 (d; 2H; J = 8.8 Hz; H3’, H5’); 10.02 (s; 1H; NH); 10.30 (s; 1H; SO2NH). C NMR (100
MHz, DMSO-d6, TMS, δ in ppm): 14.1 (CH3’); 24.4 (CH3); 87.6 (C3); 93.7 (C6); 119.6 (C2’’, C6’’);
122.0 (C2’, C6’); 123.8 (C4’’); 128.0 (C3’, C5’); 129.1 (C3’’, C5’’); 134.3 (C1’); 137.6 (C1’’); 142.1
(C4’); 142.5 (C3a); 149.4 (C5); 152.9 (C2); 158.7 (C7). HRMS (ESI): calc. for C20H19N5O2S 394.1330;
found [M+1]+ 394.1331. HPLC: 96.4%.

N-(4-chlorophenyl)-4-((2,5-dimethylpyrazolo[1,5-a]pyrimidin-7-yl)amino)benzenesulfonamide
hydrochloride (26) Yield: 25%. m.p. 218-220 °C. IR (cm-1): 1612; 1551; 1328-1154; 827. 1H NMR (400
MHz, DMSO-d6, TMS, δ in ppm): 2.37 (s; 3H; CH3’) 2.42 (s; 3H; CH3); 6.22 (s; 1H; H3); 6.42 (s; 1H;
H6); 7.14 (d; 2H; J = 8.9 Hz; H2’’, H6’’); 7.32 (d; 2H; J = 8.9 Hz; H3’’, H5’’); 7.62 (d; 2H; J = 8.6 Hz;
H2’, H6’); 7.78 (d; 2H; J = 8.7 Hz; H3’, H5’); 10.04 (s; 1H; NH); 10.46 (s; 1H; SO2NH). 13C NMR (100
MHz, DMSO-d6, TMS, δ in ppm): 14.1 (CH3’); 24.4 (CH3); 87.7 (C3); 93.8 (C6); 121.2 (C2’’, C6’’);
122.0 (C2’, C6’); 127.9 (C4’’); 128.0 (C3’, C5’); 129.0 (C3’’, C5’’); 133.9 (C1’); 136.6 (C1’’); 142.3
(C4’); 142.4 (C3a); 149.4 (C5); 152.9 (C2); 158.7 (C7). HRMS (ESI): calc. for C20H18ClN5O2S
428.0924; found [M+1]+ 428.0925. HPLC: 99.2%.
4-((2,5-dimethylpyrazolo[1,5-a]pyrimidin-7-yl)amino)-N-(4-fluorophenyl)benzenesulfonamide
1
hydrochloride (27) Yield: 33%. m.p. 225-227 °C. IR (cm-1): 3073; 1622; 1552; 1326-1152; 1091. H
NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.36 (s; 3H; CH3’); 2.42 (s; 3H; CH3); 6.21 (s; 1H; H3);
6.36 (s; 1H; H6); 7.07-7.01 (m; 4H; H2’’, H3’’, H5’’; H6’’); 7.57 (d; 2H; J = 8.7 Hz; H2’, H6’); 7.73 (d;
2H; J = 8.7 Hz; H3’, H5’); 9.98 (s; 1H; NH). 13C NMR (100 MHz, DMSO-d6): 14.1 (CH3’); 24.3 (CH3);
87.4 (C3); 93.7 (C6); 115.5 (d; J = 22.1 Hz; C3’’, C5’’) 122.0 (C2’, C6’); 122.1 (d; J = 8.0 Hz; C2’’,
C6’’); 127.9 (C3’, C5’); 135.8 (C1’); 136.1 (C1’’); 141.5 (C4’); 142.7 (C3a); 149.4 (C5); 152.8 (C2);
158.1 (d; J = 237.7 Hz; C4’’); 158.6 (C7). 19F NMR (376 MHz, DMSO-d6): -120.38. HRMS (ESI): calc.
for C20H19FN5O2S 412.1247; found [M+1]+ 412.1248. HPLC: 99%.
4-((2,5-dimethylpyrazolo[1,5-a]pyrimidin-7-yl)amino)-N-(p-tolyl)benzenesulfonamide hydrochloride
(28) Yield: 31%. m.p. 253-255 °C. IR (cm-1): 3265; 1622; 1551; 1382; 1330-1153. 1H NMR (400 MHz,
DMSO-d6, TMS, δ in ppm): 2.18 (s; 3H; CH3’’); 2.36 (s; 3H; CH3’); 2.42 (s; 3H; CH3); 6.21 (s; 1H;
H3); 6.37 (s; 1H; H6); 7.04-6.98 (m; 4H; H2’’, H3’’, H5’’, H6’’); 7.58 (d; 2H; J = 8.7 Hz; H2’, H6’);
7.74 (d; 2H; J = 8.7 Hz; H3’, H5’); 10.02 (s; 2H; SO2NH; NH). 13C NMR (100 MHz, DMSO-d6, TMS,
δ in ppm): 14.1 (CH3’); 20.1 (CH3’’); 24.3 (CH3); 87.4 (C3); 93.7 (C6); 120.2 (C2’’, C6’’); 122.0 (C2’,
C6’); 127.9 (C3’, C5’); 129.4 (C3’’, C5’’) 132.6 (C4’’); 134.9 (C1’); 135.7 (C1’’); 141.9 (C4’); 142.6
(C3a); 149.4 (C5); 152.8 (C2); 158.6 (C7). HRMS (ESI): calc. for C21H22N5O2S 408.1476; found
[M+1]+ 408.1476 . HPLC: 100%
4-((2,5-dimethylpyrazolo[1,5-a]pyrimidin-7-yl)amino)-N-(4-methoxyphenyl)benzenesulfonamide
1
hydrochloride (29) Yield: 79%. m.p. 261-262 °C. IR (cm-1): 1655; 1606; 1335-1160; 1025. H NMR
(400 MHz, DMSO-d6, TMS, δ in ppm): 2.49 (s; 3H; CH3’); 2.51 (s; 3H; CH3); 3.67 (s; 3H; OCH3); 6.42
(s; 1H; H3); 6.48 (s; 1H; H6); 6.83 (d; 2H; J = 8.9 Hz; H2’’, H6’’); 7.05 (d; 2H; J = 8.9 Hz; H3’’, H5’’);
7.63 (d; 2H; J = 8.6 Hz; H2’, H6’); 7.82 (d; 2H; J = 8.6 Hz; H3’, H5’); 10.15 (s; 1H; SO2NH); 11.62
13
(s;1H; NH). C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 14.0 (CH3’); 19.7 (CH3); 55.0 (OCH3);
91.3 (C6); 114.2 (C3’’, C5’’); 123.2 (C2’, C6’); 125.1 (C2’’, C6’’); 128.1 (C3’, C5’); 129.8 (C1’); 137.6
(C1’’); 139.2 (C4’); 141.0 (C3a); 146.8 (C4’’); 155.3 (C5); 155.8 (C2); 156.4 (C7). HRMS (ESI): calc.
for C21H21N5O3S 424.1432; found [M+1]+ 424.1433. HPLC: 96.4%.
4-((2,5-dimethylpyrazolo[1,5-a]pyrimidin-7-yl)amino)-N-(4-
(trifluoromethyl)phenyl)benzenesulfonamide hydrochloride (30) Yield: 29%. m.p. 230-232 °C. IR (cm-
1
1): 1589; 1556; 1324-1105; 838. H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 2.34 (s; 3H; CH3’);
2.42 (s; 3H; CH3); 6.19 (s; 1H; H3); 6.30 (s; 1H; H6); 7.05 (d; 2H; J = 8.5 Hz; H2’’, H6’’); 7.35 (d; 2H;
J = 8.5 Hz; H3’’, H5’’); 7.50 (d; 2H; J = 8.6 Hz; H2’, H6’); 7.78 (d; 2H; J = 8.6 Hz; H3’, H5’); 9.87 (s;
13
1H; NH). C NMR (100 MHz, DMSO-d6, TMS, δ in ppm): 14.2 (CH3’); 24.3 (CH3); 86.9 (C3); 93.5