Design, synthesis, structural characterization and in vitro evaluation of new 1,4-disubstituted-1,2,3-triazole derivatives against glioblastoma cells

Article abstract of DOI:10.1016/j.bioorg.2018.10.003

A new series of 1,4-disubstituted-1,2,3-triazole derivatives were synthesized through the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (Click chemistry) and their inhibitory activities were evaluated against different human glioblastoma (GBM) c

Full text of DOI:10.1016/j.bioorg.2018.10.003

Accepted Manuscript
Design, synthesis, structural characterization and in vitro evaluation of new 1,4-
disubstituted-1,2,3-triazole derivatives against glioblastoma cells
Veronica D. da Silva, Bruna M. de Faria, Eduardo Colombo, Lucas Ascari,
Gabriella P.A. Freitas, Leonã S. Flores, Yraima Cordeiro, Luciana Romão,
Camilla D. Buarque
PII:
S0045-2068(18)30551-0
DOI:
https://doi.org/10.1016/j.bioorg.2018.10.003
YBIOO 2537
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
7 June 2018
25 September 2018
4 October 2018
Please cite this article as: V.D. da Silva, B.M. de Faria, E. Colombo, L. Ascari, G.P.A. Freitas, L.S. Flores, Y.
Cordeiro, L. Romão, C.D. Buarque, Design, synthesis, structural characterization and in vitro evaluation of new
1,4-disubstituted-1,2,3-triazole derivatives against glioblastoma cells, Bioorganic Chemistry (2018), doi: https://
doi.org/10.1016/j.bioorg.2018.10.003
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Design, synthesis, structural characterization and in vitro evaluation of new 1,4-
disubstituted-1,2,3-triazole derivatives against glioblastoma cells
Veronica D. da Silva1, Bruna M. de Faria2, Eduardo Colombo1, Lucas Ascari3, Gabriella
P. A. Freitas2, Leonã S. Flores4, Yraima Cordeiro3, Luciana Romão2, Camilla D.
Buarque1*
1Laboratório de Síntese orgânica, Pontifícia Universidade Católica do Rio de Janeiro, 22451-900, Rio de
2
Janeiro-RJ, Brasil; Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, 21941-
3
4
902, Rio de Janeiro- RJ, Brasil. Faculdade de Farmácia, UFRJ, RJ, Brasil, 21941-902; Laboratório de
Difração de raios X, UFJF, MG, Brasil, 36036-900.
*Corresponding author: camilla-buarque@puc-rio.br
Abstract
A new series of 1,4-disubstituted-1,2,3-triazole derivatives were synthesized
through the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (Click chemistry)
and their inhibitory activities were evaluated against different human glioblastoma
(GBM) cell lines, including highly drug-resistant human cell lines GBM02, GBM95.
The most effective compounds were 9d, containing the methylenoxy moiety linked to
triazole and the tosyl-hydrazone group, and the symmetrical bis-triazole 10a, also
containing methylenoxy moiety linked to triazole. Single crystal X-ray diffraction
analysis was employed for structural elucidation of compound 9d. In silico analyses of
physicochemical, pharmacokinetic, and toxicological properties suggest that compounds
8a, 8b, 8c, 9d, and 10a are potential candidates for central nervous system-acting drugs.
Keywords: Azide-Alkyne Cycloaddition, 1,4-disubstituted-1,2,3-triazoles, tosyl-
hydrazone, glioblastoma, drug-resistant, drug score, cancer, crystal structure.
Highlights
 Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) is the key step for
obtaining 1,4 disubstituted-1,2,3-triazoles.
 Compounds 9d and 10a presented higher potencies and the best selectivity
indices for glioblastoma.
 Methylenoxy moiety is important for the pharmacological activity.
 Compound 9d had the structural elucidation by single crystal X-ray diffraction
analysis
 In silico PK evaluation revealed that compounds 8a, 8b, 8c, 9d and 10a are
potential candidates for central nervous system-acting drugs.

1. Introduction
Glioblastoma (GBM) is one of the most aggressive human cancers in the brain [1].
Although new therapeutics are currently being tested in clinical trials, GBMs are still
highly drug-resistant, limiting the effectiveness of chemotherapy [1]. Examples of new
4-anilinoquinazolines are erlotinib, gefitinib, acting as inhibitors of epidermal growth
factor receptor (EGFR) [2], and cediranib, pazopanib and sorafenib acting as tyrosine
kinase inhibitors. Nevertheless, temozolomide (TMZ) (1) (Figure 1) is the first choice
chemotherapeutic agent for the treatment of GBM. Due to its small size and lipophilic
features, the drug is able to cross the blood-brain barrier and acts as a DNA alkylating
agent [3]. However, the therapeutic efficacy of TMZ is unsatisfactory against the
invasive and resistant nature of GBM [4], showing high IC50 values of 330 μM against
U87 cells, requiring high concentrations of the drug to current treatment of GBM [5].
The well-known scaffold 1,2,3-triazol, present in several heterocyclic compounds,
harbors a broad spectrum of biological applications [6], such as anti-tubercular [7],
antibacterial and antifungal [8], [9], anticonvulsant [10], antiviral [11], [12], anticancer
[13], [14] activities, among others. 1,2,3-triazoles are classified within the most
common amide bond isosters since they present a good overlap with the amide-binding
moiety [15]. In particular, 1,2,3-triazols can actively participate in dipole-dipole
interactions due to its strong dipole moment, are stable for oxidative and reducing
conditions and the 1,2,3-triazolic ring is not protonated in physiological pH due to its
low basicity as well as may mimic the features of different functional groups [16]
There are some examples of 1,4-disubstituted-1,2,3-triazoles with anticancer activity.
For example, the triazole-based vismodegib analogue (2) had the potency increased
against different cancer and endothelial cell lines when compared to vismodegib, a
synthetic Hedgehog signaling pathway inhibitor. This compound was over 50 times
more active against bovine aortic endothelial cells (BAEC) than original vismodegib,
due to the isosteric exchange of the amide moiety [17]. A series of 1,4-disubstituted-
1,2,3-triazoles was tested as aromatase inhibitors for the treatment of breast cancer and
compound 3 showed the best result, with IC50 = 1.36 μM [18]. Using the same chemical
approach, 1,2,3-triazole-1,4-benzoxazine hybrids were synthesized and evaluated for
anticancer activity against HeLa (cervical), MIAPACA (pancreatic), MDA-MB-231
(breast) and IMR32 (neuroblastome) cell lines. Compound 4 presented the best result

with IC50 values ranging from 0.1 to 1.1 μM, against the above cited cell lines [13]. A
series of pyrazolo-triazole hybrids were synthesized and evaluated for anticancer
activity against four tumor cell lines, HT-29 (colon), PC-3 (prostate), A549 (lung), and
U87MG (glioblastoma) cells. Compound 5 presented the best result against U87MG
cell lines [19]. Other compounds based on 1,2,3-triazoles such as 6 presented anticancer
activity against breast cancer (MCF-7) [20]. Some bis-heterocycles with wide range of
applications also have gained importance in medicinal chemistry in the literature. For
example, some sulfone/sulfonamide-linked bis(oxadiazoles), bis(thiadiazoles) and
bis(triazoles) presented antioxidant activity [21]. The 1,2,3-bis-1,2,3-triazole (7)
presented anticancer activity against B16 melanoma, where the bis-triazole moiety was
more active than mono-triazole used for its preparation [22]. Moreover, there are some
bis‐triazole‐based commercial drugs available in the market, for example Fluconazole,
Itraconazole and Posaconazole were used as antifungal drugs and vorozole as
antineoplastic drug. [23] Figure 1 describes some examples of these potentially
bioactive triazoles.
Fig. 1. Commercial drug TMZ (1) and selected examples of 1,4-disubstituted-1,2,3-
triazoles (2-7) with anticancer activity.
Triazoles with different pattern of substitution were also described to present cytotoxic
effects against GBM cell lines [19], [24], [25]. Ribavirin, for example, is a 1,3-

disubstituted-1,2,4-triazole used for the treatment of seven different GBM cell lines
presented IC50 values ranging from ~30 to 660 µM [25].
The potential of 1,4-disubstituted-1,2,3-triazoles as antineoplastic substances prompted
us to design and synthetize new compounds as prototypes for GBM therapy, including
drug-resistance cell lines. In this manuscript, we report the synthesis of 1,4
disubstituted-1,2,3-triazole based compounds 8-10 and the evaluation of their potential
anti-GBM activity.
Compounds 8a-c and 9a-d were designed to correlate the effect of exchange of
functional groups of aldehydes, an electrophilic group that can form a schiff base with
DNA [26], [27] , by classical pharmacophoric groups, designed to obtain innovative
bifunctional drugs since triazoles can work as special ligand by connecting these one.
Sulphonylhydrazones and hydrazones, on the other hand, are known to inhibit several
enzymes involved in cancer disease [2], [7], [28]–[30], and the polar group CHF2 can
act as an unusual hydrogen bond donor being a bioisoster of OH group [31].
The effect of substitutions at the A and C-rings on the cytotoxicity against human GBM
cell lines (GBM02, GBM95) and the commercial cell U87, was also evaluated for
compounds 8a-e. In order to understand the role of spacer methylenoxy moiety in
compounds 8a-e, derivative 8f was synthesized. Since 1,2,3-triazoles gained importance
for medicinal chemistry, investigation of bis-triazoles 10a, 10b and 10c easily prepared
from bis-alkynes derived from resorcinol was undertaken.

Fig. 2. 1,4-disubstituted-1,2,3- triazoles synthesized in this work
Additionally, the compounds were subjected to a computational prediction of their
physicochemical, pharmacokinetic, and toxicological properties. Compounds 8a, 8b, 8c,
9d, and 10a showed the best results both in vitro and in silico.
2. Results and discussion
2.1. Synthesis of 1,4-disubstituted-1,2,3-triazoles
The 1,4-disubstituted-1,2,3-triazoles (8a-d) were obtained in high yields by the copper-
catalyzed azide-alkyne click chemistry reaction between aryl-azides (13a-c) and
propargyl-phenols (12a-d) (Scheme 1). using one equivalent of corresponding azides
and alkynes, except for compound (10a), whereas two equivalents of the azide (13a)
was used [32]. These propargyl-phenols were prepared by alkylation of phenols (11a-d)
with propargyl bromide in the presence of K2CO3 as a base in acetonitrile (CH3CN)

affording the corresponding products in yields ranging from 30% to 80%. 2-azido-
benzaldehyde (13a) was obtained by reaction of 2-nitro-benzaldeyde with sodium azide
in 60% yield [33]. The other aryl-azides (13b-c) were prepared from commercial
anilines by diazotization reaction with NaNO2 followed by substitution with sodium
azide (NaN3) in 85 and 94% yield, respectively [34]. The chemical structures of the
1
aryl-azides were confirmed by FT-IR and H NMR. The FT-IR analysis showed a
strong absorption band near to 2100 cm-1, attributed to the stretching vibrations of the
azido group. Triazole 8f, which lacks the methylenomethoxy moiety, was also prepared
by click chemistry reaction between phenylacetylene (12e) with aryl-azide 13a [35].
1
Compounds (8a-f) were characterized by H NMR, 13C NMR and mass spectrometry.
The 1H NMR spectrum exhibited a singlet signal at δ 5.3 ppm, relative to the methylene
group (CH2). In addition, the appearance of the sharp singlet signal in the region δ 8.03
- 9.10 ppm confirmed the presence of triazole proton. In 13C NMR, carbon signals
characteristic of the methylene group and the triazole ring (C4) were observed around δ
61.14 - 62.87 and 144.05 -148.56 ppm, respectively.
Scheme 1. Synthesis of the 1,4-disubstituted-1,2,3-triazoles compounds (8a–f ).
Alcohol (9b) was prepared by reduction of 8a with NaBH4 in methanol and the product
was obtained in 65% yield (Scheme 2). The fluorinated compound 9a was obtained in
72% yield by electrophilic fluorination in the presence of diethylaminosulfur trifluoride
(DAST). Compound 9c was obtained by reaction of 8a with phenyl-hydrazine in 70%

yield and compounds 9d and 10b were obtained by reaction of 10a with tosyl-
hydrazine to afford the corresponding hydrazones in 65% and 76% yields, respectively.
Compound 10c was obtained by reaction of 10a with 3-aminopropyl-imidazole in 60%
yield.
Scheme 2. Synthesis of the compound (10a) and triazole derivatives (9a-d and 10b-c).
Single crystal X-ray diffraction analysis was employed for structural elucidation
of compound 9d. The crystal and instrumental parameters used in the unit cell
determination, the data collection, and structure refinement parameters are presented in
Table S1 while selected bond distances and angles in Table S2 (supplementary
material). The E-configuration of the tosyl-hydrazone was observed in the solid
crystalline state, based on C8/N2/N1 iminic bond and involving a C9-C8-N2-N1 torsion
angle of −175.77 (1). The extended conformation of 9d molecule is assembled on a
supramolecular environment involving considerable classical hydrogen bonds N1—

H1N···O3i;d(H···A) = 2.05 Å and <(D-H···A) = 171° :. i = -x, -y, -z. Some weak forces
are showed as C20—H20···N4i and C15—H15···O1ii non-classical hydrogen bonds (ii
= −x, 1−y, −z). The geometry adopted by approximation of toluene rings suggests a
weak π-π stacking interaction [36]–[38]. The analysis of full interaction maps for
conformation adopted by 9d (Fig.4a) indicates large H-bond acceptor peaks directing
for the tosyl and azide groups suggesting a stronger acceptor region. Parallel, a smaller
donor peak directed to toluene ring may indicate a propensity to unconventional
hydrogen bond. Additionally, the π-π stacking interaction only has been observed on
toluene ring, which is the unique ring group unsatisfied by hydrophobic region probes
(Fig. 4b). Therefore, the principal contact interactions observed are unsatisfied by the
region probes and it may suggest a possible metastability of the crystal, in the sense of
the polymorphic structure formation.
Fig.3 ORTEP representation of the asymmetric unit of 9d crystal structure. Ellipsoids
are shown at the 30% of probability level.

(a)
(b)
Fig. 4 (a) Overview of 9d full interaction maps, displaying all the preferential
interactions for the adopted conformation and showing all probability contouring of 2, 4
and 6 times random. For colors: the H-bond donor in blue, acceptor in red and
hydrophobic interaction are depicted in orange tridimensional scatterplots. (b) The
emphasis for π-π stacking interaction of toluene groups from the molecule.
2.2.Biological evaluation
2.2.1. Cytotoxicity of 8a-f, 9a-d and 10a-c compounds in GBM cells
In order to evaluate the antitumor potential of triazole compounds against GBM
cells, U87, GBM02 and GBM95 cell lines were incubated with 10, 50 or 100 µM of
each of the compounds for 48 and 72 hours. Treatments 8a, 8b, 8c, 9d, 10a and 10c
showed the most efficient cytotoxic effects (Fig. 5 and Table 1). These compounds were
able to reduce viability of the three GBM cell lines tested, in concentration- and time-
dependent manners (Fig. 5). Therefore, the most prominent results were detected in
treatments with 100 µM of compounds for 72 hours. Thus, 8a, 8b, 8c, 9d, 10a and 10c
are cytotoxic to all three GBM cell lines tested, presenting IC50 values ranging from ~20
to 190 µM for 72 hours treatments (Table 1). In order to compare the antitumor
potencial of triazoles with the current chemotherapeutic used in the treatment of GBM
patients, we treated GBM cells with 100, 500 and 1000 μM TMZ for 48 and 72 hours
and measured cell viability. We observed that TMZ treatment reduced viability of

GBM02 and GBM95 presenting an IC50 values of 624.8 and 1132 μM in the 72-hour
treatments. However, the same treatment was not able to reduce the viability of U87
lineage cells (Table 1). Our results suggest that the triazole compounds in these studies
have a higher cytotoxic potential than the current chemotherapeutic agent (TMZ).
We next investigated whether triazole compounds act selectively against GBM
cells. For that, we treated human astrocyte cells with the same set of treatments used
before. All triazole compounds, except for 10c, did not induce significant reductions in
astrocytes viability (Fig 6). The 10c compound was cytotoxic to astrocytes, reducing
58.6% of the cellular viability (Fig 6). We also observed that the treatment with TMZ
reduced the viability of human astrocytes, exhibiting an IC50 of 993.3μM 48h e 948.8
μM 72h (Table 1).
Thus, 8a, 8b, 8c, 9d, and 10a presented selectivity effect, exhibiting
antitumoral activity without being cytotoxic to healthy astrocytes at the concentrations
used in our work.
Fig 5. Cytotoxicity of 1,2,3-triazole compounds in GBM cells. Graphs show the percentage of
viable cells after treatment with 10, 50 and 100 μM of 8a-f, 9a-d and 10a-c compounds in
U87 (A and B), GBM02 (C and D) and GBM95 (E and F) for 48 and 72 hours determined by
MTT assay. The values (A-F) represent mean and standard error of three independent
experiments in triplicate. * p<0.05; ** p<0.01 *** p<0.001.

Fig 6. Cytotoxicity of 1,2,3-triazole compounds against human astrocyte cells. Graphs show
percentage of viable cells after the treatment for 48 (A) and 72 hours (B) with 8a-f, 9a-d and
10a-c compounds in astrocyte cells determined by MTT assay. Values represent mean and
standard error of three independent experiments in triplicate. *p<0.05; ** p<0.01 *** p<0.001.
Fig 7. Cytotoxicity of TMZ in GBM cells and human astrocytes. Graphs show the percentage of
viable cells after treatment with 100, 500 and 1000 μM of TMZ in AstH,U87, GBM95 and
GBM02 for 48 (A) and 72 hours (B) determined by MTT assay. The values (A-B) represent
mean and standard error of three independent experiments in triplicate.*p<0.05; **p<0.01;
****p<0.0001.

Table 1. IC50 (µM) values of the compounds tested and TMZ for GBM and astrocyte (AST)
cells treated for 48 and 72 hours.
IC50
GBM 95
48H
GBM 02
U87
ASTH
72H
59.7
95.3
70.8
952.2
247.3
391.1
-
48H
72H
70
48H
233.4
298
72H
154.6
183.5
146.5
2017
268.8
394.5
-
48H
72H
89.9
107.5
83.9
519.3
398.6
274.7
-
113.4
238
1179
2174
8a
95.8
76.3
743.5
243.5
356.5
-
685.2
1014
8b
179.9
1416
301.5
654.5
-
218.8
-
2135
1745
8c
1285
5210
8d
329.7
1414
13812
8432
-
-
-
8e
-
-
8f
-
-
9a
589
2545
57.6
28.7
30.3
1290
29.5
-
1578
263.1
44.9
38.2
402.8
30.6
-
-
-
9b
142.5
58
-
-
-
537
8753
-
-
9c
120.8
81.7
517.5
57.3
65.9
140.5
-
27.1
72.4
-
355.9
9d
44
-
-
10a
10b
10c
TMZ
754.8
40
130
57.2
-
144.1
2643
1132
1712
624.8
-
-
993.3
949.8
In agreement with our results, it has been shown that treatment with different
concentrations of 1,2,4-triazole[3,4-b]-1,3,4-thiadiazine derivative reduced T98G GBM
cell line viability [24] in concentration and time-dependent manners. Ribavirin
treatments in seven different GBM cell lines presented IC50 values ranging from ~30 to
660 µM [25]. Moreover, when we compare our results with the in vitro effect of TMZ,
the current chemotherapeutic agent used in patients with GBM, we can see that 8a, 8b,
8c, 9d, 10a and 10c showed more cytotoxic potential and, differently from TMZ, the
compounds 8a, 8b, 8c, 9d and 10a were not cytotoxic for human astrocytes. Therefore,
as we observed in our results, other studies also showed low efficiency of TMZ in vitro.
Castro and collaborators demonstrated that the IC50 of TMZ for U87 cells after 24h of
treatment is 330µM [5] and for T98G and U138 cells after 72h of treatment is higher
than 500 μM [39]. Previous studies from our group showed that 500 µM of TMZ
treatment for 48h reduced only 29.5% ± 6.7% of U87 cells viability [40] and 1000 µM
of TMZ did not reduce GBM02 and GBM95 cells viability after 24h of incubation [41],
[42].
The best biological response against human tumor cell lines (U87, GBM95 and
GBM02) is related to the symmetric aldehyde triazole linked to the methylenoxy moiety

(10a) or to unsymmetrical tosyl-hydrazone triazole linked to such moiety (9d). The
presence of the methylenoxy moiety is necessary since its removal, such as in 8f, leads
to drastic reduction of the activity of this compound in the cell lines tested. In the
symmetrical bis-triazole 10a, the presence of the aldehyde group was sufficient to
maintain the activity, and its interconversion to other functional has disturbed the
biological activity. It is possible that the bis-triazole group overcomes the need of tosyl-
hydrazone, but it may also be related to physical chemical properties such as molecular
weight or any other kind of interaction with the cell lines. On the other hand, in the case
of the unsymmetrical compounds, the aldehyde group did not result in particular good
activity, and its interconversion to other functional group was mandatory, especially
with the tosyl-hydrazone. sulfonyl-hydrazones are privileged groups present in many
compounds with anticancer activity [29]. Interestingly, other hydrazones derivatives,
which also are considered privileged groups [30], did not improve the biological activity
in the same way, reinforcing the role of the aryl-sulfone group. The other modifications
performed at aldehyde-triazole 8a did not enhance the pharmacological activity against
the tested cell lines, the same observed in relation to B-ring modifications of the
compound 8a. Since previous work have reported the anticancer activity of triazole
derivatives containing propargyl phenol ethers groups [14], [18], It seems to be an
important spacer in the analyzed 1,4-disubstituted 1,2,3-triazole series.
2.2.2. In silico analysis
Successful drugs which target the central nervous system (CNS) normally have
the following properties: molar weight ≤ 450; neutral or moderately positive charge at
pH 7.4; logD 1–3; total number of hydrogen bonds ≤ 8; polar surface area ≤ 90 Å2; and
adequate absorption in the intestines [43]–[46]. Other desirable features for most drugs
are metabolic stability [43], no hERG blockade-mediated cardiotoxicity [47], low
hepatotoxicity [48], and no mutagenesis and carcinogenesis [49], [50]. In order to assess
the feasibility of the compounds studied herein for in vivo application, we performed an
in silico analysis of physicochemical, pharmacokinetic, and toxicological properties
using ADMET Predictor™ software. Although the software includes a direct predictor
for blood-brain permeability, we also gave attention to other properties which greatly
influence brain penetration, such as ionization state in the plasma, logD, hydrogen

bonding capacity, and polar surface area. All compounds from this series were predicted
to be hepatotoxic in humans. However, this cannot be considered a major problem in a
drug discovery process, as many widely used drugs in the market are associated with
case reports of idiosyncratic liver injury. Outstandingly, none of them were predicted to
elicit lethal acute toxicity in rats. All the other results are presented in Table 2.
Table 2. Prediction of physicochemical, pharmacokinetic, and toxicological properties
of the compounds performed on ADMET Predictor™ software.
MWta Chargeb logDc HBd PSAe BPf HJPg Meth Cardi Carcj Mutk
279
295
333
0 (100%) 2.88
4
57
Yes
H
H
No
0
No
8a
8b
–1 (2%)
2.47
6
77
Yes
H
I
No
0
No
0 (98%)
0 (100%) 2.84
5
8
5
3
3
5
6
66
104
70
Yes
Yes
Yes
Yes
Yes
Yes
Yes
H
H
H
H
H
H
H
H
L
I
No
No
No
No
No
No
Yes
0
0
No
No
8c
8d
8e
8f
353 –1 (100%) –0.71
319
249
301
281
369
0 (100%) 3.45
0 (100%) 2.91
0 (100%) 4.09
0 (100%) 2.49
0 (100%) 4.33
0
Yes
No
48
H
H
H
H
0
40
0
No
9a
9b
9c
60
0
Yes
Yes
64
M
–1 (89%)
2.93
448
480
8
8
98
Yes
Yes
H
H
L
I
Yes
No
0
0
No
No
9d
0 (11%)
0 (100%) 2.83
114
10a
–2 (41%)
817
–1 (50%) 4.93
0 (9%)
16
197
140
No
No
I
I
I
Yes
Yes
0
No
No
10b
0 (60%)
695
–8.10 10
H
M
10c
+1 (40%)
a Molar weight (g/mol).
b Charge at pH 7.4.
c Logarithm of octanol-water distribution coefficient taking into account all ionization states.
d Total number of hydrogen bond donors and acceptors.
e Polar surface area (Å2).
f Brain penetration in rats (Yes or No).
g Human jejunal permeability (H, high; I, intermediate; L, low).
h Clearance rate catalyzed by CYPs (H, high; I, intermediate; L, low).
i Cardiotoxicity in humans, measured by the ability to inhibit hERG K+ channel.
j Carcinogenicity in animal models (M, mouse; R, rat; 0, none).
k Mutagenicity, measured by the ability to induce mutations in at least 2 different Salmonella
typhimurium strains in the Ames test.
Compounds 8a, 8b, and 8c were the most promising drug candidates, as they met all
desirable criteria for CNS-acting drugs, despite high risk of liver toxicity and high rates
of metabolic degradation. Additionally, compounds 10a and 9d met most of the criteria

for CNS-acting drugs and were also considered to be good drug candidates along with
8a, 8b, and 8c. Conversely, 10c was predicted to be especially unsuitable to become a
safe and active drug.
3. Conclusion
A series of 1,4-disubstituted-1,2,3-triazole derivatives was synthesized in good
yields and were evaluated for different glioblastoma cell lines (GBM02, GBM95 and
U87). Only compounds 8a, 8b, 8c, 9d, 10a, and 10c were able to reduce viability in the
cell lines tested. The best results were obtained for compounds 9d and 10a with IC50
values of 27.1 μM for U87 cell line and 30.3 μM for the drug-resistant GBM95 human
cell line, respectively. These compounds were also not toxic to astrocytes. All
compounds had the structures confirmed by spectroscopic and spectrometric analyses
and compound 9d was additionally characterized by single crystal X-ray diffraction and
the E-configuration of tosyl-hydrazone was confirmed. An in silico analysis of ADMET
properties revealed that compounds 8a, 8b, 8c, 9d, and 10a meet the criteria for central
nervous system-acting drugs and therefore are potential new drug candidates.
4. Experimental Part
4.1 Materials and Methods
1
For the structural elucidation of the synthesized compounds, H NMR and 13C NMR
spectra were recorded at ambient temperature on a Bruker Avance III spectrometer
1
13
(operating at 400 MHz for H NMR and 100 MHz for C NMR). The chemical shifts
(δ) were given in parts per million (ppm) from internal tetramethylsilane on the δ scale,
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet). All coupling
constants (J values) were given in Hz. Melting points were determined with an
Electrothermal, analog model. Infrared spectra were performed using a Varian -3100
spectrometer. High resolution mass spectra were obtained by Bruker, MicrOTOF II
instrument. Reactions were monitored by thin layer chromatography using Merck TLC
Silica gel 60 F254. Silica gel column chromatography was performed over Merck Silica
gel 60 Å (particle size: 0.040-0.063 mm, 230-400 mesh ASTM). All reagents used were

commercially obtained and, where necessary, purified prior to use, such as THF and
CH2Cl2 that were dried on a 4 Å molecular sieve.
4.2 Synthesis of aryl-azides 13a-c
4.2.1 2-azidobenzaldehyde (13a)
The 2-azidobenzaldehyde (13a) was prepared following the procedure of Feldman and
co-workers [33]. A solution of 2–nitrobenzaldehyde (1.0 g, 6.6 mmol) and sodium azide
(1.3 g, 20 mmol) in 15 mL of DMF was heated to 60 °C and stirred at that temperature
for 96 h. The reaction mixture was diluted with diethyl ether (50 mL) and washed with
water (4 x 25 mL). The combined organic layers were dried over Na2SO4, and
concentrated in vacuum.
4.2.1 2-azidobenzaldehyde (13a)
The crude product was purified by flash column chromatography, using ethyl
acetate/hexane (5:95) as eluent. The product was obtained as a pale yellow solid in 60 %
1
yield. MP: 36-37 °C. H NMR (400 MHz, CDCl3) δ 10.36 (s, 1H, CHO), 7.89 (dd, J =
7.8, 1.6 Hz, 1H, Ar-H), 7.65 – 7.60 (m, 1H, Ar-H), 7.30 – 7.21 (m, 2H, Ar-H).. IR (KBr,
νmax): 2129 (N≡N), 1688 (C=O) cm-1.
4.2.2.1. Aryl-azides (13b-c)
The aryl-azides (13b-c) were prepared following the procedure of Wilkening and co-
workers [34]. The aniline derivative (7.5 mmol) was dissolved in 5 mL of water and
concentrated sulfuric acid (98%, 1.5 mL) and additional water (1.5 mL) was added. The
suspension was cooled to 0 °C and a solution of NaNO2 (7.6 mmol) of water (1.5 mL)
was slowly added under constant stirring. After 15 min, NaN3 (9.3 mmol) was added
and the mixture was stirred for additional 0.5 – 1 h. The reaction mixture was extracted
with ethyl acetate (3x 20 mL) and the combined organic fractions were washed with
water (50 mL). The organic layer was dried over Na2SO4 and concentrated under
reduced pressure. The desired azides (13b-c) were obtained without further purification.

4.2.2.2 4-azidobenzoic acid (13b)
Yield 80 %; light yellow solid. MP: 185°C. 1H NMR (400 MHz, DMSO-d6) δ 12.97 (s,
1H, OH), 7.96 (d, J = 8.7 Hz, 2H, Ar-H), 7.22 (d, J = 8.7 Hz, 2H, Ar-H). IR (KBr, νmax):
2105 (N≡N), 1284 (C-O) cm-1.
4.2.2.3 6-azido-2H-chromen-2-one (13c)
1
Yield 94 %; orange solid. MP: 159-161°C. H NMR (400 MHz, CDCl3) δ 7.66 (d, J =
9.6 Hz, 1H, =CH), 7.34 (d, J = 8.8 Hz, 1H, Ar-H), 7.20 (dd, J = 8.8, 2.6 Hz, 1H, Ar-H),
7.12 (d, J = 2.6 Hz, 1H, Ar-H), 6.48 (d, J = 9.6 Hz, 1H, =CH). IR (KBr, νmax): 2105
(N≡N), 1711(C=O) cm-1.
4.3 Synthesis of alkynes 12a-d
Phenols (11a-d) (5.3 mmol) and anhydrous K2CO3 (10.6 mmol) were added in a 50 mL
two-necked flask and dissolved in 15 mL acetonitrile. After 15 min, propargyl bromide
(80% in toluene, 6.4 mmol) was slowly added and the reaction mixture was refluxed
under N2 atmosphere for 4.5 h. Then the mixture was diluted with water (25 mL) and
extracted with dichloromethane (3x 20 mL). The combined organic layers were dried
over Na2SO4 and the solvent was concentrated under reduced pressure.
4.3.1 (prop-2-yn-1-yloxy)benzene (12a).
Yield 75 %; yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.33 – 7.29 (m, 2H, Ar-H), 6.99
(m, 3H, Ar-H), 4.70 (d, J = 2.4 Hz, 2H, CH2), 2.52 (t, J = 2.4 Hz, 1H, CH). IR (KBr,
νmax): 3290 (≡CH), 2122 (C≡C) cm-1.
3-(prop-2-yn-1-yloxy)phenol (12b)
The crude product was purified by flash column chromatography, using ethyl acetate/
1
hexane (20:80). Yellow oil; yield 30 %. H NMR (400 MHz, CDCl3) δ 7.15 (t, J = 8.0
Hz, 1H, Ar-H), 6.56 (dd, J = 8.1, 1.8 Hz, 1H, Ar-H), 6.50 – 6.46 (m, 2H, Ar-H), 4.88 (s,

1H, OH), 4.67 (d, J = 2.4 Hz, 2H, CH2), 2.52 (t, J = 2.4 Hz, 1H, CH). IR (KBr, νmax):
3287 (≡CH), 2122 (C≡C), 1281 (C-O) cm-1.
4.3.2 1,3-bis(prop-2-yn-1-yloxy)benzene (12c)
Two equivalents of anhydrous K2CO3 and propargylic bromide were required. The
crude product was purified by flash column chromatography, using ethyl acetate/
hexane (20:80). White solid; yield 60 %. MP: 38-39 ºC. 1H NMR (400 MHz, CDCl3) δ
7.24 – 7.19 (m, 1H, Ar-H), 6.64 – 6.61 (m, 3H, Ar-H), 4.68 (d, J = 2.4 Hz, 4H, CH2),
2.53 (t, J = 2.4 Hz, 2H, CH). IR (KBr, νmax): 3288 (≡CH), 2122 (C≡C) cm-1.
4.3.3 3-methoxy-4-(prop-2-yn-1-yloxy)benzaldehyde (12d).
1
Yield 80 % ; white solid; MP: 86-87 ºC. H NMR (400 MHz, CDCl3) δ 9.87 (s, 1H,
CHO), 7.49 – 7.42 (m, 2H, Ar-H), 7.15 (d, J = 8.2 Hz, 1H, Ar-H), 4.87 (d, J = 2.4 Hz,
2H, CH2), 3.94 (s, 3H, OCH3), 2.56 (t, J = 2.4 Hz, 1H, CH). IR (KBr, νmax): 3249
(≡CH), 2127 (C≡C), 1688 (C=O) cm-1.
4.4 General procedure for the synthesis of triazoles (8a-f; 10a)
The triazoles were prepared following the procedure described by Sharpless at al [32].
The alkyne (0.6 mmol) and aryl-azide (0.6 mmol) were added to a 1:1 mixture of water
and tert-butyl alcohol (6 mL). Sodium ascorbate (0.06 mmol, in 200 µL of water) was
added, followed by copper (II) sulfate pentahydrate (0.006 mmol, in 100 µL of water).
The reaction mixture was stirred vigorously at room temperature and monitored by TLC
until the reagents were completely consumed. At the end of the reaction the mixture was
diluted with ice water (50 mL), the precipitate was collected by filtration, washed with
cold water (2 x 25 mL) and dried under vacuum. If the precipitation doesn’t work, the
reaction the mixture was extracted with 30 mL of dichloromethane and washed with
water (2 x 25 mL). The combined organic layers were dried over NaSO4 and evaporated
under reduced pressure.

4.4.1 2-(4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl)benzaldehyde (8a).
1
Yield 65 %; brown solid; Reaction time: 8h; MP: 56-58 ºC. H NMR (400 MHz,
CDCl3) δ 9.92 (s, 1H, CHO), 8.13 (dd, J = 7.7, 1.5 Hz, 1H), 8.03 (s, 1H, triazole-H),
7.78 (td, J = 7.7, 1.6 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.54 (dd, J = 7.9, 1.0 Hz, 1H),
7.36 – 7.31 (m, 2H), 7.06 – 6.98 (m, 3H), 5.35 (s, 2H).13C NMR (101 MHz, CDCl3) δ
188.41, 158.12, 138.21, 134.69, 130.45, 130.13, 129.63, 125.48, 124.66, 121.54,
114.78, 61.89. IR (KBr, νmax): 3093, 2867, 1703, 1600, 1496, 1239, 1042 cm-1.
HRMS(ESI) m/z calculated for C16H13N3O2+Na [M+Na]+, 302.0899; found 302.0900.
4.4.2 2-(4-((3-hydroxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)benzaldehyde (8b).
Yield 60 %; light brown solid; Reaction time: 8h; MP: 112-114 ºC. 1H NMR (400 MHz,
DMSO) δ 9.80 (s, 1H), 9.46 (s, 1H), 8.89 (s, 1H, triazole-H), 8.04 (dd, J = 7.9, 1.4 Hz,
1H), 7.92 (td, J = 7.8, 1.3 Hz, 1H), 7.78 (dd, J = 7.4, 5.8 Hz, 2H), 7.10 (t, J = 8.1 Hz,
1H), 6.53 (dd, J = 8.1, 2.0 Hz, 1H), 6.48 (t, J = 2.0 Hz, 1H), 6.41 (dd, J = 8.0, 1.8 Hz,
1H), 5.20 (s, 2H). 13C NMR (101 MHz, DMSO) δ 189.83, 159.73, 159.06, 144.16,
137.94, 135.40, 130.56, 130.42, 130.35, 129.57, 127.04, 126.19, 108.82, 105.75,
102.55, 61.30. IR (KBr, νmax): 3185, 3103, 2922, 1686, 1599, 1154, 1035 cm-1.
HRMS(ESI) m/z calculated for C16H13N3O3+Na [M+Na]+, 318.0849; found 318.0849.
4.4.3 2-(4-((3-(prop-2-yn-1-yloxy)phenoxy)methyl)-1H-1,2,3-triazol-1-
yl)benzaldehyde (8c)
The crude product was purified by flash column chromatography using ethyl acetate/
hexane (20:80) as eluent. The product was obtained as a white solid in 40 % yield. MP:
1
96-97ºC. H NMR (400 MHz, CDCl3) δ 9.92 (s, 1H), 8.13 (dd, J = 7.8, 1.5 Hz, 1H),
8.04 (s, 1H, triazole-H), 7.78 (td, J = 7.7, 1.6 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.54 (dd,
J = 7.9, 0.9 Hz, 1H), 7.25 – 7.21 (m, 1H), 6.73 – 6.61 (m, 3H), 5.33 (s, 2H), 4.69 (d, J =
13
2.4 Hz, 2H), 2.53 (t, J = 2.4 Hz, 1H). C NMR (101 MHz, CDCl3) δ 188.39, 159.29,
158.86, 145.01, 138.18, 134.69, 130.44, 130.16, 129.64, 125.48, 124.78, 107.90,
107.81, 102.37, 78.44, 75.65, 61.97, 55.90, 29.71. IR (KBr, νmax): 3262, 2920, 2117,
1693, 1589, 1143, 1040 cm-1. HRMS(ESI) m/z calculated for C19H15N3O3+Na
[M+Na]+, 356.1005; found 356.1006.

4.4.4 4-(4-((4-formyl-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)benzoic acid
(8d).
1
Yield 80 %; white solid; Reaction time: 8 h, MP: 238-239 ºC. H NMR (400 MHz,
DMSO) δ 13.27 (s, 1H), 9.87 (s, 1H), 9.11 (s, 1H, triazole-H), 8.18 - 8.05 (m, 4H), 7.59
(dd, J = 8.2, 1.8 Hz, 1H), 7.46 – 7.41 (m, 2H), 5.38 (s, 2H), 3.82 (s, 3H). 13C NMR (101
MHz, DMSO-d6) δ 191.65, 152.88, 149.32, 143.67, 139.51, 129.93, 125.81, 123.43,
112.72, 109.72, 61.14, 55.14. IR (KBr, νmax): 3144, 3086, 2827, 1632, 1588, 1504,
1422, 1262, 1140 cm-1. HRMS(ESI) m/z calculated for C18H15N3O5+Na [M+Na]+,
376.0903; found 376.0904.
4.4.5 6-(4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (8e)
The crude product was purified by flash column chromatography using ethyl acetate/
hexane (40:60) as eluent. The product was obtained as an orange solid in 35 % yield.
1
Reaction time: 24h; MP: 148-150 ºC. H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H,
triazole-H), 7.86 (dd, J = 8.9, 2.5 Hz, 1H), 7.77 (d, J = 9.6 Hz, 1H), 7.49 (d, J = 8.9 Hz,
1H), 7.32 (dd, J = 8.7, 7.4 Hz, 1H), 7.05 – 6.97 (m, 2H), 6.55 (d, J = 9.6 Hz, 1H), 5.32
(s, 1H). 13C NMR (101 MHz, CDCl3) δ 160.13, 158.49, 154.07, 146.02, 142.83, 133.75,
130.09, 124.11, 121.94, 121.34, 120.03, 120.01, 118.94, 118.85, 115.16, 77.16, 62.31.
IR (KBr, νmax): 3055, 2920 1725, 1491, 1235, 1043 cm-1. HRMS(ESI) m/z calculated
for C18H13N3O3+Na [M+Na]+, 342.0849; found 342.0848.
4.4.6 2-(4-phenyl-1H-1,2,3-triazol-1-yl)benzaldehyde (8f)
The crude product was purified by flash column chromatography using ethyl acetate/
hexane (20:80) as eluent. The product was obtained as a white solid in 30 % yield.
Reaction time: 24h; MP: 116-117 ºC. 1H NMR (400 MHz, CDCl3) δ 10.00 (s, 1H), 8.18
(s, 1H, triazole-H), 8.14 (dd, J = 7.7, 1.1 Hz, 1H), 7.93 (d, J = 7.1 Hz, 2H), 7.80 (td, J =
7.7, 1.5 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.48 (t, J = 7.5 Hz,
13
2H), 7.40 (t, J = 7.4 Hz, 1H). C NMR (101 MHz, CDCl3) δ 188.45, 148.56, 138.35,
134.69, 130.49, 130.10, 129.70, 129.54, 129.04, 128.77, 125.95, 125.36, 121.50. IR
(KBr, νmax): 3133, 2920 1685, 1597, 1455, 1018 cm-1. HRMS(ESI) m/z calculated for
C15H11N3O+Na [M+Na]+, 272.0794; found 272.0794.

4.4.7 2,2'-(4,4'-((1,3-phenylenebis(oxy))bis(methylene))bis(1H-1,2,3-triazole-4,1
diyl)) dibenzaldehyde (10a)
The crude product was purified by flash column chromatography using ethyl acetate/
hexane (20:80) as eluent. The product was obtained as a light yellow solid in 50 %
yield. Reaction time: 16 h, MP: 106-107 ºC. 1H NMR (400 MHz, CDCl3) δ 9.91 (s, 2H),
8.11 (dd, J = 7.8, 1.5 Hz, 2H), 8.05 (s, 2H, triazole-H), 7.77 (td, J = 7.7, 1.6 Hz, 2H),
7.68 (t, J = 7.5 Hz, 2H), 7.54 (dd, J = 7.9, 0.9 Hz, 2H), 7.25 (t, J = 8.2 Hz, 1H), 6.73 (t,
J = 2.3 Hz, 1H), 6.69 (dd, J = 8.2, 2.4 Hz, 1H), 5.33 (s, 4H). 13C NMR (101 MHz,
CDCl3) δ 189.17, 160.18, 145.69, 138.87, 135.30, 131.14, 131.08, 130.83, 130.34,
126.18, 125.51, 108.51, 102.95, 62.87. IR (KBr, νmax): 3117, 2918, 1688, 1598, 1154,
1036 cm-1. HRMS(ESI) m/z calculated for C26H20N6O4+Na [M+Na]+, 503.1438; found
503.1438.
4.5 Synthesis
triazole (9a)
of
1-(2-(difluoromethyl)phenyl)-4-(phenoxymethyl)-1H-1,2,3-
The compound 9a was prepared following the procedure described by Boechat [7].
To a solution of compound 8a (0.06 g, 0.2 mmol) in anhydrous dichloromethane (2
mL), DAST (0.08 g, 0.52 mmol) was added dropwise under N2 atmosphere. The
reaction mixture was stirred for 24 h at room temperature. At the end of the reaction, it
was added a saturated solution of NaHCO3 (4 mL). The mixture was extracted with
dichloromethane (2 x 10 mL), washed with brine (2 x 10 mL), dried (Na2SO4), and
filtered. The solvent was evaporated under reduced pressure and the residue was
purified by flash chromatography using ethyl acetate as eluent to give the product as an
oil in 72 % yield. 1H NMR (400 MHz, CDCl3) δ 7.93 (s, 1H, triazole-H), 7.87 (dd, J =
5.6, 3.7 Hz, 1H), 7.66 (dd, J = 5.8, 3.4 Hz, 2H), 7.48 (dd, J = 5.5, 3.6 Hz, 1H), 7.32 (dd,
J = 8.7, 7.4 Hz, 2H), 7.05 – 6.98 (m, 3H,), 6.79 (t, J = 54.8 Hz, 1H, CHF2), 5.32 (s, 2H).
13C NMR (101 MHz, CDCl3) δ 158.12, 144.94, 131.81, 130.33, 129.63, 127.14, 127.08,
127.01, 125.97, 124.78, 121.49, 114.83, 113.68, 111.30, 108.93, 61.89. R (KBr, νmax):
1598, 1493, 1235, 1215, 1029 cm-1. HRMS(ESI) m/z calculated for C16H13F2N3O+Na
[M+Na]+, 324.0918; found 324.0919.

4.6 Synthesis of 2-(4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl)phenyl)methanol (9b)
A mixture of compound 8a (0.06g, 0.21 mmol) in methanol (3 mL) was stirred until
completely dissolved and then it was cooled in an ice bath. NaBH4 (0.02g, 0.54 mmol)
was added portionwise. After 30 minutes, the reaction mixture became clear, and TLC
indicated the end of the reaction. The mixture was extracted with dichloromethane (15
mL), washed with water (2 x 10 mL). The organic phase was dried with Na2SO4,
filtered and the solvent was evaporated under reduced pressure. The product (9b) was
1
obtained as a white solid in 65 % yield. MP: 94-96 ºC. H NMR (400 MHz, CDCl3) δ
8.03 (s, 1H, triazole-H), 7.66 (d, J = 7.1 Hz, 1H), 7.59 – 7.47 (m, 1H), 7.42 (d, J = 7.7
Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H), 7.04 (dd, J = 15.7, 7.8 Hz, 1H), 5.35 (s, J = 11.2 Hz,
1H), 4.51 (d, J = 5.3 Hz, 1H), 3.39 (t, J = 5.8 Hz, 1H).13C NMR (101 MHz, CDCl3) δ
158.13, 144.75, 135.92, 135.62, 131.61, 130.12, 129.65, 129.18, 124.45, 124.11,
121.49, 114.82, 61.93, 61.88. IR (KBr, νmax): 3290, 2922, 1597, 1496, 1237, 1045 cm-1.
HRMS(ESI) m/z calculated for C16H15N3O2+Na [M+Na]+, 304.1056; found 304.1056.
4.7 General procedure for the synthesis of compounds (9c, 9d, 10b and 10c)
Compounds 8a or 10a (0.40 mmol, 1 eq) and hydrazine or imidazole (0.48 mmol, 1.2
eq) were added to a 10 mL round-bottomed flask and completely dissolved in methanol
(3 mL). The reaction mixture was refluxed for 2 hours at 60 ºC. At the end of the
reaction, the mixture was cooled in an ice bath. The precipitate was filtered and washed
with cold water and ethanol.

4.7.1 (E)-4-(phenoxymethyl)-1-(2-((2-phenylhydrazono)methyl)phenyl)-1H-1,2,3-
triazole (9c)
1
Yield 70 %; white solid; MP: 128-129 ºC. H NMR (400 MHz, CDCl3) δ 8.25 (dd, J =
8.0, 1.1 Hz, 1H), 7.89 (s, 1H), 7.77 (s, 1H), 7.56 (t, J = 7.1 Hz, 1H), 7.44 (td, J = 7.6,
1.4 Hz, 1H), 7.39 – 7.33 (m, 4H), 7.32 – 7.29 (m, 1H), 7.11 – 7.01 (m, 5H), 6.92 (t, J =
7.3 Hz, 1H), 5.37 (s, 2H).13C NMR (101 MHz, CDCl3) δ 158.11, 144.05, 134.23,
131.29, 130.81, 130.14, 129.66, 129.32, 128.51, 126.57, 126.07, 121.45, 120.61,
114.92, 112.87, 61.85. IR (KBr, νmax): 3226, 3036, 1738, 1598, 1493, 1237 cm-1.
HRMS(ESI) m/z calculated for C22H19N5O+Na [M+Na]+, 392.1481; found 392.1482.
4.7.2 (E)-4-methyl-N'-(2-(4-(phenoxymethyl)-1H-1,2,3
triazol1yl)benzylidene)benzenesulfonohydrazide (9d)
1
Yield 65 %; white solid; MP: 167-169 ºC. H NMR (400 MHz, CDCl3) δ 8.57 (s, 1H),
8.09 (dd, J = 7.3, 2.1 Hz, 1H), 7.82 – 7.79 (m, 3H), 7.54 – 7.48 (m, 3H), 7.35 – 7.27 (m,
4H), 7.05 – 6.95 (m, 3H), 5.26 (s, 2H), 2.39 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
157.97, 144.63, 144.14, 141.29, 135.47, 130.66, 130.21, 129.65, 129.12, 127.84,
127.32, 125.74, 125.29, 121.51, 114.82, 61.58, 21.58. IR (KBr, νmax): 3145, 1738, 1595,
1491, 1333, 1165 cm-1. HRMS(ESI) m/z calculated for C23H21N5O3S+Na [M+Na]+,
470.1265; found 470.1261.
4.7.3 (N',N''E,N',N''E)-N',N''-(((4,4'-((1,3-phenylenebis(oxy))bis(methylene))bis(1H-
1,2,3-triazole-4,1-diyl))bis(2,1-phenylene))bis(methanylylidene))bis(4-
methylbenzenesulfonohydrazide) product (10b)
Compound 10a (0.1g, 0.20 mmol) was treated with 2 equivalents p-toluenosufonyl-
hydrazine (0.08g, 0.45 mmol). Compound 10b was obtained as white solid in 76%
1
yield. MP: 135-137 ºC. H NMR (400 MHz, CDCl3) δ 9.96 (s, 1H), 8.08 (dd, J = 7.5,
1.7 Hz, 1H), 7.84 (s, 1H), 7.77 (d, J = 8.3 Hz, 1H), 7.55 – 7.47 (m, 1H), 7.33 (dd, J =
7.6, 1.3 Hz, 1H), 7.22 (d, J = 8.1 Hz, 1H), 6.67 (dd, J = 8.2, 2.0 Hz, 1H), 6.55 (s, 1H),
13
5.28 (s, 1H), 3.51 (s, 1H), 2.37 (s, 1H). C NMR (101 MHz, CDCl3) δ 159.03, 144.26,
143.94, 141.13, 135.56, 135.28, 130.55, 130.30, 130.29, 129.58, 129.35, 127.76,
127.13, 125.81, 125.64, 109.44, 102.85, 62.06, 21.55. IR (KBr, νmax): 3571, 3130, 1738,

1595, 1490, 1329, 1160 cm-1. HRMS(ESI) m/z calculated for C40H36N10O6S2+Na
[M+Na]+, 839.2152; found 839.2153.
4.7.4 (N,N'E,N,N'E)-N,N'-(((4,4'-((1,3-phenylenebis(oxy))bis(methylene))bis(1H-
1,2,3-triazole-4,1-diyl))bis(2,1-phenylene))bis(methanylylidene))bis(3-(1H-
imidazol-1-yl)propan-1-amine) (10c)
The compound 10a (0.1g, 0.20 mmol) was treated with 2 equivalents 3-aminopropyl-
imidazole (0.05g, 0.42 mmol). Compound 10c was obtained as a brown viscous oil in
60 % yield. 1H NMR (400 MHz, CDCl3) δ 8.16 (dd, J = 5.8, 3.6 Hz, 2H), 8.07 (s, 2H),
7.94 (s, 1H), 7.63 – 7.57 (m, 4H), 7.49 (s, 2H), 7.42 (dd, J = 5.8, 3.4 Hz, 2H), 7.03 (s,
2H), 6.94 (s, 2H), 6.73 (t, J = 2.3 Hz, 1H), 6.69 (d, J = 2.4 Hz, 1H), 6.67 (d, J = 2.4 Hz,
1H), 5.31 (s, 4H), 4.05 (t, J = 6.9 Hz, 4H), 3.48 (t, J = 5.8 Hz, 4H), 2.15 – 2.07 (m, 4H).
13C NMR (101 MHz, CDCl3) δ 159.54, 157.24, 144.58, 137.32, 136.45, 131.42, 131.24,
130.39, 130.30, 129.47, 128.70, 125.80, 125.12, 119.02, 107.83, 102.39, 77.16, 62.06,
57.84, 44.67, 31.94. IR (KBr, νmax): 3111, 2930, 1641, 1592, 1490, 1455, 1379, 1148,
1033 cm-1.
4.8 BIOLOGICAL EVALUATION
4.8.1 Glioblastoma and astrocyte cell culture
U87 cell line was obtained from the American Type Culture Collection (ATCC).
GBM02 and GBM95 cell lines were established in the Laboratory of Cell Morphology
(LMC) from the Federal University of Rio de Janeiro (UFRJ), as previously described
[51] (Ethics Committee – CONEP 2340). Human astrocyte cells was prepared and
cultured as previously described [52]. Those cells were cultivated in Dulbecco’s
modified eagle’s medium (DMEM F12) supplemented with 10% of fetal bovine serum
(SFB) and were maintained at 37 ºC in 5% CO2 and 95% air atmosphere.
4.8.2 MTT – viability assay
Cellular viability was accessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
4
bromide (MTT) assay. Cells were cultured into 96-well plates at 1.5 x 10 cells/well

concentration and then treated with 10, 50 and 100 µM of the compounds for 48 and 72
4
hours. For the TMZ analysis cells were plated into 96-well plates at 1.5 x 10 cells/well
concentration and then treated with 100,500 and 1000 µM of TMZ for 48 and 72h.
Dimethyl sulfoxide (DMSO) at the same dilution was used as control. In the end of the
treatment MTT was added in each well in a final concentration of 5 mg/ml and
incubated for 2 hours at 37 ºC. The formazan crystals produced were dissolved with
DMSO and then the absorbance was measured at 570 nm using Victor 3 Perkin Elmer
UV spectrophotometer. To calculate IC50 values a dose response-curve was plotted on a
semi-log scale with concentrations (X-axis) versus percentage of cellular viability (Y-
axis) and was fitted to a sigmoidal function. The curve fitting was performed using
GraphPad Prism 6 [41].
4.9 In silico analysis
The 2D chemical structures of the compounds were drawn using ChemDraw (version
Ultra 12.0) and were subjected to physicochemical, pharmacokinetic, and toxicological
analyses on ADMET Predictor™ software (version 7.1; Simulations Plus, CA).
Acknowledgements
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior - Brasil (CAPES) - Finance Code 001
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