Synthesis and evaluation of a large library of nitroxoline derivatives as pancreatic cancer antiproliferative agents

Article abstract of DOI:10.1080/14756366.2020.1780228

Pancreatic cancer (PC) is one of the deadliest carcinomas and in most cases, which are diagnosed with locally advanced or metastatic disease, current therapeutic options are highly unsatisfactory. Based on the anti-proliferative effects shown by nitroxoline, an old urinary antibacterial agent, we explored a large library of newly synthesised derivatives to unravel the importance of the OH moiety and pyridine ring of the parent compound. The new derivatives showed a valuable anti-proliferative effect and some displayed a greater effect as compared to nitroxoline against three pancreatic cancer cell lines with different genetic profiles. In particular, in silico pharmacokinetic data, clonogenicity assays and selectivity indexes of the most promising compounds showed several advantages for such derivatives, as compared to nitroxoline. Moreover, some of these novel compounds had stronger effects on cell viability and/or clonogenic capacity in PC cells as compared to erlotinib, a targeted agent approved for PC treatment.

Full text of DOI:10.1080/14756366.2020.1780228

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis and evaluation of a large library of
nitroxoline derivatives as pancreatic cancer
antiproliferative agents
Serena Veschi, Simone Carradori, Laura De Lellis, Rosalba Florio, Davide
Brocco, Daniela Secci, Paolo Guglielmi, Mattia Spano, Anatoly P. Sobolev &
Alessandro Cama
To cite this article: Serena Veschi, Simone Carradori, Laura De Lellis, Rosalba Florio, Davide
Brocco, Daniela Secci, Paolo Guglielmi, Mattia Spano, Anatoly P. Sobolev & Alessandro Cama
(2020) Synthesis and evaluation of a large library of nitroxoline derivatives as pancreatic cancer
antiproliferative agents, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 1331-1344,
DOI: 10.1080/14756366.2020.1780228
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1331–1344
https://doi.org/10.1080/14756366.2020.1780228
RESEARCH PAPER
Synthesis and evaluation of a large library of nitroxoline derivatives as pancreatic
cancer antiproliferative agents
Serena Veschia, Simone Carradoria , Laura De Lellisa, Rosalba Florioa, Davide Broccoa, Daniela Seccib, Paolo
Guglielmib, Mattia Spanob, Anatoly P. Sobolevc and Alessandro Camaa
b
aDepartment of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara, Chieti, Italy; Dipartimento di Chimica e Tecnologie del Farmaco,
c
ꢀ
Sapienza Universita di Roma, Rome, Italy; Istituto per i Sistemi Biologici, Laboratorio di Risonanza Magnetica “Segre-Capitani”, CNR,
Monterotondo (Rome), Italy
ABSTRACT
ARTICLE HISTORY
Received 17 May 2020
Accepted 22 May 2020
Pancreatic cancer (PC) is one of the deadliest carcinomas and in most cases, which are diagnosed with
locally advanced or metastatic disease, current therapeutic options are highly unsatisfactory. Based on the
anti-proliferative effects shown by nitroxoline, an old urinary antibacterial agent, we explored a large
library of newly synthesised derivatives to unravel the importance of the OH moiety and pyridine ring of
the parent compound. The new derivatives showed a valuable anti-proliferative effect and some displayed
a greater effect as compared to nitroxoline against three pancreatic cancer cell lines with different genetic
profiles. In particular, in silico pharmacokinetic data, clonogenicity assays and selectivity indexes of the
most promising compounds showed several advantages for such derivatives, as compared to nitroxoline.
Moreover, some of these novel compounds had stronger effects on cell viability and/or clonogenic cap-
acity in PC cells as compared to erlotinib, a targeted agent approved for PC treatment.
KEYWORDS
Nitroxoline derivatives; drug
repurposing; pancreatic
cancer; Erlotinib;
clonogenicity; 4-
nitro(thio)phenyl
1. Introduction
direct action on tumour cells, nitroxoline may activate antitumour
immune response, which is recognised to be crucial in novel and
more effective anticancer strategies14,15. Indeed, the immune sys-
tem plays a crucial role in long-term cancer response to chemo-
therapy16. Overall, these data indicate a marked antitumour effect
of nitroxoline on different cancers including PC, suggesting that
this drug is a promising candidate for repositioning in the treat-
ment of PC.
Pancreatic cancer (PC) is one of the deadliest neoplasms, with a sur-
vival rate at 5 years of less than 6%1. Incidence and mortality rates
for PC are increasing and its unfavourable prognosis is due to poor
response to current therapies2,3. Thus, it is of paramount import-
ance to find novel and more effective therapeutic approaches. In
this respect, it has been reported that several non-anticancer agents
approved for the treatment of different human diseases may have
anticancer properties4. Our recent studies provided the first evi-
dence that the non-antitumor drug nitroxoline has antitumor
effects on PC cells5,6. Previously, this antibiotic showed antitumour
effects in preclinical cancer models including xenograft and genetic-
ally modified mice models of glioblastoma, myeloma, fibrosarcoma
as well as bladder, prostate, kidney and breast cancers7–13. In line
with the results obtained in other tumours, we showed that nitro-
xoline significantly reduces cell viability, affects cell cycle, promotes
apoptosis and markedly decreases clonogenic activity in PC cells5.
Moreover, using integrative proteomic and functional approaches,
we evaluated nitroxoline effects on protein expression in PC cells,
showing that this drug affects several biological pathways and
oncogenic proteins, previously unknown to play a role in nitroxo-
line anticancer activity6. Notably, our study revealed a pleiotropic
mechanism of nitroxoline anticancer action that involves proc-
esses playing a crucial role in growth, migration, invasion and cell
death, ROS production and DNA damage6. Remarkably, this drug
induced a previously unknown deregulation of molecules that are
involved in cell bioenergetics, leading to mitochondrial depolar-
For these reasons, nitroxoline structure has prompted the
Medicinal chemists to explore the structural requirements suitable
to improve antitumor effects. In previous papers, taking advantage
of the reactivity of the quinoline nucleus, most authors studied the
introduction of halogens and additional side chains as well as mod-
ifications of the nitro group17,18. A limitation of most previous
efforts was that they were directed towards the cathepsin B inhibi-
tory activity, disregarding the other mechanisms of action demon-
strated for nitroxoline as an anticancer compound so far. The aim
of our study was the hitherto unexplored modification of the OH
group. This could lead to a strong alteration of the chemical–physi-
cal characteristics of this 8-hydroxyquinoline. Indeed, this functional
group endowed the chemical structure with a discrete acidity (rein-
forced by the p-NO2 moiety) and chelating ability. Furthermore,
this phenolic compound is characterised by proton and electron-
donating capacity and, as a consequence, antioxidant properties
which can occur during several important biological processes19.
Starting from these premises and pursuing our efforts towards
the discovery of new anti-proliferative heterocyclic agents20–22, we
functionalised the OH of nitroxoline in order to obtain a large
isation6. Moreover, recent evidence suggests that, besides its library of ether analogues introducing linear and branched (C2-C15),
CONTACT Simone Carradori
simone.carradori@unich.it
Department of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara, Chieti 66100, Italy
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1332
S. VESCHI ET AL.
saturated and unsaturated alkyl chains, and substituted and glassware. All the solvents and reagents were directly used as
unsubstituted aryl and bicyclic rings. Moreover, we inserted a
carbonyl spacer between the benzylic methylene and the aryl
ring to evaluate the impact of this elongation. Aliphatic ethers
were also decorated with COOH, CN, and COOEt to further
explore the chemical space around these positions. Benzylic
ethers were characterised by the presence of electron-donating
(methyl, methoxyl, thiomethyl) and electron-withdrawing (halo-
gens, nitro, cyano, trifluoromethyl) substituents in ortho, meta
and para positions. Polysubstitutions were also provided. Lastly,
to better understand the importance of the quinoline nucleus,
we aimed at simplifying the nitroxoline structure to 4-nitro(-
thio)phenol. On these two scaffolds, we added the same sub-
stituents that resulted to be more active in the first series giving
the possibility to extrapolate robust structure-activity relation-
ships (SARs).
All the gathered compounds have been first tested in a one-
point concentration screening against three human PC cell lines
to assess the best-in-class compounds for further cell-based
experiments. Specifically, we selected AsPC-1, Capan-2 and BxPC-3
pancreatic cancer cell lines that display different genetic profiles:
AsPC-1 and Capan-2 are KRAS mutated, while BxPC-3 and AsPC-1
carry p53 mutations.
supplied by Sigma-Aldrich (Milan, Italy) without further purifica-
tion unless otherwise noted. Where mixtures of solvents are
specified, the stated ratios are volume:volume. All melting points
R
were measured on a StuartV melting point apparatus SMP1 and
are uncorrected (temperatures are reported in ꢀC). 1H and 13 C
NMR spectra were recorded at 300 MHz and 75 MHz (Varian
Mercury spectrometer), or at 400.13 MHz and 101.03 MHz on a
Bruker spectrometer, using CDCl3 and DMSO-d6, as the solvents
at room temperature. 19 F spectra were recorded on a Bruker
AVANCE 600 spectrometer at 564.7 MHz, using CDCl3 as solvent.
All the compounds were analysed with a final concentration of
ꢁ25 mg/mL. 1H and 13 C chemical shifts are expressed as d units
(parts per millions) relative to the solvent signal (DMSO-d6: 1H
2.50 and 13 C 39.5; CDCl3: 1H 7.26 and 13 C 77.4), whereas 19 F
chemical shifts are expressed as d units relative to an external
standard (CF3COOH, d ꢂ 76.55 ppm). 1H spectra are described as
follows: dH (spectrometer frequency, solvent): chemical shift/ppm
(multiplicity, J-coupling constant(s), number of protons, assign-
ment). 13 C spectra are described as follows: dC (spectrometer fre-
quency, solvent): chemical shift/ppm (assignment) and are fully
proton decoupled. 19 F spectra are described as follows: dF (spec-
trometer frequency, solvent): chemical shift/ppm (multiplicity, J-
coupling constant(s), number of fluorine, assignment). Multiplets
are abbreviated as follows: br – broad; s – singlet; d – doublet; t –
triplet; q – quartette; td – triplet of doublets; m – multiplet.
Coupling constants J are valued in Hertz.
2. Chemistry
For the synthesis of compounds 1–61, we followed the synthetic
approaches outlined in Figure 1. Derivatives 1–12, 14–46 (Figure
1(A)) have been easily synthesised by reacting nitroxoline with the
proper alkyl/benzyl bromides or a-bromoacetophenones; these
reactions were performed in N,N’-dimethylformamide (DMF), in the
presence of potassium carbonate (K2CO3) and under nitrogen (N2)
atmosphere. In addition, compound 12 was hydrolysed in mild
conditions using lithium hydroxide (LiOH) in a mixture of water
and methanol (in the ratio 50:50, v:v) at room temperature, to pro-
vide the carboxylic acid derivative 13. For compounds 47–61, the
same reactions involving 4-nitrophenol or 4-nitrothiophenol were
performed (Figure 1(B)). The structures were confirmed by spectral
studies (1H/13C/19F NMR), whereas the purity of these compounds
was confirmed by combustion analysis, TLC parameters, crystallo-
graphic studies (for compound 16) and melting point evaluation.
In silico analysis of the most active compounds was performed by
using the online free software SwissADME, a web tool that allows
to appraise pharmacokinetics, as well as drug-likeness (the prob-
ability to be an oral drug) and medicinal chemistry friendliness
(PAINS) of small molecules23. Target prediction was attempted tak-
ing advantage of the SwissTargetPrediction web tool24.
The processing and analyses of the NMR data were carried out
with MestreNova. Elemental analyses for C, H, and N were recorded
on a Perkin-Elmer 240 B microanalyzer obtaining analytical results
within
0.4% of the theoretical values for the final compounds.
Preparative chromatography was carried out employing Sigma-
R
AldrichV silica gel (high purity grade, pore size 60 Å, 230–400 mesh
particle size). All the purifications and reactions were carried out
by TLC performed on 0.2 mm thick silica gel-aluminium backed
plates (60 F254, Merck). Spot visualisation was performed under
short- and long-wavelength (254 and 365nm, respectively) ultra-
violet irradiation. Where given, systematic compound names and
ClogP values are those generated by ChemBioDraw Ultra 14.0 fol-
lowing IUPAC conventions.
4.2. Synthesis of nitroxoline derivatives
4.2.1. General procedure for the synthesis of compounds 1–12
and 14–46
Freshly ground potassium carbonate (K2CO3, 1.1 equiv.) was added
to a stirring solution of nitroxoline (1 equiv.) in DMF (20 ml). The
yellow suspension was stirred for 30 min at room temperature;
then, the proper (substituted)benzyl, diaryl or alkyl bromide or
a-bromoacetophenone (1.5 equiv.) was added and the reaction
stirred until disappearance of the starting reagents, as detected by
thin layer chromatography (TLC). Then, the mixture was poured
into ice-cold water (150ml) and partitioned with dichloromethane
(DCM, 3 ꢃ 40 ml). The organics were separated, reunited and added
with anhydrous sodium sulphate (Na2SO4) to remove water. The
salt was filtered and washed two times with 5 ml of dry DCM. The
organic phase was evaporated in vacuo to afford the crude extract
containing the target molecule that was recovered through col-
umn chromatography, employing silica gel (SiO2) and proper mix-
3. X-ray diffraction analysis
Crystals of compound 16 (Figure 2) were obtained by slow evap-
oration from an ethyl acetate/n-hexane mixture. Information about
the crystal data, experimental collection conditions and refine-
ment as well as the structural geometric parameters are available
in the Cambridge Crystallographic Data Centre in CIF format
(CCDC 2001211).
4. Materials and methods
4.1. Chemistry
Unless otherwise indicated, all reactions were carried out under
a positive pressure of nitrogen in washed and oven-dried tures of n-hexane/ethyl acetate.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1333
1
2
8
9
, R= Et
, R= Pr
, R= Prenyl
, R= Pentyl
3,
10,
R= CH2CH=CH2
R= Geranyl
1, R= Farnesyl
2, R= CH2CO2Et
NO
OR
2
1
1
4
, R= CH2C
, R= CH2CN
, R= Bu
CH
LiOH
MeOH, H O
2
5
6
7
N
13
R= CH2COOH
, R= CH2CH=CHCH3
R-X
1
1
4, Cy= Ph
A
5, Cy= 2-CH3Ph
3
3
3
3
3
3
3
3
3
3
4
4
4
0, Cy= 4-ClPh
NO
OH
2
NO
O
2
16,
Cy= 2-CF3Ph
7, Cy= 2-CNPh
8, Cy= 2-NO2Ph
9, Cy= 2-BrPh
0, Cy= 3-CH3Ph
1, Cy= 3-OCH3Ph
2, Cy= 3-FPh
1, Cy= 4-BrPh
CyCH X
1
1
1
2
2
2
2
2
2
2
2
2
2
2, Cy= 4-IPh
2
3, Cy= 4-CNPh
N
N
K2CO3
DMF
4, Cy= 4-CF3Ph
5, Cy= 4-NO2Ph
6, Cy= 2,6-diFPh
7, Cy= 3,5-diCH3Ph
8, Cy= 3,5-diFPh
9, Cy= 3-Cl-5-OCH3Ph
0, Cy= 3,4-diClPh
1, Cy= naphthalen-1-yl
2, Cy= phthalimide
nitroxoline
Cy
CyCOCH 2Br
3, Cy= 3-ClPh
4, Cy= 3-BrPh
5, Cy= 3-NO2Ph
6, Cy= 3-CF3Ph
7, Cy= 4-CH3Ph
8, Cy= 4-SCH3Ph
9, Cy= 4-FPh
NO
2
N
O
O
4
4
3, Cy= Ph
Cy
4, Cy= 3-NO2Ph
45,
Cy= 4-NO2Ph
4
6, Cy= thiophen-3-yl
B H
3
4
4
7, Y= O; Z= 2-BrPh
8, Y= O; Z= 2-NO2Ph
5
5
5
5, Y= O; Z= 4-BrPh
6, Y= O; Z= 4-CNPh
7, Y= S; Z= 4-BrPh
NO
NO
Y
2
2
Bz/R-X or
ArCOCH2Br
49,
Y= O; Z= 3-BrPh
0, Y= O; Z= 3-NO2Ph
1, Y= O; Z= 3-CF3Ph
2, Y= S; Z= 3-NO2Ph
3, Y= O; Z= 4-FPh
4, Y= O; Z= 4-ClPh
5
5
5
5
5
K2CO3
DMF
58,
Y= O; Z= 2,6-diFPh
9, Y= O; Z= 3,4-diClPh
0, Y= O; Z= phthalimide
YH
Z
5
6
6
4-nitro(thio)phenol
1, Y= O; Z= 3-NO2PhC=O
Y = O, S
Figure 1. Synthesis of compounds 1–61.
4.2.2. Synthesis of compound 13
poured into ice-cold water (100 ml) and extracted with dichlorome-
Lithium hydroxide (1.1 equiv.) dissolved in 15 ml of water was thane (DCM, 3 ꢃ 25 ml). The organics were reunited and added
added dropwise to a stirring solution of ethyl 2-((5-nitroquinolin- with anhydrous sodium sulphate to remove water. The salt was fil-
8-yl)oxy)acetate (12, 1.0 equiv.) in 30 ml of methanol. The reaction tered and washed three times with 5 ml of dry DCM. The organic
was stirred at room temperature for 24 h; then, the mixture was phase was evaporated in vacuo to afford the crude extract contain-
concentrated in vacuo to remove methanol and quenched with ing the target molecule that was recovered through column chro-
3 N HCl (15 ml). The precipitate was collected by filtration and matography, employing silica gel and proper mixtures of n-hexane/
ethyl acetate.
washed with n-hexane to give the title compound 13, without fur-
ther purification requirements.
4.3. Characterisation data for nitroxoline and
4-nitro(thio)phenol derivatives
4.2.3. General procedure for the synthesis of compounds 47–61
Freshly ground potassium carbonate (K2CO3, 1.1 equiv.) was added
to a stirring solution of 4-nitro(thio)phenol (1 equiv.) in DMF 8-ethoxy-5-nitroquinoline (1). Yellow solid, 66% yield, ClogP 2.71,
(10 ml). The yellow suspension was stirred for 30 min at room tem- mp ¼ 126–128 ꢀC. 1H NMR (400 MHz, CDCl3): d 1.68–1.71 (t,
perature; then, the proper (substituted)benzyl, diaryl and alkyl J ¼ 7.0 Hz, 3H, CH3), 4.44–4.49 (q, J ¼ 7.0 Hz, 2H, OCH2), 7.08–7.10
bromide or a-bromoacetophenone (1.5 equiv.) was added and the (d, J ¼ 8.9 Hz, 1H, ArH), 7.69–7.72 (m, 1H, ArH), 8.53–8.55 (d,
reaction stirred until disappearance of the starting reagents, as J ¼ 8.9 Hz, 1H, ArH), 9.07–9.08 (m, 1H, ArH), 9.24–9.27 (m, 1H, ArH);
detected by thin layer chromatography. Then, the mixture was 13 C NMR (100 MHz, CDCl3): d 14.4 (CH3), 65.6 (CH2), 106.0 (Ar),

1334
S. VESCHI ET AL.
Figure 2. Crystal structure of nitroxoline-based compound 16.
123.1 (Ar), 124.5 (Ar), 127.7 (Ar), 132.7 (Ar), 137.4 (Ar), 139.3 (Ar), Characterisation data are in agreement with those reported in
150.1 (Ar), 160.2 (Ar). Anal. calcd for C11H10N2O3: C, 60.55; H, 4.62; the literature18
.
8-butoxy-5-nitroquinoline (6). Brown solid, 78% yield, ClogP
N, 12.84, found: C, 60.51; H, 4.60; N, 12.82.
5-nitro-8-propoxyquinoline (2). Yellow solid, 72% yield, ClogP 3.77, mp ¼ 104–106 ꢀC. 1H NMR (300 MHz, CDCl3): d 1.00–1.05 (t,
3.24, mp ¼ 110–112 ꢀC. 1H NMR (300 MHz, CDCl3): d 1.08–1.13 (t, J ¼ 7.5 Hz, 3H, CH3), 1.52–1.65 (m, 2H, CH2), 2.00–2.10 (m, 2H, CH2),
J ¼ 7.6 Hz, 3H, CH3), 2.00–2.12 (m, 2H, CH2), 4.26–4.31 (t, J ¼ 7.2 Hz, 4.34–4.39 (t, J ¼ 7.2 Hz, 2H, OCH2), 7.07–7.10 (d, J ¼ 9.0 Hz, 1H,
2H, OCH2), 7.02–7.05 (d, J ¼ 9.0 Hz, 1H, ArH), 7.63–7.68 (m, 1H, ArH), 7.68–7.72 (m, 1H, ArH), 8.52–8.55 (d, J ¼ 9.0 Hz, 1H, ArH),
ArH), 8.47–8.50 (d, J ¼ 8.7 Hz, 1H, ArH), 9.00–9.03 (m, 1H, ArH), 9.06–9.08 (m, 1H, ArH), 9.25–9.28 (m, 1H, ArH); 13 C NMR (75 MHz,
9.18–9.21 (m, 1H, ArH); 13 C NMR (75 MHz, CDCl3): d 10.4 (CH3), CDCl3): d 13.8 (CH3), 19.2 (CH2), 30.6 (CH2), 70.0 (OCH2), 106.4 (Ar),
22.0 (CH2), 71.5 (OCH2), 123.1 (Ar), 124.5 (Ar), 127.7 (Ar), 132.7 (Ar), 123.2 (Ar), 124.5 (Ar), 128.0 (Ar), 133.5 (Ar), 137.2 (Ar), 149.7 (Ar),
106.1 (Ar), 137.3 (Ar), 139.1 (Ar), 150.0 (Ar), 160.3 (Ar). Anal. calcd 160.0 (Ar). Anal. calcd for C13H14N2O3: C, 63.40; H, 5.73; N, 11.38,
for C12H12N2O3: C, 62.06; H, 5.21; N, 12.06, found: C, 62.00; H, 5.19; found: C, 63.20; H, 5.63; N, 11.12.
(E)-8-(but-2-en-1-yloxy)-5-nitroquinoline (7). This compound exists in a
N, 12.00.
8-(allyloxy)-5-nitroquinoline (3). Brown solid, 81% yield, ClogP mixture of E/Z isomers with the following ratio (4.4/1) as extrapolated
1
2.96, mp ¼ 102–103 ꢀC. 1H NMR (300 MHz, CDCl3): d 4.97–4.99 (d,
by their OCH2 signal integration in the H NMR spectrum (peaks listed
J ¼ 1.2 Hz, 2H, OCH2), 5.39–5.54 (m, 2H, ¼CH2), 6.12–6.25 (m, 1H, only for the major isomer). Brown solid; 79% yield, ClogP 3.49, mp ¼
87–89 ꢀC; 1H NMR (300 MHz, CDCl3): d 1.70–1.72 (d, J¼ 6.6 Hz, 3H,
CH3), 4.79–4.81 (d, J¼ 6.4 Hz, 2H, OCH2), 5.76–5.96 (m, 2H, 2 x¼ CH),
6.99–7.02 (d, J¼ 9.0 Hz, 1H, ArH), 7.57–7.61 (m, 1H, ArH), 8.40–8.43 (d,
J¼ 9.0 Hz, 1H, ArH), 8.94–8.96 (m, 1H, ArH), 9.10–9.13 (m, 1H, ArH); 13 C
NMR (75 MHz, CDCl3): d 17.9 (CH3), 70.5 (OCH2), 106.5 (Ar), 123.0 (Ar),
124.1 (Ar), 124.4 (CH¼), 127.5 (Ar), 129.9 (CH¼), 132.5 (Ar), 137.3 (Ar),
139.3 (Ar), 150.1 (Ar), 159.9 (Ar). Anal. calcd for C13H12N2O3: C, 63.93; H,
¼CH), 7.07–7.10 (d, J ¼ 9.0 Hz, 1H, ArH), 7.68–7.72 (m, 1H, ArH),
8.49–8.52 (d, J ¼ 8.7 Hz, 1H, ArH), 9.05–9.07 (m, 1H, ArH), 9.22–9.26
(m, 1H, ArH); 13 C NMR (75 MHz, CDCl3): d 70.6 (CH2), 106.9 (Ar),
119.7 (CH2¼), 123.1 (Ar), 124.6 (Ar), 127.5 (Ar), 131.5 (Ar), 133.0
(CH¼), 150.0 (Ar), 159.6 (Ar). Anal. calcd for C12H10N2O3: C, 62.61;
H, 4.38; N, 12.17, found: C, 62.66; H, 4.40; N, 12.21.
5-nitro-8-(prop-2-yn-1-yloxy)quinoline (4). Brown solid, 68% yield,
CLogP 2.60, mp ¼ 157–158 ꢀC. 1H NMR (300 MHz, CDCl3): d 4.95; N, 11.47, found: C, 63.69; H, 4.80; N, 11.22.
8-((3-methylbut-2-en-1-yl)oxy)-5-nitroquinoline (8). Yellow solid,
2.62–2.63 (bs, 1H, CꢄCH), 5.14–5.15 (bs, 2H, OCH2), 7.31–7.34 (d,
J ¼ 8.7 Hz, 1H, ArH), 7.72–7.77 (m, 1H, ArH), 8.55–8.58 (d, J ¼ 8.7 Hz,
1H, ArH), 9.07–9.09 (bs, 1H, ArH), 9.26–9.29 (d, J ¼ 9.3 Hz, 1H, ArH);
13 C NMR (75 MHz, CDCl3): d 57.2 (OCH2), 76.6 (HCꢄ, overlapping
with the solvent signals), 77.8 (Cꢄ), 107.9 (Ar), 123.1 (Ar), 124.7
(Ar), 127.5 (Ar), 134.0 (Ar), 138.2 (Ar), 149.7 (Ar), 157.7 (Ar). Anal.
calcd for C12H8N2O3: C, 63.16; H, 3.53; N, 12.28, found: C, 63.06; H,
3.50; N, 12.20.
82% yield, ClogP 3.88, mp ¼ 98–99 ꢀC. 1H NMR (300 MHz, CDCl3):
d 1.81 (s, 6H, 2 x CH3), 4.94–4.96 (d, J ¼ 6.3 Hz, 2H, OCH2),
5.60–5.64 (m, 1H, ¼CH), 7.05–7.08 (d, J ¼ 8.7 Hz, 1H, ArH),
7.67–7.13 (m, 1H, ArH), 8.51–8.54 (d, J ¼ 8.7 Hz, 1H, ArH), 9.05–9.07
(m, 1H, ArH), 9.24–9.27 (m, 1H, ArH); 13 C NMR (75 MHz, CDCl3): d
18.4 (CH3), 25.9 (CH3), 66.9 (OCH2), 106.6 (Ar), 118.3 (CH¼), 123.1
(Ar), 124.5 (Ar), 127.8 (Ar), 133.1 (Ar), 139.5 (C), 149.9 (Ar), 160.0
2-((5-nitroquinolin-8-yl)oxy)acetonitrile (5). Orange solid, 65% (Ar). Anal. calcd for C14H14N2O3: C, 65.11; H, 5.46; N, 10.85, found:
yield, ClogP 1.22, mp ¼ 198–200 ꢀC [Lit. mp ¼ 194–197 ꢀC]. C, 65.33; H, 5.60; N, 11.00.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1335
5-nitro-8-(pentyloxy)quinoline (9). Brown sticky oil, 83% yield, 133.0 (Ar), 136.4 (Ar), 137.8 (Ar), 139.4 (Ar), 150.2 (Ar), 159.8 (Ar).
ClogP 4.30.1H NMR (300 MHz, CDCl3): d 0.90–0-94 (t, J ¼ 6.9 Hz, 3H, Anal. calcd for C17H14N2O3: C, 69.38; H, 4.79; N, 9.52, found: C,
CH3), 1.39–1.53 (m, 4H, 2 ꢃ CH2), 2.01–2.06 (m, 2H, CH2), 4.30–4.34 69.01; H, 4.59; N, 9.27.
5-nitro-8-((2-(trifluoromethyl)benzyl)oxy)quinoline (16). Yellow
(t, J ¼ 7.2 Hz, 2H, OCH2), 7.02–7.05 (d, J ¼ 9.6 Hz, 1H, ArH),
1
solid, 82% yield, ClogP 4.83, mp ¼ 145–147 ꢀC. H NMR (300 MHz,
7.63–7.67 (m, 1H, ArH), 8.47–8.50 (d, J ¼ 8.7 Hz, 1H, ArH), 9.01–9.02
(bd, J ¼ 2.7 Hz, 1H, ArH), 9.18–9.21 (d, J ¼ 8.7 Hz, 1H, ArH); 13 C CDCl3): d 5.66 (s, 2H, OCH2), 6.93–6.96 (m, 1H, ArH), 7.36–7.41 (t,
J ¼ 7.5 Hz, 1H, ArH), 7.47–7.52 (t, J ¼ 7.5 Hz, 1H, ArH), 7.63–7.69 (m,
NMR (75 MHz, CDCl3): d 14.0 (CH3), 22.4 (CH2), 28.0 (CH2), 28.4
2H, ArH), 7.75–7.77 (d, J ¼ 7.5 Hz, 1H, ArH), 8.35–8.38 (m, 1H, ArH),
(CH2), 70.1 (OCH2), 106.1 (Ar), 123.1 (Ar), 124.5 (Ar), 127.7 (Ar),
132.7 (Ar), 137.2 (Ar), 139.2 (Ar), 150.1 (Ar), 160.4 (Ar). Anal. calcd 9.02–9.04 (m, 1H, ArH), 9.12–9.16 (m, 1H, ArH); 13 C NMR (100 MHz,
CDCl3): d 67.3 (OCH2), 107.2 (Ar), 123.0 (d, JC-F ¼ 2.2 Hz, Ar), 124.3
for C14H16N2O3: C, 64.60; H, 6.20; N, 10.76, found: C, 64.39; H, 6.09;
N, 10.52.
1
(d, JC-F ¼ 280.3 Hz, CF3), 124.6 (Ar), 126.0 (Ar), 126.1 (Ar), 127.3
3
(E)-8-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-5-nitroquinoline
(10). (Ar), 127.9 (Ar), 128.2 (Ar), 132.6 (d, JC-F ¼ 11.5 Hz, Ar), 132.8 (Ar),
Brown sticky oil, 91% yield, ClogP 5.92. 1H NMR (300 MHz, CDCl3): 133.9 (Ar), 138.0 (Ar), 139.3 (Ar), 150.3 (Ar), 159.0 (Ar); 19 F NMR
d 1.54 (s, 3H, CH3), 1.60 (s, 3H, CH3), 1.78 (s, 3H, CH3), 2.07 (bs, 4H, (564.7 MHz CDCl3): d ꢂ 58.93 (s, 3 F, ArCF3). Anal. calcd for
2 ꢃ CH2), 4.94–5.02 (m, 3H, OCH2 þ ¼CH), 5.55–5.59 (bs, 1H, ¼CH), C17H11F3N2O3: C, 58.63; H, 3.18; N, 8.04, found: C, 58.40; H, 3.02;
7.00–7.03 (d, J ¼ 8.7 Hz, 1H, ArH), 7.61–7.66 (m, 1H, ArH), 8.45–8.48 N, 7.90.
2-(((5-nitroquinolin-8-yl)oxy)methyl)benzonitrile (17). Yellowish
(d, J ¼ 8.7 Hz, 1H, ArH), 8.99–9.00 (d, J ¼ 3.6 Hz, 1H, ArH), 9.17–9.20
1
(d, J ¼ 8.7 Hz, 1H, ArH); 13 C NMR (75 MHz, CDCl3): d 16.9 (CH3),
solid, 82% yield, ClogP 3.52, mp ¼ 201–203 ꢀC. H NMR (300 MHz,
17.7 (CH3), 25.6 (CH3), 26.1 (CH2), 39.5 (CH2), 67.0 (OCH2), 106.5 CDCl3): d 5.68 (s, 2H, OCH2), 7.09–7.13 (m, 1H, ArH), 7.44–7.49 (t,
J ¼ 7.5 Hz, 1H, ArH), 7.61–7.81 (m, 4H, 4 ꢃ ArH), 8.44–8.48 (m, 1H,
(Ar), 118.2 (Ar), 123.1 (CH¼), 123.5 (Ar), 124.4 (Ar), 127.6 (Ar), 131.9
ArH), 9.05–9.07 (m, 1H, ArH), 9.18–9.22 (m, 1H, ArH); 13 C NMR
(CH¼), 132.7 (C), 137.2 (Ar), 139.3 (C), 142.3 (Ar), 150.0 (Ar), 160.1
(Ar). Anal. calcd for C19H22N2O3: C, 69.92; H, 6.79; N, 8.58, found: C, (75 MHz, CDCl3): d 68.8 (OCH2), 107.2 (Ar), 111.1 (Ar), 116.9 (CꢄN),
69.45; H, 6.58; N, 8.22.
123.1 (Ar), 124.7 (Ar), 127.2 (Ar), 128.5 (Ar), 129.0 (Ar), 132.7 (Ar),
133.0 (Ar), 133.5 (Ar), 138.4 (Ar), 138.8 (Ar), 139.3 (Ar), 150.4 (Ar),
5-nitro-8-(((3,7,11-trimethyldodeca-2,6,10-trien-1-yl)oxy)quinoline
(11). The compound exists as a mixture of E/Z isomers (peaks 158.9 (Ar). Anal. calcd for C17H11N3O3: C, 66.88; H, 3.63; N, 13.76,
listed only for the major isomer). Dark brown sticky oil, 77% yield, found: C, 66.40; H, 3.50; N, 13.61.
ClogP 7.95. 1H NMR (300 MHz, CDCl3): d 1.50 (s, 3H, CH3), 1.52 (s,
3H, CH3), 1.59 (s, 3H, CH3), 1.77 (s, 3H, CH3), 1.87–2.07 (m, 8H,
4 ꢃ CH2), 4.91–5.02 (m, 4H, OCH2 þ 2 x ¼ CH), 5.56 (s, 1H, CH¼),
5-nitro-8-((2-nitrobenzyl)oxy)quinoline (18). Yellow solid, 82%
yield, ClogP 3.61, mp ¼ 208–210 ꢀC. 1H NMR (300 MHz, CDCl3): d
5.94 (s, 2H, OCH2), 7.07–7.10 (m, 1H, ArH), 7.55–7.57 (m, 1H, ArH),
6.97–7.00 (d, J ¼ 8.7 Hz, 1H, ArH), 7.57–7.60 (bs, 1H, ArH), 8.41–8.44 7.72–7.77 (m, 2H, 2 ꢃ ArH), 8.02–8.04 (d, J ¼ 7.5 Hz, 1H, ArH), 8.25
8.28 (d, J ¼ 8.1 Hz, 1H, ArH), 8.46–8.49 (m, 1H, ArH), 9.10 (s, 1H,
ArH), 9.23–9.26 (d, J ¼ 9.0 Hz, 2H, 2 ꢃ ArH); 13 C NMR (75 MHz,
(d, J ¼ 8.7 Hz, 1H, ArH), 8.95 (s, 1H, ArH), 9.12–9.15 (d, J ¼ 8.7 Hz,
1H, ArH); 13 C NMR (75 MHz, CDCl3): d 16.0 (CH3), 16.8 (CH3), 17.6
(CH3), 25.4 (CH3), 26.0 (CH2), 26.6 (CH2), 39.2 (CH2), 39.6 (CH2), 66.7 CDCl3): d 68.3 (OCH2), 107.2 (Ar), 124.7 (Ar), 125.4 (Ar), 128.4 (Ar),
128.9 (Ar), 132.8 (Ar), 134.6 (Ar), 127.2 (Ar), 150.5 (Ar). Anal. calcd
for C16H11N3O5: C, 59.08; H, 3.41; N, 12.92, found: C, 59.29; H, 3.57;
139.4 (C), 142.3 (Ar), 150.0 (Ar), 160.1 (Ar). Anal. calcd for N, 13.20.
(OCH2), 106.3 (Ar), 118.0 (CH¼), 123.0 (Ar), 123.4 (CH¼), 124.0 (Ar),
124.3 (CH¼), 127.4 (C), 131.2 (Ar), 132.4 (C), 135.5 (C), 137.2 (Ar),
8-((2-bromobenzyl)oxy)-5-nitroquinoline (19). Yellow solid, 74%
yield, ClogP 4.81, mp ¼ 144–146 ꢀC. 1H NMR (300 MHz, CDCl3): d
5.61 (s, 2H, OCH2), 7.00–7.03 (d, J ¼ 8.7 Hz, 1H, ArH), 7.20–7.31 (m,
C24H30N2O3: C, 73.07; H, 7.67; N, 7.10, found: C, 72.80; H, 7.49;
N, 6.88.
ethyl 2-((5-nitroquinolin-8-yl)oxy)acetate (12). Ochre solid, 88%
yield, ClogP 2.33, mp ¼ 178–179 ꢀC [Lit. mp ¼ 174–175 ꢀC]. 2H, 2 ꢃ ArH), 7.57–7.64 (m, 2H, 2 ꢃ ArH), 7.71–7.75 (m, H, ArH),
8.45–8.48 (d, J ¼ 8.7 Hz, 1H, ArH), 9.10–9.11 (bs, 1H, ArH), 9.23–9.26
Characterisation data are in agreement with those reported in
(d, J ¼ 8.7 Hz, 1H, ArH); 13 C NMR (75 MHz, CDCl3): d 70.7 (OCH2),
the literature18
.
2-((5-nitroquinolin-8-yl)oxy)acetic acid (13). Yellow solid; 81% 107.3 (Ar), 122.0 (Ar), 123.1 (Ar), 124.7 (Ar), 127.4 (Ar), 127.9 (Ar),
yield, ClogP 1.47, mp ¼ 205–207 ꢀC [Lit. mp ¼ 205–210 ꢀC]. 128.6 (Ar), 129.8 (Ar), 132.7 (Ar), 132.9 (Ar), 134.5 (Ar), 138.0 (Ar),
Characterisation data are in agreement with those reported in 139.5 (Ar), 150.4 (Ar), 159.3 (Ar). Anal. calcd for C16H11BrN2O3: C,
the literature18
8-(benzyloxy)-5-nitroquinoline (14). Dark red sticky solid, 82%
.
53.50; H, 3.09; N, 7.80, found: C, 53.12; H, 2.91; N, 7.55.
8-((3-methylbenzyl)oxy)-5-nitroquinoline (20). Yellow solid, 78%
yield, CloP 3.95. 1H NMR (300 MHz, CDCl3): d 5.48 (bs, 2H, OCH2), yield, ClogP 4.45, mp ¼ 94–96 ꢀC. 1H NMR (300 MHz, CDCl3): d
6.97–7.01 (m, 1H, ArH), 7.26–7.37 (m, 3H, 3 ꢃ ArH), 7.45–7.48 (m, 2.35 (s, 3H, CH3), 5.51 (s, 2H, OCH2), 7.04–7.07 (d, J ¼ 8.7 Hz, 1H,
2H, 2 ꢃ ArH), 7.60–7.64 (m, 1H, ArH), 8.32–8.37 (m, 1H, ArH), ArH), 7.14–7.15 (bs, 1H, ArH), 7.26–7.31 (ms, 3H, 3 ꢃ ArH),
8.99–9.01 (bs, 1H, ArH), 9.09–9.15 (m, 1H, ArH); 13 C NMR (75 MHz, 7.68–7.73 (m, 1H, ArH), 8.42–8.45 (m, 1H, ArH), 9.07–9.08 (bs, 1H,
CDCl3): d 71.4 (OCH2), 107.2 (Ar), 123.0 (Ar), 124.5 (Ar), 127.1 (Ar), ArH), 9.22–9.24 (m, 1H, ArH); 13 C NMR (75 MHz, CDCl3): d 21.4
127.3 (Ar), 128.4 (Ar), 128.9 (Ar), 132.4 (Ar), 135.3 (Ar), 137.6 (Ar), (CH3), 71.6 (OCH2), 107.4 (Ar), 123.1 (Ar), 124.2 (Ar), 124.6 (Ar),
139.5 (Ar), 150.2 (Ar), 159.7 (Ar). Anal. calcd for C16H12N2O3: C, 127.5 (Ar), 127.8 (Ar), 128.8 (Ar), 129.3 (Ar), 132.9 (Ar), 135.2 (Ar),
68.56; H, 4.32; N, 9.99, found: C, 68.82; H, 4.41; N, 10.16.
138.7 (Ar), 150.1 (Ar). Anal. calcd for C17H14N2O3: C, 69.38; H, 4.79;
N, 9.52, found: C, 69.00; H, 4.52; N, 9.29.
8-((2-methylbenzyl)oxy)-5-nitroquinoline (15). Yellow solid, 93%
yield, ClogP 4.40, mp ¼ 126–128 ꢀC. 1H NMR (300 MHz, CDCl3): d
8-((3-methoxybenzyl)oxy)-5-nitroquinoline (21). White solid, 85%
2.45 (s, 3H, CH3), 5.50 (s, 2H, OCH2), 7.06–7.10 (d, J ¼ 9.0 Hz, 1H, yield, ClogP 3.87, mp ¼ 100–101 ꢀC. 1H NMR (400 MHz, CDCl3): d
ArH), 7.19–7.26 (m, 3H, ArH), 7.44–7.46 (d, J ¼ 6.9 Hz, 1H, ArH), 3.80 (bs, 3H, OCH3), 5.55 (s, 2H, OCH2), 6.88–6.90 (d, J ¼ 8.0 Hz, 1H,
7.68–7.72 (m, 1H, ArH), 8.46–8.49 (d, J ¼ 8.7 Hz, 1H, ArH), 9.05–9.06 ArH), 7.07–7.10 (m, 3H, 3 x ArH), 7.28–7.34 (m, 1H, ArH), 8.43–8.45
(bs, 1H, ArH), 9.22–9.25 (d, J ¼ 8.7 Hz, 1H ArH); 13 C NMR (75 MHz, (m, 1H, ArH), 9.09 (bs, 1H, ArH), 9.22–9.24 (m, 1H, ArH); 13 C NMR
CDCl3): d 19.1 (CH3), 70.2 (OCH2), 107.2 (Ar), 123.2 (Ar), 124.6 (Ar), (100 MHz, CDCl3): d 55.3 (OCH3), 71.4 (OCH2), 107.4 (Ar), 112.7 (Ar),
126.2 (Ar), 127.4 (Ar), 128.2 (Ar), 128.6 (Ar), 130.7 (Ar), 132.8 (Ar), 113.8 (Ar), 119.2 (Ar), 123.1 (Ar), 124.6 (Ar), 127.3 (Ar), 130.0 (Ar),

1336
S. VESCHI ET AL.
132.6 (Ar), 137.0 (Ar), 137.8 (Ar), 139.7 (Ar), 150.3 (Ar), 159.8 (Ar), (75 MHz, CDCl3): d 21.1 (CH3), 71.3 (OCH2), 107.2 (Ar), 122.8 (Ar),
160.1 (Ar). Anal. calcd for C17H14N2O4: C, 65.80; H, 4.55; N, 9.03,
found: C, 65.99; H, 4.60; N, 9.19.
124.4 (Ar), 127.3 (Ar), 129.4 (Ar), 132.2 (Ar), 137.3 (Ar), 138.1 (Ar),
139.3 (Ar), 150.0 (Ar), 159.7 (Ar). Anal. calcd for C17H14N2O3: C,
69.38; H, 4.79; N, 9.52, found: C, 69.59; H, 4.87; N, 9.69.
8-((3-fluorobenzyl)oxy)-5-nitroquinoline (22). Yellow sticky oil,
65% yield, ClogP 4.09. 1H NMR (400 MHz, CDCl3): d 5.55 (s, 2H,
OCH2), 7.03–7.08 (m, 2H, 2 ꢃ ArH), 7.24–7.31 (m, 2H, 2 ꢃ ArH),
7.36–7.40 (m, 1H, ArH), 7.71–7.75 (m, 1H, ArH), 8.45–8.47 (d,
J ¼ 8.8 Hz, 1H, ArH), 9.09–9.11 (m, 1H, ArH), 9.23–9.25 (m, 1H, ArH);
8-((4-(methylthio)benzyl)oxy)-5-nitroquinoline (28). Brown solid,
71% yield, ClogP 4.51, mp ¼ 123–128 ꢀC. 1H NMR (300 MHz,
CDCl3): d 2.43 (s, 3H, SCH3), 5.46 (s, 2H, OCH2), 7.02–7.05 (d,
J ¼ 9.0 Hz, 1H, ArH), 7.20–7.23 (d, J ¼ 8.4 Hz, 2H, 2 ꢃ ArH), 7.38–7.41
(d, J ¼ 8.1 Hz, 2H, 2 ꢃ ArH), 7.66–7.70 (m, 1H, ArH), 8.39–8.42 (d,
J ¼ 8.7 Hz, 1H, ArH), 9.03 (s, 1H, ArH), 9.18–9.21 (d, J ¼ 8.1 Hz, 1H,
ArH); 13 C NMR (75 MHz, CDCl3): d 22.0 (SCH3), 77.6 (OCH2), 114.0
(Ar), 129.6 (Ar), 131.0 (Ar), 133.0 (Ar), 134.1 (Ar), 134.5 (Ar), 138.2
(Ar), 139.0 (Ar), 140.2 (Ar), 144.1 (Ar), 145.8 (Ar), 155.8 (Ar), 156.3
(Ar), 157.8 (Ar), 166.0 (Ar). Anal. calcd for C17H14N2O3S: C, 62.56; H,
4.32; N, 8.58, found: C, 62.82; H, 4.41; N, 8.69.
2
13 C NMR (100 MHz, CDCl3): d 70.7 (OCH2), 107.2 (Ar), 114.1 (d, JC-F
2
4
¼ 22.3 Hz, Ar), 115.5 (d, JC-F ¼ 21.2 Hz, Ar), 122.6 (d, JC-F
¼
3
3.1 Hz, Ar), 123.1 (Ar), 124.7 (Ar), 127.2 (Ar), 130.6 (d, JC-F ¼ 8.3 Hz,
Ar), 132.6 (Ar), 137.9 (Ar), 139.6 (Ar), 146.0 (Ar), 150.4 (Ar), 159.5
(Ar), 164.1 (Ar); 19 F NMR (564.7 MHz, CDCl3): d ꢂ 110.63 (td, F-H
J ¼ 9.0 Hz, 6.0 Hz, 1 F, ArF). Anal. calcd for C16H11FN2O3: C, 64.43; H,
3.72; N, 9.39, found: C, 64.69; H, 3.79; N, 9.48.
8-((3-chlorobenzyl)oxy)-5-nitroquinoline (23). Yellow solid, 82%
yield, ClogP 4.66, mp ¼ 126–128 ꢀC. 1H NMR (300 MHz, CDCl3): d
5.50 (s, 2H, OCH2), 7.01–7.04 (d, J ¼ 8.7 Hz, 1H, ArH), 7.31–7.33 (m,
2H, 2 ꢃ ArH), 7.36–7.39 (m, 1H, ArH), 7.50 (s, 1H, ArH), 7.69–7.73
(m, 1H, ArH), 8.42–8.45 (d, J ¼ 8.7 Hz, 1H, ArH), 9.06–9.08 (m, 1H,
ArH), 9.20–9.23 (m, 1H, ArH); 13 C NMR (75 MHz, CDCl3): d 70.6
(OCH2), 107.2 (Ar), 123.1 (Ar), 124.7 (Ar), 125.2 (Ar), 127.2 (Ar),
128.7 (Ar), 130.3 (Ar), 132.7 (Ar), 134.9 (Ar), 137.3 (Ar), 139.5 (Ar),
150.4 (Ar), 159.4 (Ar). Anal. calcd for C16H11ClN2O3: C, 61.06; H,
3.52; N, 8.90, found: C, 61.48; H, 3.59; N, 8.99.
8-((3-bromobenzyl)oxy)-5-nitroquinoline (24). Beige solid, 87%
yield, ClogP 4.81, mp ¼ 141–143 ꢀC. 1H NMR (300 MHz, CDCl3): d
5.50 (s, 2H, OCH2), 7.04 (d, J ¼ 8.7 Hz, 1H, ArH), 7.24–7.29 (m, 1H,
ArH), 7.43–7.49 (t, J ¼ 9.3 Hz, 2H, 2 ꢃ ArH), 7.66 (s, 1H, ArH),
7.70–7.74 (m, 1H, ArH), 8.44–8.47 (m, 1H, ArH), 9.08–9.09 (d,
J ¼ 3.0 Hz, 1H, ArH), 9.22–9.25 (d, J ¼ 8.7 Hz, 1H, ArH); 13 C NMR
(75 MHz, CDCl3): d 70.6 (OCH2), 107.2 (Ar), 123.0 (Ar), 123.1 (Ar),
124.7 (Ar), 125.7 (Ar), 127.2 (Ar), 130.1 (Ar), 130.5 (Ar), 131.6 (Ar),
132.7 (Ar), 137.6 (Ar), 138.0 (Ar), 139.5 (Ar), 150.4 (Ar), 151.3 (Ar),
159.4 (Ar). Anal. calcd for C16H11BrN2O3: C, 53.50; H, 3.09; N, 7.80,
found: C, 53.19; H, 3.00; N, 7.67.
5-nitro-8-((3-nitrobenzyl)oxy)quinoline (25). Yellow sticky oil, 78%
yield, ClogP 3.69. 1H NMR (300 MHz, CDCl3): d 5.60 (s, 2H, OCH2),
7.06–7.09 (d, J ¼ 8.7 Hz, 1H, ArH), 7.59–7.64 (t, J ¼ 8.1 Hz, 1H, ArH),
7.72–7.76 (m, 1H, ArH), 7.88–7.91 (d, J ¼ 7.5 Hz, 1H, ArH), 8.21–8.25
(m, 1H, ArH), 8.41 (s, 1H, ArH), 8.45–4.48 (d, J ¼ 8.7 Hz, 1H, ArH),
9.08–9.10 (m, 1H, ArH), 9.21–9.25 (m, 1H, ArH); 13 C NMR (75 MHz,
CDCl3): d 70.2 (OCH2), 107.2 (Ar), 122.3 (Ar), 123.6 (Ar), 124.8 (Ar),
127.1 (Ar), 130.1 (Ar), 132.8 (Ar), 133.2 (Ar), 137.4 (Ar), 150.5 (Ar).
Anal. calcd for C16H11N3O5: C, 59.08; H, 3.41; N, 12.92, found: C,
59.49; H, 3.61; N, 13.07.
8-((4-fluorobenzyl)oxy)-5-nitroquinoline (29). Yellow solid, 69% yield,
ClogP 4.09, mp ¼ 177–179 ꢀC. 1H NMR (300 MHz, CDCl3): d 5.48 (s,
2H, OCH2), 7.04–7.11 (m, 3H, ArH), 7.47–7.52 (t, J ¼ 6.9 Hz, 2H, ArH),
7.68–7.72 (m, 1H, ArH), 8.43–8.46 (d, J ¼ 8.7 Hz, 1H, ArH), 9.05–9.06 (s,
J ¼ 2.1 Hz, 1H, ArH), 9.20–9.22 (d, J¼ 9.0 Hz, 1H, ArH); 13 C NMR
2
(75 MHz, CDCl3): d 70.9 (OCH2), 107.5 (Ar), 115.9 (d, JC-F ¼ 21.75 Hz,
3
2ꢃ Ar), 123.1 (Ar), 124.7 (Ar), 127.5 (Ar), 129.3 (d, JC-F ¼ 7.95 Hz, 2 x
Ar), 130.9 (Ar), 133.4 (Ar), 137.8 (Ar), 150.0 (Ar), 159.2 (Ar), 161.1 (Ar-
F), 164.4 (Ar); 19 F NMR (564.7 MHz, CDCl3): d ꢂ111.83 (m, 1F, ArF).
Anal. calcd for C16H11FN2O3: C, 64.43; H, 3.72; N, 9.39, found: C,
64.02; H, 3.62; N, 9.25.
8-((4-chlorobenzyl)oxy)-5-nitroquinoline (30). Yellow solid, 81%
yield, ClogP 4.66, mp ¼ 152–153 ꢀC. 1H NMR (300 MHz, DMSO-
d6): d 5.46 (s, 2H, OCH2), 7.42–7.60 (m, 5H, 5 ꢃ ArH), 7.81–7.86
(m, 1H, ArH), 8.54–8.57 (d, J ¼ 8.7 Hz, 1H, ArH), 9.00–9.03 (m, 2H,
2 ꢃ ArH); 13 C NMR (75 MHz, DMSO-d6): d 70.3 (OCH2), 108.3 (Ar),
125.3 (Ar), 128.1 (Ar), 129.1 (Ar), 130.3 (Ar), 132.2 (Ar), 135.5 (Ar),
150.8 (Ar). Anal. calcd for C16H11ClN2O3: C, 61.06; H, 3.52; N,
8.90, found: C, 61.38; H, 3.65; N, 9.12.
8-((4-bromobenzyl)oxy)-5-nitroquinoline (31). Beige solid, 85% yield,
ClogP 4.81, mp ¼ 165–167 ꢀC. 1H NMR (300 MHz, CDCl3): d 5.48 (s,
2H, OCH2), 7.00–7.04 (m, 1H, ArH), 7.37–7.40 (m, 2H, 2ꢃ ArH),
7.51–7.54 (m, 2H, 2ꢃ ArH), 7.69–7.74 (m, 1H, ArH), 8.42–8.46 (m, 1H,
ArH), 9.07–9.08 (m, 1H, ArH), 9.21–9.25 (m, 1H, ArH); 13 C NMR
(75 MHz, CDCl3): d 70.7 (OCH2), 107.2 (Ar), 122.4 (Ar), 123.1 (Ar), 124.6
(Ar), 127.2 (Ar), 128.9 (Ar), 132.1 (Ar), 132.6 (Ar), 134.3 (Ar), 137.9 (Ar),
139.5 (Ar), 150.3 (Ar), 159.4 (Ar). Anal. calcd for C16H11BrN2O3: C,
53.50; H, 3.09; N, 7.80, found: C, 53.19; H, 3.00; N, 7.69.
8-((4-iodobenzyl)oxy)-5-nitroquinoline (32). Brown solid, 78% yield,
1
ClogP 5.07, mp ¼ 195–197ꢀC. H NMR (400 MHz, DMSO-d6): d 5.44
(s, 2H, OCH2), 7.37–7.44 (m, 3H, ArH), 7.81–7.86 (m, 3H, ArH),
8.55–8.57 (d, J ¼ 8.8 Hz, 1H, ArH), 9.02–9.04 (d, J ¼ 8.8 Hz, 2H, ArH);
13 C NMR (100 MHz, DMSO-d6): d 70.5 (OCH2), 108.2 (Ar), 122.6 (Ar),
125.3 (Ar), 128.1 (Ar), 129.2 (Ar), 130.6 (Ar), 132.2 (Ar), 136.2 (Ar),
137.2 (Ar), 137.7 (Ar), 137.8 (Ar), 139.2 (Ar), 150.8 (Ar), 159.8 (Ar).
Anal. calcd for C16H11IN2O3: C, 47.31; H, 2.73; N, 6.90, found: C,
47.58; H, 2.80; N, 6.99.
5-nitro-8-((3-(trifluoromethyl)benzyl)oxy)quinoline (26). Pink solid,
76% yield, ClogP 4.83, mp ¼ 106–109 ꢀC. 1H NMR (300 MHz,
CDCl3): d 5.56 (s, 2H, OCH2), 7.04–7.07 (d, J ¼ 9.3 Hz, 1H, ArH),
7.51–7.56 (m, 1H, ArH), 7.60–7.62 (m, 1H, ArH), 7.70–7.78 (m, 3H,
3 ꢃ ArH), 8.44–8.47 (d, J ¼ 9.0 Hz, 1H, ArH), 9.08–9.09 (m, 1H, ArH),
9.22–9.25 (m, 1H, ArH); 13 C NMR (75 MHz, CDCl3): d 70.8 (OCH2),
4
107.3 (Ar), 123.15 (Ar), 124.0 (d, JC-F ¼ 3.4 Hz, Ar), 124.7 (Ar),
4-(((5-nitroquinolin-8-yl)oxy)methyl)benzonitrile (33). Brown solid,
94% yield, ClogP 3.38, mp ¼ 208–212 ꢀC. 1H NMR (300 MHz,
DMSO-d6): d 5.57 (s, 2H, OCH2), 7.39–7.43 (m, 1H, ArH), 7.73–7.76
(d, J ¼ 8.4 Hz, 2H, 2 ꢃ ArH), 7.81–7.86 (m, 1H, ArH), 7.90–7.93 (m,
2H, 2 ꢃ ArH), 8.52–8.55 (m, 1H, ArH), 8.99–9.03 (m, 2H, 2 ꢃ ArH);
13 C NMR (75 MHz, DMSO-d6): d 70.1 (OCH2), 108.3 (Ar), 111.3 (Ar),
119.2 (CN), 122.6 (Ar), 125.3 (Ar), 128.0 (Ar), 128.7 (Ar), 132.2 (Ar),
133.0 (Ar), 138.0 (Ar), 139.2 (Ar), 142.2 (Ar), 150.9 (Ar), 159.6 (Ar).
Anal. calcd for C17H11N3O3: C, 66.88; H, 3.63; N, 13.76, found: C,
125.4 (Ar), 125.5 (Ar), 127.2 (Ar), 129.5 (Ar), 130.6 (Ar), 132.9 (Ar),
136.3 (Ar), 150.3 (Ar), 159.2 (Ar); 19 F NMR (564.7 MHz CDCl3):
d ꢂ 60.92 (s, 3 F, ArCF3). Anal. calcd for C17H11F3N2O3: C, 58.63; H,
3.18; N, 8.04, found: C, 58.96; H, 3.26; N, 8.15.
8-((4-methylbenzyl)oxy)-5-nitroquinoline (27). Yellow solid, 66%
yield, ClogP 4.45, mp ¼ 95–97 ꢀC. 1H NMR (300 MHz, CDCl3): d
2.20 (s, 3H, CH3), 5.36 (s, 2H, OCH2), 6.91–6.94 (d, J ¼ 8.7 Hz, 1H,
ArH), 7.05–7.07 (d, J ¼ 7.5 Hz, 2H, 2 ꢃ ArH), 7.27–7.30 (d, J ¼ 7.8 Hz,
2H, 2 ꢃ ArH), 7.49–7.52 (m, 1H, ArH), 8.23–8.26 (d, J ¼ 9.0 Hz, 1H,
ArH), 8.88 (s, 1H, ArH), 8.99–9.02 (d, J ¼ 9.0 Hz, 1H, ArH); 13 C NMR 67.00; H, 3.70; N, 13.82.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1337
5-nitro-8-((4-(trifluoromethyl)benzyl)oxy)quinoline (34). Brown C17H13ClN2O4: C, 59.23; H, 3.80; N, 8.13, found: C, 59.39; H, 3.86;
1
solid, 81% yield, ClogP 4.83, mp ¼ 128–130 ꢀC. H NMR (300 MHz, N, 8.20.
CDCl3): d 5.57 (s, 2H, CH2), 6.99–7.02 (d, J ¼ 8.7 Hz, 1H, ArH),
7.64–7.71 (m, 5H, 5 ꢃ ArH), 8.40–8.43 (d, J ¼ 9.0 Hz, 1H, ArH),
8-((3,4-dichlorobenzyl)oxy)-5-nitroquinoline (40). Red solid, 78%
yield, ClogP 5.26, mp ¼ 122–132 ꢀC. 1H NMR (300 MHz, CDCl3): d
9.05–9.06 (m, 1H, ArH), 9.17–9.20 (m, 1H, ArH); 13 C NMR (75 MHz, 5.44 (s, 2H, OCH2), 6.99–7.02 (d, J ¼ 9.0 Hz, 1H, ArH), 7.32–7.34 (d,
1
CDCl3): d 70.6 (OCH2), 107.3 (Ar), 123.1 (Ar), 123.9 (d, JC-F
¼
J ¼ 8.4 Hz, 1H, ArH), 7.43–7.46 (d, J ¼ 8.1 Hz, 1H, ArH), 7.59 (s, 1H,
3
270.0 Hz, CF3), 124.7 (Ar), 125.9 (q, JC-F ¼ 4.01 Hz, 2 ꢃ Ar), 126.6 ArH), 7.67–7.71 (m, 1H, ArH), 8.40–8.43 (d, J ¼ 9.0 Hz, 1H, ArH),
(Ar), 127.2 (d, JC-F ¼ 4.6 Hz, 2 x Ar), 130.6 (q, JC-F ¼ 31.2 Hz, Ar), 9.03–9.04 (m, 1H, ArH), 9.16–9.19 (d, J ¼ 8.7 Hz, 1H; ArH); 13 C NMR
132.9 (Ar), 138.1 (Ar), 139.2 (Ar), 139.3 (Ar), 150.3 (Ar), 159.1 (Ar). (75 MHz, CDCl3): d 70.0 (OCH2), 107.1 (Ar), 123.1 (Ar), 124.7 (Ar),
19 F NMR (564.7 MHz, CDCl3): d ꢂ 61.32 (s, 3 F, ArCF3). Anal. calcd 126.5 (Ar), 127.2 (Ar), 129.1 (Ar), 130.9 (Ar), 132.6 (Ar), 133.1 (Ar),
for C17H11F3N2O3: C, 58.63; H, 3.18; N, 8.04, found: C, 58.49; H, 135.5 (Ar), 138.1 (Ar), 139.4 (Ar), 150.5 (Ar), 159.1 (Ar). Anal. calcd
4
2
3.15; N, 7.96.
for C16H10Cl2N2O3: C, 55.04; H, 2.89; N, 8.02, found: C, 55.29; H,
5-nitro-8-((4-nitrobenzyl)oxy)quinoline (35). Yellow sticky oil, 98% 2.98; N, 8.11.
yield, ClogP 3.69. 1H NMR (300 MHz, CDCl3): d 5.52 (s, 2H, OCH2),
8-(naphthalen-1-ylmethoxy)-5-nitroquinoline (41). Red solid,
7.35–7.38 (m, 5H, 5 ꢃ ArH), 8.05–8.08 (m, 4H, 4 ꢃ ArH); 13 C NMR 81% yield, ClogP 5.12, mp ¼ 177.0–178.0 ꢀC. 1H NMR (300 MHz,
(75 MHz, CDCl3): d 70.1 (OCH2), 108.4 (Ar), 111.4 (Ar), 122.5 (Ar), CDCl3): d 5.87 (s, 2H, OCH2), 7.52–7.57 (m, 3H, 3 ꢃ ArH), 7.61–7.64
125.4 (Ar), 128.7 (Ar), 133.1 (Ar), 138.2 (Ar), 139.0 (Ar), 142.4 (Ar), (d, J ¼ 9.0 Hz, 1H, ArH), 7.74–7.81 (m, 2H, 2 ꢃ ArH), 7.95–7.99 (m,
150.7 (Ar), 159.9 (Ar). Anal. calcd for C16H11N3O5: C, 59.08; H, 3.41; 2H, 2 ꢃ ArH), 8.11–8.14 (m, 1H, ArH), 8.55–8.58 (d, J ¼ 8.7 Hz, 1H,
N, 12.92, found: C, 59.28; H, 3.49; N, 12.99.
ArH), 8.89–8.90 (d, J ¼ 3.9 Hz, 1H, ArH), 8.97–9.00 (m, 1H, ArH);
8-((2,6-difluorobenzyl)oxy)-5-nitroquinoline (36). Yellow solid, 13 C NMR (75 MHz, CDCl3): d 69.8 (OCH2), 108.3 (Ar), 122.6 (Ar),
88% yield, ClogP 4.24, mp ¼ 193–195 ꢀC. 1H NMR (300 MHz, 124.4 (Ar), 125.3 (Ar), 125.9 (Ar), 126.6 (Ar), 127.0 (Ar), 127.6 (Ar),
CDCl3): d 5.52 (s, 2H, OCH2), 6.94–6.99 (t, J ¼ 7.8 Hz, 2H, 2 ꢃ ArH), 128.2 (Ar), 129.0 (Ar), 129.5 (Ar), 131.7 (Ar), 131.9 (Ar), 132.1 (Ar),
7.26–7.29 (d, J ¼ 9.3 Hz, 1H, ArH), 7.32–7.40 (m, 1H, ArH), 7.65–7.69 133.8 (Ar), 137.7 (Ar), 139.2 (Ar), 150.6 (Ar), 160.1 (Ar). Anal. calcd
(m, 1H, ArH), 8.52–8.55 (d, J ¼ 8.7 Hz, 1H, ArH), 9.03–9.05 (d, for C20H14N2O3: C, 72.72; H, 4.27; N, 8.48, found: C, 72.89; H, 4.31;
J ¼ 4.2 Hz, 1H, ArH), 9.20 (d, J ¼ 9.0 Hz, 1H, ArH); 13 C NMR (75 MHz, N, 8.59.
2
CDCl3) d 59.7 (OCH2), 106.6 (Ar), 111.6 (Ar), 111.8 (d, JC-F
¼
2-(((5-nitroquinolin-8-yl)oxy)methyl)isoindoline-1,3-dione (42). Yellow
1
22.9 Hz, 2 x Ar), 123.1 (Ar), 124.5 (Ar), 127.3 (Ar), 131.4 (Ar), 131.6 solid, 77% yield, ClogP 3.07, mp ¼ 210ꢀC (dec.); H NMR (400 MHz,
3
(d, JC-F ¼ 10.3 Hz, Ar), 132.6 (Ar), 139.8 (Ar), 143.4 (Ar), 150.3 (Ar), CDCl3): d 5.92 (s, 2H, OCH2), 7.64–7.67 (d, J ¼ 8.7 Hz, 1H, ArH),
159.7 (Ar), 160.4 (ArC-F
x
2). 19 F NMR (564.7 MHz, CDCl3): 7.80–7.84 (m, 1H, ArH), 7.91–8.00 (m, 4H, 4 ꢃ ArH), 8.53–8.56 (d,
d ꢂ 111.74 (t, J ¼ 6.6 Hz, 2 F, ArF). Anal. calcd for C16H10F2N2O3: C, J ¼ 8.7 Hz, 1H, ArH), 8.94–8.99 (m, 2H, 2ꢃ ArH); 13 C NMR (100 MHz,
60.76; H, 3.19; N, 8.86, found: C, 60.89; H, 3.22; N, 8.95.
CDCl3): d 67.0 (OCH2), 110.5 (Ar), 122.5 (Ar), 124.3 (Ar), 127.5 (Ar),
8-((3,5-dimethylbenzyl)oxy)-5-nitroquinoline (37). Brownish sticky 131.7 (Ar), 132.3 (Ar), 135.7 (Ar), 139.1 (Ar), 151.0 (Ar), 157.8 (Ar),
1
oil, 81% yield, ClogP 4.95. H NMR (400 MHz, CDCl3): d 2.32 (s, 6H,
167.3 (2 x N-C ¼ O). Anal. calcd for C18H11N3O5: C, 61.89; H, 3.17; N,
2 x CH3), 5.49 (s, 2H, OCH2), 6.98 (s, 1H, ArH), 7.07–7.09 (d,
J ¼ 8.8 Hz, 1H, ArH), 7.12 (s, 1H, ArH), 7.69–7.73 (m, 1H, ArH),
12.03, found: C, 61.70; H, 3.13; N, 11.96.
2-((5-nitroquinolin-8-yl)oxy)-1-phenylethan-1-one (43). Yellow-brow
8.44–8.46 (d, J ¼ 8.8 Hz, 1H, ArH), 9.08–9.09 (m, 1H, ArH), 9.22–9.25 solid, 81% yield, ClogP 3.14, mp ¼ 196–198 ꢀC. 1H NMR (300 MHz,
(m 1H, ArH); 13 C NMR (100 MHz, CDCl3): d 21.3 (2 ꢃ CH3), 71.7 CDCl3): d 5.80 (s, 2H, OCH2), 6.90–6.92 (d, J ¼ 9.0 Hz, 1H, ArH),
(OCH2), 107.3 (Ar), 123.1 (Ar), 124.5 (Ar), 124.9 (Ar), 127.5 (Ar), 7.51–7.56 (t, J ¼ 7.8 Hz, 2H, 2 ꢃ ArH), 7.63–7.66 (d, J ¼ 6.9 Hz, 1H,
130.1 (Ar), 132.6 (Ar), 135.2 (Ar), 137.7 (Ar), 138.6 (Ar), 139.6 (Ar), ArH), 7.69–7.74 (m, 1H, ArH), 8.03–8.06 (d, J ¼ 7.8 Hz, 2H, 2 ꢃ ArH),
150.3 (Ar), 160.0 (Ar). Anal. calcd for C18H16N2O3: C, 70.12; H, 5.23; 8.42–8.45 (d, J ¼ 8.7 Hz, 1H, ArH), 9.06–9.08 (d, J ¼ 3.6 Hz, 1H, ArH),
N, 9.09, found: C, 70.29; H, 5.27; N, 9.18.
9.22–9.25 (d, J¼ 8.7 Hz, 1H, ArH); 13 C NMR (75MHz, DMSO-d6): d
71.8 (OCH2), 108.5 (Ar), 122.6 (Ar), 123.3 (Ar), 125.3 (Ar), 127.9 (Ar),
128.5 (Ar), 129.4 (Ar), 132.2 (Ar), 134.5 (Ar), 137.8 (Ar), 138.9 (Ar),
150.7 (Ar), 159.8 (Ar), 193.7 (C ¼ O). Anal. calcd for C17H12N2O4: C,
66.23; H, 3.92; N, 9.09, found: C, 66.36; H, 3.99; N, 9.19.
8-((3,5-difluorobenzyl)oxy)-5-nitroquinoline (38). Yellow solid,
71% yield, ClogP 4.24, mp ¼ 135–136 ꢀC. 1H NMR (400 MHz,
CDCl3): d 5.51 (s, 2H, OCH2), 6.78–6.83 (m, 1H, ArH), 7.02–7.09 (m,
3H, 3 ꢃ ArH), 7.72–7.75 (m, 1H, ArH), 8.45–8.47 (d, J ¼ 8.8 Hz, 1H,
ArH), 9.09–9.11 (m, 1H, ArH), 9.22–9.24 (dd, J ¼ 1.5 Hz, J ¼ 8.8 Hz,
1–(3-nitrophenyl)-2-((5-nitroquinolin-8-yl)oxy)ethan-1-one (44). Dark
2
1H, ArH); 13 C NMR (100 MHz, CDCl3): d 70.1 (OCH2), 103.9 (t, JC-F brown solid, 65% yield, ClogP 3.05, mp ¼ 203–205 ꢀC. 1H NMR
2
¼ 25.3 Hz, Ar), 107.1 (Ar), 109.8 (d, JC-F ¼ 26.1 Hz, 2 ꢃ Ar), 123.1 (300 MHz, DMSO-d6, slightly soluble): d 5.82 (s, 2H, OCH2), 7.07–7.10
3
(Ar), 124.7 (Ar), 127.0 (Ar), 132.6 (Ar), 138.3 (Ar), 139.4 (t, JC-F
¼
¼
(d, J¼ 8.4 Hz, 1H, ArH), 7.76–7.85 (m, 2H, 2 ꢃ ArH), 8.48–8.53 (m, 3H,
1
9.1 Hz, Ar), 139.6 (Ar), 150.5 (Ar), 159.1 (Ar), 163.3 (d, JC-F
3 x ArH), 8.92 (s, 1H, ArH), 9.15 (s, 1H, ArH), 9.37–9.39 (m, 1H, ArH).
1
249.7 Hz, ArC-F), 163.4 (d, JC-F ¼ 249.8 Hz, ArC-F). Anal. calcd for Anal. calcd for C17H11N3O6: C, 57.80; H, 3.14; N, 11.89, found: C,
C16H10F2N2O3: C, 60.76; H, 3.19; N, 8.86, found: C, 60.50; H, 3.13; 57.96; H, 3.18; N, 11.99.
N, 8.77.
1–(4-nitrophenyl)-2-((5-nitroquinolin-8-yl)oxy)ethan-1-one
(45).
8-((3-chloro-5-methoxybenzyl)oxy)-5-nitroquinoline (39). Yellow Brown solid, 73% yield, ClogP 3.05, mp ¼ 190 ꢀC (dec.). 1H NMR
1
solid, 72% yield, ClogP 4.72, mp ¼ 154–155 ꢀC. H NMR (400 MHz, (300 MHz, DMSO-d6, slightly soluble): d 6.11 (s, 2H, OCH2), 7.38–7.41
DMSO-d6): d 3.82 (s, 3H, OCH3), 5.42 (s, 2H, OCH2), 7.01–7.03 (d, (d, J¼ 8.7 Hz, 1H, ArH), 7.83–7.87 (m, 1H, ArH), 8.27–8.30 (d,
J ¼ 8.8 Hz, 1H, ArH), 7.15–7.16 (d, J ¼ 2.8 Hz, 1H, ArH), 7.50–7.52 (d, J ¼ 9.0 Hz, 2H, 2 ꢃ ArH), 8.39–8.42 (d, J ¼ 8.7 Hz, 2H, 2 ꢃ ArH),
J ¼ 8.8 Hz, 1H, ArH), 7.65–7.67 (d, J ¼ 8.4 Hz, 1H, ArH), 7.82–7.85 (m, 8.46–8.49 (d, J¼ 9.3 Hz, 1H, ArH), 9.01–9.03 (m, 2H, 2ꢃ ArH). Anal.
1H, ArH), 8.56–8.59 (d, J ¼ 9.2 Hz, 1H, ArH), 8.98–9.04 (m 2H,
calcd for C17H11N3O6: C, 57.80; H, 3.14; N, 11.89, found: C, 57.62; H,
2 ꢃ ArH); 13 C NMR (100 MHz, DMSO-d6): d 56.2 (OCH3), 68.8 3.10; N, 11.78.
(OCH2), 108.0 (Ar), 113.9 (Ar), 115.3 (Ar), 122.6 (Ar), 125.3 (Ar),
2-((5-nitroquinolin-8-yl)oxy)-1-(thiophen-3-yl)ethan-1-one (46). Brown
125.6 (Ar), 128.2 (Ar), 132.2 (Ar), 132.6 (Ar), 134.7 (Ar), 137.8 (Ar), solid, 86% yield, ClogP 2.92, mp ¼ 128–130 ꢀC. 1H NMR (400 MHz,
139.2 (Ar), 150.7 (Ar), 160.0 (Ar), 160.8 (Ar). Anal. calcd for DMSO-d6): d 5.89 (s, 2H, OCH2), 7.29–7.31 (d, J ¼ 9.2 Hz, 1H, ArH),

1338
S. VESCHI ET AL.
7.63–7.64 (d, J ¼ 5.2 Hz, 1H, ArH), 7.72–74 (m, 1H, ArH), 7.85–7.89 (m, 2H, OCH2), 7.03–7.07 (m, 2H, 2 ꢃ ArH), 7.57–7.59 (d, J ¼ 8.4 Hz, 2H,
1H, ArH), 8.50–8.52 (d, J ¼ 8.8 Hz, 1H, ArH), 8.79–8.80 (d, J¼ 1.6 Hz,
1H, ArH), 9.03–7.06 (m, 2H, 2 x ArH); 13 C NMR (100 MHz, DMSO-d6): d
72.0 (OCH2), 108.4 (Ar), 122.6 (Ar), 125.3 (Ar), 127.0 (thiophene), 127.9
(thiophene), 128.2 (Ar), 132.2 (thiophene), 135.0 (Ar), 137.9 (Ar), 139.0
(Ar), 139.1 (thiophene), 150.7 (Ar), 159.7 (Ar), 188.4 (C ¼ O). Anal.
calcd for C15H10N2O4S: C, 57.32; H, 3.21; N, 8.91, found: C, 57.50; H,
3.26; N, 8.99.
1-bromo-2-((4-nitrophenoxy)methyl)benzene (47). White solid, 89%
yield, ClogP 4.73, mp ¼ 122–123 ꢀC. Characterisation data are in
agreement with those reported in the literature25.
1-nitro-2-((4-nitrophenoxy)methyl)benzene (48). Pinkish solid, 97%
yield, ClogP 3.53, mp ¼ 130–132ꢀC. 1H NMR (400 MHz, CDCl3): d
5.62 (s, 2H, OCH2), 7.08–7.12 (m, 2H, 2 ꢃ ArH), 7.55–7.59 (t,
J ¼ 8.0 Hz, 1H, ArH), 7.73–7.77 (t, J ¼ 7.6 Hz, 1H, ArH), 7.85–7.87 (d,
J ¼ 8.0 Hz, 1H, ArH), 8.22–8.27 (t, J ¼ 9.2 Hz, 3H, 3 ꢃ ArH); 13 C NMR
(100 MHz, CDCl3): d 67.4 (OCH2), 115.0 (Ar), 125.3 (Ar), 126.1 (Ar),
129.0 (Ar), 132.2 (Ar), 134.2 (Ar), 142:2 (Ar), 147.0 (Ar), 163.0 (Ar).
Anal. calcd for C13H10N2O5: C, 56.94; H, 3.68; N, 10.22, found: C,
56.74; H, 3.61; N, 10.14.
2 ꢃ ArH), 7.72–7.74 (d, J ¼ 8.4 Hz, 2H, 2 ꢃ ArH), 8.22–8.26 (m, 2H,
2 ꢃ ArH); 13 C NMR (100 MHz, CDCl3): d 69.5 (OCH2), 112.4 (Ar), 114.8
(Ar), 118.4 (CꢄN), 126.0 (Ar), 127.7 (Ar), 132.6 (Ar), 140.9 (Ar), 142.1
(Ar), 163.0 (Ar). Anal. calcd for C14H10N2O3: C, 66.14; H, 3.96; N,
11.02, found: C, 66.26; H, 3.99; N, 11.11.
(4-bromobenzyl)(4-nitrophenyl)sulfane (57). Yellow solid, 87%
yield, ClogP 4.99, mp ¼ 133–134ꢀ C. 1H NMR (400 MHz, CDCl3): d
4.21 (s, 2H, SCH2), 7.27–7.29 (d, J ¼ 8.4 Hz, 2H, 2 ꢃ ArH), 7.33–7.7.35
(m, 2H, 2 ꢃ ArH), 7.47–7.49 (m, 2H, 2 ꢃ ArH), 8.11–8.13 (m, 2H,
2 ꢃ ArH); 13 C NMR (100 MHz, CDCl3): d 36.5 (SCH2), 121.8 (Ar),
124.0 (Ar), 126.9 (Ar), 130.4 (Ar), 132.0 (Ar), 134.6 (Ar), 145.5 (Ar),
146.5 (Ar). Anal. calcd for C13H10BrNO2S: C, 48.16; H, 3.11; N, 4.32,
found: C, 48.29; H, 3.14; N, 4.39.
1,3-difluoro-2-((4-nitrophenoxy)methyl)benzene (58). White solid,
95% yield, ClogP 4.15, mp ¼ 125–127 ꢀC. 1H NMR (400 MHz,
DMSO-d6): d 5.29 (s, 2H, OCH2), 7.18–7.23 (m, 2H, 2 ꢃ ArH),
7.26–7.29 (d, J ¼ 9.2 Hz, 2H, 2 ꢃ ArH), 7.53–7.61 (m, 1H, ArH),
8.23–8.25 (d, J ¼ 9.2 Hz, 2H, 2 ꢃ ArH); 13 C NMR (100 MHz, DMSO-
2
2
d6): d 59.0 (OCH2), 111.9 (t, JC-F ¼ 19.1 Hz, Ar), 112.4 (d, JC-F
¼
3
1-bromo-3-((4-nitrophenoxy)methyl)benzene (49). White solid, 90%
yield, ClogP 4.73, mp ¼ 91–92 ꢀC. Characterisation data are in agree-
ment with those reported in the literature26.
24.8 Hz, Ar), 115.7 (2 x Ar), 126.4 (2 x Ar), 132.6 (t, JC-F ¼ 10.5 Hz,
1
1
Ar), 141.8 (Ar), 161.6 (d, JC-F ¼ 249.6 Hz, ArC-F), 161.7 (d, JC-F
¼
249.6 Hz, ArC-F), 163.7 (Ar); 19 F NMR (564.7 MHz, CDCl3): d ꢂ 118.37
(t, J ¼ 6.6 Hz, 2 F, ArF). Anal. calcd for C13H9F2NO3: C, 58.87; H, 3.42;
N, 5.28, found: C, 58.99; H, 3.46; N, 5.32.
1-nitro-3-((4-nitrophenoxy)methyl)benzene (50). Yellow solid, 60%
yield, ClogP 3.61, mp ¼ 125–128 ꢀC. 1H NMR (400 MHz, CDCl3): d
5.28 (s, 2H, OCH2), 7.07–7.11 (m, 2H, 2 ꢃ ArH), 7.64–7.66 (t,
J ¼ 8.0 Hz, 1H, ArH), 7.79–7.81 (d, J ¼ 7.6 Hz, 1H, ArH), 8.25–8.27 (d,
J ¼ 9.2 Hz, 3H, 3 ꢃ ArH), 8.35 (s, 1H, ArH); 13 C NMR (100 MHz,
CDCl3): d 69.2 (OCH2), 114.9 (Ar), 122.3 (Ar), 123.5 (Ar), 126.1 (Ar),
129.9 (Ar), 133.1 (Ar), 137.7 (Ar), 142.2 (Ar), 148.6 (Ar), 163.0 (Ar).
Anal. calcd for C13H10N2O5: C, 56.94; H, 3.68; N, 10.22, found: C,
56.74; H, 3.60; N, 10.16.
1-((4-nitrophenoxy)methyl)-3-(trifluoromethyl)benzene (51). White
solid, 72% yield, ClogP 4.75, mp ¼ 70–74 ꢀC. 1H NMR (400 MHz,
CDCl3): d 5.23 (s, 2H, OCH2), 7.05–7.09 (m, 2H, 2 x ArH), 7.57–7.73
(m, 4H, 4 x ArH), 8.23–8.27 (m, 2H, 2 x ArH);19F NMR (564.7 MHz,
CDCl3): d ꢂ 66.64 (s, 3 F, ArCF3) Anal. calcd for C14H10F3NO3: C,
56.57; H, 3.39; N, 4.71, found: C, 56.64; H, 3.41; N, 4.73.
(3-nitrobenzyl)(4-nitrophenyl)sulfane (52). Yellow sticky oil, 70%
yield, ClogP 3.87. 1H NMR (400 MHz, CDCl3): d 4.26 (s, 2H, SCH2),
7.26–7.30 (m, 2H, 2 ꢃ ArH), 7.43–7.47 (t, J ¼ 8.0 Hz, 1H, ArH),
7.63–7.65 (dd, J ¼ 7.6 Hz, J ¼ 0.4 Hz, 1H, ArH), 8.02–8.08 (m, 1H,
ArH), 8.19–8.20 (t, J ¼ 1.6 Hz, 3H, 3 ꢃ ArH); 13 C NMR (100 MHz,
CDCl3): d 36.5 (SCH2), 122.9 (Ar), 123.6 (Ar), 124.1 (Ar), 127.3 (Ar),
129.9 (Ar), 134.7 (Ar), 138.0 (Ar), 145.5 (Ar), 145.7 (Ar), 148.5 (Ar).
Anal. calcd for C13H10N2O4S: C, 53.79; H, 3.47; N, 9.65, found: C,
53.27; H, 3.43; N, 9.60.
1-fluoro-4-((4-nitrophenoxy)methyl)benzene (53). White solid, 92%
yield, ClogP 4.01, mp ¼ 123–124 ꢀC. [Lit. mp ¼ 122.4 ꢀC]. 19 F NMR
(564.7 MHz, CDCl3): d ꢂ 110.05 (m, 1 F, ArF). Characterisation data
are in agreement with those reported in the literature27.
1-chloro-4-((4-nitrophenoxy)methyl)benzene (54). White solid,
98% yield, ClogP 4.58, mp ¼ 113–115 ꢀC. [Lit. mp ¼ 114–115 ꢀC].
Characterisation data are in agreement with those reported in the
literature28.
1,2-dichloro-4-((4-nitrophenoxy)methyl)benzene (59). White solid,
89% yield, ClogP 5.17, 146–147 ꢀC. 1H NMR (400 MHz, CDCl3): d
5.13 (s, 2H, OCH2), 7.03–7.05 (d, J ¼ 6.8 Hz, 2H, 2 x ArH), 7.29 (s,
1H, ArH), 7.50–7.56 (m, 2H, 2 x ArH), 8.23–8.25 (d, J ¼ 6.8 Hz, 2H, 2
x ArH); 13 C NMR (100 MHz, CDCl3): d 69.1 (OCH2), 114.8 (Ar), 124.8
(Ar), 126.0 (Ar), 126.6 (Ar), 129.3 (Ar), 130.8 (Ar), 132.6 (Ar), 133.1
(Ar), 135.7 (Ar), 142.0 (Ar), 163.1 (Ar). Anal. calcd for C13H9Cl2NO3:
C, 52.38; H, 3.04; N, 4.70, found: C, 52.42; H, 3.06; N, 4.75.
2-((4-nitrophenoxy)methyl)isoindoline-1,3-dione (60). Yellow solid,
75% yield, ClogP 2.99, mp ¼ 152–154 ꢀC. [Lit. mp ¼ 160–161 ꢀC].
Characterisation data are in agreement with those reported in the
literature29.
2–(4-nitrophenoxy)-1–(3-nitrophenyl)ethan-1-one
(61).
beige
1
solid, 72% yield, ClogP 2.96, mp ¼ 144–147 ꢀC. H NMR (400 MHz,
CDCl3): d 5.46 (s, 2H, OCH2), 7.03–7.05 (d, J ¼ 9.2 Hz, 2H, 2 x ArH),
7.77–7.81 (t, J ¼ 8.0 Hz, 1H, ArH), 8.22–8.25 (d, J ¼ 8.8 Hz, 2H,
2 ꢃ ArH), 8.35–8.37 (d, J ¼ 7.6 Hz, 1H, ArH), 8.50–8.52 (d, J ¼ 7.8 Hz,
1H, ArH), 8.85 (s, 1H, ArH); 13 C NMR (100 MHz, CDCl3): d 70.9
(OCH2), 114.8 (Ar), 123.1 (Ar), 126.1 (Ar), 128.5 (Ar), 130.4 (Ar),
133.8 (Ar), 135.2 (Ar), 142.5 (Ar), 148.6 (Ar), 162.4 (Ar), 191.4
(C ¼ O). Anal. calcd for C14H10N2O6: C, 55.64; H, 3.34; N, 9.27,
found: C, 55.78; H, 3.39; N, 9.33.
4.4. Cell lines and treatments
Human PC cell lines (AsPC-1, Capan-2 and BxPC-3) were cultured
as previously described5. The three cell lines display different gen-
etic profiles5. Human fibroblast cell line HFF-1 was purchased
from American Type Culture Collection (ATCC; Manassas, VA, USA)
and it was cultured in DMEM high glucose (4.5 g/L), supplemented
with 15% FBS.
8-Hydroxy-5-nitroquinoline (nitroxoline) was purchased from
Sigma (St. Louis, MO, USA). All compounds were dissolved in
DMSO (stock solutions of 40 mM) and then in culture media for
the final working concentrations. The final concentration of
DMSO in the experiments was at most 0.27% and showed no
1-bromo-4-((4-nitrophenoxy)methyl)benzene (55). White solid, 89%
yield, ClogP 4.73, mp ¼ 120–121 ꢀC. 1H NMR (400 MHz, CDCl3): d
5.13 (s, 2H, OCH2), 7.03–7.05 (m, 2H, 2ꢃ ArH), 7.32–7.34 (d,
J ¼ 8.4 Hz, 2H, 2 x ArH), 7.56–7.58 (d, J¼ 8.4 Hz, 2H, 2 ꢃ ArH),
8.22–8.25 (m, 2H, 2 ꢃ ArH). Anal. calcd for C13H10BrNO3: C, 50.67; H,
3.27; N, 4.55, found: C, 50.69; H, 3.29; N, 4.58.
4-((4-nitrophenoxy)methyl)benzonitrile (56). White solid, 94% yield,
ClogP 3.30, mp ¼ 157–158 ꢀC. 1H NMR (400 MHz, CDCl3): d 5.25 (s, cell toxicity.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1339
4.5. Cell viability assay
double/triple bonds, functional groups, branched and long chains.
Conversely, the insertion of a benzyl group (11–40) provided com-
pounds of similar potency with respect to nitroxoline on AsPC-1
(24, 33, 37, 40) and Capan-2 (24, 40) cells. Preferred positions and
substituents on the aryl ring were 3-Br, 4-CN, 3,5-diCH3 and 3,4-
diCl. On BxPC-3 cells, these compounds displayed a more interest-
ing scenario being some compounds (22, 26, 31, 32, 39) equipo-
tent with respect to nitroxoline and compounds 18, 19, 24, 33, 36,
40 even more potent at the same tested concentration. More in
detail, only electron-withdrawing substituents (2-NO2, 2-Br, 3-Br, 4-
CN, 2,6-diF, 3,4-diCl) on specific positions at the benzyl ring fav-
oured the inhibitory activity. The presence of a carbonyl spacer
between the OCH2 and the phenyl ring (43–46) furnished nitroxo-
line analogues with discrete effect on the three cell lines, whereas
the molecular simplification to 4-nitro(thio)phenol derivatives
(47–61) was detrimental for the inhibitory activity against AsPC-1
and Capan-2 cell lines. On BxPC-3 cells, compounds 48, 50, 55, 57
and 59 were endowed with promising activity, despite the substi-
tution of the oxygen with the sulphur led to a loss of biological
activity (compare the couples 50/52 and 55/57). Collectively, the
deletion of the pyridine nucleus of the parent compound gave
worse results. Thus, we selected the best-in-class compounds (24,
33, 36, 40 and 44) for further characterisation of their chemical-
physical properties and antiproliferative effects.
Cell viability was evaluated by MTT assay (Sigma-Aldrich, St. Louis,
MO, USA), essentially as previously described30. In particular, for
initial screening all compounds were used at a fixed concentration
of 40 lM for 48 h in the three PC cell lines. In concentration-
response curves, MTT assays were carried out by incubating PC
cell lines, or the normal fibroblast cell line with the most active
molecules, at concentrations ranging from 0 lM to 54 lM for 48 h.
4.6. Clonogenic assay
Clonogenic assay was performed essentially as previously described
(5). Capan-2 and BxPC-3 were seeded in 6-well plates at 103 cells/
well, while AsPC-1 were seeded in 6-well plates at 0.5 ꢃ 103 cells/
well. After cell attachment, PC cell lines were treated for 48h with
compounds 33, 40, or with nitroxoline as indicated. Colonies were
fixed when cells in the control vehicle formed colonies consisting of
at least 30 cells31.
4.7. Half maximal inhibitory concentration (IC50) calculation and
statistical analysis
IC50 values were calculated by CompuSyn software32. Comparisons
of mean values were performed using an unpaired Student’s
t-test or, for multiple comparisons, a one-way ANOVA followed
by Dunnett’s test. A p values ꢅ0.05 was estimated as statistically
significant.
To evaluate the concentration-dependent effects of the most
promising compounds, PC cell lines were treated with 24, 33, 36,
40 and 44 for 48 h at concentrations of 2, 6, 18 and 54 lM, or with
vehicle (control) (Figure S1). Selected compounds affected PC cell
viability in a concentration-dependent manner (Figure S1). IC50 val-
ues were calculated by CompuSyn software (Table 1). Compounds
33 and 40 had the lowest IC50 values as compared to other deriva-
tives and to nitroxoline, displaying the most consistent antiprolifer-
ative effects across the three PC cell lines. Interestingly, in AsPC-1
and Capan-2 compounds 33 and 40 had IC50 values lower than
5. Results and discussion
5.1. Antiproliferative effects and SAR studies
Starting from the results published on the anti-cancer potential of
nitroxoline, our approach was oriented towards a wide exploration
of the chemical spare around the phenolic moiety in the nitroxo-
line skeleton in order to attain newly synthesised derivatives with
improved inhibitory activity on the viability of PC cell lines and
with different chemical-physical characteristics to finely tune the
predicted pharmacokinetic properties. Recent developments have
started to address this issue17,18,33, but poor information remains,
considering that those modifications could be important to better
define the target of these compounds.
Based on the previously reported antiproliferative activity of
nitroxoline on human PC cell lines (AsPC-1, Capan-2 and BxPC-3)5,
we explored the effects of these novel compounds on cell viability
in the same PC cells. To identify the most active compounds in
the series, preliminary MTT experiments were performed by incu-
bating the three PC cell lines with the 61 derivatives and with the
lead compound nitroxoline, for 48 h at one-point screening con-
centration (40 lM) (Figure 3). Several novel derivatives inhibited
viability more potently than nitroxoline and this effect was more
evident in BXPC3 where compounds 18, 19, 24, 33, 36, 40, 48
and 55 were more effective than nitroxoline (Figure 3(C)). A simi-
lar potency was observed in Capan-2 with compound 44, affecting
a greater fraction of cells as compared to nitroxoline (Figure 3(B)).
Notably, 24, 33, 36, 40 and 44 were the most active compounds
also in AsPC-1 cells (Figure 3(A)), although with a potency compar-
able to nitroxoline.
those previously obtained with erlotinib5,
a targeted agent
approved for PC treatment. Moreover, in BxPC-3, the least sensitive
PC cell line to nitroxoline, but sensitive to erlotinib, the IC50 of
compound 40 was lower as compared to both agents5.
The two compounds with the lowest IC50 values in MTT assays
were also tested for their ability to interfere with clonogenicity in
PC cells. In particular, we compared the effects of 33, 40 and
nitroxoline on colony formation ability of the three PC cell lines
(Figure 4). At 1 lM only compound 33 had a significant effect,
reducing clonogenicity of AsPC-1. Conversely, when used at 5 lM
the effects of the three compounds on clonogenicity appeared
more consistent. In particular compound 33 markedly reduced
clonogenicity of AsPC-1, nitroxoline affected both AsPC-1 and
BxPC3, and compound 40 dramatically reduced clonogenicity of
the three PC cell lines, consistently showing a greater effect as
compared to 33 and nitroxoline used at the same concentrations.
Remarkably, the effects of these novel compounds on cell self-
renewal capacity in PC cells were stronger than those that we pre-
viously reported with erlotinib at higher concentrations5.
We next tested the effects of the most active compound on
viability of normal HFF-1 fibroblast cells. Notably, compound 40
showed low toxicity against normal HFF-1 cells (IC50 23.1 lM) and
selectivity index (SI) values comparable or superior to nitroxoline
in the three PC cell lines (Table 2 and Figure S2). In particular,
compound 40 has similar selectivity as nitroxoline in Capan-2, but
is much more selective in AsPC-1 and BxPC-3, suggesting that
Structure-activity relationships (SARs) within this library sug-
gested that the functionalization of the OH with aliphatic chains
(1–13) led to a reduced inhibitory activity against the three cell
lines with respect to nitroxoline disregarding the presence of compound 40 may be more effective and safer than nitroxoline.

1340
S. VESCHI ET AL.
Figure 3. Screening of novel nitroxoline derivatives (1–61) on PC cell viability. Effects of novel derivatives on the viability of AsPC-1 (A), Capan-2 (B) and BxPC-3 (C) PC
cell lines were assessed by MTT assays. The lead compound nitroxoline was included as a reference. MTT assays were performed by using compounds at 40lM for
48 h and the histograms show the relative decrease of cell viability induced by treatments, as compared to nitroxoline. Data shown are the means SD of duplicate
MTT experiments, each with quintuplicate determinations and are calculated as ratio relative to the lead compound nitroxoline (identified by a dashed line).
Table 1. IC50 values for compounds 24, 33, 36, 40, 44 and nitroxoline in PC
cell lines.
5.2. In silico pharmacokinetic parameters and target prediction
IC50 (mM)
In Table 3, the physical–chemical, pharmacokinetics and medicinal
Compound
AsPC-1
Capan-2
BxPC-3 chemistry parameters obtained from the commercially available
41.2
web tool SwissADME23 are reported, aiming to describe some prop-
ꢆ
Nitroxoline
26.8
26.1
13.7
17.7
4.9
20.1
22.8
16.9
40.2
17.8
21.6
9.8
27.1
30.5
24
33
36
40
44
47.1
20.7
24.5
9.7
erties of the tested compounds that are fundamental for a proper
drug development. On the basis of the well-known Lipinski’s rule
of five is possible to evaluate the drug-likeness of one compound,
i.e., the probability that the molecule will be effective as an oral
drug. As one can see, all the evaluated compounds fully comply
with the limits of the Lipinski’s rule, supporting their feasible oral
39.5
10.9
ꢆ
Erlotinib
IC50 values for Nitroxoline and Erlotinib were previously reported by us5.
ꢆ

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1341
Figure 4. Effect of compounds 33, 40 and nitroxoline on the clonogenic capacity of AsPC-1, Capan-2 and BxPC-3PC cell lines. Representative plates of colony forma-
tion assays for the three PC cell lines exposed to tested compounds at 1 lM, 5 lM, or vehicle (control) are shown. Data shown in the histograms are the means SD
ꢆ
ꢆꢆ
ꢆꢆꢆ
p < 0.001;
ꢆꢆꢆꢆ
p < 0.0001).
of two independent experiments and are expressed as fold change relative to control ( p < 0.05; p < 0.01;
use (Lipinski violation is equal 0 for all the analogues), similarly to substrate of the permeability glycoprotein (P-gp). This protein pro-
the lead compound nitroxoline. motes the energy-dependent efflux of cytotoxic drugs out of the
The capability of these compounds to be passively adsorbed in cytosol and its overexpression in tumour cells leads to multi-drug
the gastrointestinal tract is an important information influencing resistance (MDS), which reduces the efficacy of antitumor treat-
their bioavailability after oral administration; even in this case all ments. The outcomes extrapolated by in silico studies rule out the
the examined compounds exhibited promising results. One of the possibility that these compounds could be substrate of P-gp, rein-
Achilles’ heels of some antitumor drugs is the propensity to be forcing their role as antitumor drugs. Finally, the presence of PAINS

1342
S. VESCHI ET AL.
(Pan Assay Interference Compounds) that are substructures able to data reported in Table 4. The boiled-egg graph is obtained by
elicit promiscuous pharmacological behaviour, was also evaluated considering the two parameters WLOGP, as a lipophilic index, and
showing as all the compounds are devoid of this attribute.
TPSA, as a measure of apparent polarity, enriched with the infor-
For the most active analogue (40), we also reported (Figure 5) mation about the risk to be substrate of P-gp. It allows, at first
the boiled-egg and bioavailability radar pictures, the two graphical glance, to predict simultaneously two ADME parameters that are
outputs of the SwissADME tool. These graphs are based on the the passive absorption at the gastrointestinal tract (white area)
and the ability to permeate the blood brain barrier (BBB, yellow
area), along with the susceptibility to P-gp depending on the col-
our of the dot (the red dot means that the compound is not a
substrate of the P-gp; on the contrary, the blue dot indicates a
putative substrate of the P-gp). As one can see, the most active
compound 40 is contained in the yellow area (even if it is to the
limit) and is pictured with a red dot, signifying that it is likely able
to cross the BBB, it is passively absorbed at the GI level and is not
effluxed by P-gp.
Table 2. Selectivity index (SI) values for compound 40 and nitroxoline.
Selectivity index (SI)
Compound
AsPC-1
Capan-2
BxPC-3
40
4.71
1.69
2.36
2.69
2.38
1.10
Nitroxoline
SI values are calculated for each compound as follows: SI ¼ IC50 on normal fibro-
blast cells (HFF-1)/IC50 on cancer cell line. IC50 value of nitroxoline on HFF-1 is
45.4 lM (Figure S2).
Furthermore, the bioavailability radar provides the drug-likeness
representation of the compound. The pink area is indicative for
the optimal range of each physicochemical property (lipophilicity,
size, polarity, solubility, saturation, flexibility) required to be bio-
available after oral administration. For the analogue 40 only the
insaturation parameter is out of the desired range, while all the
others fit into the red-depicted area, accounting for a putative
moderate oral bioavailability.
With the aim to identify the putative target/s of our com-
pounds we also exploited the web-tool SwissTargetPrediction24,
performing the analysis on the lead nitroxoline and compound
40. In Tables 5 and 6, we reported the data concerning the most
probable targets found by the web-tool, that is, the ones exhibit-
ing the highest values of probability to be a target. Remarkably,
Table 3. In silico evaluated physicochemical properties of nitroxoline and the
most potent compounds 24, 33, 36, 40, and 44.
Compound
Nitroxoline
24
33
36
40
44
Molecular weight (MW)
H-bond acceptors (HBA)
H-bond donators (HBD)
190.16
4
1
1.17
0
High
No
0
359.17 305.29 316.26 349.17 353.29
4
0
3.22
0
5
0
2.37
0
6
0
3.21
0
4
0
3.67
0
7
0
1.83
0
ꢆ
Consensus Log P
Lipinski violations
GI absorption
P-gp substrate
PAINS alerts
ꢆ
High
No
0
High
No
0
High
No
0
High
No
0
High
No
0
Arithmetic mean of the values predicted by five in silico methods: XLOGP3,
WLOGP, MLOGP, SILICOS-IT, iLOGP. Parameters range required to satisfy the
Lipinski’s rule of five: MW ꢅ 500 g/mol, HBD ꢅ 5, HBA ꢅ 10, log P ꢅ 5.
Boiled-egg
Bioavailability radar
White region: high probability of passive
absorption by the gastrointestinal tract.
Yellow region: high probability of brain
penetration.
Compounds that are substrates of P-gp
Compounds that are not substrates of P-gp
Lipophilicity: -0.7 < XLOGP3 < 5.0
Size: 150 < MW (g/mol) < 500
2
°
Polarity: 150 < TPSA (Α ) < 500
Insolubility: 0 < Log S (ESOL) < 6
Insaturation: 0.25 < Fraction Csp3 < 1
Flexibility: 150 < N° rotatable bonds < 500
Figure 5. Representation of the boiled-egg graph and bioavailability radar calculated by SwissADME web-tool.
Table 4. In silico estimated physicochemical parameters of compound 40, used to device the boiled-egg graph and bioavailability radar.
Cmpd
WLOGPa
TPSA (Ð2)a,b
XLOGP3b
Log S (ESOL)b
MWb
Csp3b
Nꢀ of rotatable bondsb
40
4.88
67.94
5.07
ꢂ5.45
349.17
0.06
4
aParameters used for the boiled-egg graph.
bParameters used for the bioavailability radar. Bioavailability radar parameters functional ranges: XLOGP3 between ꢂ0.7 and þ5.0, MW
between 150 and 500 g/mol, TPSA between 20 and 130Å2, log S not higher than 6, saturation: fraction of carbons in the sp3 hybridisation
not less than 0.25, and flexibility: no more than 9 rotatable bonds.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1343
Table 5. Protein target prediction for nitroxoline.
Target
Common name
Uniprot ID
ChEMBL ID
Target Class
Probability
Cyclooxygenase-2
PTGS2
METAP2
ERCC5
RNASEH1
PARP1
PSMB5
IDO1
P35354
P50579
P28715
O60930
P09874
P28074
P14902
P48775
CHEMBL230
CHEMBL3922
CHEMBL4736
CHEMBL5893
CHEMBL3105
CHEMBL4662
CHEMBL4685
CHEMBL2140
Oxidoreductase
Protease
Other nuclear protein
Hydrolase
Transferase
Protease
Oxidoreductase
Oxidoreductase
1
1
Methionine aminopeptidase 2
DNA excision repair protein ERCC-5
Ribonuclease H1
Poly [ADP-ribose] polymerase-1
Proteasome Macropain subunit MB1
Indoleamine 2,3-dioxygenase
Tryptophan 2,3-dioxygenase (by homology)
0.03123
0.03123
0.03123
0.03123
0.03123
0.03123
TDO2
Table 6. Protein target prediction for compound 40.
Target
Common name
Uniprot ID
ChEMBL ID
Target Class
Probability
Cyclooxygenase-1 (by homology)
TGF-beta receptor type I
Macrophage migration inhibitory factor
Carbonic anhydrase XII
Phosphodiesterase 5A
ATP-binding cassette sub-family G member 2
Arachidonate 15-lipoxygenase
Phosphatidylinositol-5-phosphate 4-kinase type-2 gamma
DNA topoisomerase II alpha
PTGS1
TGFBR1
MIF
P23219
P36897
P14174
O43570
O76074
Q9UNQ0
P16050
Q8TBX8
P11388
P19320
CHEMBL221
Oxidoreductase
Kinase
Isomerase
0.10161
0.10161
0.10161
0.10161
0.10161
0.10161
0.10161
0.10161
0.10161
0.10161
CHEMBL4439
CHEMBL2085
CHEMBL3242
CHEMBL1827
CHEMBL5393
CHEMBL2903
CHEMBL1770034
CHEMBL1806
CHEMBL3735
CA12
Lyase
PDE5A
ABCG2
ALOX15
PIP4K2C
TOP2A
VCAM1
Phosphodiesterase
Primary active transporter
Oxidoreductase
Kinase
Isomerase
Adhesion
Vascular cell adhesion protein 1
among targets with the highest probability score, cyclooxygenase-
2 was previously unknown to be a target of nitroxoline, while
Type-2 methionine aminopeptidase (METAP2) activity was previ-
ously reported to be inhibited by nitroxoline12. Moreover, among
the molecules with a lower probability score PARP1 is a known
target of nitroxoline, which induces relevant effects on PARP
cleavage5. On the contrary, for compound 40 we did not obtain a
main preference for one protein; thus, further and deep studies
could shed light on its putative targets.
ORCID
Simone Carradori
http://orcid.org/0000-0002-8698-9440
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6. Conclusion
Our study explored the impact of the chemical modifications of
the core nucleus of a well-known antibiotic, nitroxoline, that
recently emerged as a promising candidate for its antiproliferative
effects on PC cell lines. The functionalization of the phenolic OH
with substituted benzyl rings led to an improvement of the bio-
logical activity with respect to the parent compound and erlotinib,
whereas the introduction of alkyl groups, the deletion of the pyri-
dine ring or the bioisosteric change from oxygen to sulphur were
detrimental for the reported activity. Moreover, the most promis-
ing compounds were further evaluated in terms of clonogenicity
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Disclosure statement
No potential conflict of interest was reported by the authors.
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Funding
ꢀ
This work was supported by Ministero dell’Istruzione, dell’Universita
e della Ricerca (PRIN funds to Alessandro Cama - grant number
2017EKMFTN_005).

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