Flavoenzyme-mediated reduction reactions and antitumor activity of nitrogen-containing tetracyclic ortho-quinone compounds and their nitrated derivatives

Article abstract of DOI:10.17179/excli2017-273

Nitrogen-based tetracyclic ortho-quinones (naphtho[1'2':4.5]imidazo[1,2-a]pyridine-5,6-diones, NPDOs) and their nitro-substituted derivatives (nitro-(P)NPDOs) were obtained by condensation of substituted 2,3-dichloro-1,4-naphthoquinones with 2-amino-pyridine and -pyrimidine and nitration at an elevated temperature. The structural features of the compounds as well as their global and regional electrophilic potency were characterized by means of DFT computation. The compounds were highly reactive substrates of single- and two-electron (hydride) - transferring P-450R (CPR; EC 1.6.2.4) and NQO-1 (DTD; EC 1.6.99.2), respectively, concomitantly producing reactive oxygen species. Their catalytic efficiency defined in terms of the apparent second-order rate constant (kcat/KM (Q)) values in P-450R- and NQO-1-mediated reactions varied in the range of 3-6 × 107 M-1 s-1 and 1.6-7.4 × 108 M-1 s-1, respectively. The cytotoxic activities of the compounds on tumor cell lines followed the concentration-dependent manner exhibiting relatively high cytotoxic potency against breast cancer MCF-7, with CL50 values of 0.08-2.02 μM L-1 and lower potency against lung cancer A-549 (CL50 = 0.28-7.66 μM L-1). 3-nitro-pyrimidino-NPDO quinone was the most active compound against MCF-7 with CL50 of 0.08 ± 0.01 μM L-1 (0.02 μg mL-1)) which was followed by 3-nitro-NPDO with CL50 of 0.12 ± 0.03 μM L-1 (0.035 μg mL-1)) and 0.28 ± 0.08 μM L-1 (0.08 μg mL-1) on A-549 and MCF-7 cells, respectively, while 1- and 4-nitro-quinoidals produced the least cytotoxic effects. Tumor cells quantified by AO/EB staining showed that the cell death induced by the compounds occurs primarily through apoptosis.

Full text of DOI:10.17179/excli2017-273

EXCLI Journal 2017;16:663-678 – ISSN 1611-2156
Received: March 14, 2017, accepted: April 29, 2017, published: May 11, 2017
Original article:
FLAVOENZYME-MEDIATED REDUCTION REACTIONS AND
ANTITUMOR ACTIVITY OF NITROGEN-CONTAINING
TETRACYCLIC ORTHO-QUINONE COMPOUNDS AND THEIR
NITRATED DERIVATIVES
Milda Pečiukaitytė-Alksnė, Jonas Šarlauskas*, Lina Misevičienė, Audronė Marozienė,
Narimantas Čėnas, Kastis Krikštopaitis, Zita Staniulytė, Žilvinas Anusevičius*
Institute of Biochemistry, Life Sciences Center, Vilnius University, Saulėtekio av. 7,
Vilnius, LT-10257, Lithuania
* Corresponding authors: E-mail: jonas.sarlauskas@bchi.vu.lt (J. Šarlauskas);
zilvinas.anusevicius@bchi.vu.lt (Ž. Anusevičius)
http://dx.doi.org/10.17179/excli2017-273
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/).
ABSTRACT
Nitrogen-based tetracyclic ortho-quinones (naphtho[1'2':4.5]imidazo[1,2-a]pyridine-5,6-diones, NPDOs) and
their nitro-substituted derivatives (nitro-(P)NPDOs) were obtained by condensation of substituted 2,3-dichloro-
1,4-naphthoquinones with 2-amino-pyridine and -pyrimidine and nitration at an elevated temperature. The struc-
tural features of the compounds as well as their global and regional electrophilic potency were characterized by
means of DFT computation. The compounds were highly reactive substrates of single- and two-electron (hydride)
- transferring P-450R (CPR; EC 1.6.2.4) and NQO-1 (DTD; EC 1.6.99.2), respectively, concomitantly producing
reactive oxygen species. Their catalytic efficiency defined in terms of the apparent second-order rate constant
(kcat/KM (Q)) values in P-450R- and NQO-1-mediated reactions varied in the range of 3-6 × 107 M-1 s-1 and 1.6-7.4
× 108 M-1 s-1, respectively. The cytotoxic activities of the compounds on tumor cell lines followed the concentra-
tion-dependent manner exhibiting relatively high cytotoxic potency against breast cancer MCF-7, with CL50 values
of 0.08-2.02 µM L-1 and lower potency against lung cancer A-549 (CL50 = 0.28-7.66 µM L-1). 3-nitro-pyrimidino-
NPDO quinone was the most active compound against MCF-7 with CL50 of 0.08 ± 0.01 µM L-1 (0.02 µg mL-1))
which was followed by 3-nitro-NPDO with CL50 of 0.12 ± 0.03 µM L-1 (0.035 µg mL-1)) and 0.28 ± 0.08 µM L-1
(0.08 µg mL-1) on A-549 and MCF-7 cells, respectively, while 1- and 4-nitro-quinoidals produced the least cyto-
toxic effects. Tumor cells quantified by AO/EB staining showed that the cell death induced by the compounds
occurs primarily through apoptosis.
Keywords: heterocycle quinones, ortho-quinones, enzymatic reactivity, antitumor activity, apoptosis, DFT
computation
INTRODUCTION
also represent a large class of redox active
xenobiotics which may induce a variety of
hazardous effects including cardiotoxicity,
neurotoxicity, immunotoxicity and carcino-
genesis (Bolton et al., 2000; Monks and
Jones, 2002; Bolton and Dunlap, 2017, and
refs. therein). On the other hand, quinoid
Quinones constitute an important class of
naturally occurring compounds which play a
pivotal role in the diverse biological systems
(Nohl et al., 1986; Monks et al., 1992; Bolton
et al., 2000; Monks and Jones, 2002). They
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EXCLI Journal 2017;16:663-678 – ISSN 1611-2156
Received: March 14, 2017, accepted: April 29, 2017, published: May 11, 2017
compounds are widely used for treatment of tumor cells, this enzyme has become one of
viral and microbial infections, and comprise the most attractive targets for bioreductive ac-
one of the major classes of chemotherapeutic tivation of various redox active xenobiotics
agents (Riffel et al., 2002; Asche, 2005; and drug agents including quinoid-based
Garuti et al., 2007, and refs. therein). Owing compounds (Danson et al., 2004; Rooseboom
to electrophilic (electron-accepting) capacity et al., 2004, Siegel et al., 2012; Bian et al.,
of quinones, one of the primary stages of their 2014, 2015 and refs. therein). The quinone-
(cyto)toxic and/or therapeutic action is related mediated enhanced generation of ROS was
to their bioreductive activation mediated by proved to contribute to the "non-specific" cel-
oxidoreductases producing semiquinone radi- lular damage and/or induction of specific
cal and/or hydroquinone (diol) species ROS-sensing signaling pathways thus caus-
(Rooseboom et al., 2004; Siegel et al., 2012). ing cell cycle arrest, senescence, apoptosis
The single-electron reduction of quinones is and other events (Mates and Sanchez-
typically catalyzed by single-electron trans- Jimenez, 2000; Liou and Storz, 2010, and
ferring flavoenzymes such as NADPH-cyto- refs. therein).
chrome P-450 reductase (P-450R, CPR),
Among a broad variety of quinoid-based
NADH-cytochrome b5 reductase (CB5R), compounds, the natural and synthetic hetero-
NADPH:ubiquinone oxidoreductase (comp- cyclic quinones were shown to be efficient
lex I), etc. (Bachur et al., 1979; Ross et al., enzymatic substrates and potential cyto-
1996; Čėnas et al., 1994; Matsuda et al., 2000; toxic/anti-tumor agents (Pan et al., 1993;
Rooseboom et al., 2004, and refs. therein). Rooseboom et al., 2004; Deegan et al., 2006;
The semiquinone radicals formed are readily Colucci et al., 2008; Garuti et al., 2007; Val-
back-oxidized by molecular oxygen (the so- derrama et al., 2008; Chen and Hu 2009; Vás-
called futile redox cycling) generating super- quez et al., 2010; Koyama et al., 2010, 2011;
oxide along with other reactive oxygen spe- Brandy et al., 2012; Delgrado et al., 2012;
cies (ROS) (Powis, 1989; Rooseboom et al., Zhang et al., 2012; Bian et al., 2014, 2015;
2004). The obligatory two-electron (hydride) Cai et al., 2016). Compared to the heterocy-
reduction of quinones to their diol forms, typ- clic para-quinoidals, the enzymatic reactivity
ically mediated by two-electron transferring and/or cytotoxic/antitumor activity studies of
flavoenzyme NAD(P)H: quinone acceptor ortho-quinoid compounds are still limited to a
oxidoreductase (DT-diaphorase (DTD), few groups of the natural and synthetic ortho-
NQO-1), affords the protection against cyto- quinoid compounds such as 9,10-phenanthro-
toxic effects of quinones diverting them from line-5,6-dione (phendione, PD) (Deegan et
their redox cycling (Joseph et al., 2000). al., 2006), pyrroloquinoline quinone (PQQ)
NQO-1 is well-known for being a major xe- (Shen et al., 2009; Shankar et al., 2010), tet-
nobiotic metabolizing enzyme, whose induc-
tion is frequently associated with the protec-
tion against toxic and/or carcinogenic effects
of a large variety of electrophiles. On the
other hand, the two-electron reduction of cer-
tain structure quinoid xenobiotics and drug
agents to unstable diol species is accompanied
by their chemical rearrangement to highly re-
active intermediates and/or spontaneous auto-
oxidation generating ROS (Gutierrez, 2000;
Beall and Winski, 2000; Colucci et al., 2008;
Rooseboom et al., 2004; Siegel et al., 2012;
Bian et al., 2014, 2015). Because of the en-
hanced expression of NQO-1 in tumors and
rahydropyran-fused ortho-napthoquinone (-
lapachone) and its ortho-quinone analogues
(Bian et al., 2014, 2015, and refs. therein), tet-
rahydrophenanthrofuran-diones (tanshinon-
es) (Zhang et al., 2012; Dang et al., 2011; Cai
et al., 2016, and refs. therein). The ortho-quin-
oid compounds have proved to be in general
more effective electrophilic and redox active
agents than para-quinoid compounds due to
the enhanced electrophilic character of the
vicinal carbonyl moieties of the former com-
pounds (Tonholo et al., 1998; Campodonico
et al., 2009; Borges et al., 2014).
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EXCLI Journal 2017;16:663-678 – ISSN 1611-2156
Received: March 14, 2017, accepted: April 29, 2017, published: May 11, 2017
Herein we report the study of enzymatic by silica gel Wakogel C-200 (Wako Chemi-
reactivity and antitumor activity of the syn- cal, Osaka, Japan). The melting points were
thesized fused N-containing tetracyclic ortho- defined in open capillaries by using MEL-
quinoid compounds (naphtho[1'2':4.5] imid- TEMP equipment (Barnstead Thermolyne
azo[1,2-a]pyridine-5,6-diones, (P)NPDOs) Corp, Dubuque, IA, USA). UV-VIS spectra
and their nitrated derivatives. The chemical were obtained by Lambda 25 UV-VIS (Per-
structures of the compounds are given in Fig- kin-Elmer, Waltham, MA, USA) spectropho-
ure 1 (for NPDO compounds, the numbers de- tometer. IR spectra were recorded in KBr on
note the relative positions of the nitro groups). a Perkin-Elmer spectrophotometer (FT-IR
Their structural features as well as the global Spectrum BX II). NMR spectra were obtained
and the regional (fractional) electrophilic po- with Bruker spectrometer (1H - 400 MHz and
tencies were characterized by means of DFT 13C - 100 MHz) in d-chloroform or d6-DMSO
computation. Their enzymatic reduction reac- by using residual solvent signal as an internal
tions and redox cycling ability were assessed standard. HRMS spectra were recorded by
applying single-electron transferring P-450R Dual-ESI Q-TOF 6520 (Agilent Technolo-
(CPR) and two-electron-transferring NQO-1 gies, Santa Clara, CA, USA) mass spectrom-
(DTD) as the main flavoenzymes being re- eter.
sponsible for bioreductive activation of a
large variety of redox active xenobiotics in- Synthesis
cluding nitroaromate- and quinoid-based
Naphtho[1‘,2‘:4,5]imidazo[1,2-a]pyridi-
compounds. The cytotoxic activities of ne-5,6-dione (NPDO), 9-nitro-NPDO, and
(P)NPDO and nitro-(P)NPDO quinoidals naphtho[1‘,2‘:4,5]imidazo[1,2-a]pyrimidine-
were estimated against the human A-549, 5,6-dione (PNPDO) were synthesized by con-
MCF-7 and HL-60 tumor cell lines.
densation of 2,3-dichloro-1,4-naphthoqui-
none (0.01 M) with 2-amino-N-heterocycles
(0.02 M) in dry 2-butanol (300 mM) (Figure
2). The reaction mixture was stirred under ar-
gon and heated at 100 °C in a closed vessel
for 36 h (for synthesis of 9-nitro-NPDO, the
time was increased to 96 h). The reaction mix-
ture was concentrated, allowed to cool to
10 °C, filtered and washed with cold 1-propa-
nol (3x20 ml), hexane, and dried. The result-
ant products were re-crystallized from 1,2-di-
chlorobenzene or purified by flash chroma-
tography (CH3OH/CH2Cl2 (1/10)).
MATERIALS AND METHODS
General
All chemical reagents and solvents were
obtained from commercial suppliers (Sigma-
Aldrich (St. Louis, MO, USA), TCI-Europe
(Zwijdrecht, Belgium) and Merck (Darm-
stadt, Germany)). The purity of the com-
pounds was checked by TLC (silica gel 60
F254 aluminium plates (Merck, Darmstadt,
Germany)) and visualized by UV light. The
flash column chromatography was performed
Figure 1: Chemical structures of N-tetracyclic ortho-quinoidals and their nitrated derivatives used in this
work: A) NPDOs and B) pyrimidino-NPDOs (PNPDOs)
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EXCLI Journal 2017;16:663-678 – ISSN 1611-2156
Received: March 14, 2017, accepted: April 29, 2017, published: May 11, 2017
Figure 2: The condensation of 2,3-dichloro-1,4-naphthoquinone with 2-amino-N-heterocycles to obtain
NPDO quinoids ( X=CH, R=H), 9-nitro-NPDO ( X=CH, R= NO2) and pyrimidine-NPDO (X=N, R=H) com-
pounds
3-Nitro-naphtho[1‘,2‘:4,5]imidazo[1,2-
reaction of 5-nitro-2,3-dichloro-1,4-naphtho-
a] pyridine-5,6-dione (3-nitro-NPDO), and 3- quinone (NCNQ) with 2-aminopyridine dur-
nitro-naphtho[1‘,2‘:4,5]imidazo[1,2-a] py- ing the prolonged heating in a polar solvent
rimidine-5,6-dione (3-nitro-PNPDO) were (Figure 3). 2-Aminopyridine (1.88 g, 20 mM)
obtained by the nitration reactions of NPDO was slowly added to the stirred NCNQ solu-
and pyrimidino-NPDO (PNPDO) quinoids, tion (2.27 g, 10 mM) in dry 1-butanol
applying fuming nitric acid in 98 % H2SO4 at (200 ml). The suspension was stirred under
reflux. 1 g (4 mM) pymd-NPDO was added to argon and heated at 110 °C for 56 h, then con-
the stirred ice-cold mixture of 0.8 ml 95 % centrated and allowed to cool to 5 °C. The ob-
HNO3 and 25 ml 98 % H2SO4, heated to tained product was filtered and washed with
110 °C and refluxed for 3 h, then allowed to cold methanol (3x20 ml), then hexane, and
cool to room temperature and poured on 200 g dried. The resultant mixture of 1-nitro- and 4-
of ice. The precipitate was filtered, washed by nitro-NPDO isomers was fractionally crystal-
5 % sodium hydrocarbonate solution and sub- lized from hot chloroform, producing two
sequently by distilled water to the neutral re- crystal fractions, which were purified by flash
action. After drying, the final product, re- chromatography on SiO2 with ethyl acetate.
crystallized from CH2Cl2, yielded a small red After re-crystallization, the more and less sol-
crystalline powder.
uble fractions yielded 1-nitro- and 4-nitro-
1- and 4-nitro-naphtho[1‘,2‘:4,5] imida- NPDO isomers, respectively, with molar ratio
zo [1,2-a]pyridine-5,6-diones (1- and 4-nitro- of 1:3.
NPDOs) were synthesized by condensation
Figure 3: The synthesis of 1- and 4-nitro-NPDOs by condensation reaction of 2-amino-pyridine with 5-
nitro-2,3-dichloro-1,4-napthoquinone (110 °C/56 h)
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Received: March 14, 2017, accepted: April 29, 2017, published: May 11, 2017
1
Characterization
Naphtho[1‘,2‘:4,5]imidazo[1,2-a]pyridi-
1115, 1072, 1010, 942, 910, 736. H-NMR
(DMSO-d6): δ (ppm) 9.36 (d, 1H, H-8, JH8-
ne-5,6-dione (NPDO). Orange solid. Yield: H9=7.62 Hz) 8.96 (s, 1H, H-4) 8.57 (d, 1H, H-
43%. Melting point: 304-305 °C. UV-Vis 1, JH1-H2= 8.54 Hz) 8.43 (d, 1H, H-2, JH1-
λmax (nm): 216, 242, 261, 283, 295, 396. FT- H2=8.54 Hz) 7.93 (d, 1H; H-11, JH10-H11=7.63
IR (KBr): ν (cm-1) 1687, 1651 (C═O), 1629, Hz) 7.75 (t, 1H, H-10, JH9-H10=JH10-H11=7.63
1602, 1498, 1482, 1418, 1326, 1257, 1197, Hz) 7.32 (t, 1H, H-9, JH8-H9 =JH9-H10 =7.63
1151, 1089, 927, 899, 766, 723, 699, 622, Hz). HRMS m/z [M+H]+. 294.0505 (calc.:
1
576. H-NMR (d6-DMSO): δ (ppm) 9.25 (d, 294.0509).
1H, H-8, JH8-H9=5.00 Hz) 8.12 (d, 1H, H-10,
9-Nitro-naphtho[1‘,2‘:4,5]imidazo[1,2-
JH9-H10=5.00 Hz) 7.70, 7.46 (2m, 4H, H-1, H- a]pyridine-5,6-dione (9-nitro-NPDO). Re-
13
2, H-3, H-4) 7.14 (m, 1H, H-9). C-NMR dish orange solid. Yield: 11 %. M.p. 342-3 °C
(CDCl3): δ (ppm) 181.82 (1C, C-5) 169.33 (with decomposition). FT-IR (KBr): ν (cm-1)
(1C, C-6) 155.58 (1C, Cquat) 150.56 (1C, 1694, 1652 (C=O), 1640, 1599, 1551, 1513,
Cquat) 135.74 (1C, C-2) 131.71 (1C, Cquat) 1413, 1344, 1304, 1270, 1240, 1220, 907,
131.32 (1C, C-10) 130.79 (1C, C- Cquat) 822. 1H-NMR (CDCl3): δ (ppm) 9.45 (s, 1H,
130.55 (1C, C-3) 130.00 (1C, C-4) 128.73 H-8) 8.17, 7.62 (2m, 4H, H-1, H-2, H-3, H-4)
(1C, Cquat) 126.72 (1C, C-8) 124.75 (1C, C- 7.75 (dd, 1H, H-11, JH10-H11=9.6 Hz, JH8-
1) 118.03 (1C, C-11) 116.65 (1C, C-9). H11=0.73 Hz) 7.58 (dd, 1H, H-10, JH10-H11=9.6
13
HRMS: λmaxm/z [M+H]+. 249.0652, (calc.: Hz, JH8-H10=2.06 Hz). C-NMR (CDCl3): δ
249.0657).
(ppm) 182.13 (1C, C-5), 164.04 (1C, C-6),
Naphtho[1‘,2‘:4,5]imidazo[1,2-a]pyrimi- 155.40 (C-quat), 135.6 (1C, C-3) 133.5 (1C,
dine-5,6-dione (PNPDO). Bright yellow C-4) 131.10 (1C, C-8) 127.8 (1C, C-1) 122.3
solid. Yield: 17 %. Melting point: 345 °C. (1C, C-11) 114.4 (1C, C-9). HRMS m/z
UV/ Vis λmax (nm): 241, 263, 293. FT-IR [M+H]+ 294.0512 (calc.: 294.0509).
spectrum ν (cm-1): 3049, 3066, 1700, 1654,
1-nitro-naphtho[1‘,2‘:4,5]imidazo[1,2-
1610, 1540, 1523, 1479, 1423, 1396, 1375, a]pyridine-5,6-dione (1-nitro-NPDO). Or-
1347, 1241, 1220, 1186, 1153, 1119, 1073, ange red solid. Yield: 17 %, m.p. 292 °C. UV/
1014, 938, 920, 887, 850, 832, 815, 784, 757, Vis spectrum (nm): 240, 296, 398. FT-IR (cm-
736, 723, 696, 669, 648, 610, 578. H-NMR 1): 3080, 1701, 1654, 1601, 1539, 1497, 1445,
1
(CDCl3): δ (ppm) 9.53 (dd, 1H, H-8, J JH8- 1415, 1405, 1377, 1320, 1285, 1244, 1218,
H9=6.41 Hz) 8.88 (d, 1H, H-10, JH1-H2 =7.93 1186, 1145, 1003, 929, 905, 868, 833, 768,
1
Hz) 8.36 (dd, 2H, H-1, H-4, JH3-H4=9.16 Hz), 750, 715, 699. H NMR: (400 MHz, d-chlo-
7.75 (m, 2H, H-2, H-3), 7.27 (t, 1H, H-9, JH8- roform): δ (ppm) 9.34 (d, 1H, H-8, JH8-
H9=JH9-H10=6.09 Hz). 13C-NMR (CDCl3): δ H9=6,43Hz); 8.32 (d. 1H, H-2, JH2-H3 =7.85Hz;
(ppm) 187.68 (1C, C-5), 182.16 (1C, C-6) 7.87 (m. 3H, H-2, H3, H-11); 7.74 (t. 1H, H-
177.00 (1C, Cquat) 150.16 (1C, Cquat) 10, JH9-H10=JH10-H11=7.76Hz); 7.54 (d, 1H, H-
149.40 (1C, Cquat) 144.90 (1C, C-4) 136.06 4, JH3-H4=7.95Hz); 7.31 (t, 1H, H-9, JH8-
(1C, C-2) 132.86 (1C, C-10) 130.84 (1C, H9=JH9-H10=6. 24Hz). 13C-NMR (100 MHz, d-
Cquat) 130.82 (1C, Cquat) 129.17 (1C, C-8) chloroform) ): δ (ppm) 190.20, 184.52, (2C,
126.99 (1C, C-1) 124.54 (1C, C-3) 117.40 C-5, C-6), 175.06 (1C, Cquat), 141.92 (1C, C-
(1C,
C-9).
HRMS:
(C14H6N4O4): 1), 133.02 (1C, C-2), 130.67 (1C, C-10),
[M+H]+.:250.0608 (found), 250.0611(calc.).
126,85 (1C, C-8), 125.10 (1C, C-4), 122,36
(1C, C-3), 117.11 (1C, C-11), 116.71 (1C, C-
3-Nitro-naphtho[1‘,2‘:4,5]imidazo[1,2-
a]pyridine-5,6-dione (3-nitro-NPDO). Red 9). HRMS: (C15H7N3O4) [M+H]+.: 294.0510
solid. Yield: 57 %. Melting point >360 °C. (found), 294.0509 (calc.).
FT-IR spectrum ν (cm-1): 1702, 1651 (C═O),
1628, 1609, 1544, 1521 (NO2), 1504, 1409,
1348, 1325, 1287, 1245, 1210, 1191, 1153,
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EXCLI Journal 2017;16:663-678 – ISSN 1611-2156
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4-nitro-naphtho[1‘,2‘:4,5]imidazo[1,2-
(Wavefunction Inc., version 1.1.0, 2011, Ir-
a]pyridine-5,6-dione (4-nitro-NPDO). Red vine, CA, USA) software package. Their ge-
solid. Yield 21 %, m.p. 323 °C. UV/ Vis spec- ometries were initially refined by the mechan-
trum (nm): 241, 264, 297, 400. FT-IR (cm-1): ical MMFF94 method and further optimized
3073, 3038, 1695, 1652, 1602, 1541, 1498, by the semi-empirical PM6 method. The final
1474, 1450, 1416, 1371, 1325, 1285, 1256, reoptimization and computation of the com-
1240, 1218, 1185, 1256, 1241, 1218, 1186, pounds were performed by DFT-B3LYP
1150, 1110, 1006, 931, 905, 826, 806, 771, functional with 6-31+G (d) basis set. The ge-
718, 703, 686, 574. HRMS: (C15H7N3O4) ometries of the compounds were globally op-
[M+H]+.: 294.0508 (found), 294.0509 (calc.) timized without symmetry constraints and
1H NMR: (400 MHz, d-chloroform): δ (ppm) their stationary points were confirmed by vi-
9.29 (d, 1H, H-8, JH8-H9=6,40Hz); 8.42 (d. 1H, brational frequency analysis. The electron af-
H-10, JH10-H11=JH9-H10 =7.94Hz; 7.83 (m. 2H, finity (EA) of the compounds was assessed as
H-11); 7.74 (t. 1H, H-10, JH9-H10=JH10- the Gibbs' energy difference between their op-
H11=7.64Hz); 7.51 (d, 1H, H-3, JH3- timized neutral and anion free radical states
H4=7.95Hz); 7.27 (t, 1H, H-9, JH8-H9=JH9- (at 298.15 K). The cDFT-based global reac-
H10=6.12Hz). 13C-NMR (100 MHz, d-chloro- tivity indices of the compounds were calcu-
form) ): δ (ppm) 187.74, 192.19 (2C, C-5, C- lated applying their LUMO and HOMO ei-
6), 177.02 (1C, Cquat), 144.95 (1C, C-4), genvalues, and their regional electrophilic re-
136.12 (1C, C-2), 132.98 (1C, C-10), 129,21 activity was assessed in terms of the electro-
(1C, C-8), 127.13 (1C, C-1), 124,56 (1C, C- philic Fukui function (f +) values by using the
3), 118.63 (1C, C-11), 117.41 (1C, C-9).
3-nitro-naphtho[1‘,2‘:4,5]imidazo[1,2-
a]pyrimidine-5,6-dione (3-nitro-PNPDO).
UV/Vis spectrum λmax (nm): 239, 319,
single-point FMO approach (Contreras et al.,
1999) employing the PYTHON scripting lan-
guage.
430. FT-IR spectrum (cm-1): 3049, 3066, Enzymes and enzymatic assay
1702, 1654, 1610, 1540, 1523, 1479, 1423,
NADPH:cytochrome P-450 reductase
1396, 1375, 1347, 1241, 1220, 1186, 1153, (CPR; EC 1.6.2.4) was purified by the method
1119, 1073, 1014, 938, 920, 887, 850, 832, described in Pechurskaja et al. (2007), and its
815, 784, 757, 736, 723, 696, 669, 648, 610, concentrations was quantified spectrophoto-
1
578. H-NMR (d-chloroform): δ (ppm) 9.58 metrically at 455 nm (Δε456 = 21.4 mM-1
(dd, 1H, H-8, J JH8-H9=6.43 Hz) 8.93 (s, 1H, cm-1). NAD(P)H: quinone oxidoreductase
H-4) 8.36 (dd, 2H, H-1, H-4, JH1-H2=9.13 Hz), (DTD, NQO1; EC 1.6.99.2) was purified by
7.83 (m, 2H, H-2, H-4), 7.27 (t, 1H, H-9, JH8- the established method (Prochaska, 1988),
H9=JH9-H10=6.43
Hz).
13C-NMR
(d- and its concentration was quantified at 460
chloroform): δ (ppm) 188.18 (1C, C-5), nm applying Δε460 = 11.0 mM-1 cm-1. Bovine
183.23 (1C, C-6) 179.40 (1C, Cquat) 153.04 cytochrome c, superoxide dismutase (SOD;
(1C, Cquat) 147.18 (1C, Cquat) 145.98 (1C, EC 1. 15.1.1) and catalase (CAT; EC
C-4) 138.46 (1C, C-2) 133.89 (1C, C-10) 1.11.1.6) were obtained from suppliers
132.54 (1C, Cquat) 131.12 (1C, Cquat) (Sigma-Aldrich (Saint Louis, MO, USA)).
129.96 (1C, C-8) 127.19 (1C, C-1) 125.23
The enzymatic reactivity of the com-
(1C, C-3) 119.10 (1C, C-9). HRMS: pounds was measured spectrophotometrically
(C14H6N4O4): [M+H]+. : 295.0460 (found), by Carry-60 spectrophotometer (Agilent
295.0462 (calc.)
Technologies) in 0.1 M K-phosphate buffer
(pH 7.0) containing 1 mM EDTA, at 25 °C.
The enzymatic rates were defined according
Computational details
The quantum mechanical computation of to NADPH oxidation at 340 nm (Δε340 = 6.22
the compounds was performed by Spartan 10' mM−1 cm−1) and corrected for the auto-oxida-
tion of the enzymes in the absence of quinoid
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EXCLI Journal 2017;16:663-678 – ISSN 1611-2156
Received: March 14, 2017, accepted: April 29, 2017, published: May 11, 2017
substrates (0.11 s-1 and 0.05 s-1 for CPR and was removed, and formazan crystals were dis-
DTD, respectively). Typically, 10-15 concen- solved by 96 % EtOH. The absorbance den-
trations of the compounds were used, and sity was measured at 570 nm (Varioskan
each measurement was performed in tripli- Flash microplate spectrophotometer; Thermo
cate. In separate experiments, quinone-medi- Scientific, USA). At least three independent
ated single-electron reduction of 50 µM fer- experiments were performed for each concen-
ricytochrome
c
was
monitored tration of the compounds and the cell survival
spectrophotometrically at 550 nm (Δε550
=
was assessed as a percentage in respect to
20.0 mM-1 cm-1), and O2 uptake was recorded 0.1 % DMSO-treated cells. The CL50 values
polarographically applying a Clark electrode were defined by using a four-parameter lo-
(Digital Model 10, Rank Brothers Ltd., Cam- gistic equation (SigmaPlot software, Inc., San
bridge, UK), at 25 °C.
Jose, CA).
Assessment of kinetic parameters
Double fluorescence Acridine Orange
The apparent turnover number (kcat) and (AO)/Ethidium Bromide(EB) staining
the apparent Michaelis constant (KM,Q) values The type of cell death was assessed by
were obtained by using Michaelis-Menten means of the double AO/EB fluorescence
equation of v0/[E0] (s-1) vs. [Q], where v0, [E0] staining. After 24 h treatment by ortho-
and [Q] denote the enzymatic rate, the total quinoid compounds at their CL50 concent-
enzyme concentration, and the concentration rations, the cells were washed with PBS,
of the compound, respectively. The apparent stained with AO (100 μg/ml in PBS) and EB
second-order rate constant (kcat/KM,Q ) values (100 μg/ml in PBS) and analysed by
were calculated applying the re-parameter- fluorescence microscope (Olympus IX51,
ized form of Michaelis-Menten equation Japan). 0.1 % DMSO-treated cells served as a
(Kolm et al., 1995; Šarlauskas et al., 2016).
negative contol.
Tumor cell lines and cell viability assay
RESULTS AND DISCUSSION
Human pulmonary epithelial A-549 and
breast tumor MCF-7 cells were cultured in
DMEM media (Life Technologies, USA),
containing 10 % (v/v) fetal bovine serum
(Life Technologies, USA), 100 U/ml penicil-
lin, and 100 μg/ml streptomycin (Biological
Industries, Israel), in a humidified atmosphere
with 5 % CO2 at 37 °C. The adherent cells
were detached by 0.25 % trypsin-EDTA. The
target cells, at density of 2 × 105/ml, were
seeded into 96-well plates to adhere for 24 h.
After reaching 60–80 % confluence, they
were treated by the compounds for 24 h. Cell
survival was quantified by colorimetric assay
based on the conversion of MTT to MTT-
formazan product. The stock solutions of the
compounds were prepared in DMSO (0.1 %
(v/v) in culture media). After the 24 h treat-
ment with the compounds, the cells were
The computational study
The quantum mechanical computation
showed that the structures of optimized tetra-
cyclic systems of NPDO and PNPDO quin-
oids have a planar geometry and the orienta-
tion of the nitro groups depends on the posi-
tions of their attachment. The nitro groups at-
tached at the 3 and 9 positions of 3- and 9-
nitro-quinoids were almost parallel to the tet-
racyclic plane, pointing to their electron-at-
tracting -inductive and -resonance effects,
while the dihedral angles of the nitro groups
introduced at the 1 and 4 positions were de-
fined to be almost at the perpendicular orien-
tation ( ~89o), thus pointing to their elec-
tron-attracting inductive effects. Note that the
close dihedral angle of the nitro group was de-
fined for 4-nitro-NPDO quinone by means of
washed twice with 0.1 M PBS, and MTT so- the X-ray diffraction analysis ( ~84o) (un-
lution (0.2 mg/ml PBS) was added. Following published data). The twisting of the nitro
1 h incubation at 37 °C, the overlying media
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EXCLI Journal 2017;16:663-678 – ISSN 1611-2156
Received: March 14, 2017, accepted: April 29, 2017, published: May 11, 2017
groups is most likely to be caused by the re-
As shown in Table 1, the PNPDO, which
pulsive interaction of the lone electron pair of differs from NPDO by the existence of an ad-
the nitrogen atom (N-12) of the tetracyclic ditional nitrogen atom in the tetracyclic sys-
system with the oxygen atom of the nitro tem (Figure 1), possesses a higher electron-
group attached to position 1 in the 1-nitro- accepting potency compared to NPDO
quinoid compound, and by the repulsive inter- quinoidal. The attachment of nitro groups to
action of the oxygen atom of quinoid moiety NPDO and PNPDO compounds substantially
with the oxygen atom of the nitro group added increases the electrophilic potency for nitro-
to position 4 in the 4-nitro-quinoid com- quinoidals as reflected in terms of the as-
pound.
sessed EA, ϵLUMO and ω index values (Table
The electrophilic (electron-accepting) po- 1), which vary depending on the positions of
tencies of the compounds assessed in terms of the nitro groups, i.e, 3-nitro- and 9-nitro-
their LUMO energy (ϵLUMO) along with the quinoidals exhibited a markedly higher elec-
electron affinity (EA) values are listed in Ta- trophilic potency compared to that of 1- and
ble 1. This table also provides a set of reactiv- 4-nitro-quinoidals. For the whole set of the
ity indices of the compounds estimated apply- compounds, the approximate order of the sin-
ing the conceptual DFT (cDFT) approach gle- and two-electron accepting potencies
(Geerling et al., 2003, and refs. therein). Fol- were as follows: 3-nitro-PNPDO > 3-nitro-
lowing this approach, the electrophilicity in- NPDO ~ 9-nitro-NPDO > 1-nitro-NPDO > 4-
dex (ω), representing the global electrophilic nitro-NPDO > PNPDO > NPDO (Table 1).
power of molecules, is defined by ω = µ2/(2η)
Since, in contrast to the unsubstituted
= (µ2/2)S where µ and η denote the electronic quinoidals, the nitro-quinoid compounds con-
chemical potential and electronic chemical tain two electron-accepting/redox-active
hardness (or softness, S = 1/η), respectively, sites, namely the quinone moiety and the nitro
which were approached in terms of HOMO group, their regional electrophilic potencies
and LUMO eigenvalues of the compounds, µ were assessed in terms of the electrophilic Fu-
= (ϵLUMO + ϵHOMO)/2 and η = 1/S = ϵLUMO
-
kui function (FF) values grouped over C=O
ϵHOMO. Using this approach, the quantitative atoms of the quinone moiety ( f + ) and N and
q
+
relationships between the hydride affinity and O atoms of the nitro group ( f -nitro), which
the ω index values have been defined for a may reflect the propensity to accept nucleo-
range of para- and ortho-quinoids phile (an electron and/or an hydride ion) at the
(Campodonico et al., 2009), which shows that initial stage of the redox conversion of the
this quantity can be applied to predict the ease compounds. It should be noted that for the as-
of the two-electron (hydride)-reduction of sessment of the FF values, the single
quinoid oxidants.
Table 1: The electron affinity (EA), the LUMO energy (ϵLUMO), the electronic chemical potential index
(µ), the chemical hardness index (η), and the electrophilic ω index values of the ortho-quinoid com-
pounds calculated by means of DFT-B3LYP functional with 6-31+G(d) basis set
No. Compound
EA
ϵLUMO
µ
η
ω
eV
1.
2.
3.
NPDO
PNPDO
1-NO2-NPDO
1.779
1.951
2.181
-3.232
-3.420
-3.622
-4.926 3.387
-5.289 3.537
-5.264 3.286
3.583
3.955
4.216
4.
5.
6.
7.
3-NO2-NPDO
4-NO2-NPDO
9-NO2-NPDO
3-NO2-PNPDO
2.407
1.941
2.434
2.564
-3.780
-3.622
-3.770
-3.970
-5.425 3.284
-5.292 3.342
-5.452 3.367
-5.664 3.414
4.481
4.191
4.414
4.699
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point frontier molecular orbital (FMO) ap- accompanied by concomitant oxygen uptake
proach (Contreras et al., 1999) was used in and its initial rate was supressed by
this study as one of the most appropriate superoxide dismutase (SOD, 150 µg mL-1) by
methods for redox active compounds which, 30-45 %, while the introduction of catalase
in contrast to the frequently used finite-differ- (CAT, 100 U mL-1) showed a marginal
ence approximation methods, yields the posi- inhibiting effect, indicating the quinone/semi-
tive FF values and obeys the normalization quinone redox cycling which yields
condition (Šarlauskas et al., 2014, and refer- superoxide. For all the compounds examined,
ences therein). As expected, the highest frac- the initial NADPH oxidation rates followed
tion of the global electrophilic potency of the hyperbolic dependence on the varying con-
nitro-quinoidals resides upon the C=O atoms centrations of the compounds (Figure not
of the quinone moiety (the f values varied shown), with kcat of 28-35 s-1 being close to
+
q
in the range of 0.42 (9-nitro-NPDO) - 0.56 (4- the previously reported turnover number
nitro-NPDO)) as the most preferential site for (~ 25 s-1) for FMNH--mediated reduction of
the reduction of compounds, while the f+
ferricytochrome c (Čėnas et al., 1994). The
values of the nitro groups were assessed to be catalytic efficiency of the reduction of the
-nitro
highly dependent upon their position: the f+
compounds, estimated in terms of kcat/KM, Q
values, varied in the range of 3.1-7.2 × 107
M-1 s-1 (Table 2) which tentatively increased
with an increase in their electrophilic potency
(Table 1).
-
nitro values of the coplanar (-conjugative) ni-
tro groups of 3-, 9-nitro-NPDO and 3-nitro-
PNPDO compounds (0.100, 0.149 and 0.146,
respectively) were much higher than those of
the twisted nitro groups of 1- and 4-nitro-
quinoidals, with f+-nitro of 0.003 and 0.004, re-
spectively, implying that the nitro groups of
1- and 4-nitro-quinoidals, compared to 3- and
9-nitro-quinoidals, possess a much lower ten-
dency to undergo reductive conversion.
The
NQO-1-mediated
two-electron
(hydride) reduction of the compounds
proceeded with kcat of 170 - 350 s-1 and
kcat/KM,Q values equaled to 1.6-7.4 × 108 M-1
s-1, which were approximately ten-folds
higher than those of CPR-mediated reduction
of the compounds (Table 2). The kcat/KM,Q of
these quinoidals were defined to be far higher
than those of ortho-quinoid compounds such
as phenanthrene ortho-quinone (9,10-PQ)
(kcat/KM,Q of 1× 106 M-1 s-1 (Anusevičius et al.,
Enzymatic reactivity
During NADPH-cytochrome P-450 re-
ductase (CPR)-mediated reactions, the time
course of oxidation of NADPH (150 µM) by
(P)NPDOs as well as by nitrated quinoidals 2002)), -lapachone and its synthetic
(5-10 µM) proceeded in a single continuous analoques (kcat/KM,Q of ~ 1-3 × 106 M-1 s-1
phase (Figure not shown). The reactions were (Bian et al., 2014, 2015)).
Table 2: The apparent second-order rate constant (kcat / KM(Q)) values of CPR and DT-diaphorase-me-
diated reduction reactions of (P)NPDO quinone compounds
Compound
CPR
DDT
No.
kcat/KM,Q (M-1 s-1)
1.
2.
3.
4.
5.
6.
7.
NPDOa
(3.1 ± 0.2) × 107
(3.4 ± 0.2) × 107
(3.3 ± 0.5) × 107
(7.2 ± 0.5) × 107
(4.9 ± 0.2) × 107
(5.9 ± 0.2) × 107
(6.1 ± 0.4) × 107
(2.4 ± 0.1) × 108
(1.6 ± 0.4) × 108
(4.6 ± 0.7) × 108
(7.4 ± 0.3 ) × 108
(4.7 ± 0.3) × 108
(3.2 ± 0.2) × 108
(5.1 ± 0.4) × 108
PNPDOa
1-NO2-NPDO
3-NO2-NPDOa
4-NO2-NPDO
9-NO2-NPDOa
3-NO2-PNPDOb
aTaken from (Šarlauskas et al., 2016)
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Like in CPR-mediated single-electron to be much faster than the reoxidation of sem-
.-
tranferring reactions, the NQO-1-mediated iquinones by O2, with kEX and E1 (O2/O2 ) of
7
oxidation of NADPH (200 µM) by ~100-450 s-1 (Zahir et al., 1988) and -155 mV
unsubstituted as well as nitrated quinoidals (Wardman, 1989), respectively. Thus the re-
(5-10 µM) proceeded in a single continuous duction of ferricytochrome c by semiquinone
phase, far exceeding the concentrations of the is more likely to be indirectly suppressed by
compounds (Figure not shown); the reactions SOD by scavenging the superoxide and thus
were followed by the concomitant consump- lowering the concentration of semiquinone
tion of O2 with rates close those of NADPH radical, owing to its reoxidation by O2.
oxidation. The rates of O2 uptake were
suppressed by the introduction of CAT (by ca. Antitumor activity
30-65 %), while SOD showed marginal
The in vitro antitumor effects of the
supressing effects (~3-5 %), clearly indicating quinoid compounds against human tumor cell
the generation of peroxide as a main reaction lines, lung carcinoma A-549, breast cancer
product.
MCF-7, and leukemia HL-60 cell lines were
Measuring NQO-1-mediated reduction of assessed after their 24-h treatment by varying
the compounds in the presence of cytochrome concentrations of the compounds up to the
c (50 µM) as a terminal single-electron limits of their solubility (~12.5-15 µM). The
acceptor, the initial rates of its reduction were viability of A-549 and MCF-7 cell lines
close to the double rates of NADPH versus NPDO quinoidals decreased in a
oxidation. In introduction of SOD partially concentration-dependent manner, and the
supressed the rates (by ca. 30-50 %), implying application of four-parametric logistic
the superoxide-mediating reduction of function provided appropriate fits to the
cytochrome c. In this reaction system, the experimental data (Figure 4). The estimated
superoxide is likely to be formed through the CL50 values of the compounds, causing a
single-electron oxidation of the hydroquinon- 50 % lethal effect, are given in Table 3.
es by cytochrome c producing semiquinone Unlike A-549 and MCF-7 cells, the cytotoxic
radicals and their reoxidation by O2. It should potency of NPDO quinoids towards HL-60
be noted that semiquinone radicals can also be leukemic cells was markedly lower (CL50
directly reoxidized by ferricytochrome c as it > 15 µM L-1), which hampered the establish-
has previously been reported by Winterbourn ment of the concentration-response curves
(1981). The reoxidation of semiquinones by and, consequently, the assessment of CL50
O2 and cytochrome c has been defined to fol- values (Figure not shown). As shown in Table
low an "outer-sphere" electron transfer (ET) 3, the cytotoxic potency of the whole set of
mechanism governed by the single-electron the quinoid compounds towards MCF-7
potential (E1 ) and the self-exchange rate con- (CL50 = 0.08 - 2.74 µM L-1) was in general
7
stant (kEX) values of the reactive species higher than their potency against A-549 cell
(Meyer et al., 1983; Tollin et al., 1986; Mar- line, with CL50 values of 0.28-7.66 µM L-1,
cus & Sutin, 1985). The reoxidation of sem- except for 3-nitro-NPDO, whose activity
iquinones by ferricytochrome c, whose kSE against A-549 (CL50 = 0.12 ± 0.03 µM L-1)
and E1 (cyt c+3/cyt c+2) are equal to 5 × 105 M- was approximately twice as high as that on
7
1 s-1 (Dixon et al., 1989) and + 260 mV (Rod- MCF-7 cell line (CL50 = 0.27 ± 0.08 µM L-1).
key and Ball, 1950), respectively, is predicted
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Figure 4: The dependence of viability of A-549 and MCF-7 tumor cells upon the varying concentrations
of NPDO and PNPDO quinoidals. The cell survival was assessed by means of MTT assay and
expressed as percentages relative to the 0.1 % DMSO -treated cells used as control. Each point
represents the mean of the experiments performed in triplicate and the vertical bars the standard error
of the mean (S.E.M). The data were analyzed by nonlinear regression analysis by using the logistic type
concentration-responce curve.
Table 3: Cytotoxic effects of ortho-quinoids against A-549, MCF-7 and HL-60 cell lines (results are
shown as the mean ± S.E.M.)
No. Compound
A-549
MCF-7
HL-60
CL50 (µM L-1)
1.
2.
3.
4.
5.
6.
7.
NPDOa
0.67 ± 0.16
1.59 ± 0.28
7.01 ± 0.50
0.12 ± 0.03
7.66 ± 0.73
2.30 ±0.65
1.15 ± 0.48
0.51 ± 0.13
0.78 ± 0.27
2.02 ± 0.47
0.28 ± 0.08
2.74 ± 0.85
1.70 ± 0.10
0.076 ± 0.010
> 15
>15
PNPDOa
1-NO2-NPDO
3-NO2-NPDOa
4-NO2-NPDO
9-NO2-NPDOa
3-NO2-PNPDO
> 15
> 15
> 15
> 15
> 15
aTaken from (Šarlauskas et al., 2016)
Compared to NPDO quinoidal, PNPDO, 0.48 µM L-1) was only slightly higher com-
possessing a higher electrophilic potency, ex- pared to its precursor (CL50 =1.59 ± 0.28 µM
hibited lower antitumor activity against both L-1). Virtually the same was observed for 3-
A-549 and MCF-7 cell lines (Table 3). The at- nitro-NPDO quinoidal, whose cytotoxic ef-
tachment of nitro group at position 3 of the fects on A-549 cell line (CL50 = 0.120 ± 0.03
benzene ring of PNPDO quinoidal signifi- µM L-1) and MCF-7 cells (CL50 = 0.280 ±
cantly enhanced the cytotoxic potency of 3- 0.08 µM L-1) were approximately 6- and 2-
nitro-PNPDO against MCF-7 cell line, with folds higher, respectively, than those of
CL50 value of 0.076 ± 0.010 µM L-1 being 10- NPDO compound. In contrast to 3-nitro- and
folds higher than that of its precursor (CL50 = 9-nitro-NPDOs, the attachment of the nitro
0.78 ± 0.27 µM L-1), while activity of 3-nitro- group at the positions 1 and 4 of NPDO ben-
PNPDO on A-549 cell line (CL50 of 1.15 ± zene ring markedly decreased the cytotoxic
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Received: March 14, 2017, accepted: April 29, 2017, published: May 11, 2017
potency of 1-nitro- and 4-nitro-quinoidals to- treated with the most active quinoid com-
wards A-549 cell line and to a lesser degree pounds were quantified applying the dual
on MCF-7 cell line when compared to NPDO, EB/EO fluorescence staining approach. As
as evidenced in Figure 4 and listed in Table 3. shown in Figure 5A and B, 0.1 % DMSO-
These data show, that, in general, the 3- and treated A-549 and MCF-7 tumor cells used as
9-nitro substituted quinoids with planar nitro controls showed 93-95 % viable cells (highly
groups and thus higher electrophilic potency condensed bright green chromatin), 2-3 %
for their single- and two-electron (hydride) re- viable apoptotic (bright green nucleus with
duction, as defined in terms of EA, ϵLUMO and condensed/fragmented chromatin), ~ 3 % late
ω index values, respectively, exhibited mark- apoptotic (condensed and fragmented orange
edly higher antitumor activity as compared to chromatin), and 1-2 % necrotic cells (non-
that of the 1- and 4-nitro-quinoidals with non- fragmented red nucleous), respectively. After
planar nitro groups and lower electrophilic 24 h treatment, NPDO and 3-nitro-NPDO
potency. In contrast to the unsubstituted or- quinoidals, at their CL50 concentrations
tho-quinones, the nitro-quinone compounds (Table 3), diminished the viable cells to 47-
bearing a nitro group as an additional electro- 50 % (A-549) and 30-45 % (MCF-7), respect-
philic site may experience more complex met- ively, and increased the apoptotic cells (34-
abolic conversion. The (cyto)toxic action of 38 % (A-549) and 30-45 % (MCF-7) viable
nitro-based xenobiotics and drug agents is fre- apoptotic and 11-15 % (A-549) and 9-12 %
quently related to their reductive activation, (MCF-7) nonviable apoptotic), while the
i.e, single-electron reduction to the free radi- percentages of the necrotic cells in both cell
cals followed by ROS generation and/or two- lines were virtually the same as those in the
/four-electron reduction of the nitro groups untreated cells, implying that the tumor cell
yielding highly reactive nitroso-/hydroxyla- death induced by the treatment of NPDO
mine intermediates (Tocher, 1997; Knox and quinoidals occurs primarily through apop-
Chen, 2004; Chen and Hu, 2009, and refs. tosis.
therein). The position and the geometry of the
nitro groups have been reported to be im-
portant structural features of the nitro-based
xenobiotics in their cytotoxic activity, i.e, the
compounds with planar nitro groups have
been observed to be more effective cytotoxic
agents than their isomers bearing non-planar
nitro groups (Onchoke et al., 2004; Taka-
mura-Enya et al., 2006). As reported above,
the assessed electrophilic Fukui function val-
ues showed that the highest fraction of the
electrophilic potency of nitro-quinoidals re-
sides upon the C=O atoms, suggesting that the
cytotoxic activity of the compounds is most
likely to be due to the redox action of their
quinone moiety; however, a relatively high
regional electrophilic potency was also de-
fined for the nitro groups of the 3- and 9-nitro-
quinoidals, which implies that their nitro
groups can be involved in cytotoxic action.
To estimate the mode of cell death in-
duced by the ortho-quinoidals, the tumor cells
CONCLUDING REMARKS
The nitrogen-containing tetracyclic ortho-
quinones and their nitrated derivatives serve
as excellent electron-accepting and redox ac-
tive substrates of single- and two-electron
(hydride)-transferring electron transferring
flavoenzymes CPR and NQO-1, respectively,
generating reactive oxygen species, and dis-
played relatively high antitumor potency. The
antitumor activities of nitro-quinoidals were
observed to be dependent upon the position of
nitro groups added to the tetracyclic system;
3- and 9-nitro-quinoidals having the highest
electron-accepting potency and being virtu-
ally the most efficient enzymatic substrates,
showed the highest antitumor effects while 1-
and 4-nitro-quinoidals produced the least
anti-tumor activities. The assessment of the
electrophilic Fukui function values implies
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Received: March 14, 2017, accepted: April 29, 2017, published: May 11, 2017
Figure 5: A) Morphological changes of A-54 tumor cells upon 24-h treatment by CL50 concentrations of
NPDO and 3-nitro-NPDO quinoidals, quantified by means of AO/EB staining (viable cells (solid arrows),
viable apoptotic (dashed-dot arrows), nonviable apoptotic (dotted arrows), and necrosis cells (dashed
arrows)). B) The percentage of viable, apoptotic and necrotic cells of A-549 and MCF-7 tumor cells upon
the treatment by NPDO and 3-nitro-NPDO. Data represent the mean of at least three independent ex-
periments.
that ortho-quinone moiety of the compounds Acknowledgement
is essential for their redox action inducing cy-
The authors are highly grateful for the fi-
totoxic effects; however, a relatively high lo- nancial support of the Scientific Council of
cal electrophilic potency defined for nitro Lithuania (the project No. MIP-032/2014).
groups of the 3- and 9-nitro-quinoidals sug- The authors thank Audrius Laurynėnas (Vil-
gests that their nitro groups might be also in- nius University, Center of Life Science) for
volved in cytotoxic action. The data obtained his excellent assistance in DFT computation.
about the structures of (P)NPDOs, global and The authors also thank Lina Marčiulionytė
regional reactivities, and their antitumor (Lecturer at Vilnius University) for proof-
activities provide some guide for synthesis of reading the manuscript.
related structure compounds as antitumor
agents.
Conflict of interest
The authors declare no conflict of interest.
675

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Received: March 14, 2017, accepted: April 29, 2017, published: May 11, 2017
Chen Y, Hu L. Drug of anticancer prodrugs for reduc-
tive activation. Med Chem Rev. 2009;1:29-37.
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