Discovery of anti-cancer activity for benzo[1,2,4]triazin-7-ones: Very strong correlation to pleurotin and thioredoxin reductase inhibition

Article abstract of DOI:10.1016/j.bmc.2016.05.066

The thioredoxin (Trx)–thioredoxin reductase (TrxR) system plays a key role in maintaining the cellular redox balance with Trx being over-expressed in a number of cancers. Inhibition of TrxR is an important strategy for anti-cancer drug discovery. The natu

Full text of DOI:10.1016/j.bmc.2016.05.066

Accepted Manuscript
Discovery of anti-cancer activity for benzo[1,2,4]triazin-7-ones: Very strong
correlation to pleurotin and thioredoxin reductase inhibition
Martin Sweeney, Robert Coyle, Paul Kavanagh, Andrey A. Berezin, Daniele Lo
Re, Georgia A. Zissimou, Panayiotis A. Koutentis, Michael P. Carty, Fawaz
Aldabbagh
PII:
S0968-0896(16)30410-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.05.066
BMC 13050
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
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25 May 2016
29 May 2016
Please cite this article as: Sweeney, M., Coyle, R., Kavanagh, P., Berezin, A.A., Re, D.L., Zissimou, G.A., Koutentis,
P.A., Carty, M.P., Aldabbagh, F., Discovery of anti-cancer activity for benzo[1,2,4]triazin-7-ones: Very strong
correlation to pleurotin and thioredoxin reductase inhibition, Bioorganic & Medicinal Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bmc.2016.05.066
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Bioorganic & Medicinal Chemistry
Discovery of anti-cancer activity for benzo[1,2,4]triazin-7-ones: Very strong
correlation to pleurotin and thioredoxin reductase inhibition
Martin Sweeneya, , Robert Coylea, , Paul Kavanagha, Andrey A. Berezinb, Daniele Lo Reb, Georgia A.
Zissimoub, Panayiotis A. Koutentisb, Michael P. Cartyc, and Fawaz Aldabbagha,
a School of Chemistry, National University of Ireland Galway, University Road, Galway, Ireland
b Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus
c Centre of Chromosome Biology, Biochemistry, School of Natural Sciences, National University of Ireland Galway, University Road, Galway, Ireland
 These authors contributed equally
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The thioredoxin (Trx)-thioredoxin reductase (TrxR) system plays a key role in maintaining the
cellular redox balance with Trx being over-expressed in a number of cancers. Inhibition of TrxR
is an important strategy for anti-cancer drug discovery. The natural product pleurotin is a well-
known irreversible inhibitor of TrxR. The cytotoxicity data for benzo[1,2,4]triazin-7-ones
showed very strong correlation (Pearson correlation coefficients  0.8) to pleurotin using
National Cancer Institute COMPARE analysis. A new 3-CF3 substituted benzo[1,2,4]triazin-7-
one gave submicromolar inhibition of TrxR, although the parent compound 1,3-
diphenylbenzo[1,2,4]triazin-7-one was more cytotoxic against cancer cell lines.
Benzo[1,2,4]triazin-7-ones exhibited different types of reversible inhibition of TrxR, and cyclic
voltammetry showed characteristic quasi-reversible redox processes. Cell viability studies
indicated strong dependence of cytotoxicity on substitution at the 6-position of the 1,3-
diphenylbenzo[1,2,4]triazin-7-one ring.
Available online
Keywords:
Anti-tumor
Bioreduction
Heterocyclic compound
NCI-DTP COMPARE program
2016 Elsevier Ltd. All rights reserved.
1. Introduction
First identified in the late 1960s,1 and isolated in 1980,2 the rich chemistry of 1,3-diphenylbenzo[1,2,4]triazin-7-ones 1 (Figure 1)
has more recently been explored.3-6 Benzotriazinone 1a (R = H) and derivatives have been implicated as multi-target inhibitors in
Alzheimer’s disease of beta-amyloid (Aβ) aggregation and acetyl-(AChE)/butyryl- (BChE) cholinesterase.7 Scaffold 1 contains a
highly conjugated iminoquinone motif and an iminoquinone derivative of imidazo[5,4-f]benzimidazoles was shown to have good
specificity (Pearson correlation coefficient of 0.51) towards NAD(P)H:quinone oxidoreductase 1 (NQO1) expression using
COMPARE analysis of toxicity towards the 60 cell lines at the National Cancer Institute (NCI) Development Therapeutics Program
(DTP).8
We now report COMPARE analysis of the toxicity of benzotriazinones leading to the discovery of very strong correlations to
pleurotin. The latter naturally occurring antibiotic is a para-quinone with a perhydroanthracene core, which was first isolated in the
1940s from the basidiomycete, Pleurotus griseus.9 Though pleurotin has been synthesized,10 a multi-gram fermentation process
using Hohenbuehelia atrocaerulea for supply of pleurotin to the NCI has been reported.11 Pleurotin possesses antibacterial,9
antifungal,12 and anti-cancer activity, including inhibiting the hypoxia-induced factor (HIF-1α), a transcription factor associated
with many aspects of tumor growth.13 The underlying pathway to much of this biological activity is pleurotin’s ability to act as a
potent inhibitor (IC50 0.17 μM) of the thioredoxin (Trx)-thioredoxin reductase (TrxR) system.14
Earlier reports more specifically describe pleurotin as an irreversible inhibitor of TrxR with a Ki of 0.28 μM.13,15 TrxR is a
flavoprotein homodimer with each monomer containing a FAD prosthetic group, NADPH binding domain, and a redox-active
selenothiol active site.16,17 TrxR is the only known enzyme to reduce Trx protein, which in turn provides reducing equivalents for a
———
*Corresponding author. Tel.: +353 91 49 3120; fax: +353 91 49 5576
E-mail address: fawaz.aldabbagh@nuigalway.ie (F. Aldabbagh)
E-mail address: michael.carty@nuigalway.ie (M. P. Carty)

number of essential redox-dependent cellular processes, such as H2O2 metabolism, sulfate assimilation, and signal transduction.18
Trx protein is, however, over-expressed in a number of cancers, and is associated with increased cell proliferation, inhibition of
apoptosis, and decreased patient survival.19 The reduced form of Trx protein inhibits the tumor suppressor protein PTEN (protein
tyrosine phosphatase and tensin homolog).20 Therefore negative regulation of Trx protein through TrxR inhibition is a significant
strategy for the discovery of new anti-cancer agents. Herein, we investigate the structural requirements of the 1,3-
diphenylbenzo[1,2,4]triazin-7-ones 1a-1l, and the new compound 1-phenyl-3-(trifluoromethyl)benzo-[1,2,4]triazin-7-one 2 for
cytotoxicity against cancer cell lines, and inhibitory activity towards TrxR using a purified enzyme assay.
Figure 1
2. Results and discussion
2.1. Synthesis
The synthesis of 1,3-diphenylbenzo[1,2,4]triazin-7-ones 1a-1l was previously reported starting from 1,3-
diphenylbenzo[1,2,4]triazin-4-yl radical 3,3-6 and 1-phenyl-3-(trifluoro-methyl)benzo[1,2,4]triazin-7-one 2 is similarly accessible by
mild MnO2 oxidation of the analogous trifluoromethyl substituted radical 4 (Scheme 1).4
Scheme 1
2.2. Development Therapeutic Program (DTP) National Cancer Institute (NCI) 60 human tumor cell line screen and
COMPARE analysis
NCI one dose (10 µM) mean graph data of parent 1,3-diphenylbenzo[1,2,4]triazin-7-one 1a showed strong growth inhibition
against a number of cancer cell lines, particularly leukemia, colon, melanoma and renal cancers (Figure 2). Significant activity was
apparent towards the prostate cancer line DU-145 and breast cancer cell line MCF-7, and these cell lines were available to us at our
laboratory in Galway. We investigated the toxicity profile of 1a, following selection of the compound for five-dose testing by the
NCI, which established the GI50, LC50 and TGI. The stated parameters provide the seed vector through which the NCI COMPARE
algorithm determines closely matching cytotoxicity profiles.21 Complete single and five dose data for 1a can be found in the
supplementary data accompanying this article. COMPARE allows comparisons to be carried out towards all the synthetic
compounds in the NCI database. The program applies the commercially available SAS statistical package to calculate a Pearson
product-moment correlation coefficient (0 to ±1) to establish the degree of similarity between two cytotoxicity profiles. Coefficients
in the range (±0.3 to ±0.5) are considered medium strength, while those above (±0.5) are considered strong. Out of ~70,000
compounds in the NCI database, pleurotin had one of the highest correlation coefficients to benzotriazinone 1a at 0.84, which
represents a very strong correlation (Table 1).
Figure 2
1-Phenyl-3-(trifluoromethyl)benzo[1,2,4]triazin-7-one 2 is structurally distinct from benzotriazinones 1a-1l since the 3-phenyl is
replaced by a strong inductively electron-withdrawing trifluoromethyl (CF3) group. Elevated toxicity towards the MCF-7 cell line
supported trends in our later obtained MTT assay data (Table 2). Compound 2 appeared overall somewhat less cytotoxic against the
main cancer types in comparison to 1a (Figure 2), but with a very strong correlation to compound 1a and pleurotin using
COMPARE analysis after the five dose testing (Table 1). The large pearson correlation coefficients (0.8) indicated possible
common pathways for anti-cancer activity.
Table 1
2.3. Cytotoxicity against normal and cancer cell lines using the MTT assay
To determine the specificity and overall cytotoxicity towards selected cancer cell lines, the effect of substitution at the 6-position
of the 1,3-diphenylbenzo[1,2,4]triazin-7-one scaffold with amine, amide and alkoxy substituents was investigated (compounds 1a-
1l in Figure 1). Cytotoxicity was determined against the prostate cancer cell line DU-145 and the breast cancer cell line MCF-7
using the MTT assay (Table 2). These cell lines form part of the NCI-60 human tumor cell line screen. As a control the response of
a normal human-skin fibroblast cell line GM00637 was determined.
Pleurotin displayed submicromolar toxicity towards all three cell lines investigated, with parent 1a showing comparable toxicity.
Substitution with methoxy- and ethoxy-groups in compounds 1k and 1l and amide in 1j decreased toxicity to negligible values. The
methoxy substituent (OMe) on heterocyclic quinones has been shown to lead to significant reductions in toxicity towards cell lines
in comparison to its replacement with an aziridinyl substituent. The trends in toxicity correlate to a reduced electron-affinity
property as a result of the electron donation by the alkoxy substitutent.22,23 Cyclic amine substituents in 1f-1i led to a general
reduction in toxicity values compared to the parent 1a, with the exception of pyrrolo-substituted benzotriazinone 1f. Compound 1f
showed the greatest specificity toward the MCF-7 cell line similar to the toxicity of pleurotin. Among the acyclic amines 1b-1e,
secondary amine substituents in 1c and 1d gave submicromolar toxicity. The ethylamino-substituent in 1d has a similar potency to

the parent 1a with a comparative approximate two-fold reduction in toxicity towards the MCF-7 cell line. 1-Phenyl-3-
(trifluoromethyl)benzo[1,2,4]triazin-7-one 2 with a strong electron-withdrawing substituent at the 3-position was evaluated due to
the lack of availability of benzotriazinones with electron-withdrawing substituents at the 6-position. Compound 2 showed
submicromolar toxicity towards the two cancer cell lines (DU-145 and MCF-7) with a 2- to 3-fold decrease in toxicity towards the
normal skin fibroblast cell line (GM00637).
Table 2
2.4. Cyclic voltammetry
Cyclic voltammetry on compounds 1a and 2 were carried out in anticipation that bioreductive activation may be implicated in
cytotoxicity (Figure 3). Redox response experiments were carried out in dichloromethane, which showed that both 1a and 2 undergo
two characteristic quasi-reversible one-electron redox processes corresponding to the 0/-1 redox transition (I and Iʹ) and -1/-2 redox
transition (II and IIʹ). Benzotriazinone 1a displayed a redox potential (E°ʹ) of -1.12 V vs. Fc/Fc+ for the 0/-1 redox transition (I) and
-1.75 V vs. Fc/Fc+ for the -1/-2 redox transition (II). Benzotriazinone 2 produced a similar redox response to 1a, although E°ʹ values
for equivalent redox couples (Iʹ and IIʹ) are shifted by approximately +0.23 V in the positive direction. This can be attributed to the
strong inductive withdrawal by the 3-CF3 of 2 relative to the 3-phenyl substituent present in 1a resulting in an enhanced
susceptibility of 2 to undergo reduction.
Figure 3
Table 3
2.5. Thioredoxin reductase (TrxR) inhibition assays
To determine whether 1a and 2 inhibited TrxR, and to identify the mode of inhibition, enzyme activity was estimated in the
absence or presence of varying concentrations of inhibitor. Data was analysed using the Lineweaver-Burk plot (Figure 4). In the
case of 1a Vmax decreased and Km increased with increasing inhibitor, characteristic of mixed inhibition. However, 2 is more
characteristic of uncompetitive inhibition where Vmax decreased with increasing inhibitor and Km was lower in the presence of
inhibitor, than in the uninhibited condition.
To estimate the Ki value, a secondary plot (Figure 5) of the slopes of the Lineweaver-Burk plot against inhibitor concentrations
was generated. Ki values were found to be 3.90 and 0.78 µM for compounds 1a and 2, respectively. The lower Ki for 2 towards the
reductase enzyme may be attributed to it being more reducible than 1a as determined by cyclic voltammetry (Figure 3). Therefore
benzotriazinones 1a and 2 clearly inhibit TrxR, but unlike pleurotin the mode of inhibition is reversible rather than irreversible. The
Ki value for the electron-deficient iminoquinone 2 is closer in magnitude to that of pleurotin (Ki 0.28 μM).13,15
Figure 4
Figure 5
3. Conclusions
Using NCI COMPARE program, parent benzotriazinone 1a and 3-CF3 substituted analogue 2 were found to have very strong
correlations to a naturally occurring anti-cancer agent pleurotin. Benzotriazinones 1a and 2 are TrxR inhibitors, although the
mechanism for inhibition is reversible rather than irreversible as for pleurotin. The latter may be attributed to differences in
chemical structure with pleurotin containing an isolated non-conjugated quinone motif.
Varying the substituent at the 6-position of the 1,3-diphenylbenzo[1,2,4]triazin-7-ones has a profound effect on toxicity.
Benzotriazinone 1a is generally more cytotoxic towards cancer cell lines, although compound 2 shows greater specificity towards
solid tumor cell lines in comparison to a normal fibroblast cell line. The latter CF3-substituted compound is more easily reducible
and shows a more than five-fold greater inhibition of TrxR. Differences in the extent and type of TrxR inhibition as well as other
cellular interactions may modulate the cytotoxicity of 1a and 2 towards normal and cancer cell lines.

4. Experimental section
4.1. Synthetic materials and methods
4.1.1. General
1,4-Dioxane was dried over CaH2. Reactions were protected from atmospheric moisture by CaCl2 drying tubes. All volatiles
were removed under reduced pressure. All reaction mixtures and column eluents were monitored by TLC using commercial glass
backed thin layer chromatography (TLC) plates (Merck Kieselgel 60 F254). The plates were observed under UV light at 254 and 365
nm. The technique of dry flash chromatography was used throughout for all non-TLC scale chromatographic separations using
Merck Silica Gel 60 (less than 0.063 mm).24 Melting and decomposition points were determined using a TA Instruments DSC
Q1000 with samples hermetically sealed in aluminium pans under an argon atmosphere using heating rates of 5 °C/min. IR spectra
were recorded on a Shimadzu FTIR-NIR Prestige-21 spectrometer with a Pike Miracle Ge ATR accessory and strong, medium and
1
weak peaks are represented by s, m and w, respectively. H and 13C NMR spectra were recorded on a BrukerAvance 300 machine
(at 300 and 75 MHz, respectively). Deuterated solvents were used for homonuclear lock and the signals are referenced to the
deuterated solvent peaks. MALDI-TOF MS were conducted on a Bruker BIFLEX III time-of-flight (TOF) mass spectrometer. 1,3-
Diphenylbenzo[1,2,4]triazin-7-one
(1a),3
6-amino-1,3-diphenylbenzo-[1,2,4]triazin-7-one
(1b),6
6-(methylamino)-1,3-
diphenylbenzo[1,2,4]triazin-7-one (1c),5 6-(ethylamino)-1,3-diphenylbenzo[1,2,4]triazin-7-one (1d),6 6-(diethylamino)-1,3-
diphenyl-benzo[1,2,4]triazin-7-one (1e),7 1,3-diphenyl-6-(pyrrolidin-1-yl)benzo[1,2,4]triazin-7-one (1f),7 1,3-diphenyl-6-(piperidin-
1-yl)benzo[1,2,4]triazin-7-one
(1g),6
6-morpholino-1,3-diphenylbenzo[1,2,4]triazin-7-one
(1h),7
1,3-diphenyl-6-
thiomorpholinobenzo[1,2,4]triazin-7-one (1i),7 N-(7-oxo-1,3-diphenyl-1,7-dihydrobenzo[1,2,4]triazin-6-yl)acetamide (1j),5 6-
methoxy-1,3-diphenylbenzo[1,2,4]triazin-7-one (1k),7 and 6-ethoxy-1,3-diphenylbenzo-[1,2,4]triazin-7-one (1l)6 were synthesized
according to literature procedures.
4.1.2. 1-Phenyl-3-(trifluoromethyl)benzo[1,2,4]triazin-7-one (2)
To a stirred solution of 1-phenyl-3(trifluoromethyl)-1,4-dihydro-1,2,4-benzotriazin-4-yl (4)4 (44.2 mg, 0.16 mmol) in dry 1,4-
dioxane (4 mL) at ca. 20 °C was added MnO2 (0.696 g, 50 equiv) and the reaction mixture stirred vigorously for 3 h. Upon
completion, the reaction was poured onto a short silica pad and washed with n-hexane. Chromatography (n-hexane/diethyl ether,
1:1) afforded the title compound 2 as dark purple shiny needles (43.3 mg, 93%); mp (DSC) onset: 140.5 °C, peak max: 144.9 °C
(from n-hexane); Rf 0.58 (t-BuOMe); (found: C, 57.65; H, 2.67; N, 14.35. C14H8F3N3O requires C, 57.74; H, 2.77; N, 14.43%);
λmax(DCM)/nm 281 (log ε 3.36), 291 inf (3.25), 481 inf (2.40), 528 (2.56), 561 (2.48), 607 inf (2.12); νmax/cm-1 3061w (Ar C- H),
1620m, 1605m, 1597m, 1549s (C=O), 1495m, 1466w, 1458w, 1422w, 1400s, 1356s, 1333m, 1319w, 1304w, 1294w, 1246w,
1217s, 1200s, 1177m, 1150s, 1136s, 1113m, 1096s, 1074w, 1030w, 1001w, 984m, 926w, 908w, 853s, 824m, 775m, 733w; δH(300
MHz, CDCl3) 7.72 (1H, d, J 9.8 Hz), 7.67-7.58 (3H, m), 7.56-7.48 (2H, m), 7.33 (1H, dd, J 9.8, 2.1 Hz), 6.12 (1H, d, J 1.9 Hz);
2
δC(75 MHz, CDCl3) 182.6 (s), 156.3 (s), 143.1 (d), 142.4 (q, JFC 39.0 Hz, CCF3), 139.9 (s), 136.6 (s), 132.2 (d), 130.8 (d), 130.3
(d), 125.2 (d), 119.2 (q, 1JFC 273.6 Hz, CF3), 100.0 (d); MALDI-TOF (m/z): 293 (MH++1, 4%), 292 (MH+, 100), 264 (7), 257 (70),
239 (1), 209 (6), 196 (2), 172 (1), 133 (3).
4.2. Cell culture and cytotoxicity evaluation
4.2.1. Cell culture
An SV40-transformed normal human fibroblast cell line (repository number GM00637) was obtained from the National Institute
for General Medical Sciences (NIGMS) Human Genetic Cell Repository (Coriell Institute for Medical Research, New Jersey,
USA). The DU-145 prostate cancer cell line (ATCC repository number HTB-81) was obtained from Prof. R.W.G. Watson, School
of Medicine & Medical Sciences, University College Dublin, Ireland. The MCF-7 cell line, a human breast cancer cell line was
obtained from Dr. Adrienne Gorman, Biochemistry, School of Natural Sciences, National University of Ireland Galway.
The SV40-transformed normal human skin fibroblast cell line (GM00637) was grown in minimum essential media (MEM)
Eagle-Earle BSS supplemented with 15% heat-inactivated fetal bovine serum (FBS), penicillin-streptomycin, 2 mM L-glutamine,
2X essential and non-essential amino acids and vitamins. DU-145 prostate cancer cells were grown in RPMI-1640 medium
supplemented with 10% heat-inactivated fetal bovine serum, penicillin-streptomycin and 2 mM L-glutamine. MCF-7 cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing high glucose (4.5 mg/mL) and supplemented with 10% heat-
inactivated FBS and 1% penicillin-streptomycin.
4.2.2. Cytotoxicity measurement
Cell viability was determined using the MTT colorimetric assay.25 Normal cells were plated into 96-well plates at a density of
10,000 cells per well (200 µL per well) and allowed to adhere over a period of 24 h. DU-145 cells were plated into 96-well plates at
a density of 2,000 cells per well (200 µL per well) and allowed to adhere over a period of 24 h. MCF-7 cells were plated into 96-
well plates at a density of 1,000 cells per well (200 µL per well) and allowed to adhere over a period of 24 h. Compound solutions
were applied in DMSO (1% v/v final concentration in well). Cells were then incubated at 37 °C under a humified atmosphere
containing 5% CO2 for 72 h. Control cells were exposed to an equivalent concentration of DMSO alone. MTT (20 µL, 5 mg/mL
solution) was added, and the cells were incubated for another 4 h. The supernatant was removed carefully by pipette. The resultant
MTT formazan crystals were dissolved in 100 µL of DMSO and absorbance was determined on a plate reader at 550 nm with a
reference at 690 nm. Cell viability is expressed as a percentage of the DMSO-only treated value. Dose response curves were

analyzed by non-linear regression analysis and IC50 values were estimated using GraphPad Prism software, v 6.03 (GraphPad Inc.,
San Diego, CA, USA).
4.3. Electrochemistry
Cyclic voltammograms were recorded using a PalmsSens EmStat3+ potentiostat. Samples (~2 mmol) were dissolved in
dichloromethane containing 0.1 M tetrabutylammonium perchlorate (TBAP) as supporting electrolyte and 1 mM ferrocene (Fc) as
an internal reference. Voltammograms were recorded in a single compartment electrochemical cell (0.5 mL volume) containing a
glassy carbon disk working electrode (3 mm diameter), an Ag/AgCl reference electrode and a Pt wire counter electrode. All
measurements were recorded under nitrogen at room temperature.
4.4. Thioredoxin reductase (TrxR) inhibition studies
4.4.1. Materials
The thioredoxin reductase assay kit (Sigma Aldrich) contained all reagents and enzyme required for the thioredoxin reductase
assay and was used as received. Rat liver thioredoxin reductase was in 50 mM Tris-HCl, pH 7.4, containing 1 mM EDTA, 300 mM
NaCl, and 10% glycerol. The working buffer contained 100 mM potassium phosphate with 10 mM EDTA, 0.24 mM NADPH and
the assay buffer 5× contained 500 mM potassium phosphate, pH 7.0 and 50 mM EDTA.
4.4.2. Enzyme kinetics
The kinetics of rat liver thioredoxin reductase (TrxR) inhibition was analysed in 96-well microtiter plates. The experimental
setup follows manufacturer’s instructions. The reduction of 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) with NADPH to 5-thio-2-
nitro-benzoic acid (TNB) gave a linear increase in absorbance at 405 nm and 27 °C. Each assay point represents an average from
three independent experiments carried out in duplicate. The absorbance was measured at intervals of 5 minutes for a period of 50
minutes. Absorbance was measured using a Biotek Powerwave XS2 plate reader. Taking ΔA405 from the linear portion of the
absorbance plot, TrxR activity was quantified using equation 1.
Equation 1
A unit of mammalian TrxR is the amount of enzyme that will cause an increase in A405 of 1.0 min-1mL-1 at pH 7.0 and 25 °C,
where ΔA405 min-1(TrxR) = [ΔA405 min-1 (sample) - ΔA405 min-1 (sample + inhibitor)]; dil = sample dilution factor; vol = total
reaction volume; enzvol = enzyme volume.
The enzyme specific activity (Units/µg protein) or initial velocity (V0) is calculated by dividing the UnitmL-1 by TrxR
concentration (2.5 × 10-3 µg/mL) in the well. The double reciprocal Lineweaver-Burk is plotted using GraphPad Prism software, v
6.03 (GraphPad Inc., San Diego, CA, USA). The final concentration of DMSO in the assay did not exceed 3.2% (v/v).
Acknowledgements
FA thanks the Irish Research Council (IRC) for a Government of Ireland Postgraduate Scholarship for Martin Sweeney and
College of Science, National University of Ireland Galway (NUI Galway) for a postgraduate scholarship for Robert Coyle. We
thank the National Cancer Institute (USA), Development Therapeutic Program for providing us with a small quantity of pleurotin.
PAK thanks the Cyprus Research Promotion Foundation [Grants: NEAYPODOMH/NEKYP/0308/02 and
YGEIA/BIOS/0308(BIE)/13], the University of Cyprus (Medium Sized Grant), and the following organizations in Cyprus for
generous donations of chemicals and glassware: the State General Laboratory, the Agricultural Research Institute, the Ministry of
Agriculture, Medochemie Ltd and Biotronics Ltd. Furthermore, PAK thanks the A. G. Leventis Foundation for helping to establish
the NMR facility in the University of Cyprus.
Supplementary data
Supplementary data includes 1H and 13C NMR spectra of compound 2, cell viability plots and NCI COMPARE analysis.
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Figure 1. Benzo[1,2,4]triazin-7-ones 1a-1l, 2 and pleurotin

Figure 2. Summary of DTP NCI-60 single dose (10 µM) screening results for benzotriazinones 1a and 2 expressed as average
percent growth of each cancer cell type relative to untreated cells

Figure 3. Cyclic voltammograms of 1a (blue line) and 2 (red line) recorded in dichloromethane (0.1 M TBAP) at a glassy carbon
electrode (scan rate: 0.1 V s-1). Arrow indicates direction of scan

Figure 4. Kinetics of thioredoxin reductase (TrxR) inhibition by 1,3-diphenylbenzo[1,2,4]-triazin-7-one 1a and 1-phenyl-3-
(trifluoromethyl)benzo[1,2,4]triazin-7-one 2 using 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) as the substrate: (a) Lineweaver-
Burk plot with inhibitor 1a concentrations of 0 ( ), 0.15 ( ), 0.30 ( ) and 0.50 ( ) µM. (b) Lineweaver-Burk plot with
inhibitor 2 concentrations of 0 ( ), 0.30 ( ) and 1 ( ) µM

Figure 5. Graphical determination of Ki for 1,3-diphenylbenzo[1,2,4]triazin-7-one 1a and 1-phenyl-3-
(trifluoromethyl)benzo[1,2,4]triazin-7-one 2 using the slopes of the Lineweaver-Burk plots in Figure 4 versus inhibitor
concentrations.

Scheme 1. Synthesis of benzo[1,2,4]triazin-7-ones

Table 1. Pearson correlation coefficients obtained by COMPARE analysis
Compound
Pleurotin
0.842
2
0.803
-
1a
2
0.793

Compd
R
Cell Lines
DU-145
GM00637
MCF-7
Pleurotin
0.51 ± 0.12
0.43 ± 0.06
0.23 ± 0.03
0.28 ± 0.03
H
0.23 ± 0.01
2.04 ± 0.21
0.81 ± 0.08
0.95 ± 0.03
1a
Table 2. Cytotoxicity evaluation using the MTT assay: IC50 values (µM)a
NH2
1.83 ± 0.08
1b
NHMe
NHEt
NEt2
0.93 ± 0.03
0.24 ± 0.01
2.73 ± 0.36
0.98 ± 0.06
0.22 ± 0.01
3.11 ± 0.08
0.69 ± 0.12
1.62 ± 0.24
>5.0
1c
1d
1e
1.79 ± 0.12
1.19 ± 0.02
2.29 ± 0.06
1.63 ± 0.31
2.46 ± 0.19
0.61 ± 0.05
3.21 ± 0.37
1.22 ± 0.06
0.36 ± 0.08
1.98 ± 0.06
2.37 ± 0.07
0.97 ± 0.16
1f
1g
1h
1i
>5.0
>5.0
>5.0
1j
1k
1l
OMe
OEt
>5.0
>5.0
>5.0
>5.0
>5.0
>5.0
1.61 ± 0.21
0.85 ± 0.04
0.60 ± 0.13
2
a
IC50 represents the compound concentration required for the reduction
of the mean cell viability to 50% of the control value after incubation for
72 h at 37 oC

Compound
E°ʹ [V] versus Fc/Fc+
E°ʹ (I)
E°ʹ(II)
-1.75
1a
2
-1.12
-0.86
-1.55
Table 3. Formal Potentials (E°ʹ) (±0.010 V) calculated as (Epc + Epa)/2 from cyclic voltammograms recorded at 100 mVs-1

Equation 1

Graphical Abstract
Discovery of anti-cancer activity for
benzo[1,2,4]triazin-7-ones: Very strong
correlation to pleurotin and thioredoxin
reductase inhibition
Leave this area blank for abstract info.
Martin Sweeneya, , Robert Coylea, , Paul Kavanagha,
Andrey A. Berezinb, Daniele Lo Reb,
Georgia A. Zissimoub, Panayiotis A. Koutentisb,
Michael P. Cartyc, * and Fawaz Aldabbagha,*
aSchool of Chemistry, National University of Ireland
Galway, University Road, Galway, Ireland
b Department of Chemistry, University of Cyprus,
P.O. Box 20537, 1678 Nicosia, Cyprus
c Centre of Chromosome Biology, Biochemistry,
School of Natural Sciences, National University of
Ireland Galway, University Road, Galway, Ireland