PRODRUGS FOR NITROREDUCTASE BASED CANCER THERAPY- 2: Novel amide/Ntr combinations targeting PC3 cancer cells

Article abstract of DOI:10.1016/j.ejmech.2019.03.035

The use of nitroreductases (NTR) that catalyze the reduction of nitro compounds by using NAD(P)H in GDEPT (Gene-directed enzyme prodrug therapy) studies which minimize toxicity at healthy cells and increases concentration of drugs at cancer cells is remarkable. Discovery of new prodrug/NTR combinations is necessary to be an alternative to known prodrug candidates such as CB1954, SN23862, PR-104A. For this aim, nitro containing aromatic amides (A1-A23) were designed, synthesized, performed in silico ADMET and molecular docking techniques in this study. Prodrug candidates were studied on reduction potentials with Ssap-NtrB by HPLC system. Also, cyototoxic properties and prodrug ability of these amides were investigated using different cancer cell lines such as Hep3B and PC3. As a result of theoretical and biological studies, combinations of A5, A6 and A20 with Ssap-NtrB can be suggested as potential prodrugs/enzyme combinations at NTR based cancer therapy compared with CB1954/NfsB.

Full text of DOI:10.1016/j.ejmech.2019.03.035

Accepted Manuscript
Prodrugs for nitroreductase based cancer therapy- 2 novel amide/Ntr combinations
targeting PC3 cancer cells
Tuğba Güngör, Ferah Cömert Önder, Esra Tokay, Ünzile Güven Gülhan, Nelin
Hacıoğlu, Tuğba Taşkın Tok, Ayhan Çelik, Feray Köçkar, Mehmet Ay
PII:
S0223-5234(19)30253-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.035
EJMECH 11205
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 December 2018
Revised Date: 26 February 2019
Accepted Date: 14 March 2019
Please cite this article as: Tuğ. Güngör, Ferah.Cö. Önder, E. Tokay, Ü.Gü. Gülhan, N. Hacıoğlu,
Tuğ.Taşı. Tok, A. Çelik, F. Köçkar, M. Ay, Prodrugs for nitroreductase based cancer therapy- 2 novel
amide/Ntr combinations targeting PC3 cancer cells, European Journal of Medicinal Chemistry (2019),
doi: https://doi.org/10.1016/j.ejmech.2019.03.035.
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PRODRUGS FOR NITROREDUCTASE BASED CANCER THERAPY- 2
Novel Amide/Ntr Combinations Targeting PC3 Cancer Cells
a
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c
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Tuğba Güngör , Ferah Cömert Önder , Esra Tokay , Ünzile Güven Gülhan , Nelin Hacıoğlu ,
c, #
b
a*
d
Tuğba Taşkın Tok , Ayhan Çelik Feray Köçkar , Mehmet Ay
a
Department of Chemistry, Faculty of Sciences and Arts, Natural Products and Drug
Research Laboratory, Çanakkale Onsekiz Mart University, Çanakkale 17020, Turkey
Department of Molecular Biology and Genetic, Faculty of Sciences and Arts, Balıkesir
University, Balıkesir 10145, Turkey
b
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Department of Chemistry, Faculty of Science, Gebze Technical University, Gebze-Kocaeli
41400, Turkey
dDepartment of Chemistry, Faculty of Sciences and Arts, Gaziantep University, Gaziantep
27310, Turkey
*Corresponding author
a*
E-mail addresses: mehmetay06@comu.edu.tr; mehmetay06@yahoo.com (Mehmet Ay )
Çanakkale Onsekiz Mart University, Faculty of Sciences and Arts, Department of Chemistry,
Natural Products and Drug Research Laboratory, 17020 Çanakkale, Turkey.
#
Ayhan Çelik was left GTU after September, 2016.
≠
Patent pending in preperation

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ABSTRACT
The use of nitroreductases (NTR) that catalyze the reduction of nitro compounds by using
NAD(P)H in GDEPT (Gene-directed enzyme prodrug therapy) studies which minimize
toxicity at healthy cells and increases concentration of drugs at cancer cells is remarkable.
Discovery of new prodrug/NTR combinations is necessary to be an alternative to known
prodrug candidates such as CB1954, SN23862, PR-104A. For this aim, nitro containing
≠
aromatic amides (A1-A23) were designed, synthesized, performed in silico ADMET and
molecular docking techniques in this study. Prodrug candidates were studied on reduction
potentials with Ssap-NtrB by HPLC system. Also, cyototoxic properties and prodrug ability
of these amides were investigated using different cancer cell lines such as Hep3B and PC3.
As a result of theoretical and biological studies, combinations of A5, A6 and A20 with Ssap-
NtrB can be suggested as potential prodrugs/enzyme combinations at NTR based cancer
therapy compared with CB1954/NfsB.
Keywords: Nitro aromatic amides, Prodrug, Ssap-NtrB, Enzymatic activity, Cytotoxicity,
Molecular docking.
1. INTRODUCTION
Gene-directed enzyme prodrug therapy (GDEPT) have captured the attention of the
biology-medicine community since it limits toxicity at healthy cells and increases amount of
drug in cancer cells for cancer therapies [1]. GDEPT mainly consists of two steps. The first
step is transfer of the enzyme gene to the tumor cell by loading the carrier vector and
expression of this gene in the tumor cell [2, 3]. The second step is the conversion of nontoxic
prodrug to cytotoxic drug by enzyme catalysis. Finally, the active drug is transported to other
cancer cells with bystander effect [4].

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The use of nitroreductases (NTR) that catalyze the reduction of nitro compounds by
using NAD(P)H in GDEPT studies is remarkable [2]. Reduction with NTRs can be occured in
two types to form nitroso, hydroxylamine and amine metabolites [5]. Type I NTRs are oxygen
insensitive and catalayze two electron reduction of the nitro group, type II NTRs are oxygen
sensitive and performs single electron reduction.
5-Aziridinyl-2,4-dinitrobenzamide (CB1954), which is the prototype of the
dinitrobenzamide prodrug family, has been the most successful prodrug for nitroreductases of
GDEPT [2]. When CB1954 is reduced by NTR, 2-hydroxylamine and 4-hydroxylamine
metabolites occur at equal amounts [6]. While 4-hydroxylamine metabolite is more cytotoxic
and shows good bystander effect, 2-amino metabolite exhibits better diffusion and higher
stability than the 4-hydroxylamine metabolite [7-9]. It is known that 2-hydroxylamine
metabolite provides inhibitory effect on the growth of cancer cells [10, 11]. The efficacy of
CB1954 in GDEPT system has been exhibited by clinical studies on prostate cancer and liver
cancer [12,13]. However, due to the hepatotoxic effects of the prodrug CB1954 and low
efficiency of E. coli NTR on CB1954, clinical trials were terminated [14,15], emphasizing the
need for better prodrug alternatives and new nitroreductase enzymes [16-23]. A mustard
derivative of CB1954, SN23862 (5-[N,N-bis(2-chloroethyl)amino]-2-hydroxyamino-4-
nitrobenzamide) has higher K and k values and better bystander effect as compared to
M
cat
CB1954 and is more cytotoxic when interacts with NfsB (E.coli nitroreductase) [24, 25]. The
limitation at in vivo studies of first generation of dinitrobenzamide mustards (DNBM) due to
their low solubility, DNBM phosphate esters have been developed as pre-prodrug. These
compounds (e.g. PR-104) were firstly converted to corresponding prodrug alcohol (e.g. PR-
104A) with systemic phosphate effect and the next step is reduction of prodrugs by NfsB to
give active drugs (e.g. PR-104H) [26, 27] PR-104A has been found more cytotoxic in
hypoxic and aerobic conditions against different cancer cells when compared to other DNBM

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compounds. Preclinical studies of PR-104A has been successful and proceeded to clinical
studies [28] (Figure 1). Later, it was discovered that PR-104 had a myelotoxic effect and
clinical trials did not proceed further [29]. However, new studies continue to be conducted on
the second generation of PR-104A analogues for the treatment of cancer [15, 19, 31].
Design and synthesis of nitro containing compounds as fluorescent imaging probes for
NTR detection and diagnosis of tumor cells at hypoxic conditions have come to the fore in
recent years [31-34].
8
4
NO2
NO2
NO2
CONH2
CONH2
H
85
OH
N
O N
2
O N
2
O N
2
N
N
O
8
6
N
Cl
Cl
Br
OSO CH3
2
87
CB1954
SN23862
PR-104A
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9
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9
9
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0
1
2
3
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5
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Figure 1. The structures of a few reported prodrug candidates
Also it is known that benzamide derivatives have superior pharmacological properties
and used as drugs (vismodegib, sonidegib, taladegib, parsalmide, tiapride etc.) in the
treatment of various diseases [35-38]. In this study, nitro-containing amide prodrugs were
designed on the basis of model prodrug CB1954, SN23862, PR-104A and other known
benzamides.
In our previous work, we synthesized three benzamide and one hydrazide derivative as
nitro bearing prodrug and investigated their enzymatic activities on Ssap-NtrB a new
nitroreductase developed by our research group [18], antiproliferative effects on different
cancer cells [39]. Promising results prompted us to diversify the nitro containing amides (23
compounds) and investigate more amide prodrugs to be used for Ntr-based cancer therapies.
Four different amide core structures such as phenylacetamide (A1-A4),
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4-(4-nitrobenzoyl)morpholine/piperidine (A5-A6), N-substitutedphenyl benzamide (A7-A14),
bis(4-nitrobenzamide) (A15-A23) were designed in this study. The main reason selecting
these skeletons is to discover the most active structure among these four type compounds that
more interacting with the Ssap-NtrB enzyme through different pharmacophore groups. The
effects of -Br and -NO groups at different positions in phenylacetamide (A1-A4), morpholine
2
or piperidine heterocycles in A5-A6, -NO , -Cl, -CH , -N(CH ) , -N(n-Bu) functional groups
2
3
3 2
2
at different positions in N-substituted phenylbenzamide (A7-A14) and two benzamide groups
and various linkers such as -Et, -Bu as short/long alkyl chains, piperazine as a heterocyclic
group, 1,2-, 1,3-, 1,4- cyclohexyl, phenyl as alicylic/aromatic rings at bis(4-nitrobenzamide)
(A15-A23) were investigated. Herein, we report synthesis data, enzymatic and cytotoxic
results of a series of nitro containing amide prodrugs with both experimental and in silico
methods. Detailed structure-activity relationship (SAR) results were also discussed (Figure 2).
a) our previous work: 4 prodrug
O
O
NO2
NO2
H
N
N
N
H
H
O N
2
O N
2
NO2
2-nitro
4
-nitro
,4-dinitro
2
b) this work: 23 prodrug
R6
O
O
O
O
O
O N
N
O
2
N
R7
2
R1
N
N
N
N
H
H
H
R5
X
R2
O N
2
R4
O N
NO2
2
R3
NO2
1
4
7
R : -H, -Et
R : -H, -CH N(CH3)2, N(Bu)2
R : Et, Bu, piperazine,
1,2- 1,3- 1,4-cyclohexyl,
1,2- 1,3- 1,4-phenyl
3,
X: C or O
2
5
R : -H, -Br
R : -H, -NO
2
3
6
R : -H, -NO
R : -Cl, -NO
2
2
A1-A4
A5-A6
A7-A14
A15-A23
1
14
15
1
Figure 2. An overview of amide based prodrug structures studied (a) previously [39] and (b)
1
1
1
16
17
18
in this work

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2. RESULT AND DISCUSSION
2.1. Chemistry
All of the known products were confirmed by comparison of their spectral data with
those reported in the literature. The prodrugs were fully characterized by their melting point,
1
13
FT-IR, H NMR, C NMR and MS spectras.
A1 and A3 prodrugs were synthesized from aniline derivatives via acetylation reaction in
good yields 92% and 81%, respectively. Prodrugs A2 and A4 were obtained from A1 or A3
and ethyl iodide at the conditions that used NaH as base in DMF solvent at room temperature
in 42 % and 82 % yields, respectively [40] (Figure 3).
O
O
O N
2
NH2
R2
Ac
2
O/Pyridine
or
O N
2
N
O N
2
N
EtI, NaH
DMF, r.t.
H
R2
R2
Ac O/AcOH:H SO
4
2
2
R3
R3
R3
2
3
R :- H, R : -NO
A1 (92%)
A3 (81%)
A2 (42%)
A4 (82%)
2
2
3
R :- Br, R : -H
1
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Figure 3. Synthesis of A1-A4
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As revealed in Figures 4 and 5, different reaction conditions were applied to obtain A5-
A23 prodrugs by amidation reaction. Prodrugs A7-A14 were synthesized from different
aniline derivatives (4-chloroaniline, 4-nitroaniline and 2,4-dinitroaniline) and various 3,5-
dinitrobenzoic acid derivatives that contain some functional groups like as -H, -CH3,
-N(CH ) , -N(n-Bu) at 4-position by using DCC and DMAP at Steglish esterification
3
2
2
conditions (Figure 4).

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R6
O
O
O N
2
R6
N
O N
2
DCC/DMAP
R4
H
OH
R5
+
R4
DCM, r.t., 12-24h
H N
2
NO2
A7-A14
R5
NO2
4
5
6
R : -H, R , R : -NO
A7 (38%)
A8 (20%)
A9 (48%)
A10 (74%)
A11 (53%)
A12 (50%)
A13 (79%)
A14 (67%)
2
4
5
6
R : -CH , R :-H, R : -NO
3
2
4
5
6
R : -CH , R , R : -NO
3
2
4
5
6
R : -N(CH ) , R :-H, R : -Cl
3
2
4
5
6
R : -N(CH ) , R :-H, R : -NO
2
3
2
4
5
6
R : -N(CH ) , R , R : -NO
2
3
2
4
5
6
R : -N(n-Bu) , R :-H, R : -Cl
2
4
5
6
R : -N(n-Bu) , R :-H, R : -NO
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Figure 4. Synthesis of A7-A14
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A5 and A6 were synthesized from 4-nitrobenzoyl chloride and piperidine (A5) or
morpholine (A6) according to known methods with some modifications (Figure 5). Prodrugs
A15-A23 were obtained from 4-nitrobenzoyl chloride (1.0 equiv.) and various aliphatic,
aromatic or heterocyclic diamino compounds (2.0 equiv. or more) at traditional conditions or
by using microwave irradiation (Figure 5). Prodrugs A15-A17 were prepared from
4-nitrobenzoyl chloride and diamino derivatives (ethylenediamine, 1,4-diaminobutane or
piperazine) with triethylamine in DMF at room temperature or 50 ˚C for 5-6 h in moderate
yields (Figure 5). Prodrugs A18-A20 were synthesized by using 1,2-, 1,3- or
1,4-diaminocyclohexane with triethylamine in chloroform at reflux temperature for 8-14 h in
65-75% yields (Figure 5). Prodrugs A21-A23 were obtained from 1,2-, 1,3- or
1,4-diaminobenzene with DMAP in pyridine at 70 °C by using microwave energy for 20-25
min (Figure 5).

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O
H
N
N
X: C
X: O
A5 (63%)
A6 (90%)
O
X
X
O N
2
Cl
i, ii, iii or iv
+
O N
2
O
O
R7
R7
H N
2
NH2
N
N
H
H
O N
2
NO2
A15-A23
7
H N
NH2 H N
N H
A17 (55%)
NH2
NH R NH :
2
2
2
NH2 H N
2
A15 (50%)
A16 (62%)
NH2
NH2
NH2
NH2
NH2
H N
2
NH2
NH2
NH2
NH2
NH2
A18
A19
A20
(75%)
A21
(50%)
A22
(65%)
A23
(71%)
(
70%) (65%)
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1
50
51
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Figure 5. Synthesis of A5, A6 and A15-A23
(i. K CO /MeCN/reflux/6h (for A5); ii. Et N/DCM/reflux/8h (for A6); iii. Et N/DMF/r.t or 50
2
3
3
3
°C/5-6h (for A15-A17); iv. Et N/CHCl /reflux/8-14h (for A18-A20); v. Microwave
3
3
irradiation/DMAP/pyridine/70 °C/20-25 min (for A21-A23)
Many of the products (especially A15-A23) had very low solubility in common organic
solvents resulting in difficulties at the purification steps of the corresponding products.
Therefore, purification was carried out by crystallization or hot and/or cold washing with
solvents that have different polarity.
2.2. In silico process
Generally, in silico methods including computational calculation and molecular modeling
techniques are used to design and develop an overview and/or new solutions in biomedical
applications. So those, in silico ADMET and molecular docking studies were performed to
support the evaluation and illumination of this study.

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2.2.1. ADMET studies
Understanding of ADMET properties of prodrug candidates are immensely essential
process to solve or eliminate the undesired pharmacokinetics and toxicitiy effects. Therefore,
the entitled compounds were selected and drug-like properties were determined according to
Lipinski (rule of five) [41] and Veber rules, [42] (Table 1). Two compounds, A13 and A14
which are remarked as red color in Table 1 were removed from the data since they violated
Veber and Lipinski rules. After this part, ADMET parameters were calculated by using DS
2017, [43] as given in Table 2. Additionally, ADMET plot was built by using calculated
AlogP98 versus PSA_2D properties, indicating the confidence level of the predictions for the
Blood Brain Barrier Penetration (BBB) model and the Human Intestinal Absorption (HIA)
model (Figure 6). This plot represents the two analogous 95% and 99% confidence ellipses
corresponding to HIA and BBB models. PSA value of any compound has an inverse
relationship with human intestinal absorption value of any compound and so cell wall
permeability [44]. AlogP98 is shown to lipophilicity the fact that this value is a raito, used to
estimate hydrophilicity and ahydrophobicity. For this reason, the information of H-bonding
characteristics as obtained by calculating PSA could be taken into consideration along with
AlogP98 calculation [45]. The 95% confidence ellipse indicates the region of chemical space
including well-absorbed compounds (≥90%). However 99% confidence ellipse demonstrates
the region of chemical space that includes the compounds with excellent absorption through
cell membrane. As it is well known, any compound with an optimum cell permeability should
2
follow these criteria (PSA < 140 Å and AlogP98 < 5) [45]. The compounds (A1-A6, A10,
2
A17, SN23862 and CB1954) showed polar surface area (PSA) < 140 Å , except compounds
(A7-A9, A11-A12, A15-A16 and A18-A23) that have also in turn violated the 99% and 95%
confidence ellipse for both HIA and BBB (Figure 6). When we consider the AlogP98 criteria,
all compounds had AlogP98 value <5. In Table 2, absorption level of most of the compounds

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is 0 which indicates a very good human intestinal absorption except compounds (A7-A9,
A11-A12, A15,A16, A18-A23) all have fallen outside the 99% absorption ellipse (level 2 and
3; poor or very poor). Compounds SN23862, CB1954, A17 and A10 also poses moderate
HIA value, level 1. The aqueous solubility has an important role in the bioavailability of the
optimal drugs, with the exception of compounds A3-A6, A17, SN23862 and CB1954 that had
good aqueous solubility level (level 3) as referred in DS 2017, all other compounds had low
but possible aqueous solubility levels such as level 2. Table 2 revealed that generality of the
compounds had undefined values for BBB penetration with the exception of compounds A2,
A3, A4, A5 and A6 that had medium BBB penetration level. On the other hand, all
compounds had predicted hepatotoxicity values. Our results indicate that all compounds were
found toxic to liver. Similarly, all ligands exhibited satisfactory effects with respect to
CYP2D6 liver (with reference to DS 2017), suggesting that compounds are inhibitors of
CYP2D6 (Table 2). This demonstrates that all studied compounds were not metabolized well
in Phase-I metabolism. Finally, the ADMET plasma protein binding property prediction
remarked that the compounds (A1-A6, A10, A17, SN23862 and CB1954) had binding ≥ 90%,
respectively, (refer to DS 2017), clearly suggesting that compounds (A1-A6, A10, A17,
SN23862 and CB1954) have good bioavailability and are highly bound to carrier proteins in
the blood.

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08 Table 1. The Lipinski and Veber rules analysis of the compounds (A1-A23) and reference compounds (CB1954 and SN23862) for Ssap-NtrB.
AlogP
≤5)
MW
(≤500 g/mol)
MPSA
(≤140 A )
HA_Lipinski
HD_Lipinski
NRB
(≤10)
HA_Veber
HD_Veber
Comp.
2
(
(≤10)
(≤5)
(≤12)
(≤12)
A1
A2
0.739
1.293
1.593
2.147
2.055
0.826
2.192
2.784
2.678
3.23
225.158
253.211
259.057
287.11
120.73
8
8
1
0
1
0
0
0
1
1
1
1
1
1
1
1
2
2
0
2
2
3
4
5
5
3
3
3
4
9
7
9
6
8
10
6
8
6
6
6
6
6
1
0
1
0
0
0
1
1
1
1
1
1
1
1
2
2
0
2
2
111.94
74.92
A3
5
2
A4
66.13
5
3
A5
234.251
236.224
377.223
346.252
391.249
364.741
375.293
420.291
448.900
459.452
358.306
386.359
384.343
412.396
412.396
66.13
5
2
A6
75.36
6
2
A7
212.37
166.55
212.37
123.97
169.8
14
11
14
9
6
A8
5
A9
6
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
5
2.46
12
15
9
6
2.354
5.887
5.117
1.683
2.326
1.913
3.114
2.72
215.61
123.97
169.8
7
11
12
7
12
10
10
10
10
10
149.83
149.83
132.25
149.83
149.83
9
4
6
6

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A20
A21
2.844
3.188
3.188
3.188
0.601
2.113
412.396
406.348
406.348
406.348
252.184
351.143
149.83
149.83
149.83
149.83
137.73
137.97
10
10
10
10
9
2
2
2
2
2
2
6
6
6
6
4
8
6
6
6
6
6
6
2
2
2
2
1
1
A22
A23
CB1954
SN23862
9
2
2
2
2
2
09 Footnote: Comp, compound; ALogP, octanol/water partition coefficient, a measure for lipophilicity; MW, molecular weight; MPSA, molecular
10 polar surface area; HA_Lipinski, Number of hydrogen bond acceptors; HD_Lipinski, Num_H_Donors_Lipinski; Number of hydrogen bond
11 donors; NRB, Number of Rotatable bonds; HA_Veber, Number of hydrogen bond acceptors based on Veber; HD_Veber, Number of hydrogen
12 bond donors based on Veber.
13
2
2
2
2
14 Table 2. In silico ADMET analysis of the 21 compounds and reference compounds (CB1954 and SN23862) for Ssap-NtrB. The entitled
15 compounds were divided into three parts and ordered from right to left and from top to bottom on the plot. The compounds (A7-A9, A11-A12,
16 A15-A16 and A18-A23) outside of the all ellipses in the ADMET plot were colored grey. In the second part, compounds (A1-A2, A10, A17,
17 SN23862 and CB1954) were colored blue. In the last part, compounds (A3-A6) inside of all ellipses in the graphic were shown red.
2
Comp.
A1
PSA_2D(< 140 Å )
115.757
AlogP98(< 5)
0.739
HIA
Solubility
3(good)
3(good)
3(good)
3(good)
3(good)
BBB
CYP2D6
false
Hepatotoxic
true
PPB
true
true
true
true
true
0(good)
0(good)
0(good)
0(good)
0(good)
4 (undefined)
2(medium)
2(medium)
2(medium)
2(medium)
A2
106.299
1.293
false
true
A3
72.934
1.593
false
true
A4
63.476
2.147
false
true
A5
63.476
2.055
false
true

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A6
A7
72.406
201.403
158.58
0.826
2.192
2.784
2.678
3.23
0(good)
3 (very low)
3 (very low)
3 (very low)
1 (moderate)
3 (very low)
3 (very low)
2 (low)
3(good)
2(low)
2(low)
2(low)
2(low)
2(low)
2(low)
3(good)
3(good)
3(good)
2(low)
2(low)
2(low)
2(low)
2(low)
2(low)
3(good)
3(good)
2 (medium)
4(undefined)
4(undefined)
4(undefined)
4(undefined)
4(undefined)
4(undefined)
4(undefined)
4(undefined)
4(undefined)
4(undefined)
4(undefined)
4(undefined)
4(undefined)
4(undefined)
4(undefined)
4(undefined)
4(undefined)
false
false
false
false
false
false
false
false
false
false
false
false
false
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
false
false
false
true
A8
A9
201.403
119.109
161.932
204.755
145.868
145.868
126.952
145.868
145.868
145.868
145.868
145.868
145.868
132.839
132.839
A10
A11
A12
A15
A16
A17
A18
A19
A20
A21
A22
A23
CB1954
SN23862
2.46
false
false
false
false
true
2.354
1.683
2.325
1.914
3.114
2.72
2 (low)
1(moderate)
2 (low)
false
false
false
false
false
false
true
2 (low)
2.844
3.188
3.188
3.188
0.601
2.113
2 (low)
2 (low)
2 (low)
true
2 (low)
true
1(moderate)
1(moderate)
true
true
true
2
2
2
2
18
19
20
21
Abbreviations: PSA, polar surface area; AlogP98, the logarithm of the partition coefficient between n-octanol and water; HIA, Human Intestinal Absorption,
BBB blood brain barrier; CYP2D6 cytochrome P450 2D6 binding, false for CYP2D6: non-inhibitor; True for Hepatotoxic: toxic; PPB plasma protein binding,
more than 90% for PPB value is true: Chemicals strongly bound. Less than 90% for PPB value is false: Chemicals weakly bound.
2
22

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38 Figure 6. ADMET plot for various nitro amide derivatives as anticancer prodrugs

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2.2.2. Molecular docking results
According to the biological activity studies and ADMET analysis, the selected five
compounds (A2, A5, A6, A17 and A20) were docked with Ssap-NtrB model. In the
meantime, CB1954 and SN23862 as reference compounds were redocked with Ssap-NtrB
model which was modelled in our previous study [39]. The results of redocking revealed that
obtained results were compatible with the literature [18, 39].
Based on the redocking process, molecular docking was applied between the chosen three
compounds and Ssap-NtrB model. To clarify the binding forms of the chosen compounds,
binding affinities were also predicted by using molecular docking. Furthermore, interactions
of each compound-enzyme complex were defined and the interactions that are dominant or
not in active site of the enzyme model were specified. As shown in Figure 7, Compound A5
formed two hydrogen bonds with A: Arg13 (2.068 Å), B: Ser43 (1.89 Å) and one
hydrophobic interaction with FMN (4.34 Å). On the other hand, compound A6 was shown to
have also same interactions with compound A5 except that the same nitro group in compound
A6 showed hydrogen bond interaction with B: Val44 instead of B: Ser43 residue of the
enzyme. The compound A2 has a similar interaction with SN23862 as the reference
compound. The other ones, A17 and A20 have different framework than others, but bound on
the active site of the enzyme. Compound A17 formed two hydrogen bond with B: Ser43 (2.79
Å), B: Pro41 (2.75 Å) and one π-π stacking with FMN like compound A5. However, it was
shown to have unfavorable interactions with A: Phe162, A: Val204 and B: Pro41 residues that
are displayed as red dash line and spheres in Figure 7. The last compound A20 was shown to
have hydrogen bonds and hydrophobic interactions with different residues of the binding site
in Ssap-NtrB. It was also observed that prodrug A2, A5 and A20 formed stronger
hydrophobic interactions with hydrophobic pocket than other compounds A6 and A17. The
binding forms are demonstrated that the para nitro and carbonyl groups in phenyl ring were

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able to affect the interaction on Ssap-NtrB significantly. Interaction types and distances of the
2
studied compounds and standard prodrugs were given in SI-Table S1.

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2
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77
78 Figure 7. Two-dimensional docking poses of compound A5 (A); compound A6 (B); compound A20 (C) in the binding site of Ssap-NtrB
79 *Hydrogen bonds were shown with yellow dash lines and dark green spheres. The pink sphere and dash lines presented cofactor FMN and pi-pi
80 stacked.The hydrophobic interactions which is van der Waals were represented by light green spheres. Unfavorable interactions like steric bumps
81 were displayed as red dash line and spheres.

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Additionally, binding energy (∆G) and inhibition constant (Ki) values of the compounds
were computed using DS 2017 program are represented in Table 3. The molecular docking
results showed that compounds A5, A17 and A20 displayed lower binding energy values and
higher affinities for target enzyme than did CB1954 and SN23862. Topological polar surface
area (TPSA) [46] and AlogP [47] of each compound was also computed using DS 2017
demonstrating the membrane permeability and lipophilicity of the compounds, respectively.
The results are exhibited in Table 3. These parameters are very important properties for drug
2
discovery. If TPSA value is greater than 140 Å , a compound has no enough level for
membrane permeability. The obtained values of the compounds (152.959-257.443) except A5
2
(130.459) are greater than 140 Å . These results reveal that A5 compound can be a suitable
prodrug, having hydrophobic property, good membrane permeability and specifically
targeting Ssap-NtrB.
Table 3. The binding energy (∆G), inhibition constant (Ki), Topological polar surface area
(TPSA) and ALogP values of the compounds and CB1954 and SN23862 as standard prodrugs
with that of Ssap-NtrB.
Prodrugs
A2
∆G (kcal/mol)
-6.98
Ki (µM)
7.67
TPSA
AlogP
1.293
2.055
0.826
1.913
2.844
0.601
2.113
224.312
130.459
152.959
257.443
253.935
213.711
195.360
A5
-7.56
2.85
A6
-6.88
9.06
A17
-7.95
1.49
A20
-7.05
6.82
CB1954
SN23862
-7.13
5.96
-7.01
7.28
2
2
98
99

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2.3. Enzymatic reduction via Ssap-NtrB
2.3.1. Metabolite profiles of prodrugs A1-A23
Initial activation screening of all synthesized amide prodrugs A1-A23 by Ssap-NtrB was
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310
performed in accordance with the assay condition described in the experimental section.
Reduction profiles of the prodrugs A1-A23 were assessed according to HPLC
chromatograms.
All the prodrugs except A10, A13-A14, A16-A17 and A20-A23 were readily reduced by
Ssap-NtrB with half-lives of up to 3 hours. However, there were some product formation
upon longer reaction times of A10, A13-A14, A17 and A20-A21 with Ssap-NtrB in cofactor
regeneration system (Table 4).
Table 4. Ssap-NtrB activation of compounds A1-A23
Compound
Retention
time
Half life
t (h)*
Amount of
product
Retention
times of major
metabolites
(min)
HPLC
program
***
1
/2
(
min)
after 10 min
(%)**
A1
A2
14.4
< 0.1
88
8.15, 12.06,
I
15.84
14.55
<0.02
<0.02
< 0.2
< 0.1
100
100
56.4
78
11.65
I
II
I
1
6.85
11.8, 14.2
7.12, 15.74
12.28, 15.55,
A3
A4
13.88
15.18
I
18.39
A5
A6
18.22
< 1.5
<0.02
< 0.25
5.57
100
15.99
14
I
II
I
1
7.81
15.17
65.4
11.71

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A7
A8
14.51,
7.52
18.04
<0.37
23
9.76
I
1
< 0.38
< 0.26
< 0.23
22
15.94
I
II
I
2
3.93
8.07
32.96
36.6
20.16, 20.79
14.45, 17.61,
15.35
1
A9
2
1
2
4.11
9.22
5.86
< 0.18
48
-
17.44, 21.6,
II
I
23.88
-
-
A10
A11
< 3.15
< 0.3
2.65
28
17.02, 20.14,
2.67
15.83, 16.24,
6.84
II
I
2
18.43
1
1
8.51
< 0.42
< 0.1
20.3
90.1
2.97, 11.5
I
A12
A13
A14
A15
A16
24.85
12.04, 19.19,
II
2
1.58
20.12
-
< 2.4
-
-
3.6
-
-
I
II
I
3
4,2
29.05
26.5
-
3
1.96
< 1.56
< 2.54
< 1.2
-
5.34
6.57
7.2
-
28.34
II
II
I
17.56
10.39, 14.06
1
8.65
19.02
7.28
15.8
-
-
I
1
-
-
I

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A17
A18
A19
17.6
< 1.42
5.9
11.53, 14.47,
4.85
II
II
1
2
0.85
< 1.8
9.33
15.55, 17.99,
17.7
2
0.91
< 0.52
< 0.26
16.2
32.7
19.43
I
1
9.40,
20.15
9.44,
0.6
19.96
9.2
22.08
1.38
21.98
1.88
16.61
14.03, 14.58,
16.83, 17.4
17.87
II
1
< 2
4.25
I
2
A20
A21
A22
A23
-
-
-
I
II
II
I
1
< 1.5
11.2
13.48
< 3.2
5.25
16.49
2
-
-
-
-
-
-
-
-
-
-
-
-
II
I
2
I
3
3
3
3
3
3
3
3
3
3
3
3
11
12
13
14
15
16
17
18
19
20
21
22
*approximate time needed for reduction of the substrate amount up to 50% upon Ssap-NtrB
activation.
**Percent of products produced upon reduction of the substrate by Ssap-NtrB (initial amount of
substrate was taken as 100%). In a typical solution, 50-100 µM substrate was dissolved in 25 mM, pH
7.5 Tris/Cl buffer with 5-10% DMSO concentration and incubated with NADH (200-500 µM) and
Ssap-NtrB (2.5-30 ug/mL) at 21-25°C. The amounts were calculated from the HPLC chromatogram
peak areas as a function of time.
*** HPLC program I: 0-5 min at 20% ACN, 15-22 min at 80% ACN, 27-32 min at 20% ACN
HPLC program II: 0-5 min at 20% ACN, 25-35 min at 80% ACN, 40-45 min at 20% ACN, used for
coupled reaction analysis.
A2, A5, A7-A10, A12-A15, A17-A22 reactions with Ssap-NtrB were monitored against
time by using cofactor regeneration system with recombinant formate dehydrogenase (FDH)
from Candida methylica (CmFDH) (Figure S59). In cofactor regeneration system reactions,
the FDH enzyme converts NAD to NADH, thus sufficient amount of NADH will be available
3
3
3
23
24
25

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for nitroreductase reduction reactions. By this coupled enzyme system (Ssap-NtrB and FDH),
the time dependent profile of reactions can be monitored for longer periods without the
limititation of the cofactor since Ssap-NtrB is a NAD(P)H dependent enzyme and needs
NAD(P)H in order to convert the substrate into its products.
HPLC results indicated that A2, A5, A6 and A19 were faster reduced by Ssap-NtrB while
A2 was the fastest reduced substrate by Ssap-NtrB with only around one minute half-life.
100% conversion of the parent compound were observed in A2/Ssap-NtrB reaction and
A5/Ssap-NtrB coupled reaction involving cofactor regeneration (Table 4). For A2/Ssap-NtrB
reaction, multiple product peaks were detected at 11.8 min and 14.2 min after coupled
reaction while only one product were detected from the reduction reaction with limited
amount of cofactor (Fig. 8 and SI). Similarly, there were two metabolites produced upon A5
/Ssap-NtrB-FDH coupled reaction since there was only one metabolite produced after 10
minutes of reaction with limited NADH. This states that the amount of NADH was not
sufficient for the reduction reaction to take place until the last step. However, when there was
enough cofactor, all the A5 substrate (100%) was reduced to its metabolites (14.0 and 22.3
min retention times).
The product profiles of reduction reactions exhibited time-dependent properties. As an
example, the release of products from Ssap-NtrB/A2 reaction as a function of time is shown
in Figure 8. Products’ profiles versus time shown in Figure 8B and 8C indicated that five
products were observed after enzymatic reduction with retention times of 11.8 min, 14.2 min,
21.2 min, 8.0 min and 15.2 min. At the end of 2 min reaction time, all of the product A2 was
converted to corresponding metabolites. The main product P1 formation increased with time
upon the increased production of P2 product. At the end of 30 min, there were no P1 and P3
products stable, predicted to be converted to prodrugs P4 and P5.

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51
3
Figure 8. A) HPLC chromatogram of reduction reaction of prodrug A2 catalyzed by Ssap-
NtrB, (a) Control without Ssap-NtrB, (b) Enzymatic reduction reaction at 5 min, (B) HPLC
chromatogram of reactions, (C) Time dependent product profiles.
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Aromatic nitro compounds have been indicated to be reduced by different nitroreductases
[48]. Dinitrobenzamide derivatives such as CB1954 and derivatives, SN23862 and PR-104
[24, 25, 26] have been approved to be effective against different nitroreductases especially
E. coli nitroreductases. In this study, nitroaromatic prodrug candidates A1-A23 were
synthesized and their probability of reduction by a different nitroreductase (Ssap-NtrB) was
examined. Among amide derivatives, A1-A9, A11-A12, A15 and A18-A19 compounds were
easily reduced while Ssap-NtrB was not reactive for A16, A22 and A23 compounds even after
long reaction times with cofactor regeneration system.
2.3.2. Kinetic studies
Due to low solubility of the compounds, not all compounds were able to be assessed to
undergo reduction with Ssap-NtrB. Steady-state kinetic experiments were performed for
compounds A1 and A2 with calculating specific activities of a range of prodrug
concentrations (0-800 µM) dissolved in 5% DMSO. Catalytic efficiencies (k /K ) of Ssap-
cat
M

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-1
-1
-1
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NtrB with A1 and A2 were calculated as 98284 s M and 117278 s M respectively. When
compared with the catalytic activity of well-known CB1954/E.coli NfsB combination, Ssap-
NtrB/A1 and Ssap-NtrB/A2 were found to be much more effective (17.55 and 20.94 fold
respectively) representing to be better NTR/prodrug combinations for cancer therapy (Table
5). In addition, the catalytic efficiencies of our NTR/prodrug combinations are higher than the
NfsB/PR-104A combination, which was found to be more effective alternative prodrug than
CB1954 [19].
Since there are many mechanisms of reduction in the living cells, it is not possible to
assess all enzymatic reduction modes in vitro in tubes. Thus, further in vitro cell culture
experiments were conducted in order to see the effect of prodrugs and reduction products in
cancer cell lines.
Table 5. Comparison of kinetic parameters of nitroreductases toward CB1954, SN23862 and
prodrugs A1-A23
Relative to
-1
-1
-1
Enzyme
Substrate
kcat (s )
K (µM)
M
k /K
cat M
(s M )
Cofactor
CB1954/NfsB
NfsB
NfsB
CB1954
62 ± 11
11000 ± 2600
2500 ± 100
5600
10560
1
NADH
NADH
SN23862
26.4 ± 7.2
1.89
NfsB
PR-104A
60
4500
13000 ±7800
2.32
NADH
Ssap-NtrB
Ssap-NtrB
Ssap-NtrB
Ssap-NtrB
CB1954
SN23862
A1
2.26 ± 0.07 1065.2 ± 53.1
2120
9350
0.38
1.67
NADPH
NADPH
NADPH
NADPH
0.8 ± 0.1
9.9 ± 0.7
1.37 ± 1.1
82.4±16.9
100.6 ±17.2
96.9 ± 21.4
98284
117278
17.55
20.94
A2
3
80
3
81
82
2.4. Cytotoxic activity
2.4.1. Growth inhibitory effects of amides
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383
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385
Prodrug candidates were evaluated for cytotoxic properties against to Human Hepatoma
(Hep3B), Human Prostate Cancer (PC3) cells and Human Umbilical Vein (HUVEC) cells as a
healthy control model. The cells were treated with five different concentrations (150, 75, 39,

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18 and 9 µM) of prodrugs and exposed for 48h. After 48h exposure, the cell viability results
were obtained by using MTT method. % cell viability was calculated using
spectrophotometric absorbance value at 550 nm wavelength. The results were divided into
three groups: NT: Not Toxic (% Cell viability value > 80%; LT: Low Toxic (80% > % cell
viability value > 51%) and T: Toxic (% Cell viability value < 50%). For toxic compounds,
IC (50 % inhibition concentration) value was determined using Origin pro 8.5 program.
5
0
As shown in Table 6, A1, A3-A6, A8, A11 and A16-A22 were not toxic; A2, A9, A15, and
A23 were low toxic; A7, A10 and A12-A14 were toxic with 51.76 µM, 110.64 µM, 43.74
µM, 11.62 µM and 30.87 µM of IC values, respectively against Hep3B cells. For HUVEC
5
0
cells, especially A13 and A14 were toxic with 22.60 µM and 22.24 µM of 50% inhibition
concentration. A7 and A10 were low toxic. The rest of the compounds showed no toxic effect
against HUVEC cells as chosen a healthy model. A10, A13, and A14 compounds inhibited
50% cell proliferation as indicated concentrations 43.23 µM, 17.10 µM and 19.73 µM,
respectively for PC3 cells. A17 and A19 were low toxic on PC3 cells.
The cytotoxicity results of the toxic compounds (A7, A10 and A12-A14) are given in
Figure 9. The rest of results are represented in SI-Figure S60.
Table 6. Growth inhibitory effects of prodrugs at concentration range between 150-9 µM
(NT* referred as not toxic (% Cell viability value>80%). LT* referred as low toxic (80%> %
cell viability value>51). T* referred as Toxic. (50% < % Cell viability value).
Cytotoxic properties (IC -µM)
5
0
Prodrugs
A1
Hep3B
NT
HUVEC
NT
PC3
NT
NT
NT
A2
LT
NT
A3
NT
NT

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A4
A5
NT
NT
NT
NT
NT
NT
A6
NT
NT
NT
A7
T (51.16 µM)
LT
NT
A8
NT
NT
NT
A9
LT
NT
NT
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
T (119.64 µM)
LT
T (43.23 µM)
NT
NT
NT
T (43.74 µM)
NT
NT
T (11.62 µM)
T (22.60 µM)
T (17.10 µM)
T (30.87 µM)
T (22.24 µM)
T (19.73 µM)
LT
NT
NT
NT
NT
NT
NT
NT
LT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
LT
NT
LT
NT
NT
NT
NT
4
06

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Figure 9. The cytotoxic properties of prodrugs (A7, A10 and A12-A14) were determined
using MTT assay against Hep3B, PC3 and HUVEC cells. 50.000 cells/well were plated out in
96 well plates. Compounds were applied on plates at five different concentrations (9, 18, 39,
78, 150 µM). DMSO (1%) was used as vehicle control. After 48 h incubation, MTT assay was
carried out as described in ref 49. The results were obtained using spectrophotometer at 550
nm optic density. The graphics were illustrated using Origin pro 8.5 program.
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415
416
417
418
419
420
2.4.2. The prodrug abilities of nitro aromatic amides
Compounds were chosen according to cytotoxic properties. Prodrug ability was searched
for nontoxic compounds. Hence, Ssap-NtrB/prodrug (A1-A6, A8, A11 and A15-A22)
combinations were applied on PC3 cells. Also, CB1954, previously known to be a suitable
substrate for E.coli nitroreductase was used as a positive control to compare the prodrug
ability of compounds.

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In this experimental design, the cells were treated with prodrug as NTR- (alone/180 nM at
final concentration) and prodrug (three different concentrations of 180, 18 and 1.8 nM)
+NADH(cofactor)+extracellular Ssap-NTR in 25mM Tris-HCl pH 7.5. After 48h of
treatment, the growth inhibitory effects of combinations were analyzed using SRB method.
Previously, it was determined that NADH had no toxic effect for PC3 cells (Data not shown).
The IC50 values of amides with Ssap-NtrB (prodrug+NADH+Ssap-NTR) were shown in
Table 7.
Our results indicated that A5, A6 and A20 were potential candidates as a prodrug.
Combinations with Ssap-NtrB of these prodrugs, which were not toxic alone, have shown
significant toxic effects. For these prodrugs, IC values were 1.806 nM, 1.808 nM and 1.793
5
0
nM, respectively (Figure 10). In addition, prodrug A20 had toxic effect comparable with
CB1954. The rest of the graphics of compounds were shown in SI-Figure 61.
Table 7. The prodrug abilities of amides. NTR (-) represents only 150 µM or 180 nM prodrug
application. NTR (+) are values obtained from the application of final products of
nitroaromatic amides/Ssap-NtrB reaction.
Prostate cancer cell line (PC3)
Prodrugs
A1
NTR(-)
NTR(+) IC value
50
non toxic
non toxic
non toxic
non toxic
non toxic
non toxic
non toxic
10.72 µM
10.24 µM
12.59 µM
1.806 nM
1.808 nM
17.11 µM
29.12 µM
A3
A4
A5
A6
A8
A11

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4
4
4
4
4
4
4
4
4
4
4
4
4
4
37
38
39
40
41
42
43
44
45
46
47
48
49
50
A15
A16
non toxic
non toxic
non toxic
non toxic
non toxic
non toxic
non toxic
non toxic
non toxic
31.10 nM
36.83 nM
17.98 nM
15.47 nM
30.50 nM
1.793 nM
14.30 nM
33.43 nM
1.792 nM
A17
A18
A19
A20
A21
A22
CB1954