From human immunodeficiency virus non-nucleoside reverse transcriptase inhibitors to potent and selective antitrypanosomal compounds

Article abstract of DOI:10.1016/j.bmc.2014.08.005

The presence of a structural recognition motif for the nucleoside P2 transporter in a library of pyrimidine and triazine non-nucleoside HIV-1 reverse transcriptase inhibitors, prompted for the evaluation of antitrypanosomal activity. It was demonstrated that the structure-activity relationship for anti-HIV and antitrypanosomal activity was different. Optimization in the diaryl triazine series led to 6-(mesityloxy)-N₂-phenyl-1,3,5-triazine-2,4-diamine (69), a compound with potent in vitro and moderate in vivo antitrypanosomal activity.

Full text of DOI:10.1016/j.bmc.2014.08.005

Bioorganic & Medicinal Chemistry 22 (2014) 5241–5248
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
From human immunodeficiency virus non-nucleoside reverse
transcriptase inhibitors to potent and selective antitrypanosomal
compounds
Muthusamy Venkatraj a, Kevin K. Ariën b, Jan Heeres a, Jurgen Joossens a, Bertrand Dirié a, Sophie Lyssens a,
Johan Michiels b, Paul Cos c, Paul J. Lewi a, , Guido Vanham b,d, Louis Maes c, Pieter Van der Veken a,
Koen Augustyns a,
⇑
a Laboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium
b Virology Unit, Department of Microbiology, Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium
c Laboratory for Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium
d Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The presence of a structural recognition motif for the nucleoside P2 transporter in a library of pyrimidine
and triazine non-nucleoside HIV-1 reverse transcriptase inhibitors, prompted for the evaluation of
antitrypanosomal activity. It was demonstrated that the structure–activity relationship for anti-HIV
and antitrypanosomal activity was different. Optimization in the diaryl triazine series led to 6-(mesityl-
oxy)-N2-phenyl-1,3,5-triazine-2,4-diamine (69), a compound with potent in vitro and moderate in vivo
antitrypanosomal activity.
Received 17 May 2014
Revised 6 August 2014
Accepted 7 August 2014
Available online 14 August 2014
Keywords:
Ó 2014 Elsevier Ltd. All rights reserved.
African trypanosomiasis
Trypanosoma brucei
Antitrypanosomal activity
NNRTIs
Triazines
1. Introduction
nificant economic damage to cattle farming. Chemotherapy cur-
rently remains the only treatment option. However the available
After malaria and lymphatic filariasis, human African trypano-
somiasis (HAT, sleeping sickness) is estimated to have one of the
highest disease burdens of the neglected parasitic diseases
(NPD).1 HAT is endemic in 36 sub-Saharan countries and approxi-
mately 70 million people are estimated to be at risk, with at least
10,000 mortalities annually.2,3 However, gross underreporting
due to difficulties in diagnosis and remoteness of some affected
areas makes estimation of accurate rates of morbidity and mortal-
ity difficult. African trypanosomiasis is transmitted by the tsetse fly
and caused by Trypanosoma brucei gambiense, causing endemic or
chronic disease in Central and West Africa and Trypanosoma brucei
rhodesiense, causing the more acute disease in East and Southern
Africa. The animal form of African trypanosomiasis is called
Nagana and caused by Trypanosoma brucei brucei.4 Together with
HAT, Nagana is a major cause of rural underdevelopment with sig-
drugs that were developed decades ago show limited efficacy, are
not affordable, cause severe adverse effects and are threatened
by increasing drug resistance.4,5 Currently used drugs are pentam-
idine, suramin, melarsoprol, eflornithine and nifurtimox. HAT is
classified as a neglected disease since investments are negligible
compared to its impact on human and veterinary health. In the last
decades, little progress has been made in addressing this lack of
effective treatments and the initiative to tackle this pressing med-
ical need has largely moved to academic research in collaboration
with public–private partnerships, such as the Drugs for Neglected
Diseases initiative (DNDi).
It has been well established that phenotypic screening is a suc-
cessful approach for the discovery of first-in-class drugs, which is
especially valid for drug discovery in infectious diseases, since
the ‘whole micro-organism’ is the best possible target.6,7 Hence,
the screening/evaluation of existing libraries of drug-like mole-
cules for novel anti-infectives is the most logical and straightfor-
ward approach. In the framework of a drug discovery programme
for new anti-HIV microbicides,8–11 we developed a library of novel
⇑
Corresponding author. Tel.: +32 3 265 27 17; fax: +32 3 265 27 39.
E-mail address: Koen.Augustyns@uantwerpen.be (K. Augustyns).
Deceased.
http://dx.doi.org/10.1016/j.bmc.2014.08.005
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

5242
M. Venkatraj et al. / Bioorg. Med. Chem. 22 (2014) 5241–5248
pyrimidines and triazines11 related to TMC12012 (1) as non-nucle-
oside reverse transcriptase inhibitors (NNRTIs). The antiprotozoal
activity of NNRTIs has never been described, but the triazine core
is a substrate for the unique nucleoside P2 transporter in T. brucei
and is present in melarsoprol.13 The triazine core has been used in
drug delivery to trypanosomes for compounds such as polyamine
analogues and nitroheterocycles.14–19 However, since there are
no reports of diarylsubstituted pyrimidines and triazines as
antitrypanosomal agents, we broadly screened and optimized our
compounds against T. b. brucei and T. b. rhodesiense (Fig. 1).
contrast, among the diaryltriazines several compounds exhibited
submicromolar activity against both T. b. brucei and T. b. rhodes-
iense (Tables 2–5), with 69 being equipotent to suramin and
melarsoprol.
Based on the activities of the 60 diaryltriazines, we were able to
deduce a structure–activity relationship (SAR) (Fig. 2). At R1, the
presence of NH2 (e.g., 29) is clearly favoured, whereas hydrogen
(e.g., 14), chloro- (e.g., 16) and some alkylamino- (37–44) substit-
uents are less active and/or cytotoxic. Other small substituents at
this position such as cyano- (e.g., 24) and methoxy- (e.g., 26) are
not tolerated. The presence of methyl groups at R2, R3 and R4 orig-
inating from the SAR of the anti-HIV-1 NNRTIs (TMC120, 1)
resulted in potent antitrypanosomal compounds. Generally, these
methyl groups can be replaced with Br or Cl, especially at R2 and
R4, but removal of one of the methyls is less well tolerated. The
para-cyano group on the other aryl group (R5), which is known
to be crucial for anti-HIV-1 activity can be removed and offers a
way to dissociate antitrypanosomal and anti-HIV-1 activity. For
instance, in the series 60 (para-cyano), 70 (meta-cyano) and 69
(no cyano group), the antiviral activity decreases whereas the anti-
trypanosomal activity increases. Replacement of one aryl group
with a pyridyl group (45–48) was not tolerated. Changing the
arylamino substituent (X = NH) to an aryloxy substituent (X = O,
Table 5) was more favourable in almost all cases (e.g., 60 vs 28).
X = NMe did not seem to alter the activity much (36 vs 28).
Concludingly, 16, 29, 63 and 69 are the four most potent antitry-
2. Results and discussion
The target compounds were synthesized from 2,4-dichloropyr-
imidines, 2,4-dichlorotriazines and 2,4,6-trichlorotriazines by
nucleophilic aromatic substitution (SNAr) reactions (Schemes 1–5
in Supplementary content).
The activity of compounds 1–5, 6–10 and 11–70 toward T. b.
brucei and T. b. rhodesiense and their cytotoxicity on a human cell
line (MRC5) is presented in comparison with anti-HIV-1 activity
(Tables 1–5). The compounds were also tested against Trypanoso-
ma cruzi, Leishmania infantum and Plasmodium falciparum (Tables
1–3 in Supplementary content). As a control, we used standard
anti-parasitic compounds as well as known NNRTIs. The antipara-
sitic potential of all compounds was analyzed by applying the
WHO/TDR screening activity criteria specified for each parasite.20
All the disubstituted pyrimidines including TMC120 (1) as well
as the monosubstituted triazines 6–10 were inactive (Table 1). In
panosomal compounds (IC50 = <0.25 lM) from the library of disub-
stituted triazines 11–70.
Figure 1. Structures of target compounds.

M. Venkatraj et al. / Bioorg. Med. Chem. 22 (2014) 5241–5248
5243
Table 1
Antitrypanosomal and anti-HIV-1 activity of disubstituted pyrimidines 1–5 and monosubstituted triazines 6–10
NC
R3
R2
NH2
NH
NH
N
N
R1
N
N
N
N
HN
N
NH2
R1
R2
HN
HN
R1
R2
R3
R3
1 (TMC120)
2-5
6-10
Compd
R1
R2
R3
Antitrypanosomal activity and cytotoxicity IC50
(
lM)
Anti-HIV-1 activity and cytotoxicity (lM)
T. b. bruceia
T. b. rhodesienseb
MRC-5c
EC50
CC50
d
e
1
2
3
4
>16
7.19
7.27
>64
19.5
3.84
2.53
4
>64
>64
13.2
>64
0.002
>10
>10
2.47
ndf
ndf
ndf
Me
H
H
Me
H
H
Me
H
CN
>10
5
6
7
8
9
10
H
Me
Me
Br
Me
H
CN
Me
Me
Me
Me
CN
>64
>32
>32
>32
>32
>32
0.02
0.03
39.77
>32
>32
>32
>32
>32
0.02
0.02
>64
>32
>32
>32
>32
>32
>64
7.4
4.5
4.70
ndf
ndf
ndf
ndf
ndf
Me
Br
Br
Br
H
>10
>10
>10
>10
>10
H
Suraming
Melarsoprolg
Lersivirineg
MIV170g
0.003
0.001
>100
>100
a
b
c
d
e
f
Antitrypanosomal activity against the suramin-sensitive strain T. b. brucei Squib 427.
Antitrypanosomal activity against T. b. rhodesiense strain STIB-900.
Cytotoxicity measurement using human lung fibroblast MRC-5 SV2 cells.
50% effective concentration from TZM-bl virus inhibition assay.
50% cytotoxic concentration from the Water Soluble Tetrazolium-1 (WST-1) Cell Proliferation Assay.
Not determined.
g
Reference compounds.
Table 2
Antitrypanosomal and anti-HIV-1 activity of disubstituted triazines 11–36
R6
R3
R2
R5
NH
NH
R4
X
N
N
N
N
N
R1
X
N
R1
R4
R2
R4
R2
R3
R3
11, 12
13-36
Compd
X
R1
R2
R3
R4
R5
R6
Antitrypanosomal activity and cytotoxicity IC50
(l
M)
Anti-HIV-1 activity and cytotoxicity (lM)
T. b. bruceia
T. b. rhodesienseb
MRC-5c
EC50
CC50
d
e
11
12
13
14
15
16
17
18
19
20
21
22
24
25
26
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
Cl
NH2
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
CN
CN
OMe
Me
Me
Me
Br
Me
Br
Br
Br
Me
Me
Br
Me
Me
Me
Me
Me
Me
Me
Me
H
Me
Me
Me
Br
Me
Br
Me
H
Me
Me
Br
—
—
—
—
H
H
H
H
H
H
H
H
H
CN
H
H
H
>64
>64
0.58
8.01
ndf
6.66
0.11
2.96
>32
5.53
2.4
>32
4.01
>32
13.27
2.84
>64
>64
>64
43.07
>64
5.58
>32
>32
>64
5.84
>32
>64
>32
>32
0.52
4.35
7.82
0.002
0.003
0.002
0.01
0.004
0.49
0.008
0.008
0.16
>100
>100
29.65
1
2.33
22.79
0.96
45.74
0.21
2.86
>32
32.46
1.99
>32
CN
CN
CN
CN
CN
CN
CN
CN
H
>10
1.73
2.15
10.24
5
9.14
9.98
6.61
0.15
0.67
0.19
Br
Me
Me
Me
Me
Me
Br
Br
Br
Br
Br
Br
Me
Br
H
8.06
>32
11.61
4.16
0.065
0.008
0.009
0.003
CN
CN
CN
(continued on next page)

5244
M. Venkatraj et al. / Bioorg. Med. Chem. 22 (2014) 5241–5248
Table 2 (continued)
Compd
X
R1
R2
R3
R4
R5
R6
Antitrypanosomal activity and cytotoxicity IC50
(l
M)
Anti-HIV-1 activity and cytotoxicity (lM)
d
e
T. b. bruceia
T. b. rhodesienseb
MRC-5c
EC50
CC50
27
28
29
30
31
32
33
34
NH
NH
NH
NH
NH
NH
NH
NH
NH
NMe
OMe
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
Br
Me
Br
Br
Br
Me
Me
Br
Br
Me
Me
Me
Me
Me
Me
H
Me
Me
Br
Me
H
Me
Me
Br
CN
CN
CN
CN
CN
CN
CN
H
H
H
H
H
H
H
H
H
CN
H
11.60
4.84
0.12
6.15
4.47
16.28
7.01
6.44
6.84
4.47
0.02
0.03
11.97
2.49
0.08
3.21
30.82
8.68
7.5
3.89
1.12
1.45
0.02
0.02
>32
0.002
0.004
0.002
0.001
0.004
0.009
0.003
0.13
0.19
44
5.9
2.63
>100
22.23
15.16
26.46
18.51
24.63
20.51
16.61
>32
>64
>64
7.51
33.94
6.74
23.57
>64
Br
Me
Me
Me
35
Br
Me
H
CN
0.033
0.007
36
Suraming
Melarsoprolg
Lersivirineg
MIV170g
7.4
0.003
0.001
>100
>100
a
b
c
d
e
f
Antitrypanosomal activity against the suramin-sensitive strain T. b. brucei Squib 427.
Antitrypanosomal activity against T. b. rhodesiense strain STIB-900.
Cytotoxicity measurement using human lung fibroblast MRC-5 SV2 cells.
50% effective concentration from TZM-bl virus inhibition assay.
50% cytotoxic concentration from the Water Soluble Tetrazolium-1 (WST-1) Cell Proliferation Assay.
Not determined.
g
Reference compounds.
Table 3
Antitrypanosomal and anti-HIV-1 activity of disubstituted triazines 37–44
NC
Br
NH
N
N
HN
N
R1
Br
37-44
Antitrypanosomal activity and cytotoxicity IC50
Compd
R1
(l
M)
Anti-HIV-1 activity and cytotoxicity (lM)
T. b. bruceia
T. b. rhodesienseb
MRC-5c
EC50
CC50
d
e
37
38
39
40
41
42
43
44
NHMe
NMe2
0.59
1.54
1.54
2.05
12.16
0.42
1.68
1.88
0.02
0.03
0.45
1.01
2.05
0.40
11.18
0.37
1.22
0.82
0.02
0.02
1.03
53.50
>64
2.12
22.92
2.11
>64
5.64
>64
7.4
0.002
0.005
0.036
0.011
0.011
0.15
1.07
12.55
17.59
2.90
35.53
2.72
NHCHMe2
NHcyclopropyl
NH(CH2)3OMe
NHPh
Piperidinyl
N-Me piperazinyl
0.12
0.011
71.85
8.66
Suraminf
Melarsoprolf
Lersivirinef
MIV170f
0.003
0.001
>100
>100
a
b
c
d
e
f
Antitrypanosomal activity against the suramin-sensitive strain T. b. brucei Squib 427.
Antitrypanosomal activity against T. b. rhodesiense strain STIB-900.
Cytotoxicity measurement using human lung fibroblast MRC-5 SV2 cells.
50% effective concentration from TZM-bl virus inhibition assay.
50% cytotoxic concentration from the Water Soluble Tetrazolium-1 (WST-1) Cell Proliferation Assay.
Reference compounds.
As could be expected, several diaryltriazines also demonstrated
After the extensive SAR study to optimize the antitrypanosomal
activity, we selected the most potent diaryltriazine from the
arylamino series (29) and from the aryloxy series (69) for evalua-
tion of their in vitro ADME properties in comparison with melarso-
prol (Table 6). Compounds 29 and 69 are highly lipophilic
(logD = >4) with a rather low solubility. Both compounds show
excellent stability in a buffer of pH 7.4 and in plasma. Compared
to melarsoprol 29 has a similar (human) to slightly improved
(mouse) microsomal stability, whereas the metabolism of 69 is
clearly faster.
low nanomolar activity against HIV-1. However, the structure–
activity relationship for the anti-HIV-1 and antitrypanosomal com-
pounds is different (e.g., compare 60 and 69). Since this is the first
report of antitrypanosomal activity of non-nucleoside reverse
transcriptase inhibitors (NNRTIs), we decided to test several other
NNRTI drugs and drug candidates (Table 4 in Supplementary con-
tent). None of the other NNRTIs demonstrated antitrypanosomal
activity, indicating that the antitrypanosomal activity is uniquely
linked to the diaryl triazine structural motif.

M. Venkatraj et al. / Bioorg. Med. Chem. 22 (2014) 5241–5248
5245
Table 4
Antitrypanosomal and anti-HIV-1 activity of disubstituted triazines 45–48
NC
NH
N
N
N
N
X
R1
45-48
Compd
X
R1
Antitrypanosomal activity and cytotoxicity IC50
(lM)
Anti-HIV-1 activity and cytotoxicity (lM)
b
d
e
T. b. bruceia
T. b. rhodesiense
MRC-5c
EC50
CC50
45
47
48
NH
NH
NMe
Cl
NH2
NH2
>64
50.55
>64
0.02
0.03
5.82
12.57
29.32
0.02
4.38
>64
>64
>64
7.4
1.42
0.41
0.06
11.87
>100
>100
Suraminf
Melarsoprolf
Lersivirinef
MIV170f
0.02
0.003
0.001
>100
>100
a
b
c
d
e
f
Antitrypanosomal activity against the suramin-sensitive strain T. b. brucei Squib 427.
Antitrypanosomal activity against T. b. rhodesiense strain STIB-900.
Cytotoxicity measurement using human lung fibroblast MRC-5 SV2 cells.
50% effective concentration from TZM-bl virus inhibition assay.
50% cytotoxic concentration from the Water Soluble Tetrazolium-1 (WST-1) Cell Proliferation Assay.
Reference compounds.
Table 5
Antitrypanosomal and anti-HIV-1 activity of disubstituted triazines 49–70
R6
R5
NH
N
N
O
N
R1
R4
R2
R3
49-70
Compd
R1
R2
R3
R4
R5
R6
Antitrypanosomal activity and cytotoxicity IC50
(lM)
Anti-HIV-1 activity and cytotoxicity (lM)
T. b. bruceia
T. b. rhodesienseb
MRC-5c
EC50
CC50
d
e
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Me
Br
Br
Me
Me
Me
Me
H
Me
Br
H
CN
CN
CN
CN
CN
CN
CN
CN
CN
H
H
H
H
H
H
H
H
H
H
H
CN
H
H
H
H
H
H
H
H
H
H
CN
2.12
1.45
5.18
5.51
4.10
5.32
2.61
5.20
9.12
15.36
5.2
1.17
1.37
3.52
0.05
1.13
0.52
0.78
2.03
0.79
0.015
0.21
1.05
0.78
8.50
4.53
2.15
2.11
1.65
1.46
7.09
19.75
21.4
0.59
0.43
2.37
0.1
1.15
0.35
0.46
1.93
0.71
0.24
0.84
7.32
>64
8.17
8.92
>64
6.36
10
17.41
>64
25.5
8.01
7.48
2.82
>64
1.04
2.02
3.82
2.14
5.36
2.57
2.69
5.21
1.92
ndf
17.38
15.89
20.84
21.18
16.38
18.55
16.56
19.30
18.81
ndf
Cl
H
Me
Me
Me
Me
OMe
Me
Me
Me
Br
Me
Me
Me
Me
OMe
Me
Me
Me
Br
Br
Cl
CHO
Me
Me
Me
Me
Me
Me
Me
H
Br
Cl
CHO
Me
Me
Me
Cl
Cl
Cl
H
ndf
ndf
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
CN
CN
CN
CN
CN
CN
CN
CN
CN
H
0.002
0.002
0.002
0.002
0.008
0.002
0.003
0.012
0.003
0.158
0.098
17.27
2.31
>100
94.76
20.56
6.48
Br
Cl
H
H
>64
>64
Me
Me
Me
Me
OMe
Me
Me
Me
Me
Me
Me
OMe
Me
Me
7.63
5.25
>64
34.05
>64
4.49
26.70
19.68
20.63
23.08
H
25.92
(continued on next page)

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M. Venkatraj et al. / Bioorg. Med. Chem. 22 (2014) 5241–5248
Table 5 (continued)
Compd
R1
R2
R3
R4
R5
R6
Antitrypanosomal activity and cytotoxicity IC50
(
lM)
Anti-HIV-1 activity and cytotoxicity (lM)
d
e
T. b. bruceia
T. b. rhodesienseb
MRC-5c
EC50
CC50
Suraming
0.02
0.03
0.02
0.02
>64
7.4
Melarsoprolg
Lersivirineg
MIV170g
0.003
0.001
>100
>100
a
b
c
d
e
f
Antitrypanosomal activity against the suramin-sensitive strain T. b. brucei Squib 427.
Antitrypanosomal activity against T. b. rhodesiense strain STIB-900.
Cytotoxicity measurement using Human lung fibroblast MRC-5 SV2 cells.
50% effective concentration from TZM-bl virus inhibition assay.
50% cytotoxic concentration from the Water Soluble Tetrazolium-1 (WST-1) Cell Proliferation Assay.
Not determined.
g
Reference compounds.
Figure 2. SAR derived from screening of target compounds against Trypanosoma brucei.
Table 6
In vitro ADME of selected compounds
Solubilitya
(l
M)
logD7.4
Stability at pH 7.4%
recovery after 24 hc
Microsomal stability % recovery after 15 mind
Plasma stability % recovery after 1 he
b
Compd
Human
Mouse
Human
Mouse
29
69
25–50
25–50
>200
4.51
4.14
2.11
100
100
100
84
26
95
72
18
45
100
100
100
100
100
100
Melarsoprol
a
b
c
Final test compound concentration range of between 3.125
The partition coefficient between octanol and aqueous phase (10 mM PBS pH 7.4).
Stability measurement using a 100 l aliquot of the 10 M solution in DMSO+1900 ll of PBS (10 mM PBS, pH 7.4).
The metabolism by microsomes (CYP450 and other NADPH-dependent enzymes) was monitored and expressed as percentage of remaining parent compound.
Percentage of remaining parent compound.
l
M and 200
l
M [4
l
l DMSO solution in 196
l
l buffer solution (10 mM PBS pH 7.4)].
l
l
d
e
The two selected compounds 29 and 69 were also evaluated
tion and the number of survivors at day 14 post-infection were
measured as end points (Table 7). With 29, we noted a dose-depen-
dent increase of survival, with a doubling of the MST compared to
vehicle at a dose of 50 mg/kg twice daily. However, a complete
against T. b. brucei (suramin-sensitive Squib 427 strain) in Swiss
mice, with suramin as positive control compound. The mean sur-
vival time (MST), the reduction in parasitemia at day 4 post infec-

M. Venkatraj et al. / Bioorg. Med. Chem. 22 (2014) 5241–5248
5247
Table 7
Activities of compounds in the T. b. brucei mouse model
Compd
Dose (mg/kg)
Freq.
Formulation
MSTa
Reduction (%) in
Survivors on
14 dpi
parasitemia on 4 dpib
Controlc
29
7.2
7
8
0/4
0/4
0/4
0/4
0/4
2/4
0/6
6/6
25
50
80
25
50
25
10
SID Â 5
SID Â 5
SID Â 5
BID Â 5
BID Â 5
SID Â 5
SID Â 5
100% DMSO
100% DMSO
100% DMSO
100% DMSO
100% DMSO
100% DMSO/PEG200 (1:50)
100% PBS
ndd
41
9
ndd
ndd
ndd
66
10.5
15.5
8.7
>21
69
Suramin
100
a
b
c
Mean Survival time.
Day post-infection.
Vehicle-treated infected control.
Not determined.
d
cure as with suramin was not observed. A similar result was
observed for 69. Since 29 and 69 are equipotent with suramin in
the in vitro assay against T. b. brucei, the most likely reasons for
the weaker in vivo profile must be linked to the pharmacokinetic
properties. As indicated in Table 6, the solubility of 29 and 69
and the metabolic stability of 69 are suboptimal.
acquity UPLC system coupled to a waters TQD ESI mass spectrom-
eter and waters TUV detector was used with UPLC BEH C18 1.7
l
m
2.1 Â 50 mm column. Water (A) and MeCN (B) were used as elu-
ents. Flow: 0.4 mL/min, 0.25 min 95% A, 5% B, then in 4.75 min to
95% B, 5% A, then 0.25 min 95% B, 5% A, followed by 0.75 min
95% A, 5% B. Formic acid 0.1% was added to solvents A and B. The
wavelength for UV detection was 214 nm. The products were puri-
fied with flash chromatography on a Flashmaster II (Jones chroma-
tography) or on a BiotageÒ ISOLERA One flash system equipped
with a internal variable dual-wavelength diode array detector
(200–400 nm), with a 30 min gradient of 0–80% EtOAc in hexanes
or 0–10% DCM in MeOH in necessary cases. The purity of all the
target compounds were P95%, except for compound 14 (88%)
which could not be purified further. As a representative example
the experimental and analytical data for 29 and 69 were given.
Detailed experimental and analytical data for all intermediates
and remaining target compounds can be found in Supplementary
content of this publication.
3. Conclusion
The antiprotozoal activity of NNRTIs has never been described,
but the triazine core is a substrate for the unique nucleoside P2
transporter in T. b. brucei and is present in melarsoprol. This
prompted us to evaluate our drug-like library of pyrimidine and
triazine NNRTIs. For the first time, it was demonstrated that diaryl-
substituted triazines show potent antitrypanosomal activity, a
property that is not present in other NNRTI drug classes. A struc-
ture–antitrypanosomal activity relationship could be established
that was different from the SAR of the anti-HIV-1 activity. Several
compounds displayed nanomolar antitrypanosomal activity with
69 being equipotent to suramin and melarsoprol. Two selected
compounds showed a dose-dependent anti-trypanosomal activity
in vivo, but complete cure as for suramin was not observed. The
most likely reason for the suboptimal in vivo properties was found
in their pharmacokinetic properties. Hence, optimization of the
diarylsubstituted triazines with enhanced solubility and metabolic
stability is now in progress.
4.2. General procedure for synthesis of 29 and 69
Compounds 16 or 58 (1 mmol) was taken in a pressure tube and
dissolved in 2.0 M ammonia in isopropanol (10 mL) and allowed to
stir at 100 °C overnight. Solvents were evaporated and the crude
product was purified by flash chromatography using 70% EtOAc
in hexanes or 10% DCM in methanol as eluent. After evaporation,
the product was obtained as white powder.
4. Experimental section
4.1. Chemistry
4.2.1. 4-((4-Chloro-6-((2,6-dibromo-4-methylphenyl)amino)-
1,3,5-triazin-2-yl)amino)benzonitrile (29)
Yield: 0.24 g, 51%. 1H NMR (MeOD, 400 MHz) d 7.96 (d, 1H,
J = 8.7 Hz), 7.62 (d, 2H, J = 8.5 Hz), 7.55 (s, 2H), 7.40 (br s, 1H),
2.38 (br s, 3H). 13C NMR (DMSO-d6, 400 MHz) d 167, 165.3,
164.4, 145.1, 139.9, 134.1, 132.7, 132.4, 125.2, 119.5, 118.9,
102.2, 19.8. MS (ESI) m/z 476 [M+H]+. HPLC (214 nm) tr 19.6 min,
100%. LC–MS (214 nm) tr 16.9 min, 100%.
Reagents were obtained from Sigma–Aldrich or Acros. Charac-
terization of all the compounds was done with NMR and mass
spectrometry. 1H and 13C NMR spectra were recorded on a
400 MHz Bruker Avance DRX-400 and 400 MHz Bruker Avance III
nanobay spectrometer with ultrashield. Chemical shifts were in
ppm and coupling constants were in Hertz (Hz). ES mass spectra
were obtained from an Esquire 3000plus iontrap mass spectrome-
ter from Bruker Daltonics. LC–MS spectra were recorded on an
Agilent 1100 Series HPLC system using a Alltech Prevail C18 col-
umn (2.1 Â 50 mm, 5 mm) coupled with an Esquire 3000plus as
MS detector; solvent A: H2O+0.1% formic acid, solvent B: CH3-
CN+0.1% formic acid; gradient: 5% B ? 100% B over 30 min at
0.2 mL minÀ1. A wavelength of 214 nm was used. HPLC was run
on a Gilson instrument equipped with an Ultrasphere ODS column
(4.6 Â 250 mm, 5 mm); solvent A: H2O+0.1% trifluoroacetic acid
(TFA), solvent B: CH3CN+0.1% TFA; gradient: 10% B ? 100% B over
36 min at 1 mL minÀ1. A wavelength of 214 nm was used. Waters
4.2.2. 6-(Mesityloxy)-N2-phenyl-1,3,5-triazine-2,4-diamine (69)
Yield: 0.19 g, 59%. 1H NMR (MeOD, 400 MHz) d 7.47 (br s, 2H),
7.15 (br s, 2H), 6.90 (br s, 3H), 2.33 (br s, 3H), 2.09 (br s, 6H). 13C
NMR (DMSO-d6, 400 MHz) d 170, 168.4, 165.6, 147.2, 139.8,
133.9, 129.6, 128.9, 128.2, 121.9, 119.7, 20.3, 16.1. MS (ESI) m/z
322 [M+H]+. UPLC–MS (214 nm) tr 4.8 min, 100%. LC–MS
(214 nm) tr 18.1 min, 100%.
Acknowledgments
The research leading to these results has received funding from
the European Community’s Seventh Framework Programme (FP7/

5248
M. Venkatraj et al. / Bioorg. Med. Chem. 22 (2014) 5241–5248
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2007–2013) under Grant agreement No. 242135 (CHAARM). This
work was supported by BOF GOA (Research Council of University
of Antwerp), IOF-POC (Industrial Research Fund-Proof of Concept)
and Hercules Foundation. K.K.A. and G.V. are indebted to the Flem-
ish Fund for Scientific research (FWO-Vlaanderen) for financial
support. The Laboratory of Medicinal Chemistry belongs to the
Department of Pharmaceutical Sciences and is also a partner of
the Antwerp Drug Discovery Network (ADDN). The excellent tech-
nical assistance of An Matheeussen, Wim Deckers and Margot Des-
met is greatly appreciated.
Supplementary data
Supplementary data (chemistry and synthetic schemes, anti-
protozoal and antiviral activity, spectroscopic details of the inter-
mediate and target compounds, in vitro antiprotozoal assays,
in vivo drug screening model against Trypanosoma brucei brucei
Squib 427, in vitro antiviral assays, in vitro ADME assays and addi-
tional references) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmc.2014.08.005.
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Chem. 2009, 17, 2512.
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