Naphtho[1′,2′:4,5]imidazo[1,2-a]pyridine-5,6-diones: Synthesis, enzymatic reduction and cytotoxic activity

Article abstract of DOI:10.1016/j.bmcl.2015.11.084

Naphtho[1′,2′:4,5]imidazo[1,2-a]pyridine-5,6-diones (NPDOs), a new type of N-heterocycle-fused o-quinones, have been synthesized. They have been found to be efficient electron-accepting substrates of NADPH-dependent single-electron-transferring P-450R and two-electron transferring NQO1, generating reactive oxygen species (ROS) with a concomitant decrease in NADPH, which is consistent with redox-cycling. The reactivity of NPDOs toward P-450R (in terms of k_cat/K_m) varied in the range of 10⁶-10⁷ M^-1 s^-1, while their reduction by NQO1 proceeded much faster, approaching the diffusion control limit (k_cat/K_m ～ 10⁸-10⁹ M^-1 s^-1). NPDOs exhibited relatively high cytotoxic activity against human lung carcinoma (A-549) and breast tumor (MCF-7) cell lines (LC₅₀ = 0.1-8.3 μM), while promyelocytic leukemia cells (HL-60) were less sensitive to NPDOs (LC₅₀ ≥ 10 μM). 3-Nitro-substituted NPDO (11) revealed the highest potency against both A-549 and MCF-7 cell lines, with LC₅₀ of 0.12 ± 0.03 μM and 0.28 ± 0.08 μM, respectively. Dicoumarol partly suppressed the activity of the compounds against A-594 and MCF-7 cell lines, suggesting that their cytotoxic action might be partially influenced by NQO1-mediated bioreductive activation.

Full text of DOI:10.1016/j.bmcl.2015.11.084

Accepted Manuscript
Naphtho[1’,2’:4,5]imidazo[1,2-a]pyridine-5,6-diones: Synthesis, enzymatic re-
duction and cytotoxic activity
Jonas Šarlauskas, Milda Pečiukaitytė-Alksnė, Lina Misevičienė, Audronė
Marozienė, Evelina Polmickaitė, Zita Staniulytė, Narimantas Č ėnas, Žilvinas
Anusevičius
PII:
S0960-894X(15)30292-4
DOI:
Reference:
http://dx.doi.org/10.1016/j.bmcl.2015.11.084
BMCL 23340
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 October 2015
22 November 2015
23 November 2015
Please cite this article as: Šarlauskas, J., Pečiukaitytė-Alksnė, M., Misevičienė, L., Marozienė, A., Polmickaitė, E.,
Staniulytė, Z., Č ėnas, N., Anusevičius, Z., Naphtho[1’,2’:4,5]imidazo[1,2-a]pyridine-5,6-diones: Synthesis,
enzymatic reduction and cytotoxic activity, Bioorganic & Medicinal ChemistryLetters (2015), doi: http://dx.doi.org/
10.1016/j.bmcl.2015.11.084
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Naphtho[1',2':4,5]imidazo[1,2-a]pyridine-5,6-diones: Synthesis, enzymatic
reduction and cytotoxic activity
Jonas Šarlauskas*, Milda Pečiukaitytė-Alksnė, Lina Misevičienė, Audronė Marozienė, Evelina
Polmickaitė, Zita Staniulytė, Narimantas Čėnas, Žilvinas Anusevičius*
Institute of Biochemistry of Vilnius University, Mokslininku 12, LT-08662 Vilnius, Lithuania
* Corresponding authors.
E-mail addresses: jonas.sarlauskas@bchi.vu.lt (J. Šarlauskas), zilvinas.anusevicius@bchi.vu.lt
(Ž. Anusevičius).
___________________________________________________________________________
ABSTRACT
Naphtho[1',2':4,5]imidazo[1,2-a]pyridine-5,6-diones (NPDOs), a new type of N-heterocycle-fused
o-quinones, have been synthesized. They have been found to be efficient electron-accepting
substrates of NADPH-dependent single-electron-transferring P-450R and two-electron transferring
NQO1, generating reactive oxygen species (ROS) with a concomitant decrease in NADPH, which
is consistent with redox-cycling. The reactivity of NPDOs towards P-450R (in terms of kcat/Km )
varied in the range of 106-107 M-1 s-1, while their reduction by NQO1 proceeded much faster,
approaching the diffusion control limit ( kcat/Km ~ 108-109 M-1 s-1). NPDOs exhibited relatively
high cytotoxic activity against human lung carcinoma (A-549) and breast tumor (MCF-7) cell lines
(LC50 = 0.1-8.3 µM), while promeolytic leukemia cells (HL-60) were less sensitive to NPDOs
(LC50 ≥ 10 µM). 3-Nitro-substituted NPDO (11) revealed the highest potency against both A-549
and MCF-7 cell lines, with LC50 of 0.12 ± 0.03 µM and 0.28 ± 0.08 µM, respectively. Dicoumarol
partly suppressed the activity of the compounds against A-594 and MCF-7 cell lines, suggesting
that their cytotoxic action might be partially influenced by NQO1-mediated bioreductive activation.
_________________________________________________________________________
The quinoidal compounds are widely used for treatment of various infectious diseases and
comprise one of the largest classes of antitumor agents.1-5 Depending on their chemical structure,
they may exert their cytotoxic (or therapeutic) effects through different mechanisms. For instance,
1

they can directly interact with cellular nucleophiles,6-7 participate as DNA intercalating compounds
8 and alkylating agents 9-12 or act as inhibitors of specific enzymes such as topoisomerases.13 In
many cases, the cytotoxic activity of quinones is related to their enzymatic single- or two-electron
reduction/activation in the presence of suitable electron donors. The single-electron reduction of
quinones to semiquinone species is typically performed by flavoenzymes electrontransferases-
dehydrogenases (e.g., NADPH-cytochrome P-450 reductase (P-450R), NADH-cytochrome b5
reductase (b-5R), ubiquinone-oxidoreductase (complex I), etc.).14-15 Unstable semiquinones undergo
.-
rapid reoxidation by O2 (futile redox cycling), generating superoxide (O2 ) and other reactive
oxygen species (ROS) that lead to oxidative stress toxicity and other events.14-15 In contrast, the
two-electron (hydride) reduction of quinones to their stable hydroquinones is commonly thought to
be a detoxification mechanism. 16-17 This reaction is typically catalyzed by two-electron transferring
flavoenzyme NAD(P)H: quinone oxidoreductase (NQO1). However, NQO1 can also be involved
in two-electron reduction/activation of certain quinones, yielding unstable hydroquinones, capable
of alkylating DNA and/or undergoing redox-cycling, which produces ROS.18-20 Owing to the
elevated expression of NQO1 in many tumor tissues,21-26 this enzyme is believed to be an attractive
target for bioreductive activation of certain quinone-based compounds.
Natural and synthetic heterocyclic quinones, such as para-quinoid alkylating agents (mitomycin
C or its analoque EO9),9-27 para-quinoid redox-cycling agents (streptonigrin (STN),28
deoxynyboquinone (DNQ)),5,19,29 redox-cycling pyran-fused para- and ortho-naphthoquinones (α-,
-lapachones) and their synthetic analogues,5,19,20 furan-fused ortho-napthoquinone tanshinone
(TSA),30,31 as well as a number of N-heterocyclic-fused ortho-/para-quinones such as
[1,2,3]triazolo[4,5-g]phthalazine-4,9-diones,13 benzo[g]isoquinoline-5,10-diones32 and 6,11-
dihydro-pyrido[2,3-b]phenazine-6,11-diones,33 1H-imidazo[4,5-g]phthalazine-4,9-diones,34 and
even structurally much simpler ortho-quinone compound phenanthroline-5,6-dione (PD),35 have
been reported to be excellent antibiotic and/or anticancer agents. Some of them have proved to be
cytotoxic to cancer cells following bioreduction by the aforementioned flavoenzymes.
Herein we report the synthesis of naphtho[1',2':4,5]imidazo[1,2-a]pyridine-5,6-diones
(NPDOs) as a new type of N-heterocycle-fused ortho-quinone compounds. To provide some
insight into possible action of these compounds, their enzymatic reactivity and redox-cycling ability
were examined applying single-electron transferring P-450R (EC 1.6.2.4) and two-electron
transferring NQO1 (EC 1.6.99.2). In addition, the antitumor activity of NPDOs was estimated
against a panel of human cancer cell lines, and their antibacterial activity was tested on several
Gram-negative and Gram-positive bacteria strains.
2

As shown in Scheme 1, NPDOs were synthesized through a condensation reaction of 2-amino-
N-heterocycle compounds with 2,3-dichloro-1,4-naphthoquinone derivatives. The reaction pathway
involved the substitution of 2-Cl- atom of naphthoquinone derivative by N-atom of 2-amino-
heterocycle resulting in the formation of an intermediate complex that underwent cyclization under
a high temperature with the formation of N-heterocyclic ortho-quinones as final products. The
yields of the final ortho-quinoidal products were low, varying in the range of 7-61 %. The electron-
donating groups in the aromatic ring of 2-amino-N-heterocycle facilitated the condensation process,
while the electron-withdrawing groups (i.e., -nitro and -CF3) aggravated cyclization process,
prolonging the reaction time and reducing yields of final products. All details about the synthetic
procedures of the compounds and their spectral data are provided in the Supporting Information.
Scheme 1. Synthesis of N-heterocyclic ortho-quinones.
After achieving the synthesis of NPDOs, their enzymatic reactivity towards NADPH-dependent
single-electron transferring P-450R and two-electron transferring NQO1 was examined by
measuring the rates of their reduction with respect to NADPH oxidation at 340 nm (Δε340 = 6.22
3

mM-1 cm-1) at varied concentrations of the compounds and fixed concentrations of NADPH (150
µM and 200 µM for P-450R- and NQO1-catalyzed reactions, respectively). The reactivity of the
compounds was defined in terms of kcat/Km ratio values. Since both flavoenzymes are known to
follow a 'ping-pong' reaction scheme, 14,36-38 the kcat/Km is an apparent second-order rate constant
that reflects the efficiency of the reduction of the compounds by a reduced free enzyme form. As
shown in Figure 1, the rates of P-450R-catalyzed single-electron reduction of NPDOs followed
hyperbolic dependences upon varied concentrations of the compounds. The defined kcat/Km values
varied in the range of 8.9 × 106 M-1 s-1 - 7.2 × 107 M-1 s-1 (Table 1), which were close to those of the
reported P-450R-catalyzed reduction of highly reactive napthoquinoidal compounds.14 The two-
electron (hydride) reduction of NPDOs by NQO1 proceeded much faster than by P-450R (Figure
1B), with an apparent kcat/Km approaching the diffussion control limit (kcat/Km ~ 108-109 M-1 s-1
(Table 1)), except for quinoline-NPDO (13), whose kcat/Km was by two-orders of magnitude lower.
NQO1-catalyzed rates decreased in the regions of higher NPDOs concentrations (Figure 2B),
suggesting substrate inhibition of NQO1 by the compounds. Measuring their reactivity at various
app
fixed concentrations of NADPH, the obtained apparent substrate inhibition constant (Kiq
)
decreased linearly with a decrease in concentrations of NADPH (Figure 1C), indicating that NPDOs
bind to the same or closely overlapping binding site of NADPH substrate.
Fig. 1. The initial enzymatic rates (v0/[E0]) of reduction of NPDOs by (A) P-450 R and (B) NQO1
on varied concentrations of the compounds and fixed concentrations of NADPH (150 µM (P-450R)
and 200 µM (NQO1). (C) The dependence of the apparent substrate inhibition constants (Kiapp) of
compounds 1 and 5 in their NQO1-catalyzed reduction reactions on various fixed concentrations of
NADPH (200 µM, 100 µM, 25 µM and 12.5 µM).
4

Table 1.
The apparent second-order rate constant (kcat/Km) values of the reduction of NPDOs by
NADPH:cytochrome P-450 reductase (P-450R) and NADPH:quinone oxidoreductase (NQO1), and
LUMO energies (ELUMO) of the compounds as calculated by DFT/B3LYP/6-31+G(d) method.
Compound
P-450R
NOQ1
ELUMO
(eV)
kcat/Km (M-1 s-1)
NPDO (1)
3.1 ± 0.2 × 107
8.9 ± 1.1 × 106
1.4 ± 0.3 × 107
1.3 ± 0.1 × 107
2.9 ± 0.2 × 107
2.4 ± 0.4 × 107
5.4 ± 0.3 × 107
3.7 ± 0.1 × 107
3.5 ± 0.4 × 107
5.9 ± 0.2 × 107
7.2 ± 0.5 × 107
3.4 ± 0.2 × 107
5.6 ± 0.4 × 106
2.4 ± 0.1 × 108
1.0 ± 0.1 × 108
1.3 ± 0.2 × 108
2.3 ± 0.1 × 108
1.6 ± 0.1 × 108
3.1 ± 0.1 × 108
3.7 ± 0.4 × 108
3.5 ± 0.2 × 108
4.8 ± 0.4 ×108
3.2 ± 0.2 × 108
7.4 ± 0.3 ×108
1.6 ± 0.4 ×108
8.7 ± 0.7 ×105
-3.232
-3.168
-3.162
-3.188
-3.165
-3.352
-3.370
-3.360
-3.526
-3.770
-3.780
-3.420
-3.156
9-CH3-NPDO (2)
10-CH3-NPDO (3)
11-CH3-NPDO (4)
3-CH3O-NPDO (5)
9-F-NPDO (6)
9-Cl-NPDO (7)
9-Br-NPDO (8)
9-F3C-NPDO (9)
9-NO2-NPDO (10)
3-NO2-NPDO (11)
Pyrimidine-NPDO (12)
Quinoline-NPDO (13)
As shown in Figure 2, the catalytic efficiency of P-450R (in terms of log kcat/Km ) tentatively
increased with an increase in the electrophilic potency of the compounds, expressed in terms of
LUMO energy values (ELUMO). This is possibly consistent with an "outer-sphere" electron transfer
mechanism39 where the reactivity of NPDOs is governed mainly by the difference in potential
between P-450R active center and the compounds. Such a reaction mechanism is characteristic of
the reduction of quinones by P-450R and other NAD(P)H-dependent single-electron transferring
flavoenzymes.14 For NQO1-catalyzed reactions, log kcat/Km of NPDOs tentatively correlates with
ELUMO of NPDOs, omitting quinoline-NPDO (13) (Figure 2). The low reactivity of 13 might be
5

caused by its unfavourable binding and/or orientation within the active center of NQO1. The
efficiency of two-electron reduction of quinoidal substrates by NQO1 is known to be highly
dependent upon suitable - stacking with the isoalloxazine of FAD-cofactor. 20,40 The
NPDOs/NQO1 interaction modes, applying in silico docking and a molecular dinamics simulation,
will be studied in our forthcoming work.
Fig. 2. The dependence of catalytic efficiency (in terms of log kcat/Km) of reduction of NPDOs by
P-450R (closed squares) and NQO1 (open squares) on LUMO energy values of the compounds, as
calculated by DFT-B3LYP/6-31+G(d) method. The numbers of the compounds are the same as in Table 1.
In paralell experiments, we checked the redox-cycling ability of NPDOs. In P-450R- and NQO1-
catalyzed reactions, the excess amount of NADPH (150-200 µM) over ortho-quinones (5-15 µM)
was oxidized in a continuous single phase. The reactions were followed by rapid O2 uptake. During
P-450R-catalyzed reduction of NPDOs, the rate of O2 uptake was suppressed by superoxide
dismutase (SOD, 150 µg/ml ) by 35-50 %, while the catalase (CAT, 100 U/ml) had a marginal
effect, indicating that the redox-cycling of NPDOs is accompanied by concomitant production of
superoxide. In NQO1-catalyzed reactions, O2 uptake was inhibited by CAT by 45-70 %, indicating
that peroxide is produced owing to the autoxidation of the hydroquinones of NPDOs. Finally, we
checked the NQO1-catalyzed reduction of NPDOs, using cytochrome c (50 µM) as a terminal
single-electron acceptor. This assay was not applied to P-450R-mediated reduction of NPDOs, since
this enzyme catalyzes the direct reduction of cytochrome c, with kcat/Km of 1.6-5.0 × 106 M-1 s-1.
6

For all NPDOs tested, the rates of the reduction of cytochrome c accounted for 180-200 % of
NADPH oxidation rate, and the reactions were supressed by SOD by 30-45 %, indicating that
superoxide is generated during this reaction. These results are in line with general features of P-
450R- and NQO1-catalyzed redox-cycling of low-potential quinoidal compounds.14,20,41
Next, we examined the cytotoxic activity of the whole set of NPDOs against a panel of three
human tumour cells, i.e., adenocarcinomic alveolar basal epithelial (A-549), breast cancer (MCF-7)
and promyelocytic leukemia (HL-60). After 24-h exposure by ortho-quinones, cell survival was
evaluated by standard MTT assay, and the cell viability was expressed as a percentage of solvent
treated control cells. The concentrations of the compounds, causing 50 % lethal effect (LC50), were
obtained by using logistical dose-response curves. Doxorubicin was used in this study as a positive
control. As shown in Table 2, NPDOs exhibited relatively high antitumor activity against NQO1-
rich A-549 and MCF-7 cell lines, with LC50 values of 0.12-8.34 µM and 0.50-3.8 µM, respectively,
whereas NQO1-defficient HL-60 cell line was markedly less sensitive to ortho-quinones, with LC50
values of over 10 µM. Meanwhile, doxorubicin showed LC50 values of 1.17 ± 0.14, 1.56 ± 0.21 and
0.37 ± 0.10 µM against A-594, MCF-7 and HL-60 cell lines, respectively, which were close to
those reported in previous publications.42-44
Table 2
The cytotoxic activity of the ortho-quinoidal compounds against a panel of tumour cell lines.
Compound
Cell line
MCF-7
A-594
HL-60
LC50 ± SD (µM)
0.51 ± 0.10
3.94 ± 0.46
1.25 ± 0.06
1.70 ± 0.31
2.10 ± 0.31
1.32 ± 0.18
3.41 ± 1.71
1.39 ± 0.04
3.78 ± 0.48
NPDO (1)
0.67 ± 0.16
8.34 ± 0.64
1.50 ± 0.06
3.40 ± 0.15
3.35 ± 0.30
2.03 ± 0.04
2.06 ± 0.95
3.93 ± 0.50
0.90 ± 0.16
>15
>15
>15
>15
>15
~13
~10
>15
>15
9-CH3-NPDO (2)
10-CH3-NPDO (3)
11-CH3-NPDO (4)
9-CH3O-NPDO (5)
9-F-NPDO (6)
9-Cl-NPDO (7)
9-Br-NPDO (8)
9-CF3-NPDO (9)
7

9-NO2-NPDO (10)
3-NO2-NPDO (11)
Pyrimidine-NPDO (12)
Quinoline-NPDO (13)
Doxorubicin
2.30 ± 0.70
0.12 ± 0.03
1.60 ± 0.30
>15
1.70 ± 0.10
0.28 ± 0.08
0.80 ±0.40
>15
>15
>15
>15
>15
1.17 ± 0.14
1.56 ± 0.21
0.37 ± 0.10
The cytotoxic activity of NPDOs (in terms of -log LC50 values) showed some tentative positive
dependence upon ELUMO values against A-549 cell line (R = 0.623, P < 0.023), and tentative
positive correlations were observed between -log LC50 of NPDOs and their defined reactivity
towards NQO1 (in terms of log kcat/Km-values) against both A-549 (R = 0.683, P < 0.010) and
MCF-7 (R = 0.630, P < 0.010) cells. Among the NPDOs examined, 3-nitro-substituted NPDO (11),
possessing the highest electrophilic potency and being the most reactive substrate of NQO1 (Table
1), displayed the strongest cytotoxic potency against both A-459 and MCF-7 cell lines, with LC50
values of 0.12 ± 0.03 µM and 0.28 ± 0.08 µM, respectively. The unsubstituted NPDO (1) was the
second most potent agent, with LC50 values of 0.67 ± 0.16 (A-549) and 0.51 ± 0.10 (MCF-7), which
was followed by 9-CF3-substituted NPDO (9), with LC50 value of 0.90 ± 0.16 µM against A-549 cell
line, and pyrimidine-NPDO (12), with LC50 value of 0.80 ± 0.40 µM against MCF-7 cells. As
compared to unsubstituted NPDO (1), the alkyl-, alkoxy- and halide-substituted NPDOs (2-8)
showed markedly lower activity against both A-549 and MCF-7 cell lines, with LC50 values in the
range of 1.5 to 8.3 µM (Table 2). Possessing almost the same electrophilic potency as the most
active 3-nitro-NPDO (11) but being a less specific substrate of NQO1 (Table 1), the 9-nitro-NPDO
(10) exhibited 19- and 6-fold lower cytotoxic activity on A-549 and MCF-7 cells, respectively, as
compared to 11, suggesting that their activity might be determined by the position of nitro-group
introduced in the heterocyclic system.
We checked the cytotoxic activity of the most potent NPDOs (compounds 1 and 11) in the
presence of dicoumarol (DIC, 20 µM), as an efective NQO1 inhibitor that blocks the catalytic
efficiency of the enzyme by interacting with the NAD(P)H-binding site of the oxidized enzyme
form (Ki(DIC) = 1-10 nM).20-22 DIC was used for prediction of quinone-induced NQO1-mediated cell
death.10,19,20 As shown in Figure 3, the coincubation with DIC led to lower sensitivity of A-549 and
MCF-7 cell lines to compound 1, increasing LC50 values 3.1- and 2.3-fold, respectively, as well as
to compound 11, raising LC50 values 3.3- and 3.6-fold, respectively, which implies that they might
be partially influenced by their NQO1-catalyzed bioreductive activation.
8

Fig. 3. Cell death of A-549 and MCF-7 after 24-h exposure by compounds 1 and 11 in the presence and
absence of NQO1 inhibitor (DIC, 20 µM).
The mechanism of bioreduction and cytotoxic action of nitro-substituted NPDOs (compounds 10
and 11) may generate a special interest, because these compounds contain two electron-accepting
moieties, quinone and nitro-group. The cytotoxicity of nitroaromatic compounds (NACs) is
frequently manifested through their bioreduction. The single-electron reduction of NACs to their
anion radicals is accompanied by ROS formation, initiating oxidative stress,14,45 whereas their two-
electron reduction yields alkylating nitroso- and/or hydroxylamine species.46,47 However, we expect
that the cytotoxic activity of nitro-NPDOs may arise mainly from the action of their quinone
moiety. This can be partially supported by our DFT computation, assessing the electrophilicity of
the compounds condensed to atoms of quinone moiety and nitro-group (the computational details
are given in the Supporting Information). As shown in Figure 4, the highest density of absolute
LUMO (|LUMO|) and the highest electrophilic Fukui function (f+ ) values, reflecting the tendency
k
9

of the reactive sites to accept the nucleophile (an electron and/or a hydride ion), are mainly reside
on the quinone moiety of the compounds, implying that this moiety is the most probable site of their
reduction.
Fig. 4. The |LUMO| maps (izo-values = 0.04) and the electrophilic Fukui function (f+ ) values, localized
k
upon k-atoms of quinone moiety and nitro-group of 9-nitro-NPDO (compound 10) and 3-nitro-NPDO
(compound 11). The calculations were performed applying DFT-B3LYP/6-31+G(d) method.
Finally, we tested the whole set of NPDOs for their in vitro antibacterial activity against Gram-
negative Escherichia coli (ATCC 25922), Salmonella enterica (SL 5676) and Gram-positive
Staphylococcus aureus (ATCC 25923) bacterial strains by two-fold micro-dilution method, and the
MIC values, which are the lowest concentrations of the compounds that completely inhibit the
bacterial growth after 16 h incubation at 37º C, were determined. NPDOs were found to be active
against Gram-positive S. aureus strain, with MIC values ranging from 6.1 µM to 15.3 µM. The
Gram-negative bacteria strains were not affected by the ortho-quinones as observed near the limits
of their solubility in culture media (~ 15-20 µM). Amoxicillin and streptomycin, used in this study
against S. aureus as reference drugs, showed MIC values of 0.3 µM and 5.4 µM, respectively.
Among the NPDOs, Cl-, Br-, CF3- substituted NPDOs (7-9) and pyrimidine-NPDO (12) exhibited
the highest antibacterial activity (MIC values of 7.0 µM (7), 6.1 µM (8), 6.3 µM (9) and 8.0 µM
(12)), being close to that of streptomycin. The unsubstituted NPDO (1), alkyl-, alkoxy-NPDOs (2-5)
and the quinoline-NPDO (13) showed approximately 2-fold lower activity, with MIC values of 16.1
µM (1) 15.2 µM (2-4) and 14.3 µM (5). In contrast to the most potent halide-substituted NPDOs (7-
9), the activity of 9-F-NPDO (6) was 2-fold lower (MIC = 15.0 µM). S. aureus was not affected by
10

3-nitro- and 9-nitro-substituted NPDOs (10, 11) as examined up to the limits of their solubility in
culture media.
In summary, the synthesized naphtho[1',2':4,5]imidazo[1,2-a]pyridine-5,6-diones (NPDOs)
have been found to be highly reactive substrates of NADPH-dependent single- and two-electron
transferring flavoenzymes. Their reduction was accompanied by redox-cycling, producing ROS.
NPDOs exhibited relatively high antitumor activity against NQO1-rich A-549 and MCF-7 cell
lines, and 3-nitro substituted NPDO (compound 11) has been found to be the most active
compound. Partial suppression of cytotoxic activity of NPDOs in the presence of dicoumarol
suggests that their cytotoxic action might be partially influenced by NQO1-mediated bioreductive
activation of the compounds. Since it is known that planar polycyclic aromatic molecules
containing quinoid moiety tend to be the DNA-intercalating TOP inhibitors,8,48 this action mode for
our molecules will be elucidated in our forthcoming work. In addition, our further efforts will be
devoted to the synthesis and elucidation of enzymatic and antitumor activity of structural isomers of
the most active compound 11 and its analogues, containing amino- and hydroxy-functional groups.
Acknowledgement
This work was funded by the Scientific Council of Lithuania (the project No. MIP-032/2014). The
authors thank Dr. Gema Mikulskienė for helping in NMR interpretation. The authors also thank
Lina Marčiulionytė (Lecturer at Vilnius University) for proofreading the manuscript.
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