Optimization of the pharmacokinetic properties of potent anti-trypanosomal triazine derivatives

Article abstract of DOI:10.1016/j.ejmech.2018.03.048

Human African trypanosomiasis is causing thousands of deaths every year in the rural areas of sub-saharan Africa. There is a high unmet medical need since the approved drugs are poorly efficacious, show considerable toxicity and are not easy to administer

Full text of DOI:10.1016/j.ejmech.2018.03.048

Accepted Manuscript
Optimization of the pharmacokinetic properties of potent anti-trypanosomal triazine
derivatives
Irene G. Salado, Adrienn Baán, Tomas Verdeyen, An Matheeussen, Guy Caljon,
Pieter Van der Veken, Filip Kiekens, Louis Maes, Koen Augustyns
PII:
S0223-5234(18)30291-5
10.1016/j.ejmech.2018.03.048
EJMECH 10313
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 February 2018
Revised Date: 16 March 2018
Accepted Date: 17 March 2018
Please cite this article as: I.G. Salado, A. Baán, T. Verdeyen, A. Matheeussen, G. Caljon, P. Van der
Veken, F. Kiekens, L. Maes, K. Augustyns, Optimization of the pharmacokinetic properties of potent
anti-trypanosomal triazine derivatives, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.03.048.
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ACCEPTED MANUSCRIPT
Novel lead compound for African trypanosomiasis
IC50 (T. bruc) = 0.63 µM
IC50 (MRC5) = ˃ 64 µM
% parent compound at 30 min in mouse microsomes: 80%
% parent compound at 30 min in human microsomes: 85%
Cmax after an oral dose of 50 mg/kg = 5.66 µM
Compound 35
t1/2 after an oral dose of 50 mg/kg = 6.51 h

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Optimization of the pharmacokinetic properties of
potent anti-trypanosomal triazine derivatives
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Irene G. Salado1, Adrienn Baán2, Tomas Verdeyen1, An Matheeussen3, Guy Caljon3, Pieter
Van der Veken1, Filip Kiekens2, Louis Maes3, Koen Augustyns1,*
Laboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, B-2610
Antwerp, Belgium
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Laboratory of Pharmaceutical Technology and Biopharmacy, University of Antwerp,
Universiteitsplein 1, B-2610 Antwerp, Belgium
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Laboratory for Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp,
Universiteitsplein 1, B-2610 Antwerp, Belgium
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ABSTRACT
Human African trypanosomiasis is causing thousands of deaths every year in the rural areas
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of sub-saharan Africa. There is a high unmet medical need since the approved drugs are
poorly efficacious, show considerable toxicity and are not easy to administer. This work
describes the optimization of the pharmacokinetic properties of a previously published family
of triazine lead compounds. One compound (35 (UAMC-03011)) with potent anti-
trypanosomal activity and no cytotoxicity was selected for further study because of its good
microsomal stability and high selectivity for Trypanosoma brucei over a panel including
Trypanosoma cruzi, L.eishmania infantum, and Plasmodium falciparum. In vivo
pharmacokinetic parameters were determined and the compound was studied in an acute in
vivo mouse disease model. One of the important learnings of this study was that the rate of
trypanocidal activity is an important parameter during the lead optimization process.
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KEYWORDS
Trypanosoma brucei, triazine, phenotypic screening, microsomal stability, cidal efficacy
INTRODUCTION
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Neglected tropical diseases have a significant impact on human health each year in countries
with a less developed economic status and particularly suffer from a lack of research due to a
limited return on investment. Among these disorders, African trypanosomiasis affects the
rural areas of sub-Saharan Africa and affects either farm-animals named Nagana caused by
Trypanosoma brucei brucei and human beings caused by T. b. rhodesiense and T. b.
gambiense and share the same vector spreading the disease, e.g. the tse-tse fly. The current
drugs are pentamidine and suramin for the first-stage, while melarsoprol, eflornithine and
nifurtimox are used during the second stage when the parasite has spread to the central
nervous system.[1] New and safer drugs that are easily administered are needed since the
existing drugs show serious side effects and in some cases have fatal consequences.[2]
Triazine derivatives have been extensively investigated in our group. In the framework of a
drug discovery programme for new-anti HIV microbicides, about 50 triazine and pyrimidine
derivatives were synthesized and evaluated for anti-HIV-1 activity while one compound was
further developed for prevention of sexual HIV transmission.[3-7] Although the antiprotozoal
activity of anti-HIV-1 compounds had never been reported, the triazine core is known for its
ability to act as substrate for the nucleoside P2 transporter[8, 9]. These compounds were
therefore evaluated against T. brucei producing several interesting hits.[10, 11] During lead
optimization, more than 100 monomers and dimers were evaluated for which it could be
concluded that the triazine scaffold yields more potent compounds than the pyrimidine core.
Compound a (6-(mesityloxy)-N2-phenyl-1,3,5-triazine-2,4-diamine) showed sub-micromolar

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activity on T. brucei, no cytotoxicity on MRC5 cells, and was not active against T. cruzi,
Leishmania infantum and Plasmodium falciparum. Furthermore, the anti-HIV activity was
considerably reduced, indicating selective anti-trypanosomal activity. However, due to a low
in vitro metabolic stability (only 20% of parent compound left after 15 min upon incubation
with mouse microsomal S9 fraction), in vivo activity could not be demonstrated in a mouse
model of acute T. brucei infection. We report here on the design, synthesis and
characterization of compounds with improved in vivo pharmacokinetic properties while
maintaining the selective anti-trypanosomal activity.
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Figure 1. Previously reported compound a with antitrypanosomal activity.
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RESULTS AND DISCUSSION
Synthesis of new T. brucei inhibitors
The design of the molecules was made based on the SAR obtained from the previously
synthesized molecules.[10] Since the most likely metabolic hotspots are the benzylic methyl
groups and the para-position of the phenyl ring, we introduced modifications such as halogen
atoms to block aromatic hydroxylations.[12] Different linkers such as thioether or ether in
both linker positions (X and Y) between the triazine ring and the aromatic moieties were also
studied.

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Figure 2. Different structural modifications based on compound a.
The synthesis was performed by subsequent nucleophilic aromatic substitutions. Different
pathways were followed depending on the nature of the linker (Scheme 1).
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Reaction conditions: i) When X = NH: DIPEA or K2CO3, aniline derivative, dioxane, 25 -
101°C, 40 min – 48 h. When X = O: (NBu4)HSO4, NaOH, phenol derivative, toluen/water
(10:1), 0 – 25°C, 40 min – 24 h. ii) When Y = NH: DIPEA or K2CO3, aniline derivative,
dioxane, 25 – 101°C, 45 min – 5 days. When Y = O: (NBu4)HSO4, NaOH, phenol derivative,
toluen/water (10:1), 0 – 25°C, 5 mim – 5 days. When Y = S: DIPEA, dioxane, 0 °C, 1 h. iii)
When Z = NH2, NH3 in methanol or dioxane, 25 – 101°C, 16 h – 8 days. When Z = NHMe,
NH2Me, DIPEA, dioxane, 101°C, 16 h. When Z = piperidinyl, piperidine, DIPEA, dioxane,
101 °C, 16 h.
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Scheme 1. Synthetic pathway to obtain different trisubstituted triazine derivatives.
In addition, two disubstituted triazines were synthesized to study the importance of the free
amino group on the potency of the compound (Scheme 2).

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Reaction conditions i) DIPEA or K2CO3, aniline derivative, dioxane, 25°C, 5 min. ii)
tetrabutylammoniumhydrogensulfate, sodium hydroxide, dioxane, 25°C, 30 min.
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Scheme 2. Synthetic pathway to obtain different disubstituted triazine derivatives.
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In vitro parasitology assays. The compounds were evaluated on a panel including T. brucei,
T. cruzi, L. infantum and P. falciparum (results presented in supporting information).
Cytotoxicity on a human cell line (MRC-5) and on primary peritoneal mouse macrophages
(PMM) was also tested. The activity of all the compounds against T. brucei and MRC-5 are
shown in Table 1.
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Table 1. In vitro antitrypanosomal activity and cytotoxicity of the final compounds and
derivative b.
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Antitrypanosomal activity
and cytotoxicity IC50 (µM)
Compd.
X
Y
Z
R
R’
T. b. bruc.a
MRC5b
˃ 64
˃ 64
˃ 64
6.39
6.5
NH
NH
NH
NH
NH
NH
NH
NH
O
O
O
O
O
O
O
O
NH2
NH2
H
4-F
2,4,6-trimethyl
2,4,6-trimethyl
2,4,6-trimethyl
2,4,6-trimethyl
2,4,6-trimethyl
2,4,6-trimethyl
2,4,6-trimethyl
2,4,6-trimethyl
0.88
a
0.31
27
28
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31
32
33
piperidinyl
NH2
4-F
8.17
4-Cl
1.52
NH2
4-CF3
3,5-diF
4-F
6.94
NH2
1.29
4.44
˃ 64
˃ 64
NHMe
NH2
10.86
˃ 64
2,6-diF

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O
O
O
NH2
4-F
4-F
2,4,6-trimethyl
4.27
0.63
0.39
1.08
0.51
0.96
˃64
˃ 64
˃ 64
˃ 64
˃ 64
˃ 64
˃ 64
˃64
34
35
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NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
H
4-F
4-Me
O
4-F
O
H
4-Me
O
4-F
2-Cl,4-Me
4-Me
O
4-CN
4-F
S
4-F
NH
NH
NH
NH
NH
NH
NH
O
4-Me
4-F
2,6-diBr-4-Me
2,6-diBr-4-Me
4-F
9.13
7.1
8.06
6.7
4-F
2.77
39.83
˃64
˃ 64
˃ 64
˃64
4-CN
4-CN
4-CN
4-CN
4-F
4-F
4-Me
2-Cl,4-Me
2,6-diMe-4-F
2,4,6-trimethyl
2,4,6-trimethyl
7.7
˃ 64
23.74
8.00
17.76
7.87
31.05
32.46
0.03
O
H
H
Suraminc
a
b
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Each value is the mean of at least two independent determinations. Cytotoxicity
measurement using human lung fibroblast MRC-5 cells. c reference compound
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The structure-activity relationship follows the same pattern as the one previously found for
this type of compounds.[10] The nature of the linker atom Y considerably influences the
potency. If Y = O, the compounds are more potent than when Y = NH or Y = S (Compound
35 vs 40 and vs 43 or compound 39 vs 45). When X = NH, the compound is more potent than
when X = O (compound 27 vs 34). The nature of Z also plays an important role in the
potency. If Z = NH2 the compound is 25-100 fold more potent than a piperidinyl, NHMe or H
(compound 27 vs 28, 32 and 50, respectively).
Microsomal stability. The more potent compounds 27, 35, 36 and 38 were studied for in
vitro metabolic stability with human and mouse microsomal fraction (S9) (Figure 3).

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120.00
100.00
80.00
60.00
40.00
20.00
0.00
27
35
36
38
diclofenac
0.00
20.00
40.00
60.00
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Figure 3. In vitro mouse microsomal stability of compounds 27, 35, 36 and 38. Diclofenac
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was used as a reference compound.
As could be expected, the substituents on the two phenyl rings play an important role in the
metabolic stability. Compound 35 (UAMC-03011) with a para-F in both phenyl rings is the
most stable with 76% and 93% of remaining parent compound after 15 min upon incubation
with mouse and human microsomes, respectively. All compounds had a (slightly) better
metabolic stability compared to the starting lead compound a, which showed only 20%
remaining parent compound after 15 min incubation with mouse microsomes. However,
compounds 27, 36 and 38 showed insufficient stability in mouse microsomes (remaining
parent compound at 15 min of 22%, 57% and 25% respectively) to allow further study in
mouse disease models. The plasma stability was also evaluated and compounds 27, 35, 36
and 38 remained fully stable after incubation in human plasma for 24 h at 37°C (Supporting
information). Mouse plasma protein binding of 35 was 96% and plasma protein binding in
the T. brucei culture medium was 50%, demonstrating a sufficiently high unbound fraction to
exert anti-trypanosomal effect.

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Pharmacokinetics
in view of its potent and selective anti-trypanosomal activity, and excellent microsomal and
plasma stability, compound 35 was selected first for in vivo pharmacokinetic properties in
mice and subsequent study in the acute T. brucei disease model. Compound 35 was
administered orally to groups of mice using a single dose of either 50 mg/kg or 25 mg/kg.
Figure 4 shows the plasma concentrations of compound 35 vs time for the 50 mg/kg dose.
Peak concentrations rise to about 10-fold the in vitro anti-trypanosomal IC50, and then fall to
about the IC50 of 0.63 µM at 24 h with a T1/2 of about 6 h (Table 2).
Compound 35
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5
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2
1
0
0
5
10
15
20
25
30
Time (h)
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Figure 4. Concentration in blood of compound 35 after a single oral dose of 50 mg/kg in
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mice.
Table 2. Pharmacokinetic parameters of derivative 35.
T1/2
(h)
AUC0-˃4
Cl
Tmax (h)
Cmax (µM)
Vz (l)
(ng.h/mL)
(mL/min)

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50 mg/kg PO
0.5
5.66
6.51
28609
15.6
27.8
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Acute mouse model of T. b. brucei
The encouraging pharmacokinetic data and the high potency and selectivity of 35 motivated
to study this compound in an acute mouse disease model of T. b. brucei. The compound was
administered orally (PO) or injected intraperitoneally (IP) at doses differing from 10 to
50 mg/kg once (s.i.d.) or twice daily (b.i.d.) for 5 days after infection. Disappointingly, no in
vivo activity was demonstrated since the treated animals showed the same mean survival time
(MST ˂ 7 days) as the vehicle-treated infected controls (Table 3).
Table 3. In vivo activities of compound 35 on the acute T. b. brucei model.
dose
survivors
on 14 dpi
0/3
Compd.
freq.
MST
(mg/kg)
Vehicle
Suramin
7
˃ 7
6
10
25
50
50
10
SID × 5 (IP)
BID × 5 (PO)
BID × 5 (PO)
SID × 5 (PO)
SID × 5 (IP)
3/3
0/3
5.7
7
0/3
35
0/3
7
0/3
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Rate of trypanocidal activity of compound 35 compared to suramin
To explain the lack of potency in the in vivo model for a compound of high potency and with
good pharmacokinetic properties, we decided to study the rate at which the compound is able

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to kill the parasites.[13, 14] Based on the in vivo Cmax that was around 10-fold the anti-
trypanosomal IC50, we investigated the parasite reduction over time using concentrations that
were seven (Figure 5A) and forteen (Figure 5B) times higher than the IC50 of 35 and
compared this with the reference compound suramin at concentrations which exceeded its
IC50 in a similar way. Whereas suramin gave a very significant reduction after 18 h and a
complete eradication after 48 h at both 7 and 14 times the IC50, compound 35 only gave a
very minor reduction of parasites after 48 h. It is clear that 35 is much slower acting than
suramin.
A
120
100
80
60
Compound 35 (5 µM)
40
Suramin (0,125 µM)
20
0
0
10
20
30
40
50
60
Time (h)
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B
120
100
80
60
40
20
0
Compound 35 (10 µM)
Suramin (0,25 µM)
0
10
20
30
40
50
60
Time (h)
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Figure 5. Graph A: % parasite growth vs time of compound 35 at 5 µM and suramin at
0.125 µM. Graph B: % parasite growth vs time of compound 35 at 10 µM and
suramin at 0.25 µM.
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CONCLUSIONS
A novel compound series was synthesized presenting a potent and selective anti-
trypanosomal activity. By careful design considering the previously determined SAR and
metabolic hotspots in the starting compound, we developed compound 35 (UAMC-03011)
with a high in vitro microsomal stability and good pharmacokinetic properties while
maintaining the excellent potency and selectivity. After oral administration at 50 mg/kg, 35
reached blood concentrations exceeding the IC50 by 10-fold and keeping the concentration in
blood above or at the IC50 for 24 h until the next dose was administered. Despite these
properties, compound 35 did not result in any activity in the acute mouse model of T. b.
brucei, whereas the reference drug suramin at 100 mg/kg resulted in complete cure.
Published pharmacokinetic data of suramin in rats treated with a single oral dose of 100
mg/kg showed a plasma concentration after 24 h of 2.3 µM, which is 76-fold its IC50.[15].
Although rats and mice are not comparable, this indicates that the slightly higher plasma

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concentrations of suramin compared to 35, combined with its superior potency against
T. b. brucei demonstrated by the 20-fold lower IC50 is probably responsible for the failure to
demonstrate of in vivo activity of 35. Furthermore, the rate of trypanocidal activity of 35 is
much slower compared to suramin. In conclusion, the present study demonstrated that next to
potency, selectivity, lack of toxicity and excellent pharmacokinetics, the rate of trypanocidal
activity should be considered as an important parameter in lead selection and lead
optimization.
EXPERIMENTAL PART
Chemistry. Reagents were purchased from commercial sources and without further
purification. The products were purified with flash chromatography on a Flashmaster II
(Jones chromatography) or on or IsoleraOne flash purification system from Biotage.
Compounds were detected with UV light (254 nm). 1H NMR spectra were obtained on a 400
MHz Bruker Avance DRX-400 and 400 MHz Bruker Avance III nanobay spectrometer with
ultrashield. Typical spectral parameters: special width 16 ppm, pulse width 9 µs (57 °), data
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size 32 K. C NMR experiments were carried out on the Bruker 400 MHz Bruker Avance
DRX-400 and 400 MHz Bruker Avance III nanobay spectrometer with ultrashield operating
at 100 MHz. The acquisition parameters: special width 16 ppm, pulse width 9 µs (57 °), data
size 32 K. Chemical shifts are reported in values (ppm) relative to internal Me4Si, and J
values are reported in Hz. The UPLC (Ultra Performance liquid chromatography), used to
quantify the purity of the products was an ACQUITY UPLC H-Class System with an TUV
detector Waters coupled to a MS detector Waters QDa. An Acquity UPLC BEH C18 1.7µm
(2.1 x 50 mm) column was used and as eluent a mixture of 0.1% FA in water, 0.1% FA in
acetonitrile, water, acetonitrile. Final compounds were analyzed by high resolution mass. 10
µL of 10-5 M solution of each sample was injected using the CapLC system (Waters,

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Manchester, UK) and electrosprayed using a standard electrospray source. Samples were
injected with an interval of 5 min. Positive ion mode accurate mass spectra were acquired
using a Q-TOF II instrument (Waters, Manchester, UK). The MS was calibrated prior to use
with a 0.2% H3PO4 solution. The spectra were lock mass corrected using the know mass of
the nearest H3PO4 cluster. All the compounds were obtained as amorphous solids. All the
final compounds presented a purity at least 95% determined by UPLC and HNMR.
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Synthesis of intermediates:
General procedure for Compounds 1 and 2, 14 - 19:
A solution of NaOH (1 equiv) in water (12 mL for 36.7 mmol) was added to a solution of
phenol derivative (1 equiv), monoaryl-bischloro-triazine derivative (1 equiv) and
tetrabutylammoniumhydrogensulfate (0.1 equiv) in toluene (120 mL for 36.7 mmol) at 0 °C.
The reaction was stirred for a certain time at a fixed temperature specified in each case. The
solvent was evaporated, dichloromethane (50 mL) and brine (50 mL) were added. The
organic solvent was evaporated and the crude was used in the next step without further
purification.
2,4-Dichloro-6-(mesityloxy)-1,3,5-triazine (1). Reagents: NaOH (1.45 g, 36.7 mmol), mesitol
(5 g, 36.7 mmol), 2,4,6-trichloro-1,3,5-triazine (6.77 g, 36.7 mmol) and
tetrabutylammoniumhydrogensulfate (1.25 g, 3.67 mmol). Reaction conditions: 2 h at 0 °C
and 24 h at room temperature. Yield: 7.70 g, 65%. UPLC: purity 88%. m/z (ES) 284 [M], 286
[M + 2], 288 [M + 4].
2,4-Dichloro-6-(4-fluorophenoxy)-1,3,5-triazine (2). Reagents: NaOH (76 mg, 1.89 mmol),
4-fluorophenol (213 mg, 1.89 mmol), 2,4,6-trichloro-1,3,5-triazine (350 mg, 1.89 mmol) and
tetrabutylammoniumhydrogensulfate (64.4 mg, 0.19 mmol). Reaction conditions: 2 h at 0 °C
and 40 min at room temperature. Yield: 321.1 mg, 65%. UPLC: purity 70%. m/z (ES) 260
[M], 262 [M + 2], 264 [M + 4].

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2-Chloro-4-(4-fluorophenoxy)-6-(mesityloxy)-1,3,5-triazine (14). Reagents: NaOH (23.07
mg, 0.57 mmol), mesitol (79 mg, 0.57 mmol), intermediate 2 (150 mg, 0.57 mmol) and
tetrabutylammoniumhydrogensulfate (19.58 mg, 0.06 mmol). Reaction conditions: 5 min at 0
°C. Yield: 125 mg, 60%. UPLC: purity 50%. m/z (ES) 360 [M + 1], 362 [M + 3].
4-Chloro-6-(4-fluorophenoxy)-N-phenyl-1,3,5-triazin-2-amine (15). Reagents: NaOH
(19.91 mg, 0.49 mmol), 4-fluorophenol (55.8 mg, 0.49 mmol), intermediate 3 (120 mg, 0.49
mmol) and tetrabutylammoniumhydrogensulfate (16.9 mg, 0.05 mmol). Reaction conditions:
5 min at 0 °C and after that, 1 h at room temperature. Yield: 102.7 mg, 65%. UPLC: purity
65%. m/z (ES) 317 [M + 1], 319 [M + 3].
4-Chloro-N-(4-fluorophenyl)-6-(p-tolyloxy)-1,3,5-triazin-2-amine (16). Reagents: NaOH
(18.53 mg, 0.46 mmol), p-cresol (50.1 mg, 0.46 mmol), intermediate 3 (120 mg, 0.46 mmol)
and tetrabutylammoniumhydrogensulfate (15.73 mg, 0.05 mmol). Reaction conditions: 5 min
at 0 °C and after that 1 h at room temperature. Yield: 96.3 mg, 63%. UPLC: purity 63%. m/z
(ES) 331 [M + 1], 333 [M + 3].
4-Chloro-N-phenyl-6-(p-tolyloxy)-1,3,5-triazin-2-amine (17). Reagents: NaOH (33.2 mg,
0.83 mmol), p-cresol (90 mg, 0.83 mmol), intermediate 4 (200 mg, 0.83 mmol) and
tetrabutylammoniumhydrogensulfate (28.2 mg, 0.08 mmol). Reaction conditions: 5 min at 0
°C and after that 40 min at room temperature. Yield: 176 mg, 68%. UPLC: purity 68%. m/z
(ES) 313 [M + 1], 315 [M + 3].
4-Chloro-6-(2-chloro-4-methylphenoxy)-N-(4-fluorophenyl)-1,3,5-triazin-2-amine
(18).
Reagents: NaOH (18.5 mg, 0.46 mmol), 2-chloro-4-methylphenol (66 mg, 0.46 mmol),
intermediate 3 (120 mg, 0.46 mmol) and tetrabutylammoniumhydrogensulfate (15.7 mg, 0.04
mmol). Reaction conditions: 5 min at 0 °C and after that 1 h at room temperature. Yield:
109.8 mg, 68%.

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4-((4-Chloro-6-(p-tolyloxy)-1,3,5-triazin-2-yl)amino)benzonitrile (19). Reagents: NaOH
(29.6 mg, 0.74 mmol), p-cresol (0,08 ml, 0,74 mmol), intermediate 5 (250 mg, 0,74 mmol),
and tetrabutylammonium hydrogensulfate (252 mg, 0,74 mmol). Reaction conditions: 5 min
at 0 °C and after that 5 days at room temperature. Yield: 0,13 g, 52%. UPLC: purity 50%.
m/z 338 [M], 340 [M + 2].
General procedures for compounds 3 – 13, 21 - 26:
A solution of the specified aniline (1 equiv), 2,4,6-trichloro-1,3,5-triazine (1 equiv) and a
determined base (1 – 1.4 equiv) in dioxane (12 mL for 5.42 mmol) was stirred for a specified
time at fixed temperature. The solvent was evaporated and DCM (50 mL) was added, the
crude was extracted with a satured solution of NaHCO3 (50 mL) and a satured solution of
NaCl (50 mL). The organic phase was dried and the solvent was evaporated under vacuum.
The product was purified by IsoleraOne using the eluents specified for each reaction.
4,6-Dichloro-N-(4-fluorophenyl)-1,3,5-triazin-2-amine (3). Reagents: 4-fluorobenzenamine
(603 mg, 5.42 mmol), 2,4,6-trichloro-1,3,5-triazine (1 g, 5.42 mmol) and potassium carbonate
(1.05 g, 7.59 mmol). Reaction conditions: 40 min at 25 °C. Purification: heptane/ ethyl
acetate (0 – 100 ethyl acetate). Yield: 270 mg, 20%. UPLC: purity > 99%. m/z (ES) 259 [M],
261 [M + 2], 263 [M + 4].
4,6-Dichloro-N-phenyl-1,3,5-triazin-2-amine (4). Reagents: aniline (177 mg, 1.89 mmol),
2,4,6-trichloro-1,3,5-triazine (350 mg, 1.89 mmol) and potassium carbonate (367 mg, 2.66
mmol. Reaction conditions: 5 min at 25 °C. Purification: heptane/ ethyl acetate (0 – 100 ethyl
acetate). Yield: 371.9 mg, 57%. UPLC: purity > 99%. m/z (ES) 241 [M], 243 [M + 2], 245
[M + 4].
4-((4,6-Dichloro-1,3,5-triazin-2-yl)amino)benzonitrile (5). Reagents: 2,4,6-trichloro-1,3,5-
triazine (5 g, 27,1 mmol), 4-aminobenzonitrile (3,36 g, 28,45 mmol) and potassium carbonate
(3,93 g, 28,5 mmol). Reaction conditions: 48 h at 25 °C and 2 h extra at 101 °C. Purification:

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heptane/ ethyl acetate (0 – 100 ethyl acetate). Yield: 1,7 g, 23%. UPLC: purity 79%. m/z (ES)
266 [M], 268 [M + 2], 270 [M + 4].
4,6-Dichloro-N-(4-fluorophenyl)-1,3,5-triazin-2-amine
(6).
Reagents:
N-ethyl-N-
isopropylpropan-2-amine (2,26 ml, 13,01 mmol), 4-fluorobenzenamine (1,03 ml, 10,85
mmol) and 2,4,6-trichloro-1,3,5-triazine (2g, 10,85 mmol). Reaction conditions: 25 °C for 2
h. Purification: heptane/ ethyl acetate (0 – 100 ethyl acetate). Yield: 1,67 g, 60%. UPLC:
purity > 99%. m/z (ES) 259 [M], 261 [M + 2], [M + 4].
4,6-Dichloro-N-(2,6-dibromo-4-methylphenyl)-1,3,5-triazin-2-amine (7). Reagents: 2,4,6-
trichloro-1,3,5-triazine (1 g, 5,42 mmol), 2,6-dibromo-4-methylaniline (1,51 g, 5,69 mmol)
and potassium carbonate (0,78 g, 5,70 mmol). Reaction conditions: 25 °C for 48 h.
Purification: heptane/ ethyl acetate (0 – 100 ethyl acetate). Yield: 0,79 g, 34%. UPLC: purity:
84%. m/z (ES) [M - 2], [M + 2], [M + 4].
6-Chloro-N2,N4-bis(4-fluorophenyl)-1,3,5-triazine-2,4-diamine (8). Reagents: N-ethyl-N-
isopropylpropan-2-amine (0,34 ml, 1,95 mmol), 4-fluorobenzenamine (0,31 ml, 3,25 mmol)
and 2,4,6-trichloro-1,3,5-triazine (300 mg, 1,63 mmol). Reaction conditions: 25 °C for 24 h.
Purification: heptane/ ethyl acetate (0 – 100 ethyl acetate). Yield: 224,5 mg, 41%. UPLC:
purity: >99%. m/z (ES) 334 [M + 1], 336 [M + 3].
4-Chloro-N-(4-fluorophenyl)-6-(mesityloxy)-1,3,5-triazin-2-amine
(9).
Reagents:
intermediate 1, (2 g, 7.04 mmol), 4-fluoroaniline (0.78 g, 7.04 mmol), N-ethyl-N-
isopropylpropan-2-amine (0.9 g, 7.04 mmol). Reaction conditions: 4 days at room
temperature. Purification: hexane/ ethyl acetate (0 – 100 ethyl acetate). Yield: 1.3 g, 52%.
UPLC: purity 88%. m/z (ES) 359 [M + 1], 361 [M + 3].
4-Chloro-N-(4-chlorophenyl)-6-(mesityloxy)-1,3,5-triazin-2-amine
(10).
Reagents:
intermediate 1 (1 g, 3.5 mmol), 4-chloroaniline (0.45 g, 3.5 mmol), N-ethyl-N-
isopropylpropan-2-amine (0.45 g, 3.5 mmol). Reaction conditions: 4 days at room

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temperature. Purification: hexane/ ethyl acetate (0 – 100 ethyl acetate). Yield: 638.1 mg,
49%. UPLC: purity 99%. m/z (ES) 375 [M], 377 [M + 2], 379 [M + 4].
4-Chloro-6-(mesityloxy)-N-(4-(trifluoromethyl)phenyl)-1,3,5-triazin-2-amine
(11).
Reagents: intermediate 1 (500 mg, 1.76 mmol), 4-(trifluoromethyl)aniline (284 mg, 1.76
mmol), N-ethyl-N-isopropylpropan-2-amine (273 mg, 2.1 mmol). Reaction conditions: 45
min at 101 °C. Purification: hexane/ ethyl acetate (0 – 100 ethyl acetate). Yield: 409 mg,
57%. UPLC: purity > 99%. m/z (ES) 407 [M - 1], 309 [M + 1].
4-Chloro-N-(3,5-difluorophenyl)-6-(mesityloxy)-1,3,5-triazin-2-amine
(12).
Reagents:
intermediate 1 (500 mg, 1.76 mmol), 3,5-difluoroaniline (227 mg, 1.76 mmol), N-ethyl-N-
isopropylpropan-2-amine (273 mg, 2.1 mmol). Reaction conditions: 45 min at 101 °C.
Purification: hexane/ ethyl acetate (0 – 100 ethyl acetate). Yield: 500.8 mg, 76%. UPLC:
purity 85%. m/z (ES) 377 [M + 1], 379 [M + 3].
4-Chloro-N-(2,6-difluorophenyl)-6-(mesityloxy)-1,3,5-triazin-2-amine
(13).
Reagents:
intermediate 1 (616 mg, 2.17 mmol), 2,6-difluoroaniline (280.2 mg, 2.17 mmol), N-ethyl-N-
isopropylpropan-2-amine (280.2 mg, 2.17 mmol). Reaction conditions: 6 h at 101 °C.
Purification: hexane/ ethyl acetate (0 – 100 ethyl acetate). Yield: 570 mg, 70%. UPLC: purity
>99%. m/z (ES) 377 [M + 1], 379 [M + 3].
6-Chloro-N2-(2,6-dibromo-4-methylphenyl)-N4-(p-tolyl)-1,3,5-triazine-2,4-diamine
(21).
Reagents: intermediate 7 (300 mg, 0,61 mmol), p-toluidine (65,4 mg, 0,61 mmol), N-ethyl-N-
isopropylpropan-2-amine (0,12 ml, 0,67 mmol). Reaction conditions: 19 h at 101 °C.
Purification: heptane/ ethyl acetate (0 – 100 ethyl acetate). Yield: 216 mg, 73%. UPLC:
purity > 99%. m/z (ES) [M - 2], [M + 2], [M + 4].
6-Chloro-N2-(2,6-dibromo-4-methylphenyl)-N4-(4-fluorophenyl)-1,3,5-triazine-2,4-diamine
(22). Reagents: intermediate 6 (300 mg, 0,62 mmol), 4-fluoroaniline (0,06 ml, 0,62 mmol),
N-ethyl-N-isopropylpropan-2-amine (0,11 ml, 0,62 mmol). Reaction conditions: 19 h at 101

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°C. Purification: heptane/ ethyl acetate (0 – 100 ethyl acetate). Yield: 0,21 g, 69%. UPLC:
purity > 99%. m/z (ES) 488 [M - 2], 487 [M], 489 [M + 2], 491 [M + 4].
4-((4-Chloro-6-((4-fluorophenyl)amino)-1,3,5-triazin-2-yl)amino)benzonitrile
(23).
Reagents: intermediate 5 (300 mg, 1,13 mmol), 4-fluoroaniline (0,11 ml, 1,13 mmol), N-
ethyl-N-isopropylpropan-2-amine (0,21 ml, 1,24 mmol). Reaction conditions: 1 h at 101 °C.
The organic crude was used in the next step without further purification (270.6 mg, 71%).
UPLC: purity: 97%. m/z (ES) 341 [M + 1], 343 [M + 3].
4-((4-Chloro-6-(p-tolylamino)-1,3,5-triazin-2-yl)amino)benzonitrile
(24).
Reagents:
intermediate 5 (300 mg, 1,13 mmol), p-toluidine (121 mg, 1,127 mmol), N-ethyl-N-
isopropylpropan-2-amine (0,21 ml, 1,24 mmol) and. Reaction conditions: 1 h at 101 °C. The
organic crude was used in the next step without further purification (342 mg, 90%). UPLC:
purity: 90%. m/z (ES) 337[M + 1], 339 [M + 3].
4-((4-Chloro-6-((2-chloro-4-methylphenyl)amino)-1,3,5-triazin-2-yl)amino)benzonitrile
(25). Reagents: intermediate 5 (250 mg, 0,74 mmol), 2-chloro-4-methylaniline (0,09 ml, 0,74
mmol), N-ethyl-N-isopropylpropan-2-amine (0,13 ml, 0,74 mmol). Reaction conditions: 22 h
at 101 °C. The organic crude was used in the next step without further purification (0,3 g,
99% yield). UPLC: purity: 92%. m/z (ES) 371 [M], 373 [M + 2], 375 [M + 4].
4-((4-Chloro-6-((4-fluoro-2,6-dimethylphenyl)amino)-1,3,5-triazin-2-yl)amino)benzonitrile
(26). Reagents: intermediate 5 (250 mg, 0,74 mmol), 4-fluoro-2,6-dimethylaniline (103 mg,
0,74 mmol), N-ethyl-N-isopropylpropan-2-amine (0,12 mL, 0,74 mmol). Reaction conditions:
4 h at 101 °C. The crude was used in the next reaction without further purification (0,29 g,
99%). UPLC: purity 91%. m/z (ES) 369 [M], 371 [M + 2].
4-Chloro-N-(4-fluorophenyl)-6-((4-fluorophenyl)thio)-1,3,5-triazin-2-amine
(20).
A
solution of 4,6-dichloro-N-(4-fluorophenyl)-1,3,5-triazin-2-amine (300 mg, 1,16 mmol) in
THF (4,2 mL) was cooled down to 0 °C. A second solution containing 4-fluorobenzenethiol

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(135 mg, 1,05 mmol) and N-ethyl-N-isopropylpropan-2-amine (0,18 ml, 1,05 mmol) in THF
(9 mL) was added dropwise to this first solution. The final solution was stirred for 1 h at 0
°C. The solution was concentrated in vacuo and the residue was redissolved in ethyl acetate
(25 mL) and washed with water (25 mL), a saturated solution of NaHCO3 (25 mL) and brine
(25 mL).The crude was purified by IsoleraOne using heptane and ethyl acetate (0 – 100%
ethyl acetate) as eluents (0,31 g, 56%). UPLC: purity: 42%. m/z (ES) 351 [M + 2].
4-Chloro-N-(4-fluorophenyl)-1,3,5-triazin-2-amine (48). A solution of 2,4-dichloro-1,3,5-
triazine (300 mg, 2.0 mmol), 4-fluorobenzenamine (222 mg, 2.0 mmol) and N-ethyl-N-
isopropylpropan-2-amine (284 mg, 2.2 mmol) in dioxane (4 mL) was stirred at room
temperature during 5 min. Dichloromethane (50 mL) and water (50 mL) were added and the
organic phase was dried over magnesium sulphate and the organic solvent was evaporated
under vacuum. The crude was used in the next step without further purification (449 mg,
99%). UPLC: purity 99%. m/z (ES) 225 [M + 1], 227 [M + 3].
4-Chloro-N-phenyl-1,3,5-triazin-2-amine (49). A solution of 2,4-dichloro-1,3,5-triazine (300
mg, 2.0 mmol), aniline (186 mg, 2.0 mmol) and N-ethyl-N-isopropylpropan-2-amine (284
mg, 2.2 mmol) in dioxane (4 mL) was stirred at room temperature during 5 min.
Dichloromethane (50 mL) and water (50 mL) were added and the organic phase was dried
over magnesium sulphate and the organic solvent was evaporated under vacuum. The crude
was used in the next step without further purification (413 mg, 99%). UPLC: purity 99%. m/z
(ES) 207 [M + 1], 209 [M + 3].
Synthesis of final compounds:
General procedure for compounds 27 - 47:
The triazine derivative (1 equiv) was dissolved in a solution of ammonia in a specified
solvent with a determined normality. The solution was heated at a specified temperature for a

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different time, given for each reaction. The solvent was evaporated and the crude was
purified by IsoleraOne using different eluents for each reaction.
N2-(4-Fluorophenyl)-6-(mesityloxy)-1,3,5-triazine-2,4-diamine (27): Reagents: 4-chloro-N-
(4-fluorophenyl)-6-(mesityloxy)-1,3,5-triazin-2-amine (300 mg, 0.8 mmol) and ammonia 7 N
in methanol (40 mL). Reaction conditions: 65 °C for 4 h. Purification: IsoleraOne using
1
DCM/MeOH (0 – 10% MeOH). Yield: 171 mg, 60%. H-NMR (400 MHz, DMSO-d6) δ:
398
399
9.51 (s, 1H), 7.65 (s, 2H), 7.22 - 6.84 (m, 6H), 2.24 (s, 3H), 2.04 (s, 6H). 13C-NMR (100
MHz, DMSO-d6) δ: 169.9, 168.4, 165.5, 157.41 (d, J = 239.1 Hz), 147.2, 136.2 (d, J = 2.3
400
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Hz), 134.0, 129.7, 129.0, 121.4, 114.77 (d, J = 21.8 Hz), 20.3, 16.1. UPLC: purity > 99%.
m/z (ES) 340.1 [M + 1]. HRMS: Calc: 339.15 Found: 340.1584 [M + 1].
N2-(4-Chlorophenyl)-6-(mesityloxy)-1,3,5-triazine-2,4-diamine (29): Reagents: 4-chloro-N-
(4-chlorophenyl)-6-(mesityloxy)-1,3,5-triazin-2-amine (300 mg, 0.8 mmol) and ammonia 7 N
in methanol (40 mL). Reaction conditions: 65 °C for 16 h. Purification: IsoleraOne using
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DCM/MeOH (0 – 10% MeOH). Yield: 184.1 mg, 65%. H NMR (400 MHz, DMSO-d6) δ:
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9.63 (s, 1H), 7.68 (s, 2H), 7.36 - 6.99 (m, 4H), 6.90 (s, 2H), 2.24 (s, 3H), 2.03 (s, 6H). C
NMR (100 MHz, DMSO-d6) δ: 170.0, 168.4, 165.5, 147.2, 138.9, 134.0, 129.7, 129.0, 128.1,
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125.5, 121.1, 20.3, 16.1. UPLC: purity > 99%. m/z (ES) 356.1 [M + 1], 358.1 [M + 3].
HRMS: Calc: 355.12 Found: 356.1278 [M + 1].
6-(Mesityloxy)-N2-(4-(trifluoromethyl)phenyl)-1,3,5-triazine-2,4-diamine (30): Reagents: 4-
chloro-6-(mesityloxy)-N-(4-(trifluoromethyl)phenyl)-1,3,5-triazin-2-amine (409 mg, 1.0
mmol) and ammonia 7 N in methanol (40 mL). Reaction conditions: 65 °C for 2 h.
Purification: IsoleraOne using hexane/ethyl acetate (0 – 100% ethyl acetate). Yield: 209.7
1
mg, 54%. H NMR (400 MHz, DMSO-d6) δ: 9.89 (s, 1H), 7.85 (s, 2H), 7.50 (d, J = 7.9 Hz,
415
2H), 7.25 (d, J = 34.8 Hz, 2H), 6.92 (s, 2H), 2.25 (s, 3H), 2.04 (s, 6H). 13C NMR (100 MHz,

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DMSO-d6) δ: 170.1, 168.5, 165.6, 147.2, 143.62 (c, J = 1.0 Hz), 134.1, 129.7, 129.0, 125.9
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(c, J = 271.1 Hz), 125.5 (c, J = 3.6 Hz), 121.9 (c, J = 32.0 Hz), 119.3, 20.3, 16.1. UPLC:
purity > 99%. m/z (ES) 390.2 [M + 1]. HRMS: Calc: 389.15 Found: 390.1542 [M + 1].
N2-(3,5-Difluorophenyl)-6-(mesityloxy)-1,3,5-triazine-2,4-diamine (31): Reagents: 4-chloro-
N-(3,5-difluorophenyl)-6-(mesityloxy)-1,3,5-triazin-2-amine (409 mg, 1.0 mmol) and
ammonia 7 N in methanol (40 mL). Reaction conditions: 65 °C for 2 h. Purification:
1
IsoleraOne using hexane/ethyl acetate (0 – 100% ethyl acetate). Yield: 173.2 mg, 36%. H
NMR (400 MHz, DMSO-d6) δ: 9.85 (s, 1H), 7.77 - 7.01 (m, 4H), 6.90 (s, 2H), 6.70 (t, J = 8.9
13
424
Hz, 1H), 2.23 (s, 3H), 2.03 (s, 6H). C NMR (100 MHz, DMSO-d6) δ: 170.0, 168.4, 165.5,
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162.31 (dd, J = 241.6, 15.7 Hz), 147.0, 142.6 (t, J = 14.2 Hz), 134.2, 129.5, 129.0, 102.0 (d, J
= 29.3 Hz), 96.7 (t, J = 26.3 Hz), 20.3, 16.1. UPLC: purity > 99%. m/z (ES) 358.1 [M + 1].
HRMS: Calc: 357.14 Found: 358.1479 [M + 1].
N2-(2,6-Difluorophenyl)-6-(mesityloxy)-1,3,5-triazine-2,4-diamine (33): Reagents: 4-chloro-
N-(2,6-difluorophenyl)-6-(mesityloxy)-1,3,5-triazin-2-amine (500 mg, 1.3 mmol) and
ammonia 7 N in methanol (40 mL). Reaction conditions: 65 °C for 16 h. Purification:
1
IsoleraOne using hexane/ethyl acetate (0 – 100% ethyl acetate). Yield: 248 mg, 52%. H
NMR (400 MHz, DMSO-d6) δ: 9.00 (s, 1H), 7.32 (tt, J = 7.9, 6.3 Hz, 1H), 7.13 (t, J = 8 Hz,
2H), 7.01 (d, J = 13.2 Hz, 2H), 6.87 (s, 2H), 2.22 (s, 3H), 2.03 (s, 6H). 13C NMR (100 MHz,
DMSO-d6) δ: 170.2, 168.6, 167.1, 158.6 (d, J = 248.4 Hz), 147.1, 133.9, 129.6, 128.9, 127.7
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(t, J = 5.5 Hz), 115.5 (t, J = 16.4 Hz), 111.8 (dd, J = 18.1, 5.4 Hz), 20.3, 16.1. UPLC: purity
> 99%. m/z (ES) 358.2 [M + 1]. HRMS: Calc: 357.14 Found: 358.1485 [M + 1].
4-(4-Fluorophenoxy)-6-(mesityloxy)-1,3,5-triazin-2-amine (34): Reagents: 2-chloro-4-(4-
fluorophenoxy)-6-(mesityloxy)-1,3,5-triazine (205 mg, 0.57 mmol) and ammonia 7 N in
methanol (20 mL). Reaction conditions: 65 °C for 16 h. Purification: IsoleraOne using
1
hexane/ethyl acetate (0 – 100% ethyl acetate). Yield: 38.1 mg, 20%. H NMR (400 MHz,

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CDCl3) δ: 7.15 - 7.02 (m, 4H), 6.88 (d, J = 0.5 Hz, 2H), 2.29 (s, 3H), 2.11 (s, 6H). 13C NMR
(100 MHz, CDCl3) δ: 172.8, 172.1, 169.5, 160.3 (d, J = 244.2 Hz), 147.8, 147.1, 135.5,
130.0, 129.3, 123.2 (d, J = 8.5 Hz), 116.1 (d, J = 23.5 Hz), 20.9, 16.5. UPLC: purity > 99%.
m/z (ES) 341.1 [M + 1]. HRMS: Calc: 340.13 Found: 341.1412 [M + 1].
6-(4-Fluorophenoxy)-N2-(4-fluorophenyl)-1,3,5-triazine-2,4-diamine (35): Reagents: 4-
chloro-6-(4-fluorophenoxy)-N-(4-fluorophenyl)-1,3,5-triazin-2-amine (167 mg, 0.5 mmol)
and ammonia 7 N in methanol (20 mL). Reaction conditions: 65 °C for 16 h. Purification:
1
IsoleraOne using hexane/ethyl acetate (0 – 100% ethyl acetate). Yield: 46.7 mg, 30%. H
NMR (400 MHz, MeOD-d4) δ: 7.51 (s, 2H), 7.24 - 7.08 (m, 4H), 6.92 (s, 2H). 13C NMR (100
MHz, MeOD-d4) δ: 172.5, 169.9, 166.9, 161.5 (d, J = 242.3 Hz), 160.1 (d, J = 240.6 Hz),
149.8 (d, J = 2.8 Hz), 136.5 (d, J = 2.3 Hz), 124.8 (d, J = 8.5 Hz), 123.5 (d, J = 6.2 Hz),
116.9 (d, J = 23.7 Hz), 115.8 (d, J = 22.5 Hz). UPLC: purity > 99%. m/z (ES) 316.1 [M + 1].
HRMS: Calc: 315.09 Found: 316.1016 [M + 1].
N2-(4-Fluorophenyl)-6-(p-tolyloxy)-1,3,5-triazine-2,4-diamine (36): Reagents: 4-chloro-N-
(4-fluorophenyl)-6-(p-tolyloxy)-1,3,5-triazin-2-amine (165 mg, 0.5 mmol) and ammonia 7 N
in methanol (20 mL). Reaction conditions: 65 °C for 16 h. Purification: IsoleraOne using
1
hexane/ethyl acetate (0 – 100% ethyl acetate). Yield: 49.6 mg, 32%. H NMR (400 MHz,
DMSO-d4) δ: 9.49 (s, 1H), 7.65 (s, 2H), 7.21 (d, J = 8.3 Hz, 2H), 7.12 (s, 1H), 7.08 – 6.96
(m, 4H), 2.32 (s, 3H). 13C NMR (100 MHz, DMSO) δ 170.95, 168.29, 165.36, 157.45 (d, J =
242.1 Hz), 150.10, 136.07 (d, J = 2.3 Hz), 134.18, 129.78, 121.79, 121.59 (d, J = 7.5 Hz),
114.77 (d, J = 21.8 Hz), 20.40. UPLC: purity > 99%. m/z (ES) 312.1 [M + 1]. HRMS: Calc:
311.12 Found: 312.1257 [M + 1].
N2-Phenyl-6-(p-tolyloxy)-1,3,5-triazine-2,4-diamine (37): Reagents: 4-Chloro-N-phenyl-6-
(p-tolyloxy)-1,3,5-triazin-2-amine (0.3 g, 0.8 mmol) and ammonia 7 N in methanol (20 mL).
Reaction conditions: 65 °C for 16 h. Purification: IsoleraOne using hexane/ethyl acetate (0 –

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100% ethyl acetate). Yield: 79.9 mg, 33%. H NMR (400 MHz, DMSO-d6) δ: 9.45 (s, 1H),
7.64 (s, 2H), 7.29 – 6.98 (m, 8H), 6.93 (t, J = 7.3 Hz, 1H), 2.32 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ: 170.9, 168.3, 165.5, 150.1, 139.7, 134.2, 129.8, 128.3, 122.0, 121.8, 119.9,
20.4. UPLC: purity > 99%. m/z (ES) 294.1 [M + 1]. HRMS: Calc: 293.13 Found: 394.1345
[M + 1].
6-(2-Chloro-4-methylphenoxy)-N2-(4-fluorophenyl)-1,3,5-triazine-2,4-diamine
(38):
Reagents: 4-chloro-6-(2-chloro-4-methylphenoxy)-N-(4-fluorophenyl)-1,3,5-triazin-2-amine
(183 mg, 0.5 mmol) and ammonia 7 N in methanol (20 mL). Reaction conditions: 65 °C for
16 h. Purification: IsoleraOne using hexane/ethyl acetate (0 – 100% ethyl acetate). Yield:
56.8 mg, 33%. 1H NMR (400 MHz, MeOD) δ 7.48 (s, 2H), 7.32 (s, 1H), 7.17 (dd, J = 8.3, 1.4
Hz, 1H), 7.12 (d, J = 8.2 Hz, 1H) 6.90 (s, 2H), 2.37 (s, 3H). 13C NMR (100 MHz, MeOD-d4)
δ: 172.0, 169.9, 166.9, 160.1 (d, J = 240.6 Hz), 147.6, 138.3, 136.5 (d, J = 2.7 Hz), 131.5,
129.6, 128.0, 124.9, 123.4, 115.8 (d, J = 22.6 Hz), 20.7. UPLC: purity > 99%. m/z (ES) 346.1
[M + 1]. HRMS: Calc: 345.08 Found: 346.0874 [M + 1].
4-((4-Amino-6-(p-tolyloxy)-1,3,5-triazin-2-yl)amino)benzonitrile (39): Reagents: 4-((4-
chloro-6-(p-tolyloxy)-1,3,5-triazin-2-yl)amino)benzonitrile (130 mg, 0,4 mmol) and ammonia
0.5 N in dioxane (100 ml). Reaction conditions: 101 °C for 24 h. Purification: IsoleraOne
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using heptane/ethyl acetate (0 – 100% ethyl acetate). Yield: 9 mg, 7%. H NMR (400 MHz,
Acetone-d6) δ: 9.02 (s, 1H), 7.95 (d, J = 7.3 Hz, 2H), 7.58 (d, J = 8.6 Hz, 2H), 7.26 (d, J = 8.3
Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 6.65 (s, 2H), 2.39 (s, 3H). UPLC: purity: 97%. m/z (ES)
319.1 [M + 1].
N2-(4-Fluorophenyl)-6-((4-fluorophenyl)thio)-1,3,5-triazine-2,4-diamine (40): Reagents: 4-
chloro-N-(4-fluorophenyl)-6-((4-fluorophenyl)thio)-1,3,5-triazin-2-amine (310.8 mg, 0,6
mmol) and ammonia 0.5 N in dioxane (50 ml). Reaction conditions: 101 °C for 17 h.
Purification: IsoleraOne using heptane/ethyl acetate (0 – 100% ethyl acetate). Yield: 177.4

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mg, 90%. H NMR (400 MHz, DMSO-d6) δ: 9.45 (s, 1H), 7.71 – 7.58 (m, 2H), 7.42 – 6.72
(m, 8H). 13C NMR (100 MHz, DMSO-d6) δ: 165.4, 162.9 (d, J = 244.9 Hz), 162.7, 157.3 (d,
J = 233.7 Hz), 138.4, 135.9 (d, J = 2.4 Hz), 124.1 (d, J = 3.0 Hz), 121.2, 116.3 (d, J = 21.9
Hz), 114.6 (d, J = 23.8 Hz). UPLC: purity > 99%. m/z (ES) 332.1 [M + 1]. HRMS: Calc:
331.07 Found: 332.0779 [M + 1].
N2-(2,6-Dibromo-4-methylphenyl)-N4-(p-tolyl)-1,3,5-triazine-2,4,6-triamine (41): Reagents:
6-chloro-N2-(2,6-dibromo-4-methylphenyl)-N4-(p-tolyl)-1,3,5-triazine-2,4-diamine
(144.5
mg, 0,3 mmol) and ammonia 7 N in methanol (100 ml). Reaction conditions: 65 °C for 8
days. Purification: IsoleraOne using heptane/ethyl acetate (0 – 100% ethyl acetate). Yield:
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71.7 mg, 52%. H NMR (400 MHz, DMSO-d6) δ: 8.95 – 8.75 (m, 1H), 8.75 – 8.50 (m, 1H),
7.78 – 7.21 (m, 4H), 7.18 – 6.69 (m, 2H), 6.43 (s, 2H), 2.39 – 2.28 (m, 3H), 2.28 – 2.12 (m,
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3H). C NMR (100 MHz, DMSO-d6) δ: 167.1, 165.4, 139.7, 138.0, 134.4, 132.4, 129.8,
128.6, 125.4, 119.5, 20.4, 19.8. UPLC: purity > 99%. m/z (ES) 463.1 [M - 1], 465.1 [M + 1],
467.1 [M + 3]. HRMS: Calc: 463.98 Found: 462.9895 [M - 1].
N2-(2,6-Dibromo-4-methylphenyl)-N4-(4-fluorophenyl)-1,3,5-triazine-2,4,6-triamine (42):
Reagents: 6-chloro-N2-(2,6-dibromo-4-methylphenyl)-N4-(4-fluorophenyl)-1,3,5-triazine-2,4-
diamine (207.7 mg, 0,4 mmol) and ammonia 0.5 N in dioxane (75 ml). Reaction conditions:
101 °C for 5 days. Purification: IsoleraOne using DCM/methanol (0 – 10% methanol). Yield:
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45 mg, 23%. H NMR (400 MHz, DMSO-d6) δ: 9.28 – 8.60 (m, 2H), 7.94 – 7.36 (m, 4H),
7.26 – 6.34 (m, 4H), 2.34 (s, 3H). 13C NMR (100 MHz, Acetone) δ: 167.5, 165.8, 164.8,
157.9 (d, J = 238.8 Hz), 140.1, 136.7, 134.3, 132.6, 125.3, 121.1, 114.5 (d, J = 27.4 Hz),
19.5. UPLC: purity > 99%. m/z (ES) 467.0 [M - 1], 469.0 [M + 1], 471.0 [M + 3]. HRMS:
Calc: 467.95 Found: 466.9628 [M - 1].
N2,N4-bis(4-Fluorophenyl)-1,3,5-triazine-2,4,6-triamine (43): Reagents: 6-chloro-N2,N4-
bis(4-fluorophenyl)-1,3,5-triazine-2,4-diamine (224 mg, 0,7 mmol) and ammonia 7 N in

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methanol (200 mL). Reaction conditions: 65 °C for 8 days. Purification: IsoleraOne using
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heptane/ethyl acetate (0 – 100% ethyl acetate). Yield: 52.3 mg, 25%. H NMR (400 MHz,
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DMSO-d6) δ: 9.10 (s, 2H), 7.76 (s, 4H), 7.12 - 7.03 (m, 4H), 6.61 (s, 2H). C NMR (100
MHz, DMSO-d6) δ: 166.7, 164.3, 157.3 (d, J = 238.1 Hz), 136.9, 121.5 (d, J = 5.5 Hz), 114.8
(d, J = 21.9 Hz). UPLC: purity > 99%. m/z (ES) 315.1 [M + 1]. HRMS: Calc: 314.11 Found:
315.1169 [M + 1].
4-((4-Amino-6-((4-fluorophenyl)amino)-1,3,5-triazin-2-yl)amino)benzonitrile
(44):
Reagents: 4-((4-chloro-6-((4-fluorophenyl)amino)-1,3,5-triazin-2-yl)amino)benzonitrile
(338.3 mg, 0,8 mmol) and ammonia 7 N in methanol (200 ml). Reaction conditions: 65 °C
for 24 h. Purification: IsoleraOne using water/methanol (0 – 100% methanol). Yield: 26.1
mg, 10% yield. 1H NMR (400 MHz, DMSO-d6) δ: 9.59 (s, 1H), 9.24 (s, 1H), 8.02 (d, J = 8.7
Hz, 2H), 7.82 – 7.64 (m, 4H), 7.19 – 7.05 (m, 2H), 6.79 (s, 2H). 13C NMR (100 MHz,
DMSO-d6) δ: 166.7, 164.3, 164.2, 157.4 (d, J = 237.6 Hz), 144.9, 136.3, 132.8, 132.7, 121.8
(d, J = 6.9 Hz), 119.6, 119.2, 114.8 (d, J = 22.0 Hz), 102.5. UPLC: purity > 99%. m/z (ES)
322.1 [M + 1]. HRMS: Calc: 321.11 Found: 322.1234 [M + 1].
4-((4-Amino-6-(p-tolylamino)-1,3,5-triazin-2-yl)amino)benzonitrile (45): Reagents: 4-((4-
chloro-6-(p-tolylamino)-1,3,5-triazin-2-yl)amino)benzonitrile (269,9 mg, 0,8 mmol) and
ammonia 7 N in methanol (200 ml). Reaction conditions: 65 °C for 48 h. Purification:
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IsoleraOne using water/methanol (0 – 100% methanol). Yield: 14 mg, 6%. H NMR (400
MHz, DMSO-d6) δ: 9.59 (s, 1H), 9.13 (s, 1H), 8.03 (d, J = 8.6 Hz, 2H), 7.76 – 7.56 (m, 4H),
7.09 (d, J = 8.3 Hz, 2H), 6.77 (s, 2H), 2.26 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ: 166.7,
164.30, 164.2, 145.0, 137.4, 132.8, 128.8, 120.4, 119.6, 119.2, 102.5, 20.5. UPLC: purity >
99%. m/z (ES) 318.2 [M + 1]. HRMS: Calc: 317.14 Found: 318.1465 [M + 1].
4-((4-Amino-6-((2-chloro-4-methylphenyl)amino)-1,3,5-triazin-2-yl)amino)benzonitrile
(46):
Reagents:
4-((4-chloro-6-((2-chloro-4-methylphenyl)amino)-1,3,5-triazin-2-

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yl)amino)benzonitrile (300.3 mg, 0,7 mmol) and ammonia 0.5 N in dioxane (100 ml).
Reaction conditions: 101 °C for 48 h. Purification: IsoleraOne using heptane/ethyl acetate (0
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– 100% ethyl acetate). Yield: 164 mg, 63%. H NMR (400 MHz, Acetone-d6) δ: 8.82 (s,
1H,), 8.09 (s, 1H), 8.05 (d, J = 8.7 Hz, 2H), 7.63 – 7.60 (m, 3H), 7.29 (d, J = 1.2 Hz, 1H),
7.16 (d, J = 8.3 Hz, 1H), 6.36 (s, 2H), 2.33 (s, 3H). 13C NMR (100 MHz, Acetone-d6) δ:
168.6, 166.2, 165.8, 145.6, 135.5, 134.2, 133.5, 130.2, 128.7, 125.6, 120.3, 120.2, 119.9,
104.9, 20.5. UPLC: purity > 99 %. m/z (ES) 352.1 [M], 354.1 [M + 2]. HRMS: Calc: 351.10
Found: 352.1096 [M + 1].
4-((4-Amino-6-((4-fluoro-2,6-dimethylphenyl)amino)-1,3,5-triazin-2-yl)amino)benzonitrile
(47):
Reagents:
4-((4-chloro-6-((4-fluoro-2,6-dimethylphenyl)amino)-1,3,5-triazin-2-
yl)amino)benzonitrile (0,2972 g, 0,733 mmol) and ammonia 0.5 N in dioxane (100 ml).
Reaction conditions: 101 °C for 6 days. Purification: IsoleraOne using heptane/ethyl acetate
(0 – 100% ethyl acetate). Yield: 24.7 mg, 10%. 1H NMR (400 MHz, MeOD-d4) δ: 7.96 (t, J =
8.1 Hz, 1H), 7.66 – 7.57 (m, 2H), 7.37 (d, J = 8.6 Hz, 1H), 6.86 (dd, J = 15.2, 9.3 Hz, 2H),
2.22 (s, 5H). UPLC: purity: 95%. m/z (ES) 350.1 [M + 1]. HRMS: Calc: 345.15 Found:
350.1548 [M + 1].
N-(4-Fluorophenyl)-4-(mesityloxy)-6-(piperidin-1-yl)-1,3,5-triazin-2-amine (28): 4-Chloro-
N-(4-fluorophenyl)-6-(mesityloxy)-1,3,5-triazin-2-amine (300 mg, 0.8 mmol) was stirred
with piperidine (85 mg, 1 mmol) and DIPEA (216 mg, 1.6 mmol) in dioxane (10 mL) at 101
°C overnight. After that, the solvent was evaporated under vacuum. The crude was purified
by IsoleraOne using DCM and MeOH (0 – 10% methanol) as eluents obtaining the final
compound (267.9 mg, 79%). 1H NMR (400 MHz, DMSO-d6) δ: 9.55 (s, 1H), 7.91 - 6.70 (m,
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6H), 3.87 - 3.49 (m, 4H), 2.25 (s, 3H), 2.03 (s, 6H), 1.69 - 1.36 (m, 6H). 13C NMR (100 MHz,
DMSO-d6) δ: 169.9, 165.5, 165.1, 157.4 (d, J = 231.7 Hz), 147.2, 136.1, 133.9, 129.6, 128.9,

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121.4, 114.8 (d, J = 20.8 Hz), 43.8, 25.3, 24.2, 20.4, 16.2. UPLC: purity >99%. m/z (ES)
408.2 [M + 1]. HRMS: Calc: 407.21 Found: 408.2200 [M + 1].
N2-(4-Fluorophenyl)-6-(mesityloxy)-N4-methyl-1,3,5-triazine-2,4-diamine (32): 4-Chloro-
N-(4-fluorophenyl)-6-(mesityloxy)-1,3,5-triazin-2-amine (300 mg, 0.8 mmol) was stirred
with a solution 2 M of methanamine in THF (31.2 mg, 1 mmol) and DIPEA (216 mg, 1.6
mmol) in dioxane (10 mL) at reflux temperature for 16 h. After that, the solvent was
evaporated under vacuum. The crude was purified by IsoleraOne using hexane and ethyl
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acetate (0 – 100 ethyl acetate) as eluents obtaining the final compound (162.1 mg, 22%). H
NMR (400 MHz, DMSO-d6) δ: 9.64 – 9.41 (m, 1H), 7.94 – 7.39 (m, 17H), 7.26 – 6.80 (m,
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4H), 2.76 (d, J = 4.6 Hz, 3H), 2.25 (d, J = 4.5 Hz, 3H), 2.04 (d, J = 4.6 Hz, 6H). C NMR
(100 MHz, DMSO-d6) δ: 169.4, 167.4, 164.7, 157.4 (d, J = 237.9 Hz), 147.2, 136.2, 134.0,
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129.7, 128.9, 121.3 (d, J = 6.9 Hz), 114.8 (d, J = 17.7 Hz), 27.5, 20.3, 16.1. UPLC: purity >
99%. m/z (ES) 354.2 [M + 1]. HRMS: Calc: 357.17 Found: 358.1505 [M + 1].
N-(4-Fluorophenyl)-4-(mesityloxy)-1,3,5-triazin-2-amine (50): To a solution of 2,4,6-
trimethylphenol (272 mg, 2 mmol), 4-chloro-N-(4-fluorophenyl)-1,3,5-triazin-2-amine (449
mg, 2 mmol) and tetrabutylammoniumhydrogensulfate (67.9 mg, 0.2 mmol) in Toluene (6.5
mL); a solution of NaOH (80 mg, 2 mmol) in water (0.6 mL) was added at 0 °C. The solution
was stirred at 0 °C for 30 min. The solvent was evaporated, and the crude was purified by
IsoleraOne using hexane and ethyl acetate (0 – 100 ethyl acetate) as eluents obtaining the
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final product (132.6 mg, 20%). H NMR (400 MHz, DMSO-d6) δ: 10.58 – 10.11 (m, 1H),
8.52 (s, 1H), 7.77 – 7.38 (m, 2H), 7.24 – 6.86 (m, 4H), 2.28 (s, 3H), 2.02 (s, 6H). 13C NMR
(100 MHz, DMSO-d6) δ: 169.8, 168.3, 164.8, 158.1 (d, J = 243.2 Hz), 146.9, 134.8, 134.6,
129.5, 129.1, 121.6, 115.0 (d, J = 24.2 Hz), 20.3, 16.0. UPLC: purity > 99%. m/z (ES) 325.2
[M + 1]. HRMS: Calc: 324.14 Found: 325.1459 [M + 1].

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4-(Mesityloxy)-N-phenyl-1,3,5-triazin-2-amine (51): To a solution of 2,4,6-trimethylphenol
(272 mg, 2 mmol), 4-chloro-N-phenyl-1,3,5-triazin-2-amine (413 mg, 2 mmol) and
tetrabutylammoniumhydrogensulfate (67.9 mg, 0.2 mmol) in Toluene (6.5 mL); a solution of
NaOH (80 mg, 2 mmol) in water (0.6 mL) was added at 0 °C. The solution was stirred at 0 °C
for 30 min. The solvent was evaporated, and the crude was purified by IsoleraOne using
hexane and ethyl acetate (0 – 100 ethyl acetate) as eluents obtaining the final product (69.7
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mg, 11%). H NMR (400 MHz, DMSO-d6) δ: 10.57 – 10.08 (m, 1H), 8.53 (s, 1H), 7.81 –
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6.88 (m, 7H), 2.28 (s, 3H), 2.03 (s, 6H). C NMR (100 MHz, DMSO-d6) δ: 169.6, 168.5,
164.9, 146.9, 138.4, 134.6, 129.5, 129.1, 128.5, 123.2, 119.9, 39.5, 20.4, 16.0. UPLC: purity
> 99%. m/z (ES) 307.2 [M + 1]. HRMS: Calc: 306.15 Found: 307.1546 [M + 1].
ASSOCIATED CONTENT
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Full panel of parasitology screens for the final compounds, antiprotozoal in vitro and in vivo
assays such as cytotoxicity, T. cruzi, L. infantum, P. falciparum, microsomal and plasma
stability assays and results. Protocols to evaluate the pharmacokinetics of compound 35 and
the pharmacokinetic parameters. Protocol to evaluate the rate of action is also included.
AUTHOR INFORMATION
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Corresponding Author
*Phone: + 32 3 265 27 17. Fax: + 32 3 265 27 39. E-mail: Koen.Augustyns@uantwerpen.be
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Author Contributions
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The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript.
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