Synthesis, antileishmanial activity and mechanism of action studies of novel β-carboline-1,3,5-triazine hybrids

Article abstract of DOI:10.1016/j.ejmech.2018.03.014

A series of novel hybrids β-carboline-1,3,5-triazine were synthesized and evaluated for their in vitro antileishmanial activity against promastigote and amastigote forms of Leishmania amazonensis. Among the compounds tested, the hybrids 9d, 9e, 16a and 16b showed potent activity against the promastigote forms with IC₅₀ values less than 8 μM. Compounds 9e and 16b were also active against amastigote forms, displaying IC₅₀ values of 1.0 ± 0.1 μM and 1.2 ± 0.5 μM, respectively. Besides that, the hybrid 16b bearing the 4-methoxyphenyl group at C-1 of β-carboline and isopropylamino group at 1,3,5-triazine, showed low toxicity, being 23.5 and 121.4 times more toxic for promastigotes and axenic amastigotes, respectively, than for macrophage J774-A1 cell lines. Investigation of action mechanism in promastigotes showed that compound 16b caused alterations in cell division cycle and an increase of lipid-storage bodies, leading the cells to death through various factors. The accumulation of lipid bodies may be associated with apoptotic cell death.

Full text of DOI:10.1016/j.ejmech.2018.03.014

Accepted Manuscript
Synthesis, antileishmanial activity and mechanism of action studies of novel β-
carboline-1,3,5-triazine hybrids
Paula Baréa, Valéria Aquilino Barbosa, Danielle Lazarin Bidóia, Jéssica Carreira
de Paula, Talitha Fernandes Stefanello, Willian Ferreira da Costa, Celso Vataru
Nakamura, Maria Helena Sarragiotto
PII:
S0223-5234(18)30249-6
10.1016/j.ejmech.2018.03.014
EJMECH 10279
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 December 2017
Revised Date: 15 February 2018
Accepted Date: 2 March 2018
Please cite this article as: P. Baréa, Valé.Aquilino. Barbosa, D.L. Bidóia, Jé. Carreira de Paula, T.F.
Stefanello, W. Ferreira da Costa, C.V. Nakamura, M.H. Sarragiotto, Synthesis, antileishmanial activity
and mechanism of action studies of novel β-carboline-1,3,5-triazine hybrids, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.014.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, antileishmanial activity and mechanism of action studies of
novel β-carboline-1,3,5-triazine hybrids
Paula Baréaa, Valéria Aquilino Barbosaa, Danielle Lazarin Bidóiab, Jéssica Carreira de Paulab,
Talitha Fernandes Stefanellob, Willian Ferreira da Costaa, Celso Vataru Nakamurab and Maria
Helena Sarragiottoa,*
a Universidade Estadual de Maringá (UEM), Departamento de Química, Av. Colombo, 5790,
CEP: 87020-900 Maringá, PR, Brazil
b
Universidade Estadual de Maringá (UEM), Departamento de Análises Clínicas, Universidade
Estadual de Maringá, Av. Colombo, 5790, CEP: 87020-900 Maringá, PR, Brazil
*corresponding author. E-mail: mhsarragiotto@uem.br (M. H. Sarrragiotto)

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ABSTRACT
A series of novel hybrids β-carboline-1,3,5-triazine were synthesized and evaluated for
their in vitro antileishmanial activity against promastigote and amastigote forms of
Leishmania amazonensis. Among the compounds tested, the hybrids 9d, 9e, 16a and
16b showed potent activity against the promastigote forms with IC50 values less than 8
µM. Compounds 9e and 16b were also active against amastigote forms, displaying IC50
values of 1.0 ± 0.1 µM and 1.2 ± 0.5 µM, respectively. Besides that, the hybrid 16b
bearing the 4-methoxyphenyl group at C-1 of β-carboline and isopropylamino group at
1,3,5-triazine, showed low toxicity, being 23.5 and 121.4 times more toxic for
promastigotes and axenic amastigotes, respectively, than for macrophage J774-A1 cell
lines. Investigation of action mechanism in promastigotes showed that compound 16b
caused alterations in cell division cycle and an increase of lipid-storage bodies, leading
the cells to death through various factors. The accumulation of lipid bodies may be
associated with apoptotic cell death.
Keywords: β-carboline, 1,3,5-triazine, antileishmanial, Leishmania amazonensis, cell
death mechanism

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1. Introduction
Leishmaniasis is considered a neglected disease, and it is caused by protozoan
parasites of the genus Leishmania. According to World Health Organization (WHO),
leishmaniasis is endemic in 97 countries and territories, affecting mainly people in
Africa, Asia and Latin American [1]. Leishmania infections are manifested in three
clinical different forms: visceral (VL), cutaneous (CL) and mucocutaneous [1]. In South
America, the species L. amazonensis is one of the etiological agents of cutaneous
leishmaniasis, which may progress to diffuse cutaneous leishmaniasis in
immunosuppressed individuals [2]. Current chemotherapies for leishmaniasis include
treatment with pentavalent antimonials as first-line drugs, and amphotericin B,
pentamidine, paromomycin, and miltefosine as second-line drugs [3]. However, the
current drugs used clinically exhibit high toxicity, resistance, various side effects, and
an increased incidence of treatment failure [2-5]. Beside this, no vaccines against
Leishmania infections are available [6]. Therefore, there is a significant need to develop
new more effective and less toxic antileishmanial agents.
In this context, several classes of compounds [7], including natural and synthetic
β-carboline alkaloids [8-16], were described as potential antileishmanial agents.
In our previous work we have demonstrated that β-carbolines containing
substituents at 1- and 3-positions of β-carboline nucleus have shown potent
antileishmanial activity [17-22]. For instance, Tonin et al. [19] and Pedroso et al. [20]
demonstrated the potentiality of N-alkyl-(1-phenylsubstituted-β-carboline)-3-
carboxamides as antileishmanial agents. The compound N-butyl-1-(4-methoxy)-phenyl-
β-carboline-3-carboxamide (I, Fig. 1), for example, showed IC50 of 0.25 µM against
L. amazonensis promastigotes, displaying a selectivity index ratio (SI) 2,084 times
higher for the parasite than for macrophages J774G8 cells. Further studies showed that
this compound was also active against axenic and intracellular amastigote forms of L.
amazonensis, exhibiting high selectivity for the parasite. Studies on mechanism of
action demonstrated that compound I exerts its antileishmanial activity through a
cytostatic effect, thus preventing cellular proliferation [21]. In the same away, the
analog containing the N-benzyl instead N-butyl group was active against promastigote
and axenic amastigote forms with IC50 of 2.6 and 1.0 µM, respectively, and killed L.
amazonensis promastigotes through different cell death pathways, including apoptosis
and autophagy [20, 22].

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Additionally, compounds having a 1,3,5-triazine motif possess a broad range of
biological activities, which makes this heterocycle an important scaffold in the search
and development of new drugs [23]. The potentiality of 1,3,5-triazine derivatives as
antileishmanial agents has been demonstrated in several studies [24-28]. Studies on
structure-activity relationship have pointed that the presence of appropriated
substituents at 2-, 4- and 6-positions of the 1,3,5-triazine nucleus can enhance the
antileishmanial activity. For instance, the 1,2,4-triazino[5,6-b]indol-3-ylthio-1,3,5-
triazines derivatives (II, Fig. 1) containing N-ethylpiperazine, isopropylamino and
tertbutylamino substituents in 1,3,5-triazine nucleus showed more than 90% inhibition
against promastigotes form of L. donovani while compounds with the N-
methylpiperazine and morpholine substituents showed no inhibition or less than 50% of
inhibition [27]. Also, the importance of the chain length between the triazine units of
triazine mimetic III (Fig. 1) was demonstrated by Chauhan et al. [28]; the dimer with 5-
aminopentan-1-ol linker was found to be inactive while the dimer with 4-aminobutan-1-
ol linker exhibited potent antiamastigote activity for L. donovani. This compound also
showed significant in vivo inhibition (74.41%) in L. donovani/hambster model, and
prevent the progression of leishmania parasite. Besides this, it was demonstrated that
the incorporation of 1,3,5-triazine ring, bearing different amino groups, into β-carboline
nucleus led to compounds (IV, Fig. 1) with significant in vivo inhibition of L. donovani
amastigotes [14].
The inefficacy and high toxicity of the drugs used to treat leishmaniasis
infection, and the antileishmanial properties of both β-carboline and 1,3,5-triazine
nucleus, motivated us to synthetize β-carboline-1,3,5-triazine hybrids (V, Fig. 1) in
order to obtain new compounds with potent antileishmanial activity and low toxicity.
The novel hybrids were evaluated against promastigote and amastigote forms of L.
amazonensis and their cytotoxicity were determined. Also, studies on the
antileishmanial mechanism of action were carried out for the most active derivative
synthetized.

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O
R
N
N
N
N
N
N
N
R
H
N
S
N
N
II
OCH3
> 90% inhibition (promastigotes)
IC50 = 4.01 - 57.78 µM (amastigotes)
L. donovani
I
IC50 = 0.25 µM IS = 2,084
L. amazonensis promastigotes
COOCH3
R
N
N
N
NH
HN
N
H
N
N
N
N
N
R
N
N
O
n N
H
N
N
H
H
CH3
III
IV
IC50 = 0.77 to 10.32 µM
L. donovani intracellular amastigotes
68.0 - 78.6 % in vivo inhibition
L. donovani amastigotes
R2
N
N
O
N
N
R2
N
n
H
H
N
N
H
R1
V
β
-carboline-1,3,5-triazine hybrids proposed
Figure 1: Structures of β-carboline and 1,3,5-triazine derivatives with antileishmanial activity
and of β-carboline-1,3,5-triazine hybrids proposed.
2. Results and discussion
2.1. Chemistry
The novel designed β-carboline-1,3,5-triazine hybrids were synthesized by
reaction of appropriate β-carboline intermediates with cyanuric chloride, a
commercially useful reagent due the reactivity of its chlorine atoms toward nucleophiles
[29], according the synthetic routes illustrated in Schemes 1 and 2. The hybrids 8a-d,
9a-e and 10a, bearing a phenyl substituted group at 1-position of β-carboline nucleus
and chlorine atoms at 6- and 4-positions of 1,3,5-triazine ring, were obtained from the
intermediates 5a-d, 6a-e and 7a, respectively (Scheme 1). These intermediates were
prepared by a Pictet-Spengler condensation of the L-tryptophan methyl ester (2) with
benzaldehyde (a), 4-methoxybenzaldeyde (b), 4-fluorobenzaldeyde (c), 2-
chlorobenzaldeyde (d) and 3-nitrobenzaldehyde (e) followed by oxidation of the
cis/trans mixture of 3a-e in the presence of sulfur, in refluxing xylene, which afforded
the derivatives 4a-e [17-22]. Reaction of the methyl β-carboline-3-carboxylates (4) with
hydrazine hydrate, ethylenediamine and hexamethylenediamine afforded the

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corresponding β-carboline-3-carboxamides 5a-e, 6a-e and 7a. Treatment of 5a-d, 6a-d
and 7a with a suspension of cyanuric chloride in a mixture of water: acetonitrile 1:1, at
0 °C, followed by addition of sodium hydroxide in THF, gave the compounds 8a-d, 9a-
d and 10a, respectively, in good yields (45-98%). The hybrid 9e was prepared from the
reaction the intermediate 6e with cyanuric chloride, at 0 ºC, using K2CO3 as basis and
tetrahydrofuran as solvent. The intermediate 5e was also submitted to reaction with
cyanuric chloride, under the same conditions; however, in this case was observed the
formation of a complex mixture of products.
O
CH3
O
O
O
CH3
b
OH
O
c
a
NH
NH2
NH2
N
H
N
N
R1
H
H
1
2
3a-e
O
H
O
Cl
N
N
NH2
N
N
N
g
d
e
H
N
H
N
N
N
H
N
H
Cl
R1
R1
8a-d
5a-e
O
H
O
Cl
CH3
N
O
N
O
NH2
g
N
N
N
N
H
N
H
N
N
H
N
N
Cl
N
H
H
R1
R1
R1
9a-e
6a-e
4a-e
H
N
Cl
N
O
O
NH2
g
f
N
N
N
N
H
H
N
N
Cl
N
N
H
H
R1
R1
10a
7a
R1 = a) H; b) 4-OCH3; c) 4-F; d) 2-Cl; e) 3-NO2;
Scheme 1: Synthesis of compounds 8a-d, 9a-e and 10a. Reagents and conditions: (a) MeOH,
H2SO4 (cat.), reflux, 48 h; (b) R1Ph-CHO, CH2Cl2, TFA, rt, 48 h; (c) Sulfur, xylene, reflux, 48
h; (d) Hydrazine hydrate, EtOH, reflux, 48 h; (e) Ethylenediamine, rt, 24 h; (f)
Hexamethylenediamine, MeOH, CHCl3, reflux, 36 h; (g) i) Cyanuric chloride/H2O: CH3CN 1:1,
NaOH (1 M)/THF, 0 ºC, 1 h for 8a-d and 9a-d; ii) Cyanuric chloride, K2CO3, THF, 0 ºC, 1 h for
9e.
In order to evaluate the effect of other substituents than chlorine into 1,3,5-
triazine portion on antileishmanial activity, we synthetized the β-carboline-1,3,5-triazine
hybrids 11a-16a, bearing different amino substituents in the 4- and 6-positions of 1,3,5-

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triazine ring (Scheme 2). For this, the intermediate 6a (n = 2) was subjected to reaction
with cyanuric chloride, at 0 °C for 1 hour, using NaOH as basis and water-acetonitrile
as solvent, followed by addition to the reaction mixture, without previous treatment, of
10 equivalents of an appropriate amine. Heating of the reaction mixture at 70 °C for 48
hours afforded the compounds 11a-16a (Scheme 2).
The antileishmanial activity results for 11a-16a (Table 1) showed that the
hybrid 16a, bearing the isopropylamino group at 1,3,5-triazine ring, was the only one
active compound of this series. Therefore, compounds 16b-d (n = 2) and 17a-d (n = 0),
containing the isopropylamino group at 1,3,5-triazine unity and the phenyl substituted
group at position-1 of the β-carboline nucleus, were synthetized from the reaction of the
respective intermediates 6b-d and 5a-d with cyanuric chloride in basic medium,
followed by addition of 10 equivalents of isopropylamine (Scheme 2). The reaction of
6e with cyanuric chloride in the presence of K2CO3 and excess isopropylamine afforded
the compound 16e in 40% yield.
R2
4"'
O
N
6"'
N
2'"
O
6
8
1) Cyanuric chloride,
H2O/CH3CN,NaOH
(1M), 0 ºC, 1h
5
NH2
4
1
n
N
R2
3
N
7
N
N
H
n
H
H
N
N
2) Amines (R2NH e R2NH2)
70 °C, 48 h
N
N
H
H
R1
R1
11a-16a (n = 2); 16b-e (n = 2)
17a-d (n = 0)
5a-d (n = 0)
6a-e (n = 2)
1
1
2
2
R
R
Comp.
11a
Comp.
R
R
N
H
Ph
Ph
Ph
Ph
16a; 17a
16b; 17b
16c; 17c
16d; 17d
H2NNH
H
N
N
H
4-OCH -Ph
3
12a
HN
4-F-Ph
13a
N
H
N
H
Ph H3C
2-Cl-Ph
N
N
14a
a
O
N
Ph
3-NO -Ph
2
15a
16e
N
H
aFor 16e was used K2CO3 as basis and THF as solvent
Scheme 2: Synthesis of compounds 11a-16a, 16b-e and 17a-d.
All hybrids were characterized by their spectral data (HRMS, 1H NMR and 13C).
The characterization of the β-carboline-1,3,5-triazine hybrids 8a-d, 9a-e and 10a
bearing chlorine at 6- and 4-positions of 1,3,5-triazine ring was mainly supported by the
signals at δC 167.1 – 170.3 (C0), characteristic of C-Cl bonds, and at δC 163.9 – 165.3
(C0) related to C-2 of the1,3,5-triazine ring, in 13C NMR spectra. The hybrids 11a-16a,
16b-e and 17a-d were characterized by the signal of aminoalkyl substituents linked to

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6- and 4-positions of 1,3,5-triazine. For example, the compounds containing the
isopropylamino substituent (16a-e and 17a-d) showed the presence of signals at δH 0.86
1
– 1.12 (12H, CH3), δH 4.01 – 4.09 (2H, CH), in H NMR spectra, and δC 22.5 – 22.7
13
(CH3) and δC 40.1 – 41.6 (CH), in C NMR spectra, related to methylene hydrogens
and carbons, respectively.
2.2. Antileishmanial Activity
In this work, novel β-carboline-1,3,5-triazine hybrids synthesized (8a-d, 9a-e,
10a-15a, 16a-e and 17a-d) were evaluated in vitro against the promastigote and
amastigote forms of L. amazonensis. The antileishmanial assay results are shown in
Table 1. The compounds that showed IC50 (50% Inhibitory Concentration) values
greater than 100 µM were considered inactive. The toxic effects on the host cells were
determined by the selectivity index (SI). The SI for each active compound was
calculated as the ratio between the cytotoxicity (CC50) for the macrophage J774-A1 cell
lines and IC50 against the promastigote and amastigote forms of L. amazonensis.
In order to evaluate the influence of the linkers between the β-carboline and
1,3,5-triazine units, firstly, the β-carboline-1,3,5-triazine hybrids 8a (n=0), 9a (n=2) and
10a (n=6), bearing the phenyl group at 1-position of β-carboline nucleus and chlorine
atoms at 4- and 6-positions of triazine ring, were assayed for their antileishmanial
activity. As showed in the Table 1, compounds 8a (n = 0) and 9a (n = 2) were
moderately active for L. amazonensis promastigotes, with C50 values of 43.3 µM and
30.9 µM, respectively, while the compound 10a (n = 6) was inactive. The hybrids 8a
and 9a showed also potent activity and high selectivity against amastigote forms of L.
amazonensis, with IC50 values minor than 2 µM and SI of 85.9 and 70.8, respectively.
Aiming to increase the activity of compounds 8a and 9a against L. amazonensis
promastigotes we introduced electron-donating and electron-withdrawing groups at the
phenyl group in the 1-position of the β-carboline nucleus. The 4-methoxy, 2-chloro and
3-nitro substituents were chosen based on our previous studies, which demonstrated that
these groups at 1-phenyl provided β-carboline derivatives with promising
antileishmanial activities [19-21]. Besides that, a new withdrawing substituent (4-
fluoro) was chosen in order to compare its effects on the antileishmanial activity with
those of the other groups.
In this work, the introduction of 4-methoxy (8b and 9b) and 4-fluoro (8c and 9c)
at 1-phenyl group led to a decrease or loss of activity against promastigote forms in

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comparison to 8a and 9a. On the other hand, the presence of 2-chloro and 3-nitro
substituents at 1-phenyl group in 9d and 9e resulted in a 4- and 6-fold increase,
respectively, in the activity towards promastigotes. The hybrids 9b and 9e, as well as 9a
presented potent activity and high selectivity index for the amastigote forms.
From the IC50 data for the β-carboline-1,3,5-triazine derivatives 11a-16a we
verified that only the compound 16a, with the isopropylamino group in 1,3,5-triazine
ring, was active, showing IC50 values of 7.5 ± 2.5 µM and 35.0 ± 0.5 µM, respectively,
against promastigotes and amastigotes forms of L. amazonensis. Comparison of these
results with those obtained for 9a show that replacement of the chlorine atoms at C-4
and C-6 of 1,3,5-triazine ring by isopropylamino group increases about 4-fold the
activity to the promastigotes form. On the other hand, the insertion of isopropylamino
group in 16a resulted in a decrease of activity for the amastigote forms by
approximately 18 times in comparison with those of 9a. The introduction of the others
amino substituents in 1,3,5-triazine ring (11a-15a) also led to loss of activity for
promastigote forms.
Finally, we evaluated the antileishmanial activity for the hybrids bearing the
isopropylamino group at C-4 and C-6 of 1,3,5-triazine and the phenyl substituted groups
at the β-carboline nucleus 16b-e (n = 2) and 17a-d (n=0).
Analysis of IC50 data for the series where n=2 showed that the insertion of the
isopropylamino group led to a loss and decrease of the activity towards the
promastigotes for compounds 16d (IC50 > 100 µM) and 16e (IC50=44.6 ± 1.2 µM)
compared to 9d (IC50 = 7.6 ± 2.02 µM) and 9e (IC50 = 5.1 ± 0.1 µM), respectively. On
the other hand, the replacement of chlorine atoms of 9b (IC50 = 67.5 ± 0.0 µM) by
isopropylamino group in compound 16b (IC50 = 6.2 ± 1.4 µM) increased the
antileishmanial activity against to the promastigote forms by about 10 times. Compound
16b was also active for amastigotes (IC50 = 1.2 ± 0.5 µM), with increase of the activity
by about 30-fold when compared with the compound 16a (IC50 = 35.0 ± 0.5 µM).
Besides that, the hybrid 16b showed a decreasing toxicity, being 23.5 and 121.4 times
more toxic for promastigotes and axenic amastigotes of the parasites, respectively, than
for the host cells (Table 1). The positive control miltefosine, a drug used for treatment
of cutaneous leishmaniasis [30], showed IC50 of 18.5 and 2.4 µM, and SI of 2.2 and
16.9, for promastigotes and amastigotes, respectively (Table 1). Thus, 16b becomes
promising for other studies.

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Compounds 17a-d (n=0) were moderately active or inactive against L.
amazonensis promastigotes, showing that the best linker between the β-carboline and
1,3,5-triazine moieties is those having the ethylene unity (n=2).

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Table 1: Antileishmanial activity data for compounds 8a-d, 9a-e, 11a-15a, 16a-e and
17a-d against L. amazonensis.
Promastigotes Amastigotes
J774A1
CC50 (µM)
94.5 ± 7.8
IS
Comp.
n
R1
R2
IC50 (µM)
IC50 (µM)
PRO
2.2
AMA
85.9
0
0
0
0
2
2
2
2
2
6
2
Ph
4-OCH3-Ph
4-F-Ph
2-Cl-Ph
Ph
Cl
Cl
43.3 ± 10.3
8a
8b
8c
1.1 ± 0.1
>100
>100
n.t
n.t
n.t
n.t
n.d
n.d
0.6
4.4
3.0
n.d
3.8
16.3
n.d
n.d
n.d
n.d
Cl
Cl
34.1 ± 10.6
30.9 ± 0.9
67.5 ± 0.1
>100
n.t
22.0 ± 4.2
134.6 ± 11.8
201.3 ± 3.9
n.t
n.d
8d
9a
Cl
70.8
201.3
n.d
1.9 ± 0.4
1.0 ± 0.1
n.t
4-OCH3-Ph
4-F-Ph
2-Cl-Ph
3-NO2-Ph
Ph
Cl
9b
9c
Cl
Cl
n.t
28.5 ± 3.6
83.1 ± 7.8
n.t
n.d
9d
9e
7.6 ± 2.02
5.1 ± 0.1
>100
Cl
1.1 ± 0.2
n.t
75.5
n.d
Cl
10a
11a
Ph
-NHNH2
>100
n.t
n.t
n.d
n.t
n.t
n.t
n.t
2
2
Ph
Ph
>100
>100
n.d
n.d
n.d
n.d
12a
13a
n.t
n.t
n.t
n.t
2
2
Ph
Ph
>100
>100
n.d
n.d
n.d
n.d
14a
15a
2
2
2
2
2
0
0
0
Ph
35.0 ± 0.5
1.2 ± 0.5
n.t
98.1 ± 6.5
145.7 ± 10.1
n.t
13.1
23.5
n.d
n.d
1.9
2.6
n.d
2.80
121.4
n.d
16a
16b
16c
16d
16e
17a
17b
17c
7.5 ± 2.5
6.2 ± 1.4
>100
4-OCH3-Ph
4-F-Ph
n.t
n.t
2-Cl-Ph
3-NO2-Ph
Ph
>100
n.d
44.6 ± 1.2
39.7 ± 2.9
>100
n.t
32.5 ± 3.4
n.t
86.3 ± 11.8
102.7 ± 9.9
n.t
n.d
3.16
n.d
4-OCH3-Ph
4-F-Ph
n .t
43.4 ± 1.2
50.0 ± 14.1
1.2
n.d
0
-
2-Cl-Ph
-
>100
n.t
n.t
n.d
2.2
n.d
17d
Miltefosinea
-
18.5 ± 1.1
2.4 ± 0.1
40.5 ± 1.7
16.9
a positive control; n.t: not tested; n.d: not determined.

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2.3.Transmission Electron Microscopy
As the compound 16b showed potent activity against promastigotes and axenic
amastigotes of L. amazonensis, and higher selectivity index than the other derivatives
synthetized for both forms, it was submitted to studies for investigate their mechanism
of protozoan cell death.
The treatment of L. amazonensis promastigotes with compound 16b during 24 h
caused ultrastructural alterations, mainly in mitochondria and plasma membrane. After
48 h of treatment, it was observed storage-lipic bodies, mitochondrial swelling and
plasma membrane alterations. The treatment with 72 h showed alterations in flagellar
pocket with vacuoles and mitochondrial swelling. These results initially demonstrated
that in promastigotes the compound 16b caused alterations in cell division cycle and,
due to stress, it started a process of energy storage (storage-lipid bodies) [31], leading
the cells to death thought various factors (Figure 2). For scanning electron microscopy
(SEM) the treatment after 24, 48 and 72 h caused cell rounding and cell volume
reduction (Figure 3).
Figure 2. Ultrastructural alterations in promastigotes of Leishmania amazonensis
treated with 16b, visualized by transmission electron microscopy. Control cells (A-C)
and parasites treated with IC50 (6.2 µM) (D-F) for 24 h (A; D), 48 h (B; E) and 72 h (C;
F). (n) nucleus; (m) mitochondrion; paraflagellar pocket; (*) lipid-storage bodies. Bars
= 1 µm.

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Figure 3. Morphological alterations in promastigotes of Leishmania amazonensis
treated with 16b, visualized by scanning electron microscopy. Control cells (A-C)
and parasites treated with IC50 (6.2 µM) (D-F) for 24 h (A;D), 48 h (B; E) and 72 h
(C;F). Bars = 5 µm.
2.4. Biochemical assays
Leishmania amazonensis promastigotes were treated with compound 16b at the
concentrations corresponding to IC50 and IC90, for 24 h, and loaded with 2',7'-
dichlorodihydrofluorescein diacetate (H2DCFDA), a probe that is permeable to the
plasma membrane and chemically reduced in 2',7'-dichlorofluorescein (DCF) when in
contact with reactive oxygen species (ROS), producing fluorescence. The parasites
treated with positive control (0.5 and 4 µM of H2O2) showed a fluorescence increase of
45.9 and 230.8%, while treatment with 16b with concentrations corresponding to IC50
and IC90 showed increase 81.9 and 184.8%. The parasites pre-incubated with N-acetyl-
cysteine (NAC) and treatment with H2O2 (0.5 and 4 µM) showed reversible effects, but
the treatment with 16b not showed such effects (Fig. 4A).
Additionally, the evaluation of lipid bodies was performed by using the probe
Nile red. The positive control (H2O2) at concentrations of 0.5 and 4 µM showed an
increase in fluorescence of 19% and 241.2%, respectively. The treatment with 16b at
concentrations corresponding to IC50 and IC90 showed an increase in fluorescence of 9%
and 428.6%, respectively, confirming the increase of lipid-storage bodies. However, this
accumulates of lipid-storage bodies is not related with the ROS generation, initially
observed. The accumulation of lipid bodies is caused for cellular stress, mitochondrial
dysfunction and may be associated with apoptotic cell death [21, 32] (Fig. 4B).

ACCEPTED MANUSCRIPT
µM
µM
µM
µM
Figure 4. Total ROS production using the fluorescent probe H2DCFDA (A) and Nile
Red accumulation (B) in promastigotes of Leishmania amazonensis treated with 16b for
24 h. IC50 (6.2 µM); IC90 (9.9 µM). The data are expressed as relative fluorescence of at
least three independent experiments. H2O2 was used as a positive control. Two-way
ANOVA followed by Bonferroni post hoc test. *p ≤ 0.05 compared to untreated
parasites (control).
3. Conclusions
In this work, novel β-carboline-1,3,5-triazine hybrids containing different
spacers between the β-carboline and 1,3,5-triazine units, chlorine and amino groups at
6- and 4-position of 1,3,5-triazine ring, and electron-donating and electron-withdrawing
substituents at the 1-phenyl group of β-carboline nucleus have been synthesized and
evaluated in promastigote and amastigote forms of Leishmania amazonenis. The assay
results showed that the nature of substituents attached at β-carboline and 1,3,5-triazine
moieties influenced the activity. The hybrid 9e, containing the 3-nitrophenyl at C-1 of
β-carboline was the most active among the compounds bearing chlorine atoms at 1,3,5-
triazine ring, displaying potent activity for both promastigote and amastigote forms.
The presence of amino groups at 1,3,5-triazine nucleus led to loss of activity for
almost all compounds, except for those bearing the isopropylamino group (16a, 16b,
16e, 17a and 17c). Compound 16b containing the 4-methoxyphenyl at C-1 of β-
carboline nucleus showed potent activity and low toxicity to amastigote and
promastigote forms of L. amazonenis, being a promising candidate for antileishmanial
agent.
Studies on mechanism of action showed that in promastigotes the compound 16b
caused alterations in cell division cycle and an increase of lipid-storage bodies, leading
the cells to death thought various factors. This accumulation of lipid bodies is caused of
cellular stress, mitochondrial dysfunction and may be associated with apoptotic cell
death.

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In summary, from our results it was possible to obtain new hybrids β-carboline-
1,3,5-triazine with remarkable antileishmanial activity, which can be considered as
potential compounds for future studies in view to development of new antileishmanial
agents.
4. Experimental section
4.1.General methods
All reagents were purchased from commercial suppliers. The reactions were
monitored by thin layer chromatography conducted on Whatman TLC plates (Silica Gel
60 F254). NMR spectra were recorded in a in Varian spectrometer model Mercury plus
1
13
BB at 300 MHz (for H) and 75 MHz (for C), and in a Bruker spectrometer model
1
13
Avance III HD at 500 MHz (for H) and 125 MHz (for C), with TMS and deuterated
solvents, dimethyl sulfoxide (DMSO-d6) and chloroform (CDCl3), as internal standard.
Mass spectra (ESI/MS) were recorded on Thermoelectron Corporation Focus-DSQ II
spectrometer. Melting points were determined in Microquímica apparatus model
MQAPF-301 and are uncorrected.
The following chemicals were used in biological assay: Warren medium; fetal
bovine serum (FBS); carbonylcyanide m-chlorophenylhydrazone (CCCP),
dimethylsulfoxide (DMSO), Roswell Park Memorial Institute Medium (RPMI 1640;
Gibco 99 Invitrogen, Grand Island, NY, USA), 3-[4,5-dimethylthiazol-2-yl]-2,5-100
diphenyltetrazolium bromide (MTT), potassium sodium buffer; Nile red (Sigma); 2',7'-
dichlorodihydrofluorescein diacetate (H2DCFDA-ThermoFisher); sodium cacodilate
(Electron Microscopy Scanning - EMS); glutaraldehyde 25% (EMS); potassium
ferrocyanide; osmium tetroxide; EPON resin; acetone P.A.; uranyl acetate; lead citrate.
4.2.Chemistry
4.2.1. Synthesis of L-tryptophan methyl ester (2)
To a suspension of commercial L-tryptophan (1) (5.00 g, 24.5 mmol) in
methanol (50 mL) was added dropwise concentrated sulfuric acid until complete
solubilization. The reaction mixture was kept under reflux for 48 hours. The solution
was cooled and neutralized with a solution of 5% sodium carbonate, and extracted with
ethyl acetate (4 x 30 mL). The organic phase was dried with anhydrous sodium sulfate
and after filtration the solvent was removed in a rotary evaporator. The pure product
was obtained in 91% yield.

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4.2.2. Synthesis of cis e trans 1-(substituted-phenyl)-3-carbomethoxy-1,2,3,4-
tetrahydro-β-carboline (3a-e)
To a solution of L-tryptophan methyl ester (2) (4.6 mmol) in dichloromethane
(20 mL) was added 4.6 mmol of aldehyde [benzaldehyde (a), 4-methoxy-benzaldehyde
(b), 4-fluoro-benzaldeyde (c), 2-chloro-benzaldeyde (d) and 3-nitro-benzaldehyde (e)]
and trifluoroacetic acid (9.20 mmol). The reaction mixture was kept at room
temperature and its progress monitored by TLC. After complete consumption of starting
materials (48 h), the solvent was evaporated, and the crude product solubilized in ethyl
acetate and neutralized with a solution of 5% sodium carbonate. The solution was
extracted with ethyl acetate (3 x 30 mL). The organic phase was dried with anhydrous
sodium sulfate, filtered and the solvent was removed in a rotary evaporator. The solid
obtained was washed with methanol, providing a mixture of cis and trans 1-
(substituted-phenyl)-3-carbomethoxy-tetrahydro-β-carboline (3a-e) in yields in the
range 75-85%.
4.2.3. Synthesis of methyl 1-(substituted-phenyl)-β-carboline-3-carboxylates
(4a-e)
To the mixture of cis and trans 1-(substituted-phenyl)-3-carbomethoxy-1,2,3,4-
tetrahydro-β-carbolines 3a-e (6.53 mmol) suspended in xylene (30 mL) was added 20
mmol of sulfur. The mixture was refluxed and the reaction progress monitored by TLC
every 12 h. After complete consumption of starting materials (48 h), the solution was
cooled and left on ice bath at 0 °C for approximately 1 h. The precipitate formed was
filtered and washed with petroleum ether to afford the β-carbolines 4a-e in yields in the
range of 85 - 93%.
4.2.4. Synthesis of 1-(substituted-phenyl)-β-carboline-3-carbohydrazides (5a-e)
To a solution of compounds 4a-e (3.31 mmol) in ethanol (30 mL) was added
hydrazine hydrate 50% (66 mmol). The solution was kept under reflux and the reaction
progress monitored by TLC every 12 h. After complete consumption of starting
materials (48 h), the solution was cooled and left on ice bath at 0 °C for 1 hour. The
precipitate formed was filtered in vacuum and washed with ethanol to afford the
compounds 5a-e in yields in the range of 53-85%.
4.2.5. Synthesis of of N-(2-aminoethyl)-1-(substituted-phenyl)-β-carboline-3-
carboxamides (6a-e)

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The compounds 4a-e (1 mmol) were solubilized in 1 mL of ethylenediamine and
the resulting solution was stirred at room temperature. The reaction progress was
monitored by TLC every 8 h, and after complete consumption of starting materials (24
h), the solution was treated with 2 mL of distilled water and kept at 0 °C for
approximately 1 hour. The precipitates formed were filtered in vacuum and washed with
distilled water to furnish the compounds 6a-e in yields in the range of 60-90%.
4.2.6. Synthesis of N-(6-aminohexyl)-1-phenyl-β-carboline-3-carboxamide (7a)
To a solution of hexamethylenediamine (0.70 g) in chloroform (2 mL), under
reflux, was added a solution of 4a (0.3 mmol in 0.5 mL of methanol). The solution was
kept under reflux, and the reaction progress monitored by TLC every 12 h. After
complete consumption of starting materials (36 h), the solution was cooled and treated
with 2 mL of distilled water and the precipitate formed was filtered and washed with a
mixture of water-ethanol (1:1). The compound 7a was obtained in 70% yield.
4.2.7. Procedure for the preparation of β-carboline-4,6-dichloro-1,3,5-triazine
derivatives (8a-d, 9a-d and 10a)
To a suspension of cyanuric chloride (0.2 mmol) in water-acetonitrile 1:1 (0.5
mL), at 0 °C, was added a suspension of 5a-d (0.2 mmol), 6a-d (0.2 mmol) or 7a (0.2
mmol) in THF (3 mL). The pH was adjusted to 9-10 with sodium hydroxide 1 mol L-1.
The reaction mixture was stirred at 0 ºC for 1 hour and treated with distilled water (1
mL). The precipitates formed were filtered and washed with distilled water to afford
compounds 8a-d, 9a-d or 10a in yields in the range of 45-98%.
4.2.7.1.
N'-(4,6-dichloro-1,3,5-triazin-2-yl)-1-phenyl-β-carboline-3-
1
carbohydrazide (8a): Yellow crystals; Yield: 50%; M.p. > 350 °C (decomp.); H NMR
(300 MHz): δ ppm: 7.33 (t, J = 7.3, 1H, H-6), 7.56-7.74 (m, 5H, H-7, H-8, H-3’, H-4’,
H-5’), 8.25 (d, J = 7.2, 2H, H-2’, H-6’), 8.44 (d, J = 7.8, 1H, H-5), 8.87 (s, 1 H, H-4),
10.98 (s, 1H, CO-NH), 11.98 (s, 1H, NH, H-9). 13C NMR (75 MHz): δ ppm: 112.8 (CH,
C-8), 114.1 (CH, C-4), 120.4 (CH, C-6), 121.2 (C0, C-4b), 122.1 (CH, C-5), 128.8
(3CH, C-2’, C-4’, C-6’), 129.0 (CH, C-3’, C-5’), 129.1 (CH, C-7), 129.8 (C0, C-4a),
134.5 (C0, C-9a), 137.2 (C0, C-1’), 138.8 (C0, C-3), 141.0 (C0, C-1), 141.6 (C0, C-8a),
163.9 (C0, C-2'"), 164.5 (C=O), 169.1 and 170.3 (C0, C-4'", C-6'"); HRMS-ESI calcd for
C21H14Cl2N7O [M+H]+ 450.0631, found: 450.0599, error 7.1 ppm..

ACCEPTED MANUSCRIPT
4.2.7.2.
N'-(4,6-dichloro-1,3,5-triazin-2-yl)-1-(4-methoxyphenyl)-β-carboline-3-
1
carbohydrazide (8b). Yellow crystals; Yield: 98%; M.p. > 350 °C (decomp.); H NMR
(300 MHz): δ ppm: 3.90 (s, 3H, OCH3), 7.20 (d, J = 8.9, 2H, H-3’, H-5’), 7.32 (t, J =
7.5, 1H, H-6), 7.61 (t, J = 7.6, 1H, H-7), 7.72 (d, J = 8.2, 1H, H-8), 8.23 (d, J = 8.9, 2H,
H-2’, H-6’), 8.43 (d, J = 7.8, 1H, H-5), 8.83 (s, 1 H, H-4), 10.89 (s, 1H, CO-NH), 11.94
13
(s, 1H, NH, H-9). C NMR (75 MHz): δ ppm: 55.4 (OCH3), 112.8 (CH, C-8), 113.3
(CH, C-4), 114.1 (2CH, C-3’, C-5’), 120.3 (CH, C-6), 121.2 (C0, C-4b), 122.0 (CH, C-
5), 130.4 (2CH, C-2’, C-6’), 129.6 (CH, C-7), 129.5 (C0, C-4a), 128.7 (C0, C-9a), 134.0
(C0, C-1’), 138.5 (C0, C-3), 140.5 (C0, C-1), 141.6 (C0, C-8a), 160.0 (C0, C-4’), 164.5
(C0, C-2'"), 164.5 (C=O), 166.1 and 167.1 (C0, C-4'", C-6'"); HRMS-ESI calcd for
C22H16Cl2N7O2 [M+H]+ 480.0737, found: 480.0731, error 1.2 ppm.
4.2.7.3.
N'-(4,6-dichloro-1,3,5-triazin-2-yl)-1-(4-fluorophenyl)-β-carboline-3-
1
carbohydrazide (8c). Yellow crystals; Yield: 47%; M.p. > 298 °C (decomp.); H NMR
(300 MHz): δ ppm: 7.32 (td, J = 7.5, 1.1, 1H, H-6), 7.47 (t, J = 8.9, 2H, H-3’, H-5’),
7.61 (td, J = 7.6, 1.1, 1H, H-7), 7.71 (d, J = 8.2, 1H, H-8), 8.28 (dd, J = 8.9, 5.5, 2H, H-
2’, H-6’), 8.42 (d, J = 7.8, 1H, H-5), 8.79 (s, 1 H, H-4), 11.53 (s, 1H, CO-NH), 11.94 (s,
13
1H, NH, H-9). C NMR (75 MHz): δ ppm: 112.7 (CH, C-8), 112.7 (CH, C-4), 115.5
and 115.8 (2 CH, C-3’, C-5’), 120.3 (CH, C-6), 121.2 (C0, C-4b), 122.1 (CH, C-5),
128.7 (CH, C-7), 130.3 (C0, C-4a), 130.7 and 130.8 (2 CH, C-2’, C-6’), 130.9 (C0, C-
9a), 133.8 (C0, C-1’), 133.8 (C0, C-3), 141.6 (C0, C-1), 141.6 (C0, C-8a), 161.1 (C0, C-
4’), 164.3 (C0, C-2'"), 164.3 (C=O); HRMS-ESI calcd for C21H13Cl2FN7O [M+H]+
468.0537, found: 468.0534, error 0.6 ppm.
4.2.7.4.
N'-(4,6-dichloro-1,3,5-triazin-2-yl)-1-(2-chlorophenyl)-β-carboline-3-
1
carbohydrazide (8d). Yellow crystals; Yield: 45%; M.p. > 345 °C (decomp.); H NMR
(300 MHz): δ ppm: 7.31 (m, 1H, H-6), 7.73 (m, 2H, H-7, H-8), 7.60 (m, 4H, H-3’, H-4’,
H-5’, H-6’), 8.43 (d, J = 8.0, 1H, H-5), 8.88 (s, 1 H, H-4), 11.04 (s, 1H, CO-NH), 11.71
(s, 1H, NH, H-9). 13C NMR (75 MHz): δ ppm: 112.4 (CH, C-8), 114.6 (CH, C-4), 120.2
(CH, C-6), 121.0 (C0, C-4b), 122.3 (CH, C-5), 127.5 (CH, C-7), 128.8 (CH, C-5’),
129.8 (C0, C-4a), 130.7 (C0, C-2’), 132.2 (CH, C-3’), 132.3 (CH, C-6’), 132.6 (C0, C-
4’), 135.4 (C0, C-1’), 136.3 (C0, C-3), 136.4 (C0, C-9a), 140.1 (C0, C-1), 141.4 (C0, C-
8a); HRMS-ESI calcd for C21H13Cl3N7O [M+H]+ 484.0242, found: 484.0214, error 5.8
ppm.

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4.2.7.5.
N-{2-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]ethyl}-1-phenyl-β-
carboline-3-carboxamide (9a): White crystals; Yield: 75%; M.p: 146-150 °C; 1H NMR
(500 MHz): δ ppm: 3.54 (m, 2H, H-2”), 3.60 (m, 2H, H-1”), 7.32 (ddd, J = 7.9, 7.0, 0.9,
1H, H-6), 7.56-7.70 (m, 5H, H-7, H-8, H-3’, H-4’, H-5’), 8.13 (dd, J = 8.2, 1.2, 2H, H-
2’, H-6’), 8.42 (d, J = 8.1, 1H, H-5), 8.83 (s, 1 H, H-4), 8.87 (t, J = 6.2, CONH), 9.25 (t,
J = 5.6, 1H, NH-C2”), 11.84 (s, 1H, NH, H-9). 13C NMR (125 MHz): δ ppm: 37.8 (CH2,
C-1"), 40.9 (CH2, C-2"), 112.7 (CH, C-8), 113.1 (CH, C-4), 120.2 (CH, C-6), 121.2 (C0,
C-4b), 122.0 (CH, C-5), 128.6 (CH, C-4’), 128.7 (2CH, C-2’, C-6’), 128.8 (CH, C-3’,
C-5’), 128.9 (CH, C-7), 129.8 (C0, C-4a), 134.2 (C0, C-9a), 137.5 (C0, C-1’), 139.7 (C0,
C-3), 140.6 (C0, C-1), 141.6 (C0, C-8a), 165.3 (C0, C-2'"), 165.5 (C=O), 168.4 and 169.4
(C0, C-4'", C-6'"); HRMS-ESI calcd for C23H18Cl2N7O [M+H]+ 478.0944, found:
478.0946, error 0.4 ppm.
4.2.7.6.
N-{2-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]ethyl}-1-(4-
methoxyphenyl)-β-carboline-3-carboxamide (9b). White crystals; Yield: 87%; M.p:
194-196 °C; 1H NMR (500 MHz): δ ppm: 3.54 (m, 2H, H-2”), 3.60 (m, 2H, H-1”), 3.89
(s, 3H, OCH3), 7.19 (d, J = 8.9, 2H, H-3’, H-5’), 7.30 (t, J = 7.9, 1H, H-6), 7.59 (ddd, J
= 8.2, 7.1, 1.1, 1H, H-7), 7.69 (d, J = 8.2, 1H, H-8), 8.10 (d, J = 8.9, 2H, H-2’, H-6’),
8.39 (d, J = 7.9, 1H, H-5), 8.77 (s, 1 H, H-4), 8.86 (t, J = 6.1, CONH), 9.26 (t, J = 5.6,
1H, NH-C2”), 11.79 (s, 1H, NH, H-9). 13C NMR (125 MHz): δ ppm: 37.8 (CH2, C-1"),
40.9 (CH2, C-2"), 55.4 (OCH3), 112.5 (CH, C-8), 112.7 (CH, C-4), 114.2 (2CH, H-3’,
H-5’), 120.1 (CH, C-6), 121.3 (C0, C-4b), 122.0 (CH, C-5), 129.6 (C0, C-4a), 128.5
(CH, C-7), 129.9 (C0, C-9a), 130. 1 (2CH, C-2’, C-6’), 134.0 (C0, C-1’), 139.5 (C0, C-
3), 140.5 (C0, C-1), 141.5 (C0, C-8a), 159.9 (C0, C-4’), 165.3 (C0, C-2'"), 165.5 (C=O),
168.4 and 169.4 (C0, C-4'", C-6'"); HRMS-ESI calcd for C24H20Cl2N7O2 [M+H]+
508.1050, found: 508.1047, error 0.6 ppm.
4.2.7.7.
N-{2-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]ethyl}-1-(4-fluorophenyl)-
1
β-carboline-3-carboxamide (9c). White crystals; Yield: 65%; M.p: 153-157 °C; H
NMR (300 MHz): δ ppm: 3.54 (m, 2H, H-2”), 3.60 (m, 2H, H-1”), 3.89 (s, 3H, OCH3),
7.19 (d, J = 8.9, 2H, H-3’, H-5’), 7.30 (t, J = 7.9, 1H, H-6), 7.59 (ddd, J = 8.2, 7.1, 1.1,
1H, H-7), 7.69 (d, J = 8.2, 1H, H-8), 8.10 (d, J = 8.9, 2H, H-2’, H-6’), 8.39 (d, J = 7.9,
1H, H-5), 8.77 (s, 1 H, H-4), 8.86 (t, J = 6.1, CONH), 9.26 (t, J = 5.6, 1H, NH-C2”),
11.79 (s, 1H, NH, H-9). 13C NMR (75 MHz): δ ppm: 37.8 (CH2, C-1"), 40.9 (CH2, C-
2"), 112.7 (CH, C-8), 113.2 (CH, C-4), 115.5 and 115.8 (2CH, H-3’, H-5’), 120.3 (CH,

ACCEPTED MANUSCRIPT
C-6), 121.2 (C0, C-4b), 122.1 (CH, C-5), 128.7 (C0, C-4a), 130.0 (CH, C-7), 131.0 and
131.1 (2 CH, C-2’, C-6’), 131.1 (C0, C-9a), 133.8 (C0, C-1’), 134.1 (C0, C-3), 139.6
(C0, C-1), 141.6 (C0, C-8a), 165.2 (C0, C-4’), 165.2 (C0, C-2'"), 165.5 (C=O), 168.4 and
169.4 (C0, C-4'", C-6'"); HRMS-ESI calcd for C23H17Cl2FN7O [M+H]+ 496.0850,
found: 496.0849, error 0.2 ppm.
4.2.7.8.
N-{2-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]ethyl}-1-(2-chlorophenyl)-
1
β-carboline-3-carboxamide (9d). White crystals; Yield: 55%; M.p: 160-164 °C; H
NMR (300 MHz): δ ppm: 3.54 (m, 2H, H-2”), 3.60 (m, 2H, H-1”), 7.31 (dt, J = 7.9, 4.1,
4.1, 1H, H-6), 7.55-7.72 (m, 6H, H-7, H-8, H-3’, H-4’, H-5’, H-6’), 8.43 (d, J = 7.9, 1H,
H-5), 8.73 (t, J = 5.8, CONH), 8.91 (s, 1 H, H-4), 9.21 (t, J = 5.2, 1H, NH-C2”), 11.67
13
(s, 1H, NH, H-9). C NMR (75 MHz): δ ppm: 37.7 (CH2, C-1"), 40.9 (CH2, C-2"),
112.4 (CH, C-8), 113.8 (CH, C-4), 120.1 (CH, C-6), 121.0 (C0, C-4b), 122.2 (CH, C-5),
127.5 (CH, C-7), 128.7 (CH, C-5’), 128.9 (C0, C-4a), 129.8 (C0, C-2’), 130.6 (CH, C-
3’), 132.1 (CH, C-6’), 132.7 (C0, C-4’), 135.2 (C0, C-1’), 136.4 (C0, C-3), 139.2 (C0, C-
9a), 139.7 (C0, C-1), 141.5 (C0, C-8a), 165.1 (C0, C-2'"), 165.5 (C=O), 168.3 and 169.3
(C0, C-4'", C-6'"); HRMS-ESI calcd for C23H17Cl3N7O [M+H]+ 512.0555, found:
512.0555, error 0 ppm.
4.2.7.9.
N-{6-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]hexyl}-1-phenyl-β-
1
carboline-3-carboxamide (10a). White crystals; Yield: 74%; M.p: 101-102 °C; H
NMR (300 MHz): δ ppm: 1.36 (m, 4H, H-3”, H-4”), 1.51-1.62 (m, 4H, H-2”, H-5”),
3.24-3.29 (m, 2H, H-6”), 3.38-4.42 (m, 2H, H-1”), 7.32 (t, J = 7.1, 1H, H-6), 7.55-7.71
(m, 5H, H-7, H-8, H-3’, H-4’, H-5’), 8.16 (d, J = 7.0, 2H, H-2’, H-6’), 8.41 (d, J = 7.9,
1H, H-5), 8.81 (s, 1H, H-4), 8.68 (t, J = 6.2, CONH), 9.14 (t, J = 5.5, 1H, NH-C2”). 13C
NMR (75 MHz): δ ppm: 25.9 (CH2, C-4"), 26.1 (CH2, C-3"), 28.1 (CH2, C-5"), 29.4
(CH2, C-2"), 38.8 (CH2, C-1"), 40.7 (CH2, C-6"), 112.7 (CH, C-8), 112.9 (CH, C-4),
120.2 (CH, C-6), 121.2 (C0, C-4b), 122.0 (CH, C-5), 128.6 (CH, C-4’), 128.7 (2CH, C-
2’, C-6’), 128.8 (CH, C-3’, C-5’), 128.9 (CH, C-7), 129.9 (C0, C-4a), 134.1 (C0, C-9a),
137.5 (C0, C-1’), 140.0 (C0, C-3), 140.5 (C0, C-1), 141.6 (C0, C-8a), 164.7 (C0, C-2'"),
165.1 (C=O), 168.4 and 169.4 (C0, C-4'", C-6'"); HRMS-ESI calcd for C27H26Cl2N7O
[M+H]+ 534.1570, found: 534.1565, error 0.9 ppm.
4.2.8. Procedure for the preparation of β-carboline-4,6-dichloro-1,3,5-triazine
derivative (9e)

ACCEPTED MANUSCRIPT
A suspension of cyanuric chloride (0.2 mmol) and potassium carbonate (0.2
mmol) in anhydrous THF (1 mL) was stirred for 10 minutes at 0 °C, followed by the
addition of 6c (0.2 mmol) suspended in anhydrous THF (2 mL). The reaction mixture
was stirred at 0 ºC for 1 hour, treated with 1 mL of distilled water and maintained in ice
bath for 1 hour. The precipitate formed was filtered and washed with distilled water
providing 9e in 93 % yield.
4.2.8.1.
N-{2-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]ethyl}-1-(3-nitrophenyl)-β-
carboline-3-carboxamide (9e). Yellow crystals; Yield: 93%; M.p: 316-318 °C; 1H NMR
(300 MHz): δ ppm: 3.56 (m, 2H, H-2”), 3.63 (m, 2H, H-1”), 7.34 (td, J = 7.4; 1.2, 1H,
H-6), 7.60-7.71 (m, 2H, H-7, H-8), 7.94 (t, J = 8.0, 1H, H-5’), 8.39-8.47 (m, 2H, H-5,
H-4’), 8.56 (d, J = 7.8, 1H, H-6’), 8.83 (m, 1H, H-2’), 8.91 (s, 1 H, H-4), 8.94 (m, 1H,
NH-C2”), 9.25 (t, J = 5.2, 1H, CO-NH), 12.06 (s, 1H, NH, H-9). 13C NMR (75 MHz): δ
ppm: 37.9 (CH2, C-1"), 40.8 (CH2, C-2"), 112.6 (CH, C-8), 114.1 (CH, C-4), 120.5
(CH, C-6), 121.2 (C0, C-4b), 122.3 (CH, C-5), 123.5 (2CH, C-2’, C-4’), 130.4 (C0, C-
4a), 129.0 (CH, C-7), 135.4 (C0, C-9a), 130.4 (CH, C-5’), 138.2 (C0, C-1’), 134.5 (CH,
C-6’), 138.9 (C0, C-3), 140.0 (C0, C-1), 141.7 (C0, C-8a), 148.3 (C0, H-3’), 165.1 (C0,
C-2'"), 165.6 (C=O), 168.4 and 169.4 (C0, C-4'", C-6'"); HRMS-ESI calcd for
C23H17Cl2N8O3 [M+H]+ 523.0795, found: 523.0791, error 0.8 ppm.
4.2.9. Procedure general for preparation of (β-carboline)-1,3,5-triazine
derivatives 11a-15a, 16a-d and 17a-d.
To a suspension of cyanuric chloride (0.2 mmol) in water-acetonitrile 1:1 (0.5
mL), at 0 °C, was added 5a-d (0.2 mmol) or 6a-d (0.2 mmol) suspended in THF (3
mL). The pH was adjusted to 9-10 with sodium hydroxide 1 mol L-1. The reaction
mixture was stirred at 0 ºC, for 1 hour followed by addition of 10 equivalents of the
appropriate amine. The mixture was kept at 70 °C, and the reaction progress monitored
by TLC every 12 h. After complete consumption of starting materials (48 h), the
solution was cooled and treated with 2 mL of distilled water. The precipitates formed
were filtered, washed with distilled water and recrystallized with ethanol to afford
compounds 11a-15a, 16a-d and 17a-d in yields in range of 55-94%.
4.2.9.1.
N-{2-[(4,6-dihydrazinyl-1,3,5-triazin-2-yl)amino]ethyl}-1-phenyl-β-
1
carboline-3-carboxamide (11a). Yellow crystals; Yield: 86%; M.p: 186-189 °C; H
NMR (500 MHz): δ ppm: 3.42-3.57 (m, 4H, H-1”, H-2”), 4.12 (sl, 2H, NH2), 6.88 (sl,

ACCEPTED MANUSCRIPT
1H, NH-C2”), 7.31 (t, J = 7.3, 1H, H-6), 7.58-7.70 (m, 5H, H-7, H-8, H-3’, H-4’, H-5’),
8.12 (d, J = 7.3, 2H, H-2’, H-6’), 8.41 (d, J = 7.8, 1H, H-5), 8.83 (s, 1 H, H-4), 8.80 (d, J
= 5.3, CONH), 11.83 (s, 1H, NH, H-9). 13C NMR (75 MHz): δ ppm: 38.9 (CH2, C-1"),
40.4 (CH2, C-2"), 112.7 (CH, C-8), 113.0 (CH, C-4), 120.2 (CH, C-6), 121.2 (C0, C-4b),
122.1 (CH, C-5), 128.6 (CH, C-4’), 128.7 (2CH, C-2’, C-6’), 128.8 (CH, C-3’, C-5’),
129.0 (CH, C-7), 129.9 (C0, C-4a), 134.2 (C0, C-9a), 137.5 (C0, C-1’), 139.8 (C0, C-3),
140.6 (C0, C-1), 141.6 (C0, C-8a), 165.1 (3C0, C-2”’, C-4'", C-6'"), 167.6 (C=O);
HRMS-ESI calcd for C23H24N11O [M+H]+ 470.2160, found: 470.2160, error 0 ppm.
4.2.9.2.
N-{2[(4,6-bis(cyclohexylamino)-1,3,5-triazin-2-yl)amino]ethyl}-1-phenyl-β-
1
carboline-3-carboxamide (12a). White crystals; Yield: 74%; M.p: 201-202 °C; H
NMR (500 MHz): δ ppm: 1.07-1.82 (m, 10H, H-2””, H-3””, H-4””, H-5””, H-6””),
3.54-3.67 (m, 5H, H-1”, H-2”, H-1””), 7.32 ( m, 1H, H-6), 7.55-7.70 (m, 4H, H-7, H-3’,
H-4’, H-5’), 7.69 (d, J = 8.2, 1H, H-8), 8.10 (sl, 2H, H-2’, H-6’), 8.41 (d, J = 7.9, 1H, H-
5), 8.82 (s, 1H, H-4), 8.73 (sl, CONH), 11.84 (s, 1H, NH, H-9). 13C NMR (125 MHz): δ
ppm: 25.0 (2CH2, C-3””, C-5””), 25.4 (CH2, C-4””), 32.9 (2CH2, C-2””, C-6””), 39.4
(CH2, C-1"), 41.4 (CH2, C-2"), 48.6 (CH, C-1””), 112.7 (CH, C-8), 112.9 (CH, C-4),
120.2 (CH, C-6), 121.2 (C0, C-4b), 122.0 (CH, C-5), 128.7 (3CH, C-2’, C-4’, C-6’),
128.9 (3CH, C-7, C-3’, C-5’), 129.9 (C0, C-4a), 134.2 (C0, C-9a), 137.5 (C0, C-1’),
139.8 (C0, C-3), 140.6 (C0, C-1), 141.6 (C0, C-8a), 165.0 (C0, C-2”’), 165.0 (C=O),
166.0 (C0, C-4'", C-6'"). HRMS-ESI calcd for C35H42N9O [M+H]+ 604.3507, found:
604.3496, error 1.8 ppm.
4.2.9.3.
N-{2-[(4,6-bis(benzylamino)-1,3,5-triazin-2-yl)amino]ethyl}-1-phenyl-β-
1
carboline-3-carboxamide (13a). White crystals; Yield: 65%; M.p: 144-145 °C; H
NMR (300 MHz): δ ppm: 3.43-3.52 (m, 4H, H-1”, H-2”), 6.63-6.94 (m, 1H, NHC2”’),
7.21-7.35 (m, 6H, H-6, H-2””, H-3””, H-4””, H-5””, H-6””), 7.53-7.71 (m, 5H, H-7, H-
8, H-3’, H-4’, H-5’), 8.11 (d, J = 7.2, 2H, H-2’, H-6’), 8.42 (d, J = 7.9, 1H, H-5), 8.84
(s, 1H, H-4), 8.71-8.74 (m, CONH), 11.84 (s, 1H, NH, H-9). 13C NMR (75 MHz): δ
ppm: 39.7 (CH2, C-1"), 40.2 (CH2, C-2"), 43.0 and 43.3 (2CH2, CH2-Ph), 112.7 (CH, C-
8), 113.0 (CH, C-4), 120.2 (CH, C-6), 121.3 (C0, C-4b), 122.1 (CH, C-5), 126.4 (CH, C-
2””, C-6””), 126.9 (CH, C-4””), 127.4 (CH, C-1””), 128.0 (CH, C-3””, C-5””), 128.7
(3CH, C-2’, C-4’, C-6’), 128.9 (C-3’, C-5’), 129.0 (CH, C-7), 129.9 (C0, C-4a), 134.2
(C0, C-9a), 137.5 (C0, C-1’), 139.8 (C0, C-3), 140.7 (C0, C-1), 141.6 (C0, C-8a), 165.1

ACCEPTED MANUSCRIPT
(C=O), 165.9 (C0, C-2'"), 166.0 (C0, C-4”’, C-6”’); HRMS-ESI calcd for C37H34N9O
[M+H]+ 620.2881, found: 620.2859, error 3.5 ppm.
4.2.9.4.
N-{2-[(4,6-bis(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)amino]ethyl}-1-
phenyl-β-carboline-3-carboxamide (14a). Beige crystals; Yield: 55%; M.p: 158-160 °C;
1H NMR (300 MHz): δ ppm: 2.00-2.27 (m, 10H, H-3””, H-5””, 2CH3), 3.44-3.63 (m,
8H, H-1”, H-2”, H-2””, H-6””), 6.88 (t, J = 5.1, 1H, NHC2”’), 7.32 ( t, J = 7.3, 1H, H-
6), 7.56-7.71 (m, 5H, H-7, H-8, H-3’, H-4’, H-5’), 8.07 (d, J = 6.7, 2H, H-2’, H-6’),
8.41 (d, J = 7.8, 1H, H-5), 8.82 (s, 1H, H-4), 8.74 (t, J = 5.5, CONH), 11.84 (s, 1H, NH,
H-9). 13C NMR (75 MHz): δ ppm: 39.4 (CH2, C-1"), 40.1 (CH2, C-2"), 42.4 (CH2, C-
2””, H-6””), 45.7 (CH3), 45.9 (CH3), 54.3 (CH2, C-3””), 54.5 (CH2, C-5””), 112.7 (CH,
C-8), 113.0 (CH, C-4), 120.2 (CH, C-6), 121.2 (C0, C-4b), 122.0 (CH, C-5), 128.7
(3CH, C-2’, C-4’, C-6’), 128.8 (C-3’, C-5’), 128.9 (CH, C-7), 129.9 (C0, C-4a), 134.2
(C0, C-9a), 137.5 (C0, C-1’), 139.8 (C0, C-3), 140.5 (C0, C-1), 141.6 (C0, C-8a), 164.6
(C0, C-4”’, C-6”’), 165.0 (C=O), 166.1 (C0, C-2'"). HRMS-ESI calcd for C33H40N11O
[M+H]+ 606.3412, found: 606.3409, error 0.5 ppm.
4.2.9.5.
N-{2-[(4,6-dimorpholino-1,3,5-triazin-2-yl)amino]ethyl}-1-phenyl-β-
carboline-3-carboxamide (15a). Beige crystals; Yield: 84%; M.p: 193-194 °C; 1H NMR
(500 MHz): δ ppm: 3.50-3.60 (m, 12H, H-1”, H-2”, H-2””, H-3””, H-5””, H-6””), 6.97
(t, J = 5.4, 1H, NHC2”’), 7.31 ( t, J = 7.5, 1H, H-6), 7.55-7.64 (m, 4H, H-7, H-3’, H-4’,
H-5’), 7.69 (d, J = 8.1, 1H, H-8), 8.10 (d, J = 7.2, 2H, H-2’, H-6’), 8.41 (d, J = 7.8, 1H,
H-5), 8.83 (s, 1H, H-4), 8.76 (t, J = 5.8, CONH), 11.83 (s, 1H, NH, H-9). 13C NMR (125
MHz): δ ppm: 39.4 (CH2, C-1"), 40.1 (CH2, C-2"), 43.1 (CH2, C-2””), 43.2 (CH2, C-
6””), 66.0 (CH2, C-3””), 66.1 (CH2, C-5””), 112.7 (CH, C-8), 113.0 (CH, C-4), 120.2
(CH, C-6), 121.2 (C0, C-4b), 122.0 (CH, C-5), 128.6 (CH, C-4’), 128.7 (2CH, C-2’, C-
6’), 128.8 (C-3’, C-5’), 128.9 (CH, C-7), 129.9 (C0, C-4a), 134.2 (C0, C-9a), 137.5 (C0,
C-1’), 139.8 (C0, C-3), 140.5 (C0, C-1), 141.6 (C0, C-8a), 164.7 (C0, C-4”’, C-6”’),
165.0 (C=O), 166.0 (C0, C-2'"). HRMS-ESI calcd for C31H34N9O3 [M+H]+ 580.2779,
found: 580.2761, error 3.1 ppm.
4.2.9.6.
N-{2-[(4,6-bis(isopropylamino)-1,3,5-triazin-2-yl)amino]ethyl}-1-phenyl-β-
1
carboline-3-carboxamide (16a). White crystals; Yield: 94%; M.p: 200-201 °C; H
NMR (500 MHz): δ ppm: 1.05 (sl, 12 H, CH3), 3.45-3.55 (m, 4H, H-1”, H-2”), 4.02 (dq,
J = 13.0, 6.7, 2H, CHCH3), 6.06-6.64 (m, 3H, NH), 7.31 (t, J = 7.4, 1H, H-6), 7.55-7.70

ACCEPTED MANUSCRIPT
(m, 5H, H-7, H-8, H-3’, H-4’, H-5’), 8.11 (d, J = 7.0, 2H, H-2’, H-6’), 8.41 (d, J = 7.9,
1H, H-5), 8.82 (s, 1 H, H-4), 8.74 (t, J = 5.6, CONH), 11.83 (s, 1H, NH, H-9). 13C NMR
(125 MHz): δ ppm: 22.7 (CHCH3), 39.8 (CH2, C-1"), 39.9 (CH2, C-2"), 40.1 (CHCH3),
112.9 (CH, C-8), 112.7 (CH, C-4), 120.2 (CH, C-6), 121.2 (C0, C-4b), 122.0 (CH, C-5),
128.6 (CH, C-4’), 128.7 (2CH, C-2’, C-6’), 128.8 (2CH, C-3’, C-5’), 128.9 (CH, C-7),
129.9 (C0, C-4a), 134.2 (C0, C-9a), 137.5 (C0, C-1’), 139.8 (C0, C-3), 140.6 (C0, C-1),
141.6 (C0, C-8a), 164.9 (C0, C-2”’), 165.0 (2C0, C-4'", C-6'"), 165.9 (C=O); HRMS-ESI
calcd for C29H34N9O [M+H]+ 524.2881, found: 524.2868, error 2.5 ppm.
4.2.9.7.
N-{2-[(4,6-bis(isopropylamino)-1,3,5-triazin-2-yl)amino]ethyl}-1-(4-
methoxyphenyl)-β-carboline-3-carboxamide (16b). White crystals; Yield: 75 %; M.p:
1
262-264 °C; H NMR (300 MHz): δ ppm: 1.05 (sl, 12H, CHCH3), 3.55 (m, 4H, H-1”,
H-2”), 3.89 (s, 3H, OCH3), 4.03 (m, 2H, CHCH3), 7.18 (d, J = 8.4, 2H, H-3’, H-5’),
7.30 (t, J = 7.3, 1H, H-6), 7.58 (t, J = 7.3, 1H, H-7), 7.69 (m, 1H, H-8), 8.07 (d, J = 8.1,
2H, H-2’, H-6’), 8.38 (d, J = 7.7, 1H, H-5), 8.77 (s, 1 H, H-4), 8.73 (sl, CONH), 11.79
(s, 1H, NH, H-9). 13C NMR (75 MHz): δ ppm: 22.7 (CHCH3), 38.6 (CH2, C-1"), 41.2
(CHCH3), 41.7 (CH2, C-2"), 55.4 (OCH3), 112.4 (CH, C-8), 112.7 (CH, C-4), 114.3
(2CH, H-3’, H-5’), 120.2 (CH, C-6), 121.3 (C0, C-4b), 122.0 (CH, C-5), 128.5 (C0, C-
9a), 129.7 (C0, C-4a), 130.0 (CH, C-7), 130. 1 (2CH, C-2’, C-6’), 134.0 (C0, C-1’),
139.6 (C0, C-3), 140.5 (C0, C-1), 141.5 (C0, C-8a), 160.0 (C0, C-4’), 165.0 (C0, C-2'"),
165.1 (C=O), 166.0 (C0, C-4'", C-6'"); HRMS-ESI calcd for C30H36N9O2 [M+H]+
554.2986, found: 554.2990, error 0.7 ppm.
4.2.9.8.
N-{2-[(4,6-bis(isopropylamino)-1,3,5-triazin-2-yl)amino]ethyl}-1-(4-
fluorophenyl)-β-carboline-3-carboxamide (16c). White crystals; Yield: 48 %; M.p: 190-
1
195 °C; H NMR (500 MHz): δ ppm: 1.05 (d, J = 6.90, 12H, CHCH3), 3.30-3.60 (m,
4H, H-1”, H-2”), 4.03 (dd, J = 13.5; 6.8, 2H, CHCH3), 7.46 (t, J = 8.8, 2H, H-3’, H-5’),
7.32 (t, J = 7.5, 1H, H-6), 7.60 (t, J = 7.5, 1H, H-7), 7.68 (d, J = 8.2, 1H, H-8), 8.15 (sl,
2H, H-2’, H-6’), 8.42 (d, J = 7.8, 1H, H-5), 8.73 (sl, CONH), 8.82 (s, 1 H, H-4), 11.86
(sl, 1H, NH, H-9). 13C NMR (125 MHz): δ ppm: 22.6 (CHCH3), 39.3 (CH2, C-1"), 40.9
(CH2, C-2"), 42.1 (CHCH3), 112.6 (CH, C-8), 113.0 (CH, C-4), 115.6 (CH, H-3’) 115.8
(CH, H-5’), 120.2 (CH, C-6), 121.2 (C0, C-4b), 122.1 (CH, C-5), 128.7 (C0, C-9a),
130.0 (C0, C-4a), 130.8 (CH, C-2’), 130.9 (CH, C-6’), 133.9 (CH, C-7), 134.1 (C0, C-
1’), 139.5 (C0, C-3), 139.7 (C0, C-1), 141.6 (C0, C-8a), 161.6 (C0, C-4’), 163.6 (C=O),

ACCEPTED MANUSCRIPT
164.9 (C0, C-2'", C-4'", C-6'"); HRMS-ESI calcd for C29H33FN9O [M+H]+ 542.2787,
found: 542.2802, error 2.8 ppm.
4.2.9.9.
N-{2-[(4,6-bis(isopropylamino)-1,3,5-triazin-2-yl)amino]ethyl}-1-(2-
chlorophenyl)-β-carboline-3-carboxamide (16d). White crystals; Yield: 69 %; M.p:
152-157 °C; 1H NMR (300 MHz): δ ppm: 1.05 (d, J = 4.3, 12H, CHCH3), 3.38-3.52 (m,
4H, H-1”, H-2”), 4.01 (d, J = 6.0, 2H, CHCH3), 7.31 (ddd, J = 7.9; 4.9; 3.1, 1H, H-6),
7.56-7.71 (m, 7H, H-7, H-8, H-3’, H-4’, H-5’, H-6’, NH), 8.42 (d, J = 7.8, 1H, H-5),
8.62 (sl, CONH), 8.90 (s, 1 H, H-4), 11.66 (sl, 1H, NH, H-9). HRMS-ESI calcd for
C29H33ClN9O [M+H]+ 558.2491, found: 558.2486, error 0.9 ppm.
4.2.9.10. N'-[4,6-bis(isopropylamino)-1,3,5-triazin-2-yl]-1-phenyl-β-carboline-3-
1
carbohydrazide (17a). White crystals; Yield: 49 %; M.p: 204-206 °C; H NMR (300
MHz): δ ppm: 1.12 (sl, 12H, CH3), 4.05 (sl, 2H, CHCH3), 6.58 (sl, 2H, NH), 7.33 (t, J =
7.2, 1H, H-6), 7.59-7.73 (m, 5H, H-7, H-8, H-3’, H-4’, H-5’), 8.25 (sl, H-2’, H-6’), 8.45
(d, J = 7.6, 1H, H-5), 8.84 (s, 1 H, H-4), 10.17 (s, CONH), 11.91 (s, 1H, NH, H-9). 13C
NMR (75 MHz): δ ppm: 22.5 (CHCH3), 40.7 (CHCH3), 112.7 (CH, C-8), 113.4 (CH, C-
4), 120.3 (CH, C-6), 121.2 (C0, C-4b), 122.1 (CH, C-5), 128.7 (CH, C-4’), 128.8 (4CH,
C-2’, C-3’, C-5’, C-6’), 129.0 (CH, C-7), 129.8 (C0, C-4a), 134.2 (C0, C-9a), 137.4 (C0,
C-1’), 139.3 (C0, C-3), 140.6 (C0, C-1), 141.5 (C0, C-8a), 164.0 (C=O), 165.0 (3C0, C-
2”’, C-4'", C-6'"); HRMS-ESI calcd for C27H30N9O [M+H]+ 496.2568, found: 496.2553,
error 3.0 ppm.
4.2.9.11.
N'-[4,6-bis(isopropylamino)-1,3,5-triazin-2-yl]-1-(4-methoxyphenyl)-β-
1
carboline-3-carbohydrazide (17b). White crystals; Yield: 98 %; M.p: 182-183 °C; H
NMR (500 MHz): δ ppm: 0.86-1.13 (m, 12H, CHCH3), 3.90 (s, 3H, OCH3), 4.04-4.09
(m, 2H, CHCH3), 7.19 (d, J = 8.9, 2H, H-3’, H-5’), 7.32 (t, J = 7.4, 1H, H-6), 7.60 (td, J
= 7.7; 1.1, 1H, H-7), 7.71 (d, J = 8.2, 1H, H-8), 8.23 (d, J = 6.4, 2H, H-2’, H-6’), 8.42
(d, J = 7.9, 1H, H-5), 8.79 (s, 1 H, H-4), 10.15 (s, CONH), 11.85 (s, 1H, NH, H-9). 13C
NMR (125 MHz): δ ppm: 22.5 (CHCH3), 41.6 (CHCH3), 55.4 (OCH3), 112.7 (CH, C-
8), 112.9 (CH, C-4), 114.1 (2CH, H-3’, H-5’), 120.2 (CH, C-6), 121.3 (C0, C-4b), 122.0
(CH, C-5), 129.6 (C0, C-4a), 128.5 (CH, C-7), 129.8 (C0, C-9a), 130.2 (2CH, C-2’, C-
6’), 134.0 (C0, C-1’), 139.1 (C0, C-3), 140.6 (C0, C-1), 141.5 (C0, C-8a), 160.0 (C0, C-
4’), 164.1 (C0, C-2'", C-4”’, C-6’”), 165.0 (C=O); HRMS-ESI calcd for C28H32N9O2
[M+H]+ 526.2673, found: 526.2639, error 6.5 ppm.

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4.2.9.12.
N'-[4,6-bis(isopropylamino)-1,3,5-triazin-2-yl]-1-(4-fluorophenyl)-β-
1
carboline-3-carbohydrazide (17c). White crystals; Yield: 60 %; M.p: 176-180 °C; H
NMR (300 MHz): δ ppm: 1.03-1.11 (m, 12H, CHCH3), 4.04 (sl, 2H, CHCH3), 6.57 (sl,
2H, NH), 7.32 (td, 7.2, 1.0, 1H, H-6), 7.46 (t, J = 8.9, 2H, H-3’, H-5’), 7.61 (td, J = 7.7;
1.1, 1H, H-7), 7.71 (d, J = 8.2, 1H, H-8), 8.32 (sl, 2H, H-2’, H-6’), 8.44 (d, J = 7.9, 1H,
H-5), 8.84 (s, 1 H, H-4), 10.22 (s, CONH), 11.92 (s, 1H, NH, H-9). 13C NMR (75 MHz):
δ ppm: 22.6 (CHCH3), 41.3 (CHCH3), 112.7 (CH, C-8), 113.5 (CH, C-4), 115.5 and
115.7 (2CH, C-3’, C-5’), 120.3 (CH, C-6), 121.2 (C0, C-4b), 122.1 (CH, C-5), 128.7
(C0, C-4a), 129.9 (CH, C-7), 131.1 and 131.2 (2 CH, C-2’, C-6’), 133.8 (C0, C-9a),
134.1 (C0, C-1’), 139.3 (C0, C-3), 139.6 (C0, C-1), 141.5 (C0, C-8a), 161.0 (C0, C-4’),
164.1 (C0, C-2'"), 164.3 (C=O), 165.0 (2 C0, C-4”’, C-6’”); HRMS-ESI calcd for
C27H29FN9O [M+H]+ 514.2474, found: 514.2460, error 2.7 ppm.
4.2.9.13.
N'-[4,6-bis(isopropylamino)-1,3,5-triazin-2-yl]-1-(2-chlorophenyl)-β-
1
carboline-3-carbohydrazide (17d). White crystals; Yield: 56 %; M.p: 177-181 °C; H
NMR (300 MHz): δ ppm: 1.08 (sl, 12H, CHCH3), 4.04 (sl, 2H, CHCH3), 6.56 (sl, 1H,
NH), 7.32 (ddd, J = 7.9; 4.8; 3.2, 1H, H-6), 7.59-7.74 (m, 4H, H-3’, H-4’, H-5’, H-6’),
7.71-7.74 (m, 2H, H-7, H-8), 8.45 (d, J = 7.9, 1H, H-5), 8.91 (s, 1 H, H-4), 9.91 (sl,
13
CONH), 11.73 (s, 1H, NH, H-9). C NMR (75 MHz): δ ppm: 22.5 (CHCH3), 41.2
(CHCH3), 112.4 (CH, C-8), 114.2 (CH, C-4), 120.2 (CH, C-6), 121.1 (C0, C-4b), 122.3
(CH, C-5), 127.5 (CH, C-7), 128.8 (CH, C-5’), 129.8 (C0, C-4a), 130.7 (C0, C-2’), 132.2
(CH, C-3’), 132.5 (CH, C-6’), 132.5 (C0, C-4’), 135.3 (C0, C-1’), 136.3 (C0, C-3), 138.8
(C0, C-9a), 139.8 (C0, C-1), 141.4 (C0, C-8a), 163.8 (C=O), 164.9 (2 C0, C-4”’, C-6’”),
167.3 (C0, C-2'"); HRMS-ESI calcd for C27H29ClN9O [M+H]+ 530.2178, found:
530.2162, error 3.0 ppm.
4.2.10. Procedure for the preparation of β-carboline-4,6-bis(isopropylamino)-1,3,5-
triazine derivative (16e)
A suspension of cyanuric chloride (0.2 mmol) and potassium carbonate (0.2
mmol) in anhydrous THF (1 mL) was stirred for 10 minutes at 0 °C, followed by the
addition of 6e (0.2 mmol) suspended in anhydrous THF (2 mL). The reaction mixture
was stirred at 0 ºC for 1 hour, followed by addition of 10 equivalents of isopropylamine.
The mixture was kept at 70 °C, and the reaction progress monitored by TLC every 12 h.
After complete consumption of starting materials (48 h), the solution was cooled and

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treated with 2 mL of distilled water. The precipitate formed was filtered and washed
with distilled water, providing 16e in 40% yield.
4.2.10.1.
N-{2-[(4,6-bis(isopropylamino)-1,3,5-triazin-2-yl)amino]ethyl}-1-(3-
nitrophenyl)-β-carboline-3-carboxamide (16e). Yellow crystals; M.p. > 350 °C; Yield:
1
40 %; H NMR (300 MHz): δ ppm: 1.10 (dd, J = 7.8; 6.7, 12H, CHCH3), 2.78 (t, J =
6.1, 2H, H-2”), 3.41 (q, J = 6.1, 2H, H-1”), 3.94 - 4.05 (m, 2H, CHCH3), 7.34 (m, 1.2,
1H, H-6), 7.60-7.70 (m, 3H, H-7, H-8, NH), 7.94 (t, J = 8.0, 1H, H-5’), 8.39-8.46 (m,
2H, H-5, H-4’), 8.58 (d, J = 7.8, 1H, H-6’), 8.80 – 8.86 (m, 2H, H-2’, NH), 8.91 (s, 1 H,
H-4). 13C NMR (75 MHz): δ ppm: 22.0 (CH3CH), 41.2 (CH2, C-1"), 41.6 (CH2, C-2"),
42.0 (CHCH3), 112.6 (CH, C-8), 113.9 (CH, C-4), 120.4 (CH, C-6), 121.2 (C0, C-4b),
122.2 (CH, C-5), 123.5 (CH, C-2’), 123.6 (CH, C-4’), 129.0 (C0, C-4a), 130.4 (CH, C-
7), 130.4 (C0, C-9a), 134.4 (CH, C-5’), 135.4 (C0, C-1’), 138.1 (CH, C-6’), 139.0 (C0,
C-3), 140.1 (C0, C-1), 141.7 (C0, C-8a), 148.3 (C0, H-3’), 164.5 (C0, C-2'"), 164.5
(C=O), 164.7 (C0, C-4'", C-6'"); HRMS-ESI calcd for C29H33N10O3 [M+H]+ 569.2732,
found: 569.2702, error 5.3 ppm.
4.3. Antileishmanial activity
4.3.1. Parasites and cell culture
Leishmania amazonensis promastigotes were maintained at 25 °C in Warren
medium and axenic amastigotes forms were maintained at 25 °C in Schneider’s
medium, supplemented with 10% FBS. J774-A1 macrophages were maintained at 37 °C
under 5% CO2 atmosphere in RPMI 1640 medium (pH 7.2) supplemented with 10%
FBS.
4.3.2. Antiprotozoal activity
The effects of synthesized compounds were evaluated in promastigotes and
axenic amastigotes forms of L. amazonensis in log phase of growth (48 h and 72 h,
respectively) at concentration of 1 x 10-6 cells mL-1. The inoculants were introduced into
sterile 96-well micro plates containing increasing concentrations of compounds 8a-d,
9a-e, 10a-15a, 16a-d and 17a-d. After incubation for 72 h at 25 °C and 32 °C for
promastigotes and axenic amastigotes forms, was added 50 µL of solution of XTT/
PMS (0.5 and 0.3 mg/mL) in the absence of light. After 4 h, the absorbance was read in
a spectrophotometer at 450 nm. The concentration that decreased 50% (IC50) of the

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absorbance values compared with the negative control was determined by regression
analysis of the data. To 16b also was determined the concentration that decreased 90%
(IC90) of the absorbance values. Miltefosine was used as positive control.
4.3.3. Cytotoxicity assay
The cytotoxicity was evaluated in J774-A1 macrophages. To a suspension of
macrophages in log phase growth (72 h) at concentration of 5 x 10-5 cells mL-1 was
cultured in RPMI 1640 medium supplemented with 10% FBS, were introduced into
sterile 96-well micro plates and incubated for 24 h at 37 °C and 5 % of CO2 tension.
After this period, the supernatant was removed and increasing concentrations of the
substances were added. After 48 h of incubation under the same conditions mentioned
above, the cells were washed with phosphate buffered saline 0.01 M and 50 µL of MTT
(2 mg mL-1) was added to each well and incubated at absence of light at 25 °C. After 4
h, 150 µL of DMSO was added in order to disrupt the cells and solubilize purple
formazan crystals. The absorbance was read at 570 nm in microplate reader (Biotek
Power Wave XS spectrofluorometer). The concentration that decreased 50% (CC50) of
the absorbance value compared with the negative control was determined by regression
analysis of the data.
4.3.4. Electron Microscopy
Promastigotes (1 x 106 parasites/mL) were treated with IC50 value of 16b and
incubated for 24, 48 and 72 h at 25 °C. The cells were fixed with 0.1 M cacodylate
buffer buffer and 2.5% of glutaraldehyde. For the ultrastructural analysis, promastigotes
fixed were post-fixed with 1% OsO4 and 0.8% potassium ferrocyanide in 0.1 M
cacodylate buffer for 1 h. After that, the samples were dehydrated in acetone and
included in EPON resin. Ultrathin sections of 60 nm were obtained and contrasted with
uranyl acetate and lead citrate. The samples were observed in Microscopy Jeol JM 1400
TEM. For scanning electron microscopy (SEM), promastigotes were placed on a glass
support with poly-L-lysin, dehydrated with ethanol and submitted at critical point
(replacement of ethanol by CO2). After that, the samples were metalized with gold and
observed in FEI Quanta 250 SEM.
4.3.5. Oxidative stress analysis