Tuning NO release of organelle-targeted furoxan derivatives and their cytotoxicity against lung cancer cells

Article abstract of DOI:10.1016/j.bioorg.2021.104911

We herein report a study on a set of hybrid compounds in which 3-R-substituted furoxan moieties (R = CH₃, CONH₂, CN, SO₂C₆H₅), endowed with varying NO-releasing capacities, are joined to a mitochondri

Full text of DOI:10.1016/j.bioorg.2021.104911

Bioorganic Chemistry 111 (2021) 104911
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Tuning NO release of organelle-targeted furoxan derivatives and their
cytotoxicity against lung cancer cells
a
b
a
a
a,*
Federica Sodano , Elena Gazzano , Barbara Rolando , Elisabetta Marini , Loretta Lazzarato
,
a
c
a
Roberta Fruttero , Chiara Riganti , Alberto Gasco
a
Department of Drug Science and Technology, University of Torino, 10125 Torino, Italy
Department of Life Sciences and Systems Biology, University of Torino, 10123 Torino, Italy
Department of Oncology, University of Torino, 10126 Torino, Italy
b
c
A R T I C L E I N F O
A B S T R A C T
We herein report a study on a set of hybrid compounds in which 3-R-substituted furoxan moieties (R = CH ,
Keywords:
3
NO-donors
2 2 6 5
CONH , CN, SO C H ), endowed with varying NO-releasing capacities, are joined to a mitochondrial probe,
Furoxans
rhodamine B. Each product has been investigated for its ability to release NO both in physiological solution, in
the presence of cysteine, and in A549 lung adenocarcinoma cancer cells. The cytotoxicity of all the products
against the aforementioned cancer cells has been assessed, including the structurally related compounds with no
Mitochondrial Targeting
Anticancer Activity
Lysosomal Targeting
mitochondrial targeting, which were taken as a reference. In the case of the models bearing the –CH
CONH groups at the 3-position on the furoxan, only the targeted models showed a significant cytotoxic ac-
tivity, and only at the highest concentrations, in accordance with their weak NO-releasing properties. On the
contrary, the presence of the strong electron-withdrawing groups, as –CN and -SO , at the 3-position gave
rise to anticancer agents, likely because of the high NO-releasing and of their capability of inhibiting cellular
proteins by covalent binding. In detail, the rhodamine hybrid containing the 3-SO substituted furoxan
3
and
–
2
2 6 5
C H
2 6 5
C H
moiety emerged as the most interesting product as it showed high cytotoxicity over the entire concentration
range tested. This substructure was also linked to a phenothiazine scaffold that is able to accumulate in lyso-
somes. Nevertheless, mitochondrial targeting for these NO-donor furoxan substructures was found to be the most
efficient.
1
. Introduction
Furoxan (1,2,5-oxadiazole 2-oxide, Chart 1) is a heterocycle system
inflammatory drugs, enzyme inhibitors, multi-drug resistance (MDR)
reversal agents and active natural products [14–15].
The most important cellular functions are localised inside cell or-
ganelles, thus, cytotoxic agents with innate tropism for these sites have
increased efficacy. Drugs frequently lack affinity for a specific organelle
and diffuse randomly within cells. The design of hybrid compounds
obtained by joining drugs with vectors that can accumulate into one
specific site, with particular attention to mitochondrion and lysosome, is
an interesting strategy for overcoming this problem [16–20].
whose complex chemistry has been discussed in several reviews [1–4].
There is currently great interest in furoxan derivatives due to their
ability to release nitric oxide (NO) in physiological conditions under the
action of thiol cofactors [5–7]. As NO-prodrugs, furoxans display a wide
range of biological activities, including anticancer properties [8]. The
conjugation of NO-donor furoxan moieties with appropriate native
drugs has been used as a strategy to generate a variety of hybrid struc-
tures, which act against more than one target simultaneously [8–13].
The 3-phenylsulfonylfuroxan moiety, a potent NO-donor, has been
frequently used in the design of hybrid anticancer structures. It has been
combined with antibiotics, cytotoxic drugs, non-steroidal anti-
Mitochondria are key organelles that play a main role in the regu-
lation of the cell cycle. As they exert a variety of functions, including
energy production, redox metabolism and apoptosis, mitochondria are
considered important targets for cancer drugs [21–22]. The design of
hybrid molecules in which cytotoxic agents are conjugated with
Abbreviations: MDR, multi-drug resistance; LCD, lysosomal-mediated cellular death; LMP, lysosomal membrane permeabilization; ROS, reactive oxygen species;
FR, folate receptors; PBS, phosphate buffer saline; LDH, lactate dehydrogenase.
*
Corresponding author.
E-mail address: loretta.lazzarato@unito.it (L. Lazzarato).
https://doi.org/10.1016/j.bioorg.2021.104911
Received 20 November 2020; Received in revised form 5 January 2021; Accepted 7 April 2021
Available online 20 April 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

F. Sodano et al.
Bioorganic Chemistry 111 (2021) 104911
Chart 1. Chemical structure of hybrid and reference compounds.
mitochondrial-targeting lipophilic cations, such as triphenylphospho-
nium and rhodamine moieties, [23–25] represents a promising approach
for the development of new antitumor drugs.
mediates toxicity [31,32]. NO exerts profound effects on mitochondria.
Low NO concentrations modulate the mitochondrial electron transport
chain and stimulate mitochondrial biogenesis. By contrast, high NO
concentrations induce toxicity, principally via the inhibition of the
tricarboxylic acid cycle and mitochondrial respiration [33]. NO is also
involved in the regulation of lysosomal functions, although this aspect is
still in need of thorough investigation. For this reason, there is great
interest in NO tracking across lysosomes [34–35].
On this basis, we have designed a set of hybrid compounds 1, 2, 3 and
4 (Chart 1), in which furoxan substructures that are endowed with
different NO-releasing capacities are joined to rhodamine B, a mito-
chondrial probe recently investigated by us [25]. The related non-
mitochondrion-targeting furoxan derivatives 5, 6, 7, 8 and the rhoda-
mine derivative 9 (Chart 1) were synthesised as reference compounds. In
the chemical structure of compounds 5–8, the furoxan substructure was
linked, through an appropriate bridge, to a phenyl substituted moiety
bearing a bromine atom in the o-position. The bromine substituent is
important to maintain a comparable lipophilic/hydrophilic balance
with the rhodamine final derivatives.
Lysosomes are acidic organelles (pH ≤ 5) and the site of hydrolytic
enzymes, which are implicated in cellular digestion, immune responses,
inflammasome activation, and drug resistance in cancer [26–27]. Also,
they are involved in lysosomal-mediated cellular death (LCD), a process
caused by lysosomal membrane permeabilisation (LMP) [26]. Depend-
ing on the degree of permeabilization, lysosomes can induce the release
of enzymes in the cytosol, and thus give rise to apoptosis, autophagy and
necrosis [26–28]. Cancer cells have large lysosomes that are endowed
with a more fragile membrane than non-transformed cells, and are,
therefore, more inclined to undergo LMP [28]. For these reasons, lyso-
somes are considered to be important targets for cancer therapy,
[
26,28–30] and the conjugation of agents that can provoke LCD to
lipophilic vectors that display tropism for these loci has high interest.
NO is an important ubiquitous gaseous messenger, whose role de-
pends on the dosage. At low concentrations (pM, nM), it displays pro-
tective and regulatory effects, while, at high concentrations ( M), it
μ
2

F. Sodano et al.
Bioorganic Chemistry 111 (2021) 104911
The promising data regarding the mitochondrial targeting of the 3-
NMR (150 MHz, CDCl
3
) δ = 157.2, 113.0, 44.4, 43.8, 15.2, 7.9.
phenylsulfonylfuroxan substructure has also driven us to link this moi-
ety to a lysosomal probe. Compound 10, in which the 3-phenylsulfonyl-
furoxan moiety was linked to a phenothiazine vector that displays
lysosomal tropism, [35] and its reference compound 11, were syn-
thesised and evaluated (Chart 1).
4-((2-Bromo-N-ethylbenzamido)methyl)-3-methyl-1,2,5-oxadiazole 2-
oxide (5). A solution of 2-bromobenzoic acid 16 (1.35 mmol, 272 mg) in
2
dry DCM (10 mL) was refluxed with SOCl (4.05 mmol, 0.1 mL) for 12 h.
The solvent was evaporated and the residue was taken up with toluene
(30 mL), concentrated under reduced pressure three times in order to
obtain 17 as a yellow oil. This intermediate was dissolved in dry DCM
(10 mL), and compound 14 (1.35 mmol, 212 mg) and an excess of
triethylamine (0.6 mL) were added, and the resulting solution was
stirred for 3 h. The reaction mixture was washed with saturated sodium
bicarbonate solution (2 × 15 mL), water (2 × 15 mL) and 1 M HCl (2 ×
The synthesis, lipophilicity and NO release of all compounds have
been reported in this paper, togheter with the study of the reactivity of
the furoxan ring towards thiol cofactors. The cytotoxicity of the hybrid
molecules against A549 lung adenocarcinoma cancer cells had been
compared with that of their reference compounds. Moreover, intracel-
lular localisation in the same cell type was studied via confocal micro-
scopy for compound 3, and by fluorescence microscopy for compounds 4
and 10.
2 4
15 mL), then dried over anhydrous Na SO and concentrated to dryness.
Purification of the residue via silica gel chromatography, using DCM/
Acetone (99/1, v/v) as the eluent, gave the target compound 5 as a white
1
solid (300 mg, 65%). H NMR (600 MHz, CDCl
3
) δ = 7.57 (d, J = 8.1 Hz,
2
. Experimental section
1H), 7.40 – 7.36 (m, 1H), 7.30 – 7.23 (m, 2H), 5.07 (d, J = 14.9 Hz, 1H),
.54 (d, J = 14.9 Hz, 1H), 3.28 – 3.18 (m, 2H), 2.33 (s, 3H), 1.11 (t, J =
4
1
3
2
.1. Synthesis
7.1 Hz, 3H). C NMR (150 MHz, CDCl
3
) δ = 169.5, 154.7, 137.3, 133.1,
1
30.8, 127.9, 127.7, 119.1, 113.2, 42.8, 37.8, 13.4, 8.3. ESI-MS [M +
+
+
All reactions involving air-sensitive reagents were performed under
H] : m/z 340.3 and [M + H + 2] : m/z 342.3. HPLC purity ≥ 95%
nitrogen in oven-dried glassware using the syringe-septum cap tech-
nique. All solvents were purified and degassed before use. Chromato-
graphic separation was carried out under pressure on Merck silica gel 60
using flash-column techniques. Reactions were monitored using thin-
layer chromatography (TLC) carried out on 0.25 mm silica-gel-coated
aluminium plates (60 Merck F254) and using UV light (254 nm) as
visualising agent. Unless otherwise specified, all reagents were used as
received without further purification. Dichloromethane was dried over
(CH CN 0.1% TFA/H O 0.1% TFA 70:30 (v/v), flow = 1.0 mL/min, t
3.7 min), at 226 and 254 nm.
4-((2-(6-(Diethylamino)-3-(diethyliminio)–3H-xanthen-9-yl)-N-ethyl-
3
2
R
=
benzamido)methyl)-3-methyl-1,2,5-oxadiazole 2-oxide (1). A solution of
N-(9-(2-(chlorocarbonyl)phenyl)-6-(diethylamino)–3H-xanthen-3-yli-
dene)-N-ethylethanaminium 18 (1.29 mmol, 642 mg) [25] in dry DCM
(30 mL) was treated with compound 14 (1.29 mmol, 203 mg) and an
excess of triethylamine (0.6 mL) and the resulting solution was stirred
for 24 h. The reaction mixture was washed with saturated sodium bi-
carbonate solution (2 × 15 mL), water (2 × 15 mL) and 1 M HCl (2 × 15
2 5
P O and freshly distilled under nitrogen prior to use. DMF was stored
over 3 Å molecular sieves. Anhydrous THF was freshly distilled under
nitrogen from Na/benzophenone ketyl. Chemical shifts (δ) are given in
parts per million (ppm) and the coupling constants (J) in Hertz (Hz). The
following abbreviations were used to designate the multiplicities: s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet, bs = broad
singlet. ESI spectra were recorded on a Micromass Quattro API micro
2 4
mL), then dried over anhydrous Na SO and concentrated to dryness.
Purification of the residue via silica gel chromatography, using DCM/
MeOH (90/10, v/v) as the eluent, gave target compound 1 as a mirrored
1
purple solid (600 mg, 75%). H NMR (600 MHz, CDCl
3
) δ = 7.72 – 7.69
(m, 2H), 7.59 – 7.56 (m, 1H), 7.44 – 7.34 (m, 1H), 7.17 (d, J = 9.5 Hz,
2H), 6.90 – 6.86 (m, 2H), 6.73 (d, J = 2.4 Hz, 2H), 4.41 (s, 2H), 3.67 –
6.57 (m, 8H), 3.28 (q, J = 6.9 Hz, 2H), 1.87 (s, 3H), 1.33 (t, J = 7.0 Hz,
(
Waters Corporation, Milford, MA, USA) mass spectrometer. Data were
processed using a MassLynx System (Waters).
1
3
The purity of the final compounds was determined by RP-HPLC.
Analyses were performed with a HP1100 chromatograph system (Agi-
lent Technologies, Palo Alto, CA, USA) equipped with a quaternary
pump (G1311A), a membrane degasser (G1379A), and a diode-array
detector (DAD) (G1315B) integrated into the HP1100 system. Data an-
alyses were processed using a HP ChemStation system (Agilent Tech-
nologies). The analytical column was a LiChrospher® 100 C18-e (250 ×
12H), 1.21 (t, J = 7.1 Hz, 3H). C NMR (150 MHz, CDCl ) δ = 169.0,
3
157.7, 155.7, 155.1, 154.2, 135.4, 132.0, 130.4, 130.2, 126.6, 114.0,
+
113.7, 112.6, 96.3, 46.2, 44.5, 37.9, 13.8, 12.7, 7.8. ESI-MS [M] : m/z
582.7. HPLC purity ≥ 95% (CH
3 2
CN 0.1% TFA/H O 0.1% TFA 70:30 (v/
v), flow = 1.0 mL/min, t = 9.3 min), at 226, 254 and 580 nm.
R
3-Carbamoyl-4-((ethylamino)methyl)-1,2,5-oxadiazole 2-oxide (15). A
solution of 4-(bromomethyl)-3-carbamoyl-1,2,5-oxadiazole 2-oxide 13
4
.6 mm, 5 µm) (Merck KGaA, 64,271 Darmstadt, Germany) eluted with
(4.50 mmol, 1 g)[37] in CH
ethylamine solution 70 wt% in H
room temperature. After the reaction was complete, the mixture was
extracted with DCM, washed with H O, brine and dried over anhydrous
Na SO . The organic layer was concentrated to dryness to give target
compound 15 (502 mg, 60%). H NMR (600 MHz, CDCl
2H), 2.68 (q, J = 7.1 Hz, 2H), 1.95 (s, 1H), 1.12 (t, J = 7.1 Hz, 3H).
NMR (150 MHz, CDCl
) δ = 157.0, 156.8, 111.1, 44.7, 43.1, 15.1.
3
CN (12 mL) was treated with an excess of an
CH CN 0.1% TFA/H O 0.1% TFA in a ratio that depended on the
3
2
2
O (22.5 mmol, 1.81 mL) for 2 h at
characteristics of the compound. All compounds were dissolved in the
mobile phase at a concentration of about 0.01 mg/ml and injected
2
through a 20
μ
L loop. HPLC retention times (t
R
) were obtained at flow
2
4
1
rates of either 1.0 or 1.2 mL/min and the column effluent was monitored
using the DAD. The DAD acquired the UV spectra in the range from 190
to 800 nm, and the HPLC chromatogram was recorded at 226, 254, 580
and 660 nm (with 800 nm as the reference wavelength). The purity of
the test samples was evaluated as the percentage ratio between the areas
of the main peak and of possible impurities at the three wavelengths,
and also using a DAD purity analysis of the chromatographic peak. The
purity of all the target compounds was found to be ≥ 95%.
3
) δ = 4.08 (s,
13
C
3
4-((2-Bromo-N-ethylbenzamido)methyl)-3-carbamoyl-1,2,5-oxadiazole
2-oxide (6). The intermediate 17 (2.29 mmol, 501 mg) was dissolved in
dry DCM (15 mL), and compound 15 (1.8 mmol, 335 mg) and an excess
of triethylamine (0.7 mL) were added and the resulting solution was
stirred for 2 h. The reaction mixture was washed with a saturated so-
dium bicarbonate solution (2 × 15 mL), water (2 × 15 mL) and 1 M HCl
4
-((Ethylamino)methyl)-3-methyl-1,2,5-oxadiazole 2-oxide (14). A so-
lution of 4-(bromomethyl)-3-methyl-1,2,5-oxadiazole 2-oxide 12 (4.14
mmol, 800 mg)[36] in CH CN (10 mL) was stirred for 3 h at room
temperature with an ethylamine solution 70 wt% in H O (20.7 mmol,
.68 mL). After the reaction was complete, the mixture was extracted
(2 × 15 mL), then dried over anhydrous Na
2 4
SO and concentrated to
dryness. Purification of the residue via silica gel chromatography, using
DCM/Acetone (96/4, v/v) as the eluent, gave the target compound 6 as a
white solid (300 mg, 65%). The 1H and C NMR spectra of the com-
3
2
13
1
with DCM, washed with H
2
O, brine and dried over anhydrous Na
2
SO
4
.
pound showed the presence of two rotamers, which are caused by the
1
The organic layer was concentrated to dryness to give target compound
carbamoyl function. H NMR (600 MHz, CDCl
3
) δ = 7.62 – 7.58 (m, 1H),
1
1
4 (450 mg, 69%). H NMR (600 MHz, CDCl
3
) δ = 3.83 (s, 2H), 2.68 (q,
7.42 – 7.37 (m, 1H), 7.36 – 7.33 (m, 1H), 7.32 – 7.28 (m, 1H), 6.06 (s) &
5.97 (s) (2H), 5.25 (d, J = 17.4 Hz) & 4.92 (d, J = 17.5 Hz) (1H), 4.85 (d,
1
3
J = 7.1 Hz, 2H), 2.18 (s, 3H), 1.28 (s, 1H), 1.10 (t, J = 7.1 Hz, 3H).
C
3

F. Sodano et al.
Bioorganic Chemistry 111 (2021) 104911
J = 18.4 Hz) & 4.61 (d, J = 18.5 Hz) (1H), 3.43 – 3.29 (m) & 3.26 – 3.21
126.8, 114.3, 113.6, 105.0, 97.0, 96.2, 46.2, 45.1, 39.0, 13.9, 12.6. ESI-
1
3
+
(
m) (2H), 1.32 (t, J = 7.1 Hz) & 1.16 (t, J = 7.2 Hz) (3H). C NMR (150
MS [M] : m/z 593.5. HPLC purity ≥ 95% (CH
3
CN 0.1% TFA/H
2
O 0.1%
MHz, CDCl
3
) δ = 169.7, 156.3, 155.4, 137.7 & 137.5, 133.1 & 133.0,
TFA 70:30 (v/v), flow = 1.0 mL/min, t
R
= 9.5 min), at 226, 254 and 580
1
4
30.8 & 130.6, 128.1 & 128.0, 127.8 & 127.7, 119.3 & 119.2, 111.0,
nm.
+
4.8 & 44.4, 41.0, 14.0 & 12.4. ESI-MS [M + H] : m/z 369.3 and [M +
2-Bromo-N-ethyl-N-(2-hydroxyethyl)benzamide (19). Intermediate 17
(2.29 mmol, 501 mg), dissolved in dry DCM (15 mL), was treated with 2-
(ethylamino)ethanol (22.9 mmol, 2.24 mL) and stirred for 3 h at room
temperature. After the reaction was complete, the mixture was washed
with water (2 × 15 mL), a saturated sodium bicarbonate solution (2 ×
+
H + 2] : m/z 371.3. HPLC purity ≥ 95% (CH
3 2
CN 0.1% TFA/H O 0.1%
TFA 70:30 (v/v), flow = 1.0 mL/min, t
-Carbamoyl-4-((2-(6-(diethylamino)-3-(diethyliminio)–3H-xanthen-
-yl)-N-ethylbenzamido)methyl)-1,2,5-oxadiazole 2-oxide (2). A solution
of
N-(9-(2-(chlorocarbonyl)phenyl)-6-(diethylamino)–3H-xanthen-3-
ylidene)-N-ethylethanaminium (1.29 mmol, 642 mg) [25] in dry DCM
30 mL) was treated with compound 15 (240 mg, 1.29 mmol) and an
R
= 4.2 min), at 226 and 254 nm.
3
9
15 mL) and 1 M HCl (2 × 15 mL), then dried over anhydrous Na
2 4
SO .
The organic layer was concentrated to dryness to give target compound
19 (311 mg, 50%). 1H and C NMR spectra of the compound showed
13
(
excess of triethylamine (0.5 mL), and the resulting solution was stirred
overnight. The reaction mixture was washed with saturated sodium bi-
carbonate solution (2 × 15 mL), water (2 × 15 mL) and 1 M HCl (2 × 15
the presence of two rotamers, which are caused by the carbamoyl
1
function. H NMR (600 MHz, CDCl
3
) δ = 7.59 – 7.56 (m) & 7.54 – 7.52
(m) (1H), 7.37 – 7.30 (m, 1H), 7.28 – 7.19 (m, 2H), 3.94 – 3.88 (m) &
3.88 – 3.81 (m) (2H), 3.67 – 3.51 (m, 1H), 3.38 – 3.29 (m) & 3.28 – 3.16
2 4
mL), then dried over anhydrous Na SO and concentrated to dryness.
1
3
Purification of the residue via silica gel chromatography, using DCM/
(m) (2H), 1.26 (t, J = 7.1 Hz) & 1.07 (t, J = 7.2 Hz) (3H). C NMR (150
MeOH (98/2, v/v) as the eluent, gave target compound 2 as a mirrored
MHz, CDCl
3
) δ = 170.9 & 169.3, 138.5 & 138.2, 133.0 & 132.8, 130.5 &
1
13
purple solid (144 mg, 18%). H and C NMR spectra of the compound
130.2, 128.3 & 127.8, 127.7 & 127.6, 119.3 & 119.3, 62.2 & 60.3, 49.9
& 48.5, 45.2 & 40.2, 14.0 & 12.4.
showed the presence of two rotamers, which are caused by the carba-
1
moyl function. H NMR (600 MHz, CDCl
3
) δ = 7.71 – 7.64 (m, 2H), 7.63 –
4-(2-(2-Bromo-N-ethylbenzamido)ethoxy)-3-(phenylsulfonyl)-1,2,5-
oxadiazole 2-oxide (8). 3,4-bis(phenylsulfonyl)furoxan 20 (1.10 mmol,
403 mg) and 1,8-diazabicyclo[5.4.0]undec-7-ene (2.20 mmol, 0.328
mL) were added to a solution of compound 19 (1.10 mmol, 300 mg) in
dry DCM (10 mL). The resulting mixture was stirred for 4 h at room
temperature, subsequently washed with water (3 × 15 mL) and dried
7
.59 (m, 1H), 7.38 – 7.35 (m, 1H), 7.20 (d, J = 9.5 Hz, 2H), 6.95 – 6.89
(
m, 2H), 6.87 – 6.83 (m, 2H), 4.63 (s) & 4.58 (s) (2H), 3.73 – 3.55 (m,
8
7
H), 3.31 – 3.28 (m) & 3.21 – 3.16 (m) (2H), 1.67 (bs, 2H), 1.31 (t, J =
13
.0 Hz, 12H), 1.06 (t, J = 7.1 Hz) & 0.78 (t, J = 7.0 Hz) (3H). C NMR
(
150 MHz, CDCl
3
) δ = 169.6, 157.8, 155.6, 155.4, 154.5, 153.8, 136.1,
1
4
0
32.0, 130.4, 130.0, 129.8, 127.3, 113.8, 113.7, 110.6, 96.7, 46.2, 45.3,
2 4
over anhydrous Na SO . The organic layer was concentrated to dryness.
+
0.4, 13.9, 12.7. ESI-MS [M] : m/z 611.6. HPLC purity ≥ 95% (CH
3
CN
The crude product was purified via silica gel chromatography using
DCM/Acetone (98/2, v/v) and the desired compound 8 was obtained as
a colourless oil (430 mg, 79%). 1H and C NMR spectra of the com-
.1% TFA/H
2
O 0.1% TFA 70:30 (v/v), flow = 1.0 mL/min, t
R
= 7.1
13
min), at 226, 254 and 580 nm.
-((2-Bromo-N-ethylbenzamido)methyl)-3-cyano-1,2,5-oxadiazole 2-
4
pound showed the presence of two rotamers, which are caused by the
1
oxide (7). Trifluoroacetic anhydride (2.04 mmol, 0.290 mL) and pyri-
dine (1.02 mmol, 0.083 mL) were added to a solution of compound 6
carbamoyl function. H NMR (600 MHz, CDCl
3
) δ = 8.08 – 8.02 (m, 1H),
7.80 – 7.73 (m, 1H), 7.67 – 7.54 (m, 1H), 7.43 – 7.37 (m, 1H), 4.85 –
4.70 (m) & 4.52 – 4.45 (m) & 4.42 – 4.35 (m) (2H), 4.20 – 4.12 (m) &
4.08 – 4.01 (m) (1H), 3.90 – 3.82 (m) & 3.76 – 3.69 (m) (1H), 3.63 – 3.56
(m) & 3.50 – 3.42 (m) & 3.36 – 3.29 (m) (1H), 1.34 (t, J = 7.1 Hz) & 1.15
(
1.02 mmol, 377 mg) in dry DCM (20 mL), under positive N
2
pressure at
◦
0
C. The resulting solution was stirred for 3 h and subsequently washed
with saturated sodium bicarbonate solution (2 × 15 mL), water (1 × 15
mL) and brine (1 × 15 mL). The organic layer was dried over anhydrous
1
3
(t, J = 7.1 Hz) (3H). C NMR (150 MHz, CDCl ) δ = 169.6, 158.9, 138.1
3
Na
2
SO
4
and concentrated to dryness. The purification of the residue via
& 137.90, 136.0 & 135.8, 133.0 & 132.9, 130.6 & 130.5, 129.9 & 129.8,
128.8 & 128.7, 128.3 & 128.0, 127.9 & 127.6, 119.3, 110.7, 69.4 &
silica gel chromatography, using petroleum ether/EtOAc (80/20, v/v) as
1
+
the eluent, gave target compound 7 as a white solid (144 mg, 40%). H
68.89, 46.0 & 45.0, 43.4 & 40.4, 14.16 & 12.5. ESI-MS [M + H] : m/z
1
3
+
and C NMR spectra of the compound showed the presence of two
496.3 and [M + H + 2] : m/z 498.3. HPLC purity ≥ 95% (CH
3
CN 0.1%
1
rotamers, which are caused by the carbamoyl function. H NMR (600
TFA/H
2
O 0.1% TFA 70:30 (v/v), flow = 1.0 mL/min, t
R
= 7.0 min), at
MHz, CDCl
3
) δ = 7.62 – 7.58 (m, 1H), 7.42 – 7.37 (m, 1H), 7.36 – 7.33
226 and 254 nm.
(
m, 1H), 7.32 – 7.28 (m, 1H) 4.96 (d, J = 15.2 Hz, 1H), 4.71 (d, J = 15.2
4-(2-(2-(6-(Diethylamino)-3-(diethyliminio)–3H-xanthen-9-yl)-N-eth-
ylbenzamido)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (4). A
solution of N-(9-(2-(chlorocarbonyl)phenyl)-6-(diethylamino)–3H-
xanthen-3-ylidene)-N-ethylethanaminium 18 (0.96 mmol, 477 mg) [25]
in dry DCM (10 mL) was treated with 2-(((3-phenylsulfonylfuroxan-4-yl)
oxy)ethyl)ethylamine 24 (0.96 mmol, 300 mg) [38] and an excess of
triethylamine (0.4 mL), and the resulting solution was stirred for 24 h.
The reaction mixture was washed with water (2 × 15 mL) and a satu-
rated sodium bicarbonate solution (4 × 15 mL), then dried over anhy-
Hz, 1H), 3.45 – 3.28 (m) & 3.24 – 3.18 (m) (2H), 1.33 (t, J = 7.1H) &
1
3
1
.19 (t, J = 7.2 Hz) (3H). C NMR (150 MHz, CDCl
3
) δ = 169.9, 153.9,
1
36.7, 133.1, 131.0, 127.9, 119.2, 105.5, 97.0, 44.8 & 43.2, 39.9, 13.9
+
+
&
11.9. ESI-MS [M + Na] : m/z 373.3 and [M + Na + 2] : m/z 375.3.
CN 0.1% TFA/H O 0.1% TFA 70:30 (v/v), flow
= 5.06 min), at 226 and 254 nm.
HPLC purity ≥ 95% (CH
1.0 mL/min, t
3
2
=
R
3
-Cyano-4-((2-(6-(diethylamino)-3-(diethyliminio)–3H-xanthen-9-yl)-
N-ethylbenzamido)methyl)-1,2,5-oxadiazole 2-oxide (3). Trifluoroacetic
anhydride (0.346 mmol, 0.050 mL) and pyridine (0.173 mmol, 0.017
2 4
drous Na SO and concentrated to dryness. Purification of the residue
mL) were added to a solution of compound 2 (0.173 mmol, 112 mg) in
via silica gel chromatography, using DCM/Acetone (80/20, v/v) as the
◦
dry DCM (15 mL), under positive N
2
pressure at 0 C. The resulting
eluent, gave target compound 4 as a mirrored purple solid (200 mg,
1
13
solution was stirred for 3 h and subsequently washed with a saturated
27%). H and C NMR spectra of the compound showed the presence of
1
sodium bicarbonate solution (2 × 15 mL), water (2 × 15 mL) and brine
two rotamers, which are caused by the carbamoyl function. H NMR
(
2 × 15 mL). The organic layer was dried over anhydrous Na
2
SO
4
and
(600 MHz, CDCl
3
) δ = 8.05 (d, J = 7.8 Hz) & 7.99 (d, J = 7.7 Hz) & 7.93
concentrated to dryness. Purification of the residue via silica gel chro-
matography, using DCM/MeOH (98/2, v/v) as the eluent, gave target
compound 3 as a mirrored purple solid (54 mg, 50%). 1H NMR (600
(d, J = 7.7 Hz) (2H), 7.78 – 7.67 (m) & 7.65 – 7.60 (m) (6H), 7.38 – 7.29
(m, 3H), 6.95 – 6.90 (m) & 6.80 – 6.74 (m) (2H), 6.70 (d, J = 2.1 Hz,
2H), 3.99 (t, J = 4.8 Hz, 2H), 3.72 – 3.55 (m, 8H), 3.33 (q, J = 6.9 Hz,
2H), 1.34 – 1.23 (m, 12H), 1.17 (t, J = 7.0 Hz) & 0.63 (t, J = 6.9 Hz)
MHz, CDCl
m, 1H), 7.20 (d, J = 9.5 Hz, 2H), 6.98 – 6.94 (m, 2H), 6.72 (d, J = 2.4
Hz, 2H), 4.50 (s, 2H), 3.69 – 3.55 (m, 8H), 3.36 (q, J = 6.9 Hz, 2H), 1.32
3
) δ = 7.74 – 7.69 (m, 2H), 7.62 – 7.59 (m, 1H), 7.41 – 7.38
(
(3H). 13C NMR (150 MHz, CDCl
3
) δ = 169.3, 158.4, 157.7, 155.7, 137.8,
136.1 & 136.0, 132.3, 130.6 & 130.6, 130.1 & 130.0, 129.9, 128.6,
128.3, 127.2, 114.2, 113.4, 110.3, 96.4 and 96.2, 69.6 & 69.5, 53.9,
1
3
(
t, J = 7.0 Hz, 12H), 1.22 (t, J = 7.0 Hz, 3H). C NMR (150 MHz, CDCl
3
)
+
δ = 169.4, 157.7, 155.7, 155.0, 152.9, 132.0, 130.5, 130.4, 130.3,
46.2 & 45.5, 43.0, 31.9 & 31.1, 29.4, 14.2, 12.7. ESI-MS [M] : m/z
4

F. Sodano et al.
Bioorganic Chemistry 111 (2021) 104911
1
7
38.6. HPLC purity ≥ 95% (CH
v), flow = 1.0 mL/min, t = 10.4 min), at 226, 254 and 580 nm.
-(2-Aminoethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (21).
3
CN 0.1% TFA/H
2
O 0.1% TFA 70:30 (v/
blue solid (55 mg, 86%). H NMR (600 MHz, CD
3
OD) δ = 8.82 (d, J =
R
8.1 Hz, 1H), 8.20 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 9.4 Hz, 1H), 7.79 (t, J
= 7.5 Hz, 1H), 7.63 – 7.68 (m, 1H), 7.30 – 7.26 (m, 1H), 7.13 (s, 1H),
7.09 (d, J = 2.7 Hz, 1H), 3.66 (q, J = 7.2 Hz, 4H), 3.60 (t, J = 6.9 Hz,
2H), 3.25 – 3.16 (m, 2H), 2.33 (t, J = 7.0 Hz, 2H), 1.88 – 1.72 (m, 4H),
4
Ethanolamine (3.0 mmol, 0.180 mL) and sodium hydride (2.0 mmol, 80
◦
mg) at 0 C were added to a solution of 3,4-bis(phenylsulfonyl)furoxan
1
3
2
0 (1.0 mmol, 366 mg) in anhydrous THF (15 mL). The solution was
1.34 (t, J = 7.2 Hz, 6H), 1.13 (t, J = 7.3 Hz, 3H). C NMR (150 MHz,
slowly warmed to room temperature and stirred for 2 h. The reaction
mixture was concentrated in vacuo, dissolved in 15 mL of water and
extracted with EtOAc (3 × 10 mL). The organic layer was collected,
washed with water (25 mL) and brine (25 mL) sequentially, then dried
CD
3
OD) δ = 175.5 & 175.4, 154.6, 152.7, 141.4, 138.5, 135.2, 134.8,
134.1, 133.3, 132.2, 130.8, 126.3, 125.5, 123.5, 118.7, 106.1, 103.3,
+
46.9, 45.2, 36.3, 35.4 & 35.2, 29.1, 24.3, 14.9, 13.1. ESI-MS [M] : m/z
461.6. HPLC purity ≥ 95% (CH
3 2
CN 0.1% TFA/H O 0.1% TFA 80:20 (v/
over anhydrous Na
SO
2 4
and concentrated to dryness. The crude product
sat)
90/10, v/v) and the desired compound 21 was obtained as a white solid
v), flow = 1.2 mL/min, t = 16.9 min), at 226, 254 and 660 nm.
R
was purified via silica gel chromatography using DCM/MeOH (NH
3
(
(
2.2. Determination of lipophilicity descriptor (log P)
1
60 mg, 56%). H NMR (600 MHz, CDCl
3
) δ = 8.08 – 8.05 (m, 2H), 7.79 –
m, 2H), 1.25 (bs, 2H). 1 C NMR (150 MHz, CDCl
) δ = 159.1, 138.1,
7
.74 (m, 1H), 7.65 – 7.60 (m, 2H), 4.46 (t, J = 5.2 Hz, 2H), 3.20 – 3.17
The partition coefficients (log P), between n-octanol and PBS (pH
7.4), of the target and reference compounds were obtained using the
shake-flask technique at room temperature. In the shake-flask experi-
ments, 50 mM PBS (pH 7.4) was used as the aqueous phase and ionic
strength was adjusted to 0.15 M using KCl. The organic (n-octanol), and
aqueous phases were mutually saturated via shaking for 4 h. The com-
pounds were solubilised in the buffered aqueous phase at concentrations
of either 0.1 mM or 0.05 mM, depending on their solubility, and
appropriate amounts of n-octanol were added. The two phases were
shaken for about 20 min, by which time the partitioning equilibrium of
the solutes had been reached, and then centrifuged (10000 rpm, 10
min). The concentration of the solutes was measured in the aqueous
phase using a UV spectrophotometer (UV-2501PC, Shimadzu). Each log
P value is an average of at least six measurements. All experiments were
performed avoiding exposure to light.
3
(
3
1
35.8, 129.8, 128.7, 73.6, 40.8.
Z)-4-Carboxy-N-(9-(diethylamino)-5H-benzo[a]phenothiazin-5-yli-
dene)butan-1-aminium (23). (Z)-N-(9-(diethylamino)-5H-benzo[a]phe-
nothiazin-5-ylidene)-5-ethoxy-5-oxopentan-1-aminium 22 was
(
synthesised as described by Sarika Verma et al. [39] starting from the
respective naphthyl derivative (0.59 mmol, 308 mg). Intermediate 22
was dissolved in a solution of dioxane/6 M HCl (50/50, v/v, 12 mL) and
kept under stirring at room temperature overnight. The solvent was
evaporated and the residue was taken up with DCM (20 mL), and
concentrated under reduced pressure twice. The crude product was
purified via silica gel chromatography using DCM/MeOH (95/5, v/v)
and the desired compound 23 was obtained as a blue solid (40 mg, 15%).
1
H NMR (600 MHz, CD
3
OD) δ = 8.43 – 8.31 (m, 1H), 8.04 – 7.90 (m,
1
–
5
1
3
H), 7.62 – 7.43 (m, 3H), 7.19 – 6.99 (m, 1H), 6.89 – 6.64 (m, 2H), 3.61
3.53 (m, 4H), 3.40 – 3.32 (m, 2H), 2.36 – 2.28 (m, 2H), 1.79 – 1.68 (m,
2.3. NO release in physiological solution
1
3
4), 1.32 – 1.21 (m, 6H). C NMR (150 MHz, CD OD) δ = 179.7, 152.8,
3
51.1, 130.7, 129.2, 124.6, 123.9, 117.1, 104.6, 101.7, 99.4, 45.5, 44.0,
5.9, 27.9, 23.1, 11.9.
NO release was evaluated as nitrite, produced during incubation in
the following conditions, and detected using the Griess reaction. All of
the compounds, except derivative 10, were incubated at 100 µM in a
mixture 50 mM pH 7.4 PBS solution/MeOH (1% DMSO) 50/50 v/v. The
compound with lysosomal targeting, 10, and its non-targeted reference,
8, however, were incubated at 100 µM in a mixture 50 mM pH 5.0 citrate
buffer solution / MeOH (1% DMSO) 50/50 v/v. The incubation of all
compounds was performed in the presence of L-cysteine at a 0.5 mM
concentration (a 5-fold excess compared to the NO-donor derivative).
(
Z)-4-(2-(5-((9-(Diethylamino)-5H-benzo[a]phenothiazin-5-ylidene)
ammonio)pentanamido)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole
2-
′
oxide (10). N-(3-dimethylaminopropyl)-N -ethylcarbodiimide hydro-
.
chloride (EDC HCl, 0.55 mmol, 106 mg), 1-hydroxybenzotriazole hy-
drate (HOBt, 0.63 mmol, 89 mg), 4-(dimethylamino)pyridine (DMAP,
0
.55 mmol, 69 mg) and intermediate 21 (0.55 mmol, 157 mg) were
added to a solution of compound 23 (0.42 mmol, 197 mg) in dry DMF
20 mL). The resulting mixture was stirred for 24 h at room temperature
◦
(
After 1 h at 37 C, the presence of nitrite in the sample was determined
and subsequently concentrated to dryness. Purification of the residue via
silica gel chromatography, using DCM/MeOH (97/3, v/v) as the eluent,
using the Griess assay: 1 mL of the reaction mixture was treated with
250 µL of the Griess reagent (4% w/v sulphanilamide, 0.2% w/v N-
naphthylethylenediamine dihydrochloride, 1.47 M phosphoric acid);
after 10 min at room temperature, the reaction mixture was analysed via
RP-HPLC to detect the azo dye.
1
gave the target compound 10 as a blue solid (155 mg, 50%). H NMR
(
600 MHz, CDCl
3
) δ = 8.96 – 8.91 (m, 1H), 8.78 (d, J = 8.1 Hz, 1H), 8.11
–
8.04 (m, 2H), 7.96 (d, J = 9.4 Hz, 1H), 7.78 – 7.66 (m, 3H), 7.60 (t, J =
7
4
3
1
.8 Hz, 2H), 7.13 – 7.09 (m, 1H), 7.03 (s, 1H), 6.85 (d, J = 2.7 Hz, 1H),
.53 (t, J = 5.6 Hz, 2H), 3.84 – 3.78 (m, 2H), 3.75 (q, J = 5.6 Hz, 2H),
.61 (q, J = 7.2 Hz, 4H), 2.48 (t, J = 7.3 Hz, 2H), 1.99 – 1.91 (m, 2H),
HPLC analyses were performed using a HP 1200 chromatograph
system (Agilent Technologies, Palo Alto, CA, USA) equipped with a
quaternary pump (model G1311A), a membrane degasser (G1322A),
and a multiple wavelength UV detector (MWD, model G1365D) inte-
grated into the HP1200 system. Data analysis was performed using a HP
ChemStation system (Agilent Technologies). The sample was eluted on a
1
3
.91 – 1.84 (m, 2H), 1.35 (t, J = 7.2 Hz, 6H). C NMR (150 MHz, CDCl
3
)
δ = 173.8, 158.8, 154.5, 148.4, 137.9, 135.7, 134.3, 133.1, 129.8,
1
2
29.5, 128.6, 124.7, 124.0, 110.5, 105.1, 70.4, 44.9, 38.1, 36.3, 30.2,
+
3.8, 12.8. ESI-MS [M] : m/z 701.7. HPLC purity ≥ 95% (CH
3
CN 0.1%
HyPURITY Elite C18 column (250 × 4.6 mm, 5
μ
m, Hypersil, Thermo-
TFA/H
2
O 0.1% TFA 80:20 (v/v), flow = 1.2 mL/min, t
R
= 11.1 min), at
Quest Corporation, UK). The injection volume was 20
μ
L (Rheodyne,
2
26, 254 and 660 nm.
Cotati, CA). The mobile phase consisted of CH CN 0.1% TFA (solvent A)
3
(
Z)-N-(9-(Diethylamino)-5H-benzo[a]phenothiazin-5-ylidene)-5-(ethyl-
and H O 0.1% TFA (solvent B) at flow-rate = 1.0 mL/min with gradient
2
′
amino)-5-oxopentan-1-aminium (11). N-(3-dimethylaminopropyl)-N -
conditions: 50% A to 4 min; from 50 to 90% A between 4 and 8 min; 90%
.
ethylcarbodiimide hydrochloride (EDC HCl, 0.17 mmol, 32 mg), 1-
A between 8 and 12 min; and from 90 to 50% A between 12 and 15 min.
The column effluent was monitored at 540 mn and referenced against a
800 nm wavelength. Data analysis was performed using Agilent Chem-
Station. The values obtained from the integration of the peak of azo dye
were interpolated in a calibration curve obtained using standard solu-
hydroxybenzotriazole hydrate (HOBt, 0.19 mmol, 26 mg), 4-(dimethy-
lamino)pyridine (DMAP, 0.17 mmol, 21 mg) and a 2 M ethylamine so-
lution in THF (0.64 mmol, 34 µL) were added to a solution of compound
2
3 (0.13 mmol, 60 mg) in dry DMF (10 mL). The resulting mixture was
2
stirred for 24 h at room temperature and subsequently concentrated to
dryness. Purification of the residue via silica gel chromatography, using
DCM/MeOH (98/2, v/v) as the eluent, gave the target compound 11 as a
tions of sodium nitrite at 0.5 µM to 50 µM (r = 0.996). The yield in
–
nitrite was expressed as percentage NO (mol/mol, relative to the initial
2
compound concentration) ± SEM.
5

F. Sodano et al.
Bioorganic Chemistry 111 (2021) 104911
2
.4. UPLC-MS analysis
was prepared for all compounds; this stock solution was diluted in cul-
ture medium to achieve the final concentration. The concentration of
DMSO in the culture medium was<0.1% under each experimental
condition. Control cells were treated with 0.1% DMSO. Preliminary
experiments showed that cells treated with 0.1% DMSO did not differ
from cells treated with culture medium without DMSO in any biological
assay (data not shown).
In order to detect the products formed in the reaction between the
furoxan derivatives and L-cysteine, hybrid compounds 3 and 4 were
incubated at 100 µM in a mixture of 50 mM pH 7.4 PBS solution/MeOH
(
1% DMSO) 50/50 v/v. The incubation of the two compounds was
◦
performed at 37 C in the presence of L-cysteine at a 0.5 mM concen-
tration (a 5-fold excess compared to the NO-donor derivative). At reg-
ular time intervals, the reaction mixture was filtered through a 13 mm
3.2. Cells
Syringe Filter w/0.45 m polytetrafluoroethylene membrane and the
μ
filtrate was analysed on a Acquity Ultra Performance LC, Waters Cor-
poration Milford MA, USA, equipped with BSM, SM, CM and a PDA
detector. The analytical column was a Phenomenex Synergi 4U POLAR-
Human lung adenocarcinoma A549 cells, provided by Istituto Zoo-
profilattico Sperimentale “Bruno Ubertini” (Brescia, Italy), were
cultured in Ham’s F12 medium supplemented with 10% foetal bovine
serum and 1% penicillin–streptomycin. Cell cultures were maintained in
RP80A, 150 × 2 mm⋅4
μ
3
m. The mobile phase consisted of CH CN 0.1%
◦
HCOOH and H O 0.1% HCOOH 80/20 v/v. The UPLC retention time (t
2
R
)
a humidified atmosphere at 37 C and 5% CO . When indicated, cells
2
ꢀ 1
was obtained at a flow rate of 0.4 mL min , and the column effluent was
monitored using a Micromass Quattro micro API, with Esci multimode
were incubated for 24 h with the compounds in Ham’s F12 before the
experimental procedures described below.
+
ionisation enabled, as the detector. The molecular ion [M] was
employed for quantitative measurements of the analytes. The MS con-
3.3. NO release in cells
◦
ditions were: drying gas (nitrogen) heated at 350 C at a flow rate of 800
L/h; nebuliser gas (nitrogen) at 80 L/h; capillary voltage in positive
mode at 3000 V; fragmentor voltage at 30.
After incubation, 1 mL of cell supernatant was collected, centrifuged
for 10 min at 13000g, and then the presence of nitrite in the reaction
mixture was determined using the Griess assay: 0.5 mL of the reaction
mixture was treated with 125 µL of the Griess reagent (4% w/v sul-
phanilamide, 0.2% w/v N-naphthylethylenediamine dihydrochloride,
1.47 M phosphoric acid); after 10 min at room temperature, the sample
was analysed using RP-HPLC to detect the azo dye, as previously
described in paragraph 2.3.
3
. Biological studies
3
.1. Chemicals
Cell culture medium was supplied by Invitrogen Life Technologies
(
Carlsbad, CA) and plasticware for cell cultures was obtained from
Falcon (Becton Dickinson, Franklin Lakes, NJ). The protein content of
cell monolayers and lysates was assessed using the BCA kit from Sigma
Chemical Co (St. Louis, MO). Unless otherwise specified, all reagents
were obtained from Sigma Chemical Co. A 5 mM stock solution in DMSO
3.4. Cytotoxicity
The cytotoxic effect of the compounds was measured as the leakage
of lactate dehydrogenase (LDH) activity into the extracellular medium
Scheme 1. Synthesis of target compounds 1–3 and related reference compounds 5–7. Reagents and conditions: a) CH
3 2 2 2 3 2
CH NH 70% wt. in H O, CH CN, rt; b) SOCl ,
◦
DCM, Δ; c) 14 or 15, Et
3
N, DCM, rt; d) (CF
3
CO)
2
O, pyridine, dry DCM, 0 C.
6

F. Sodano et al.
Bioorganic Chemistry 111 (2021) 104911
using a Synergy HT microplate reader (Bio-Tek Instruments, Winooski,
VT) [40]. This method is a sensitive index of cell necrosis – the type of
cell death expected from a damage in lysosome and mitochondria –
verified in different cancer types treated with antitumor agents releasing
NO [17,24,25,41]. Both intracellular and extracellular LDH were
measured, and the extracellular LDH activity (LDH out) was calculated
as a percentage of the total (intracellular + extracellular) LDH activity
60X). For compounds 4 and 10, slides were analysed using a Leica
DC100 microscope (Leica Microsystems GmbH, Wetzlar, Germany; 10X
ocular lens, 63X objective). For each experimental condition, a mini-
mum of 5 microscopic fields were examined. The ratio of yellow pixels/
green pixels (i.e., mitochondria containing compound 4/total mito-
chondria or lysosomes containing compound 10/total lysosomes) was
calculated with the ImageJ software (https://imagej.nih.gov/ij/).
(
LDH tot) in the dish. All compounds were tested in a concentration
range 0.1–50 µM, except for compounds 10 and 11, due to their poor
solubility in the cellular medium at 50 µM.
3
.6. Statistical analysis
All data are provided as means ± SEM. The results were analysed
using one-way Analysis of Variance (ANOVA) and Tukey’s test (soft-
ware: SPSS 21.0 for Windows, SPSS Inc., Chicago, IL). p < 0.05 was
considered significant.
3
.5. Intracellular localisation studies
5
5
× 10 A549 cells were grown on sterile glass coverslips and
transfected with the GFP-E1
CellLight™ Mitochondria-GFP, BacMam 2.0, Invitrogen Life Technol-
ogies) to label mitochondria, or with the GFP-Lamp1 expression vector
CellLight™ Lysosomes-GFP, BacMam 2.0, Invitrogen Life Technolo-
gies) to label lysosomes. After 24 h, mitochondrion-labelled cells were
incubated with 5 M of either compound 3 or compound 4 for 4 h, and
α
pyruvate dehydrogenase expression vector
(
4. Results and discussion
(
4.1. Chemistry
μ
The hybrid rhodamine derivative 1 and its reference compound 5
were prepared as depicted in Scheme 1. The treatment of 4-bromome-
thy-3-methylfuroxan (12) [36] with a 70% ethylamine water solution
afforded the formation of 4-ethylaminoethyl-3-methylfuroxan (14). A
dichloromethane (DCM) solution of this intermediate, containing an
excess of triethylamine, was treated with acyl chloride 17, which was, in
turn, obtained from the reaction between 16 and thionyl chloride,
lysosome-labelled cells with 5 µM of compound 10 for 4 h. Samples were
then rinsed with PBS, fixed with 4% w/v paraformaldehyde for 15 min,
washed three times with PBS and once with water, and mounted with 4
μ
L of Gel Mount Aqueous Mounting. For compound 3, slides were ana-
lysed using an Olympus FV300 laser scanning confocal microscope
Olympus Biosystems, Hamburg, Germany; ocular lens: 10X; objective:
(
Scheme 2. Synthesis of target compounds 4, 8, 10 and 11. Reagents and conditions: a) CH
3
CH
2
NHCH
CH
2
CH
CH
2
OH, DCM, rt; b) 20 (3,4-bis(phenylsulfonyl)furoxan),
◦
DBU, DCM, rt; c) 24 (2-(((3-phenylsulfonylfuroxan-4-yl)oxy)ethyl)ethylamine), Et N, DCM, rt; d) NH
3
2
2
2
OH, NaH, THF, 0 C; e) dioxane/6 M HCl (50/50, v/v)
.
.
rt; f) 21, EDC HCl, HOBt, DMAP, DMF, rt; g) 2 M CH
3
2
CH NH
2
in THF, EDC HCl, HOBt, DMAP, DMF, rt.
7

F. Sodano et al.
Bioorganic Chemistry 111 (2021) 104911
Table 1
yielding reference product 5. Under these same conditions, the final
product 1 was prepared starting from 18 [25]. Analogous procedures
were used to prepare 3-carbamoyl-4-ethylaminomethyl (15) from 13,
a
NO release and lipophilicity of the target compounds.
COMPOUND
NO release
Lipophilicity
[
37] and the final products 2 and 6 from 18 and 17, respectively
–
a
%
NO
2
(mol/mol) ± SEM
CLOGP (BioLoom)
log P ± SEM
(
Scheme 1).
5
1
6
2
7
3
8
4
0
0
2.13
2.24
1.56
1.67
2.84
2.94
4.66
4.65
6.94
1.96 ± 0.02
1.90 ± 0.07
1.63 ± 0.06
1.55 ± 0.03
2.60 ± 0.05
2.68 ± 0.09
nd
The cyano-substituted final products 3 and 7 were obtained via the
dehydration of the related amides 2 and 6 under the action of tri-
fluoroacetic anhydride and pyridine in dry DCM solutions (Scheme 1).
3-Phenylsulfonylfuroxan derivatives 4 and 8 were synthesised ac-
cording the pathways depicted in Scheme 2. Intermediate 17 was dis-
solved in dry DCM and allowed to react with an excess of 2-(ethylamino)
ethanol to afford 19. The nucleophilic displacement of the 4-phenylsul-
fonyl group of 3,4-diphenylfuroxan (20) by 19 in a dry DCM solution, in
the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), gave 8.
Finally, coupling between 18 and (2-{[(3-phenylsulfonylfuroxan-4-yl)
oxy]ethyl}ethylamine (24)[38] in dry DCM, which contained an excess
of trimethylamine, produced the last derivative of our series 4.
< 1
< 1
25 ± 1
17 ± 1
26 ± 7
24 ± 4
20 ± 1
nd
1
0
nd
a
calculated by BioLoom (BioLoom for windows v. 1.5, BioByte Corp. Clar-
emont, CA 91711–4707); nd = not determinable by this method (log P > 4).
Fig. 1. a-b. NO release in A549 cells after 24 h incubation with compounds 3 vs 7 (a) and 4 vs 8 vs 10 (b). Measurements were performed in duplicate and data are
presented as means ± SEM (n = 3) Vs untreated cells (CTRL): *p < 0.05; **p < 0.01, ***p < 0.001.
8

F. Sodano et al.
Bioorganic Chemistry 111 (2021) 104911
Fig. 2. a-c. Cytotoxicity of compounds 3 vs 7 (a), 4 vs 8 (b) and 10 vs 8 (c) against A549 lung adenocarcinoma cancer cells. Measurements were performed in
duplicate and data are presented as means ± SEM (n = 3). Vs untreated cells (CTRL): *p < 0.05, **p < 0.01, ***p < 0.001.
9

F. Sodano et al.
Bioorganic Chemistry 111 (2021) 104911
The synthesis of target compounds 10 and 11, which contain the
phenothiazine chromophore, is reported in Scheme 2. The addition of
detected in the supernatant using the Griess assay. The results are re-
ported in Fig. 1a-b and Figures S3a-b. The NO-release profiles of all the
hybrid derivatives were comparable with those of their reference com-
pounds; in the case of compounds 1 and 5, the release of a small amount
of NO was observed only at the highest concentrations (Figure S3a). The
same situation was observed with products 2 and 6 (Figure S3b). By
contrast, models 3 and 7, which bear the strongly electron-withdrawing
–CN group at the 3-position of the furoxan scaffold, showed a high
amount of NO release (Fig. 1a). The NO-releasing capability of 4, 8 and
3
,4-bis (phenylsulfonyl)furoxan (20) to a THF solution of ethanolamine
◦
and NaH at 0 C generated 21. The acid hydrolysis of the phenothiazine
ester 22 carried out in dioxane gave intermediate 23 [39]. This latter,
coupled with 21 in dry DMF under the action of 4-(dimethylamino)
′
pyridine (DMAP), N-ethyl-N -(3-dimethylaminopropyl)carbodiimide
.
hydrochloride (EDC HCl) and 1-hydroxybenzotriazole hydrate (HOBt),
afforded 10. Under these same conditions, the final product 11 was
prepared from 23 under the action of a THF solution of ethylamine.
6 5 2
10 was lower than that of 3 and 7, despite the -C H SO group being
endowed with electron-withdrawing properties comparable to those of
4
.2. Lipophilicity
the –CN group (Fig. 1b).
Lipophilicity is an important molecular descriptor that plays a
4.5. Cytotoxicity
leading role in the passive cellular absorption of a substance. The
partition coefficient (Log P) between n-octanol and water is normally
used to describe a product’s lipophilicity [42]. Log P of the final com-
pounds 1–3 and 5–7 was measured using the shake-flask technique and
Cytotoxicity was assessed by measuring the release of lactate dehy-
drogenase (LDH) to evaluate cell necrosis in A549 cells, incubated for
24 h with the compounds in the 0.1–50 µM concentration range
(Figures S4a-d and Fig. 2a-b). In cells, the thiol cofactors responsible for
NO release are both free thiols and the cysteinyl residues of proteins. In
the former, there is a depletion of the free cellular thiols, resulting in
increased oxidative stress, whereas in the latter, protein function is
altered (Figures S1 and S2). Thus, the cellular toxicity of the products
may be due to both their ability to covalently bind thiol biomolecules
and their NO-release.
5
0 mM PBS (pH 7.4) as the aqueous phase. The results are reported in
Table 1 and are compared with the corresponding CLOGP values,
showing a good agreement between experimental and calculated data.
The compounds bearing the mitochondrial targeting moiety (1–4) dis-
played a hydrophilic-lipophilic balance comparable to that of the cor-
responding reference compounds 5–8.
4
.3. NO release in physiological solution
Rhodamine derivative 9, which was taken as a reference for an
evaluation of the toxicity of the simple rhodamine ring (Figures S4a),
showed very low toxicity over the whole range of concentrations tested.
The furoxans 5 and 6, substituted with –CH and –CONH , respectively,
did not trigger any significant anticancer activity (Figures S4b-c). Their
respective mitochondrial-targeting hybrids 1 and 2 only showed slightly
higher cytotoxicity (see Figures S4b-c). This behaviour is in keeping
with their poor reactivity with biomolecules and, consequenty, the
inefficient NO release of all the products.
The NO-releasing capacity of all the final furoxan derivatives in the
presence of thiol cofactors was evaluated in a PBS solution that con-
tained an excess of cysteine (1:5). NO release was assessed by the
3
2
2
detection of nitrite, the primarily product of NO/O oxidation in aerobic
aqueous solution, using Griess reaction. The formation of nitrite in these
conditions is governed by a third-order rate law and can be accompanied
by the production of a very little amount of nitrate.
The results, reported in Table 1, show that furoxans 1 and 5 did not
release a detectable amount of NO after 1 h. A small amount of NO was
produced by 2 and 6, which bear the electron withdrawing carbamoyl
group at position 3. By contrast, a large amount of NO was generated by
By contrast, while the cyano furoxan derivative 7 showed significant
cytotoxic activity only at the highest concentration (50 µM), its rhoda-
mine analogue 3 was markedly more active, from 10 µM concentration
(Fig. 2a). As the NO-donation capacity of the two cyano derivatives was
comparable, we hypothesised that the difference in toxicity can be
explained by the potential mitochondrial accumulation of compound 3.
The 3-phenylsulfonylfuroxan 8 triggered high cellular toxicity from
10 µM concentration, as shown in Fig. 2b, whereas its rhodamine
analogue 4, which possesses similar NO-releasing capacity, caused
remarkable cytotoxicity already at 1 µM. This behaviour may be
reasonably attributed to its capability of accumulating in mitochondria.
Although the NO-releasing ability of two 3-phenylsulfonyl products, 4
and 8, is certainly lower than that of the related cyano derivatives 3 and
7, the formers were found to be much more cytotoxic than the latter.
Hence, we may infer that the activity of 4 and 8 is mainly due to the
covalent labelling of thiol biomolecules via the nucleophilic displace-
ment of the phenylsulfonyl group during diffusion within the cell, and,
in particular, after accumulation within the mitochondria.
4
,8,10 and 3,7, in which the furoxan rings are substituted at position 3
by strongly electron withdrawing -SO
2
C
6
H
5
and –CN groups, respec-
tively [43]. An analysis of the data also showed that the NO-releasing
capacity of the organelle-targeting products 3,4,10 is comparable to
that of the related references 7 and 8.
The UPLC/mass study of the reaction of 4 with cysteine showed that
a relevant amount of the corresponding 3-cysteinyl derivative is formed
after 5 min (Figure S1a, see SI), in keeping with the nucleophilic
displacement of the 3-phenylsulfonyl moiety in the furoxan system [44].
A thioimidate adduct between cysteine and the strong electrophilic CN
group was detected, after 5 min, in an analogous UPLC/mass study
carried out on 3 (Figure S2a). It is possible that the corresponding 3-thio
substituted derivatives and thiocyano adducts may play some role in the
NO release of the 3-phenylsulfonyl and the 3-CN furoxans, respectively.
The mechanism of NO release from furoxans in presence of thiol
cofactors is complex and still needs further investigation (5a,6,7).
Generally speaking, the release is favoured by basic pH and by the low
The cytotoxicity of the hybrid with lysosomal targeting 10 and of the
phenothiazine derivative 11 was assessed. Compound 11, which was
used to evaluate the toxicity of the phenothiazine scaffold, showed very
low toxicity only at 25 µM (Figure S4d, see SI). Compound 10 and its
reference 8, which displayed similar NO donor ability in cells due to the
presence of the 3-phenylsulfonylfuroxan moiety (Fig. 1b), did not
exhibit any significant activity in the 0.1–2.5 µM concentration range,
while good activity was observed at 10 and 25 µM (Fig. 2c). As the
cytotoxicity of compounds 8 and 10 was comparable (Fig. 2c), we can
speculate that the accumulation of the 3-phenylsulfonylfuroxan sub-
structure in the lysosomes did not lead to any anticancer-activity ben-
pK
a
of the thiol reagent, allowing thiolate anions to perform a nucleo-
philic attack on the hetero-ring. In Figures S1 and S2, possible mecha-
nisms of NO production by 3-SO
reported.
2 6 5
C H and 3-CN substituted furoxans are
4
.4. NO release in A549 cells
NO release in A549 lung adenocarcinoma cancer cells was studied
for all furoxan derivatives 1–8 in the concentration range 0.1–50 µM,
and for 10 in the concentration range 0.1–25 µM. The cells were incu-
bated for 24 h with the abovementioned compounds and then nitrite was
efits. A possible reason for this could be the acidic lysosomal
environment, which strongly limits the nucleophilicity of thiol co-
factors, and therefore the capacity of the product to release NO and to
1
0

F. Sodano et al.
Bioorganic Chemistry 111 (2021) 104911
Fig. 3. Fluorescence microscopy images of A549 cells incubated for 24 h with the GFP-E1
α
pyruvate dehydrogenase expression vector to label mitochondria (green)
and then treated with compound 4 (red). Nuclei were counterstained with DAPI. The merged image is shown in the right panel. Scale bars: 10 m. Micrographs are
μ
representative of three experiments with similar results. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article.)
Fig. 4. Fluorescence microscopy images of A549 cells incubated for 24 h with the GFP-Lamp1 expression vector to label lysosomes (green), and then treated with
compound 10 (red). Nuclei were counterstained with DAPI. The merged image is shown in the right panel. Scale bars: 10 m. Micrographs are representative of three
μ
experiments with similar results.
covalently bind, and inhibit, proteins. The limited NO-releasing capa-
bility of compound 10 in an acidic buffer solution was confirmed (data
not shown).
6 5 2
performed. The 3-CN and 3-C H SO substituted products showed effi-
cient NO-donation capabilities both in physiological solution and in
A549 lung adenocarcinoma cancer cells. These products displayed
interesting cytotoxic activity when tested in the abovementioned cells in
an LDH assay. In detail, the mitochondria-targeting molecules were
clearly more active than their related reference compounds, thus
demonstrating the benefits that mitochondrial accumulation has on
their activity. Their cellular toxicity may be due to both NO release and
the covalent inhibition of thiol biomolecules. The rhodamine hybrid 4,
4
.6. Intracellular localisation studies in A549 lung adenocarcinoma
cancer cells
To demonstrate that intracellular localisation occurred, we carried
out a confocal analysis of 3-phenylsulfonylfuroxan derivatives 4 and 10,
used as prototypes of compounds bearing a cellular-organelle targeting
moiety. The remarkable fluorescence of the rhodamine moiety is a
powerful tool for studying the intracellular localisation of its derivatives.
Cells were treated with compound 4 after 24 h of incubation with the
6 5 2
which contains the 3-C H SO -substituted furoxan moiety, was
observed to be the most promising member of the series. It triggered
high activity across the whole tested concentration range (1–50 µM). A
comparative analysis between its toxicity and that of the related 3-CN
derivative suggested that its capacity to covalently bind cellular thiols
via the nucleophilic displacement of the phenylsulfonyl group plays a
paramount role in its cytotoxic properties. An attempt to improve the
activity of the 3-phenylsulfonylfuroxan toxophore by accumulating it in
lysosomes via conjugation with a phenothiazine scaffold was performed.
However, its rhodamine analogue showed higher effectiveness, con-
firming that mitochondrial targeting remains the best choice for these
furoxan substructures.
GFP-E1 pyruvate dehydrogenase expression vector, which is used to
α
label mitochondria. As expected, compound 4 was identified in mito-
chondria (Fig. 3) in the fluorescence analysis. The percentage of mito-
chondria accumulating compound
4 over the total number of
mitochondria was 57 ± 6%, suggesting a good mitochondrial targeting.
By exploiting the fluorescence of the rhodamine moiety, we also eval-
uated the accumulation of the cyano derivative 3 via confocal micro-
scopy. This rhodamine derivative was also confirmed to be able to
selectively accumulate in mitochondria, as shown in Figure S5.
The fluorescence of the phenothiazine scaffold revealed the accu-
mulation of compound 10 within lysosomes, as shown in Fig. 4. The
percentage of lysosomes accumulating compound 10 over the total
number of lysosomes was 39 ± 5%, indicating that the vectorisation
toward lysosome – although less efficient than the vectorisation toward
the mitochondria – was anyway achieved.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
5
. Conclusions
This work was supported by Ricerca Locale from the University of
Turin. We would like to thank dr. Joanna Kopecka and Costanzo Cos-
tamagna for the technical assistance.
A study of a set of 3-R-substituted furoxan derivatives and their
conjugates with the mitochondrial probe rhodamine B has been
1
1

F. Sodano et al.
Bioorganic Chemistry 111 (2021) 104911
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