Prodrugs for nitroreductase-based cancer therapy-3: Antitumor activity of the novel dinitroaniline prodrugs/Ssap-NtrB enzyme suicide gene system: Synthesis, in vitro and in silico evaluation in prostate cancer

Article abstract of DOI:10.1016/j.ejmech.2019.111937

Prodrugs for targeted tumor therapies have been extensively studied in recent years due to not only maximising therapeutic effects on tumor cells but also reducing or eliminating serious side effects on healthy cells. This strategy uses prodrugs which are safe for normal cells and form toxic metabolites (drugs) after selective reduction by enzymes in tumor tissues. In this study, prodrug candidates (1-36) containing nitro were designed, synthesized and characterized within the scope of chemical experiments. Drug-likeness properties of prodrug candidates were analyzed using DS 2018 to investigate undesired toxicity effects. In vitro cytotoxic effects of prodrug canditates were performed with MTT assay for human hepatoma cells (Hep3B) and prostate cancer cells (PC3) and human umbilical vein endothelial cells (HUVEC) as healthy control. Non-toxic compounds (3, 5, 7, 10, 12, 15, 17, 19 and 21–23), and also compounds (1, 2, 5, 6, 9, 11, 14, 16, 20 and 24) which had low toxic effects, were selected to examine their suitability as prodrug canditates. The reduction profiles and kinetic studies of prodrug/Ssap-NtrB combinations were performed with biochemical analyses. Then, selected prodrug/Ssap-NtrB combinations were applied to prostate cancer cells to determine toxicity. The results of theoretical, in vitro cytotoxic and biochemical studies suggest 14/Ssap-NtrB, 22/Ssap-NtrB and 24/Ssap-NtrB may be potential prodrug/enzyme combinations for nitroreductase (Ntr)-based prostate cancer therapy.

Full text of DOI:10.1016/j.ejmech.2019.111937

Journal Pre-proof
Prodrugs for nitroreductase-based cancer therapy-3: Antitumor activity of the novel
dinitroaniline prodrugs/Ssap-NtrB enzyme suicide gene system: Synthesis, in vitro
and in silico evaluation in prostate cancer
Esra Tokay, Tuğba Güngör, Nelin Hacıoğlu, Ferah Cömert Önder, Ünzile Güven
Gülhan, Tuğba Taşkın Tok, Ayhan Çelik, Mehmet Ay, Feray Köçkar
PII:
S0223-5234(19)31089-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.111937
EJMECH 111937
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 September 2019
Revised Date: 29 November 2019
Accepted Date: 30 November 2019
Please cite this article as: E. Tokay, Tuğ. Güngör, N. Hacıoğlu, Ferah.Cö. Önder, Ü.Gü. Gülhan,
Tuğ.Taşı. Tok, A. Çelik, M. Ay, F. Köçkar, Prodrugs for nitroreductase-based cancer therapy-3:
Antitumor activity of the novel dinitroaniline prodrugs/Ssap-NtrB enzyme suicide gene system:
Synthesis, in vitro and in silico evaluation in prostate cancer, European Journal of Medicinal Chemistry
(2020), doi: https://doi.org/10.1016/j.ejmech.2019.111937.
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2019 Published by Elsevier Masson SAS.

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PRODRUGS FOR NITROREDUCTASE-BASED CANCER THERAPY-3:
Antitumor activity of the novel dinitroaniline prodrugs/Ssap-NtrB enzyme suicide gene
system: Synthesis, in vitro and in silico evaluation in prostate cancer
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Esra Tokay , Tuğba Güngör , Nelin Hacıoğlu , Ferah Cömert Önder , Ünzile Güven Gülhan ,
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, #
2*
1*
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Tuğba Taşkın Tok , Ayhan Çelik Mehmet Ay , Feray Köçkar
1
Department of Molecular Biology and Genetic, Faculty of Sciences and Arts, Balıkesir
University, Balıkesir 10145, Turkey
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Department of Chemistry, Faculty of Sciences and Arts, Natural Products and Drug
Research Laboratory, Çanakkale Onsekiz Mart University, Çanakkale 17020, Turkey
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Department of Chemistry, Faculty of Science, Gebze Technical University, Gebze-Kocaeli
41400, Turkey
4
Department of Chemistry, Faculty of Sciences and Arts, Gaziantep University, Gaziantep
27310, Turkey
*Corresponding authors
E-mail addresses: fkockar@balikesir.edu.tr; feraykockar@hotmail.com (Feray Köçkar) and
mehmetay06@comu.edu.tr; mehmetay06@yahoo.com (Mehmet Ay).
#
Ayhan Çelik left GTU after September, 2016.

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Abstract
Prodrugs for targeted tumor therapies have been extensively studied in recent years
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due to not only maximising therapeutic effects on tumor cells but also reducing or eliminating
serious side effects on healthy cells. This strategy uses prodrugs which are safe for normal
cells and form toxic metabolites (drugs) after selective reduction by enzymes in tumor tissues.
In this study, prodrug candidates (1-36) containing nitro were designed, synthesized
and characterized within the scope of chemical experiments. Drug-likeness properties of
prodrug candidates were analyzed using DS 2018 to investigate undesired toxicity effects. In
vitro cytotoxic effects of prodrug canditates were performed with MTT assay for human
hepatoma cells (Hep3B) and prostate cancer cells (PC-3) and human umbilical vein
endothelial cells (HUVEC) as healthy control. Non-toxic compounds (3, 5, 7, 10, 12, 15, 17,
19 and 21-23), and also compounds (1, 2, 5, 6, 9, 11, 14, 16, 20 and 24) which had low toxic
effects, were selected to examine their suitability as prodrug canditates. The reduction profiles
and kinetic studies of prodrug/Ssap-NtrB combinations were performed with biochemical
analyses. Then, selected prodrug/Ssap-NtrB combinations were applied to prostate cancer
cells to determine toxicity. The results of theoretical, in vitro cytotoxic and biochemical
studies suggest 14/Ssap-NtrB, 22/Ssap-NtrB and 24/Ssap-NtrB may be potential
prodrug/enzyme combinations for nitroreductase (Ntr)-based prostate cancer therapy.
Keywords: Dinitroaniline, Prodrug, Ssap-NtrB, Nitroreductase, Enzymatic activity,
Cytotoxicity, Prostate cancer, in silico studies
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1. Introduction

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Chemotherapeutic drugs in clinical use cause very harmful side effects. To overcome this
problem, nowadays some cancer therapy approaches were evaluated namely GDEPT (Gene-
Directed Enzyme Prodrug Therapy) and ADEPT (Antibody-Directed Enzyme Prodrug
therapy). In these strategies, prodrugs that are not toxic are selectively activated by certain
biochemical mechanisms which are unique to tumor cells and induce their cytotoxic effects in
tumor tissues [1]. In GDEPT, a gene encoding non-human prodrug-activating enzyme is
selectively delivered to tumor tissue [2]. Some non-human prodrug-activating enzymes, also
known as suicide genes, include herpes simplex virus thymidine kinase, cytosine deaminase
(CD), cytochrome P450 and nitroreductases [3]. Nitroreductases are able to catalyze the
reduction of nitro-substituted compounds using flavin mononucleotide (FMN) or flavin
adenine dinucleotide (FAD) as prosthetic groups and nicotinamide adenine dinucleotide
(NADH) or nicotinamide adenine dinucleotide phosphate (NADPH) as reducing agents [4].
Various enzyme/prodrug combinations are in development and have been studied [5]. The
best-known examples are CB1954/E. coli Nitroreductase and SN23862/E. coli Nitroreductase.
In addition, the nitroreductases from other organisms such as YwrO isolated from B.
amyloliquefaciens and NbzA isolated from P. pseudoalcaligenes were also investigated [6-8]
due to the disadvantages of the CB1954/E.coli nitroreductase combination [9]. Although new
nitroreductases were isolated from different organisms, there is still a requirement to discover
new enzyme/prodrug combinations that work with high efficiency. In our previous works, we
originally discovered a novel nitroreductase named Ssap-NtrB, from Staphylococcus
saprophyticus [10]. Then we worked on a study that includes modification of existing
antibiotics such as sulfamethoxazole (SMX) in the form of precursor prodrugs that can be
activated by Ssap-NtrB nitroreductase [11]. The following studies of our group aim to find
better prodrugs that can interact with Ssap-NtrB effectively and can show selective
cytotoxicity for cancer cells. For this purpose, we reported the synthesis of some nitro-
containing amide derivatives and their computational, enzymatic and cytotoxic applications
[12,13].
Further in this direction, herein we present the synthesis of dinitroaniline derivatives
(1-36) as prodrug candidates for GDEPT applications from different readily available
reactants and dinitrohalobenzenes (Fig. 2-5). Four different dinitroaniline cores (Fig. 1. A-D)
of N-(substituted)-2,4-dinitroaniline (1-13), symmetrical bis(2,4-dinitrophenyl)diamine
1
3
derivatives (14-24) that have minimum four nitro groups, N ,N -bis(substitutedphenyl)-4,6-
dinitrobenzene-1,3-diamines (25-29), N-(5-morpholino-2,4-dinitro phenyl)alkanamides and 5-
morpholino-2,4-dinitrophenyl alkanoates (30-36) were designed on the basis of model

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dinitroaniline prodrugs such as CB1954, SN23862, PR-104A and then synthesized. The main
aim of studying these different types of dinitroanilines is to highlight the best skeleton that
shows good enzymatic and cytotoxic results through different pharmacophore groups. It was
decided that morpholino-2,4-dinitrophenyl derivatives (30-36) should be added to the prodrug
series after the observation that prodrug 13 named 4-(2,4-dinitrophenyl)morpholine was
reduced to its metabolites by Ssap-NtrB with high conversion rate in a very short reaction
time. The effects of aliphatic, aromatic, and heterocyclic functional groups on prodrugs 1-13,
various linkers such as short/long alkyl chains, alicylic/aromatic/heterocyclic rings on
prodrugs 14-24, various functional groups such as -NO , -OH, -SO H, -Cl at different
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3
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positions on phenyl ring in prodrugs 25-29 and different R groups of ester or amide structures
on prodrugs 30-36 were discussed in terms of enzyme interactions and cytotoxic assays.
Also, molecular docking, molecular dynamic simulation, in silico predictions of drug-
likeness and pharmacokinetic properties of compounds were used for definition and
elucidation of the molecular interactions between Ssap-NtrB and compounds 14 and 22.
Further, the data about in silico prediction, drug likeness and pharmacokinetic properties are
given in the supplementary information part of this research. Cytotoxic analyses of
dinitroaniline-based prodrugs were performed using MTT assay against cancer cells, Hep3B,
PC3 and healthy HUVEC cells. Non-toxic nitro compounds were used for reaction assays.
Active metabolites resulting in reactions with Ssap-NtrB were applied to PC3 cells to mimic
the bystander effect. In this effect, active metabolites (drug) diffuse to kill neighboring cells.
Death of the cells was determined with NADH independent SRB analyses. In the light of
computational data and all experimental results from enzymatic and cytotoxic studies,
structure-activity relationships were examined in detail.
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14
15

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Fig. 1. An overview of dinitroaniline-based prodrugs 1-36.
2. Results and discussion
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2.1 Chemistry
The reaction schemes and scopes of compounds 1-36 are presented in Fig. 2-5. Since the
final aim of the study is to determine the prodrug capacity of the mentioned compounds, they
were designed as prodrugs 1-36. Structures of all the prodrugs were characterized by melting
1
13
point, FT-IR, H NMR, C NMR, MS and elemental analysis. Known intermediates and
products were confirmed by comparison to their reported spectral data in the literature.
According to our literature knowledge, prodrugs 8, 17, 18, 22, 23, 25, 28, and 30-36 (14
compounds) were synthesized in this study for the first time. Although prodrugs 14-16, 19-21,
27 and 29 (8 compounds) were described in the literature, there is no information available
about synthetic procedures or spectroscopic data. Prodrugs 1-7, 9-13, 24 and 26 (14
compound) are known compounds in the literature. While 7 of them (1 [14], 4 [14], 10 [15],
11[16], 12 [15], 13 [17] and 26 [18]) were synthesized according to literature methods for our
application, prodrugs 2, 3, 5-7, 9 and 24 were obtained with more advantageous experimental
procedures compared to the literature methods. It is well known that no biological data related
to Ntr-based cancer therapy is available for all studied prodrug candidates (1-36). So, this is
the first time Ssap-NtrB-activated prodrugs (1-36) and their in silico and in vitro applications
were examined.
Prodrugs 1-13, N-(substituted)-2,4-dinitroaniline, were obtained from 2,4-dinitro-1-
chlorobenzene or 2,4-dinitro-1-fluorobenzene and various amines including aliphatic amines
such as ethylamine, isopropylamine and cyclohexylamine, aromatic amines such as aniline, 3-
nitroaniline, 4-nitroaniline, 3-methoxyaniline, 3,5-difluoroaniline, and 2-naphthylamine,
heterocyclic amines such as (S)-2-pyrrolidinemethanol, piperidine, 3-piperidinol, and
morpholine via aromatic nucleophilic substitution reaction (S Ar) in moderate to good yields
N
(41-93%) (Fig. 2).
In the same manner, bis(2,4-dinitrophenyl)diamine derivatives (14-24) were
synthesized from 2,4-dinitro-1-chlorobenzene and various aliphatic, alicyclic, aromatic or
heterocyclic diamino derivatives under different experimental conditions that used Et N or
3
NaH as base in DMF solvent at room temperature or 60-70 °C (only for prodrug 22) by
applying the S Ar reaction mechanism (Fig. 3). Detailed synthesis steps (Fig. 2-5) and
N

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characterization of prodrugs are described in the Experimental Section and Supporting
Information.
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Fig. 2. Structures of N-(substituted)-2,4-dinitroanilines (Prodrugs 1-13).
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Fig. 3. Structures of bis(2,4-dinitrophenyl)diamines (Prodrugs 14-24).
As revealed in Fig. 4, different reaction conditions were applied to obtain prodrugs 25-
29 by substitution reactions using 1,3-dichloro-4,6-dinitrobenzene or 1,3-difluoro-4,6-
dinitrobenzene and various aniline derivatives that contain several functional groups like -
NO , -OH, -SO H, -Cl in different positions on the phenyl ring as starting materials.
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O N
NO2
NH
2
NH2
O N
2
NO2
HN
R3
Cl
Cl
(
F)
(F)
R3
R3
3
R : -NO -OH, -SO H, -Cl
Prodrug 25-29
2,
3
O N
NO2
NH
2
O N
NO2
NH
2
HN
HN
O N
2
NO2
NO2
NO2
25
26
O N
NO2
NH
O N
NO2
NH
2
2
O N
NO2
NH
2
HN
HN
HN
SO H
SO H
Cl
Cl
3
3
OH
OH
2
7
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29
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1
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Fig. 4. Structures of N ,N -bis(substitutedphenyl)-4,6-dinitrobenzene-1,3-diamines (Prodrugs
25-29).
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Prodrugs 30-36 were designed using prodrug 13, and consist of amide (30-32) and ester
derivatives (33-36) of morpholino-2,4-dinitrobenzene (Fig. 5). The starting materials for
prodrugs 30-32 of 5-fluoro-2,4-dinitroaniline and 5-morpholino-2,4-dinitroaniline were
synthesized respectively according to the literature procedures [19, 20]. In the third step, the
starting material was reacted with various acyl chlorides such as propionyl chloride, 3,3,3-
trifluoropropionyl chloride and isovaleryl chloride using pyridine as both reagent and solvent
at reflux temperature and different reaction times with good yields, 64, 80 and 75%
respectively, for prodrugs 30-32. Similarly, the synthesis of ester derivatives (33-36) was
carried out in three steps. In the first step, 5-chloro-2,4-dinitrophenol was obtained from 1,5-
dichloro-2,4-dinitrobenzene and sodium acetate in aqueous medium using microwave energy
according to the literature procedure [21]. Then, the substitution of morpholine with another
chlorine atom by heating at reflux temperature in morpholine for 3 h resulted in 91% yield
product formation. In the last step, ester prodrugs containing targeted morpholine (33-36)

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were obtained successfully as a result of the esterification reaction of 5-morpholino-2,4-
dinitrophenol and different acyl chlorides such as propionyl chloride, isovaleryl chloride, 3-
methoxypropionyl chloride and dimethylcarbamoyl chloride using Et N in different solvents
3
(DCM, MeCN or DMF) stirring at room temperature (for 33-35) or heating to 120 °C under
microwave irradiation (for 36).
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Fig. 5. Structures of N-(5-morpholino-2,4-dinitrophenyl)alkanamides and 5-morpholino-2,4-
dinitrophenyl alkanoates (Prodrugs 30-36).

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2.2. Cytotoxic activities of potential prodrugs 1-36
The cell viability of hepatoma cells (Hep3B) and prostate cancer cells (PC-3) was determined
after being exposed to nitro compounds using the MTT assay protocol. Human umbilical vein
(HUVEC) cells were used as healthy control. Potential prodrugs 1-36 were used with different
concentrations (150, 75, 38, 19 and 9 µM) for 24 h and 48 h. Growth inhibitory effects of the
nitro compounds are illustrated as graphs in SI-Figure S99. The cytotoxic effects of prodrugs
1-36 against Hep3B, PC-3 and HUVEC cells and IC values are shown in Table 1. LT (low
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0
toxic) was represented by cell viability range between 51-80%. Over 80% cell viability value
was NT (non-toxic). As evident from this table, nitro compounds, namely 3, 5, 7, 12, 15, 17,
19 and 21-23, had non-toxic effects for the three different cell lines. Compounds 1, 2, 6, 13,
16, 20, 24, 29 and 31 had low toxicity for Hep3B cells; 9, 14 and 20 had low toxicity for PC3
cells; 11 and 30-36 had low toxicity for HUVEC cells. Compounds 4, 8, 11, 18, 30 and 32
had toxic properties on Hep3B cells with IC value of 150 µM, 89.4 µM, 50.3 µM, 131.7
5
0
µM, 90.73 µM and 150 µM over 48 h, respectively. Prodrug 25 showed the highest toxic
effect on Hep3B cells with 4.8 µM IC value over 48 h and had low toxic effect on PC3 and
5
0
HUVEC cells. The 50% inhibition concentration of prodrug 26 was 12.3 µM for Hep3B cells,
110.8 µM for PC3 cells and 35.1 µM for HUVEC cells. Prodrug 27 had toxic effect on
Hep3B, PC3 and HUVEC cells at 14.5 µM, 74.13 µM and 150 µM over 48 h, respectively.
Prodrug 28 exhibited 50% inhibitory activity on Hep3B cells at 71.9 µM concentration. The
50% inhibition concentration of prodrug 29 was 150 µM for PC3 and 86.5 µM for HUVEC
cells.

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Table 1. Cytotoxic effect of prodrugs
Cytotoxic features (IC µM)
5
0
Hep3B
Prodrugs 24h
CB1954 NT
PC-3
24h
NT
NT
NT
NT
NT
NT
NT
NT
HUVEC
24h
NT
NT
NT
NT
NT
NT
NT
NT
LT
48h
NT
LT
48h
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
-
48h
NT
NT
NT
NT
NT
NT
NT
NT
LT
1
2
3
4
5
6
7
8
9
LT
LT
NT
LT
NT
T(84.85) T (150)
NT
LT
NT
LT
NT
NT
LT
NT
T (89.4) NT
LT
LT
-
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
LT
NT
NT
LT
1
0
NT
LT
NT
11
T (50.3) NT
NT
-
1
2
NT
NT
NT
NT
NT
NT
T (150)
NT
NT
NT
NT
NT
NT
T (5.4)
NT
LT
-
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
LT
13
NT
LT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
LT
14
NT
NT
LT
15
16
17
NT
18
T (131.7) NT
19
NT
NT
LT
20
LT
21
NT
NT
NT
NT
NT
NT
22
NT
23
NT
24
LT
25
T (4.8)
26
T (12.2) T (12.3) LT
T (110.8) LT
T (35.1)
2
7
LT
LT
LT
T (14.5) NT
T (71.9) NT
T (74.13) T (66.7) T (150)
28
NT
NT
NT
29
LT
T (150)
LT
-
T (86.5) LT
30
T (90.73) LT
-
-
-
-
-
-
-
LT
NT
LT
LT
LT
LT
LT
LT
LT
LT
LT
LT
LT
LT
31
LT
LT
-
32
T (150)
NT
LT
-
33
NT
NT
NT
NT
-
34
NT
-
35
NT
-
36
NT
-
2
2
28
29

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2.3. Enzymatic reduction of prodrugs 1-36 via Ssap-NtrB
Reduction profiles of prodrugs 1-36 were assessed according to HPLC chromatograms
(see SI). Among prodrugs 1-36, compounds 11-13 were easily reduced in only 10 min with
limited cofactor and were converted to their main metabolites, while Ssap-NtrB was not very
reactive for compounds 14-18 with both limited cofactor conditions and cofactor regeneration
system. HPLC results indicated that except prodrugs 14-18, all prodrug candidates interact
with Ssap-NtrB at different rates and convert the nitro groups of substrates to some
hydroxylamine and/or amine metabolites. At the end of the reduction reaction of prodrugs 5
and 6 that have three nitro groups and prodrug 25 that has four nitro groups numerous minor
metabolite peaks formed. Also, prodrugs 30-36 were substrates reduced more quickly to give
major metabolites with Ssap-NtrB using the cofactor regeneration system (see SI).
The product profiles for catalytic reduction with the cofactor regeneration system vary
according to reaction times. So, the time dependent properties of the prodrugs (11, 13, 30-36)
with good enzyme activity were examined in detail (see SI). For example, while prodrug 11,
1-(2,4-dinitrophenyl)piperidine (r.t: 24.21 min), was converted 100% to four metabolites with
retention times of 20.11, 21.8, 22.4 and 29.02 min, prodrug 13, 4-(2,4-dinitrophenyl)
morpholine (r.t: 19.38 min), was completely finished within 30 min reaction and the two main
metabolites had retention times of 15.37 and 18.2 minutes. According to HPLC
chromatograms of prodrugs 30 and 31, amide derivatives of 2,4-dinitrophenylmorpholine,
five metabolites were observed after enzymatic reduction and at the end of 20 min reaction
time, all of the substrates were converted to corresponding hydroxylamine and/or amine
metabolites. Time-dependent metabolite profiles of other prodrugs (32-36) are given in
Supporting Information and these substrates generally were converted to their corresponding
metabolites in very short reaction times of 20-60 min.
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2.4.Kinetic analysis
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Michaelis-Menten parameters were calculated for the catalytic reduction reaction of prodrugs
2, 9, 11, 13, 19, 21, 28, 30 and 31 with Ssap-NtrB and compared with the combinations of
standart prodrugs such as CB1954, PR-104A and SN23862 and known nitroreductases such
as NfsA, NfsB and YwrO (Table 2). Also, enzymatic activity of prodrugs 15, 22, 23, 25, 26,
27 and 29 were determined as unit activity of U/mg due to their low solubility in buffer
solution. Catalytic efficiencies (k /K ) of Ssap-NtrB with prodrugs 9, 13, 28, 30 and 31 were
cat
M
-1
-1
calculated to be 8267, 6974, 13297, 26506 and 26506 s M respectively. When compared
with the catalytic activity of standard E.coli NfsB/CB1954 combination, Ssap-NtrB/9, Ssap-

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NtrB/13, Ssap-NtrB/28, Ssap-NtrB/30 and Ssap-NtrB/31 were found to be more effective with
1.48, 1.25, 2.37, 4.73 and 4.73 fold, respectively. In addition, the combinations of Ssap-
NtrB/11 (1.84 fold), Ssap-NtrB/13 (3.29 fold), Ssap-NtrB/19 (1.08 fold), Ssap-NtrB/28 (6.27
fold), Ssap-NtrB/30 and Ssap-NtrB/31 (12.5 fold) were more effective than Ssap-
NtrB/CB1954 combinations, too. So, in terms of kinetic parameters, prodrugs 28, 30 and 31
are better options among prodrug candidates because of effective and fast reactions with Ssap-
NtrB.
271
272
273
Table 2. Comparison of kinetic parameters of nitroreductases with known prodrugs and
selected prodrugs 1-36.
Relative
Specific
Wavelength
nm)
k
(s )
cat
-1
K
(µM)
M
k
cat/K
M
to
Substrate
Enzyme
Cofactor
-1
-1
Activity
U/mg)
(
(s M )
CB1954/
NfsB
(
CB1954 [5b]
CB1954 [5b]
NfsA
NfsB
NADPH
NADH
420
420
16±0.5
62 ± 11
220±18
73000
5600
13.04
1.00
11000 ± 2600
SN23862 [5c]
CB1954 [10]
NfsB
NADH
26.4±7.2
2500±100
10560
2120
1.89
0.38
Ssap-
NtrB
Ssap-
NtrB
NADPH
420
340
2.26 ± 0.07
1065.2 ± 53.1
SN23862 [10]
CB1954 [5d]
PR-104A [5e]
NADPH
NADPH
NADH
0.8 ± 0.1
8.2±1.1
60
82.4±16.9
618±169
4500
9350
1.67
2.37
2.32
YwrO
13268
1
3000 ±
800
20000 ±
4000
NfsB
400
400
7
1
PR-104A [5e]
NfsA
NADPH
12
100
21.43
1
Prodrug 2
Prodrug 9
NADPH
NADPH
NADH
340
340
410
0.3±0.0
0.1±0.0
67.5±2.5
15.7±5.8
4136
8267
3901
0.74
1.48
0.70
0.404 ± 0.13
103.6 ± 51.46
Prodrug 11
4
20
0.49±0.05
2.1 ± 0.04
Prodrug 13
Prodrug 15
Prodrug 19
NADH
400
400
420
400
3.098 ± 0.51
444.17± 94.70
6974
1.25
NADH
NADH
0.315 ± 0.03
0.016 ± 0.005
137.19 ± 27.9
301.4 ± 41.28
2296
53
0.41
0.01
Prodrug 21
4
00
0.12±0.009
2.12±0.003
0.16±0.003
0.60±0.59
0.02±0.002
1.61±0.13
Prodrug 22
Prodrug 23
Prodrug 25
Prodrug 26
Prodrug 27
400
400
400
400
420
390
Ssap-
NtrB
NADH
3.396 ± 1.99
255.4 ± 171.5
13297
2.37
Prodrug 28
3
90
2.12±0.003
5.77±0.04
Prodrug 29
Prodrug 30
420
400
400
NADH
NADH
0.37 ± 0.01
14.03 ± 1.24
9.95 ± 1.8
26506
26506
4.73
4.73
0.234 ± 0.03
Prodrug 31
4
00
0.99±0.08
2
2
2
74
75
76

277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
2.5. Metabolite analysis
It is well known that nitroreductase reduces the nitro group firstly to hydroxylamine and then
to the amine functional group. The nitroso metabolite of compounds can occur, but it is
difficult to observe due to its instability. Prodrug 6 was chosen to investigate the reduction
mechanism with Ssap-NtrB and metabolite profiles. Prodrug 6 has trinitro groups on two
phenyl rings, so it will theoretically have nine metabolites that contain different numbers of
nitro, hydroxyl amine or amine groups resulting from gradual reduction. For this purpose, the
LC-MS/MS technique was applied to prodrug 6 after the enzymatic reaction and the
experimental results were interpreted. The HPLC and LC-MS/MS spectra with the possible
metabolites occurring as a result of the enzymatic reduction of prodrug 6 are given in Fig. 6.
The MS spectrum of prodrug 6/Ssap-NtrB reaction was obtained using quick scan negative
mode because of the negative ionization of nitro groups. So 1-3 minus values of molecular
weights of metabolites were observed on MS spectra. According to MS spectra, biocatalytic
reduction of prodrug 6 in 15 minutes reaction time resulted in four metabolites with m/z of
287, 274, 241 and 212. The molecular weight of substrate was observed with m/z of 303 as
-
[M-H] . Also, HPLC spectra showed 80% conversion rate of the reduction reaction of prodrug
6 within 15 min and the formation of five metabolites with retention times of 19.15, 20.01,
21.98, 24.25 and 25.35 min. While the peak of m/z 287 corresponds to a single
hydroxylamine derivative with one step reduction of the nitro group, the peak of m/z 274 can
correspond to double hydroxylamine or single amine metabolites. Other peaks with m/z of
241 and 212 consistof double and triple amine metabolites, respectively. Although it is
difficult to determine the reduction order of nitro groups in different positions on phenyl
rings, the nitroreductase enzyme (Ssap-NtrB) can reduce all three nitro groups on the
substrate to their final amine metabolites.
3
02

3
03
04
3
Fig. 6. A) Predicted reaction steps and corresponding metabolites of the Ssap-NtrB catalyzed
reaction for prodrug 6, B) LC-MS spectrum of prodrug 6/Ssap-NtrB reaction using quick scan
negative mode (C) HPLC chromtogram of enzymatic reduction reaction of prodrug 6 for 15
min.
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
2.6. Cytotoxic effects of active metabolites resulting from the reduction of nitro
compounds by Ssap-NtrB
In order to investigate the growth inhibitory effects of toxic metabolites resulting from the
reduction reaction of nitro compounds with Ssap-NtrB enzyme,SRB assay was used. In the
beginning, three different solutions (R, C1 and C2) were applied to PC-3 cells to mimic the
by-stander effect to determine toxic reaction metabolites. Non-toxic nitro compounds (3, 5, 7,
14, 15, 17, 19 and 21-24) were reacted with the Ssap-NtrB enzyme to determine the growth
inhibitory effect of metabolites. The reaction solution (R) contained NADH (600 µM) as
cofactor and nitro compounds (150 µM) at 100 µL final volume. Control 1 (C1) solution
included nitro compounds (150 µM) at 100 µL final volume completed with 25 mM Tris/HCl
buffer. Control 2 (C2) solution contained only Ssap-NtrB enzyme (31 µg/mL). R, C1 and C2
solutions were applied to PC3 cells. 3/Ssap-NtrB, 7/SSap-NtrB, 10/Ssap-NtrB, 12/sap-NtrB,
13/Ssap-NtrB 20/Ssap-NtrB and 21/Ssap-NtrB pairs showed highly toxic effect with 50%
proliferation. Thus, these compounds were not applied at three different concentration ranges
(see SI-Fig. S100). As shown in SI-Figure S100, the rest of the non-toxic compound/Ssap-
NtrB pairs (1, 2, 5, 6, 9, 11, 15, 16, 17, 19 and 23) were added into the reaction at three
concentrations (150, 39 and 9 µM). Compounds 14, 22 and 24 were applied in the reaction at
low concentration (180, 18 and 1.8 nM) because of saturated toxicity at high concentration.

327
328
329
330
331
After 48 h incubation time, SRB assay was performed and the absorbances were read using a
spectrophotometer at 492 nm. Optical density values were used to calculate % cell viability
data. IC50 values of toxic metabolites are given in Table 3. Compounds 14, 22 and 24
exhibited highly toxic effect for PC3 cells, prodrug 14 especially showed significant toxicity
at 0.79 nM with 50% inhibition concentration compared with CB1954 (Table 3, Fig. 7).
332
333
334
Table 3. Cytotoxic effects of selected prodrugs
Prostate Cancer Cell line (PC3)
48h
+
-
Prodrugs Ssap-NtrB Ssap-NtrB
CB1954
1.792 nM
6.43 µM
9.77 µM
>150 µM
5.07 µM
4.98 µM
>150 µM
5.07 µM
>150 µM
>150 µM
>150 µM
0.79 nM
7.83 µM
5.73 µM
9.58 µM
14.96 µM
>150 µM
>150 µM
1.8 nM
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
1
2
3
5
6
7
9
10
12
13
14
15
16
17
19
20
21
22
23
7.133 µM
3.99 nM
24
3
3
35
36
3
3
3
3
3
3
3
3
3
3
37
38
39
40
41
42
43
44
45
46

3
47
48
3
Fig. 7. Cytotoxic effect of potential prodrugs in the presence of Ssap-NtrB. Potential prodrugs
were added to the reaction at three different concentrations (R-180 nM, R-18 nM, R-1.8 nM).
C1 (Control 1) contained potential prodrug at 180 nM final concentration in 25mM Tris-Cl
pH:7.5 Buffer. CB1954 was used as positive control.
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
2.7. Molecular docking
Docking calculations were performed with AutoDock 4.2 to define which effect(s) are
important for prodrugs in the interaction mechanisms of 14/Ssap-NtrB and 22/Ssap-NtrB
based on in vitro study. Additionally, CB1954 and SN23862 compounds with Ssap-NtrB were
used as reference prodrugs in this study. First of all, the validation of docking processes was
analyzed with self-docking analysis. The reference prodrugs were docked with our modelled
target. The results were compared with the experimental data from our previous studies that
revealed a compatible approach to apply in this process.
Then, prodrug candidates 14 and 22 identified in this study according to biological
activity studies were prepared for the same process. Their binding energy and binding
constant values were calculated and compared with CB1954 and SN23862 as reference
prodrugs.

365
366
367
368
369
370
371
372
373
374
375
376
The two-dimensional (2D) orientation of CB1954 and SN23862 and detailed non-
bonding interactions are given in SI, S93-S94 and Table S1.
In this study, compounds 14 and 22 were interacted with Ssap-NtrB as target.
Compound 14 had twelve interactions including eight hydrogen bonds with Arg13, Lys188,
Ser43 residues and FMN as coenzyme, one electrostatic interaction with Glu163 and three
hydrophobic interactions with Val204 residue, FMN, as coenzyme of the target. Compound
22 forms seven hydrogen bonds and four hydrophobic interactions with the different residues
and FMN molecule of the same enzyme, as seen in Fig. 8.
Besides interaction types of the complexes, docking binding energy and binding
constant values reveal the affinity and binding tendency of the compounds for the enzyme.
Even the binding energy of compound 14 displayed better trends than compound 22 and
reference prodrugs, CB1954 and SN23862 in Table 4.
3
3
77
78
379
380
381
382
383
384
385
386
Fig. 8. The best docking modes of compound 14 (A, light blue and stick) and compound 22
(B, orange and stick) in the active site of Ssap-NtrB.

3
3
87
88
Table 4. The binding energy and binding constant values of two compounds (14 and 22) and
CB1954* and SN23862* as reference prodrugs with Ssap-NtrB.
Prodrugs
∆G(kcal/mol)
Ki (µM)
1
4
2
-8.93
-8.25
-7.13
-7.01
0.28349
0.89567
5.96
2
CB1954*
SN23862*
7.28
3
89
3
3
3
90
91
92
2.8. Molecular dynamic simulation
To investigate the stability of protein-ligand complex interactions between the compounds
(14 and 22) and target (Ssap-NtrB), molecular dynamic simulations were performed for
protein ligand-free and protein-ligand for 10 ns duration. First, we evaluated the structural
stability by analyzing molecular dynamic trajectories for Ssap-NtrB/ligand (14 and 22)
complexes and ligand-unbound Ssap-NtrB including RMSD and RMSF. The RMSD of the
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
0
Ssap-NtrB was about <2 A during 10 ns (Fig. 9a). The overall agreement between the bound-
22 Ssap-NtrB and unbound-Ssap-NtrB simulation and the behaviors are also quite similar
indicating that the models considered here are stable.
RMSF values were also analyzed to identify the regions with high fluctuations. The
results are summarized in Fig. 9b. In general, a similar RMSF distribution was observed for
the ligand-bound state and ligand-unbound state of Ssap-NtrB. RMSF of the active site amino
acid residues were analyzed to check how many fluctuations occur during the simulation
period and it was found that the key amino acids in the active site did not fluctuate. This
indicates 22 was too stable inside the binding pocket of Ssap-NtrB. Furthermore, there may be
unexpected merit in Fig. 9; that is, this RMSF fluctuates a bit in the three-loop regions
(residues 64-84, 89-119, and 122-142) of Ssap-NtrB. These differences may result in
differences in the binding abilities of the ligands. However, overall, the RMSF analysis
indicated that the complexes exhibited similar dynamics during the MD simulations. This
means that 22 has a stable effect within the binding pocket of Ssap-NtrB.

4
12
4
13
14
4
Figure 9. a) RMSD trajectory of 14-Ssap-NtrB, 22-Ssap-NtrB and Ssap-NtrB during the MD
simulation. b) RMSF profile of 14-Ssap-NtrB, 22-Ssap-NtrB and Ssap-NtrB. Red color
4
15

4
4
4
16
17
18
indicates 14-Ssap-NtrB complex, green color indicates 22-Ssap-NtrB and blue color indicates
Ssap-NtrB.
4
19
2.9. Structure-activity relationships (SARs).
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
According to the in vitro data obtained by enzymatic, cytotoxic studies and theoretical
results, the preliminary structure-activity relationships of prodrug candidates 1-36 were
analyzed in a basic framework (Fig. 10).
Among the N-(substituted)-2,4-dinitroanilines (1-13), the metabolites of compounds 1, 2,
5, 6 and 9 containing ethyl, isopropyl, 3-nitrophenyl, 4-nitrophenyl and 2-naphthyl groups,
respectively, have IC values in the range of 4.98-9.77 µM and compounds 3, 7, 10, 12 and
5
0
13 have values higher than 150 µM on prostate cancer cells over 48 h. Also, compounds 11-
13, which have heterocyclic groups such as piperidine, 3-hydroxypiperidine and morpholine,
interacted with Ssap-NtrB very quickly in limited cofactor conditions and were reduced to
their corresponding hydroxylamine or amine metabolites with high conversion rate. Although
these are good enzymatic results, it was observed that these compounds were not toxic
(metabolite’s IC >150 µM) on prostate cancer cells over 48 h.
5
0
Except compound 18 (1,4-diaminocyclohexyl derivative-toxic on Hep3B), all bis (2,4-
dinitrophenyl)diamines (14-24) were non-toxic for HUVEC and two cancer cells, so their
prodrug abilities were examined in detail. While the metabolites of compounds 15-17, 19 and
23 have IC values in the range of 5.73-14.96 µM, the values forcompounds 20-21 are higher
5
0
1
2
than 150 µM for prostate cancer cells over 48 h. In addition, the IC values of 14 (N ,N -
5
0
1
2
bis(2,4-dinitrophenyl)ethane-1,2-diamine), 22 (N ,N -bis(2,4-dinitrophneyl)-4-nitro benzene-
1,2-diamine) and 24 (1,4-bis(2,4-dinitrophenyl)piperazine) metabolites were found to be 0.79,
1.8 and 3.99 nM, respectively, and these were the most toxic prodrugs for PC3 cells.
According to the molecular docking data, it was observed that whereas compound 14 has
eight hydrogen bonds, one electrostatic and three hydrophobic interactions with Ssap-NtrB,
compound 22 has seven hydrogen bonds and four hydrophobic interactions. Also, compounds
14-15, 19 and 22-24 were determined to show excellent bystander effect and high substrate
activity for Ssap-NtrB enzyme.
1
3
All N ,N -bis(substitutedphenyl)-4,6-dinitrobenzene-1,3-diamines (25-29) showed toxic
effect on Hep3B or PC3 cancer cells. So, it can be concluded that they can be used as liver or
prostate cancer drugs, but not be used as prodrugs for nitroreductase-based cancer therapy.
Especially compound 25 with two 3-nitrophenyl moieties was the most toxic compound with

4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
IC value of 4.8 µM on hepatoma cells over 48 h, while was not toxic to the healthy cell
50
model. Also, it was determined that compound 28, containing sulfonic acid groups at 4-
position on the phenyl rings, has higher k /K value and was more effective than E.coli
cat
M
NfsB/CB1954 combination (2.37 fold) and Ssap-NtrB/CB1954 combination (6.27 fold)
according to the results of kinetic experiments.
Although morpholine and nitro groups containing amide or ester derivatives (30-36) were
reduced to their major metabolites with high conversion rate under cofactor regeneration
conditions, they were found to be toxic or had low toxicity for hepatoma cancer cells and had
low toxicity for healthy HUVEC cell. So, the prodrug abilities of these derivatives lost their
importance for nitroreductase-based cancer therapy. Also it should be noted that
propionamide (30) and 3,3,3-trifluoropropionamide derivatives (31) have the highest k /K
cat
M
values (same values for both compounds) among all prodrug candidates and were more
effective than E.coli NfsB/CB1954 combination (4.73 fold) and Ssap-NtrB/CB1954
combination (12.5 fold) according to the Michaelis-Menten parameters.
As a result of all the experimental data, compounds 14, 22 and 24 can be recommended as
potential prostate cancer prodrugs to be used for nitroreductase-based cancer therapy (Fig.
10).
4
65
66
4

4
67
4
4
4
68
69
70
Fig. 10. Structure-activity relationship (SAR) analysis of prodrugs 1-36.

4
71
3. Conclusion
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
In this study, 2,4-dinitroaniline derivatives (36 prodrug candidates) classified into four
different categories were synthesized by using different reaction conditions, with moderate to
good yields and characterized by several spectroscopic techniques. While 14 compounds (8,
17, 18, 22, 23, 25, 28, 30-36) were newly synthesized in this study, synthetic procedures and
spectroscopic data of 8 compounds (14, 15, 16, 19-21, 27, 29) were introduced to the
literature and more advantageous experimental conditions were developed for the synthesis of
7 compounds (2, 3, 5-7, 9 and 24). There is no evidence about known nitro containing
compounds with nitroreductase enzymes and their applications in the literature.
According to HPLC analysis, prodrugs 11-13 had high conversion rate with the Ssap-NtrB
reduction reaction and major selective metabolites with limited cofactor. Also, prodrugs 30-36
were faster reduced substrates to give major metabolites in the cofactor regeneration system.
When the kinetic parameters of prodrugs 1-36 with Ssap-NtrB are evaluated, it was
determined that prodrugs 28, 30 and 31 have higher k /K values compared to various
cat
M
known enzyme/prodrug combinations. Predicted reaction steps and possible metabolites of the
Ssap-NtrB catalyzed reaction for prodrug 6 were examined using HPLC and LC-MS/MS
analysis.
The cytotoxic activities of the compounds were evaluated using the MTT method. Three
different cell types were selected in these studies to evaluate the specific activities of
compounds. As expected, the cytotoxic properties of the prodrug candidates differ according
to the cell type. We found that human hepatoma carcinoma cells were more sensitive than
human prostate carcinoma and endothelial cells. Potential prodrugs which showed non-toxic
effect on HUVEC cells exhibited excellent toxicity against cancer cell models. We showed
that they may play crucial role as drug candidates in cancer therapy owing to better toxicity.
1
3
We observed that compounds 25-29, known as N ,N -bis(substitutedphenyl)-4,6-
dinitrobenzene-1,3-diamines which have different functional groups (-NO , -OH, -SO H, -Cl)
2
3
on two phenyl rings, had toxic effect at different concentations on various cell lines. In
addition, although prodrugs 28, 30 and 31 have higher k /K values, they weren’t analyzed
cat
M
in SRB reaction studies because of their toxicity for Hep3B cells (71.9 µM, 90.73 µM and
low toxicity, respectively).
The rest of the compounds were analyzed for prodrug potential in the presence of Ssap-
NtrB enzyme. The reaction components and conditions were optimized as mentioned in the
material-method section. The SRB method was carried out on PC3 cells to determine

504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
cytotoxic effect of compounds that reacted with the enzyme. In our study, compounds were
reacted with the enzyme outside of the cell. Then, the products (active metabolites) were
introduced into cells. The results were compared with CB1954 as positive control. Reactive
species released upon reduction showed excellent bystander effects with IC values indicated
50
in Table 3. Prodrugs (3, 14, 15, 19 and 22-24) exhibited high substrate activity for Ssap-NtrB
enzyme. 14/Ssap-NtrB, 22/Ssap-NtrB and 24/Ssap-NtrB combinations had high toxicity at
nanomolar concentration levels compared with CB1954. Although it was observed in HPLC
chromatogram that compounds (14, 22 and 24) interacted with Ssap-NtrB at low levels and
produced minor metabolites in limited reaction times, the cytotoxic effect of the enzyme
combinations of these compounds showed excellent activity on prostate cancer cells in 48 h.
There could be two reasons for these outcomes. The first reason is long reaction times (24 and
48 h) in SRB cell experiments can change the type and/or the ratio/concentration of
metabolites. Because of different experimental conditions in enzymatic and cytotoxic studies,
it is not always expected that these two experimental results will be consistent. The second
explanation is although the corresponding metabolites have low concentration, they can show
good toxic effect on cancer cells. So, it is difficult to predict which metabolite and/or
metabolites forming at what rate kills the cancer cells. Further mechanistic studies to better
understand and interpret these outputs are continuing in our research group.
With docking studies, prodrug 14 has twelve interactions including eight hydrogen bonds
with Arg13, Lys188, Ser43 residues and FMN as coenzyme, one electrostatic interaction with
Glu163 and three hydrophobic interactions with Val204 residue, FMN as coenzyme of the
target. On the other hand, compound 22 forms seven hydrogen bonds and four hydrophobic
interactions with different residues and FMN molecule of the same enzyme.
5
28
4. Experimental section
529
530
531
532
533
534
535
536
4.1. General information
All solvents and reagents were supplied from commercial sources such as Sigma-Aldrich,
Merck, Alfa Aesar or ABCR and used in the synthesis stage directly unless otherwise stated.
NADH and NADPH were purchased from Roche Applied Science. Ssap-NtrB was purified
using Nickel HiTrap™ column (Amersham Biosciences). 1,2-Diaminocyclohexane and 1,3-
diaminocyclohexane were used in reactions as cis- and trans- mixture. The melting point was
measured with X-4 Melting-point Apparatus. Analytical thin-layer chromatography was
performed on precoated Kieselgel 60GF254 plates (Merck), visualization of the spots was

537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
done by UV light (254 nm) and/or KMnO stain. Silica gel 60 (230-400 mesh, Merck) was
4
used for column chromatography. Infrared spectra (IR) were measured using ATR techniques
1
13
on a Perkin Elmer Spectrum 100 FTIR spectrophotometer. H and C spectra were recorded
on Varian Mercury 500 MHz and Jeol 400 MHz NMR High-Performance Digital FT-NMR
spectrometer with TMS as internal standard and DMSO-d6 as the solvent. The molecular
weights of prodrugs were determined using GC-MS-QP2010 Ultragas Chromatograph-Mass
Spectrometer and Shimadzu LC-MS/MS 8040 Liquid Chromatograph Mass Spectrometer
using an ESI source. Elemental analyses for C, H and N were recorded by Thermo, Flash 200
Organic Elemental Analyzer. Enzymatic activities were obtained by using Shimadzu UV-
3600 UV-VIS-NIR spectrophotometer and Molecular Devices Spectromax Plus 384
microplate reader. Metabolites obtained during enzymatic reductions were determined with
Shimadzu High Performance Liquid Chromatography. Enzymatic and cytotoxic graphs were
drawn by using the Origin Pro 8.5 program.
4.2. Synthesis and characterization of prodrugs 1-36
N-Ethyl-2,4-dinitroaniline (1). Prodrug 1 was synthesized from 2,4-dinitro-1-chlorobenzene
and ethylamine in methanol by using pyridine as base under reflux conditions for 3 h
according to the procedure described in [14]. Bright yellow crystal; 77% yield (lit. 85%) [14];
m.p. 128 °C (lit. m.p. 113 °C [14]; IR (ATR) ϑ 3340, 3280, 3085, 2917, 2850, 1619, 1591,
-1 1
1530, 1509, 1441, 1328, 1280, 1238, 1169, 1096, 1019, 926, 771, 727, 686 cm ; H NMR
(500 MHz, DMSO-d ) δ 8.81 (m, 2H), 8.21 (d, j=9.64 Hz, 1H), 7.19 (dd, j=9.65 and 1.6 Hz,
6
13
1H), 3.50 (p, j=7.08 Hz, 2H), 1.23 (td, j=7.15 and 1.8 Hz, 3H); C NMR (125 MHz, DMSO-
d6) δ 148.39, 135.01, 130.37, 129.96, 124.06, 115.55, 38.17, 14.22.
N-Isopropyl-2,4-dinitroaniline (2). The mixture of 2,4-dinitro-1-chlorobenzene (9.8 mmol;
2.0 g) and isopropylamine (49.0 mmol; 4.24 mL; 5.0 equiv.) in 5 mL DMSO was stirred at
room temperature for 24 h. Then, the mixture was poured into 100 mL water and the resulting
solid was filtered and dried in air. The crude product was purified by crystallization with ethyl
alcohol. Yellow solid; 2.0 g; 90% yield; m.p. 94 °C (lit. m.p. 95-96 °C [22]; IR (ATR) ϑ 3330,
3124, 3113, 2982, 2943, 1615, 1593, 1509, 1418, 1388, 1276, 1229, 1161, 1140, 916, 821,
-1 1
741 cm ; H NMR (500 MHz, DMSO-d ) δ 8.81 (d, j=2.74 Hz, 1H), 8.385 (d, j=7.64 Hz,
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1H), 8.225 (dd, j=9.645 and 2.76 Hz, 1H), 7.24 (d, j=9.73 Hz, 1H), 4.05 (m, 1H), 1.285 (d,
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3
j=6.38 Hz, 6H); C NMR (125 MHz, DMSO-d ) δ 147.67, 135.12, 130.50, 129.97, 124.12,
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115.87, 45.18, 22.23.

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N-Cyclohexyl-2,4-dinitroaniline (3). The mixture of 2,4-dinitro-1-fluorobenzene (25.0
mmol; 4.65 g; 3.14 mL), cyclohexylamine (25.3 mmol; 2.50 g) and triethylamine (25.1 mmol;
2.55 g) in 20 mL DMF was stirred at room temperature for 1 h. The mixture was poured into
200 mL water and the resulting solid was filtered and dried in air. Bright yellow solid; 3.98 g;
60% yield; m.p. 166 °C (lit. m.p. 151-153°C [22]; IR (ATR) ϑ 3351, 3114, 3081, 2944, 2891,
2857, 1619, 1586, 1515, 1420, 1330, 1263, 1256, 1234, 1138, 1089, 1050, 921, 830, 745, 715
-1 1
cm ; H NMR (500 MHz, DMSO-d ) δ 8.85 (q, j=1.25 Hz, 1H), 8.47 (d, j=7.67 Hz, 1H), 8.23
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1
3
(d, j=9.6 Hz, 1H), 7.30 (d, j=9.39 Hz, 1H), 3.79 (m, broad, 1H), 1.95-1.23 (m, 10H); C
NMR (125 MHz, DMSO-d ) δ 147.71, 135.20, 130.44, 130.0, 124.23, 116.03, 51.67, 32.02,
6
25.32, 24.43.
N-Phenyl-2,4-dinitroaniline (4). Prodrug 4 was synthesized from 2,4-dinitro-1-
chlorobenzene and aniline in DMSO in room temperature conditions for 24 h according to the
procedure described in [14]. Red needle crystal; 93% yield (lit. 88.7%) [14]; m.p. 158 °C (lit.
m.p. 156-157 °C [14]; IR (ATR) ϑ 3317, 3109, 3097, 1617, 1595, 1581, 1516, 1495, 1420,
-1 1
1322, 1253, 1144, 1122, 1058, 922, 844, 742, 684 cm ; H NMR (500 MHz, DMSO-d ) δ
6
10.15 (s, 1H), 8.88 (t, j=2.59 Hz, 1H), 8.21 (d, j=9.56 Hz, 1H), 7.51 (t, j=7.21 Hz, 2H), 7.40-
13
7.35 (m, 3H), 7.10 (dd, j=9.57 and 1.78 Hz, 1H); C NMR (125 MHz, DMSO-d ) δ 147.09,
6
138.13, 136.77, 131.62, 130.25, 127.41, 126.19, 126.14, 123.84, 117.28.
2,4-Dinitro-N-(3-nitrophenyl)aniline (5). The mixture of 2,4-dinitro-1-chlorobenzene (4.9
mmol, 1.0 g) and 3-nitroaniline (4.9 mmol, 0.676 g) in 20 mL DMSO was heated to 100 °C
for 26 h. At the end of this time, the mixture was poured into 200 mL water and the resulting
solid was filtered, washed with water and dried in air. The crude product was purified by
column chromatography on silica gel (CHCl ). Yellow solid; 1.095 g; 74% yield; m.p. 190-
3
-1 1
194 °C; IR (ATR) ϑ 3300, 3092, 1623-1481, 1517, 1334, 1128, 1063, 806, 738 cm ; H NMR
(500 MHz, DMSO-d ) δ 10.23 (s, 1H), 8.895 (d, j=2.72 Hz, 1H), 8.26-8.24 (m, 2H), 8.16-8.14
6
1
3
(m, 1H,), 7.88-7.85 (m, 1H), 7.76 (t, j=8.13 Hz, 1H), 7.30 (d, j=9.5 Hz, 1H); C NMR (125
MHz, DMSO-d ) δ 149.00, 146.00, 140.04, 137.84, 133.11, 131.87, 131.41, 130.21, 123.65,
6
121.26, 120.27, 117.98.
2,4-Dinitro-N-(4-nitrophenyl)aniline (6). NaH (5.43 mmol; 0.24 g; 1.1 equiv.; 55% oil
dispersion) was added to 5 mL DMF solution of 4-nitroaniline (4.94 mmol; 0.68 g) slowly in

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an ice bath and stirred at room temperature for 3 h. In the second step, 2,4-dinitro-1-
chlorobenzene (4.94 mmol, 1.0 g) was added to the mixture and and stirred at room
temperature for 24 h. Then, the mixture was poured into 100 mL water and the resulting solid
was filtered, washed with water and dried in air. The crude product was purified by
crystallization with heptane:ethyl acetate [23]. Yellow solid; 0.78 g; 52% yield; m.p. 178 °C
(lit m.p. 185-187°C) [24]; IR (ATR) ϑ 3338, 3110, 3073, 1616, 1581, 1502, 1442, 1426, 1324,
-1
1
1279, 1230, 1108, 1064, 840, 741 cm ; H NMR (500 MHz, DMSO-d ) δ 10.20 (s, 1H),
6
8.875 (d, j=2.74 Hz, 1H), 8.33 (dd, j=9.68 and 2.77 Hz, 1H), 8.29 (d, j=9.12 Hz, 2H,), 7.58
1
3
(m, 3H); C NMR (125 MHz, DMSO-d ) δ 145.80, 143.92, 139.13, 135.25, 130.01, 125.78,
6
123.48, 122.94, 119.85.
2,4-Dinitro-N-(3-methoxyphenyl)aniline (7). The mixture of 2,4-dinitro-1-chlorobenzene
(2.45 mmol, 0.5 g), 3-methoxyaniline (2.45 mmol, 0.30 g; 0.766 mL) and pyridine (0.24 mL)
in 10 mL DMSO was heated to100 °C for 6 h. At the end of this time, the mixture was poured
into 100 mL water and extracted with chloroform (3x30 mL). The organic layer was washed
with water (2x20 mL),dried over Na SO and concentrated in vacuo. The crude product was
2
4
purified by crystallization with ethyl alcohol. Orange solid; 0.31 g; 41% yield; m.p. 133-
134°C lit. m.p. 130-132°C [25]; IR (ATR) ϑ 3333, 3110-3079, 2944, 1620-1494, 1583, 1334,
-1 1
1361, 1263, 1124, 1033, 823, 742 cm ; H NMR (500 MHz, DMSO-d ) δ 10.09 (s, 1H), 8.88
6
(d, j=2.76 Hz, 1H), 8.23 (dd, j=9.58 and 2.7 Hz, 1H), 7.41 (t, j=8.01 Hz, 1H,), 7.18 (d, j=9.58
1
3
Hz, 1H), 6.98-6.92 (m, 3H), 3.78 (s, 3H); C NMR (125 MHz, DMSO-d ) δ 160.75, 146.93,
6
139.29, 136.85, 131.73, 130.97, 130.23, 123.79, 117.92, 117.61, 113.09, 111.61, 55.7.
N-(3,5-Difluorophenyl)-2,4-dinitroaniline (8). NaH (4.64 mmol; 0.202 g; 1.5 equiv.; 55%
oil dispersion) was added to 10 mL DMF solution of 3,5-difluoroaniline (3.09 mmol; 0.4 g)
slowly in an ice bath and stirred at room temperature for 2 h. In the second step, 5 mL DMF
solution of 2,4-dinitro-1-chlorobenzene (3.09 mmol, 0.63 g) was added to the mixture and and
stirred at room temperature for 20 h. Then, the mixture was poured into 100 mL water and the
resulting solid was filtered, washed with water and dried in air. Orange solid; 0.38 g; 42%
yield; m.p. 161-162 °C; IR (ATR) ϑ 3315, 3098, 1607, 1585, 1515, 1367, 1236, 1125, 921,
-1 1
833, 742, 718 cm ; H NMR (400 MHz, DMSO-d ) δ 10.03 (s, 1H), 8.86 (d, j=2.73 Hz, 1H),
6
1
3
8.265 (dd, j=9.49 Hz and J=2.77 Hz, 1H), 7.37 (d, j= 9.49 Hz, 1H), 7.18-7.14 (m, 3H); C
NMR (100 MHz, DMSO-d ) δ 164.50, 162.05, 145.49, 141.54 138.04, 133.34, 130.24,
6
,
+
123.55, 118.57, 108.45, 102.00; MS (m/e): 295 (100) (r.t: 18,4. min) (M , C H F N O ,
1
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4