Design, synthesis and biological evaluation of quinazoline derivatives as anti-trypanosomatid and anti-plasmodial agents

Article abstract of DOI:10.1016/j.ejmech.2015.04.028

In this paper, the design, synthesis and biological evaluation of a set of quinazoline-2,4,6-triamine derivatives (1-9) as trypanocidal, antileishmanial and antiplasmodial agents are explained. The compounds were rationalized basing on docking studies of the dihydrofolate reductase (DHFR from Trypanosoma cruzi, Leishmania major and Plasmodium vivax) and pteridin reductase (PTR from T. cruzi and L. major) structures. All compounds were in vitro screened against both bloodstream trypomastigotes of T. cruzi (NINOA and INC-5 strains) and promatigotes of Leishmania mexicana (MHOM/BZ/61/M379 strain), and also for cytotoxicity using Vero cell line. Against T. cruzi, three compounds (5, 6 and 8) were the most effective showing a better activity profile than nifurtimox and benznidazole (reference drugs). Against L. mexicana, four compounds (5, 6, 8, and 9) exhibited the highest activity, even than glucantime (reference drug). In the cytotoxicity assay, protozoa were more susceptible than Vero cells. In vivo Plasmodium berghei assay (ANKA strain), the compounds 1, 5, 6 and 8 showed a more comparable activity than chloroquine and pyrimethamine (reference drugs) when they were administrated by the oral route. The antiprotozoal activity of these substances, endowed with redox properties, represented a good starting point for a medicinal chemistry program aiming for chemotherapy of Chagas' disease, leishmaniosis and malaria.

Full text of DOI:10.1016/j.ejmech.2015.04.028

European Journal of Medicinal Chemistry 96 (2015) 296e307
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of quinazoline derivatives
as anti-trypanosomatid and anti-plasmodial agents
a
,
b
c
d
ꢀ
ꢀ
Cesar Mendoza-Martínez , Jose Correa-Basurto , Rocío Nieto-Meneses ,
d
d
d
ꢀ
ꢀ
ꢀ
Adrian Marquez-Navarro , Rocío Aguilar-Suarez , Miriam Dinora Montero-Cortes ,
d
d
e
ꢀ
Benjamín Nogueda-Torres , Erick Suarez-Contreras , Norma Galindo-Sevilla ,
f
f
a, b, *
ꢀ
ꢀ
Angela Rojas-Rojas , Alejandro Rodriguez-Lezama , Francisco Hernandez-Luis
Programa de Maestría y Doctorado en Ciencias Químicas, UNAM, Mexico, DF 04510, Mexico
Facultad de Química, Departamento de Farmacia, UNAM, Mexico, DF 04510, Mexico
Escuela Superior de Medicina, Laboratorio de Modelado Molecular y Bioinformatica de la SEPI, IPN, Mexico, DF 11340, Mexico
Escuela Nacional de Ciencias Biologicas, Departamento de Parasitología, IPN, Mexico, DF 11340, Mexico
Departamento de Infectología, Instituto Nacional de Perinatología, Mexico, DF 11000, Mexico
a
ꢀ
b
ꢀ
c
ꢀ
ꢀ
d
e
f
ꢀ
ꢀ
ꢀ
ꢀ
Tecsiquim S.A. de C.V., Toluca Estado de Mexico 50233, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper, the design, synthesis and biological evaluation of a set of quinazoline-2,4,6-triamine de-
rivatives (1e9) as trypanocidal, antileishmanial and antiplasmodial agents are explained. The com-
pounds were rationalized basing on docking studies of the dihydrofolate reductase (DHFR from
Trypanosoma cruzi, Leishmania major and Plasmodium vivax) and pteridin reductase (PTR from T. cruzi and
L. major) structures. All compounds were in vitro screened against both bloodstream trypomastigotes of
T. cruzi (NINOA and INC-5 strains) and promatigotes of Leishmania mexicana (MHOM/BZ/61/M379 strain),
and also for cytotoxicity using Vero cell line. Against T. cruzi, three compounds (5, 6 and 8) were the most
effective showing a better activity profile than nifurtimox and benznidazole (reference drugs). Against
L. mexicana, four compounds (5, 6, 8, and 9) exhibited the highest activity, even than glucantime
(reference drug). In the cytotoxicity assay, protozoa were more susceptible than Vero cells. In vivo
Plasmodium berghei assay (ANKA strain), the compounds 1, 5, 6 and 8 showed a more comparable activity
than chloroquine and pyrimethamine (reference drugs) when they were administrated by the oral route.
The antiprotozoal activity of these substances, endowed with redox properties, represented a good
starting point for a medicinal chemistry program aiming for chemotherapy of Chagas' disease, leish-
maniosis and malaria.
Received 20 November 2014
Received in revised form
10 April 2015
Accepted 11 April 2015
Available online 14 April 2015
Keywords:
Trypanosoma cruzi
Leishmania mexicana
Plasmodium berghei
Quinazoline
Dihydrofolate reductase
Pteridin reductase
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
region, affecting approximately 8 million people, of whom 30e40%
either have or will develop cardiomyopathy, digestive mega-
Parasitic infectious diseases due to pathogenic protozoan still
constitute a major health problem world wide and mainly occur in
underdeveloped countries [1]. Chagas' disease (CD), caused by
Trypanosoma cruzi [2], leishmaniosis, caused by Leishmania spp. [3],
and malaria, caused by Plasmodium vivax and Plasmodium falcipa-
rum [4], are among the most serious infections in the tropical re-
gions. CD is broadly disperse in Latin America and the Caribbean
syndromes, or both [2]. Leishmaniosis are endemic in 98 countries
or territories with an estimated incidence of 0.2e0.4 million cases
of visceral leishmaniosis (VL) and 0.7e1.2 million cases of cuta-
neous leishmaniasis (CL) each year [5]. The clinical manifestations
may vary from single cutaneous lesions to fatal VL [3,5]. Meanwhile,
malaria remains in large areas of the developing world, with 40% of
the earth's population living in malaria-endangered areas. As a
direct consequence, over 1 million humans per year die of this
disease, with the estimate of 550 million clinical cases [4]. These
public health problems are accentuated by multiple factors
including the AIDS epidemic, the increase of international travel
and difficulties controlling vectors.
* Corresponding author. Facultad de Química, Departamento de Farmacia, UNAM,
ꢀ
Mexico, DF 04510, Mexico.
ꢀ
E-mail address: franher@unam.mx (F. Hernandez-Luis).
http://dx.doi.org/10.1016/j.ejmech.2015.04.028
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

C. Mendoza-Martínez et al. / European Journal of Medicinal Chemistry 96 (2015) 296e307
297
In the absence of effective vaccines, chemotherapy plays a
critical role controlling these diseases [1,6]. The treatment of CD
relies on two available drugs; nifurtimox (Nfx, Lampit™) and
benznidazole (Bnz, Rochagan™), introduced into the clinical ther-
apy over four decades ago. Although in most cases these drugs are
efficient in the acute phase of the disease, they are almost inef-
fective in the chronic phase [7]. For the treatment of leishmaniosis,
glucantime, miltefosine and amphotericine B, are in use to date;
however, these compounds have common drawbacks such as high
toxicity, high cost or emerging resistance [8]. Regarding malaria,
sulfadoxineepyrimethamine, quinine, chloroquine and their de-
rivatives, are still in use today; new therapies based on artemisinin
were recently introduced into the clinic [9]. Moreover, the emer-
gences of drug-resistant strains of the causative organisms have
seriously impaired their therapeutic value. This has frustrated at-
tempts to eradicate these diseases by means of chemotherapy, and
the currently used drugs have an increased risk of becoming
obsolete. In this context, intensive efforts have been devoted to find
novel prototype compounds among natural and synthetic sources
to develop new drugs to treat these infectious diseases.
In previous research work using quinazoline-2,4,6-triamine
(TAQ) as template, Davoll et al. [10] synthesized a series of de-
rivatives bearing different substituents at the 6-position. Several
compounds displayed strong antiparasitic activity against Plasmo-
dium berghei, and modest activity against T. cruzi in cultures of
chicken embryo cells and in mice, respectively. Subsequently, other
studies showed the ability of TAQ to co-crystallize with pteridine
reductase 1 of Leishmania major (lmPTR1) [11] and that several
quinazoline-2,4-diamine derivatives had the ability to interact with
dihydrofolate reductase (DHFR) isolated from Leishmania mexicana
(lmxDHFR) and T. cruzi (tcDHFR) [12,13]. PTR1 and DHFR are
implicated in the metabolism of folate parasitic protozoa, and they
are treatable targets for antiparasitic leads [8]. In addition, it is
known that the mechanism of antifolate resistance in L. major and
T. cruzi is mediated by PTR and its isoforms [14,15].
The sum of these results encouraged the further search for other
TAQ derivatives as trypanocidal, antileishmanial and anti-
plasmodial agents. In this context, this document presents the
design and synthesis of nine quinazoline derivatives (1e9, Table 1)
with their in vitro evaluation against bloodstream trypomastigotes
of T. cruzi (NINOA and INC-5 strains) and promastigotes of
L. mexicana (MHOM/BZ/61/M379 strain). Likewise, compounds
were evaluated in P. berghei mouse model of infection.
The design was conducted searching the best ligand arrange-
ment into tcDHFR, lmDHFR, pvDHFR, tcPTR2 and lmPTR1.
2. Results and discussion
2.1. Design
The quinazoline derivative 1 showed a good profile against
T. cruzi and P. berghei in previous studies [10]. In order to know the
factors for this behavior, the theoretical interaction of this com-
pound with tcDHFR and tcPTR2 (Fig. 1) was analyzed.
According to the molecular docking of 1 with both proteins,
three regions with different characteristics were found in tcDHFR
and two in tcPTR2. Similar findings were presented with lmDHFR,
pvDHFR, hDHFR, and lmPTR1 (Table 2). Into DHFR, region I present
a lipoid character because it is constituted by alkyl and aromatic
side chains. Region II has got a hydrophilic character mainly formed
by hydrophilic amino acids, CO and NH groups of the peptide
bonds. Region III has got arginine capable of bridging hydrogen
with the 4-methoxy substituent of 1. The three regions are present
in DHFR for both, human and parasite; however, they strongly differ
in region I, where the parasite proteins have got an aromatic ring
(Phe or Tyr) whereas the human protein is more hydrophilic
(Asn64). Selective parasite protein compounds should be directed
primarily towards region I. The best interaction of 1 with DHFR or
PTR is with region I. On the other hand, TAQ moiety interacts with
the previously reported aminoacids [11,13].
Table 1
Quinazoline derivatives.
Compound
R1
R2
R3
MW
Mp (ꢀC)
Clog Pa
HBDb
HBAc
PSA (Å2)d
1
2
3
4
5
6
7
8
9
A
A
B
H
H
H
H
H
Ethyl
A
D
D
NH2
NHCOCH3
NH2
NH2
NH2
NH2
NH2
NH2
NH2
295
371
323
366
349
377
415
469
523
215e216
187e188
134e136
198e200
206e207
177e178
106e107
121e122
133e135
2.24
2.13
1.61
2.54
3.15
3.77
3.94
4.85
5.76
5
3
5
5
5
4
4
4
4
6
8
7
7
6
6
7
7
7
99.09
105.24
108.33
102.33
99.09
90.30
99.54
99.54
99.54
C
D
D
A
A
D
a
Calculated from molinspiration (www.molinspiration.com).
HBA: Hydrogen bond-acceptor group.
HBD: Hydrogen bond-donor group.
b
c
d
PSA: Polar surface area.

298
C. Mendoza-Martínez et al. / European Journal of Medicinal Chemistry 96 (2015) 296e307
Fig. 1. Poses and interactions of compound 1 on A) tcDHFR and B) tcPTR2.
With this in mind, new derivatives were designed in order to
reduction with sodium borohydride (NaBH4) gave 1, 3e5, respec-
tively. The synthesis of compound 2 also took place via reductive
amination, however, the synthesis conditions were different. In this
case, both solvent and temperature were modified because the
acetyl group loses when this reaction is carried out in methanol.
For compounds 6e9, 1 and 5 were used, which were condensed
with the corresponding aldehyde under acidic conditions. Two
different reducing agents were used, for 6, sodium cyanoborohy-
dride (NaBH3CN), and for 7e9, sodium triacetoxyborohydride
[NaBH(OAc)3] (Scheme 2).
Target compounds 1e9 were obtained in fair yields and pure as
solids with sharp melting points (Table 1). The structures of the
synthesized compounds were confirmed by spectroscopic tech-
niques, such as 1H NMR, 13C NMR, infrared, TOF mass spectra and
FAB mass spectra or elemental analyses.
replace the 4-methoxy substituent of 1 considering the predicted
interactions for docking studies. Generally, 3e5 compounds
contain lyphophilic substituents at 6-position in order to bring
their association with target enzymes. Additionally, compound 5
was designed to prevent the metabolism by O-dealkylation,
replacing 4-methoxy group by 4-trifluoromethoxy group (Fig. 2)
and removes the density of the oxygen electron decreasing the
ability to bridge hydrogen with regions II and III, in order to in-
crease the selectivity towards the parasite proteins by preferen-
tially interacting with region I.
Compounds 6e9 were designed to interact with two regions in
the proteins. Docking analyses under DHFR and PTR show that the
hypothesis is correct, and that the two N6substituents are primarily
directed toward regions I and II (Fig. 3).
All compounds have signals in common due to similar func-
tional groups in both IR and NMR spectra. The 1H NMR of amine
group, in 2-position, was found at 5.6 ppm for compounds 1 and
3e6, and approximately 6e7 ppm for compounds 7e9. The amine
group, in 4-position, is approximately 7 ppm for compounds 1 and
3e6 and 7e8 ppm for compounds 7e9. Curiously, the substitution
degree is associated with amines' displacement. For the acetylated
compound 2, there are not any present amines, and NH groups
(amides) are at a lower field (10e11 ppm). In addition, the amine in
6-position only exists in compounds 1e5 and this occurs as a triplet
at approximately 5.6 ppm.
2.2. Chemistry
The compounds mentioned above (1e5) were synthesized ac-
cording to the sequence of reactions outlined in Scheme 1. The
cyclocondensation of commercial 5-nitroanthranilonitrile (10) with
guanidine hydrochloride led to 6-nitroquinazoline-2,4-diamine (11);
which was treated with acetic anhydride followed by hydrogenation
under a hydrogen atmosphere using palladium on charcoal to
furnish N,N0-(6-aminoquinazoline-2,4-diyl)diacetamide (12). Sub-
sequent condensation of 14 with substituted benzaldehydes and
Table 2
Analysis of interactions for compound 1 with DHFR and PTR.
Protein
Region I (hydrophobic)
Region II (hydrophilic)
Region III (hydrophilic)
TAQ interactions
lmDHFR
Trp84, Val87, Phe91, Leu94, and
Val101
Glu43, Ser44, Ser86 (hydrogen
bond with methoxy group) and
cofactor NADP
Arg97 (hydrogen bond with
methoxy group)
Val30, Phe56 (¶-stacking), Asp52,
Val156, Thr180 and cofactor NADP
(nicotinamide moiety)
lmPTR1
Met183, Leu188, Leu226,
Tyr283
Leu229, Val230 (hydrogen bond
with methoxy group), Asp232
Ser 111, Phe113 (¶-stacking), Tyr194
and cofactor NADP (¶-stacking-
nicotinamide moiety and hydrogen
bond-phosphate group)
tcDHFR
tcPTR2
Ile84, Pro85, Phe88, and Leu91
Met171, Leu176, Leu214
Ile41 (hydrogen bond with
methoxy group), Ser83, and
cofactor NADP
Leu 217, Met221, Tyr229 (hydrogen
bond with methoxy group), and
Pro218
Arg94 (hydrogen bond with
methoxy group)
Val26, Phe52 (¶-stacking), Asp48 Ile154,
Thr178 and cofactor NADP
(nicotinamide moiety)
Ser103, Tyr105 (¶-stacking), Tyr182 and
cofactor NADP (¶-stacking-
nicotinamide moiety and hydrogen
bond-phosphate group)
pvDHFR
hDHFR
Trp118, Ile121, Pro122, Tyr125,
Leu128
Gly43, Leu45 and cofactor NADP
Asp21, Leu22 and cofactor NADP
Arg131 (hydrogen bond
with methoxy group)
Ile13, Cys14, Asp53, Ile173,
Tyr179,Thr194, Phe57 (¶-stacking) and
cofactor NADP (nicotinamide moiety)
Val115, Tyr121, Ile7, Phe34 (¶-stacking),
Glu30 and cofactor NADP (nicotinamide
moiety)
Trp57, Ile60, Pro61, Asn64,
Leu67
Arg70 (hydrogen bond with
methoxy group)

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299
Fig. 2. Replacing the methoxy group of compound 1 for trifluoromethoxy group.
Fig. 3. Interaction of compounds 6e9 (green: 6, orange: 7, yellow: 8, purple: 9) on A) tcDHFR and B) tcPTR2. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
Scheme 1. Reagents and conditions: (a) guanidine hydrochloride, NaOH, EtOH/PrOH, reflux; (b) acetic anhydride, 110 ꢀC; (c) H2, 10% Pd/C, MeOH, r.t.; (d) MeOH, 60 ꢀC, 1 h; (e) 0 ꢀC,
NaBH4;(f) DMF, 85 ꢀC, 1 h; (g) 0 ꢀC, NaBH4.
The methyl protons in compounds 1e2 and 8e9 are at approxi-
mately 3.7 ppm in 1H NMR and 55 ppm in 13C NMR. The methylene
group protons linking the quinazoline nucleus with benzyl groups
are approximately at 4 ppm as doublets in compounds 1e5, and as
singlets, slightly displaced downfield (4.5 ppm), for compounds 6e9.
This signal in 13C NMR is of 46 ppm for compounds 1e6 and 53 ppm
for compounds 7e9. Interestingly, compound 8 has got two different
substituents (p-methoxybenzyl and p-trifluoromethoxybenzyl) and
there are two methylene signals; in both 1H and 13C NMR they are
very close together. On the other hand, the CF3 group signal is
approximately 120 ppm and appears as a quartet with a large J
(above 257 Hz) in all derivatives with fluorine atom.
2.3. Antiprotozoal in vitro activity
The compounds 1e9 were tested in vitro against bloodstream
trypomastigotes of two T. cruzi strains (NINOA and INC-5) (Table 3).
Anti-T. cruzi properties were expressed in terms of the LC50 (50%
lytic concentration) for each compound. This parameter was
calculated after 24 h of parasite exposition. Nfx and Bnz were used
as reference drugs. In general, most compounds were more active
than the reference drug. First, as can be seen in data of Table 3, the
NINOA strain was more susceptible to compound 5, 6 and 8 (LC50
6.42, 20.56 and 17.77 M, respectively). The INC-5 strain was more
susceptible to compound (LC50 29.23 M) and then to
:
m
9
:
m

300
C. Mendoza-Martínez et al. / European Journal of Medicinal Chemistry 96 (2015) 296e307
Scheme 2. Reagents and conditions: (a) CH3CN/CH3COOH (1:1), NaBH(OAc)3; (b) CH3CN, NaBH3CN then HCl (38%).
Table 3
9), does not have the best biological activity. Some other factors to
consider could be steric or electronic. Compound 6 has an ethyl as
second substituent on the N6 position, and it is the most active
against T. cruzi (NINOA strain) and L. mexicana; when increasing the
size of the substituent to an aromatic ring (7, 8 and 9) the activity
decreases, indicating the influence of the steric factor. The substi-
tution of amines groups, at 2 and 4-position, for acetamides (2)
decreases their biological activity, which means that they are very
important for the interaction with its receptor. Compound 4 was
the least active, which could be due to the size of the substituent in
the benzene ring. However, this substituent may not have an
electronic effect on the quinazoline nucleus and the decreased
activity is due only to size.
In vitro activity of 1e9 against bloodstream trypomastigote of T.cruzi and promati-
gotes of L. mexicana.
Compound
Trypanosoma cruzi
Leishmania mexicana
NINOA strain
INC-5 strain
MHOM/BZ/61/M379 strain
LC50 (mM) SD LC50 (mM) SD LC50 (mM) SD
1
2
3
4
5
6
7
8
9
32.74 3.40
196.89 6.71
66.51 3.98
287.81 16.54
20.56 3.37
6.28 0.65
62.64 0.27
17.77 0.79
35.01 4.21
155.00 4.78
508.61 12.32
72.47 13.71
183.67 7.61
46.06 7.59
43.82 8.96
46.52 4.53
40.67 8.96
29.23 6.34
nd
14.59 1.32
250.00 3.56
22.35 2.73
65.43 6.45
8.08 3.18
3.06 1.80
23.10 1.42
8.00 0.89
4.93 0.52
182.68 0.19
nd
Glucantime nd
Nfx
Bnz
2.4. In vitro cytotoxicity studies
600 18.92
780 12.23
310 9.45
690 6.78
nd
An important criterion in the search of active compounds with
antiprotozoal activity is their toxicity on mammalian host cells. For
this purpose, the cytotoxicity was determined using Vero cells and
expressed as the concentration required to kill 50% of the
mammalian cells (CC50). The quinazoline derivatives were evalu-
ated and the CC50 was determined. In this work, compounds with
CC50 < 100 mM were classified as toxic for the mammalian cell line.
With this criterion, none of the compounds was toxic for
mammalian cells (Table 4).
nd: not determined.
LC: lytic concentration.
SD: Standard deviation.
compounds 5, 6 and 8. Second, compounds 1, 2, 3 and 4 exhibited
low activity towards both strains. Compound 4 was the least active
with LC50 above 200 mM.
The activity against L. mexicana had a similar tendency to those
obtained against T. cruzi (Table 3). The most active compounds were
5, 6, 8 and 9. Compound 6 had the lowest LC50, indicating that the
activity had increased. Compound 2 was the least active with LC50
2.5. In vivo antiplasmodial activity
of 250
m
M.
In vivo activity of quinazolines derivatives on P. berghei murine
model was evaluated. For the P. berghei murine model, the com-
pounds were applied at 0e4 days post-inoculation (pi) and they
were dosed by oral route at a dose of 50 mg/kg during 4 days. Data
showed that compounds 1, 5, 6 and 8 were the most active in the
parasitemia reduction (98.5, 99.9, 100 and 95.2%, respectively)
(Table 5). These compounds showed almost the same activity as the
positive controls, chloroquine (CQ) and pyrimethamine (Pyr). No
discernible toxic side effects in the mice were associated with any
of the compounds. Compound 2 was a little bit less active (89.3%),
Globally considered, the potency in both parasites is as follows:
6 > 5>9 > 8>1 > 7>3 > 2>4. The variations of this order may be due
to genetic factors, virulence factors or analysis conditions.
Due to the small number of evaluated compounds, the attempt
of analyzing SAR aspects seems unreasonable, but several obser-
vations and comparisons on the results can be made. The most
active compounds are those with fluorine atoms, which could
indicate an effect of lipophilicity. However, it is not the only factor
because, the compound with the highest value of clogP (compound

C. Mendoza-Martínez et al. / European Journal of Medicinal Chemistry 96 (2015) 296e307
301
Table 5
while the other quinazoline derivatives (4, 7 and 9) showed a very
In vivo activity of 1e3, 5e6,8 in P. berghei murine models.
weak or nonexistent activity (3).
Compound
Parasitemia suppression (%)
Survival time
(days post-administration)
2.6. ABTS assay
1
2
3
4
5
6
7
8
98.5
89.3
44.2
53.2
99.9
100
0
95.2
21
100
100
0
18
6
5
2
A study of the antioxidant capacity of the compounds as an in-
dicator of the modification of the electron distribution in molecules
was performed. Results are presented as the ability of compounds to
scavenge 50% of free radical ABTSꢁþ (IC50) (Table 6). The order of
antiradical activity is as follow: 5 > 3>4 > 1>TAQ>6 > 8>9 > 7.
Interestingly, the highest antiradical power, which is compound 5, is
one of the most active against T. cruzi, L. mexicana and P. berghei.
Compounds 1, 3 and 4 have got a slightly lower antiradical capacity.
This indicates that the influence of the substituent in the benzene
ring is hardly perceived in the quinazoline nucleus. The disubsti-
tuted compound 6, has got a lower radical scavenging effect to the
above compounds, in this case, the influence of the second substit-
uent is remarkable. However, there are many more differences to
those who are disubstituted by two aromatic rings (7, 8 and 9) with
IC50 values that are much lower. That there is no obvious correlation
of these results with the biological activity, could indicate that the
electronic requirements are not as important as the steric effect or
lipophilicity (change due to fluorine atom), but still more work is
needed in order to try this hypothesis.
11
12
17
>23
7
21
14
>23
>23
0
3
4
6
1
1
2
9
CQ
Pyr
Control
Table 6
IC50 from ABTS assay.
Compound
IC50 (mM)
1
2
3
4
5
6
7
8
5.72 0.03
7.04 0.09
6.29 0.17
6.38 0.57
4.75 0.06
7.36 0.13
113.95 10.86
24.46 0.22
33.91 0.54
6.78 0.10
10.92 0.26
11.25 0.32
14.84 0.25
2.7. Exploratory DHFR inhibition
9
TAQ
In this study, only two of the most active compounds (5 and 6)
were analyzed. Folic acid and folinic acid studies were carried out to
explore the participation of DHFR inhibition on antiparasitic ac-
tivity of 5 or 6. Folic acid is an essential co-factor for the enzyme
DHFR involved in the de-novo synthesis of purines and pyrimi-
dines, the essential precursors of DNA. Folinic acid is the reduced
form of folic acid that circumvents the inhibition of DHFR, so some
normal DNA replication processes can proceed even in the presence
of DHFR inhibitor. For these studies, L. mexicana cultures were
pretreated with folic acid in order to reduce the interaction of test
compound with DHFR; if DHFR inhibition is involved, a decrease in
the antiparasitic activity of 5 and 6 should be observed. Addition-
ally, pretreatment with folinic acid provides tetrahydrofolates
intracellular and effectively bypasses the block on DHFR induced by
test compounds slowing their biological activity. Therefore, if the
mechanism of antiparasitic action involves the DHFR inhibition,
there must be a clear correlation in the responses of folic and folinic
acid studies. Finally, an antioxidant (ferulic acid) was also used with
the intention of determining if there is a redox type mechanism
involved, this will be obvious by presenting a decrease in the
leishmanicidal activity of the quinazoline derivatives.
Trolox
Vitamin C
Vitamin E
IC: Inhibitory concentration.
It is observed that the antiparasitic activity ameliorates with
respect to increasing the concentration of folic acid, folinic acid and
ferulic acid. In this sense, it is interesting to note that compound 6 is
more dependent on the concentration of these molecules compared
to compound 5. The data also indicates that there is a significant
correlation between the data obtained for folic acid and folinic acid,
this means that there is a inhibition of DHFR. Besides, the influence
of the concentration of ferulic acid on antiparasitic activity indicates
the presence of a redox mechanism of action (Fig. 4).
3. Conclusions
We have synthesized, via simple and straightforward routes,
nine quinazoline antiparasitic derivatives as potential trypanocidal,
leishmanicidal and antiplasmodial agents. Our results indicate that,
despite their significant activity against P. berghei, the bloodstream
trypomastigotes of T. cruzi and amastigotes of L. mexicana, com-
pounds 5, 6, 8 and 9 were the most active compounds compared to
compound 1, which means that rational changes were accurate to
increase biological activity. On the other hand, it is important to
note that fluoride conferred increased biological activity to the
compounds 5, 6, 8 and 9, this is probably because the lipophilicity
of the molecule is increased, which modifies its absorption. Data
also indicates that the amines groups, at 2 and 4-positions, are
important for biological activity and that the activity is lost when
acetamides are placed in those positions.
Table 4
Cytotoxicity activities.
Compound
CC50 (mM) SD
1
2
3
4
5
6
7
8
147
156
3
8
>200
>200
182
7
>200
>200
>200
>200
163
In the case of the possible mechanism of action according to the
theoretical results and the experimental data with folic acid, folinic
acid and ferulic acid, inhibition of DHFR is proposed but also a redox
mechanism could be possible, such as inhibition of PTR or oxidation
of molecules to generate a more active metabolite as demonstrated
9
Nfx
Pyr
3
4
122
CC: Cytotoxic concentration.
SD: Standard deviation.

302
C. Mendoza-Martínez et al. / European Journal of Medicinal Chemistry 96 (2015) 296e307
Fig. 4. Effect of different concentrations of folic acid, folinic acid and ferulic acid on the leishmanicidal activity of compounds 5 and 6 (A and B respectively). Comparison of the same
effects between 5 and 6 in separate graphs at different concentrations of folic acid (C), folinic acid (D) and ferulic acid (E).
in the ABTS assay. A direct enzymatic study should be conducted to
clarify this hypothesis.
F254 plates (E. Merck) with visualization by irradiation with a UV
lamp. Silica gel 60 (70e230 mesh) was used for column chroma-
tography. IR spectra were recorded with an FT Perkin Elmer 16 PC
spectrometer. 1H (300 MHz) and 13C (75 MHz) NMR spectra were
measured at room temperature using a Varian EM-390 spectrom-
eter. Solvents are indicated in the text. Data for 1H NMR spectra are
reported as follows: chemical shifts are given in ppm relative to
Additionally, given the in vitro activity of the compounds as
antiprotozoal, an in vivo study should be carried out to T. cruzi and
L. mexicana. In the case of P. berghei, doseeresponse and toxicity
in vivo studies are the next step. This study provides a simple
modification to an antiparasitic molecule reported by rational
design. This is important to progress in the fight against orphan
diseases around the world due to the urgency of new treatments for
resistant strains.
tetramethylsilane (Me4Si,
patterns have been designated as follows: s, singlet; d, doublet; q,
quartet; dd, doublet of doublet; t, triplet; m, multiplet; bs, broad
d
¼ 0); J values are given in Hz; splitting
singlet. Data for 13C NMR is reported in terms of chemical shift (
d,
ppm) relative to residual solvent peak. MS were recorded on a JEOL
JMS-SX102A spectrometer by FAB [FAB(þ)] and Water Synapt
G2Sspectrometer by TOF MS ES þ. Elemental analyses (C, H, N) for
new compounds were performed on Fisons EA 1108 instruments
and agreed with the proposed structures within 0.4% of the
theoretical values. Catalytic hydrogenations were carried out in a
Parr shaker hydrogenation apparatus. Starting materials 5-
4. Experimental
4.1. Synthesis
Melting points were determined in open capillary tubes with a
Büchi B-540 melting point apparatus and are uncorrected. Re-
actions were monitored by TLC on 0.2 mm pre-coated silica gel 60

C. Mendoza-Martínez et al. / European Journal of Medicinal Chemistry 96 (2015) 296e307
303
nitroanthranilonitrile, aldehydes, NaBH4, NaBH3CN, NaBH(OAc)3
and guanidine hydrochloride were commercially available (Sigma
Aldrich).
(dd, J1 ¼ 2, J2 ¼ 8, 1H, benzodioxane), 6.92 (m, 2H, CH quinazoline,
CH benzodioxane), 6.98 (br. s, 2H, NH2), 7.02 (br. s, 2H, CH quina-
zoline); 13C NMR (DMSO-d6): 46.39 (CH2), 64.02 (CH2eO), 100.52
(CH, quinazoline), 110.76 (C, quinazoline), 116.44 (CH, benzodiox-
ane), 116.74 (CH,benzodioxane), 120.65 (CH,benzodioxane), 123.51
(CH, quinazoline), 124.96 (CH, quinazoline), 133.20 (C, benzodiox-
ane), 142.18 (CeO, benzodioxane), 142.79 (CeNH, quinazoline),
143.11 (CeO, benzodioxane), 144.91 (C, quinazoline), 158.32
(CeNH2, quinazoline), 161.47 (CeNH2, quinazoline). MS (TOF MS
ES þ) m/z ¼ 324 (Mþ1); HRMS (TOF MS ES þ)calcd for C17H18N5O2:
324.15, found: 324.15.
4.1.1. Synthesis de compounds 1, 3e5
A solution of appropriate aldehyde (1.60 mmol) and compound
12 (0.4558 g, 1.76 mmol), in methanol (80 ml) was stirred at 60 ꢀ
C
for 2 days. Then the mixture was cold to 0 ꢀC and NaBH4 (0.11 g,
3.20 mmol) was added. Later, it was stirred at room temperature for
12 h. After the mixture was evaporated on rotavapour under
decrease pressure and cold water was poured above it. The solid
was filtered off, washed with cold water and dried.
4.1.5. N6-{4-[(Dimethylamino)propoxy]benzyl}quinazoline-2,4,6-
triamine (4)
4.1.2. N6-(4-Methoxybenzyl)quinazoline-2,4,6-triamine (1)
Yellow powder (0.19 g, 40%, Mp: 215.1e216.8 ꢀC). TLC (CHCl3/
MeOH ¼ 80/20) Rf ¼ 0.73. IR (KBr): 3450 and 3328 (NeH), 1639
(CeN), 1469 and 1563 (C]C Ar), 1243 (CeOeC); 1H NMR (DMSO-
d6) ppm: 3.72 (s, 3H, CH3), 4.20 (d., J ¼ 6, 2H, CH2), 5.53 (br. s, 2H,
NH2), 5.81 (t, J ¼ 6, 1H, NH), 6.89 (d, J ¼ 9, 2H, p-methoxybenzyl),
6.96 (br. s, 3H, NH2, CH quinazoline), 7.03 (m, 2H, CH quinazoline),
7.35 (d, J ¼ 9, 2H, p-methoxybenzyl); 13C NMR (DMSO-d6): 46.69
(CH2), 55.45 (CH3eO), 100.89 (CH, quinazoline), 111.15 (C, quina-
zoline), 114.02 (CH, p-methoxybenzyl), 123.91 (CH, quinazoline),
125.35 (CH, quinazoline), 129.42 (CH, p-methoxybenzyl), 132.31 (C,
p-methoxybenzyl), 143.26 (CeNH, quinazoline), 145.37 (C, quina-
zoline), 158.57 (CeO, p-methoxybenzyl), 158.70 (CeNH2, quinazo-
line), 161.85 (CeNH2, quinazoline). MS (þFAB) m/z ¼ 296 (Mþ1);
HRMS (þFAB) calcd for C16H18N5O: 296.15, found: 296.15.
Yellow powder (0.29 g, 49%, Mp: 198.1e200.4 ꢀC). TLC (CHCl3/
MeOH ¼ 80/20) Rf ¼ 0.02. IR (KBr): 3393 and 3347 (NeH), 1625
(CeN),1568 and 1512 (C]C Ar),1240 (CeOeC); 1H NMR (DMSO-d6)
ppm: 1.80 (q, J ¼ 7, 2H, CH2), 2.11 (s, 6H, CH3eN), 2.31 (t, J ¼ 7, 2H,
CH2eN), 3.94 (t, J ¼ 7, 2H, CH2eO), 4.19 (d., J ¼ 6, 2H, CH2), 5.53 (br.
s, 2H, NH2), 5.82 (t, J ¼ 6, 1H, NH), 6.89 (d, J ¼ 9, 2H, aromatic p-
dimethylaminopropoxybenzyl), 6.98 (br. s, 3H, NH2, CH quinazo-
line), 7.03 (m, 2H, CH quinazoline), 7.32 (d, J ¼ 9, 2H, aromatic p-
dimethylaminopropoxybenzyl); 13C NMR (DMSO-d6): 27.01 (CH2),
45.28 (CH3eN), 46.59 (CH2eNH),55.77 (CH2eN), 65.77 (CH2eO),
100.52 (CH, quinazoline), 110.79 (C, quinazoline), 114.18 (CH, aro-
matic p-dimethylaminopropoxybenzyl), 123.51 (CH, quinazoline),
125.01 (CH, quinazoline), 129.03 (CH, aromatic p-dimethylamino-
propoxybenzyl), 131.86 (C, aromatic p-dimethylaminopropox-
ybenzyl), 142.90 (CeNH, quinazoline), 145.06 (C, quinazoline),
157.60 (CeO, p-dimethylaminopropoxybenzyl), 158.36 (CeNH2,
quinazoline), 161.48 (CeNH2, quinazoline). MS (TOF MS ES þ) m/
z ¼ 367 (Mþ1); HRMS (TOF MS ES þ) calcd for C20H27N6O: 367.22,
found: 367.22.
4.1.3. N,N0-{6-[(4-Methoxybenzyl)amino]quinazoline-2,4-diyl}
diacetamide (2)
A solution of 4-methoxybenzaldehyde (0.22 g, 1.61 mmol),
compound 12 (0.5850 g, 2.26 mmol), acetic acid (one drop) in N,N-
dimethylformamide (1 mL) was stirred at 85 ꢀC for 1 h. Then the
reaction mixture was cold to 0 ꢀC and was added NaBH4 (0.09 g,
2.58 mmol). Later, it was stirred at room temperature for 12 h. After
the mixture was concentrated under decreased pressure and cold
water was poured above it, the solid was filtered off, washed with
cold water and dried.
4.1.6. N6-(4-Trifluoromethoxybenzyl)quinazoline-2,4,6-triamine (5)
White powder (0.49 g, Yield: 88%, Mp ¼ 206.1e207 ꢀC), TLC
(CHCl3/MeOH, 80:20) Rf ¼ 0.32. IR (KBr): 3444 and 3351 (NeH),
1673 (CeN),1488 and 1567 (C]C Ar),1266 (CeOeC),1143 (CeF). 1H
NMR (DMSO-d6)
d
: 4.32 (d., J ¼ 6, 2H, CH2), 5.6 (br. s, 2H, NH2), 6.05
Yellow powder (0.38 g, Yield: 45%, Mp ¼ 187.1e188.2 ꢀC), TLC
(CHCl3/MeOH, 80:20) Rf ¼ 0.73. IR (KBr): 3324 and 3221 (NeH),
1692 and 1653 (C]O), 1574 and 1511 (C]C Ar); 1H NMR (DMSO-d6)
(t, J ¼ 6, 1H, NH), 6.97 (s, 1H, CH, quinazoline), 7.08 (br. s, 3H, CH
quinazoline and NH2), 7.28 (d., J ¼ 8, 2H, p-trifluoromethoxibenzyl),
7.52 (d, J ¼ 8, 2H, p-trifluoromethoxibenzyl); 13C NMR (DMSO-d6)
d:
d: 2.18 (s, 3H, CH3), 2.45 (s, 3H, CH3), 3.72 (s, 3H, CH3), 4.28 (d,
46.55 (CH2), 101.01 (CH, quinazoline), 111.16 (C, quinazoline), 120.53
(q, J ¼ 257, CF3), 121.20 (CH, p-trifluoromethoxybenzyl), 123.82 (CH,
quinazoline), 125.44 (CH, quinazoline), 129.76 (CH, p-tri-
fluoromethoxybenzyl), 140.12 (C, p-trifluoromethoxybenzyl),
143.11 (C, quinazoline), 145.27 (C, quinazoline), 147.53 (C, p-tri-
fluoromethoxybenzyl), 147.60 (CF3), 158.72 (CeNH2, quinazoline),
161.94 (CeNH2, quinazoline); MS (þFAB) m/z ¼ 349 (Mþ1); HRMS
(þFAB) calcd for C16H14F3N5O: 349.31, found 350.12.
J ¼ 5.3 Hz, 2H, CH2), 6.62 (t, J ¼ 5.3, 1H, NH), 6.9 (d, J ¼ 8.4, 1H,
aromatic p-methoxibenzyl), 7.07 (s, 1H, aromatic quinazoline), 7.34
(d, J ¼ 8.4 Hz, 1H, aromatic p-methoxibenzyl), 7.39 (br, 1H, 1H, ar-
omatic quinazoline), 7.48 (s, J ¼ 8.3, 1H, aromatic quinazoline),
10.49 (s, 1H, NH amide), 10.25 (s, 1H, NH amide); 13C NMR (DMSO-
d6) d: 24.39 and 25.22 (CH3,amide), 46.20 (CH2), 55.05 (OeCH3),
98.44 (CH, quinazoline), 113.76 (CH, p-methoxybenzyl), 115.54 (C,
quinazoline), 125.86 (CH, quinazoline), 127.17 (CH, quinazoline),
128.92 (CH, p-methoxybenzyl),131.14 (C, p-methoxybenzyl),143.49
(CeNH, quinazoline), 145.16 (C, p-methoxybenzyl) 146.30 (C, qui-
nazoline), 149.95 (C-amide, quinazoline), 155.64 (C-amide, quina-
zoline), 158.31 (CeO, p-methoxybenzyl), 168.86 (C]O, amide),
171.54 (C]O, amide); MS (þFAB) m/z ¼ 380 (Mþ1); HRMS (þFAB)
calcd for C20H21N5O3: 380.17, found 380.17.
4.1.7. N6-Ethyl-N6-(4-trifluoromethoxybenzyl)quinazoline-2,4,6-
triamine (6)
A solution of acetaldehyde (0.84 mL, 14.95 mmol), compound 5
(0.31 g, 0.89 mmol) and sodium cyanoborohydride [NaBH3CN]
(0.26 g, 4.14 mmol) was added and subjected to constant stirring in
34 mL of acetonitrile for 2 min, then, concentrated HCl was slowly
added until pH ¼ 2. It was stirred for half hour and the reaction
mixture was poured into a cold solution of sodium carbonate and
then filtered to obtain the product. The solid was washed with
ether, filtered and the obtained solution was concentrated to get a
yellow powder. Recrystallization from methanol gave the desired
product.
4.1.4. N6-(2,3-Dihydro-1,4-benzodioxin-6-ylmethyl)quinazoline-
2,4,6-triamine (3)
Yellow powder (0.33 g, 63%, Mp: 134.5e136.2 ꢀC). TLC (CHCl3/
MeOH ¼ 80/20) Rf ¼ 0.24. IR (KBr): 3442 and 3353 (NeH), 1622
(CeN), 1567 and 1507 (C]C Ar), 1285 (CeOeC); 1H NMR (DMSO-
d6) ppm: 4.15 (d., J ¼ 6, 2H, CH2), 4.19 (s, 4H, OeCH2), 5.55 (br. s, 2H,
NH2), 5.86 (t, J ¼ 6, 1H, NH), 6.78 (d, J ¼ 8, 1H, benzodioxane), 6.86
Pale yellow needles (0.15 g, Yield: 45%, Mp ¼ 176.5e178.1 ꢀC).
TLC (CHCl3/MeOH ¼ 80/20) Rf ¼ 0.35. IR (KBr): 3322 and 3158

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C. Mendoza-Martínez et al. / European Journal of Medicinal Chemistry 96 (2015) 296e307
(CeN), 1618 (C]N), 1558 and 1507 (C]C Ar), 1253 and 1155 (CeF),
1219 (CeO); 1H NMR (DMSO-d6) ppm: 1.09 (t, J ¼ 7, 2H, CH3), 3.41
(c, J ¼ 7, 2H, CH2), 4.54 (s, 2H, CH2), 5.54 (s, 2H, NH2), 7.06 (m, 4H, CH
quinazoline and NH2), 1.06 (d, J ¼ 2, 1H, CH quinazoline), 7.26 (d,
J ¼ 8, 2H, CH p-trifluoromethoxibenzyl), 7.35 (d, J ¼ 8, 2H, p-tri-
fluoromethoxibenzyl); 13C NMR (DMSO-d6): 11.91 (CH3 ethyl),
44.93 (CH2 ethyl), 52.57 (CH2), 104.25 (CH, quinazoline), 110.87 (C,
ES þ) calcd for C24H23N5O2F3: 470.18, found: 470.18.
4.1.11. N6,N6-di(4-Trifluoromethoxybenzyl)quinazoline-2,4,6-
triamine (9)
Orange powder (0.18 g, 34%, Mp: 132.9e134.7 ꢀC). TLC (CHCl3/
MeOH ¼ 80/20) Rf ¼ 0.36. IR (KBr): 3331 and 3217 (NeH), 1630
(CeN),1527 and 1416 (C]C Ar),1320 (CeF),1160 (CeOeC); 1H NMR
(DMSO-d6) ppm: 4.69 (s, 4H, CH2), 6.62 (br. s, 2H, NH2), 7.14 (d, J ¼ 9,
1H, CH quinazoline), 7.19 (dd, J1 ¼ 9, J2 ¼ 2, 1H, CH quinazoline),
7.29(d, J ¼ 8, 4H, CH p-trifluoromethoxybenzyl), 7.37 (d, J ¼ 8, 4H,
CH p-trifluoromethoxybenzyl), 7.38 (s, 1H, CH quinazoline), 7.69
(br. s, 2H, NH2); 13C NMR (DMSO-d6): 53.17 (CH2), 105.19 (CH, qui-
nazoline), 110.91 (C, quinazoline), 120.51 (c, J ¼ 257, CF3), 121.46
(CH, p-trifluoromethoxybenzyl), 121.82 (CH, quinazoline), 122.39
(CH, quinazoline), 129.31 (CH, p-trifluoromethoxybenzyl), 138.67
(C, p-trifluoromethoxybenzyl), 143.79 (C, quinazoline), 143.82 (C,
quinazoline), 147.60 (C, p-trifluoromethoxybenzyl), 157.27 (CeNH2,
quinazoline), 162.43 (CeNH2, quinazoline). MS (TOF MS ES þ) m/
z ¼ 522 (Mþ1); HRMS (TOF MS ES þ) calcd for C24H18N5O2F6:
522.14, found: 522.17.
quinazoline), 120.01 (q,
J
¼
257, CF3), 120.89 (CH, p-tri-
fluoromethoxybenzyl), 123.82 (CH, quinazoline), 125.44 (CH, qui-
nazoline), 129.76 (CH, p-trifluoromethoxybenzyl), 140.12 (C, p-
trifluoromethoxybenzyl), 143.11 (C, quinazoline), 145.26 (C, quina-
zoline), 147.54 (C, p-trifluoromethoxybenzyl), 158.72 (CeNH2, qui-
nazoline), 161.94 (CeNH2, quinazoline); MS (TOF MS ES þ) m/
z ¼ 378 (Mþ1); HRMS (TOF MS ES þ) calcd for C18H19F3N5O 378.15,
found 378.15.
4.1.8. Synthesis of compounds 7e9
The amine 1 or 5 (1 mmol) and 4-methoxybenzaldehyde or 4-
trifluoromethoxybenzaldehyde (5 mmol) were subjected to stir-
ring at room temperature in 5 mL of a mixture of acetic acid and
acetonitrile (1:1) with 5 mmol of sodium triacetoxyborohydride
[NaBH(OAc)3] for 7 days. After this time, 15 mL of methanol and
sodium carbonate were added to neutralize the acetic acid. The
solvent was evaporated, then the solid was redissolved in acetone,
filtered and the obtained solution was subjected to flash chroma-
tography for purification eluted with chloroform: methanol 98:2.
4.1.12. 6-Nitroquinazoline-2,4-diamine (11)
Sodium hydroxide (3.4 g, 85 mmol) was added to a solution of
guanidine hydrochloride (3.65 g, 38 mmol) in ethanol, and the
reaction was stirred for 20 min at room temperature. The solution
was then stirred under reflux for 6 h with 5-nitroanthranilonitrile
(5 g, 31 mmol) in 1-propanol (40 mL). Then, the mixture was
cooled to 0 C. The solid was filtered off, washed with cold water,
ꢀ
4.1.9. N6,N6-di(4-Methoxybenzyl)quinazoline-2,4,6-triamine (7)
Brown powder (0.096 g, 23%, Mp: 106.1e107.4 ꢀC). TLC (CHCl3/
MeOH ¼ 80/20) Rf ¼ 0.43. IR (KBr): 3329 and 3147 (NeH), 1655 (C]
N), 1509 (C]C Ar), 1242 (CeOeC); 1H NMR (DMSO-d6) ppm: 3.70
(s, 6H, CH3), 4.51 (s, 4H, CH2), 6.35 (br. s, 2H, NH2), 6.85 (d, J ¼ 9, 4H,
CH p-methoxybenzyl), 7.11 (d, J ¼ 9, 1H, CH quinazoline), 7.17 (d,
J ¼ 9, 4H, CH p-methoxybenzyl), 7.21 (dd, J1 ¼ 9, J2 ¼ 3, 1H, CH
quinazoline), 7.36 (d, J ¼ 3, 1H, CH quinazoline), 7.55 (br. s, 2H,
NH2); 13C NMR (DMSO-d6): 53.10 (CH2), 55.01 (OeCH3), 105.45 (CH,
quinazoline), 110.47 (C, quinazoline), 113.77 (CH, p-methox-
ybenzyl), 114.53 (CH, quinazoline), 122.33 (CH, quinazoline), 122.87
(CH, quinazoline), 128.56 (CH, p-methoxybenzyl), 130.44 (C, p-
methoxybenzyl), 140.39 (C, quinazoline), 143.86(CeNH, quinazo-
line), 157.14(CeNH2, quinazoline), 158.19 (CeO, p-methoxybenzyl),
161.98 (CeNH2, quinazoline). MS (þFAB) m/z ¼ 416 (TOF MS ES þ);
HRMS (TOF MS ES þ) calcd for C24H25N5O2: 416.21, found: 416.21.
washed with cold ethanol and dried. An orange solid product was
obtained (5.4 g, 86%, Mp: 360 ꢀC), TLC (2-butanol/acetic acid/
water ¼ 80/20/5) Rf ¼ 0.4. IR (KBr): 3464 and 3440 (NeH), 1614
(CeH, aromatic), 1325 (NO2). 1H NMR (DMSO-d6): 6.77 (s, 2H, NH2),
7.22 (d, J ¼ 9, 1H, aromatic), 7.86 (s, 2H, NH2), 8.21 (dd, J1 ¼3, J2 ¼ 9,
1H, aromatic), 9.08 (d, J ¼ 3, 1H, aromatic); 13C NMR (DMSO-d6):
108.84, 121.92, 124.87, 126.63, 139.12, 157.28, 163.00, 163.025. Anal.
Calcdfor C12H13N5O2: C, 46.83; H, 3.44; N, 34.13.
4.1.13. N-[2-(Acetylamino)-6-aminoquinazolin-4-yl]acetamide (12)
Compound 3 (1 g, 4.87 mmol) and acetic anhydride (1 mL) were
heated at 100ꢀ C for 12 h. Then, the reaction mixture was poured
into cold water, and the solid was filtered off, washed with cold
water at pH 7 and dried. A yellow solid (3a) was obtained (0.89 g,
65%, Mp: 278 ꢀC), TLC (CHCl3/MeOH ¼ 60/40) Rf ¼ 0.8. Anal. Calcd
for C12H11N5O4: C, 49.83; H, 3.83; N, 24.21. Found: C, 49.99; H, 3.11;
N, 23.81.
The catalytic reduction of 3a (0.5 g, 1.73 mmol) with hydrogen
and Pd/C (10%) (0.05 g) was performed on a Parr assembly at 60 psi
at room temperature for 1 h. The catalyst was then removed by
filtration, and the filtrate was concentrated on a rotavapor under
reduced pressure to yield 4 (0.34 g, 76%, Mp ¼ 238 ꢀC), TLC (CHCl3/
MeOH ¼ 60/40) Rf ¼ 0.67. IR (KBr): 3353 and 3230 (NeH). 1H NMR
(DMSO-d6): 2.2 (s, 3H, CH3), 2.34 (s, 3H, CH3), 5.61 (br. s, 2H, NH2),
7.04 (d, J ¼ 3, 1H, aromatic), 7.28 (dd, J1 ¼ 8, J2 ¼ 3, 1H, aromatic), 7.5
(d, J ¼ 8, 1H, aromatic), 10.46 (br. s, 1H, NH amide), 10.22 (br. s, 1H,
NH amide); 13C NMR (DMSO-d6): 24.51, 102.37, 116.82, 126.02,
127.48, 145.16, 146.55, 150.08, 156.08, 169.18, 170.73. Anal. Calcd for
4.1.10. N6-(4-Methoxybenzyl)-N6-(4-trifluoromethoxybenzyl)
quinazoline-2,4,6-triamine (8)
Yellow powder (0.30 g, 63%, Mp: 120.8e121.9 ꢀC). TLC (CHCl3/
MeOH ¼ 80/20) Rf ¼ 0.40. IR (KBr): 3390 and 3157 (NeH), 1682
(CeN), 1555 and 1511 (C]C Ar), 1322 (CeF), 1243 (CeOeC); 1H
NMR (DMSO-d6) ppm: 3.69 (s, 3H, CH3), 4.57 (s, 2H, CH2), 4.63 (s,
2H, CH2), 6.49 (br. s, 2H, NH2), 6.85 (d, J ¼ 9, 2H, p-methoxybenzyl),
7.12 (d, J ¼ 9, 1H, CH quinazoline), 7.19 (d, J ¼ 9, 1H, CH p-
methoxybenzyl),7.20 (dd, J1 ¼ 9, J2 ¼ 3, 1H, CH quinazoline), 7.28 (d,
J ¼ 9, 2H, CH p-trifluoromethoxybenzyl), 7.36 (m, 3H, CH p-tri-
fluoromethoxybenzyland CH quinazoline), 7.63 (br. s, 2H, NH2); 13
C
NMR (DMSO-d6): 52.85 (CH2), 53.40 (CH2), 55.02 (OeCH3), 105.13
(CH, quinazoline), 110.47 (C, quinazoline), 113.83 (CH, p-methox-
C12H13N5O2: C, 55.59; H, 5.05; N, 27.01. Found:C, 57.0; H, 4.52; N,
ybenzyl), 120.11 (q,
J
¼
257, CF3), 121.03 (CH, p-tri-
25.20.
fluoromethoxybenzyl), 122.16 (CH, quinazoline), 122.44 (CH,
quinazoline), 128.52 (CH, p-methoxybenzyl), 128.94 (CH, p-tri-
fluoromethoxybenzyl),130.24 (C, p-methoxybenzyl), 138.01(C, qui-
nazoline),138.43 (C, p-trifluoromethoxybenzyl), 143.64 (CeNH,
quinazoline), 147.14 (C, p-trifluoromethoxybenzyl), 156.90 (CeNH2,
quinazoline), 158.25 (CeO, p-methoxybenzyl), 162.02 (CeNH2,
quinazoline). MS (TOF MS ES þ) m/z ¼ 470 (Mþ1); HRMS (TOF MS
4.2. In vitro trypanocidal assays
Two mexican strains of T. cruzi (INC-5 and NINOA) were used.
The parasites were maintained in the laboratory in the vector
Meccuspallidipennis (Insecta: Hemiptera) and by successive pas-
sages in NIH female mice of 25e30 g. INC-5 strain was obtained

C. Mendoza-Martínez et al. / European Journal of Medicinal Chemistry 96 (2015) 296e307
305
from a chronic case of Chagas disease in the city of Guanajuato,
Mexico; a NINOA strain was obtained from an acute case of Chagas
disease by xenodiagnosis in the city of Oaxaca, Mexico [16]. The test
was evaluated in vitro on bloodstream trypomastigotes of both
strains. Blood was obtained by cardiac puncture of a female NIH
mice at the peak of parasitaemia (4 ꢂ 106 parasites/mL). Heparin
was used as anticoagulant. Infected blood was diluted with a 0.85%
sterile saline solution to a final concentration of 1 ꢂ 106 trypo-
mastigotes/mL. For each compound, stock was prepared in
dimethyl sulfoxide (DMSO) (10 mg/mL). Then, each stock solution
was serially diluted in sterile distilled water to the desired con-
centration. For the in vitro assay, sterile 96-well culture plates were
controls with untreated cells, which should give an OD reading of 1
or more. The presented results are expressed in terms of the CC50
(50% cytotoxic concentration).
4.5. In vivo antimalarial assays
From a cryopreserved stock of P. berghei the contents of the vial
in 3 female CD1 mice were thawed and then proceeded to inoculate
initiating the transmission cycle in the mouse. Five days later, the
parasitemia was determined and the mouse that had closer to 30%
(30% of the erythrocytes are parasitized) had 45 drops of blood
extracted from the vein, which were diluted with 2 mL of physio-
logical saline. 0.2 mL of the diluted blood were administered
intraperitoneally (ip) to 5 healthy mice. The parasitemia of this new
group was revised from the second day. When parasitemia reached
30%, blood from the tail was extracted and diluted with physio-
logical saline and 0.2 mL were administered ip to a new group of
five healthy mice. This procedure was repeated until the infection
was stabilized. After reaching this stabilization, we proceeded to
carry out suppression tests during 4 days.
Mice were treated orally by administration of compounds
(50 mg/kg) every 24 h on day 0e4 post inoculation (n ¼ 5 in
duplicate). At day 4, at about the same time that the infection was
performed, blood samples from the tail vein of mice were taken, the
parasitemia was determined. The inhibitory rate of the compound
at day 4 was expressed as [(Parasitaemia in control group-
eParasitaemia in compound treated group)/(Parasitaemia in con-
trol placebo group)] ꢂ 100%. Inhibitory rate and survivaletime is
recorded in comparison to untreated groups. Mice still without
parasitemia on day 30 post infection are considered cured [21]. The
control consisted of 0.2 mL of Arabic Gum orally administered.
Positive control drugs were chloroquine (CQ) and pyrimethamine
(Pyr).
used. Each plate contained 195
trypomastigotes and 5 L of each dilution so that each compound
was evaluated at a final concentration of 100, 50, 10 and 5 g/mL. As
mL of the suspension of bloodstream
m
m
a control we used a final concentration of DMSO 1% since it has
been found that concentrations <5% have no impact on mobility,
infectivity and ultrastructural morphology. The Nfx, Bzn and Pyr
were used as reference compounds. The concentrations of each
compound were evaluated in triplicate. The plates were incubated
at 4ꢀ C for 24 h. Trypanocidal activity was evaluated by counting the
number of bloodstream trypomastigotes after the incubation time
by the Brener Method [17,18]. Briefly, 5 mL of blood were placed on
slides, covered with a coverslip and the flagellates were examined
with an optical microscope at 40ꢂ magnification. Trypanocidal
activity was expressed in terms of lysis of the flagellates after in-
cubation time compared with the control group. The IC50 (mM) was
calculated using the program GraphPadPrism®.
4.3. Antileishmania assays
The antileishmanial activity was tested in an in vitro culture of
the L. mexicana strain MHOM/BZ/61/M379 growing in Dulbecco's
modified Eagle's medium (DMEM) containing L-glutamine and
4.6. ABTS assay
glucose (4.5 mg/L), without sodium bicarbonate (GIBCO, Grand Is-
land, NY), supplemented with 10% fetal calf serum (GIBCO) [19,20].
The experiments were performed in 24-well plates for tissue cul-
ture by adding 0.5 mL of one of the serial dilutions of the compound
solubilized in a specified solvent (in a ratio of 1:10 of com-
pound:media) to each well along with 0.5 mL 2 ꢂ 106 parasites/mL.
The plates were stored at 26 ꢀC for 24 h. The parasite density was
counted with a hemacytometer in triplicate. The presented results
are expressed in terms of the LC50 (50% lytic concentration).
Experiments were performed based on the work of Re et al. with
some modifications [22]. ABTS and potassium persulfate were
dissolved in distilled water to a final concentration of 7 mM and
2.45 mM, respectively. These two solutions were mixed and the
mixture was allowed to stand in the dark at room temperature for
16 h before use in order to produce ABTS radical (ABTSꢁþ). For the
study of compounds, the ABTS radical solution was diluted with
distilled water to an absorbance of 1.00 at 734 nm. Compounds or
Trolox standards (final concentrations 0e20 mM) were added to
diluted ABTSꢁþ solution and the absorbance reading was taken
10 min after mixing using the spectrophotometer. Results are
presented as the ability of compounds to scavenge 50% of free
4.4. Cytotoxicity studies
From a bottle (t-25) with 100% of cell confluence, we performed
a cell suspension (cells were washed with 1ꢂ PBS, trypsinized
(trypsin 0.25%) and MEM culture medium added 10% FBS). In a 96-
radical ABTSꢁþ (IC50). Parameters IC50
(mM) were determined with a
relative uncertainty of less than five percent.
well microplate,100 mL of cell suspension were placed in each of the
rows. In the microplates, they were incubated at 37ꢀ C in a humid
atmosphere with 5% CO2 for 24 h. After incubation, each well was
placed in the compounds at different concentrations, using cell
rows to witness DMSO, and incubated 24 h at 37ꢀ C with 5% CO2. At
4.7. DHFR inhibition
DHFR inhibition effect was tested on the in vitro growth assay
for promastigotes of L. mexicana [23]. The evaluated concentrations
the end of incubation, 50
well of the microplate and the cells were incubated again for 2 h.
100 L of SDS-HCl solution were added to each well. They were
mL of MTT solution were placed in each
of compounds 5 and 6 on the leishmania parasites were of 8
M, respectively. To test DHFR inhibition by folinic acid, folic acid
and ferulic acid, the compounds were incubated with 20, 40, 60, 80
or 100
M with 106 leishmania at the logarithmic phase of growth,
mM and
3
m
m
mixed and incubated at 24 ꢀC, overnight. The microplate was read
using an ELISA reader at a wavelength of 540 nm. Finally, we
determined the viability percentage. The viability percentage was
determined as follows:
m
washed and resuspended with PBS and incubated for 1 h at room
temperature. Then, the parasites were centrifuged, the media was
eliminated, and the parasites were resuspended in the culture
media and distributed in a 24-well plate. Compounds were then
added at the desired concentration, and the plates were incubated
for 24 h. The percentage of living parasites was calculated using the
Viability % ¼ (OJ-treated cells/OD control cells) ꢂ 100
Each test was performed in triplicate with positive and negative

306
C. Mendoza-Martínez et al. / European Journal of Medicinal Chemistry 96 (2015) 296e307
formula % AP ¼ 100 ꢂ (Tc e Tp)/Tc, where % AP is the percentage of
growth inhibition for each period, each dosage, Tc, is the number of
parasites/mL in the control wells, and Tp is the average number of
moving parasites/mL.
UNAM, for the determination of all spectra. For theoretical studies
we are grateful to DGSCA-UNAM for allowing us the use of Kan-
Balam supercomputer.
Appendix A. Supplementary data
4.8. Modeling
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.04.028.
Molecular modeling was performed with NAMD 2.6, Modeler,
Gaussian 03, Autodock 4.0.1 and VMD programs [24e27]. The
DHFR proteins were obtained from Protein Data Bank (PDB) (ID-
PDB: 2H2Q, 2BL9 and 1KMS from T. cruzi, P. vivax and human,
respectively). To construct DHFR L. major, the T. cruzi enzyme (ID-
PDB: 2H2Q Key) was used as the template. First, the amino acids
were corrected with the Modeler program. Classical MD simula-
tions were performed using the NAMD 2.6 program with the
CHARMM27 force field, and water molecules were added using the
VMD program. The structure was neutralized with 4 sodium ions
after being immersed in a TIP3P water box containing 9249 water
molecules. The equilibration protocol began with 1500 minimiza-
tion steps followed by 30 ps of MD at 310 K with fixed protein
atoms. Then, the entire system was minimized to 1500 steps (at 0 K)
and then heated gradually from 10 to 310 K by temperature reas-
signment during the first 60 ps of 100 ps equilibration dynamics
without restraints. The final step was a 30 ps NTP dynamics using
the Nose-Hoover Langevin piston pressure control 28 at 310 K and
1.01325 bar for density (volume) fitting. From this point, the
simulation continued in the NTV ensemble for 5 ns. The periodic
boundary conditions and the particle-mesh Ewald method were
applied for a complete electrostatics calculation. The dielectric
water constant was used, and the temperature was maintained at
310 K using Langevin dynamics. The structure was allowed to
converge, which was reached at 10 ns. A snapshot was obtained at
this time for computational docking. Docking was also performed
for PTR from L. major and T. cruzi (ID-PDB: 1WOC and1MXF,
respectively). The structure for lmPTR1, which was obtained after
crystallization with TAQ, validated the study.
For docking of all proteins, water molecules were removed and
its active site was defined as all residues within a grid of
120 Å ꢂ 120 Å ꢂ 120 Å with a center in the protein for blind docking
and then within a grid of 60 Å ꢂ 60 Å ꢂ 60 Å to 6.5 Å centered at
coordinates XYZ for focused docking, with an initial population of
100 randomly placed individuals and a maximum number of
1.0 ꢂ 107 energy evaluations. The compounds for docking were
drawn in Gauss view 3.0. Before docking, the compounds were
subjected to a conformational analysis and energy minimization
using the hybrid functional B3LYP with a set of bases 6, 31 G(d,p).
Docking studies were made with three minimum energy confor-
mations for evaluation of conformational space. The values of Kd
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