Synthesis and biological evaluation of 2,4,6-trinitroaniline derivatives as potent antitumor agents

Article abstract of DOI:10.1007/s00706-020-02690-7

Abstract: Nitro group-containing compounds are well known as effective anticancer drugs. The aim of the study is to synthesize a series of trinitroaniline derivatives to determine their potential antitumor activities on diverse cancer cell models, anti-apoptotic and anti-metastatic features on hepatoma cells. The anti-proliferative studies show that IC₅₀ values of N-phenyl-2,4,6-trinitroaniline, N-(2,4,6-trinitrophenyl)naphthalen-1-amine, N-(2,4,6-trinitrophenyl)naphthalen-2-amine, N-(3-nitrophenyl)-2,4,6-trinitroaniline were similar to IC₅₀ value of cisplatin in Hep3B cells. In fact, IC₅₀ value of N-(3,5-difluorophenyl)-2,4,6-trinitroaniline is better than cisplatin. In addition, all compounds could decrease the expression of the cell cycle checkpoint protein cyclin D1. To investigate the effect of compounds on the apoptotic pathway, mRNA and protein expressions of Bcl-2 and Bax were analyzed with qRT-PCR and Western blot. Annexin V staining assay, apoptotic mRNA and protein analysis indicate that N-isopropyl-2,4,6-trinitroaniline, N-(2,4,6-trinitrophenyl)-5-methylisoxazole-3-amine, N-(3-nitrophenyl)-2,4,6-trinitroaniline, N-(4-nitrophenyl)-2,4,6-trinitroaniline induce intrinsic apoptosis by increasing the ratio of Bax/Bcl-2 expression. In addition, colony formation and wound healing assays confirmed that these compounds also inhibit the metastatic activity of Hep3B cells. 2,4,6-Trinitroaniline derivatives, especially N-(3-nitrophenyl)-2,4,6-trinitroaniline might be used as candidate for the development of new antitumor drugs. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-020-02690-7

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-020-02690-7
ORIGINAL PAPER
Synthesis and biological evaluation of 2,4,6‑trinitroaniline derivatives
as potent antitumor agents
Nelin Hacıoğlu1 · Tuğba Güngör2 · Esra Tokay1 · Ferah Cömert Önder2,3 · Mehmet Ay2 · Feray Köçkar1
Received: 7 July 2020 / Accepted: 14 September 2020
© Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract
Nitro group-containing compounds are well known as efective anticancer drugs. The aim of the study is to synthesize a series
of trinitroaniline derivatives to determine their potential antitumor activities on diverse cancer cell models, anti-apoptotic
and anti-metastatic features on hepatoma cells. The anti-proliferative studies show that IC50 values of N-phenyl-2,4,6-trini-
troaniline, N-(2,4,6-trinitrophenyl)naphthalen-1-amine, N-(2,4,6-trinitrophenyl)naphthalen-2-amine, N-(3-nitrophenyl)-2,4,6-
trinitroaniline were similar to IC50 value of cisplatin in Hep3B cells. In fact, IC50 value of N-(3,5-difuorophenyl)-2,4,6-
trinitroaniline is better than cisplatin. In addition, all compounds could decrease the expression of the cell cycle checkpoint
protein cyclin D1. To investigate the efect of compounds on the apoptotic pathway, mRNA and protein expressions of Bcl-2
and Bax were analyzed with qRT-PCR and Western blot. Annexin V staining assay, apoptotic mRNA and protein analysis
indicate that N-isopropyl-2,4,6-trinitroaniline, N-(2,4,6-trinitrophenyl)-5-methylisoxazole-3-amine, N-(3-nitrophenyl)-2,4,6-
trinitroaniline, N-(4-nitrophenyl)-2,4,6-trinitroaniline induce intrinsic apoptosis by increasing the ratio of Bax/Bcl-2 expres-
sion. In addition, colony formation and wound healing assays confrmed that these compounds also inhibit the metastatic
activity of Hep3B cells. 2,4,6-Trinitroaniline derivatives, especially N-(3-nitrophenyl)-2,4,6-trinitroaniline might be used
as candidate for the development of new antitumor drugs.
Graphic abstract
Keywords Cytotoxicity · Trinitroaniline · Synthesis · Nitro compound · Apoptosis · Hepatocellular carcinoma
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00706-020-02690-7) contains
supplementary material, which is available to authorized users.
3
* Feray Köçkar
Department of Medical Biology, Faculty of Medicine,
Canakkale Onsekiz Mart University, Canakkale 17020,
Turkey
fkockar@balikesir.edu.tr; feraykockar@hotmail.com
1
Department of Molecular Biology and Genetics, Faculty
of Sciences and Arts, Balıkesir University, Balıkesir 10145,
Turkey
2
Natural Products and Drug Research Laboratory, Department
of Chemistry, Faculty of Sciences and Arts, Çanakkale
Onsekiz Mart University, Çanakkale 17020, Turkey
Vol.:(0123456789)
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N. Hacıoğlu et al.
cancer cell lines that have different properties: Hep3B
(Human Hepatoma Cell line), PC3 (Human Prostate Can-
cer Cell line), HT-29 (Human Colorectal Adenocarcinoma
Cell line), and Saos-2 (Human Osteosarcoma Cell line)
along with healthy model HUVEC (Human Umbilical Vein
Endothelial Cell). The anti-proliferative studies are supple-
mented with their plausible mechanisms of action of cell
cycle arrest, attended by cell death via apoptosis discovered
as a major treatment result. Also, to clarify their efective-
ness in suppressing hepatoma cells, colony formation and
wound healing assays were performed.
Introduction
Cancer is a disease that has priority in the feld of medicinal
chemistry research, which is a primary concern worldwide
[1, 2]. Despite the signifcant studies and eforts to fnd can-
cer chemotherapeutic agents, toxicity and lack of sensitivity
have been determined as signifcant barriers. Consequently,
there is a remaining requirement to develop new and more
efective anticancer agents that can accomplish the problems
with currently available agents [3].
Nitro functional group-containing aromatic compounds
are a very important class of applied chemistry due to their
applications such as dyes, solvents, chemical intermediates,
polymers, pesticides, and explosives [4, 5]. They are also
used for a great number of therapeutic agents including
Parkinson’s disease, insomnia, infammatory, tranquilizers,
fungicides, insecticides, herbicides, parasitic infections, and
some type of cancer [6–9]. Chloramphenicol, nitrazepam,
metronidazole, nimesulide, pyrrolnitrin, niridazole, para-
thion, and azathioprine are some of the nitro-containing aro-
matic drugs which show the pharmacological efects based
on electron-withdrawing nitro groups or the other phar-
macophore groups at the drug molecules [10]. Also, nitro
group-containing compounds, nilutamide and futamide are
well known as efective anticancer drugs in the literature. It
was emphasized that cyano containing-nilutamide deriva-
tive is a therapeutic agent for prostate cancer with its non-
steroidal anti-androgen properties [11]. In another study,
nitro-containing coumarin compound caused the death of
cells by apoptosis, especially in colon cancer models whose
KRAS gene was wild and mutant [12]. Indirubin-based
nitro-containing compounds have particularly cell plastic-
ity and anticancer efects [13]. This compound also acts on
cell plasticity by inhibiting glycogen synthase 3 beta and
contributes to cell metastasis via the aurora kinase enzyme
that regulates carcinogenesis [14]. Additionally, the tumo-
rigenic efects of some nitroanilines have been reported and
the carcinogenic potential of dinitroanilines based on nitro
and amino groups have been emphasized by the researchers
[15, 16]. It is noted that nitro groups of a drug molecule can
cause some side-efects including mutagenicity, hepatotox-
icity, carcinogenicity or bone-marrow suppression [6, 10].
However, in some cases, these risks could be negligible for
the treatment of various illnesses.
Results and discussion
Design, synthesis, and characterization
of compounds 1–12
Various trinitroaniline derivatives 1–12 were designed in
this study. The major reason for choosing these skeletons is
to fnd the most efective structure that will cause Hep3B,
PC3, Saos-2, and HT-29 cells death. The efects of aliphatic
(ethyl, isopropyl, 4-nitrobenzyl) and various functional
group-containing aromatic/heteroaromatic groups (phenyl,
1-naphthyl, 2-naphthyl, methylisoxazole, 3-nitrophenyl,
4-nitrophenyl, 3-methoxyphenyl, 3,5-difuorophenyl) bound
to (NO2)3PhNH- were investigated for toxicity on cancer
cells.
First, the starting material of our reactions, picryl chlo-
ride, was synthesized according to the literature procedure
[21] from phosphorus oxychloride (POCl3) and picric acid
with pyridine at 120–125 °C for 2.5 h in 91% yield. At the
second step, compounds 1–11 were obtained from picryl
chloride with various aliphatic, aromatic, and heterocyclic
amines at diferent reaction conditions via aromatic nucleo-
philic substitution reaction in moderate–good yields (Fig. 1).
Compound 12 was prepared in 90% yield from ethylenedi-
amine (1.0 eq.) and picryl chloride (2.0 eq.) at the conditions
that used Et3N as base in DMF solvent at room temperature
for 6 h.
To the best of our knowledge, compound 6 was deter-
mined as a new structure in the literature. Compounds 1
and 3 [21] were synthesized according to the literature
methods. We synthesized compounds 2 [22], 4 [23], 5
[23–25], 7 [26], 8 [27, 28], 12 [29] with more advanta-
geous diferent methods compared to the literature meth-
ods. Compound 2 was obtained from picryl chloride and
isopropyl amine at room temperature conditions in DMSO
unlike literature method that includes the substitution reac-
tion of 1-dialkylamino-2,4,6-trinitrobenzenes with various
amines in DMSO at room temperature [22]. Compounds 4
and 5 were synthesized by the reaction of picryl pyridin-
ium chloride and aromatic amines (1-naphthylamine and
Our interest is to synthesize a series of trinitroaniline
derivatives 1–12 to determine their potential antitumor
activities on diverse cancer cell models, anti-apoptotic and
anti-metastatic features on hepatoma cells. The drug potency
of these compounds has been discovered under the investi-
gation of prodrugs for nitroreductase-based cancer therapy
[17–19]. Specifcally, we provide detailed proof for the high
anti-cancer efect of such compounds using several diverse
1 3

Synthesis and biological evaluation of 2,4,6‑trinitroaniline derivatives as potent antitumor…
Fig. 1 Synthesis of trinitroaniline derivatives 1–12
2-naphthylamine) in aqueous media at heating conditions
according to the Okon’s study in 1958 [23] or substitution
reaction of picryl chloride and protected amines accord-
ing to LaBarbera’s study in 2013 [24] (for compound 4) or
nitration reaction of phenyl-2-naphthylamine according to
Ryan’s study in 1918 (for compound 5) [25]. In our concept,
we obtained compounds 4 and 5 as a result of the reaction
of picryl chloride and 1-naphthylamine/2-naphthylamine in
DMSO at room temperature for 24 h in high yields (88 and
80%, respectively). Compound 8 was synthesized using pic-
ryl chloride and 3-nitroaniline the same starting materials of
the literature examples [27, 28] at diferent reaction condi-
tions using pyridine as base, heating at 100 °C for 24 h in
85% yield. Compound 9 was obtained from picryl chloride
and 4-nitroaniline according to small modifcations at reac-
tion conditions of Dreikorn’s method [28, 29] and Shizen’s
method [22]. Compound 10 has no detailed references about
synthetic and spectral data unlike some application stud-
ies such as kinetic, electrochemical, or acidity to the best
of our knowledge. Compound 11 has only one reference
in 1974 that includes kinetic study, not detailed synthetic
methodology and spectral data [30]. As compound 12 was
synthesized by nitration reaction of 1,2-bis(phenylamino)-
ethane derivative according to literature [29], the substitu-
tion reaction of picryl chloride and ethylenediamine at one
step was applied successfully in this study. All trinitroaniline
products 1–12 were characterized by melting point, IR, 1H
NMR, 13C NMR, and MS analysis (see Experimental part
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N. Hacıoğlu et al.
▶
Fig. 2 Anti-proliferative efects of trinitroaniline derivatives 1–12
in Hep3B, PC3, HT-29 Saos-2, and HUVEC cell lines at fve dif-
ferent concentrations (150, 75, 38, 19, and 9 µM). DMSO was used
as a vehicle control and indicated as NT (not treated) in graphics.
Absorbance values were measured at 550 nm with spectrophotometer.
Experiments were done in triplicate for each cell type and each dose.
Values are average SD of three independent experiments
and Supplementary data). Also, the known products were
confrmed by comparison of their spectral data with those
reported in the literature.
The spectra and values of the compounds 1–12 are
given in the Experimental section and Supplementary data.
In summary, all compounds show medium N–H stretch-
ing and medium–strong N–H bending bands of aniline at
3250–3350 cm−1 and 1650–1600 cm−1 at their FT-IR spec-
tra. Medium aromatic C–H stretching bands of trinitroani-
lines are generally at 3000–3100 cm−1 frequency range.
Also, the N–O asymmetrical and symmetrical stretch-
ing absorptions of three nitro groups were observed at
1500–1550 cm−1 and 1300–1350 cm−1 as two strong bands,
respectively. Bands of other variable groups such as aliphatic
C–H, C–O–C of ether, C–F of halogen, N–O and C=N of
isoxazole were determined at expected regions of IR spec-
tra. In the 1H NMR spectrum, all broad singlet protons for
the N–H peak of anilines were observed at 8.46–10.37 ppm.
The characteristic single aromatic C–H signals given by
the two hydrogens in trinitroanilines are in a short range
(8.91–9.04 ppm) of NMR spectrum. Additionally, aromatic
and aliphatic carbon peaks at the 13C NMR spectrum con-
frm the structures of compounds 1–12.
determines the concentration of drugs needed to inhibit
50% of cell growth. Compounds 1, 2, 6, 7, 10, and 12 show
moderate toxicity for all cell lines, ranging from IC50: 41 to
150 μM (Table 1, Fig. 2). Other compounds (3–5, 8, 9, and
11) exhibit IC50 value lower than 20 μM cytotoxicity in at
least one cell line.
According to the experimental results of 2,4,6-trinitroani-
line derivatives (compound 1–12), it was concluded that
compounds 3–5, 8, and 11 show good cytotoxicity against
the Hep3B hepatoma cancer cell, IC50: 17.02 0.66 µM,
IC50: 18.85 1.43 µM, IC50: 23.37 0.90 µM, IC50:
16.23 1.18 µM, IC50: 9.51 0.92 µM, respectively
(Table 1, Fig. 2). It is noteworthy that non-substituted
aromatic rings such as phenyl, 1-naphthyl, 2-naphthyl at
compounds 3–5 and electron-withdrawing groups such as
3-nitrophenyl and 3,5-difuorophenyl at compounds 8 and
11 increase the cytotoxic activity on Hep3B cell. Other
compounds except for 4-nitrobenzyl derivative (com-
pound 7, IC50: 108.89 0.43 µM), were moderately toxic
(41.13–58.52 µM) on Hep3B cell (Table 1). When nitro-
phenyl group-bearing trinitroaniline derivatives (compounds
7–9) were compared, compound 8 that has 3-nitrophenyl
was toxic on Hep3B cell (IC50: 16.23 1.18 µM) and shows
moderately cytotoxicity on Saos-2 (IC50: 34.03 0.64 µM);
compound 9 that has 4-nitrophenyl was toxic on PC3 (IC50:
Cytotoxic efects of compounds 1–12 against fve
diferent cell lines
The in vitro cytotoxic activities of the 2,4,6-trinitroaniline
derivatives 1–12 were determined by MTT assay against
four human cancer cell lines: Hep3B, PC3, HT-29, Saos-2
and a healthy cell model, HUVEC. The results were stated
as IC50 (μM) values and, summarized in Table 1. IC50 value
Table 1 IC50 values of all
Comp
IC50/µM
Hep3B
compounds 1–12 against fve
human cell lines
PC3
HUVEC
Saos-2
HT-29
1
44.13 0.81
45.20 1.32
17.02 0.66
18.85 1.43
23.37 0.90
58.52 0.81
108.89 0.43
16.23 1.18
56.01 1.71
55.13 1.42
9.51 0.92
33.44 1.14
52.55 0.89
>150.00
48.15 0.63
89.94 1.53
>150.00
58.44 2.04
92.44 1.48
48.88 1.14
86.90 0.57
42.01 1.72
43.21 1.29
>150.00
34.03 0.64
81.11 0.91
>150.00
54.70 1.03
66.32 0.43
>150.00
84.42 1.63
123.41 1.14
72.18 0.67
>150.00
119.81 0.61
75.08 1.10
103.18 2.09
50.03 1.18
58.18 1.01
–
2
3
4
126.99 0.77
7.02 1.01
48.80 1.28
78.02 1.09
86.88 0.48
10.97 2.01
86.61 2.18
44.13 0.43
40.80 1.52
–
>150.00
5
19.84 2.03
57.41 1.74
72.43 0.99
34.41 1.41
78.21 1.03
>150.00
9.30 1.21
78.87 1.55
–
6
7
8
9
10
11
117.43 0.81
>150.00
–
12
41.13 0.68
15.39 0.23
Cisplatin
MTT assay was used following the 48 h of treatment. The values were determined from three independent
experiments. Cisplatin was used as a positive control
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Synthesis and biological evaluation of 2,4,6‑trinitroaniline derivatives as potent antitumor…
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N. Hacıoğlu et al.
Fig. 2 (continued)
16.23 1.18 µM) and 4-nitrobenzyl derivative (compound 7)
that has an extra -CH2 as compared to compound 9 was mod-
erately toxic on PC3 and HUVEC, IC50: 78.02 1.09 µM
and 72.43 0.99 µM, respectively. When compounds 8
and 10 were examined structurally, the only diference was
to have electron-withdrawing nitro or electron-donating
methoxy group at 3-position of benzene ring. The pres-
ence of electron donating methoxy group at compound 10
increased the IC50 values on Hep3B, Saos-2, and HUVEC
cells. On the other hand, N1,N2-bis(2,4,6-trinitrophenyl)
ethane-1,2-diamine (compound 12) was moderately toxic
(IC50: 40.80–78.87 µM) on cancer cells except for Saos-2
and the healthy cell. When the 5-methylisoxazole group
(compound 6) was introduced in the parent compound, IC50
values were found between 43.21 and 72.18 µM (Table 1).
Additionally, the aliphatic group (ethyl and isopropyl)-con-
taining compounds 1 and 2 showed moderately toxic efect
(33.44–92.44 µM) on all cell types. We also determined
that compound 5 that has 2-naphthyl group shows good
toxic efect (IC50: 7.02 1.01 µM) on PC3 and compound
11 that contains 3,5-difuorophenyl (IC50: 9.51 0.92 µM)
is the most toxic one among the evaluated compounds on
Hep3B. But these two compounds were toxic against the
HUVEC healthy cell model (IC50: 19.84 2.03 µM and
IC50: 9.30 1.21 µM, respectively), so these compounds
are eliminated to become a selective drug for cancer cells
(Fig. 5). Among the compounds 1–12, no compound was
observed that shows a good toxic efect on Saos-2 and HT-29
cancer cells.
Conclusively, it was determined that all trinitroaniline
derivatives 1–12 have a toxic efect on cancer cells, espe-
cially the Hep3B cell line (Fig. 5). Cisplatin was used as a
positive control for the comparison of the cytotoxicity of
compounds in Hep3B cells. IC50 values of compounds 3–5
and 8 were similar to IC50 of cisplatin in Hep3B cells. In
fact, IC50 of compound 11 is better than cisplatin that is
a conventional chemotherapy drug. The Hep3B cell line
was chosen as an experimental model and was used in all
molecular and cellular experiments.
Efect of the compounds on apoptosis in Hep3B cells
Most of the chemotherapeutic agents ultimately promote
apoptosis in cancer cells when they thereby exert antitumor
efects [31]. Therefore, to determine whether the compounds
can induce apoptosis, Hep3B cells were treated with the
IC50 concentration of compounds for 48 h and marked with
Annexin V/7-AAD. Analyses showed that all the tested com-
pounds result in the increased rate of early and late apoptosis
of Hep3B cells (Fig. 3a). However, compounds 9, 3, 2, 6,
1 3

Synthesis and biological evaluation of 2,4,6‑trinitroaniline derivatives as potent antitumor…
Fig. 3 a Detection of apoptosis by Annexin V/7-AAD staining.
Hep3B cells were collected and stained for Annexin V and 7-AAD.
The percentages of apoptotic cells that were labeled with early apop-
tosis (EA) and late apoptosis (LA). b Western blot of Cyclin D1 level
in Hep3B cells. c The mRNA levels of Bcl-2 and Bax were analyzed
by Real-time PCR. Fold mRNA expression of Bcl-2 and Bax in
Hep3B cells with or without compounds. d The levels of Bcl-2 and
Bax proteins for compounds 1–12 were analyzed by western blot.
Cells were dissociated in RIPA bufer and lysed. These lysates were
loaded onto the prepared polyacrylamide gel. The membranes were
visualized in the UVP system
8, 10, 11, and 5 result in high apoptotic cell rate with total
apoptotic % cells of 15.0, 14.1, 13.9, 13.5, 12.9, 12.6, 11.5,
and 10.7, respectively (Fig. 3a).
groups, respectively, inhibit Hep3B cells proliferation
(Fig. 5). Compounds 2, 3, and 9 can also down-regulate
the expression of cycle checkpoint proteins cyclin D1 in
addition to compounds 4, 7, and 8 containing 1-naphthyl,
4-nitrobenzyl, and 3-nitrophenyl groups, respectively. Here-
with, results showed that these compounds could stop the
cell cycle in the G0/G1 phase by inhibiting the expression
of cyclin D1 in the Hep3B cells (Fig. 3b).
Some nitro compounds were found to cause cell cycle
arrest with analysis. Thus, cyclin D1 protein level was ana-
lyzed by Western blot. As the results are indicated in Fig. 3b,
the levels of the cyclin D1 were signifcantly reduced by
some nitro compounds 2, 3, 4, 7, 8, and 9. In the light of
fow cytometry results, compounds 2, 3, 9, and 11 that have
isopropyl, phenyl, 4-nitrophenyl, and 3,5-difuorophenyl
All compounds lead to intrinsic apoptosis that is generally
associated with down-regulation of Bcl-2 (B-cell lymphoma
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N. Hacıoğlu et al.
Fig. 4 a Colony formation ability of nitro compounds in Hep3B
cells. There are representative images of colony formation analysis in
Hep3B cells. Quantitative analysis of colony numbers is shown. The
data are expressed as numbers relative to control cells and represent
three diferent experiments. b Efect of wound healing with com-
pound treatment from 3 to 24 h. A bar graph showing the percentage
of cells that migrated after 24 h following the scratch wound in cells
treated with nitro compounds
2) and up-regulation of Bax (Bcl-2-associated X protein)
gene expression level. To confrm the apoptosis induction
and to research the action mechanism of the compounds in
detail, qRT-PCR and Western blot analysis were performed.
For qRT-PCR analysis, total RNA was purifed, and reverse
transcribed from the cell pellet. Human β-2 microglobulin
gene was used for normalization. As shown in Fig. 3c, all
compounds led to a decreased expression of the anti-apop-
totic Bcl-2 mRNA and increased the expression of the pro-
apoptotic Bax mRNA. So as to analyze the apoptotic rate,
the ratio of Bax/Bcl-2 of compounds was determined. The
increasing of the Bax/Bcl-2 ratio indicated that compounds
2, 4, 6, 7, 8, 9, and 11 could signifcantly promote intrinsic
apoptosis in Hep3B cells (p<0.05) (Fig. S44).
protein level, experiments were performed using the Bax and
Bcl-2 antibodies on fow cytometry (Figs. S45–46). Annexin
V apoptotic mRNA and protein analysis showed that com-
pound 2 having aliphatic group-isopropyl, compound 6
having heterocyclic group-5-methylisoxazole, compounds
8 and 9 having electron-withdrawing nitro groups at difer-
ent positions (3- and 4-) on phenyl ring increased the ratio
of Bax/Bcl-2 expression and cause the intrinsic apoptosis on
Hep3B cells (Fig. 5 and S40). According to the results, the
protein level of Bcl-2 decreased and Bax level increased in
correlation with Western Blot results.
Metastatic efect of compounds Hep3B cells
To investigate the efect of compounds on the apoptotic
pathway, the protein expressions of Bcl-2 and Bax were ana-
lyzed with Western blot. The protein bands are shown in
Fig. 3d. In consistent with mRNA expression, it was sighted
in the up-regulation of Bax and down-regulation of Bcl-2
protein level as shown in Fig. 3d. The increased ratio of Bax/
Bcl-2 protein levels was similarly observed for compounds
2, 6, 7, 8, and 9 (Fig. S43). To confrm the same efect at the
We performed colony formation assays to investigate the
inhibition potential of oncogenic proliferation of compounds.
As shown in Fig. 4a and b, the number of colony formation
was decreased signifcantly with all compounds (Fig. 4a).
Compounds 2, 4, 5, 7, 8, and 9 led to the drastic reduc-
tion in colony numbers. In addition, phase-contrast micro-
graphs revealed that compounds induced cell morphology
alterations, such as cell shrinkage [32]. These experiments
1 3

Synthesis and biological evaluation of 2,4,6‑trinitroaniline derivatives as potent antitumor…
Fig. 5 An overview of the biological activities of the compounds 1–12
indicated that compounds suppressed the colony formation
and inhibited the unrestricted growth of hepatoma cells.
As migration is an important characteristic for metastatic
cancers [33], wound healing assay was performed to inves-
tigate the efect of compounds on migration in Hep3B cells.
As shown in Fig. 4b, all compounds inhibited the migration
ability of Hep3B cells compared to untreated cells. Particu-
larly, the compounds 1, 3, and 5 can distinctly block the
migration compared to the other compounds. An overview
of the biological activities of the compounds 1–12 is given
in Fig. 5.
Most of the target compounds showed remarkable cytotoxic
activity, namely compounds 2, 3, 4, 6, 7, 8, 9, and 11. Amongst
them, the compounds 3, 4, 5, 8, and 11 had the more potent
inhibitory efect on Hep3B cells, IC50:17.02 µM, 18.85 µM,
23.37 µM, 16.23 µM and 9.51 µM, respectively. In addi-
tion, compounds 3 and 4 are not toxic on healthy cell model,
HUVEC. Further protein analysis of the cell cycle revealed
that compounds 2–4 and 7–9 could down-regulate expression
of the cycle checkpoint proteins cyclin D1. Annexin V apop-
totic mRNA and protein analysis indicate compounds 2, 6,
8, and 9 induce intrinsic apoptosis by increasing the ratio of
Bax/Bcl-2 expression. Furthermore, to clarify the mechanism
of anti-metastatic activity of compounds 1–12, the colony
formation and wound healing assays were performed, and it
was observed that these compounds also inhibit the metastatic
activity of Hep3B cells. In conclusion, the data resulting from
compounds 2, 6, 8, and 9 seem promising and have prompted
further biological evaluation which will lead to promising
development of new anticancer drugs.
Conclusion
In summary, a series of 2,4,6-trinitroaniline derivatives 1–12
have been synthesized and evaluated their potential antitumor
activities against four human cancer cell lines (Hep3B, PC3,
Saos-2, and HT-29) and one healthy cell model (HUVEC).
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N. Hacıoğlu et al.
(4.24 mmol), 0.7 g potassium carbonate (5.09 mmol, 1.2 eq.)
and 1.05 g picryl chloride (4.24 mmol) in 12 cm3 DMF was
heated at 60–70 °C for 10 h. After completion of the reac-
tion, the mixture was poured into 100 cm3 cold water and
the corresponding solution was left in the fridge overnight
to maturate of the solid. The precipitate was fltered, washed
several times with water, and dried at room temperature. The
crude product was washed with 20 cm3 ethyl alcohol and 20
cm3 acetone. Cream-yellow solid; yield: 0.66 g (50%); m.p.:
183–184 °C; 1H NMR (400 MHz, DMSO-d6): δ=10.20 (s,
1H), 8.95 (s, 2H), 6.21 (s, 1H), 2.35 (s, 3H) ppm; 13C NMR
(100 MHz, DMSO-d6): δ=170.98, 159.89, 141.59, 139.50,
Experimental
All reagents and solvents used were purchased from com-
mercial sources without further purification. Melting
points were determined on a digital melting point appa-
ratus (X-4). The reaction progress was monitored by TLC
on Silica gel 60GF254 plates. FT-IR spectra were obtained
on a Perkin Elmer Spectrum 100 FT-IR spectrophotom-
eter equipped with Attenuated Total Refection (ATR)
sampling accessory. The H and 13C NMR spectra were
1
recorded using DMSO-d6 solvent on Jeol 400 MHz NMR
High-Performance Digital FT-NMR and Varian Mercury
500 MHz spectrometer with tetramethylsilane as an inter-
nal standard. Mass spectra were measured on Shimadzu
LC–MS/MS 8040 Liquid Chromatograph Mass Spectrom-
eter using an ESI source.
̄
135.10, 126.31, 96.89, 12.55 ppm; IR (ATR): V = 3249,
3145, 3118, 3086, 2989, 2938, 1620, 1599, 1529, 1441,
1331, 1172, 919, 800 cm−1; LC–MS (ESI): m/z = 309.0
([M–H]−).
Compounds 1 and 3 were synthesized according to the
procedure described in [21].
N‑(4‑Nitrobenzyl)‑2,4,6‑trinitroaniline (7) The mixture of
5 cm3 DMF solution of 0.75 g 4-nitrobenzylamine hydro-
chloride (3.9 mmol) and 5 cm3 aqueous solution of 0.16 g
sodium hydroxide (4.0 mmol) was stirred at room tempera-
ture for 2 h. Then, 0.96 g picryl chloride (3.9 mmol) was
added to the reaction medium and stirred an additional 24 h.
After completion of the reaction, the mixture was poured
into 100 cm3 cold water and the corresponding solution was
left in the fridge overnight to the maturation of the solid.
The precipitate was fltered, washed with water, and dried at
room temperature. The crude product was purifed by wash-
ing with 20 cm3 ethyl alcohol. Yellow solid; yield: 0.89 g
(62%); m.p.: 178–181 °C (191 °C [26]).
General method for compounds 2, 4, and 5
DMSO solution of amine derivatives (isopropyl amine,
1-naphthylamine, or 2-naphthylamine, respectively)
(1.0 eq.) was slowly added to DMSO solution of picryl
chloride (1.0 eq.) on the ice bath. The mixture was stirred
at room temperature for 24 h and was poured over ice
pieces. The resulting precipitate was fltered, washed with
water, and air-dried. Crystallization or washing techniques
were applied to purify the crude products.
N‑(3‑Nitrophenyl)‑2,4,6‑trinitroaniline (8) 1.0 g Picryl chlo-
ride (4.03 mmol) was dissolved in 10 cm3 DMSO. A solu-
tion of 0.56 g 3-nitroaniline (4.03 mmol) and 0.38 g pyridine
(4.8 mmol, 0.4 cm3, 1.2 eq.) in 5 cm3 DMSO was added to
the reaction medium in an ice bath. The mixture was heated
at 100 °C for 24 h and then, it was poured into 200 cm3 cold
water and extracted with CHCl3 (3×50 cm3). The combined
organic layer was washed with plenty of water, dried with
anhydrous Na2SO4, and concentrated in vacuo. The crude
product was purifed by washing diethyl ether to give com-
pound 8 as bright orange crystal. Yield: 1.2 g (85%); m.p.:
205–206 °C (212–213 °C [21]).
N‑Isopropyl‑2,4,6‑trinitroaniline (2) Compound 2 was syn-
thesized from picryl chloride and isopropyl amine according
to the general procedure described above. The crude product
was purifed by crystallization with ethyl alcohol. Yellow
needle crystal; yield: 55%; m.p.: 105–106 °C (107.5–108 °C
[22]).
N‑(2,4,6‑Trinitrophenyl)naphthalen‑1‑amine (4) Compound
4 was synthesized according to the general procedure. The
crude product was purifed by washing with ethyl acetate
to give pure orange solid. Yield: 88%; m.p.: 200 °C (197–
198 °C [23]).
N‑(4‑Nitrophenyl)‑2,4,6‑trinitroaniline (9) 5 cm3 of DMF
solution of 0.42 g 4-nitroaniline (3.0 mmol) was slowly
added to 3 cm3 DMF solution of 0.75 g picryl chloride
(3.0 mmol) in an ice bath. The mixture was stirred at room
temperature for 24 h. Upon completion of the reaction as
shown by TLC, the mixture was poured into 50 cm3 cold
water and obtained solid was fltered and dried. The crude
product was purifed by column chromatography on silica
N‑(2,4,6‑Trinitrophenyl)naphthalen‑2‑amine (5) Compound
5 was synthesized according to the general procedure. The
crude product was purifed by washing with ethyl acetate
to give orange solid. Yield: 80%; m.p.: 233–234 °C (233–
234 °C [23]).
N‑(2,4,6‑Trinitrophenyl)‑5‑methylisoxazole‑3‑amine (6,
C10H7N5O7) Solution of 0.42 g 3-amino-5-methylisoxazole
1 3

Synthesis and biological evaluation of 2,4,6‑trinitroaniline derivatives as potent antitumor…
gel (CHCl3) to provide greenish solid. Yield: 0.8 g (76%);
m.p.: 221–222 °C (226–227 °C [22]).
respectively. Bright orange solid; yield: 1.45 g (90%); m.p.:
202–203 °C (dec.) (225.42–227.86 °C [35]).
N‑(3‑Methoxyphenyl)‑2,4,6‑trinitroaniline (10) 1.0 g Pic-
ryl chloride (4.03 mmol) was dissolved in 10 cm3 DMSO.
10 cm3 of DMSO solution of 0.5 g 3-methoxyaniline
(4.03 mmol; 0.45 cm3) and 0.38 g pyridine (4.8 mmol;
0.4 cm3; 1.2 eq.) was added to the reaction medium in an
ice bath. The mixture was heated at 100 °C for 6 h. The
mixture was poured into 250 cm3 cold water and extracted
with CHCl3 (3×50 cm3). The combined organic layer was
washed with plenty of water, dried with anhydrous Na2SO4
and concentrated in vacuo. The oily crude product was solid-
ifed by washing petroleum ether (40–60 °C) to give com-
pound 10 as bright orange crystal. Yield: 1.18 g (88%); m.p.:
120–121 °C (126 °C [34]); 1H NMR (400 MHz, DMSO-d6):
δ=10.21 (s, 1H), 9.00 (s, 2H), 7.27 (t, j=7.7 Hz, 1H), 6.80
(m, 3H), 3.77 (s, 3H) ppm; 13C NMR (100 MHz, DMSO-d6):
δ=160.38, 140.92, 139.89, 138.27, 136.44, 130.61, 127.39,
Cell culture and cell growth assay
The human cell lines (PC3, Hep3B, Saos-2, HUVEC, and
HT-29) were purchased from ATCC. Human cancer cell
lines (Hep3B, PC3, Saos-2, and HT-29) and human healthy
cell line (HUVEC) were cultured in DMEM (Sigma Dulbec-
co’s Modifed Eagle Medium) supplemented with 10% FCS
(Fetal Calf Serum) (inactivated at 56 °C for 1 h) (Invitrogen)
and 2 mM L-glutamine (Gibco) at 37 °C with 5% CO2 (v/v).
Approximately 5 × 104 cells, suspended in DMEM
medium, were plated in a 96-well plate. After 24 h, fve
diferent concentrations of compounds (150, 75, 38, 19, and
9 µM) were introduced into cells. DMSO as vehicle control
was included in the study and the experiment was performed
in triplicate. It was suggested the DMSO concentration
should be kept between 0.1% and 1.0% for cell-based stud-
ies [36]. Cells were then incubated for 24 and 48 h and MTT
assay was performed as described in our previous study [37].
Absorbances were measured at 550 nm with a spectropho-
tometer (Thermo Scientifc). The results, expressed as IC50,
are averages of at least three determinations.
̄
113.42, 112.28, 107.00, 55.75 ppm; IR (ATR): V = 3312,
3077, 3009, 2959, 2941, 2924, 1619, 1589, 1535, 1508,
1436, 1339, 1270, 1158, 1093, 1036, 924, 867, 791, 735,
725, 692 cm−1; LC–MS (ESI): m/z=333.0 ([M-H]−).
N‑(3,5‑Difluorophenyl)‑2,4,6‑trinitroaniline (11) 5 cm3
Methanol solution of 0.5 g 3,5-difuoroaniline (3.87 mmol)
and 0.58 g triethylamine (5.82 mmol, 0.8 cm3, 1.5 eq.) was
added dropwise to 0.96 g picryl chloride (3.87 mmol) in
5 cm3 methanol solution. The mixture was stirred at room
temperature for 3.5 h. After completion of the reaction,
the solvent was evaporated under vacuo. The oily residue
was treated with hexane/diethyl ether mixture (20 cm3);
the obtained solid was washed with 10 cm3 diethyl ether
and dried at room temperature. Bright orange solid; yield:
0.92 g (70%); %); m.p.: 185–187 °C (190–194 °C [30]);
1H NMR (400 MHz, DMSO-d6): δ = 10.14 (s, 1H), 8.99
(s, 2H), 6.98 (m, 1H), 6.90 (d, J= 8.06 Hz, 2H) ppm; 13C
NMR (100 MHz, DMSO-d6): δ = 164.24, 161.80, 143.15,
141.22, 138.21, 136.97, 127.07, 104.15, 103.86, 100.97,
Flow cytometric analysis of apoptosis
Apoptotic cells were assayed using the Guava Nexin Rea-
gent for Flow Cytometry concerning the manufacturer’s
directions. For this purpose, Hep3B cells were divided into
12-well plates. After 48 h of drug application, the cells were
removed with Trypsin–EDTA. The pellet was taken to the
media containing 1.0% BSA and adjusted according to the
kit’s recommendation. 100 mm3 of a sample was taken from
each tube and 100 mm3 of Guava Nexin Solution was added.
The mixture was kept at room temperature for 20 min.
After incubation, the mixture was analyzed with Guava®
easyCyte™.
̄
100.71, 100.44 ppm; IR (ATR):V =3312, 3082, 1597, 1505,
Western blot analysis
1343, 1286, 1115, 925, 859, 827, 714 cm−1; LC–MS (ESI):
m/z=340.0 ([M-H]−).
Hep3B cells were treated with the compounds for 48 h
using their efective concentrations, trypsinized and protein
extraction was performed using RIPA Bufer (500 mm3).
The protein concentration was calculated using the Brad-
ford method [38]. Total protein (50 µg) was loaded on SDS-
PAGE and electrophorized at 120 V for 2 h. The SDS gel
was transferred using PVDF membranes (Millipore) incu-
bated at 4 °C overnight at 15 V. Western procedure was car-
ried out as described previously [39–41]. The membranes
were incubated with the Bcl-2 and Bax antibody (sc-7382
and sc-7480 Santa Cruz Biotechnology, Inc., Santa Cruz,
CA). Immunoreactive bands were visualized using the
N1,N2‑Bis(2,4,6‑trinitrophenyl)ethane‑1,2‑diamine (12) 0.2 g
Ethylenediamine (3.33 mmol, 0.22 cm3) and 0.7 g triethyl-
amine (6.99 mmol, 0.96 cm3, 2.1 eq.) were added dropwise
to 1.65 g picryl chloride (6.66 mmol, 2 eq.) in 10 cm3 DMF
solution that cooled in an ice bath. The mixture was stirred
at room temperature for 6 h. The progress of the reaction
was followed by TLC using hexane:ethyl acetate/1:1. After
completion of the reaction, the mixture was poured into 100
cm3 cold water and the solid was fltered. The crude product
was washed with hot ethyl alcohol and cold diethyl ether,
1 3

N. Hacıoğlu et al.
110T754—113Z706, and Balıkesir University Scientifc Research Pro-
enhanced chemiluminescence system (32,106 Pierce ECL
Plus, Thermo Scientifc). The densitometry was performed
with ImageJ Software [42]. Protein bands were quantita-
tively analyzed.
ject Grant Number 2017-024.
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Acknowledgements This work was supported by Scientific and
Technological Research Council of Turkey-TUBITAK grant numbers
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