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

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

To link to this article: https://doi.org/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 Veschi^a, Simone Carradori^a, Laura De Lellis^a, Rosalba Florio^a, Davide Brocco^a, Daniela Secci^b, Paolo

Guglielmi^b, Mattia Spano^b, Anatoly P. Sobolev^cand Alessandro Cama^a

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 strategies^14,15. 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 therapies^2,3. 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 cells^5,6. 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 cancers^7–13. 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 group^17,18. 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-NO₂moiety) 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 agents^20–22, 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 Stuart^Vmelting point apparatus SMP1 and

are uncorrected (temperatures are reported in ^ꢀC). ¹H and ¹³C

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 CDCl₃and DMSO-d₆, as the solvents

at room temperature. ¹⁹F spectra were recorded on a Bruker

AVANCE 600 spectrometer at 564.7 MHz, using CDCl₃as solvent.

All the compounds were analysed with a final concentration of

ꢁ25 mg/mL. ¹H and ¹³C chemical shifts are expressed as d units

(parts per millions) relative to the solvent signal (DMSO-d₆: ¹H

2.50 and ¹³C 39.5; CDCl₃: ¹H 7.26 and ¹³C 77.4), whereas ¹⁹F

chemical shifts are expressed as d units relative to an external

standard (CF₃COOH, d ꢂ 76.55 ppm). ¹H spectra are described as

follows: d_H(spectrometer frequency, solvent): chemical shift/ppm

(multiplicity, J-coupling constant(s), number of protons, assign-

ment). ¹³C spectra are described as follows: d_C(spectrometer fre-

quency, solvent): chemical shift/ppm (assignment) and are fully

proton decoupled. ¹⁹F spectra are described as follows: d_F(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

Aldrich^Vsilica 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_,

1_0,

R= CH2_CH=CH2

R= Geranyl

₁, R= Farnesyl

₂, R= CH2_CO2_Et

NO

OR

2

1

4

, R= CH2_C

, R= CH2_CN

, R= Bu

CH

LiOH

MeOH, H O

2

5

6

7

N

1₃

R= CH2_COOH

, R= CH2_CH=CHCH3

R-X

1

₄, Cy= Ph

A

₅, Cy= 2_-CH3_Ph

3

4

₀, Cy= 4_-ClPh

NO

OH

2

NO

O

2

1_6,

Cy= 2_-CF3_Ph

₇, Cy= 2_-CNPh

₈, Cy= 2_-NO2_Ph

₉, Cy= 2_-BrPh

₀, Cy= 3_-CH3_Ph

₁, Cy= 3_-OCH3_Ph

₂, Cy= 3_-FPh

₁, Cy= 4_-BrPh

CyCH X

1

2

₂, Cy= 4_-IPh

2

₃, Cy= 4_-CNPh

N

K2_CO3

DMF

₄, Cy= 4_-CF3_Ph

₅, Cy= 4_-NO2_Ph

₆, Cy= 2_,6_-diFPh

₇, Cy= 3_,5_-diCH3_Ph

₈, Cy= 3_,5_-diFPh

₉, Cy= 3_-Cl-5_-OCH3_Ph

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_-NO2_Ph

₆, Cy= 3_-CF3_Ph

₇, Cy= 4_-CH3_Ph

₈, Cy= 4_-SCH3_Ph

₉, Cy= 4_-FPh

NO

2

N

O

4

₃, Cy= Ph

Cy

₄, Cy= 3_-NO2_Ph

4_5,

Cy= 4_-NO2_Ph

4

₆, Cy= thiophen-3_-yl

B H

3

4

₇, Y= O; Z= 2_-BrPh

₈, Y= O; Z= 2_-NO2_Ph

5

₅, Y= O; Z= 4_-BrPh

₆, Y= O; Z= 4_-CNPh

₇, Y= S; Z= 4_-BrPh

NO

Y

2

Bz/R-X or

ArCOCH2_Br

4_9,

Y= O; Z= 3_-BrPh

₀, Y= O; Z= 3_-NO2_Ph

₁, Y= O; Z= 3_-CF3_Ph

₂, Y= S; Z= 3_-NO2_Ph

₃, Y= O; Z= 4_-FPh

₄, Y= O; Z= 4_-ClPh

5

K2_CO3

DMF

5_8,

Y= O; Z= 2_,6_-diFPh

₉, Y= O; Z= 3_,4_-diClPh

₀, Y= O; Z= phthalimide

YH

Z

5

6

4-nitro(thio)phenol

₁, Y= O; Z= 3_-NO2_PhC=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 ¹³C 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); ¹³C NMR (75 MHz,

9.18–9.21 (m, 1H, ArH); ¹³C 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 OCH₂signal 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); ¹³C

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); ¹³C 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);

¹³C 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); ¹³C 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); ¹³C 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); ¹³C NMR (100 MHz,

CDCl₃): d 67.3 (OCH₂), 107.2 (Ar), 123.0 (d, J_C-F¼ 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, J_C-F¼ 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, J_C-F¼ 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); ¹⁹F 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, OCH₂þ 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); ¹³C 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); ¹³C 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, OCH₂þ 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); ¹³C 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); ¹³C 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); ¹³C 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); ¹³C 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); ¹³C 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); ¹³C NMR (75 MHz, (m, 1H, ArH), 9.09 (bs, 1H, ArH), 9.22–9.24 (m, 1H, ArH); ¹³C 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); ¹³C 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

¹³C NMR (100 MHz, CDCl₃): d 70.7 (OCH₂), 107.2 (Ar), 114.1 (d, J_C-F

2

4

¼ 22.3 Hz, Ar), 115.5 (d, J_C-F¼ 21.2 Hz, Ar), 122.6 (d, J_C-F

¼

3

3.1 Hz, Ar), 123.1 (Ar), 124.7 (Ar), 127.2 (Ar), 130.6 (d, J_C-F¼ 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); ¹⁹F 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); ¹³C 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); ¹³C 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); ¹³C 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); ¹³C NMR

2

(75 MHz, CDCl₃): d 70.9 (OCH₂), 107.5 (Ar), 115.9 (d, J_C-F¼ 21.75 Hz,

3

2ꢃ Ar), 123.1 (Ar), 124.7 (Ar), 127.5 (Ar), 129.3 (d, J_C-F¼ 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); ¹⁹F 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); ¹³C 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); ¹³C 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 195–197^ꢀC. 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);

¹³C 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); ¹³C NMR (75 MHz, CDCl₃): d 70.8 (OCH₂),

4

107.3 (Ar), 123.15 (Ar), 124.0 (d, J_C-F¼ 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);

¹³C 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); ¹⁹F 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); ¹³C 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); ¹³C 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, J_C-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, J_C-F¼ 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, J_C-F¼ 4.6 Hz, 2 x Ar), 130.6 (q, J_C-F¼ 31.2 Hz, Ar), 9.03–9.04 (m, 1H, ArH), 9.16–9.19 (d, J ¼ 8.7 Hz, 1H; ArH); ¹³C 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),

¹⁹F 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); ¹³C 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, ¹³C 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); ¹³C NMR (75 MHz, N, 8.59.

2

CDCl₃) d 59.7 (OCH₂), 106.6 (Ar), 111.6 (Ar), 111.8 (d, J_C-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 210^ꢀC (dec.); H NMR (400 MHz,

3

(d, J_C-F¼ 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). ¹⁹F 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); ¹³C 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); ¹³C 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); ¹³C 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); ¹³C NMR (100 MHz, CDCl₃): d 70.1 (OCH₂), 103.9 (t, J_C-Fbrown 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, J_C-F¼ 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, J_C-F

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, J_C-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, J_C-F¼ 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); ¹³C 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); ¹³C 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 130–132^ꢀC. 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); ¹³C 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); ¹³C 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 133–134^ꢀ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); ¹³C 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); ¹³C NMR (100 MHz, DMSO-

2

d₆): d 59.0 (OCH₂), 111.9 (t, J_C-F¼ 19.1 Hz, Ar), 112.4 (d, J_C-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, J_C-F¼ 10.5 Hz,

1

Ar), 141.8 (Ar), 161.6 (d, J_C-F¼ 249.6 Hz, ArC-F), 161.7 (d, J_C-F

¼

249.6 Hz, ArC-F), 163.7 (Ar); ¹⁹F 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); ¹³C 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); ¹³C 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]. ¹⁹F 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); ¹³C 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); ¹³C 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-diCH₃and 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 OCH₂and 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 10³cells/

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

IC₅₀values 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). IC₅₀val-

ues were calculated by CompuSyn software (Table 1). Compounds

33 and 40 had the lowest IC₅₀values 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 IC₅₀values 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 issue^17,18,33, 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 IC₅₀of

compound 40 was lower as compared to both agents⁵.

The two compounds with the lowest IC₅₀values 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 (IC₅₀23.1 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. IC₅₀values 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 SwissADME²³are 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

IC₅₀values 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 IC₅₀on normal fibro-

blast cells (HFF-1)/IC₅₀on cancer cell line. IC₅₀value 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

WLOGP^a

TPSA (Ð²)^a,^b

XLOGP3^b

Log S (ESOL)^b

MW^b

Csp3^b

N^ꢀof rotatable bonds^b

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 sp³hybridisation

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

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

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

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

References

1. Adamska A, Domenichini A, Falasca M. Pancreatic ductal

adenocarcinoma: current and evolving therapies. Int J Mol

Sci 2017;18:1338.

2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA

Cancer J Clin 2020;70:7–30.

3. Hajatdoost L, Sedaghat K, Walker EJ, et al. Chemotherapy in

pancreatic cancer: a systematic review. Medicina (Kaunas)

2018;54:48.

4. Xue H, Li J, Xie H, Wang Y. Review of drug repositioning

approaches and resources. Int J Biol Sci 2018;14:1232–44.

5. Veschi S, De Lellis L, Florio R, et al. Effects of repurposed

drug candidates nitroxoline and nelfinavir as single agents

or in combination with erlotinib in pancreatic cancer cells. J

Exp Clin Cancer Res 2018;37:236

6. Veschi S, Ronci M, Lanuti P, et al. Integrative proteomic and

functional analyses provide novel insights into the action of

the repurposed drug candidate nitroxoline in AsPC-1 cells.

Sci Rep 2020;10:2574.

7. Kumari N, Thakur N, Cho HR, Choi SH. Assessment of early

therapeutic response to nitroxoline in temozolomide-resist-

ant glioblastoma by amide proton transfer imaging: a pre-

liminary comparative study with diffusion-weighted imaging.

Sci Rep 2019;9:5585

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

reduction, in silico drug-likeness and cytotoxicity on normal

human fibroblasts. Collectively, our data supported the exploration

of this chemical scaffold to propose new compounds with alterna-

tive mechanisms of action for the treatment of pancreatic cancer.

8. Lazovic J, Guo L, Nakashima J, et al. Nitroxoline induces

apoptosis and slows glioma growth in vivo. Neuro-oncology

2015;17:53–62.

9. Mao H, Du Y, Zhang Z, et al. Nitroxoline shows antimyeloma

activity by targeting the TRIM25/p53 axle. Anticancer Drugs

2017;28:376–83.

Disclosure statement

No potential conflict of interest was reported by the authors.

10. Xu N, Huang L, Li X, et al. The novel combination of nitroxo-

line and PD-1 blockade, exerts a potent antitumor effect in a

mouse model of prostate cancer. Int J Biol Sci 2019;15:919–28.

11. Chen W, Zhang H, Chen Z, et al. Development and evalu-

ation of a novel series of nitroxoline-derived BET inhibitors

Funding

ꢀ

This work was supported by Ministero dell’Istruzione, dell’Universita

e della Ricerca (PRIN funds to Alessandro Cama - grant number

2017EKMFTN_005).

1344

S. VESCHI ET AL.

with antitumor activity in renal cell carcinoma. Oncogenesis

2018;7:83

designing isoform-selective inhibitors. Bioorg Med Chem

Lett 2013;23:6759–63.

23. Daina A, Michielin O, Zoete V. SwissADME: a free web tool

to evaluate pharmacokinetics, drug-likeness and medicinal

chemistry friendliness of small molecules. Sci Rep 2017;7:

42717.

12. Shim JS, Matsui Y, Bhat S, et al. Effect of nitroxoline on

angiogenesis and growth of human bladder cancer. J Natl

Cancer Inst 2010;102:1855–73.

ꢁ

13. Mirkovic B, Markelc B, Butinar M, et al. Nitroxoline impairs

24. Daina A, Michielin O, Zoete V. SwissTargetPrediction: updated

data and new features for efficient prediction of protein tar-

gets of small molecules. Nucleic Acids Research 2019;47:

W357–W364.

25. McMahon RE, Culp HW, Mills J, Marshall FJ. Demethylation

studies. IV. The in vitro and in vivo cleavage of alkyl- and

arylalkyl-p-nitrophenyl ethers. J Med Chem 1963;6:343–6.

26. Zhu XL, Zhang R, Wu QY, et al. Natural product Neopeltolide

as a cytochrome bc1 complex inhibitor: Mechanism of action

and structural modification. J. Agric. Food Chem 2019;67:

2774–81.

27. Debbabi K, Beji M, Baklouti A. Alkylation des esters sulfami-

ques par le bromure de 4-fluorobenzyle. Phosphorus Sulfur

Silicon Relat Elem 2005;180:1545–51.

28. Catalano A, Carocci A, Defrenza I, et al. 2-Aminobenzothiazole

derivatives: search for new antifungal agents. Eur J Med Chem

2013;64:357–64.

29. Getz JJ, Prankerd RJ, Sloan KB. Mechanism of hydrolysis of

O-imidomethyl derivatives of phenols. J Org Chem 1993;58:

4913–8.

30. Florio R, De Lellis L, Veschi S, et al. Effects of dichloroacetate

as single agent or in combination with GW6471 and metfor-

min in paraganglioma cells. Sci Rep 2018;8:13610.

31. Florio R, Veschi S, di Giacomo V, et al. The benzimidazole-

based anthelmintic parbendazole: a repurposed drug candi-

date that synergizes with gemcitabine in pancreatic cancer.

Cancers (Basel) 2019;11:pii:2042.

32. Chou TC. Drug combination studies and their synergy quan-

tification using the Chou-Talalay method. Cancer Res 2010;

70:440–6.

33. Hu S, Jin Y, Liu Y, et al. Synthesis and mechanistic studies of

quinolin-chlorobenzothioate derivatives with proteasome

inhibitory activity in pancreatic cancer cell lines. Eur J Med

Chem 2018;158:884–95.

tumor progression in vitro and in vivo by regulating cathe-

psin B activity. Oncotarget 2015;6:19027–42.

ꢁ

14. Mitrovic A, Kos J. Nitroxoline: repurposing its antimicrobial

to antitumor application. Acta Biochim Pol 2019;66:521–31.

15. Torphy RJ, Zhu Y, Schulick RD. Immunotherapy for pancre-

atic cancer: barriers and breakthroughs. Ann Gastroenterol

Surg 2018;2:274–81.

16. Graziano V, Grassadonia A, Iezzi L, et al. Combination of per-

ipheral neutrophil-to-lymphocyte ratio and platelet-to-

lymphocyte ratio is predictive of pathological complete

response after neoadjuvant chemotherapy in breast cancer

patients. Breast 2019;44:33–8.

ꢁ

17. Mirkovic B, Renko M, Turk S, et al. Novel mechanism of

cathepsin B inhibition by antibiotic nitroxoline and related

compounds. ChemMedChem 2011;6:1351–6.

ꢂ

ꢁ

18. Sosic I, Mirkovic B, Arenz K, et al. Development of new

Cathepsin B inhibitors: combining bioisosteric replacements

and structure-based design to explore the structure-activity

relationships of nitroxoline derivatives. J Med Chem 2013;56:

521–33.

19. Oliveri V, Vecchio G. 8-Hydroxyquinolines in medicinal

chemistry: a structural perspective. Eur J Med Chem 2016;

120:252–74.

€

20. Ozdemir Z, Utku S, Mathew B, et al. Synthesis and biological

evaluation of new 3(2H)-pyridazinone derivatives as non-toxic

anti-proliferative compounds against human colon carcinoma

HCT116 cells. J Enzyme Inhib Med Chem 2020;35:1100–9.

21. Lenoci A, Tomassi S, Conte M, et al. Quinoline-based p300 his-

tone acetyltransferase inhibitors with pro-apoptotic activity in

human leukemia U937 cells. ChemMedChem 2014;9:542–8.

22. Carradori S, De Monte C, D’Ascenzio M, et al. Salen and tet-

rahydrosalen derivatives act as effective inhibitors of the

tumor-associated carbonic anhydrase XII-a new scaffold for

Article Doi

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

DOI: 10.1080/14756366.2020.1780228

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Article abstract of DOI:10.1080/14756366.2020.1780228

Full text of DOI:10.1080/14756366.2020.1780228

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