Trifluoromethyl arylamides with antileukemia effect and intracellular inhibitory activity over serine/arginine-rich protein kinases (SRPKs)

Article abstract of DOI:10.1016/j.ejmech.2017.03.078

The serine/arginine-rich protein kinases (SRPKs) have frequently been found with altered activity in a number of cancers, suggesting they could serve as potential therapeutic targets in oncology. Here we describe the synthesis of a series of twenty-two tr

Full text of DOI:10.1016/j.ejmech.2017.03.078

Accepted Manuscript
Trifluoromethyl arylamides with antileukemia effect and intracellular inhibitory activity
over serine/arginine-rich protein kinases (SRPKs)
Raoni Pais Siqueira, Marcus Vinícius de Andrade Barros, Éverton de Almeida Alves
Barbosa, Thiago Souza Onofre, Victor Hugo Sousa Gonçalves, Higor Sette Pereira,
Abelardo Silva Júnior, Leandro Licursi Oliveira, Márcia Rogéria Almeida, Juliana
Lopes Rangel Fietto, Róbson Ricardo Teixeira, Gustavo Costa Bressan
PII:
S0223-5234(17)30246-5
10.1016/j.ejmech.2017.03.078
EJMECH 9338
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 December 2016
Revised Date: 28 March 2017
Accepted Date: 30 March 2017
Please cite this article as: R.P. Siqueira, M.V. de Andrade Barros, E. de Almeida Alves Barbosa, T.
Souza Onofre, V.H. Sousa Gonçalves, H.S. Pereira, A.S. Júnior, L.L. Oliveira, M.R. Almeida, J. Lopes
Rangel Fietto, R.R. Teixeira, G.C. Bressan, Trifluoromethyl arylamides with antileukemia effect and
intracellular inhibitory activity over serine/arginine-rich protein kinases (SRPKs), European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.03.078.
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Trifluoromethyl arylamides with antileukemia effect and intracellular inhibitory
activity over serine/arginine-rich protein kinases (SRPKs)
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Raoni Pais Siqueira1, Marcus Vinícius de Andrade Barros2, Éverton de Almeida Alves
Barbosa1, Thiago Souza Onofre1, Victor Hugo Sousa Gonçalves1, Higor Sette Pereira1,
Abelardo Silva Júnior3, Leandro Licursi Oliveira4, Márcia Rogéria Almeida1, Juliana
Lopes Rangel Fietto1, Róbson Ricardo Teixeira2*, Gustavo Costa Bressan1*
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1Universidade Federal de Viçosa, Departamento de Bioquímica e Biologia Molecular,
Viçosa, MG, Brazil;
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2Universidade Federal de Viçosa, Departamento de Química, Viçosa, MG, Brazil;
3Universidade Federal de Viçosa, Departamento de Veterinária, Viçosa, MG, Brazil;
4Universidade Federal de Viçosa, Departamento de Biologia Geral, Viçosa, MG, Brazil.
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*Corresponding authors: gustavo.bressan@ufv.br (GCB); robsonr.teixeira@ufv.br
(RRT)
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Abstract
The serine/arginine-rich protein kinases (SRPKs) have frequently been found with
altered activity in a number of cancers, suggesting they could serve as potential
therapeutic targets in oncology. Here we describe the synthesis of a series of twenty-
two trifluoromethyl arylamides based on the known SRPKs inhibitor N-(2-(piperidin-1-
yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) and the evaluation of their
antileukemia effects. Some derivatives presented superior cytotoxic effects against
myeloid and lymphoid leukemia cell lines compared to SRPIN340. In particular,
compounds 24, 30, and 36 presented IC50 values ranging between 6.0 – 35.7 µM. In
addition, these three compounds were able to trigger apoptosis and autophagy, and to
exhibit synergistic effects with the chemotherapeutic agent vincristine. Furthermore,
compound 30 was more efficient than SRPIN340 in impairing the intracellular
phosphorylation status of SR proteins as well as the expression of MAP2K1, MAP2K2,
VEGF, and RON oncogenic isoforms. Therefore, novel compounds with increased
intracellular effects against SRPK activity were obtained, contributing to medicinal
chemistry efforts towards the development of new anticancer agents.
Keywords
Trifluoromethyl arylamides, SRPK, SRPIN340, serine/arginine-rich protein kinase,
leukemia, pre-mRNA splicing.
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1. Introduction
Serine/arginine-rich protein kinases (SRPKs) are serine-threonine kinases related
to the phospho-regulation of serine-arginine proteins (SR proteins), a protein family
involved in pre-mRNA splicing control [1, 2]. Overexpression of the SRPK1 and SRPK2
family members has been related to tumorigenesis and to poor patient prognosis of
many human cancers including leukemia [3, 4], colon [5, 6], pancreatic [6, 7],
melanoma [8], breast [6, 9], prostate [10], and glioma [11]. In the intracellular context of
cancerous cells, dysregulated SRPKs activity promotes cell proliferation and apoptosis
escape [3, 12], suggesting that they are potential targets for the development of new
anticancer agents [13, 14].
SRPKs have also been associated with the infection mechanisms of multiple
viruses, including HIV, hepatitis, dengue, and Epstein-Barr virus [15-17]. Screening for
SRPKs inhibitors with antiviral activity, Hagiwara and colleagues identified the
isonicotinamide
compound
N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)
isonicotinamide (also called SRPIN340) (Fig. 1), which is able to selectively inhibit
SRPK1 and SRPK2 [16].
Since the identification of SRPIN340, different studies have been conducted to
evaluate its pharmacological potential in different in vitro and in vivo disease models,
including viral infection [16, 18, 19], angiogenesis [20, 21], and cancer [8]. Within this
context, in our previous studies we evaluated the cytotoxic potential of SRPIN340 in a
panel of leukemia cells with high expression levels of SRPK1 and SRPK2. This
compound was able to reduce cell viability, decrease hyperphosphorylation of SR
family members (SRSF2, SRSF4, SRSF5 and SRSF6), and to regulate the expression
of genes involved in cell proliferation and survival (MAP2K1, MAP2K2, VEGF and FAS)
[4]. Recently, other SRPK inhibitors have also been described. Similar to SRPIN340,
they displayed important biological effects (Fig. 1) [22, 23].
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Even though these reports have indicated promising results for SRPK
pharmacological inhibition in pre-clinical in vitro and in vivo assays, the search for novel
compounds with increased biological efficiency is of potential interest [8]. Here we
describe the design and synthesis of a series of twenty-two trifluoromethyl arylamides
and the assessment of their potential antileukemia effects.
2. Results and discussion
2.1. Synthesis
Trifluoromethyl arylamide SRPIN340, as well as, compounds 15-36 were
prepared in three steps. First, commercially available 1-fluoro-2-nitro-4-
(trifluoromethyl)benzene (1) was treated with amines to obtain derivatives 2-7 with
yields ranging 81% − 98% (Scheme 1).
After that, compounds 2 - 7 were submitted to reduction reactions with SnCl2/HCl
producing derivatives 8 - 13 (Scheme 2).
Finally, nucleophilic acyl substitution reactions (Scheme 3, Table 1), involving
amines 8 - 13 and aromatic acyl chlorides, produced SRPIN340 (75% yield) and
twenty-two other trifluoromethyl arylamides, compounds named 15 – 36 (30% – 91%
yield). All synthesized compounds were fully characterized by infrared (IR) and nuclear
1
magnetic resonance (NMR, H and 13C) spectroscopy techniques, as well as, by high
resolution mass spectrometry (vide infra).
The synthesis of the compounds 15 - 36 was planned so that the influence on
the biological activity of different groups attached to position 1 (see Scheme 3 and
Table 1 for numbering) could be assessed. Thus, amines containing alicyclic, aliphatic
and aromatic portions were chosen for the preparation of the compounds. In addition,
we also decided to vary the type of aromatic group attached to the carbonyl
functionality so that the impact of these modifications on biological activity could also
be evaluated. Accordingly, four types of aromatic acyl chlorides were used in the
preparation of the compounds 15 - 36. In order to compare the biological effects of
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each derivative with SRPIN340, a well known SRPK inhibitor, the latter was also
synthesized.
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2.2 Effect of compounds on cell viability
The cytotoxic activity of the synthesized trifluoromethyl arylamides 15 - 36 and
SRPIN340 was evaluated at different concentrations (0 – 200 µM) over HL60, Jurkat,
and Nalm6 human leukemic cell lines and the half-maximal inhibitory concentration
(IC50) for each compound was determined. As shown in Table 1, among the twenty-two
trifluoromethyl arylamides synthesized, ten of them were active against at least one of
the leukemia cell lines (IC50 < 100 µM). The compounds 24, 30, and 36 were the most
active ones (IC50 14.2 – 35.7 µM, 8.5 – 17.8 µM, and 6.0 – 33.8 µM, respectively) and
presented superior cytotoxicity in comparison to the SRPK inhibitor SRPIN340 (IC50
38.3 - 75.4 µM). Although further structure-activity relationship studies should be
performed, initial observations suggest that the presence of the aryl bromide group in
novel compounds may be associated with their superior activity. These aryl halide
groups (including groups with bromide or iodide) have been frequently found in the
structures of kinase inhibitors, including the anticancer agents trametinib and
vandetanib [24].
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In order to evaluate if the most active compounds affect non-tumor cells, primary
peripheral blood mononuclear cells (PBMC) were obtained and used in cytotoxic
assays. As shown in Fig. 2, PBMC cells were less sensitive to the treatments than the
evaluated leukemia lineages (Table 1). Although compound 24 slightly reduced the
lymphocytes viability at the dosage investigated, overall these compounds seem to be
selective to leukemic cells.
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2.3 Combinatorial effect with Vincristine
We further investigated potential interactions of compounds 24, 30, and 36 with
vincristine, a component of many multi-drug pediatric and adult cancer chemotherapy,
including leukemia [25]. For this purpose, Nalm6 was incubated for 48 h with two
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different doses, in isolation or in combination of compound 24 (8.9 and 17.9 µM),
compound 30 (4.3 and 8.5 µM), and compound 36 (1.5 and 3.0 µM) with vincristine
(0.5 and 1.0 nM). These doses correspond to 25% and 50% of the IC50 value
previously obtained for each compound (Table 1). After treatments, cell viability was
measured and the combination index (CI) for each drug combination was calculated
using the Chou-Talalay method [26]. According to this method, CI values significantly
lower than 1.0 (CI < 1.0) indicate synergistic effect whereas values close to 1.0 indicate
additive effect. Synergistic effects were observed for combinations containing lower
concentrations of the compounds 24, 30, and 36 (i.e., 25% of the IC50) as the
calculated CI values were 0.57, 0.45, and 0.56, respectively (Fig. 3). Moreover,
combinations performed in concentrations corresponding to 50% of the IC50 indicated
synergism for compound 30 (CI = 0.78) but additive effect for compounds 24 and 36
(CI = 1.02 and CI = 1.05, respectively). Despite this apparent incongruence, this has
been previously reported and seems to be related to the saturation of drug-target
complexes at higher concentrations or due to some interactions between compounds
[27], which is still unknown for our system. In addition, it is noteworthy that vincristine
acts on a nanomolar scale while compounds 24, 30, and 36 act on a micromolar scale,
resulting in dose-response curves with different maximum effects. Then, this can
change the synergy to additive effect when drug concentrations are increased [28].
Nevertheless, the data obtained indicates that pharmaceutical formulations containing
these compounds maybe approached to increase the potency of chemotherapeutic
agents, mainly at lower dosages, which is the overall goal of such a strategy.
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2.4 Effect of compounds on cell death and proliferation
Once compounds 24, 30, and 36 were selected as the most active derivatives,
they were used in additional experiments in order to gain insights on how they might
act in leukemic cells.
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Annexin V/PI staining assays were performed to evaluate whether the treatments
impact in Nalm6 apoptosis. After 12 or 24 h exposure, the three compounds
significantly increased annexin-V positive cells in comparison to control (Fig. 4A). After
24 h of incubation, the percentage of cells in early events of apoptosis (annexin-V+/PI-)
reached 11.9%, 14.6%, 24.7% when treated with compounds 24, 30, and 36,
respectively. Considering the percentage of total apoptotic cells (annexin-V+/PI- and
annexin-V+/PI+), it was increased practically three times by treatment with compound
36 (Fig. 4B). Importantly, necrotic cells (annexin-V-/PI+), which is considered a toxic
and degradative process of cell death [29], were barely noticed in these assays.
The effect of compounds on leukemic cells autophagy was also assessed by
fluorescence microscopy. As shown in Fig. 4C, there was an increase in red
fluorescence when Nalm6 cells were treated with 20 µM of the compounds during 24 h.
These findings indicate the presence of autophagosomes and intracellular acidification
in these cells, very similarly to the observed for cytarabine, a drug that acts on
leukemic cells by triggering apoptosis and autophagy [30], which has been considered
a complex cellular process that in some cases may increase cell death [31].
Finally, proliferation assays revealed that these three substances significantly
impaired proliferation of HL60 and Nalm6 in a time-dependent manner (Fig. 5). After 96
h of incubation, compounds 24, 30, and 36 inhibited, respectively, 33%, 38%, and 48%
of HL60 growth in comparison to control (Fig. 5A). Considering Nalm6, they inhibited
cell growth in 37%, 66%, and 72%, respectively (Fig. 5B). Thus, these data suggest
that pathways affecting cell proliferation are subjected to inhibition upon treatments.
This should be the case of the SRPK2 related activity, as it has been described to
promote leukemia cell proliferation in a previous study [3].
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2.5. Effect on intracellular SRPKs activity
The effect of compounds in altering SRPKs intracellular activity was firstly
evaluated by monitoring the expression pattern of transcripts already known to be
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modulated by SRPKs [6, 21, 32]. With this approach, compound 30 was the most
effective in impairing the expression of MAP2K1 and MAP2K2 as well as VEGF (Fig.
6A). Additionally, compounds 30 and 36 seemed to alter the splicing pattern of the
apoptosis related gene RON. Interestingly, no clear changes in gene expression was
observed in Nalm6 treated with SRPIN340, indicating the necessity of higher
concentrations of this inhibitor at the experimental conditions used [4, 8]. No effects
were observed in the expression pattern of the actin transcript, used here as
endogenous loading control.
Intracellular activity of SRPKs was also monitored by checking the SR protein
phosphorylation status through Western blotting assays. As shown in Fig. 6B,
compound 30 was efficient in decreasing phospho-SR epitopes signals in Nalm6
lysates. Again, in the experimental condition used (treatments with 20 µM for 24 h),
compound 30 was more efficient then the reference SRPK inhibitor SRPIN340. As
controls, the expression of SRPK1, SRPK2 or actin proteins were checked but no
difference was found during the treatments. These data suggest that we were able to
obtain at least one compound with increased intracellular effect over SRPK activity,
which the exact mechanism on SRPK inhibition in vitro, overall selectivity, membrane
cell penetration, or in vivo effect in disease animal models deserve to be better
elucidated in further studies.
3. Conclusions
A series of twenty-two trifluoromethyl arylamides were synthesized. Three
compounds presented superior cytotoxicity against myelogenous and lymphoid
leukemia cell lines as compared to the reference SRPK inhibitor SRPIN340. These
three compounds impaired cell proliferation, presented synergistic effect in combination
with the chemotherapeutic agent vincristine and were able to trigger apoptotic and
autophagic cell death processes. Moreover, intracellular activity of SRPKs were
affected by treatments with these compounds, mainly by compound 30, which altered
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MAP2K1, MAP2K2, VEGF, and RON gene expression as well as SR protein
phosphorylation status. Therefore, these data collectively contribute to medicinal
chemistry efforts towards the development of novel anticancer chemotherapeutic
agents based on SRPK inhibition.
4. Experimental procedures
4.1 Synthetic procedures
4.1.1 Generalities
Analytical
grade
1-fluoro-2-nitro-4-trifluoromethyl
benzene,
piperidine,
morpholine, cyclohexylamine, diethylamine, 4-bromoaniline, pyrrolidine, isonicotinoyl
chloride hydrochloride, nicotinoyl chloride hydrochloride, 2-chloropyridine-3-carboxylic
acid and benzoyl chloride were purchased from Sigma Aldrich (St. Louis, MO, USA)
and used without further purification. Anhydrous tin(II) chloride and triethylamine were
purchased from Vetec (Rio de Janeiro, Brazil) and used as received. 1H- and 13C-NMR
spectra were recorded on a Varian Mercury 300 instrument at 300 MHz and 75 MHz,
respectively, using CDCl3 and CD3OD as solvents. Infrared spectra were recorded on
either a Varian 660-IR, equipped with GladiATR scanning from 4000 to 500 cm−1 or a
Perkin Elmer Paragon 1000 FTIR spectrophotometer, using potassium bromide (1%
v/v) disks, scanning from 600 to 4000 cm-1. Melting points are uncorrected and were
obtained with a MQAPF-301 melting point apparatus (Microquimica, Campinas, Brazil).
Analytical thin layer chromatography was carried out on TLC plates covered with
60GF254 silica gel. Column chromatography was performed over silica gel (60–230
mesh). Solvents utilized as eluents were used without further purification.
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4.1.2 Synthesis of compounds 2 - 7
4.1.2.1 1-(2-nitro-4-(trifluoromethyl)phenyl)piperidine (2)
A 100 mL round bottom flask initially placed in an ice bath was charged with 8.60
mL (88.2 mmol) of piperidine, 4.10 mL of dimethylformamide (DMF), and 4.20 mL (28.7
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mmol) of 1-fluoro-2-nitro-4-trifluoromethyl benzene (1). The ice bath was removed and
the resulting mixture was magnetically stirred at room temperature for 1.5 h. After this
time, water was added and the resulting mixture was transferred to a separatory funnel.
The aqueous phase was extracted with ethyl acetate (4 x 80 mL). The organic extracts
were combined and the resulting organic layer was washed with brine, dried over
sodium sulphate, filtered and concentrated under reduced pressure. The resulting solid
was recrystallized with methanol. Compound 2 was obtained as an orange solid in 91%
yield (7.15 g, 26.1 mmol).
TLC Rf = 0.40 (ethyl acetate - hexane 16:1 v/v). mp 50.1 - 50.7 ºC. IR (ATR, cm-1)
−
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242
: 2938, 2867, 2827, 1621, 1560, 1528, 1493, 1449, 1386, 1323, 1297, 1260, 1233,
ν
max
1211, 1149, 1115, 1080, 1064, 1021, 974, 929, 906, 882, 856, 832, 789, 760, 724, 678,
629, 528.1H NMR (300 MHz, CDCl3)
δ
: 1.61-1.75 (m, 6H), 3.12 (t, 4H, J = 5.3 Hz), 7.14
(d, 1H, J = 8.7 Hz), 7.60 (dd, 1H, J = 8.7 Hz and J = 2.3 Hz), 8.03 (d, 1H,J = 2.3 Hz).
13C NMR (75 MHz, CDCl3)
δ: 24.0, 25.8, 52.3, 120.6, 120.9 (q, JC-F = 34.1 Hz), 123.7 (q, JC-
= 269.6 Hz), 124.6 (q, JC-F = 4.0 Hz), 130.1 (q, JC-F = 3.4 Hz), 139.8, 148.8. HRMS
F
(M+H+): Calculated for C12H14F3N2O2, 275.1007; found: 275.0926.
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245
Nitro compounds 3 - 7 (Scheme 1) were synthesized using a procedure similar to
that described for the preparation of compound 2. Description of experimental data that
support the structures of compounds 3-7 is provided below.
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4.1.2.2 N-cyclohexyl-2-nitro-4-(trifluoromethyl)aniline (3)
The compound was obtained as a yellow solid after recrystallization with
−
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methanol in 81% yield. TLC R = 0.10 (hexane). mp 79.3 - 80.2 ºC. IR (ATR, cm-1)
:
ν
max
f
3365, 3114, 2931, 2861, 1634, 1572, 1529, 1436, 1411, 1324, 1260, 1244, 1227, 1187,
1
1152, 1112, 1063, 976, 912, 899, 831, 763, 694, 642 . H NMR (300 MHz, CDCl3)
δ
:
1.30-2.07 (m, 10H), 3.51-3.61 (m, 1H), 6.95 (d, 1H, J = 9.3 Hz), 7.56 (dd, 1H, J = 9.3
Hz and J = 2.1 Hz), 8.34 (d 1H, J = 6.3 Hz), 8.45 (d, 1H, J = 2.1 Hz).13C NMR (75 MHz,
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CDCl3)
δ
: 24.6, 25.6, 32.7, 51.5, 115.0, 117.0 (q, JC-F= 34.1 Hz), 123.9 (q, JC-F= 269.0
Hz), 125.4 (q, J = 4.2 Hz), 132.1 (q, J = 3.0 Hz), 130.7, 146.2. HRMS (M+H+):
Calculated for C13 H15F3N2O2, 289.1086; found: 289.0994.
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4.1.2.3 1-(2-nitro-4-(trifluoromethyl)phenyl)pyrrolidine (4)
The compound was obtained as an orange solid in 97% after purification by silica
gel column chromatography eluted with hexane-ethyl acetate (5:1 v/v). TLC Rf = 0.45
−
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(hexane-ethyl acetate 5:1 v/v). mp 52.3 - 53.8 ºC. IR (ATR, cm-1)
: 2975, 2871,
ν
max
1622, 1554, 1504, 1428, 1388, 1322, 1268, 1150, 1103, 1074, 884, 808, 781, 719, 688,
634. 1H NMR (300 MHz, CDCl3)
J = 9.0 Hz), 7.53 (dd, 1H, J = 9.0 Hz and J = 2.4 Hz), 7.99 (brs, 1H). 13C NMR (75 MHz,
δ
: 1.98 - 2.03 (m, 4H), 3.23 - 3.27 (m, 4H), 6.95 (d, 1H,
CDCl3) δ: 25.8, 50.8, 116.4, 117.1 (q, JC-F = 34.2 Hz), 124.0 (q, JC-F = 269.1 Hz), 124.7
(q, JC-F = 4.0 Hz), 129.4 (q, JC-F = 3.2 Hz), 135.8, 144.4. HRMS (M+H+): Calculated for
C11H12F3N2O2, 261.0851; found: 261.0770.
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4.1.2.4 N,N-diethyl-2-nitro-4-(trifluoromethyl)aniline (5)
The compound was obtained as an orange oil in 98% yield after purification by
silica gel column chromatography eluted with hexane-ethyl acetate (8:1 v/v). TLC Rf =
−
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0.55 (hexane - ethyl acetate 8:1 v/v). IR (ATR, cm-1)
: 2979, 2939, 2877, 1621,
ν
max
1531, 1322, 1258, 1114, 1083, 903, 877, 816, 784, 717, 669, 601. 1H NMR (300 MHz,
CDCl3) : 1.16 (t, 6H, J = 7.1 Hz), 3.26 (q, 4H, J = 7.1 Hz), 7.14 (d, 1H, J = 9.0 Hz),
δ
7.58 (dd, 1H, J = 9.0 Hz and J = 2.3 Hz), 7.96 (brs, 1H). 13C NMR (75 MHz, CDCl3)
12.6, 46.1, 119.9 (q, JC-F = 34.0 Hz),120.6, 123.8 (q, JC-F = 269.3 Hz), 124.5 (q, JC-F
δ:
=
4.0 Hz), 129.4 (q, JC-F = 3.3 Hz), 139.8, 146.6. HRMS (M+H+): Calculated for
C11H14F3N2O2, 263.1007; found: 263.0944.
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4.1.2.5 4-(2-nitro-4-(trifluoromethyl)phenyl)morpholine (6)
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The compound was obtained as an orange oil in 97% yield after purification by
silica gel column chromatography eluted with hexane-ethyl acetate (3:1 v/v). TLC Rf =
−
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285
0.27 (hexane-ethyl acetate 3:1 v/v). IR (ATR, cm-1)
: 2967, 2858, 1713, 1622,
ν
max
1532, 1322, 1275, 1252, 1235, 1168, 1110, 1083, 1044, 938, 884, 824, 789, 720, 678,
1
640, 526. H NMR (300 MHz, CDCl3)
δ
: 3.13 (t, 4H, J = 4.7 Hz), 3.84 (t, 4H, J = 4.7
Hz), 7.16 (d, 1H, J = 8.7 Hz), 7.68 (dd, 1H, J = 8.7 Hz and J = 2.3 Hz), 8.05 (brs, 1H).
13C NMR (75 MHz, CDCl3)
δ
: 51.4, 66.6, 120.6, 123.4 (q, JC-F = 269.9 Hz), 122.9 (q, JC-
= 34.1 Hz), 124.4 (q, JC-F = 3.9 Hz), 130.5 (q, JC-F = 3.3 Hz), 141.0, 148.1. HRMS
F
(M+H+): Calculated for C11H12F3N2O3, 277.0800; found: 277.0727.
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288
4.1.2.6 N-(4-bromophenyl)-2-nitro-4-(trifluoromethyl)aniline (7)
The compound was obtained as an orange solid in 93% yield after purification by
silica gel column chromatography eluted with hexane - ethyl acetate (5:1 v/v). TLC Rf =
−
289
290
291
292
293
294
295
0.78 (hexane - ethylacetate 5:1 v/v). mp 89.5 - 89.9 ºC. IR (ATR, cm-1)
: 3347,
ν
max
3102, 1636, 1571, 1528, 1486, 1430, 1319, 1250, 1147, 1070, 1011, 909, 841, 805,
1
693, 632. H NMR (300 MHz, CDCl3)
δ
: 7.15-7.26 (m, 3H), 7.54-7.60 (m, 3H), 8.50
: 116.8, 120.1 (q, JC-F = 34.1 Hz),
(brs, 1H), 9,63 (brs,1H). 13C NMR (75 MHz, CDCl3)
δ
120.2, 123.5 (q, JC-F = 269.5 Hz), 125.0 (q, JC-F = 4.2 Hz), 126.8, 132.1 (q, JC-F = 3.2
Hz), 132.4, 133.3, 136.8, 144.9. HRMS (M+H+): Calculated for C13H8BrF3N2O2,
359.9721; found: 359.9648.
296
297
298
299
300
301
302
4.1.3 Synthesis of compounds 8 - 13
4.1.3.1 2-(piperidin-1-yl)-5-(trifluoromethyl)aniline (8)
A 50 mL round bottom flask initially placed in an ice bath was charged with 10.8
mL (129.6 mmol) of concentrated hydrocloric acid, 6.71 g (35.4 mmol) of tin(II) chloride,
20.0 mL of methanol, and 1.50
g
(5.47 mmol) of 1-(2-nitro-4-
(trifluoromethyl)phenyl)piperidine (2). The ice bath was removed and the resulting
mixture was continuously stirred at room temperature for 42 h. After this time, sodium
12

ACCEPTED MANUSCRIPT
303
304
305
306
307
308
309
310
hydroxide solution was added to the mixture until pH was approximately equal to 10.
Then, the mixture was transferred to a separatory funnel and extracted with ethyl
acetate (4 x 80.0 mL). The organic extracts were combined and the resulting mixture
was washed with brine, dried under sodium sulphate, filtered and concentrated under
reduced pressure. The residue was purified by silica gel column chromatography
eluted with hexane-ethyl acetate (11:1 v/v). The compound 8 was obtained as a white
solid in 78% yield (1.34 g, 5.49 mmol).
TLC Rf = 0.48 (hexane-ethyl acetate 11:1 v/v). mp 50.0 - 50.5 ºC. IR (ATR, cm-1)
−
311
312
313
: 3452, 3355, 2950, 2865, 2805, 1611, 1589, 1512, 1469, 1439, 1379, 1328, 1288,
ν
max
1256, 1227, 1205, 1160, 1104, 1064, 936, 892, 860, 810, 745, 722, 663, 643. 1H NMR
(300 MHz, CDCl3)
δ
: 1.60-1.75 (m, 6H), 2.88 (brs, 4H), 4.11 (brs, 2H, NH2), 6.93-7.03
13
314
315
316
317
318
319
(m, 3H). C NMR (75 MHz, CDCl3) δ: 24.4, 26.8, 52.4, 111.5 (q, JC-F = 3.5 Hz), 115.5
(q, JC-F = 4.1 Hz), 119.7, 124.7 (q, JC-F = 270.0 Hz), 126.1 (q, JC-F = 31.9 Hz), 141.7,
143.4. HRMS (M+H+): Calculated for C12H16F3N2, 245.1266; found: 245.1182.
The anilines 9 - 13 (Scheme 2) were synthesized from compounds 3-7 using a
similar procedure to that described for the preparation of 8. Description of experimental
data that support the structures of compounds 9 - 13 is provided below.
320
321
322
4.1.3.2 N-cyclohexyl-4-(trifluoromethyl)benzene-1,2-diamine (9)
The compound was obtained as a white solid in 56% yield after purification by
silica gel column chromatography eluted with hexane - ethyl acetate (14:1 v/v). TLC Rf
−
323
324
325
326
327
328
= 0.25 (hexane-ethyl acetate 14:1 v/v). mp 71.6 - 72.0 ºC. IR (ATR, cm-1)
: 3421,
ν
max
3350, 2928, 2855, 1625, 1601, 1528, 1470, 1440, 1362, 1324, 1300, 1240, 1217, 1146,
1
1107, 1084, 1055, 913, 885, 863, 808, 737, 668, 635. H NMR (300 MHz, CDCl3)
δ
:
1.17 - 2.15 (m, 10H), 3.25 - 3.37 (m, 4H), 6.65 (d, 1H, J = 8.4 Hz), 6.93 (d, 1H, J = 1.8
Hz), 7.08 (dd, 1H, J = 8.4 Hz and J = 1.8 Hz). 13C NMR (75 MHz, CDCl3)
δ
: 25.1, 26.0,
33.4, 51.8, 110.9, 113.9 (q, JC-F= 3.7 Hz), 118.6 (q, JC-F= 4.1 Hz), 119.3 (q, JC-F= 32.1), 125.2
13

ACCEPTED MANUSCRIPT
329
330
331
(q, JC-F= 268.8 Hz), 133.2, 139.9. HRMS (M+H+): Calculated for C13H18F3N2, 259.1422;
found: 259.1341.
332
333
334
335
4.1.3.3 2-(pyrrolidin-1-yl)-5-(trifluoromethyl)aniline (10)
The compound was obtained as a red oil in 77% yield after purification by column
chromatography eluted with hexane - ethyl acetate (3:1 v/v). TLC Rf = 0.68 (hexane -
−
336
337
338
ethyl acetate 3:1 v/v). IR (ATR, cm-1)
: 3440, 3355, 2969, 2877, 2823, 1711, 1618,
ν
max
1
1516, 1439, 1328, 1244, 1148, 1105, 954, 903, 866, 808, 661. H NMR (300 MHz,
CDCl3)
δ
: 1.91 - 1.97(m, 4H), 3.09-3.13 (m, 4H), 3.92 (brs, 2H), 6.92 - 6.98 (m, 3H).13C
: 24.3, 50.6, 112.1 (q, JC-F = 3.8 Hz), 115.8 (q, JC-F = 4.1 Hz),
339
340
341
NMR (75 MHz, CDCl3)
δ
117,8, 124.8 (q, JC-F = 269.6 Hz), 124.8 (q, JC-F = 31.8 Hz, C-5), 140.8. HRMS (M+H+):
Calculated for C11H14F3N2, 231.1109; found: 231.1026.
342
343
344
4.1.3.4 N,N-diethyl-4-(trifluoromethyl)benzene-1,2-diamine (11)
The compound was obtained as a yellow oil in 67% yield after purification by
column chromatography eluted with hexane - ethyl acetate (12:1 v/v). TLC Rf = 0.22
−
345
346
347
348
349
350
351
(hexane -ethyl acetate 30:1 v/v). IR (ATR, cm-1)
: 3469, 3362, 2973, 2933, 2870,
ν
max
2826, 1615, 1593, 1514, 1441, 1384, 1335, 1294, 1260, 1232, 1163, 1120, 928, 867,
1
817, 745, 666. H NMR (300 MHz, CDCl3)
δ
: 0.99 (t, 6H, J = 7.1 Hz), 2.99 (q, 4H, J =
7.1 Hz), 4.19 (brs, 2H), 6.94-6.97 (m, 2H), 7.05 (d, 1H, J = 8.7 Hz).13C NMR (75 MHz,
CDCl3) : 12.3, 46.6, 111.3 (q, JC-F = 3.7 Hz), 114.6 (q, JC-F = 3.9 Hz), 122.5, 124.4 (q,
δ
JC-F = 270.0 Hz), 126.3 (q, JC-F = 31.8 Hz), 139.9, 143.7. HRMS (M+H+): Calculated for:
C11H16F3N2, 233.1266; found: 233.1211.
352
4.1.3.5 2-morpholino-5-(trifluoromethyl)aniline (12)
14

ACCEPTED MANUSCRIPT
353
354
Compound was obtained as a white solid in 94% yield without any further
purification. TLC Rf = 0.48 (hexane - ethylacetate 3:1 v/v). mp 130.6 - 131.1 ºC. IR
−
355
356
357
358
359
360
(ATR, cm-1)
: 3430, 3338, 2827, 2823, 1620, 1515, 1448, 1331, 1256, 1217, 1153,
ν
max
1
1099, 938, 897, 860, 818, 651. H NMR (300 MHz, CDCl3)
δ
: 2.95 (t, 4H, J = 4.7 Hz),
3.87 (t, 4H, J = 4.7 Hz), 6.96 (brs, 1H), 6.96-7.05 (m, 2H). 13C NMR (75 MHz, CDCl3)
: 51.2, 67.6, 111.9 (q, JC-F = 3.7 Hz), 115.7 (q, JC-F = 4.0 Hz), 119.7, 124.6 (q, JC-F
δ
=
270.0 Hz), 126.9 (q, JC-F = 32.0 Hz), 141.7. HRMS (M+H+): Calculated for C11H14F3N2O,
247.1058; found: 247.0956.
361
362
363
4.1.3.6 N-(4-bromophenyl)-4-(trifluoromethyl)benzene-1,2-diamine (13)
Compound was obtained as a white solid in 79% yield after purification by silica
gel column chromatography eluted with hexane-ethyl acetate (5:1 v/v). TLC Rf = 0.25
−
364
365
366
(hexane-ethyl acetate 5:1 v/v). mp 123.5-123.8 ºC. IR (ATR, cm-1)
: 3469, 3384,
ν
max
1
1591, 1518, 1485, 1436, 1385, 1334, 1249, 1154, 1106, 928, 868, 820. H NMR (300
MHz, CDCl3)
δ: 3.73 (brs, 2H), 5.39 (brs, 1H), 6.72 (d, 2H, J = 9.0 Hz), 6.97-7.91 (m,
367
368
369
370
2H), 7.16 (d, 1H, J = 8.1 Hz), 7.34 (d, 2H, J = 9.0 Hz). 13C NMR (75 MHz, CDCl3)
δ:
113.0, 113.6 (q, JC-F = 3.7 Hz), 117.0 (q, JC-F = 3.9 Hz), 118.6, 121.9 (C-6), 124.4 (q, JC-
= 270.3 Hz), 126.7 (q, JC-F = 32.3 Hz), 132.5, 132.6, 139.5, 142.7.HRMS (M+H+):
F
Calculated for C13H11BrF3N2, 331.0058; found: 330.9987.
371
372
373
374
375
376
377
378
4.1.4 Synthesis of SRPIN340 and compounds 15 - 36
4.1.4.1 N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340)
A 25 mL round bottom flask initially placed in an ice bath was charged with 0.629
g (3.389 mmol) of isonicotinoyl chloride hydrochloride, 0.800 mL of triethylamine, 8.00
mL of dichoromethane and 0.400 (1.64 mmol) of 2-(piperidin-1-yl)-5-(trifluoromethyl)
aniline (8). The ice-bath was removed and the mixture was magnetically stirred at room
temperature for 3 h. Then, 10.0 mL of distilled water was added, and the mixture was
transferred to a separatory funnel. The aqueous layer was extracted with ethyl acetate
15

ACCEPTED MANUSCRIPT
379
380
381
382
383
384
(4 x 30.0 mL). The organic extracts were combined and the resulting organic layer was
washed with brine, dried over sodium sulphate, filtered, and concentrated under
reduced pressure. The residue was purified by silica gel column chromatography
eluted with hexane-ethyl acetate (3:1 v/v). The solid was further recrystallized with
acetone. The compound SRPIN340 was obtained as a white solid in 75% yield (430
mg, 1.23 mmol).
−
385
386
387
388
389
390
391
392
393
TLC R = 0.13 (hexane - ethyl acetate 3:1 v/v). mp 95.6 - 96.7 ºC. IR (ATR, cm-1)
:
ν
max
f
3347, 2945, 2917, 2811, 1679, 1611, 1587, 1556, 1527, 1455, 1434, 1380, 1334, 1308,
1239, 1165, 1107, 1093, 1061, 1022, 915, 895, 878, 839, 826, 751, 728, 681, 662, 644.
1H NMR (300 MHz, CDCl3)
δ: 1.65 - 1.81 (m, 6H), 2.86 (t, 4H, J = 5.1 Hz), 7.28 (d, 1H,
J = 8.4 Hz), 7.37 (dd, 1H, J = 8.4 Hz and J = 1.8 Hz), 7.76 (dd, 2H, J = 4.5 Hz and J =
1.5 Hz), 8.83 - 8.85 (m, 3H), 9.55 (s, 1H, NH). 13C NMR (75 MHz, CDCl3)
δ
: 24.0, 27.1,
53.8, 116.6, 120.8, 121.1, 121.6 (q, JC-F = 3.7 Hz), 124.2 (q, JC-F = 270.5 Hz), 127.5 (q,
JC-F = 32.3 Hz), 133.4, 141.8, 145.9, 151.1, 163.0. HRMS (M+H+): Calculated for
C18H19F3N3O, 350.1480; found: 350.1420.
394
395
396
The trifluoromethyl amides 15 - 36 (Scheme 3) were prepared by using a
similar methodology to that described for the synthesis of SRPIN340. Description of
experimental data that support the structures of compounds 15 - 36 is provided below.
397
398
399
4.1.4.2 N-(2-(cyclohexylamino)-5-(trifluoromethyl)phenyl)isonicotinamide (15)
The compound was obtained as a white solid in 82% yield after recrystallization
with ethyl acetate. TLC Rf = 0.33 (hexane - ethyl acetate 1:1 v/v). mp 159.9 -160.2 ºC.
−
400
401
402
403
404
IR (ATR, cm-1)
: 3262, 2931, 2851, 1657, 1617, 1543, 1510, 1485, 1441, 1324,
ν
max
1205, 1254, 1238, 1147, 1133, 1103, 1069, 998, 931, 880, 841, 806, 754, 709, 687,
637. 1H NMR (300 MHz, CD3OD)
δ
: 1.10 – 2.20 (m, 10H), 3.30 (quint, 1H, J = 1.8 Hz),
3.32 - 3.43 (m, 1H), 6.87 (d, 1H, J = 8.7 Hz), 7.40 (dd, 1H, J = 8.7 Hz and J = 1.7 Hz),
7.47-7.46 (m, 1H), 7.93 (dd, 2H, J = 4.7 Hz and J = 1.8 Hz), 8.73 (dd, 2H, J = 4.7 Hz
16

ACCEPTED MANUSCRIPT
405
406
407
408
409
and J = 1.8 Hz).13C NMR (75 MHz, CD3OD)
δ
: 24.9, 25.7, 32.6, 51.3, 111.2, 116.9 (q,
JC-F= 32.7 Hz), 121.6, 122.1, 124.6 (q, J = 3.8 Hz), 125.1 (q, JC-F = 3.9 Hz), 125.1 (q, J
= 267.7 Hz), 142.4, 145.8, 149.7, 165.9. HRMS (M+H+): Calculated for C19H21F3N3O,
364.1637; found: 364.1556.
410
411
412
4.1.4.3 N-(2-(pyrrolidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (16)
The compound was obtained as a white solid in 70% yield after recrystallization
with acetone. TLC Rf = 0.24 (hexane - ethyl acetate 1:1 v/v). mp 110.0 - 110.6 ºC. IR (ATR,
−
413
414
415
416
417
418
419
cm-1)
: 3242, 2976, 2872, 1654, 1613, 1538, 1512, 1489, 1436, 1409, 1370, 1327,
ν
max
1
1291, 1152, 1093, 929, 901, 849, 816, 755, 656. H NMR (300 MHz, CDCl3)
δ
: 1.94 -
1.98 (m, 4H), 3.13 - 3.17 (m, 4H), 7.10 (d, 1H, J = 8.7 Hz), 7.35 (dd, 1H, J = 8.7 Hz
and J = 1.8 Hz), 7.71 - 7.73 (m, 2H), 8.31 (brs, 1H), 8.77 (brs, 2H), 8.97 (brs, 1H).13C
NMR (75 MHz,CDCl3) δ: 25.0, 51.9, 118.4, 121.0, 121.1, 123.0 (q. JC-F = 3.6 Hz), 124.4 (q,
JC-F = 269.9 Hz), 124.3 (q, JC-F = 32.6 Hz), 129.3, 141.7, 145.1, 150.9, 163.5. HRMS
(M+H+): Calculated for C17H17F3N3O, 336.1324; found: 336.1282.
420
421
422
4.1.4.4 N-(2-(diethylamino)-5-(trifluoromethyl)phenyl)isonicotinamide (17)
The compound was obtained as a white solid in 85% yield after purification by
silica gel column chromatography eluted with hexane-ethyl acetate (2:1 v/v). TLC Rf =
−
423
424
425
426
427
428
429
430
0.60 (hexane - ethyl acetate 1:1 v/v). mp 73.8 - 74.3 ºC. IR (ATR, cm-1)
: 3326, 2976,
ν
max
2925, 2856, 1680, 1588, 1530, 1439, 1333, 1241, 1164, 1094, 1060, 922, 895, 826,
1
746, 676, 562. H NMR (300 MHz, CDCl3)
δ
: 0.98 (t, 6H, J = 7.2 Hz), 3.02 (q, 4H, J =
7.2 Hz), 7.31 - 7.40 (m, 2H), 7.72 (dd, 2H, J = 4.5 Hz and J = 1.5 Hz), 8.82 - 8.89 (m,
3H), 9.92 (brs, 1H). 13C NMR (75 MHz, CDCl3)
δ
: 13.0, 49.5, 116.3, 120.9, 121.7 (q, JC-
= 3.7 Hz), 123.7, 124.1 (q, JC-F = 270.7 Hz), 128.3 (q, JC-F = 32.8 Hz), 136.2, 141.9,
F
142.7, 151.0, 163.0. HRMS (M+H+): Calculated for C17H19F3N3O, 338.1480; found:
338.1453.
17

ACCEPTED MANUSCRIPT
431
432
433
4.1.4.5 N-(2-morpholino-5-(trifluoromethyl)phenyl)isonicotinamide (18)
The compound was obtained as a white solid in 78% yield after purification by
silica gel column chromatography eluted with hexane-ethyl acetate (2:1 v/v). TLC Rf =
−
434
435
436
437
438
439
440
441
0.18 (hexane - ethyl acetate 2:1 v/v). mp 166.5 - 168.4 ºC. IR (ATR, cm-1)
: 3351,
ν
max
2969, 2921, 2858, 1676, 1590, 1531, 1439, 1333, 1242, 1155, 1108, 918, 823, 750,
1
656. H NMR (300 MHz, CDCl3)
δ
: 2.95 (t, 4H, J = 4.5 Hz), 3.91 (t, 4H, J = 4.5 Hz),
7.32 (d, 1H, J = 8.4 Hz), 7.41 (dd, 1H, J = 8.4 Hz and J = 2.1 Hz), 7.74 (dd, 2H, J = 4.5
Hz and J = 2.8 Hz), 8.85 - 8.86 (m, 3H), 9.48 (brs, 1H).13C NMR (75 MHz, CDCl3)
δ
:
52.6, 67.7, 117.0 (q, JC-F = 3.9 Hz), 120.8, 121.3, 121.9 (q, JC-F = 3.8 Hz), 124.0 (q, JC-F
= 270.6 Hz), 128.3 (q, JC-F = 32.6 Hz), 133.5, 141.8, 144.2, 151.0, 162.9. HRMS
(M+H+): Calculated for C17H17F3N3O2, 351.1273; found: 352.1218.
442
443
444
4.1.4.6 N-(2-(4-bromophenylamino)-5-(trifluoromethyl)phenyl)isonicotinamide (19)
The compound was obtained as a yellow solid in 81% yield after purification by
silica gel column chromatography eluted with hexane-ethyl acetate (1:1 v/v). TLC Rf =
−
445
446
447
448
449
450
451
0.22 (hexane-ethylacetate 2:1 v/v). mp 203.5 - 203.9 ºC. IR (ATR, cm-1)
: 3386,
ν
max
3243, 3081, 1675, 1589, 1510, 1469, 1324, 1249, 1163, 1101, 923, 885, 821, 804, 749.
1H NMR (300 MHz, CD3OD)
(m, 3H). 8.16 (brs, 1H), 8.75-8.77 (m, 2H), 10.14 (brs, 1H). 13C NMR (75 MHz, CD3OD)
δ
: 7.06 (d, 2H, J = 8,7 Hz), 7.36-7.49 (m, 4H), 7.77-7.85
δ
: 113.4, 117.5, 120.6 (q, JC-F = 32.2 Hz), 121.8, 122.5, 124.5 (q, JC-F = 3.8 Hz), 125.1
(q, JC-F = 269.2 Hz), 125.3 (q, JC-F = 3.8 Hz), 127.0, 132.6, 142.0, 142.3, 150.8, 165.2.
HRMS (M+H+): Calculated for C19H14BrF3N3O, 436.0272; found: 436.0202.
452
453
454
4.1.4.7 2-chloro-N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)nicotinamide (20)
The compound was obtained as a white solid in 78% yield after purification by
column chromatography eluted with hexane-ethyl acetate (3:1 v/v). mp 120.3 - 121.2
455
456
ºC. IR (ATR, cm-1) −
: 3322, 2919, 2827, 1678, 1655, 1613, 1578, 1526, 1474, 1433,
ν
max
1400, 1333, 1263, 1214, 1100, 915, 893, 858, 824, 754, 662, 642, 601 . 1H NMR (300
18

ACCEPTED MANUSCRIPT
457
458
459
460
461
462
MHz, CDCl3)
δ
: 1.58 - 1.73 (m, 6H), 2.85 (t, 4H, J = 5.0 Hz), 7.30 (d, 1H, J = 8.4 Hz),
7.36 - 7.46 (m, 2H), 8.23 (dd, 1H, J = 8.4 Hz and J = 1.8 Hz), 8.54 (dd, 1H, J = 4.5 Hz
and J = 1.8 Hz), 8.87 (s, 1H), 9.73 (brs, 1H).13C NMR (75 MHz, CDCl3)
δ
: 23.9, 26.6,
54.1, 116.8 (q, JC-F = 3.7 Hz), 121.4, 121.7, 121.7 (q, JC-F = 3.9 Hz), 123.2, 124.2 (q,
JC-F = 270.4 Hz), 127.6 (q, JC-F = 32.1 Hz), 131.6, 133.8, 140.4, 146.1, 146.9, 151.6,
162.7. HRMS (M+H+): Calculated for C18H18ClF3N3O, 384.1090; found: 384.1043.
463
464
465
4.1.4.8 2-chloro-N-(2-(pyrrolidin-1-yl)-5-(trifluoromethyl)phenyl)nicotinamide (21)
The compound was obtained as a white solid in 79% yield after purification by
silica gel column chromatography eluted with hexane-ethyl acetate (2:1 v/v). TLC Rf =
−
466
467
468
469
470
471
472
473
474
475
0.52 (hexane - ethyl acetate 2:1 v/v). mp 147.5 - 148.7 ºC. IR (ATR, cm-1)
: 3232,
ν
max
2968, 2882, 2818, 1659, 1615, 1581, 1535, 1508, 1405, 1368, 1331, 1275, 1151, 1095,
818, 802, 768, 708, 540. 1H NMR (300 MHz, CDCl3)
δ
: 1.94 - 2.01 (m, 4H), 3.13 (t, 4H,
J = 6.3 Hz), 7.20 (d, 1H, J = 8.7 Hz), 7.38 (dd, 1H, J = 8.7 Hz and J = 1.5 Hz), 7.44 (dd,
1H, J = 7.8 Hz and J = 4.7 Hz), 8.32 (dd, 1H, J = 7.8 Hz and J = 1.8 Hz), 8.79 (dd, 1H,
J = 4.7 Hz and J = 1.8 Hz), 8.56 (d, J = 1.5 Hz, 1H), 9.32 (brs, 1H). 13C NMR (75 MHz,
CDCl3) δ: 24.8, 52.5, 119.3 (q, JC-F = 3.8 Hz), 119.5, 122.6 (q, JC-F = 3.7 Hz), 123.4,
125.3 (q, JC-F = 32.5 Hz), 131.1, 131.3, 140.9, 144.7, 147.0 , 151.7, 162.6. The signal
of the carbon of the CF3 group presented low intensity and it was not noticed in the
spectrum. HRMS (M+H+): Calculated for C17H16ClF3N3O, 370.0934; found: 370.0851.
476
477
478
4.1.4.9 2-chloro-N-(2-(diethylamino)-5-(trifluoromethyl)phenyl)nicotinamide (22)
The compound was obtained as a yellow solid in 59% yield after purification by
silica gel column chromatography eluted with hexane - ethyl acetate (3:1 v/v). TLC Rf =
−
479
480
481
482
0.75 hexane - ethyl acetate (1:1 v/v). mp 74.3 - 75.4 ºC. IR (ATR, cm-1)
: 3291,
ν
max
2976, 2934, 2848, 1666, 1612, 1578, 1531, 1395, 1334, 1258, 1167, 1116, 1065, 926,
899, 829, 760, 696. 1H NMR (300 MHz, CDCl3)
δ
: 0.96 (t, 6H, J = 7.1 Hz), 3.00 (q, 4H,
J = 7.1 Hz), 7.31 (d, 1H, J = 8.4 Hz), 7.38 - 7.40 (m, 2H), 8.25 (dd, 1H, J = 7.7 Hz and J
19

ACCEPTED MANUSCRIPT
483
484
485
486
487
488
489
= 2.0 Hz), 8.53 (dd, 1H, J = 4.7 Hz and J = 2.0 Hz), 8.92 (brs, 1H), 10.02 (brs, 1H). 13C
NMR (75 MHz, CDCl3) δ: 12.5, 49.1, 116.7 (brs), 121.3 (q, JC-F = 3.8 Hz), 123.2, 123.9,
124.2 (q, JC-F = 270.8 Hz), 131.5, 136.4, 140.5, 147.0, 151.6, 162.7. Signal for the
carbon attached to CF3 was of low intensity and it is not observed. The signal for the
carbon attached to the chlorine as well as the signal for the aromatic carbon attached
to the –N(Et)2 presented the same chemical shift.
C17H18ClF3N3O, 372.1090; found: 372.1016.
HRMS (M+H+): Calculated for
490
491
492
4.1.4.10 2-chloro-N-(2-morpholino-5-(trifluoromethyl)phenyl)nicotinamide (23)
The compound was obtained as a yellow solid in 91% yield after purification by
column chromatography eluted with hexane-ethyl acetate (3:1 v/v). TLC Rf = 0.43
−
493
494
495
496
497
498
499
500
hexane - ethyl acetate (1:1 v/v). mp 131.2 - 133.2 ºC. IR (ATR, cm-1)
: 3258, 2924,
ν
max
2890, 2844, 1665, 1616, 1581, 1539, 1489, 1440, 1400, 1329, 1268, 1108, 923, 895,
828, 807, 754, 648. 1H NMR (300 MHz, CDCl3)
δ
: 2.94 (t, 4H, J = 4.5 Hz), 3.87 (t, 4H, J
= 4.5 Hz), 7.36 (d, 1H, J = 8.4 Hz), 7.42-7.48 (m, 2H), 8.28 (dd, 1H, J = 7.8 Hz and J =
1.8 Hz), 8.55 (dd, 1H, J = 4.8 Hz and J = 1.8 Hz), 8.92 (brs, 1H), 9,82 (brs, 1H). 13C
NMR (75 MHz, CDCl3)
δ: 52.9, 67.3, 117.3 (q, JC-F = 3.8 Hz), 121.8, 123.4, 124.1 (q, JC-
= 270.5 Hz), 128.5 (q, JC-F = 32.6 Hz), 131.2, 134.1, 140.9, 144.3, 146.6, 151.8,
F
162.7. HRMS (M+H+): Calculated for C17H16ClF3N3O2, 386.0883; found: 386.0842.
501
502
503
4.1.4.11 N-(2-(4-bromophenylamino)-5-(trifluoromethyl)phenyl)-2-chloronicotinamide (24)
The compound was obtained as a yellow solid in 37% yield after recrystallization
with acetone. TLC Rf = 0.58 (hexane - ethyl acetate 1:1 v/v). mp 175.0 - 176.0 ºC. IR
−
504
505
506
507
508
(ATR, cm-1)
: 3404, 3217, 3048, 1644, 1592, 1529, 1489, 1401, 1334, 1098, 1073,
ν
max
1
882, 808, 751. H NMR (300 MHz, CDCl3)
δ
: 6,10 (brs, 1H), 6.81 (d, 2H, J = 8.7 Hz),
7.34 - 7.45 (m, 5H), 8.11 (s, 1H), 8.16 (dd, 1H, J = 7.8 Hz and J = 1.8 Hz), 8.49 (dd,
1H, J = 4.7 Hz and J = 2.0 Hz), 8.66 (brs, 1H). 13C NMR (75 MHz, CDCl3)
δ
: 114.5,
120.0, 121.3, 121.7 (q, JC-F = 3.8 Hz), 123.2, 124.2 (q, JC-F = 3.8 Hz), 125.6 (q, JC-F
=
20

ACCEPTED MANUSCRIPT
509
510
511
512
32.8 Hz), 129.0, 130.5, 132.7, 138.9, 140.4, 141.9, 147.1, 151.9, 163.6. HRMS (M+H+):
Calculated for C19H13BrClF3N3O, 469.9883; found: 469.9707.
513
514
515
4.1.4.12 N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)nicotinamide (25)
The compound was obtained as a white solid in 65% yield after purification by
silica gel column chromatography eluted with hexane-ethyl acetate (1:1 v/v). TLC Rf =
−
516
517
518
519
520
521
522
523
524
525
526
527
528
0.45 (hexane - ethyl acetate 1:1 v/v). mp 129.8 - 130.3 ºC. IR (ATR, cm-1)
: 3332,
ν
max
2940, 2856, 2811, 1664, 1588, 1529, 1467, 1435, 1332, 1243, 1163, 1105, 1023, 893,
834, 729, 703, 645, 584. 1H NMR (300 MHz, CDCl3)
δ
: 1.64 - 1.65 (m, 2H), 1.75 - 1.82
(m, 4H), 2.88 (t, 4H, J = 4.8 Hz), 7.28 (d, 1H, J = 8.4 Hz), 7.37 (dd, 1H, J = 8.4 Hz and
J = 1.8 Hz), 7.50 (ddd, 1H, J = 7.8 Hz, J = 4.8 Hz and J = 0.8 Hz), 8.30 (dt, 1H, J = 7.8
Hz and J = 1.8 Hz), 8.80 (dd, 1H, J = 4.8 Hz and J = 1.8 Hz), 8.84 (brs, 1H), 9.15 (d,
1H, J = 1.8 Hz), 9.55 (brs, 1H).13C NMR (75 MHz, CDCl3)
δ: 23.9, 27.0, 53.9, 116.7
(brs), 121.0, 121.4 (q, JC-F = 3.8 Hz), 124.1, 124.2 (q, JC-F = 270.6 Hz), 127.6 (q, JC-F
=
33.4 Hz), 130.6, 133.6, 135.6, 145.8, 147.7, 152.8, 163.2. HRMS (M+H+): Calculated
for C18H19F3N3O, 350.1480; found: 350.1396.
4.1.4.13 N-(2-(cyclohexylamino)-5-(trifluoromethyl)phenyl)nicotinamide (26)
The compound was obtained as a white solid in 53% yield after purification by
silica gel column chromatography eluted with hexane-ethyl acetate (1:1 v/v). TLC Rf =
−
529
530
531
532
533
534
0.38 (hexane - ethylacetate 1:1 v/v). mp 137.0 - 138.4 ºC. IR (ATR, cm-1)
: 3434,
ν
max
3234, 3046, 2930, 2852, 1643, 1615, 1591, 1532, 1456, 1331, 1105, 883, 813, 712,
636. 1H NMR (300 MHz, CDCl3)
δ
: 1.12 - 2.03 (m, 10H), 3.28 - 3.29 (m, 1H), 4.19 (brs,
1H), 6.79 (d, 1H, J = 8.4 Hz), 7.36-7.46 (m, 2H), 7.52 (brs, 1H), 8.19 - 8.25 (m, 2H),
8.72 (d, 1H, J = 4.2 Hz), 9.08 (brs, 1H). 13C NMR (75 MHz, CDCl3)
δ
: 24.7, 25.6, 32.9,
51.6, 112.5, 118.5 (q, JC-F= 32.6 Hz), 124.4 (q, J = 269.1 Hz), 122.0, 123.7, 125.1,
21

ACCEPTED MANUSCRIPT
535
536
129.6, 135.7, 144.6, 147.9, 152.6, 164.5. HRMS (M+H+): Calculated for C19H21F3N3O,
364.1638; found: 364.1549.
537
538
539
540
541
4.1.4.14 N-(2-(pyrrolidin-1-yl)-5-(trifluoromethyl)phenyl)nicotinamide (27)
The compound was obtained as a white solid in 73% yield after recrystallization
with ethyl acetate. TLC Rf = 0.33 (hexane - ethyl acetate 1:2 v/v). mp 127.8 - 128.5 ºC.
−
542
543
544
545
546
547
548
549
550
IR (ATR, cm-1)
: 3289, 3056, 2972, 2870, 2842, 1644, 1615, 1592, 1530, 1372,
ν
max
1
1332, 1266, 1249, 1152, 1109, 1082, 1024, 932, 897, 875, 827, 707, 656. H NMR
(300 MHz, CDCl3) δ: 1.96 - 2.01 (m, 4H), 3.20 (t, 4H, J = 6.2 Hz ), 7.14 (d, 1H, J = 8.4
Hz), 7.35 - 7.38 (m, 1H), 7.48 (dd, 1H, J = 7.8 Hz and 4.8 Hz), 8.26 (dt,1H, J = 7.8 Hz
and J = 1.8 Hz), 8.47 (brs, 1H), 8.79 (dd, 1H, J = 4.8 Hz and J = 1.8 Hz), 8.82 (brs,
1H), 9.10 (d,1H, J = 1.8 Hz). 13C NMR (75 MHz, CDCl3)
δ: 24.9, 52.2, 118.8, 119.6
(brs), 122.5 (q, JC-F = 3.6 Hz), 124.4 (q, JC-F = 270.2 Hz), 124.9 (q, JC-F = 32.6 Hz),
124.1, 130.5, 130.6, 135.6, 144.8, 147.8, 152.9, 163.6. HRMS (M+H+): Calculated for
C17H17F3N3O, 336.1324; found: 336.1201.
551
552
553
4.1.4.15 N-(2-(diethylamino)-5-(trifluoromethyl)phenyl)nicotinamide (28)
The compound was obtained as a white solid in 87% yield after purification by
silica gel column chromatography eluted with hexane - ethyl acetate (1:1 v/v). TLC Rf =
−
554
555
556
557
558
559
0.50 (hexane - ethyl acetate 1:1 v/v). mp 64.8 - 66.8 ºC. IR (ATR, cm-1)
: 3334,
ν
max
2975, 2932, 2854, 1678, 1614, 1586, 1534, 1483, 1440, 1336, 1247, 1167, 1150, 1114,
1062, 1021, 923, 898, 828, 716, 567. 1H NMR (300 MHz, CDCl3)
δ
: 0.99 (t, 6H, J = 7.2
Hz), 3.05 (q, 4H, J = 7.2 Hz), 7.32 (d, 1H, J = 8.1 Hz), 7.39 (dd, 1H, J = 8.1 Hz and J =
1.5 Hz), 7.49 (dd, 1H, J = 8.3 Hz and J = 4.7 Hz), 8.27 (dt, 1H, J = 8.3 Hz and J = 1.8
Hz), 8.80 (dd, 1H, J = 4.8 Hz and J = 1.8 Hz), 8.92 (d, 1H, J = 1.5 Hz), 9.12 (d, 1H, J =
22

ACCEPTED MANUSCRIPT
560
561
562
563
564
1.8 Hz), 9.90 (brs, 1H). 13C NMR (75 MHz, CDCl3)
δ: 13.0, 49.5, 116.3 (q, JC-F = 3.9
Hz), 121.0 (q, JC-F = 3.9 Hz), 123.6, 124.1, 124.2 (q, JC-F = 270.5 Hz), 128.3 (q, JC-F
=
32.3 Hz), 130.6, 135.6, 136.5, 142.6, 147.8, 152.9, 163.2. HRMS (M+H+): Calculated
for C17H19F3N3O, 338.1480; found: 338.1399.
565
566
567
4.1.4.16 N-(2-morpholino-5-(trifluoromethyl)phenyl)nicotinamide (29)
The compound was obtained as a white solid in 74% yield after purification by
silica gel column chromatography eluted with hexane - ethyl acetate (1:1 v/v). TLC Rf =
−
568
569
570
571
572
573
574
575
576
0.18 (hexane - ethyl acetate 1:1 v/v). 157.5 - 159.0 mp ºC. IR (ATR, cm-1)
: 3344,
ν
max
2970, 2846, 1674, 1588, 1534, 1469, 1441, 1339, 1247, 1198, 1156, 1114, 1022, 936,
1
918, 897, 880, 833, 734, 707, 661. H NMR (300 MHz, CDCl3)
δ
: 2.95 (t, 4H, J = 4.5
Hz), 3.91 (t, 4H, J = 4.5 Hz), 7.33 (d, 1H, J = 8.1 Hz), 7.43 (dd, 1H, 8.4 Hz and J = 2.1
Hz), 7.51 (dd, 1H, J = 7.8 Hz and J = 4.8 Hz), 8.28 (td, 1H, J = 7.8 Hz and 1.8 Hz), 8.81
- 8.88 (m, 2H), 9,13 (brs, 1H), 9.45 (brs, 1H). 13C NMR (75 MHz, CDCl3)
δ: 52.6, 67.7,
117.0 (q, JC-F = 3.9 Hz), 121.3, 121.6 (q, JC-F = 3.7 Hz), 124.1 (q, JC-F = 270.3 Hz),
124.2, 128.4 (q, JC-F = 32.5 Hz), 130.4, 133.7, 135.5, 144.1, 147.6, 153.1, 163,1.
HRMS (M+H+): Calculated for C17H17F3N3O2 352.1273; found: 352.1201.
577
578
579
4.1.4.17 N-(2-(4-bromophenylamino)-5-(trifluoromethyl)phenyl)nicotinamide (30)
The compound was obtained as a white solid in 30% yield after recrystallization
with acetone. TLC Rf = 0.38 (hexane - ethyl acetate 1:1 v/v). mp 166.7 - 167.2 ºC. IR
−
580
581
582
583
584
585
(ATR, cm-1)
: 3314, 3188, 3068, 1663, 1621, 1592, 1514, 1440, 1337, 1250, 1162,
ν
max
1
1114, 1074, 1025, 1008, 887, 808, 709. H NMR (300 MHz, CDCl3)
δ
: 6.34 (brs, 1H),
6.82 (d, 2H, J = 8.7 Hz), 7.33 - 7.46 (m, 5H), 8.08 – 8.12 (m, 2H), 8.72 (dd, 1H, J = 4.8
Hz and J = 1.5 Hz), 8.96 (d, 1H, J = 1.5 Hz), 8.53 (brs, 1H). 13C NMR (75 MHz, CDCl3)
δ
: 114.4, 120.0, 121.3, 121.6 (q, JC-F = 3.6 Hz), 123.9 (q, JC-F = 3.7 Hz), 124.1, 124.1 (q,
JC-F = 270.0 Hz), 125.5 (q, JC-F = 33.4 Hz), 129.2, 129.7, 132.7, 135.8, 138.9, 141.9,
23

ACCEPTED MANUSCRIPT
586
587
148.0, 152.9, 164.5. HRMS (M+H+): Calculated for C19H14BrF3N3O, 436.0272; found:
436.0200.
588
589
590
4.1.4.18 N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)benzamide (31)
The compound was obtained as a white solid in 88% yield after purification by
silica gel column chromatography eluted with hexane-ethyl acetate (5:1 v/v). TLC Rf =
−
591
592
593
594
595
596
597
598
0.55 (hexane - ethyl acetate 5:1 v/v). mp 116.0 - 117.0 ºC. IR (ATR, cm-1)
: 3338,
ν
max
2932, 2846, 1675, 1588, 1530, 1472, 1439, 1378, 1337, 1272, 1240, 1162, 1116, 1026,
1
932, 913, 902, 877, 828, 796, 697, 648. H NMR (300 MHz, CDCl3)
δ
: 1.64 - 1.66 (m,
2H), 1.74 - 1.82 (m, 4H), 2.88 (t, 4H, J = 5.1 Hz), 7.26 (d, 1H, J = 8.1 Hz), 7.35 (d, 1H,
J = 8.1 Hz), 7.51-7.62 (m, 3H), 7.94-7.96 (m, 2H), 8.91 (brs, 1H), 9.45 (brs, 1H).13C
NMR (75 MHz, CDCl3) δ: 24.1, 27.1, 53.7, 116.5 (q, JC-F = 3.8 Hz), 120.8, 124.4 (q, JC-F
= 269.9 Hz), 127.1, 129.2, 132.2, 134.0, 134.8, 145.8, 165.1. HRMS (M+H+):
Calculated for C19H20F3N2O, 349.1528; found: 349.1451.
599
600
601
4.1.4.19 N-(2-(cyclohexylamino)-5-(trifluoromethyl)phenyl)benzamide (32)
The compound was obtained as a white solid in 55% yield after purification by
column chromatography eluted with hexane-ethyl acetate (5:1 v/v). TLC Rf = 0.43
−
602
603
604
605
606
607
608
609
610
(hexane-ethyl acetate 5:1 v/v). mp 157.5-158.8 ºC. IR (ATR, cm-1)
:3396, 3214, 3058,
ν
max
2935, 2862, 1636, 1613, 1552, 1334, 1243, 1213, 1161, 1105, 1073, 880, 812, 707,
1
624. H NMR (300 MHz, CDCl3)
δ
: 1.13-2.04 (m, 10H), 3.27-3.33 (m, 1H), 4.10-4.22
(brs, 1H), 6.79 (d, 1H, J = 8.7 Hz), 7.38 (dd, 1H, J = 8.6 Hz andJ = 1.4 Hz), 7.46-7.60
(m, 4H), 7.76 (brs, 1H), 7.89 (d, 2H, J = 7.5 Hz). 13C NMR (75 MHz, CDCl3)
δ
: 25.0,
25.9, 33.2, 51.8, 112.7, 118.3 (q, JC-F= 32.7 Hz), 122.9, 125.1 (q, JC-F = 3.6 Hz), 127.5,
129.0, 132.5, 133.9, 145.0, 166.5. The signal of the carbono of the CF3 group was of
low intensity and it was not noticed in the spectrum. HRMS (M+H+): Calculated for
C20H22F3N2O, 363.1684; found: 363.1613.
24

ACCEPTED MANUSCRIPT
611
612
613
4.1.4.20 N-(2-(pyrrolidin-1-yl)-5-(trifluoromethyl)phenyl)benzamide (33)
The compound was obtained in 80% yield as a white solid after purification by
silica gel column chromatography eluted with hexane - ethyl acetate (5:1 v/v). TLC Rf =
−
o
614
615
616
617
618
619
620
621
0.30 (hexane-ethyl acetate 5:1 v/v). mp 122.2 - 122.8 C.IR (ATR, cm-1)
: 3242,
ν
max
2986, 2948, 2870, 1637, 1616, 1578, 1519, 1488, 1366, 1331, 1266, 1149, 1098, 1082,
1
874, 799, 695, 656. H NMR (300 MHz, CDCl3)
δ
: 1.96 - 2.01 (m, 4H), 3.13 - 3.17 (t,
4H, J = 6.3 Hz), 7.15 (d, 1H, J = 8.4 Hz), 7.34 (dd, 1H, J = 8.4 Hz and J = 1.5 Hz), 7.49
13
- 7.61 (m, 3H), 7.89 - 7.91 (m, 2H), 8.51 (brs,1H), 8.70 (brs, 1H). C NMR (75 MHz,
CDCl3) δ: 24.9, 52.0, 118.5, 119.6 (q, JC-F = 3.8 Hz), 122.0 (q, JC-F = 3.9 Hz), 124.5 (q,
JC-F = 269.9 Hz), 124.8 (q, JC-F = 32.5 Hz), 127.2, 129.2, 131.1, 132.2, 134.7, 144.6,
165.3. HRMS (M+H+): Calculated for C18H18F3N2O, 335.1371; found: 335.1277.
622
623
624
4.1.4.21 N-(2-(diethylamino)-5-(trifluoromethyl)phenyl)benzamide (34)
The compound was obtained in 84% yield as a white solid after purification by
silica gel column chromatography eluted with hexane-ethyl acetate (5:1 v/v). TLC Rf =
−
625
626
627
628
629
630
631
632
0.65 (hexane - ethyl acetate 5:1 v/v). mp 65.1 - 66.0 ºC. IR (ATR, cm-1)
: 3334, 2975,
ν
max
2932, 2858, 1678, 1586, 1534, 1483, 1440, 1336, 1247, 1167, 1150, 923, 828, 716. 1H
NMR (300 MHz, CDCl3) : 1.00 (t, 6H, J = 7.2 Hz), 3.03 (q, 4H, J = 7.2 Hz), 7.30 (d, 1H,
J = 8.4 Hz), 7,36 (dd, 1H, J = 8.4 Hz and J = 1.5 Hz), 7.50 - 7.61(m, 3H), 7.92 (dd, 2H,
δ
J = 8.1 Hz and J = 1.5 Hz), 8.97 (brs, 1H), 9.81 (brs, 1H). 13C NMR (75 MHz, CDCl3)
13.0, 49.3, 116.2 (q, JC-F = 3.9 Hz), 120.5 (q, JC-F = 3.8 Hz), 123.4, 124.3 (q, JC-F
δ:
=
270.7 Hz), 128.1 (q, JC-F = 32.3 Hz), 127.2, 129.1, 132.2, 134.9, 136.9, 142.5, 165.1.
HRMS (M+H+): Calculated for C18H20F3N2O, 337.1528; found: 337.1449.
633
634
635
4.1.4.22 N-(2-morpholino-5-(trifluoromethyl)phenyl)benzamide (35)
The compound was obtained as a white solid in 78% yield after recrystallization
with acetone. TLC Rf = 0.18 (hexane-acetate 5:1 v/v). mp 137.3 - 138.5 ºC. IR (ATR,
−
636
cm-1)
: 3369, 2967, 2896, 2851, 1668, 1589, 1534, 1465, 1438, 1335, 1238, 1157,
ν
max
25

ACCEPTED MANUSCRIPT
637
638
639
640
641
642
643
1112, 1075, 1025, 937, 917, 897, 877, 821, 801, 707, 659. 1H NMR (300 MHz, CDCl3)
δ
: 2.96 (t, 4H, J = 4.5 Hz), 3.92 (t, 4H, J = 4.5 Hz), 7.30 (d, 1H, J = 8.4 Hz), 7.38 (dd,
1H, J = 8.4 Hz and J = 1.5 Hz), 7.52 - 7.63 (m, 3H), 7.93 (dd, 2H, J = 8.1 Hz and J =
1.5 Hz), 8.91 (brs, 1H), 9.39 (brs, 1H). 13C NMR (75 MHz, CDCl3)
δ
: 52.5, 67.8, 116.9
(q, JC-F = 3.8 Hz), 121.0, 121.1 (q, JC-F = 3.9 Hz), 124.2 (q, JC-F = 270.5 Hz), 127.0,
128,2 (q, JC-F = 32.5 Hz), 129.2, 132.5, 134.1, 134.6, 144.0, 165.0. HRMS (M+H+):
Calculated for C18H18F3N2O2, 351.1320; found: 351.1266.
644
645
4.1.4.22 N-(2-(4-bromophenylamino)-5-(trifluoromethyl)phenyl)benzamide (36)
The compound was obtained as a white solid in 58% yield. TLC Rf = 0.25
−
646
647
648
649
650
651
652
653
(hexane - ethyl acetate 5:1 v/v). mp 157.3 - 158.0 ºC. IR (ATR, cm-1)
: 3366, 2967,
ν
max
2892, 2852, 1668, 1589, 1534, 1465, 1438, 1335, 1238, 1157, 1112, 937, 917, 897,
877, 821, 802, 707, 659. 1H NMR (300 MHz, CDCl3)
δ
: 6.30 (brs, 1H), 6.83 (d, 2H, J =
8.7 Hz), 7.31 - 7.47 (m, 6H), 7.55 (t, J = 7.4 Hz, 1H), 7.75 (d, 2H, J = 8.7 Hz), 8,05 (brs,
13
1H), 8.22 (brs, 1H). C NMR (75 MHz, CDCl3) δ: 113.9, 119.7, 120.9, 121.1 (q, JC-F =
3.9 Hz), 123.3 (q, JC-F = 3.7 Hz), 123.9 (q, JC-F = 270.1 Hz), 125.1 (q, JC-F = 33.0 Hz),
127.1, 128.9, 129.3, 132.4, 133.3, 138.6, 141.8, 166.3. HRMS (M+H+): Calculated for
C20H15BrF3N2O, 435.0320; found: 435.0300.
654
655
656
657
658
659
660
661
662
4.2 Biological assays
4.2.1 Cell culture
Human leukemia cell lines HL60 (acute myelogenous leukemia - AML), Nalm6
(B-cell acute lymphoblastic leukemia – ALL-B), and Jurkat (T-cell acute lymphoblastic
leukemia - ALL-T) were kindly provided by Dr. Jose Andrés Yunes (Centro Infantil
Boldrini, Campinas, São Paulo, Brazil). Cell lines were grown in RPMI-1640 medium
(Sigma) supplemented with 10% (v/v) fetal bovine serum (FBS) (LGC Biotecnologia),
100 g/mL streptomycin, and 100 units/mL penicillin (Sigma) at pH 7.2 and 37 ºC under
5% CO2 atmosphere. Peripheral blood mononuclear cells (PBMC) were isolated from
26

ACCEPTED MANUSCRIPT
663
664
665
666
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human-heparinized blood using Histopaque-1077 (Sigma) according to the
manufacturer’s protocol. The isolated lymphocytes were resuspended in complete
RPMI-1640 medium supplemented with 10% FBS and stimulated with 1% (v/v)
phytohemagglutinin (Gibco). The cells were counted using a Neubauer chamber for the
following experiments.
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4.2.2 Cell viability assay
HL60, Nalm6, and Jurkat cells (7x104 cells/well) and PBMC (1x105 cells/well)
were seeded in 96-well plates. Each well contained 100 µL of complete RPMI medium
and 100 µL of each compound solution at different concentration. The compounds
were diluted in RPMI medium with 10% FBS and 0.4% DMSO (v/v, Sigma). After 48 h
of culture, MTT (5 mg/mL, Sigma) was added to the wells. After 3 h at 37 ºC, the MTT
solution was removed and it was added 100 µL/well of DMSO to solubilize the
formazan. Absorbance was measured at 540 nm in a microplate reader (SpectraMax
M5, Molecular Devices).
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4.2.3 Drug combination studies
Cell viability of leukemia cells treated with a combination of compounds 24, 30, or
36 with vincristine was assessed by seeding 7x104 Nalm6 cells in each well of a 96-
well plate. The cells were then incubated with each compound (at concentrations
corresponding to 25 and 50% of the IC50), vincristine (0.5 or 1.0 nM, Sigma) or a
combination of each compound and vincristine for 48 h. The cell viability was
determined by MTT assay and CompuSyn software was used to calculate the
combination index (CI) as previously described [26].
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4.2.4 Apoptosis assay by flow citometry
Nalm6 cells were seeded on 96-well plate at density of 7x104 cells per well and
treated with compounds 24, 30 and 36 [20 µM]. DMSO (0.4% v/v) was used as vehicle
control. After treatments, cells were labeled by using Annexin V/FITC apoptosis
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detection kit I (BD Biosciences) according to manufacturer’s protocol. Then the cell
samples were analyzed by flow cytometry (FACS Verse, BD Bioscience).
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4.2.5 Autophagy detection with acridine orange staining
Nalm6 cells were seeded on 96-well plate at density of 7x104 cells per well and
treated with compounds 24, 30 and 36 [20 µM] or DMSO (0.4% v/v). After, cells were
washed with phosphate-buffered saline (PBS), suspended in PBS and stained by
acridine orange (1 µM, Sigma) at 37 ºC for 15 min; then the cells were washed with
PBS and resuspended in 0.5 mL of PBS. For visual examination of autophagosomes,
cells were analyzed under a fluorescence microscope Evos FL (Life technologies).
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4.2.6 Cell proliferation assay
Proliferation assays were performed in 96-well plates containing 1x104 Nalm6
cells per well or 1.5x104 HL60 cells per well. The compounds 24, 30, and 36 were
added at 20 µM and DMSO (0.4% v/v) were used as control. The effect of each
treatments on cell growth were determined by trypan blue (Invitrogen) dye exclusion.
After 24, 48, 72, and 96 h cells were loaded on a hemocytometer to obtain the viable
cell count.
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4.2.7 RT-PCR assay
Nalm6 cells were exposed to 20 µM of compounds 24, 30, and 36 or SRPIN340
for 24 h. Cells treated with DMSO (0.4% v/v) were used as control. After incubation,
mRNA was extracted using Tri Reagent (Sigma) according to the manufacturer’s
protocol. Samples were quantified by spectrophotometry (NanoDrop, Thermo
Scientific) and analyzed for integrity in 1% agarose gel. Afterwards, the RNA was used
for first-strand cDNA synthesis using the Super Script First-Strand kit (Invitrogen)
according to the manufacturer’s protocol. Then, the cDNA was used to amplify each
fragment of interest by PCR using the GoTaq Green Master Mix (Promega) kit, and the
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