Discovery of potent 4-aminoquinoline hydrazone inhibitors of NRH:quinoneoxidoreductase-2 (NQO2)

Article abstract of DOI:10.1016/j.ejmech.2019.111649

(NRH):quinone oxidoreductase 2 (NQO2) is associated with various processes involved in cancer initiation and progression probably via the production of ROS during quinone metabolism. Thus, there is a need to develop inhibitors of NQO2 that are active in v

Full text of DOI:10.1016/j.ejmech.2019.111649

Journal Pre-proof
Discovery of potent 4-aminoquinoline hydrazone inhibitors of
NRH:quinoneoxidoreductase-2 (NQO2)
Buthaina Hussein, Balqees Ikhmais, Manikandan Kadirvel, Rachael N. Magwaza,
Gavin Halbert, Richard A. Bryce, Ian J. Stratford, Sally Freeman
PII:
S0223-5234(19)30789-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.111649
EJMECH 111649
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 April 2019
Revised Date: 22 August 2019
Accepted Date: 26 August 2019
Please cite this article as: B. Hussein, B. Ikhmais, M. Kadirvel, R.N. Magwaza, G. Halbert, R.A.
Bryce, I.J. Stratford, S. Freeman, Discovery of potent 4-aminoquinoline hydrazone inhibitors of
NRH:quinoneoxidoreductase-2 (NQO2), European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111649.
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Discovery of potent 4-aminoquinoline hydrazone inhibitors of NRH:quinoneoxidoreductase-2 (NQO2)
Buthaina Husseina,§, Balqees Ikhmaisa,§, Manikandan Kadirvela, Rachael N. Magwazaa, Gavin Halbertb,
Richard A. Brycea, Ian J. Stratforda,*, Sally Freemana,*
aDivision of Pharmacy & Optometry, School of Health Sciences, Faculty of Biology, Medicine & Health,
University of Manchester, Manchester, M13 9PT, UK
bStrathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, G4 0NR,
UK
*Corresponding authors
Email: Sally.Freeman@manchester.ac.uk & Ian.Stratford@manchester.ac.uk
§ Authors have made an equal contribution
ABSTRACT
(NRH):quinone oxidoreductase 2 (NQO2) is associated with various processes involved in cancer
initiation and progression probably via the production of ROS during quinone metabolism. Thus, there is
a need to develop inhibitors of NQO2 that are active in vitro and in vivo. As part of a strategy to achieve
this we have used the 4-aminoquinoline backbone as a starting point and synthesized 21 novel
analogues. The syntheses utilised p-anisidine with Meldrum’s acid and trimethyl orthoacetate or
trimethyl orthobenzoate to give the 4-hydrazin-quinoline scaffold, which was derivatised with aldehydes
or acid chlorides to give hydrazone or hydrazide analogues, respectively. The hydrazones were the most
potent inhibitors of NQO2 in cell free systems, some with low nano-molar IC50 values. Structure-activity
analysis highlighted the importance of a small substituent at the 2-position of the 4-aminoquinoline ring,
to reduce steric hindrance and improve engagement of the scaffold within the NQO2 active site.
Cytotoxicity and NQO2–inhibitory activity in vitro was evaluated using ovarian cancer SKOV-3 and TOV-
112 cells (expressing high and low levels of NQO2, respectively). Generally, the hydrazones were more
toxic than hydrazide analogues and further, toxicity is unrelated to cellular NQO2 activity.
Pharmacological inhibition of NQO2 in cells was measured using the toxicity of CB1954 as a surrogate
end-point. Both the hydrazone and hydrazide derivatives are functionally active as inhibitors of NQO2 in
the cells, but at different inhibitory potency levels. In particular, 4-((2-(6-methoxy-2-methylquinolin-4-
yl)hydrazono)methyl)phenol has the greatest potency of any compound yet evaluated (53nM), which is
50-fold lower than its toxicity IC50. This compound and some of its analogues could serve as useful
pharmacological probes to determine the functional role of NQO2 in cancer development and response
to therapy.
Keywords
NQO2 inhibitors; anticancer; 4-aminoquinoline; hydrazine; hydrazine; ovarian cancer; SKOV-3 cells; TOV-
112D cells; CB1954.

1. Introduction
There is continued interest in dissecting the role of the flavoprotein NQO2 (Quinone oxidoreductase 2,
QR2, EC.1.10.99.2) in cancer biology and in the development of various neurological conditions.[1-9]
NQO2 is a FAD-containing protein capable of oxidizing a variety of analogues of dihydronicotinamide. It
is a structural analogue of NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase, EC.1.6.99.2) and
while the two enzymes have similar properties there are major functional differences.[10-12] In
particular, the two enzymes catalyse the reduction of various quinones into hydroquinones.[12-15] The
reduction process proceeds via a ping-pong mechanism in which two electrons are transferred from the
co-substrate to FAD molecule and subsequently to the substrate.[16, 17] However, a major difference
between the enzymes is that whereas NQO1 can use NADPH or NADH as reducing cofactors, the
enzymatic function of NQO2 can only be supported by nicotinamide derivatives such as N1-ribosyl-, N1-
methyl-, N1-benzyl-dihydronicotinamide (NRH) and 1-carbamoylmethyl-3-carbamoylpiridinium iodide
(EPR) as reducing cofactors.[12, 17-21]
There is evidence linking NQO2 activity to p53 function and degradation, to NFƙB activity and to the
induction of metastases in prostate cancers.[22-24] Also, NQO2 is reported to be highly expressed in a
number of cancer cell lines and hence needs to be evaluated as a potential therapeutic target in the
treatment of cancer.
Structurally diverse inhibitors of NQO2 are known, which include resveratrol, casimiroin, melatonin,
chloroquine and imatinib. However, none are considered as ideal NQO2 inhibitors for therapeutic
intervention as they have other pharmacological effects. Nolan et al. (2012) conducted a virtual
screening of the NCI database to identify potential new NQO2 inhibitors.[25] Lead inhibitors were
identified that belonged to the polyaromatic, ellipticine, acridine, quinoline and furan-amidine classes of
compound. This screen further identified seven quinoline compounds (Table 1) that showed potent
inhibition of NQO2.[25] We have thus used 4-aminoquinolines (Figure 1) as the basis for the design of
novel inhibitors of NQO2 and here we report the synthesis and evaluation of three different series of
compounds. These are based on hydrazine, hydrazide and hydrazone-quinolines and were chosen
because these scaffolds are emerging as important functional groups in drug design as they are present
in compounds possessing diverse biological activities, [26, 27] such as vasodilator, anti-tuberculosis,
anti-tumour, anti-microbial, anti-platelet, anti-inflammatory, anti-convulsant and anti-oxidant
activities.[27, 28]

Table 1. NSC numbers of seven aminoquinolines and their IC50 values for the inhibition of recombinant
NQO2.[25] Compound structures are given in the SI.
NSC number IC50 (µM) ± SD
13484
0.55 ± 0.05
0.50 ± 0.06
0.64 ± 0.05
0.17 ± 0.09
0.25 ± 0.05
0.04 ± 0.01
0.07 ± 0.01
76750
101984
270904
273829
617933
617939
Figure 1. The 4-aminoquinoline scaffold.
2. Results and discussion
2.1 Synthesis
The syntheses of 4-hydrazine-quinolines 6a-b are shown in Scheme 1. This first requires the synthesis of
the quinoline ring (4a-b) by reacting p-anisidine with methoxyethylidene Meldrum’s acid [29] 2a or
phenoxyethylidene Meldrum’s acid 2b,[29, 30] to give enamine intermediates 3a-b [31]. The thermal
pericyclic reaction of 3a-b takes place at high temperature (microwave, diphenyl ether, 200 ˚C, 30 min)
to give intermediates 4a-b[29, 32] with the release of acetone and carbon dioxide.[33] 6-Methoxy-
quinolin-4-ols 4a-b were then treated with phosphorus oxychloride to give 4-chloroquinolines 5a-b,[29]
which were then reacted with hydrazine monohydrate to give the 4-hydrazine-quinolines 6a-b[34, 35] in
very good yields (Scheme 1).

Me
O
Me
O
Me
O
Me
O
OMe
O
MeO
i
O
O
O
N
R
Me
H
83%
O
R
NH2
3a-b
a: R= Me
b: R= Ph
2a-b
1
ii
NHNH2
Cl
N
OH
MeO
MeO
MeO
iv
iii
N
R
R
N
93%
R
74%
65%
4a-b
5a-b
6a-b
Scheme 1. Synthesis of 4-hydrazine-quinolines 6a (R = Me) and 6b (R = Ph). Reagents and conditions: (i)
reflux, 3h; (ii) 200 °C, 30 min, MW; (iii) POCl3; (iv) NH2NH2.
Eighteen 4-hydrazone quinoline derivatives 7a-r were synthesized in good yields (30-90 %) by the
reaction of the 4-hydrazine-quinoline 6a-b with a range of aldehydes (Scheme 2) (Table 2).[35, 36] The
syntheses of the 4-hydrazide quinoline derivatives 8 were achieved by reacting the 4-hydrazinequinoline
scaffold 6b with the appropriate aryl chloride in the presence of pyridine (Scheme 2).[37] Two hydrazide
quinoline derivatives 8a-b were prepared, however they were much less active than the hydrazone
derivatives (Table 2), therefore further hydrazide analogues were not synthesised.
Scheme 2. Synthetic pathways for 4-hydrazonequinoline 7 and 4-hydrazidequinoline 8 derivatives:
Reagents: (i) R’C(O)H, (ii) R’COCl.

Table 2. Sides chains (R and R’) of the parent hydrazine- (6a), 4-hydrazone- (7a-r) and 4-hydrazide- (8a-
b) quinoline derivatives, % yields for the syntheses, IC50 values for inhibition of recombinant NQO2 and
NQO1, and toxicity towards SKOV-3 cells (following 24 h and 96 h exposure).
Entry
R
R`
Yield
%
NQO2
IC50
NQO1
IC50
Toxicity IC50 (
µ
M±SE)
(nM±SE)
(nM±SE)
24 h
96 h
6a
7a
7b
7c
-
-
80
56
52
76
2047±132
103±16.3
519±14.5
71±14.9
-
-
-
84±16
41±8.9
2.3±0.06
1.77±0.09 1.73±0.09
Me
Me
Me
4-Fluorophenyl
Phenyl
2.3±0.18
4-Imidazoyl
38700
±5200
133900
±43600
-
32.0±2.08
4.2±0.56
7d
Me 4-Hydroxyphenyl
53
55±6.6
2.75±0.26
2.3±0.26
7e
7f
Me
Me
Me
3-Pyridinyl
Benzyl
34
30
89
10±0.7
15±4.4
13±3.1
-
-
Inactive
50±1
40.67±2.3
2.0±0.06
7g
2-Hydroxy-3-
methoxyphenyl
2-Nitrofuranyl
73400
±48700
-
2.1±0.25
7h
7i
Me
Me
Me
Me
75
78
92
75
18±1.2
64±5.9
83±8.0
46±8.6
-
-
4-Benzoic acid
4-Nitrophenyl
Inactive
>100
70.0±4.93
7j
-
-
-
-
-
-
7k
3,5-
Dihydroxylphenyl
7l
Me 3-Hydroxyphenyl
33
89
137±15.9
372±109.
4
-
-
-
7m
Ph
3-Pyridinyl
Inactive
2.8±0.36
2.2±0.12
7n
Ph
4-N,N-
60
>10000
-
1.57±0.42 1.53±0.44
Dimethylaniline
4-Nitrophenyl
7o
7p
7q
Ph
Ph
Ph
40
26
91
1420±204
125±2.8
-
-
-
-
-
Benzyl
-
2-Hydroxy-3-
methoxyphenyl
4-Imidazoyl
595±178
Inactive
2.88±0.22 2.73±0.22
7r
8a
Ph
Me
Me
77
74
28
90±10.7
2906±433
472±23.3
900±7.8
440±90
425±90
2.47±0.30 2.17±0.22
4-Fluorophenyl
Phenyl
Inactive
>100
68.3±4.06 43.3±6.49
91.7±4.4 36.3±1.3
5.43±0.47 2.78±0.17
>100
8b
-
-
-
Resveratrol
9-
Aminoacridine
(9AA)

2.2 Inhibition of recombinant human NQO2 and NQO1 enzyme activity
The ability of the synthesized compounds to inhibit the NQO2 enzyme was determined by following the
reduction of 2,6-dichlorophenolindophenol (DCPIP) using EPR as a co-substrate.[18] The IC50 values for
the hydrazonequinoline derivatives 7a-r are given in Table 2. Results are also given for the standard
NQO2 inhibitor resveratrol and for 9-aminoacridine (9-AA), a compound we showed previously to be a
potent inhibitor of NQO2.[38] Many of the hydrazonequinolines are excellent NQO2 inhibitors with IC50
values less than 100 nM, being more potent than both resveratrol and 9-AA.
The nucleus of the hydrazonequinoline derivatives, 4-hydrazine-6-methoxy-2-methyl-quinoline (6a), was
also tested against recombinant human NQO2, giving an IC50 of 2047 nM. The greater activity of the 4-
hydrazonequinoline derivatives when compared with the scaffold (6a), confirmed that the inhibitory
activity can be attributed to the hydrazone functionality in the compounds.
We then went on to evaluate the NQO2-inhibitory potency of representative hydrazidequinolines 8a-b
(Table 2) which showed modest inhibitory activity. The lack of improvement in inhibitory activity of the
hydrazide- versus the hydrazonequinolines precluded any further development of this series.
To test the enzyme selectivity of the hydrazones and hydrazides, some of the compounds were assayed
for their activity against NQO1 (Table 2). Dicoumarol, the standard inhibitor of NQO1 has an IC50 value of
around 4nM whereas only compounds 7c, 7d, 7g and 7r showed any (µM) activity. Compounds 7f, 7i,
7m, 7q and 8b were completely inactive towards NQO1, therefore are NQO2 specific. Compound 7r had
an inhibitory activity towards NQO1 of 425 nM, but is still much more selective towards NQO2.
2.3 Structure-activity analysis
Probing NQO2 enzyme inhibition using the range of synthesized compounds indicate some key
structural features. Compounds 7a-7l (2-Me group), showed lower IC50 values than the corresponding
compounds 7m-7r (2-Ph group). For example, 2-methyl analogue 7e is 37-fold more potent than its
corresponding 2-phenyl analogue 7m (Table 1). This variation in potency may be explained by different
binding modes of 7e and 7m in the NQO2 active site: for the 2-methyl substituted compound 7e (Figure
2A), we find that computational docking predicts that the compound binds well in two distinct
orientations in the NQO2 site. In the first binding pose, the aryl-hydrazone moiety of 7e fully enters into
the NQO2 active site, allowing π-stacking of the 3-pyridinyl group, as well as the quinoline scaffold,
against the FAD isoalloxazine ring (yellow, Figure 2A). In the second pose, the ligand is in a 180° flipped
orientation, such that the 3-pyridinyl group can occupy a pocket formed by residues Val160, Asn161 and
Phe178’ (cyan, Figure 2A). By contrast, docking of the 2-phenyl analogue 7m leads to a markedly
different predicted orientation in the NQO2 cavity (Figure 2B): although the quinoline scaffold can π-
stack with the FAD cofactor, the additional steric bulk of the 2-phenyl group of 7m precludes the aryl-
hydrazone from entering the active site; instead the aryl-hydrazone and 3-pyridinyl moieties project out
of the active site without forming specific amino acid contacts except a weak nonpolar contact with the
sidechain of Ile128 (Figure 2B).

B
A
Ile128
Phe178’
Asn161
Asn161
Figure 2. (A) Docked poses of 2-methyl analogue 7e in the active site of NQO2. (B) Docked pose of 2-
phenyl analogue 7m in the active site of NQO2.
We further note that substituents on the aromatic ring have a noticeable effect on the inhibitory activity
of the synthesized compounds. The para-fluorine substitution in compound 7a makes the compound
five-fold more potent compared to unsubstituted-phenyl analogue 7b (Table 2). In the same manner,
compound 7d with a para-hydroxyl substituent is ten-fold more potent than unsubstituted-phenyl
analogue 7b. However, the fluorine substituent increases the hydrophobic character of the molecule
slightly, but this is not consistent with the increasing activity shown by the hydroxyl and other
hydrophilic groups as in compounds 7d, 7g, 7i, 7k and 7l. This may suggest that introducing a group
(such as F, OH) that has the ability to make hydrogen bonds with the amino acid residues, such as the
proximal polar residue Asn161 (Figure 2A), is favourable. Other potent inhibitor compounds have been
shown to form interactions with this residue, such as resveratrol analogues.[39]
2.4 Evaluation of cytotoxicity
SKOV-3 ovarian cancer cells were used to first assess the toxicity of the hydrazone- and hydrazide-
quinolines. This cell line was chosen as these cells showed relatively high levels and activity of NQO2
(unpublished data). Cells were exposed to the different drugs for 24 h or 96 h prior to an assessment of
toxicity using the MTT assay. Typical survival data is illustrated in Figure 3 for compounds 7d and 7f
given to cells for either 24 h or 96 h. It is clearly that 7d is the more toxic of the two drugs. Survival
curves were obtained for each drug and from these curves, the concentration of drug required to reduce
cell survival to 50% (IC50) was determined (Table 2). For comparison, the toxicity of the NQO2 inhibitors
resveratrol and 9-AA were also recorded. Since it was considered a possibility that the N=C double bond
of the hydrazone moiety might be labile in cellular milieu resulting in hydrolysis to give the parent
compound 6a, this was also assessed for toxicity (Table 2).

Figure 3. Toxicity of selected 4-hydrazonequinolines 7d and 7f towards SKOV-3 ovarian cancer cells as
measured by the MTT assay. Panels A and B show values of cell survival following 24 h and 96 h
exposure to the different compounds, respectively. Survival values (± SE) were derived from four
independent experiments.
For all the hydrazone-and hydrazide-quinolines evaluated, with exception of 7c, the levels of toxicity
were not dependent on exposure time. This may suggest that the drugs are degraded in the culture
medium within the first 24 h so that none is available to elicit toxicity at longer times. By comparison of
hydrazone 7b (R’ = Ph) with hydrazide 8b (R = Ph), it shows that the hydrazone shows greater toxicity
than the hydrazide. The similarity in the toxicity of structurally diverse hydrazones suggests that this
moiety might be the main contributor to the compounds’ toxicity. It is noteworthy that the parent
hydrazine 6a is less toxic than most of the hydrazones, which further suggests that the hydrazone
moiety is primarily responsible for the toxicity.
Further inspection of the toxicity data in Table 2 suggests there is no obvious correlation between the
toxicity of the compounds and their ability to inhibit the activity of recombinant NQO2. To interrogate
this further, we evaluated the toxicity of some of the hydrazones and hydrazides along with resveratrol
and 9-AA in isogenic pairs of cell lines where the expression of NQO2 was genetically altered
(unpublished data). To do this, we first used SKOV-3 cells which were stably transfected with
doxycycline-inducible shRNA directed to a portion of the NQO2 gene (SKOV-3 sh27 cells). This resulted in
a reduction of cellular activity of NQO2 from 140.5±4.0 to 93.8±2.2 nmol/min/mg of protein in cells
treated with and without doxycycline (Dox) (unpublished data). Secondly, and in contrast, we also used
TOV-112D ovarian cancer cells, which normally have barely detectable levels of NQO2 and we
constitutively over-expressed NQO2 in these cells (TOV-112D NQO2-OE cells). This resulted in a change
in activity from 5.2±0.5 nmol/min/mg of protein in the wild type cells to 128.3±4.9 nmol/min/mg of
protein in the transfected cells (unpublished data). The values of IC50 for the control and transfected cell
lines exposed to the different drugs for 96 h are given in Table 3. An important feature of this data is
that results for “control” cells are based on the following. Firstly, for the SKOV-3 cells, we used cells that
were transfected with a non-targeted vector (NTC), both NTC cells and the sh27 cells were then treated

with doxycycline (which only gives down-regulation of NQO2 in the sh27 cells). Secondly, the TOV-112D
cells were transfected with an empty vector (EV). The results show that none of the compounds have
any dependence on NQO2 activity for their toxicity and this includes those compounds that are standard
inhibitors of NQO2 (resveratrol and 9-AA).
Table 3. Toxicity of the various compounds towards isogenic cell lines where the activity of NQO2 has
been either down regulated or over-expressed (see text). Cells were exposed to compounds for 96 h
prior to measuring toxicity using the MTT assay. IC50 values (± SE) were determined from dose-response
curves generated from three independent experiments.
96 h Toxicity (IC50 (µM) ± SE)
Compound
SKOV-3
TOV-112D
EV
Number/
Name
NTC
Sh27
NQO2-OE
+Dox
+Dox
7c
6.9 ± 0.30
6.73 ± 0.41
3.65 ± 0.29
3.17 ± 0.22
42.5 ± 2.57
3.4 ± 0.10
>100
3.72 ± 0.24
2.98 ± 0.38
41.0 ± 5.0
3.77 ± 0.19
>100
7d
2.87 ± 0.15
44.67 ± 6.9
2.7 ± 0.1
2.58 ± 0.10
49.67 ± 0.88
2.78 ± 0.17
61.3 ± 3.48
>100
7f
7g
7i
65.0 ± 2.89
>100
8a
61.3 ± 3.3
10.67 ± 0.6
3.6 ± 0.21
57.3 ± 2.9
11.0 ± 0.58
3.15 ± 0.22
Resveratrol
9AA
29.0 ± 5.51
2.53 ± 0.32
39.67 ± 2.91
2.57 ± 0.20
2.5 Functional inhibition of NQO2 in cancer cells
One of the purposes for seeking novel inhibitors of NQO2 is to identify potential pharmacological probes
for the activity of NQO2, as well as its function, in living cells. The fact that none of the hydrazone- and
hydrazide- quinolines show any dependence for toxicity on NQO2 inhibitory potency (as measured by
activity against recombinant protein) means that they could be potentially useful probes. To test this we
established whether these compounds were functionally active as NQO2 inhibitors in cells. To do this,
we took advantage of a unique property of NQO2, which is to reductively activate the drug CB1954 to a
cytotoxic species.[25] Thus, cells with relatively high levels of NQO2 will be sensitive to CB1954 (as a
consequence of NQO2-mediated reductive activation), whereas in the presence of inhibitors the toxicity
of CB1954 will be much reduced.[25]
Experiments were carried out with SKOV-3 cells treated with varying concentrations of CB1954 together
with the activating co-factor EPR in the presence and absence of varying concentrations of a selection of

the putative NQO2 inhibitors. Typical survival curves are given in Figure 4. The closed circles in the
Figure show the survival of the SKOV-3 cells given CB1954 and EPR, whereas in the presence of the
inhibitors (the other symbols) it is clear that the toxicity is much reduced. Indeed, the toxicity curve
obtained in the presence of 0.5µM 7d is identical to that obtained when the exposure to CB1954 is
carried out in the absence of EPR (data not shown), which strongly suggests that the inhibitors are acting
in cells by competing with the activating co-factor for access to the enzyme active site. Importantly,
these data confirm that these compounds are functionally active as NQO2 inhibitors in cells.
Figure 4. Data showing that the 4-hydrazonequinolines can be functionally active as NQO2 inhibitors in
SKOV-3 ovarian cancer cells. Cells were exposed to the CB1954/EPR ± the inhibitors for 24 h prior to
measuring toxicity with the MTT assay 72 h later. Panels A and B show the ability of different
concentrations of the NQO2 inhibitors 7d and 7f, respectively, to reduce the toxicity of CB1954. Data
points (± SE) were derived from four independent experiments.
In order to evaluate the efficiency of the different compounds to act as inhibitors of NQO2 in cells, we
exposed SKOV-3 cells to a single concentration of CB1954 (1µM) and EPR, together with a wide range of
concentrations of the different putative inhibitors. From these experiments we were able to calculate
the concentration of inhibitor that could cause half-maximal inhibition of CB1954 toxicity. These results
are recorded in Table 4. We have already identified 9-AA as an extremely potent agent for inhibiting
NQO2 in cells.[25] This previous work was carried out with MDA-MB-468 cells and here, using SKOV-3
cells, it can be seen that 9-AA retains its potent inhibitory activity. However, it is quite apparent that
some of the hydrazonequinolines show comparable efficiency to 9-AA in the SKOV-3 cells. Indeed,
compound 7d is some ten-fold more efficient, making it one of the most potent inhibitors of NQO2 in
cells yet identified. An important feature of this set of results, which complements our previous findings
[25] is that the ability of compounds to inhibit recombinant enzyme is not necessarily a guide to the
most efficient inhibitor in cells. For example, compounds 7f and 7g show approximately four-fold
greater potency as inhibitors than 7d against recombinant NQO2 enzyme; in contrast, in cells, they are
10 to 20 fold less efficient than 7d.

Table 4. Efficiency of compounds to act as inhibitors of NQO2 in wild type SKOV-3 cells. Data are derived
from experiments similar to that given in Figure 4 with the exception that the cells were exposed to a
fixed concentration of CB1954 (1µM) plus its activating co-factor EPR in the presence of a range of non-
toxic concentrations of the different putative inhibitors of NQO2. Cells were exposed to the agents for
24 h prior to measuring toxicity with the MTT assay 72 h later.
Concentration of inhibitor
Compound
Number/ Name
required to reduce maximal
toxicity of CB1954/ EPR by
50% (µM ± SE)
7c
7d
7f
2.07 ± 0.5
0.053 ± 0.007
0.98 ± 0.22
0.44 ± 0.22
22.7 ± 8.9
7g
7i
8a
9AA
74 ± 11.4
0.45 ± 0.14
2.6 Conclusion
4-Hydrazone- and 4-hydrazide-quinoline derivatives have been prepared using multistep syntheses in
good yields. Several of the 4-hydrazone analogues showed low nanomolar inhibition against
recombinant NQO2, whereas the 4-hydrazides were less active. This activity profile was rationalized by
qualitative docking to the active site of NQO2. Further the hydrazine inhibitors are selective for NQO2,
showing little if any activity against NQO1. The hydrazones and hydrazides were toxic at micro-molar
concentrations which are, however, much higher than the concentrations required to inhibit cellular
NQO2 activity in cells. The study provides two important conclusions. Firstly, inhibitory potency against
recombinant enzyme is not necessarily the best guide to identify the inhibitors likely to be most active in
cells. Secondly, compound 7d is shown to be one of the most active NQO2 inhibitors in cells yet
reported. This compound could prove to be useful as a pharmacological probe for interrogating the role
of NQO2 in cancer and a variety of other biological conditions.
2. Experimental
3.1
Synthesis
Chemicals, solvents and deuterated solvents were purchased from Sigma-Aldrich and Fisher Scientific.
Bruker Avance 300 and 400 spectrometers were used to record 1H and 13C NMR spectra. Chemical shifts
are quoted in parts per million (ppm) and referenced to solvent peak or tetramethylsilane (TMS δ = 0).

Solvents were evaporated on a Buchi rotavapor R-3 equipped with a Buchi heating bath R-3. Microwave
reactions were conducted using a Biotage Initiator synthesiser. Thin layer chromatography (TLC) was
performed using silica gel 60 on aluminum sheets with F254. The spots were visualized using a UV GL-58
Mineral-Light lamp. Column chromatography was performed using silica gel with a particle size of 40-63
microns. Infrared spectra were recorded in the solid state using a J.A.S.C.O Fourier transform infrared
spectrometer. Melting points were measured using a Stuart melting point apparatus SMP10. A
BECKMAN DU 7400 spectrophotometer was used to determine enzyme activity. A Grant JB series water
bath was used to heat the buffer to 37 ˚C. Water was evaporated using a Christ alpha1-4 plus freeze
dryer equipped with an Edwards vacuum pump. LC-MS spectrometry was carried out using the ACQUITY
UPLC H-class system. The mass spectrometry data was acquired in the positive (ES+) and negative (ES-)
modes, scanned from 100- 1000 m/z. The LC data was obtained for Waters ACQUITY UPLC PDA detector
scanning from 210 – 400nm Mass spectrometry was carried out using a Micromass Platform II
1
instrument at the School of Chemistry, University of Manchester. The H NMR and LC-MS spectra for
most compounds are provided in Appendix A Supplementary data.
3.1.1 General procedure for the synthesis of intermediates 3a-b
A solution of Meldrum’s acid (isopropylidine malonate) (10.40 mmol) in trimethylorthoacetate for 3a or
trimethylorthobenzoate for 3b (589.2 mmol) was heated at reflux for 1 h then cooled to room
temperature. p-Methoxyaniline (2.0 g, 16.24 mmol) then was added to the mixture with DMF (2.0 ml)
and heated at reflux for a further 4 h. The mixture was then poured into ice and allowed to stir until
white crystals formed. The product was collected by suction filtration.
3.1.1.1 5-(1`-((4`-Methoxyphenyl)amino)ethylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione
(3a).
The
product was collected as white solid (2.24 g, 83%). Mp: 143-144 ᵒC; IR (cm-1): 1708 (-COO-), 1889 (C=C),
1
2942 (CH3), 3019 (2ᵒ -amine); H-NMR (400 MHz; DMSO-d6): 1.66 (s, 6H, H 7, 8), 2.44 (s, 3H, H 8`), 3.80
(s, 3H, OMe), 7.04 (d, J = 8.0 Hz, 2H, H 3`,5`), 7.32 (d, J= 8.0 Hz, 2H, H 2`, 6`), 12.49 (s, 1H, NH); 13C-NMR
(75 MHz, DMSO-d6, 1 quat. C not observed): 26.1 (C 7, 8), 55.4 (OMe), 64.3 (C 2), 84.9 (C 1`), 102.1 (C 5),
114.6 (C 3`, 5`), 127.3 (C 2`, 6`), 128.6 (C 7`), 158.7 (C 4`), 173.0 (C 4, 6); MS ES-, [M-H, 35%]: 290.1.
3.1.1.1 5-(((4-Methoxyphenyl)amino)(phenyl)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (3b). The
product was collected as a yellow solid (84%). Mp: 185-188 ᵒC; IR (cm-1): 1552 (C=C), 1650 (C=C), 1715 (-
1
COO-), 2837 (OMe), 2936 (Me), 2990 (Me), 3059 (C-H); H-NMR (300 MHz; DMSO-d6): 1.75 (s, 6H, H 7,
8), 3.65 (s, 3H, OMe), 6.91 (d, J = 9.0 Hz, 2H, H 2`, 6`), 6.91 (d, J= 9.0 Hz, 2H, H 3`, 5`), 7.21-7.32 (m, 5H,
Ph), 12.26 (s, 1H, NH); 13C-NMR (75 MHz, DMSO-d6): 26.1 (C 7,8), 55.2 (OMe), 86.2 (C 5), 102.6 (C 2),
113.8 (C 3`,5`), 122.7 (C 9), 126.8 (C 2`,6`), 127.9 (C 3``,5``), 128.5 (C 2``,6``), 129.4 (C 4``), 129.6 (C 1`,1``),
133.0 (C 4`), 157.5 (C 4,6), 170.2 (C 9’); MS ES-, [M-H, 10%]: 354.1.
3.1.2 General procedure for the synthesis of intermediates 4a-b
Intermediate 3a or 3b (2.26 mmol) was dissolved in diphenyl ether (2.0 ml) and heated by microwave
radiation at 200 ˚C for 30 min to give 4a or4b, respectively.
3.1.2.1 6-Methoxy-2-methylquinolin-4-ol (4a). The product was collected as a beige powder (400 mg,
93%). Mp: 268-270 ᵒC; IR (cm-1): 1593 (C=C), 2946 (CH3), 3099 (phenol); 1H-NMR (300 MHz; CDCl3): 2.39

(s, 3H, Me), 3.88 (s, 3H, OMe), 5.94 (s, 1H, H 3), 7.31 (dd, J = 9.0 Hz, J = 3.0 Hz, 1H, H 7), 7.51 (d, J = 3.3
Hz, 1H, H 5), 7.52 (d, J = 8.4 Hz, 1H, H 8), 11.54 (s, 1H, OH). 13C-NMR (75 MHz, DMSO-d6): 19.3 (Me), 55.2
(OMe), 104.3 (C 5), 107.4 (C 3), 119.4 (C 10), 121.6 (C 7), 125.5 (C 8), 134.7 (C 9), 148.5 (C 6), 155.2 (C 2),
176.0 (C 4). MS ES+, [M+H, 100%]:190.0.
3.1.2.2 6-Methoxy-2-phenylquinolin-4-ol (4b). The product was isolated as yellow crystals (84%). Mp:
1
>300 ᵒC; IR (cm-1): 1581 (C=C), 1591 (C=N), 2833 (OMe), 3070 (C-H); H-NMR (300 MHz; CDCl3): 3.85 (s,
3H, OMe), 6.31 (s, 1H, H 3), 7.31 (dd, J = 9.0 Hz, J = 3.0 Hz, 1H, H 7), 7.50 (d, J = 3.0 Hz, 1H, H 5), 7.57-7.59
(m, 3H, H 3`,4`,5`), 7.71 (d, J = 9.0 Hz, 1H, H 8), 7.81-7.84 (m, 2H, H 2`,6`), 11.71 (s, 1H, OH); 13C-NMR (75
MHz; DMSO-d6): 55.4 (OMe), 103.9 (C 5), 122.2 (C 7), 127.3 (C 2`,4`,6`), 128.9 (C 3`,5`), 130.6 (C 8), 155.7
(C 2). MS ES-, [M-H, 100%]: 250.1.
3.1.3 General procedure for the synthesis of intermediates 5a-b
POCl3 (1 ml, 45mmol) was added to intermediate 4a or 4b (2.6 mmol). The reaction mixture was then
heated at reflux for 3-4 h at 110 ˚C. Once the mixture was cooled, it was poured into ice and washed
with a saturated solution of NaHCO3. The resulting solid was collected by vacuum filtration to afford the
intermediate 5aor 5b.
3.1.3.1 4-Chloro-6-methoxy-2-methylquinoline (5a). The product was collected as a beige powder (560
1
mg, 86%). Mp: 98-101˚; IR (cm-1): 1594 (C=C), 1654 (C=N), 2947 (Me), 3040 (arene C-H); H-NMR (300
MHz; CDCl3): 2.60 (s, 3H, Me), 3.93 (s, 3H, OMe), 7.37 (d, J = 3.0 Hz, 1H, H 5), 7.45 (dd, J = 9.0 Hz, J = 2.8
Hz, 1H, H 7), 7.64 (s, 1H, H 3), 7.90 (d, J = 9.0 Hz, 1H, H 8); 13C-NMR (75 MHz; DMSO-d6, quat. C 4 and C 9
not observed): 23.8 (Me), 55.7 (OMe), 101.6 (C 5), 122.4 (C 3), 123.2 (C 7), 124.9 (C 10), 129.8 (C 8),
156.1 (C 2), 157.9 (C 6); MS ES+, [M+H, 100%]: 208.0.
3.1.3.24-Chloro-6-methoxy-2-phenylquinoline (5b). The product was isolated as a yellow powder (89%).
Mp: 120-123 ᵒC; IR (cm-1): 1574 ( C=C), 1620 (C=N), 2827 (OMe), 3055 (C-H); 1H-NMR (300 MHz; CDCl3):
3.92 (s, 3H, OMe), 7.32-7.47 (m, 5H, H 3,7,3`,4`,5`), 7.86 (s, 1H, H 5), 7.99-8.04 (m, 3H, H 8, 2`,6`); 13C-
NMR (75 MHz; DMSO-d6): 55.7 (OMe), 101.7 (C 5), 119.3 (C 3), 123.4 (C 10), 126.4 (C 7), 127.3 (C 2`,6`),
128.9 (C 3`,5`), 129.5 (C 4`), 131.6 (C 8), 138.6 (C 1`), 141.6 (C 4), 145.0 (C 9), 154.8 (C 2), 158.7 (C 6). MS
ES +, [M+H, 100%]: 270.0.
3.1.4 General procedure for the synthesis of intermediates 6a-b
Hydrazine monohydrate (1.16 ml, 2.4 mmol) was added to a solution of compound 5a or 5b (2.4 mmol)
in ethanol (25ml). This mixture was heated at reflux for 1-3 days and monitored by TLC until the reaction
went to completion. The resulting solid 6a or 6b was collected by suction filtration.
3.1.4.1 4-Hydrazinyl-6-methoxy-2-methylquinoline (6a). The product was collected as beige powder
80%. Mp: 243-245 ᵒC. IR (cm-1): 1247 (C-N 2° amine), 1599 (C-C), 2939 (CH3), 3026 (arene C-H), 3302
1
(NH2, 1° amine); H-NMR (400 MHz; DMSO-d6, 1 quat C not observed): 2.58 (s, 3H, CH3), 3.89 (s, 3H,
OMe), 6.92 (s, 1H, H 3), 7.44 (dd, 3J = 8.0 Hz, 4J = 4.0 Hz, 1H, H 7), 7.80 (d, J = 4.0 Hz, 1H, H 5), 7.84 (d, J =
8.0 Hz, 1H, H 8); 13C-NMR (75 MHz; DMSO-d6): 22.4 (Me), 56.3 (OMe), 98.2 (C 5), 102.2 (C 3), 116.0 (C8),

122.7 (C 7), 125.1 (C 6), 153.6 (q C), 154.3 (q C), 156.6 (C 2); MS ES-: 238.1 [M+35Cl, 100%], 240.1 [M+37Cl,
30%].
3.1.4.2 4-Hydrazinyl-6-methoxy-2-phenylquinoline (6b). The product was isolated as a yellow powder
(50%). Mp: 158-160 ᵒC; IR (cm-1): 1585 ( C=C), 1624 (C=N), 2830 (OMe), 3172 (C-H), 3298 (2ᵒ amine),
3
3349 (1ᵒ amine); 1H-NMR (300 MHz; DMSO-d6): 3.87 (s, 3H, OMe), 4.45 (s, 2H, NH2), 7.24 (dd, J = 9.3
Hz, 4J = 2.4 Hz, 1H, H 7), 7.42-7.52 (m, 5H, H 3`,4`,5`,3, 5), 7.75 (d, J = 9.3 Hz, 1H, H 8), 8.14 (d, J = 8.1 Hz,
2H, H 2`,6`), 8.30 (s, 1H, NH); 13C-NMR (75 MHz; DMSO-d6): 55.6 (OMe), 95.4 (C 5), 100.8 (C 3), 117.1 (C
8), 120.6 (C 7), 126.7 (C 2`,4`,6`), 128.4 (C 3`,5`), 130.7 (C 10), 140.4 (C 9), 143.6 (C1`), 152.9 (C 4), 154.3
(C 6), 155.8 (C2); MS ES+: [M+H, 100%]: 266.5.
3.1.5 General procedure for the synthesis of hydrazone derivatives (7a-r)
Intermediate 6a or 6b (1.0 mmol) and the appropriate aldehyde (3.0 mmol) were dissolved in methanol
(3.0 ml) and stirred for 3 h. The solid formed was then collected by filtration, washed with cold
methanol and dried by suction filtration to get the hydrazone derivative.
3.1.5.1 4-(2-(4-Fluorobenzylidene)hydrazinyl)-6-methoxy-2-methylquinoline (7a). Product isolated as a
yellow solid (90mg, 56%). Mp: 227-230 ᵒC; IR (cm-1): 1505 (C=C), 1591 (C=N), 2921 (Me), 3001 (arene C-
1
H), 3425 (-NH- 2° amine); H-NMR (400 MHz; DMSO-d6): 2.74 (s, 3H, Me), 4.00 (s, 3H, OMe), 7.33 (dd~t,
JHH = JHF = 8.0 Hz, 2 H, H 3`, 5`), 7.46 (s, 1H, H 3), 7.55 (dd, 3J = 8.0 Hz, 4J = 4.0 Hz, 1H, H 7), 7.88 (d, J = 8.0
Hz, 1H, H 8), 7.94 (dd, 3J = 8.0 Hz, 4J = 4.0 Hz, 2H, H 2`, 6`), 8.07 (d, J= 4.0 Hz, 1H, H 5), 8.86 (s, 1H, N=CH),
12.4 (s, 1H, NH). 13C-NMR (75 MHz; DMSO-d6; Quat. C 4, 4a, 8a not observed): 56.3 (OMe), 101.7 (C 5),
2
3
107.1 (C 3), 116.4 (d, JCF = 12.7 Hz, C 3`, 5`), 116.5 (C 7), 129.3 (d, JCF = 8.8 Hz, C 2`, 6`), 131.9 (C 1`),
133.4 (C=N), 156.5 (C 2), 162.3 (d, JCF = 147.0 Hz, C 4`); MS ES+, [M+H, 100%]: 310.5. Accurate mass
calculated for C18H16FN3O + H: 310.136. Found: 310.1333, error: -5.482 ppm.
3.1.5.2 4-(2-Benzylidenehydrazinyl)-6-methoxy-2-methylquinoline (7b). Product isolated as a yellow solid
(75mg, 52%). Mp: 282-285 ᵒC. IR (cm-1): 1501 (C=C), 1600 (C=N), 2845 (Me), 3121 (aromatic C-H
1
stretch), 3353 (-NH- 2° amine); H-NMR (400 MHz, DMSO-d6): 2.60 (s, 3H, CH3), 3.99 (s, 3H, OMe), 7.33
(s, 1H, H 3), 7.36 (dd, 3J = 8.0 Hz, 4J = 4.0 Hz, 1H, H 7), 7.47 (t, J = 8.0 Hz, 1H, H 4`), 7.53 (t, J = 8.0 Hz, 2H,
H 3`, 5`), 7.67 (d, J = 4.0 Hz, 1H, H 5), 7.78 (d, J = 8.0 Hz, 1H, H 8), 7.86 (d, J = 8.0 Hz, 2H, H 2`,6`), 8.45 (s,
1H, C=N), 10.92 (s, 1H, NH). 13C-NMR (75 MHz; DMSO-d6; Quat. C 4a not observed): 19.7 (Me), 56.7
(OMe), 100.1 (C5), 103.2 (C3), 115.3 (C3`,5`), 121.2 (C 6), 124.8 (C 7), 127.5 (C 1`), 128.9 (C 8), 130.7 (C
6`), 133.5 (C 2`), 133.7 (C=N), 149.4 (C 8a), 151.0 (C 4), 152.1 (C 6), 157.5 (C 2); MS ES+, [M+H, 100%]:
292.5. Accurate mass calculated for C18H17N3O + H: 292.145. Found: 292.1438, error: -2.046 ppm.
3.1.5.3 4-(2-((1H-Imidazol-5-yl)methylene)hydrazinyl)-6-methoxy-2-methylquinoline
(7c).
Product
isolated as a bright yellow solid (105 mg, 76%). Mp: 226-229 ᵒC; IR (cm-1): 1570 (C=C), 1599 (C=N), 2833
(OMe), 2928 (Me), 3121 (C-H), 3368 (2ᵒ amine); 1H-NMR (300 MHz, DMSO-d6): 2.69 (s, 3H, Me), 3.98 (s,
3H, OMe), 7.42 (s, 1H, H 3), 7.57 (d, J = 9.3 Hz, 1H, H 7), 7.68 (s, 1H, H 5`), 7.88 (s, 1H, H 5), 7.91 (d, J =
9.3 Hz, 1H, H 8), 8.25 (s, 1H, H 3`), 8.89 (s, 1H, HC=N), 12.66 (s, 2H, NH); 13C-NMR (75 MHz; DMSO-d6;
Quat. C 1`, 4a, 8a not observed): 19.8 (Me), 56.7 (OMe), 99.7 (C 5), 103.39 (C 3), 115.2 (C 5`), 121.3 (C 7),

124.4 (C 8), 133.7 (C 3`), 137.8 (C=N), 150.6 (C 4), 151.6 (C 6), 157.3 (C 2); MS ES-, [M-H, 100%]: 280.1.
Accurate mass calculated for C15H15N5O+H: 282.135. Found: 282.1329, error:-7.0888 ppm.
3.1.5.4 4-((2-(6-Methoxy-2-methylquinolin-4-yl)hydrazono)methyl)phenol (7d). Product isolated as a
yellow solid (80 mg, 53%). Mp: 208-210 ᵒC; IR (cm-1): 1570 (C=C), 1599 (C=N), 2833 (OMe), 2928 (CH3),
1
3121 (C-H), 3368 (NH), 3462 (OH); H-NMR (300 MHz; DMSO-d6): 2.71 (s, 1H, Me), 3.98 (s, 3H, OMe),
6.91 (d, J = 9.0 Hz, 2H, H 3`,5`), 7.46 (s, 1H, H 3), 7.62 (dd, 4J = 6.0 Hz, 3J = 3.0 Hz, 1H, H 7), 7.75 (d, J = 9.0
Hz, 2H, H 2`,6`), 7.89 (d, J = 9.0 Hz, 1H, H 8), 8.08 (s, 1H, H 5), 8.71 (s, 1H, C=N), 10.15 (s, 1H, OH), 12.3 (s,
1H, NH); 13C-NMR (75 MHz; DMSO-d6): 19.7 (Me), 56.6 (OMe), 99.6 (C 5), 103.1 (C 3), 115.1 (C 5`), 115.9
(C 3`), 121.1 (C 4a), 124.5 (C 7), 124.6 (C 8), 129.4 (C 2`,6`), 133.4 (C=N), 149.8 (C 8a), 150.7 (C 4), 151.7
(C 6), 157.3 (C 2), 160.1 (C1`); MS ES+, [M+H, 100%]: 308.1. Accurate mass calculated for C18H17N3O2 + H:
308.140. Found: 308.1389, error, -1.6226 ppm.
3.1.5.5 6-Methoxy-2-methyl-4-(2-(pyridin-3-ylmethylene)hydrazinyl)quinoline (7e). Product isolated as a
beige powder (50 mg, 34%). Mp: >290ᵒC (dec); IR (cm-1): 1570 (C=C), 1591 (C=N), 2830 (OMe), 2917
(CH3); 1H-NMR (300 MHz; CD3OD): 2.79 (s, 3H, CH3), 4.06 (s, 3H, OMe), 7.57-7.66 (m, 3H, H 3, 7, 5`), 7.82
(d, J = 9.3 Hz, 1H, H 8), 7.91 (s, 1H, H 5), 8.39 (d, J = 6.3 Hz, 1H, H 6`), 8.65 (s, 2H, H 2`, 4`), 8.98 (s, 1H,
HC=N); 13C-NMR (75 MHz; CD3OD): 20.2 (C Me), 56.9 (OMe), 102.1 (C 5), 102.8 (C 3), 117.1 (C 4a), 122.4
(C 7), 125.7 (C 5`), 126.7 (C 8), 131.9 (C 1`), 135.4 (C 6`), 136.2 (C=N), 134.4 (C 8a), 149.7 (C 4), 151.8 (C
2`), 153.2 (C 4`), 153.8 (C 6), 160.1 (C 2); MS ES+, [M+H, 100%]: 293.3. Accurate mass calculated for
C17H16N4O + H: 293.140. Found: 293.1378, error: -6.4816 ppm.
3.1.5.6 6-Methoxy-2-methyl-4-(2-(2-phenylethylidene)hydrazinyl)quinoline (7f). Product isolated as a
cream solid (43mg, 30%). Mp: 235-237 ᵒC; IR (cm-1): 1566 (C=C), 1610 (C=N), 2786 (OMe), 3004 (CH2),
1
3154 (C-H), 3423 (NH); H-NMR (300 MHz; DMSO-d6) δ ppm: 2.68 (s, 3H, CH3), 3.75 (d, J = 5.7 Hz, 2H,
H2`), 3.93 (s, 3H, OMe), 7.25 (s, 1H, H 3), 7.28-7.40 (m, 5H, phenyl), 7.56 (d, J = 8.7 Hz, 1H, H 7), 7.89 (d, J
= 9.0 Hz, 1H, H 8), 8.12 (s, 1H, H 5), 8.37 (t, J = 5.4 Hz, 1H, HC=N), 12.39 (s, 1H, NH). 13C-NMR (75 MHz;
DMSO-d6, Quat. C not observed): 19.9 (Me), 35.0 (C 2`, observed in DEPT), 56.5 (OMe), 99.5 (C 5), 102.9
(C 3), 114.9 (C10`), 124.6 (C 7), 126.8 (C 6`), 128.8 (C 5`, 7`), 128.91 (C 4`, 8`), 136.3 (C 3`), 152.2 (C=N),
153.1 (C 6), 157.3 (C 2); MS ES-, [M+Na, 100%]: 326.1. Accurate mass calculated for C19H19N3O + Na:
328.143. Found: 306.1581, error:-6.5325 ppm.
3.1.5.7 2-Methoxy-6-((2-(6-methoxy-2-methylquinolin-4-yl)hydrazono)methyl)phenol (7g). Product
isolated as a yellow powder (120 mg, 89%). Mp: 242-245 ᵒC. IR (cm-1): 1574 (C=C), 1603 (C=N), 2794
(OMe), 2841 (OMe), 2925 (CH3), 3016 (C-H), 3226 (NH), 3411 (OH). 1H-NMR (300 MHz; DMSO-d6) δ: 2.54
(s, 3H, CH3), 3.84 (s, 3H, OMe), 3.92 (s, 3H, OMe), 6.85 (t, J = 7.8 Hz, 1H, H 4`), 6.99 (d, J = 7.8 Hz, 2H, H 3,
3`), 7.35 (d, J = 8.5 Hz, 2H, H 7, 5`), 7.67 (d, J = 9.3 Hz, 1H, H 8), 7.80 (d, J = 2.1 Hz, 1H, H 5), 8.86 (s, 1H,
C=N), 10.12 (s, 1H, NH), 11.90 (s, 1H, OH). 13C-NMR (75 MHz; DMSO-d6, Quat. C not observed): 21.9
(Me), 55.7 (OMe), 55.9 (OMe), 102.1 (C 5), 113.0 (C 3), 113.0 (C 3`, 4`), 120.4 (C 7), 121.8 (C 5`), 146.6
(C=N), 147.9 (C 1`, 2`), 156.1 (C 2); MS ES+, [M+H, 100%]: 338.3. Accurate mass calculated for C19H19N3O3
+ H: 338.150. Found: 338.1480, error: -5.6188 ppm.
3.1.5.8 6-Methoxy-2-methyl-4-(2-((5-nitrofuran-2-yl)methylene)hydrazinyl)quinoline
(7h).
Product
isolated as a dark yellow powder (80 mg, 75%). Mp: 264-266 ᵒC; IR (cm-1): 1247 (C-O-C), 1511 (C=C),

1577 (NO), 1603 (C=N), 2722 (OMe), 3110 (C-H), 3425 (NH). 1H-NMR (300 MHz, DMSO-d6) δ: 2.76 (s, 3H,
Me), 4.00 (s, 3H, OMe), 7.45 (s, 1H, H 3), 7.49 (d, J=6.0 Hz, 1H, H 3`), 7.67 (dd, 4J= 9.0 Hz, 3J= 3.0 Hz, 1H, H
7), 7.89 (d, J= 3.0 Hz, 1H, H 4`), 7.95 (d, J = 9.0 Hz, 1H, H 8), 8.21 (s, 1H, H 5), 8.86 (s, 1H, C=N), 13.0 (s,
1H, NH); 13C-NMR (75 MHz; CD3OD): 19.7 (Me), 55.6 (OMe), 100.6 (C 5), 103.0 (C 3), 115.3 (C 4`, 3`),
121.3 (C 7), 124.0 (C 4a), 124.8 (C=N), 133.6 (C 8), 146.4 (C 8a), 148.9 (C 4), 151.0 (C 5`), 151.1 (C 6),
152.3 (C 2`), 157.5 (C 2); MS ES+, [M+H, 100%]: 327.1. Accurate mass calculated for C16H14N4O4 + H:
327.109. Found: 327.1085, error: -0.9171 ppm.
3.1.5.9 4-((2-(6-Methoxy-2-methylquinolin-4-yl)hydrazono)methyl)benzoic acid (7i). Product isolated as
a yellow solid (130 mg, 78%). Mp: 272-275 ᵒC; IR (cm-1): 1577 (C=C), 1603 (C=N), 1675 (C=O), 2648
1
(OMe), 2928 (CH3), 3004(C-H), 3314 (2ᵒ amine); H-NMR (300 MHz, DMSO-d6) δ ppm: 2.71 (s, 3H, CH3),
3.98 (s, 3H, OMe), 7.51 (s, 1H, H 3`), 7.57 (d, J = 9.3 Hz, 1H, H 7`), 7.90 (d, J= 9.3 Hz, 1H, H 8`), 7.97-8.05
(m, 5H, H 2, 3, 5, 6, 5`), 8.11 (s, 1H, N=C), 8.87 (s, 1H, NH), 13.0 (br s, 1H, OH); 13C-NMR (75 MHz; DMSO-
d6): 20.0 (Me), 56.6 (OMe), 100.6 (C 5`), 102.9 (C 3`), 115.5 (C 4`a), 121.9 (C 7`), 124.5 (C 8`), 127.4 (C 3,
5), 129.8 (C 2, 6), 132.1 (C 1), 134.3 (C 4), 137.8 (C=N), 147.5 (C 8`a), 150.7 (C 4`), 152.5 (C 6`), 157.4 (C
2`), 166.8 (C=O); MS ES+, [M+H, 100%]: 336.1. Accurate mass calculated for C19H17N3O3 + H: 336.135.
Found: 336.1339, error: -1.19 ppm.
3.1.5.10 6-Methoxy-2-methyl-4-(2-(4-nitrobenzylidene)hydrazinyl)quinoline (7j). Product isolated as a
bright yellow solid (133 mg, 92%). Mp: 220-223 ᵒC; IR (cm-1): 1341 (N=O), 1574 (C=C), 1510 (C=N), 2856
1
(OMe), 2917 (CH3), 3366 (2ᵒ amine); H-NMR (300 MHz, DMSO-d6): 2.64 (s, 3H, CH3), 3.96 (s, 3H, OMe),
7.42 (s, 1H, H 3), 7.47 (d, J = 9.0 Hz, 1H, H 7), 7.82 (d, J = 9.0 Hz, 1H, H 8), 7.95 (d, 2H, J = 8.7 Hz, H 2`,
6`), 7.98 (s, 1H, H 5), 8.03 (d, 2H, J = 8.7 Hz, H 3`, 5`), 8.70 (br s, 1H, N=CH), 12.73 (s, 1H, NH); 13C-NMR
(75 MHz; DMSO-d6; Quat. C4, 8a, 4` not observed): 21.9 (Me), 56.1 (C, OMe), 100.8 (C 5), 101.9 (C 3),
115.9 (C 4a), 126.9 (C 7, 3`, 5`), 128.5 (C 1`), 129.8 (C 2`, 6`), 131.5 (C 8), 138.3 (C=N), 156.7 (C 2), 166.9
(C 6); MS ES-, [M+H, 100%]: 337.1. Accurate mass calculated for C18H16N4O3 + H: 337.130. Found:
337.1293, error: -0.593 ppm.
3.1.5.11 5-((2-(6-Methoxy-2-methylquinolin-4-yl)hydrazono)methyl)benzene-1,3-diol (7k). Product
isolated as a bright yellow solid (0.12 g, 75%). Mp: >300 ᵒC; IR (cm-1): 1599 (C=C), 1643 (C=N), 2961
1
(OMe), 2997 (CH3), 3081(C-H), 3295 (NH); H-NMR (400 MHz, DMSO-d6): 2.72 (s, 3H, CH3), 3.99 (s, 3H,
OMe), 6.38 (s, 1H, H 6`), 6.73 (s, 2H, H 2`, 4`), 7.40 (s, 1H, H 3), 7.62 (d, J = 5.4 Hz, 1H, H 7), 7.64 (d, J = 5.7
Hz, 1H, H 8), 8.09 (s, 1H, H 5), 8.63 (s, 1H, HC=N), 9.57 (s, 2H, 2 x OH), 12.3 (s, 1H, NH); 13C-NMR (75 MHz;
DMSO-d6): 19.9 (Me), 56.5 (OMe), 99.9 (C 5), 102.9 (C 3), 105.2 (C 6`), 105.6 (C 2`, 4`), 115.3 (C 4a), 124.7
(C 7), 135.2 (C 4, 3`), 149.9 (C=N), 150.8 (C 8a), 152.2 (C 6), 157.5 (C 2), 158.8 (C 1`, 5`); MS ES+, [M+H,
100%]: 324.6. Accurate mass calculated for C18H17N3O3 + H: 324.135. Found: 324.1339, error:-1.234 ppm.
3.1.5.12
3’-((2-(6-Methoxy-2-methylquinolin-4-yl)hydrazono)methyl)phenol (7l). Product isolated
as a beige solid (0.5 g, 33%). Mp: >300 ᵒC; IR (cm-1): 1570 (C=C), 1606 (C=N), 2895 (OMe), 2965 (CH3),
3157 (C-H), 3284 (OH); 1H-NMR (400 MHz, DMSO-d6): 2.73 (s, 3H, CH3), 3.99 (s, 3H, OMe), 6.91 (d, J = 6.0
Hz, 1H, H 6`), 7.30-7.34 (m, 3H, H 2`, 3`, 5`), 7.47 (s, 1H, H 3), 7.63 (d, J = 9.3 Hz, 1H, H 7), 7.91 (d, J=9.0
Hz, 1H, H 8), 8.07 (s, 1H, H 5), 8.7 (s, 1H, N=CH), 9.75 (s, 1H, OH); 13C-NMR (75 MHz; DMSO-d6): 19.9
(Me), 56.6 (OMe), 100.0 (C 5), 103.0 (C 3), 113.5 (C 2`), 115.3 (C 6`), 117.9 (C 4a), 118.7 (C 7), 121.4 (C
4`), 124.7 (C 8), 129.9 (C 5`), 133.8 (C 3`), 134.9 (C=N), 149.5 ( C 8a), 150.8 (C 4), 152.2 ( C 6), 157.5 (C 1`),

157.8 (C 2); MS ES+, [M+H, 100%]: 308.6. Accurate mass calculated for C18H17N3O2+H: 308.140. Found:
308.1388, error -1.947 ppm.
3.1.5.13
6-Methoxy-2-phenyl-4-(2-(pyridin-3-ylmethylene)hydrazinyl)quinoline (7m). Product
isolated as a yellow solid (120 mg, 89%). Mp: 215-218 ᵒC. IR (cm-1): 1585 (C=C), 1624 (C=N), 2798 (OMe),
1
3063 (C-H), 3219 (NH); H-NMR (300 MHz, DMSO-d6): 3.97 (s, 3H, OMe), 7.37 (dd, 4J= 9.0 Hz, 3J= 1.8 Hz,
1H, H 7), 7.43-7.56 (m, 4H, H 3, 3``, 4``, 5``), 7.69 (s, 1H, H 5), 7.90 (d, J = 9.3 Hz, 1H, H 8), 7.93 (t, J = 6.3
Hz, 1H, H 5`), 8.23 (d, J = 7.8 Hz, 2H, H 2``, 6``), 8.29 (d, J = 7.8 Hz, 1H, C 6`), 8.46 (s, 1H, C=N), 8.59 (d, J =
4.8 Hz, 1H, H 4`), 8.97 (s, 1H, H 2`), 11.17 (s, 1H, NH); 13C-NMR (75 MHz; DMSO-d6): 55.8 (OMe), 98.1 (C
5), 100.5 (C 3), 116.7 (C 4a), 121.3(C 8), 123.9 (C 7), 126.8 (C 2``, 6``), 128.6 (C 3``, 5``), 128.8 (C 4``),
130.8 (C 1`), 131.0 (C 5`), 132.9 (C 6`), 139.5 (C 8a), 139.7 (C=N), 144.4 (C 4), 146.5 (C 1``), 148.2 (C 2`),
149.8 (C 4`), 154.3 (C 6), 156.4 (C 2); MS ES+, [M+H, 100%]: 355.2. Accurate mass calculated for
C22H18N4O + H: 355.156. Found 355.1551, error: -0.5631ppm.
3.1.5.14
4-((2-(6-Methoxy-2-phenylquinolin-4-yl)hydrazono)methyl)-N,N-dimethylaniline
(7n).
Product isolated as a brown powder (90 mg, 60%). Mp: 115-118 ᵒC. IR (cm-1): 1589 (C=C), 2802 (OMe),
1
2892 (OMe), 2997 (CH3), 3234 (C-H), 3513 (NH); H-NMR (300 MHz, DMSO-d6) δ ppm: 2.98 (s, 6H,
N(CH3)2), 3.95 (s, 3H, OMe), 6.74-6.82 (m, 3H, H 3, 5, 3``), 7.33 (d, J= 9.3 Hz, 1H, H 7``), 7.42-7.55 (m, 3H,
H 3`, 4`, 5`), 7.61-7.67 (m, 2H, H 2, 6), 7.82 (s, 1H, H 5``), 7.84 (d, J= 9.3 Hz, 1H, H 8``), 8.16 (d, J= 7.5 Hz,
2H, H 2`, 6`), 8.33 (s, 1H, HC=N), 10.71 (s, 1H, NH); 13C-NMR (75 MHz; DMSO-d6): 55.7 (OMe), 97.2 (C 5``),
100.6 (C 3``), 111.7 (C7``), 112.0 (C 3, 5), 121.0 (C 8``), 121.5 (C 4``a), 122.4 (C 1), 126.7 (C 2`, 6`), 127.8 (C
2, 6), 128.6 (C 4`), 128.7 (C 3`, 5`), 129.4 (C 1`), 130.9 (C 8``a), 143.6 (C=N), 146.9 (C 4``), 151.08 (C 4),
156.1 (C 2``), 160 (C 6``); MS ES+, [M+H, 100%]: 397.3. Accurate mass calculated for C25H24N4O+H:
397.203. Found: 397.2016, error: -1.7623 ppm.
3.1.5.15
6-Methoxy-4-(2-(4-nitrobenzylidene)hydrazinyl)-2-phenylquinoline (7o). Product isolated
as a red-brown powder (53 mg, 40%). Mp: 230-234 ᵒC; IR (cm-1): 1556 (NO), 1595 (C=C), 2848 (OMe),
1
2921 (C-H), 3357 (NH). H-NMR (300 MHz, DMSO-d6): 3.97 (s, 3H, OMe), 7.41 (d, J = 8.0 Hz, 1H, H 7),
7.48-7.56 (m, 3H, H 3``, 4``, 5``), 7.72 (s, 1H, H 3), 7.92 (d, J = 8.0 Hz, 1H, H 8), 7.91-7.96 (s, 1H, H 5), 8.11
(d, 2H, J = 8.1 Hz, H 2`, 6`), 8.22 (d, 2H, J = 7.2 Hz, H 2``, 6``), 8.32 (d, 2H, J = 8.1 Hz, H 3`, 5`), 8.52 (s, 1H,
N=CH), 11.37 (br s, 1H, NH). 13C-NMR (75 MHz; DMSO-d6): 55.9 (OMe), 99.5 (C 5), 100.5 (C 3), 116.8 (C
4a), 121.4 (C 7, 8), 124.1 (C 3`, 5`), 126.8 (C 2`, 6`), 127.3 (C 2``, 4``, 6``), 128.6 (C 3``, 5``), 128.9 (C 8a),
131.1 (C 1``), 139.8 (C 1`), 141.3 (C=N), 146.2 (C 4), 147.1 (C 4`), 154.3 (C 6), 156.5 (C 2); MS ES +, [M+H,
100%]: 399.1. Accurate mass calculated for C23H18N4O3+H: 399.146. Found: 399.1451, error: -0.250 ppm.
3.1.5.16
6-Methoxy-2-methyl-4-(2-(2-phenylethylidene)hydrazinyl)quinoline
(7p).
Product
isolated as a beige powder (56 mg, 26%). Mp: 180-182 ᵒC; IR (cm-1): 1585 (C=C), 1617 (C=N), 2910
(OMe), 2968 (CH2), 3026 (C-H); 1H-NMR (300 MHz, DMSO-d6): 3.75 (d, J = 5.7 Hz, 2H, H 2``), 3.90 (s, 1H,
OMe), 7.25 -7.56 (m, 10H, H 3, 7, 3`,4`,5`,4``, 5``, 6``,7``,8``), 7.71 (s, 1H, H 5), 7.82-7.86 (m, 2H, H 8,
N=CH), 8.11 (d, J = 7.5 Hz, 2H, H 2`,6`), 10.59 (s, 1H, NH); 13C-NMR (75 MHz; DMSO-d6): 38.4 (C 2``), 55.7
(OMe), 97.3 (C 5), 100.5 (C 3), 116.4 (C 4a), 121.1 (C 8), 126.5 (C 7), 126.7 (2C, 4`, 6``), 128.5 (C 5``,7``),
128.6 (C 2`,6`), 128.7 (C 8``), 128.9 (C 3`, 5`), 130.9 (C 4``), 137.4 (C 3``), 139.9 (C 8a), 144.3 (C 1`), 145.5
(C=N), 147.1 (C 4), 154.2 (C 6), 156.1 (C 2); MS ES+, [M+H, 100%]: 368.2. Accurate mass calculated for
C24H21N3O+H: 368.176. Found: 368.1763, error: 1.629 ppm.

3.1.5.17
2-Methoxy-6-((2-(6-methoxy-2-phenylquinolin-4-yl)hydrazono)methyl)phenol
(7q).
Product isolated as a bright yellow powder (132 mg, 91%). Mp: 120-123 ᵒC; IR (cm-1): 1566 (C=C), 1620
1
(C=N), 2830 (OMe), 2946 (OMe), 3059 (C-H), 3230 (NH), 3509 (OH); H-NMR (300 MHz, DMSO-d6): 3.85
(s, 3H, OMe), 3.96 (s, 3H, OMe), 6.87 (t, J=7.8 Hz, 1H, H 4`), 7.00 (d, J = 7.8 Hz, 1H, H 3`), 7.37 (d, J= 9.0
Hz, 1H, H 7), 7.46-7.56 (m, 4H, H 5`, 3``, 4``, 5``), 7.70 (s, 1H, H 3), 7.76 (s, 1H, H 5), 7.88 (d, J= 9.3 Hz, 1H,
H 8), 8.16 (d, J=7.2 Hz, 2H, H 2``, 6``), 8.81 (s, 1H, HC=N), 9.71 (s, 1H, NH), 11.00 (s, 1H, OH); 13C-NMR (75
MHz; DMSO-d6): 55.9 (2C, OMe), 97.3 (C 5), 100.5 (C 3), 112.6 (C 3`), 118.2 (C 4`), 118.3 (C6`), 119.3 (C
4a), 120.9 (C 8), 121.3 (C 7), 122.0 (C 4`), 126.7 (C2``, 6``), 128.6 (C 5, 5``), 130.9 (C 4``), 139.8 (C 8a),
140.9 (C1``), 145.6 (C=N), 147.9 (C 4, 1`), 148.5 (C 4`), 154.3 (C 6), 156.3 (C 2); MS ES+, [M+H, 100%]:
400.2. Accurate mass calculated for C24H21N3O3+H: 400.166. Found: 400.1649, error: -1.7493 ppm.
3.1.5.18
4-(2-((1H-Imidazol-4-yl)methylene)hydrazinyl)-6-methoxy-2-phenylquinoline
(7r).
Product isolated as brown solid (100 mg, 77%). Mp: 184-186 ᵒC; IR (cm-1): 1585 (C=C), 1624 (C=N), 2830
1
3
(OMe), 3063 (C-H), 3295 (2ᵒ amine); H-NMR (300 MHz, DMSO-d6): 3.96 (s, 3H, OMe), 7.38 (dd, J = 9.0
Hz, 4J = 2.4 Hz, 1H, H 7), 7.47-7.57 (m, 4H, H 3, 3``, 4``, 5``), 7.67 (d, J = 2.4 Hz, 1H, H 5), 7.84 (d, J = 6.9 Hz,
2H, H 6`, 8), 7.89 (s, 1H, H 4`), 8.20 (d, J = 7.5 Hz, 2H, H 2``, 6``), 8.40 (s, 1H, C=N), 10.90 (s, 1H, NH), 12.73
(s, 1H, NH); 13C-NMR (75 MHz; DMSO-d6, C4a not observed): 55.8 (OMe), 97.6 (C 5), 100.8 (C 3), 116.6 (C
6`), 121.3 (C 7, 8), 126.9 (C 3``, 5``), 128.5 (C 2``, 6``), 128.9 (C 4``), 130.3 (C 8a), 137.3 (C 4`), 139.4 (C 1`),
147.2 (C 1``), 153.9 (C 4), 156.3 (C 2, 6); MS ES+, [M+H, 100%]: 344.3. Accurate mass calculated for
C20H17N5O+H: 344.151. Found: 344.1487, error: -5.5208 ppm.
3.1.6 General procedure for the hydrazide derivatives 8a-b
Compound 6a (1 mmol) was dissolved in dichloromethane (10 ml), cooled in an ice bath and stirred for
15 min. Pyridine (1.5 mmol) was added slowly followed by the dropwise addition of a solution of the
appropriate acyl chloride (1 mmol) in dichloromethane (5 ml) over a period of 2 h. Once the addition
had finished, the ice bath was removed and the mixture was left to stir for another 3 h at room
temperature. The formed solid was collected by filtration, washed with water and recrystallized from
ethanol to give the final product.
3.1.6.1 4-Fluoro-N'-(6-methoxy-2-methylquinolin-4-yl)benzohydrazide (8a). Product was isolated as a
yellow solid (238 mg, 74%). Mp: 284-286 ᵒC; IR (cm-1) 1501 (C-C), 1599 (C=C), 1636 (amide), 2594 (C-H),
2939 (CH3), 3030 (arenes C-H), 3248 (NH2 1° amine); 1H-NMR (400 MHz; DMSO-d6): 2.43 (s, 3H, CH3),
3.90 (s, 3H, OMe), 6.49 (s, 1H, H 3`), 7.29 (dd, 3J = 9.2 Hz, 4J = 3.2 Hz, 1H, H 7`), 7.41 (dd~t,3 JHH = 3JHF =
8.0 Hz, 2H, H 3, 5), 7.62 (d, J = 2.8 Hz, 1H, H 5`), 7.70 (d, J = 9.0 Hz, 1H, H 8`), 8.05 (dd, 3JHH = 8.0 Hz,
4JHF = 4.0 Hz, 2H, H 2, 6), 9.10 (s, 1H, NH), 10.63 (s, 1H, NH); 13C-NMR (75 MHz; DMSO-d6): 24.9 (Me),
55.9 (OMe), 106.2 (C 5`), 107.9 (C 3`), 116.1 (d JCF = 12.7 Hz, C 3, 5), 116.3 (C 10`), 116.4 (C 7`), 127.8 (C
1), 132.5 (C 2, 6), 132.6 (C 8`), 133.0 (C 9`), 156.4 (C 4`), 164.4 (C 6`), 164.7 (C 2`), 166.4 (C=O), 166.9 (C
4); MS ES+ [M+H, 10%]: 326.1.
3.1.6.2 N'-(6-Methoxy-2-methylquinolin-4-yl)benzohydrazide (8b). Product was isolated as a beige
powder (45 mg, 28%). Mp: >285ᵒC (Dec); IR (cm-1): 1599 (C=C), 1620 (C=O amide), 2946 (CH3), 3020 (C-H
arene), 3205 (NH-amide), 3302 (NH-aryl); 1H-NMR (300 MHz; DMSO-d6): 2.65 (s, 3H, CH3), 3.94 (s, 3H,
OMe), 6.86 (s, 1H, H 3`), 7.56-7.65 (m, 4H, H 4, 5`, 7`, 8`), 7.94 (m, 2H, H 2, 6), 8.02 (d, J = 7.2 Hz, 2H, H

3,5), 8.13 (d, J = 8.1 Hz, 1H, NH), 11.20 (s, 1H, NHCO); 13C-NMR (75 MHz; DMSO-d6): 19.6 (Me), 56.1
(OMe), 102.5 (C 5`), 114.8 (C 10`), 120.9 (C 3`), 123.9 (C 7`), 127.8 (C 2, 6), 128.6 (C 3, 5), 131.8 (C 1),
132.4 (C 8`), 132.6 (C 4), 151.2 (C 9`), 152.8 (C 4`), 155.2 (C 6`), 157.0 (C 2`), 166.0 (C=O). MS ES- [M+Cl,
100%]: 238.1 (cleavage to hydrazine 6a observed).
3.2
Molecular modelling
Compounds were built using Chem 3D pro 13.0 and energy minimized using the MOE software package
[39] prior to docking. The compounds were docked into the NQO2 X-ray structure PDB code 4FGJ.[40]
Waters of crystallization were deleted from the protein and hydrogens were added, checking
protonation and tautomeric states. Docking was performed using GOLD version 5.3[41] with the ASP
scoring function.
3.3
Biology
3.3.1 Inhibition of recombinant human NQO2 and NQO1 enzyme activity
Recombinant human NQO2 and NQO1, NADH, and dichlorophenolindophenol (DCPIP) were purchased
from (Sigma-Aldrich, UK) and EPR was synthesized at the Strathclyde Institute of Pharmacy and
Biomedical Sciences.
The rates of the NQO2 and NQO1 enzyme activity were determined spectrophotometrically by recording
the rates of DCPIP colour change at 600nm, over 1 min at 37 ˚C using Beckman DU 7400
spectrophotometer. Reaction mixtures were made up to a 1 ml final volume by sequential mixing of 940
µl phosphate buffer (50 mM, pH 7.4) with 20 µl DCPIP (2 mM), 20 µl enzyme cofactor (10 mM) (either
EPR for NQO2 or NADH for NQO1) and 10 µl inhibitor (concentration ranging from 0.001 to 1000 µM) or
an appropriate concentration of DMSO or NaOH (in the control sample). The reaction was initiated by
the addition of 10 µl rhNQO2 or rhNQO1 (5 x 10-3 mg/ml).
All experiments were performed in triplicate on three independent occassions. In each independent
experiment solutions of NRH (10 mM), DCPIP (2 mM) and NQO2 and NQO1 (5 x 10-3 mg/ml) were freshly
prepared. NRH, NADH, NQO1 and NQO2 were dissolved in phosphate buffer (50 mM, pH 7.4) and kept
at 0 ˚C. DCPIP was dissolved in deionized water. The inhibitors stock solutions were prepared by
dissolving the inhibitors in DMSO or 0.13 M NaOH to give 10mM solutions. A 10-fold serial dilution was
completed using DMSO or 0.13 M NaOH to give five different concentrations of the inhibitors: 1-10000
nM for NQO2, and 0.01-100 µM for NQO1. Both NQO1 and NQO2 were reconstituted in phosphate
buffer containing 5µM of FAD and 250mM of sucrose at a stock concentration of 5 mg/ml.
IC50 values were determined using non-linear curve fitting in the Graph pad prism software. The curves
were obtained from plotting enzyme activity as a percent of the control versus the log concentration of
the inhibitor used. IC50 values were determined as 50% reduction in the enzyme activity compared to
control (100%).
3.3.2 Cell lines and cell culture

Two human ovarian cancer (OVC) cell lines, SKOV-3 and TOV-112D, were provided by the Viral Oncology
Labs, St Mary’s Hospital, and authenticated using the DNA sequencing facility at the University of
Manchester. All cells were cultured in RPMI-1640 medium (Sigma, UK) supplemented with 10% (v/v)
heat-inactivated foetal bovine serum (FBS; GIBCO, UK) and (2 mmol/L) L-glutamine (Sigma, UK). To
maintain pure cultures of NQO2 genetically modified TOV-112D and SKOV-3 cell sub-lines, the cultures
were held in RPMI medium containing 1μg/ml of puromycin (Sigma, UK) before sub-culture in fresh
medium free of antibiotics immediately prior to experimentation. For induction of NQO2 silencing in
SKOV-3 cells, Doxycycline Hyclate (Sigma, UK) was added to the culture medium at a concentration of
1μg/ml on alternate days for five days before seeding for the experiment. This ensured that the silencing
levels of NQO2 were stable throughout the experiment; DOX was also added directly to the culture
medium on alternate days throughout the experiment.
3.3.3 Toxicity Assays
SKOV-3 and TOV-112D cells (either WT or genetically derived sublines) were seeded at 1500 and 1000
cells, respectively, per well into 96-well plates and left overnight to allow cell attachment. Cells were
then treated with different compounds at various concentrations ranging between 0.001 and 100 µM
for 24 h or 96h. For the shorter exposure period, the drug-containing medium was removed after 24 h
and replenished with fresh growth medium for the remainder of the 96 h period. The number of
surviving cells was then determined using MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium
bromide) assay.[42] MTT solution (2.5 mg/ml) was prepared in sterile PBS. Following 96h incubation,
50µl MTT solution was added to the cells and incubated for 4 h at 37°C. The medium was then removed
and the remaining formazan crystals were dissolved in 200 µl dimethyl sulfoxide (DMSO) (Fisher
Scientific, UK). Optical densities were then measured on a multi-well scanning spectrophotometer at
540 nm and then analyzed using the Gen 5 Software package (BioTek). The results were then used to
calculate the surviving fraction of cells relative to solvent only controls. Dose-response curves were
generated using the GraphPad Prism 5.01 Software and nonlinear regression analysis was used to fit the
data. IC50 values (defined as the concentration of drug required to decrease cell survival by 50% relative
to controls) were determined from these curves.
To determine the ability of the novel quinoline compounds to act as pharmacological inhibitors of NQO2
in cells, we use inhibition of CB1954 toxicity as a surrogate measure of effects on NQO2. SKOV-3 cells
were treated for 24 h, with either a fixed concentration of inhibitors and varying concentrations of
CB1954 (0.001-10µM) combined with 100 µM EPR or a fixed concentration of CB1954 (1 µM) plus 100
µM EPR and varying non-toxic concentrations of the putative inhibitors. Following 24 h incubation with
the combination therapy, the medium containing treatment was replaced with fresh growth medium for
the remainder of 96 h period. Thereafter, the MTT assay was used to determine cell viability.
Percentages of surviving cells were determined and used to generate dose-response curves.

3.3.4 Statistical analysis
The primary analysis of data was performed on Microsoft Excel software (2007) and then transferred to
GraphPad Prism (version 5.01 for Windows, La Jolla California, USA, www.graphpad.com).
Acknowledgements
Viral Oncology Labs at St Mary’s Hospital, Manchester for providing the cell lines used in this paper. BH
and BI thank Al-Zaytoonah University of Jordan for sponsorship. RM thanks NRF (SA) and Aspen
Pharmacare (SA) for sponsorship.
Appendix A. Supplementary Data
References
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•
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Hydrazone analogues of 4-aminoquinolines inhibit NQO2, with nM IC50 values.
Hydrazone analogues had low cytotoxicity to ovarian cancer SKOV-3 & TOV-112 cells.
•
Useful probes to interrogate the role of NQO2 in cancer/other biological conditions.