Design, Synthesis, and Biological Activity of Hydrogen Peroxide Responsive Arylboronate Melatonin Hybrids

Article abstract of DOI:10.1021/acs.chemrestox.8b00216

Stimulus-responsive cleavage reactions have found broad use to direct drug release at a particular target disease area. Increased levels of reactive oxygen species (ROS) have been associated with the development and progression of cancer and several other disease states, motivating the development of drug conjugates that can undergo a chemoselective ROS-triggered release. Melatonin (MLT) and the reactive electrophile p-benzoquinone methide (p-QM) have evidenced either cytoprotective or cytotoxic effects in biological systems, depending on the dose, cellular targets, and time of exposure. In this study, we report the synthesis and biological activity of two MLT derivatives linked to ROS-responsive arylboronate triggers (P1 and P2), which can be activated by endogenously generated hydrogen peroxide (H₂O₂) to release MLT, or 5-methoxytryptamine (5-MeOT), and p-QM-intermediates. Their H₂O₂-induced activation mechanism was studied by HPLC-DAD-MS. P1, which rapidly releases MLT and p-QM, was able to strongly induce the Nrf2 antioxidant signaling pathway, but was ineffective to provide protection against H₂O₂-mediated oxidative damage. By contrast, P1 exhibited strong toxic effects in HeLa cancer cells, without causing significant toxicity to normal NCTC-2544 cells. Similar, although more limited, effects were exerted by P2. In both cases, cytotoxicity was accompanied by depletion of cellular glutathione (GSH), probably as a consequence of p-QM release, and increased ROS levels. A role for MLT in toxicity was also observed, suggesting that the P1 released products, MLT and p-QM, contributed additively to promote cell death.

Full text of DOI:10.1021/acs.chemrestox.8b00216

Subscriber access provided by YORK UNIV
Article
Design, synthesis and biological activity of hydrogen
peroxide responsive arylboronate melatonin hybrids
Annalida Bedini, Alessandra Fraternale, Rita Crinelli, Michele
Mari, Silvia Bartolucci, Laura Chiarantini, and Gilberto Spadoni
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00216 • Publication Date (Web): 18 Dec 2018
Downloaded from http://pubs.acs.org on December 19, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
Design, synthesis and biological activity of hydrogen peroxide responsive arylboronate
melatonin hybrids
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Annalida Bedini,†Δ Alessandra Fraternale,‡Δ Rita Crinelli,‡ Michele Mari,† Silvia Bartolucci,† Laura
Chiarantini,‡* Gilberto Spadoni†*
ΔThese authors equally contributed to this work
* Corresponding authors
†Department of Biomolecular Sciences, University of Urbino Carlo Bo, Piazza Rinascimento 6, I-
61029 Urbino, Italy.
‡Department of Biomolecular Sciences, University of Urbino Carlo Bo, Via Saffi 2, I-61029
Urbino, Italy.
1
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 2 of 38
1
2
3
4
Table of Contents graphic
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
ACS Paragon Plus Environment

Page 3 of 38
Chemical Research in Toxicology
1
2
3
4
ABSTRACT
5
6
7
8
Stimulus-responsive cleavage reactions have found broad use to direct drug release at a particular
target disease area. Increased levels of reactive oxygen species (ROS) have been associated with the
development and progression of cancer and several other disease states, motivating the development
of drug conjugates that can undergo a chemoselective ROS-triggered release. Melatonin (MLT) and
the reactive electrophile p-benzoquinone methide (p-QM) have evidenced either cytoprotective and
cytotoxic effects in biological systems, depending on the dose, cellular targets, and time of
exposure. In this study we report the synthesis and biological activity of two MLT derivatives
linked to ROS-responsive arylboronate triggers (P1 and P2), which can be activated by
endogenously generated hydrogen peroxide (H2O2) to release MLT, or 5-methoxytryptamine (5-
MeOT), and p-QM-intermediates. Their H2O2-induced activation mechanism was studied by
HPLC-DAD-MS. P1, which rapidly releases MLT and p-QM, was able to strongly induce the Nrf2
antioxidant signaling pathway, but was ineffective to provide protection against H2O2 mediated
oxidative damage. By contrast, P1 exhibited strong toxic effects in HeLa cancer cells, without
causing significant toxicity to normal NCTC-2544 cells. Similar, although more limited, effects
were exerted by P2. In both cases, cytotoxicity was accompanied by depletion of cellular
glutathione (GSH), probably as a consequence of p-QM release, and increased ROS levels. A role
for MLT in toxicity was also observed, suggesting that the P1 released products, MLT and p-QM,
contributed additively to promote cell death.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 4 of 38
1
2
3
4
INTRODUCTION
5
6
7
8
Maintenance of redox homeostasis plays a central role in health and disease prevention, and
alteration of this delicate balance can lead to pathological consequences. Overproduction of
hydrogen peroxide (H2O2) and other reactive oxygen species (ROS) in normal cells causes
oxidative stress that is thought to contribute to the onset and development of neurodegenerative1,
cancer2 and cardiovascular3 diseases, as well as ischemic stroke and diabetes4. Therefore, oxidative
stress containment is paramount for cell functionality. To counteract oxidative stress, cells have
developed endogenous defense mechanisms, including the activation of nuclear factor erythroid 2–
related factor 2 (Nrf2),5 a transcription factor controlling the expression of various cytoprotective
and antioxidant enzymes.6-8 In the last few decades, many studies have demonstrated the benefits of
several natural products in counteracting oxidative stress through modulation of the
Nrf2/Antioxidant Response Element (ARE) signaling pathway.9 Nevertheless, cancer cells,
compared with normal cells, generate higher amounts of ROS, including H2O2, and adapt to
oxidative stress by increasing their antioxidant capacity, such as enhancing the synthesis of the
antioxidant glutathione (GSH), to counteract the damaging effects of ROS.10,11 The higher levels of
ROS in tumor cells can be exploited to enhance selectivity and therapeutic activity of antitumor
agents12, and exogenous compounds increasing oxidative stress preferentially in cancer cells can
potentially be used as anticancer drugs.13
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The tryptophan-derived hormone melatonin (MLT) is a well-known multifunctional molecule
mainly secreted by the pineal gland during the night. It influences a number of functions of the
central nervous system such as the synchronization of circadian rhythms, the regulation of the
sleep-wake cycle, the control of mood and behavior, hormone secretion and many others.14
Peripherally, MLT is involved in the modulation of the immune system, of retinal functions, of the
cardiovascular system and in glucose homeostasis.15 A beneficial role of MLT administration has
also been proposed for neurodegenerative diseases, diabetes, multiple sclerosis and cancer.16,17
A
feature that characterizes MLT is the variety of mechanisms it employs to modulate the cell
4
ACS Paragon Plus Environment

Page 5 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
physiology. Its functions primarily depend on G protein-coupled transmembrane receptors, named
MT1 and MT2, mainly expressed in the central nervous system, but also detected in many peripheral
tissues.18 Apart from its receptor-mediated actions, MLT is a powerful antioxidant that scavenges
free radicals19 or activates the Nrf2/ARE signal pathway, thereby promoting anti-oxidant enzyme
expression.20-22
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Melatonin has also attracted attention as a natural oncostatic agent that can suppress neoplastic
growth in a variety of tumors.23 Although epidemiological studies concerning the association
between body circadian melatonin levels and cancer incidence have led to controversial
conclusions,23 accumulating evidence from in vitro studies and animal models has supported the
anticancer properties of MLT.24,25 Melatonin can exert its antitumor activity by induction of
apoptosis and epigenetic alteration, interaction with melatonin receptors, as well as by inhibition of
angiogenesis, invasion and metastasis.26 Preliminary clinical studies in patients with cancer have
also demonstrated that MLT can reinforce the therapeutic efficacy and/or reduce the side effects of
the cancer chemotherapeutic agents.27,28 The anticarcinogenic action of MLT has also been related,
in part, to its potent antioxidant activity, although recent evidence has indicated that MLT may exert
pro-oxidant action in tumor cells.29,30 Thus, the effects of MLT seem to be context specific allowing
normal cells to be spared while killing cancer cells.31
Quinones, including quinone imines and quinone methides represent a class of toxicological
intermediates, which can alter redox balance within cells. These compounds can induce a variety of
hazardous effects in vivo, but they could be also exploited for therapeutic purposes, predominantly
in the field of cancer chemotherapy. Bioactivation of small molecules resulting in the production of
electrophilic reactive intermediates is often considered a risk factor in drug development. However,
there are several examples of biologically active compounds containing such reactive electrophiles,
notably Michael acceptors such as p-benzoquinone methide (p-QM), which have demonstrated
promising cancer chemopreventive and chemotherapeutic activity.32,33
5
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 6 of 38
1
2
3
4
5
6
7
8
9
For example, interesting chemopreventive and antiproliferative effects in cell cultures and animal
models were reported for the hydroxybenzyl-linked NO-aspirin hybrids (pNO-ASA), or their p-
substituted-ASA congeners not containing an NO-donating group, as the result of bioactivation to
p-QM.34-36 Cytoprotection was attributed to formation of QM which covalently modifies the
repressor protein Keap1, leading to activation of Nrf2 and subsequent induction of detoxifying
enzymes. 37 Similarly, QM is able to induce toxic effects through covalent modification of a variety
of other bionucleophiles including DNA, causing genotoxicity, and GSH altering cell redox
balance. It is now becoming generally accepted that quinones have opposing effects in biological
systems which, depending on the dose, cellular targets, and time of exposure, could result in
toxicity or cytoprotection.38,39
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In light of the pleiotropic profile of MLT and the current interest in electrophilic drugs, we have
hypothesized that the combination of these compounds in one molecule could eventually result in
additive cytoprotective or cytotoxic effects. Aryl boronic esters are relatively easy to synthesize,
generally biocompatible, and react selectively with ROS in a B-C bond cleavage reaction to release
the corresponding phenolate.40 Thus, a strategy was employed to design melatonin derivatives
linked to ROS-responsive arylboronate triggers (Figure 1), which can be activated by endogenously
generated H2O2 to release MLT or 5-methoxytryptamine (5-MeOT), and p-QM intermediates.
These molecules are expected to have a potential therapeutic application for oxidative stress-related
diseases and/or cancer, acting as mediators of cytoprotective or cytotoxic responses.
6
ACS Paragon Plus Environment

Page 7 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
O
NHR
B
MeO
O
O
LG
N
H
O
Hybridization
Aryl boronic pinacol ester
MLT
, R=COCH3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5-MeOT, R=H
H
N
H
O
N
O
B
MeO
O
MeO
O
O
N
O
O
B
O
N
H
O
P1
P2
Figure 1. Design of melatonin arylboronate hybrids (P1 and P2).
EXPERIMENTAL PROCEDURES
Synthesis of hybrid compounds P1 and P2. General. All reagents, including MLT, were
purchased from commercial sources and used without further purification. Reactions were
monitored by analytical thin-layer chromatography (TLC) using Merck silica gel 60 F254 precoated
plates. Column chromatography purifications were performed under “flash” conditions using Merck
230−400 mesh silica gel.
Melting points were determined on a Buchi B-540 capillary melting point apparatus and are
uncorrected. 1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE 400 instrument.
Chemical shifts (δ scale) are reported in parts per million (ppm) relative to the central peak of the
solvent. Coupling constants (J) are given in hertz (Hz). ESI MS spectra were taken on a Waters
Micromass ZQ instrument; only molecular ions (M+H)+ are given. High-resolution mass
spectroscopy was performed on a Micromass Q-ToF Micro mass spectrometer (Micromass,
Manchester, UK) using an ESI source. The purity of tested compounds, determined by high
7
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 8 of 38
1
2
3
4
5
6
7
8
9
performance liquid chromatography (HPLC), was greater than 95%. These analyses were
performed on a Waters HPLC/DAD/MS system (separation module Alliance HT2795, Photo Diode
Array Detector 2996, mass detector Micromass ZQ; software: MassLynx 4.1) using a Gemini 5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
C6–Phenyl 110A column (150 x 4.60 mm, 5 m). Method: linear gradient from a mixture 50/50
(v/v) CH3CN/HCOOH 0.1% in H2O to a 90/10 mixture in 8 minutes and then from 90/10 mixture to
100% CH3CN in 2 min; flow rate: 1 mL min-1, injection volume 10 l, UV detection at = 254 nm
(P1, Rt = 6.13 min; P2, Rt = 6.70 min).
4-(4,4,5,5-Tetramethyl-1,3,2-dioxoborolan-2-yl)benzyl 3-(2-acetamidoethyl)-5-methoxy-1H-indole-
1-carboxylate (P1). 4-(4,4,5,5-Tetramethyl-1,3,2-dioxoborolan-2-yl)benzyl 1H-imidazole-1-
carboxylate41 (180 mg, 0.55 mmol) and DBU (40 L) were added to a solution of MLT (116 mg,
0.5 mmol) in dry CH3CN (2.5 mL). The resulting mixture was stirred under N2 atmosphere, at 50°C
for 7 h. The solvent was evaporated under reduced pressure and the crude residue was purified by
1
silica gel flash chromatography (EtOAc as eluent). Amorphous solid, mp 148-9 °C, 70% yield. H
NMR (400 MHz, CDCl3):  1.34 (s, 12H), 1.93 (s, 3H), 2.85 (t, 2H, J = 6.5 Hz), 3.55 (m, 2H), 3.84
(s, 3H), 5.41 (s, 2H), 5.80 (brs, 1H), 6.92 (dd, 1H, J = 2.0 and 9.0 Hz), 6.99 (d, 1H, J = 2.0 Hz),
13
7.41 (brs, 1H), 7.46 (d, 2H, J = 8.0 Hz), 7.86 (d, 2H, J = 8.0 Hz), 8.03 (brs, 1H). C NMR (100
MHz, CDCl3):  170.2, 156.2, 150.6, 138.0, 135.2, 134.9, 131.3, 127.6, 123.3, 118.6, 116.1, 113.3,
101.8, 83.9, 68.5, 55.7, 39.0, 25.1, 24.8, 23.3. ESI MS (m/z): 493 (M+H)+. HRMS (ESI): m/z
calculated for C27H34N2O6B, [M+H]+ 493.2510. Found: 493.2599.
4-(4,4,5,5-Tetramethyl-1,3,2-dioxoborolan-2-yl)benzyl
(2-(5-methoxy-1H-indol-3-
yl)ethyl)carbamate (P2). 4-Nitrophenyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxoborolan-2-yl)benzyl
carbonate42 (180 mg, 0.47 mmol) and TEA (0.28 mL) were added to a solution of 5-
methoxytryptamine43 (90 mg, 0.47 mmol) in dry CH2Cl2 (5 mL). The resulting mixture was stirred
8
ACS Paragon Plus Environment

Page 9 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
under N2 atmosphere, at room temperature (RT) for 16 h. The solvent was removed by distillation
under reduced pressure and the crude residue was purified by silica gel flash chromatography
(cyclohexane-EtOAc 7:3 as eluent) and crystallization. White solid, mp 161-2 °C (CH2Cl2-
petroleum ether); 52% yield. 1H NMR (400 MHz, CDCl3):  1.35 (s, 12H), 2.95 (t, 2H, J = 6.5 Hz),
3.54 (m, 2H), 3.85 (s, 3H), 4.86 (brs, 1H), 5.12 (s, 2H), 6.87 (dd, 1H, J = 2.5 and 8.5 Hz), 6.99 (m,
1H), 7.03 (m, 1H), 7.26 (d, 1H, J = 8.5 Hz), 7.34 (d, 2H, J = 8.0 Hz), 7.80 (d, 2H, J = 8.0 Hz), 7.93
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
(brs, 1H). C NMR (100 MHz, CDCl3):  156.3, 154.1, 139.7, 134.9, 131.5, 127.6, 127.1, 122.8,
112.6, 112.4, 111.9, 100.5, 83.8, 66.4, 55.9, 41.2, 29.7, 25.7, 24.8. ESI MS (m/z): 451 (M+H)+.
HRMS (ESI): m/z calculated for C25H32N2O5B, [M+H]+ 451.2404. Found: 451.2361.
Sensitivity to H2O2. The ability of P1 and P2 to release two different end-units (p-QM and MLT or
5-MeOT) in the presence of H2O2 was evaluated by HPLC-DAD-MS. Analyses were performed at
25°C on a Waters HPLC/DAD/MS system (separation module Alliance HT2795, Photo Diode
Array Detector 2996, mass detector Micromass ZQ; software: MassLynx 4.1) using a Gemini 5
C6–Phenyl 110A column (150 x 4.60 mm, 5 m). Hybrids P1 and P2 were solubilized in a 1:1 v/v
CH3CN/physiological buffer saline (PBS, 5 mM sodium phosphate buffer, 0.9% (w/v) NaCl, pH
7.5) mixture to a final concentration of 1 mg/mL and treated (or not) with 20 equivalents of 40%
m/v H2O2 at RT. At various time points the mixture was subjected to HPLC analysis. Mobile phase:
linear gradient from a 10/90 (v/v) mixture CH3CN/HCOOH 0.1% in H2O to a 90/10 mixture in 8
minutes and then 90/10 mixture for 2 min; flow rate: 1 mL min-1, injection volume 10 l, UV
detection at = 278 nm.
Cell culture and treatments. Human keratinocyte (NCTC-2544) cell line was grown in
Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 2 mM L-glutamine, 7% heat-
inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 10 µg/mL streptomycin. Human
epithelioid cervix carcinoma (HeLa) cell line was grown in RPMI supplemented with 2 mM L-
9
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 10 of 38
1
2
3
4
5
6
7
8
9
glutamine, 10% heat-inactivated FBS, 100 U/mL penicillin and 10 µg/mL streptomycin. Cells were
cultured under a humidified atmosphere containing 95% air and 5% CO2 at 37°C. Dimethyl
sulfoxide (DMSO) was used as a vehicle to enable full solubility of the tested compounds MLT, 5-
MeOT, P1 and P2 in the stock solutions (100 mM) that were stored at -80 °C.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cells were treated with various concentrations (ranging from 1 to 100 µM) of MLT, 5-MeOT, P1
and P2 and DMSO did not exceed 0.1% (v/v). Cells treated with DMSO at final concentration 0.1%
(v/v) were used as control.
Cell viability assay. Cell viability was evaluated using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) tetrazolium assay (Cell Titer 96®
Aqueous, Promega, Madison, WI, USA) as previously reported.44 This assay is based on the
reduction of the MTS tetrazolium compound by viable cells to generate a colored formazan dye that
is soluble in cell culture media. This conversion is thought to be carried out by NAD(P)H-
dependent dehydrogenase enzymes in metabolically active cells. Briefly, cells were seeded in 96-
well microtiter plates at a density of 5 x 103 and 2 x 103 cells/well for NCTC-2544 and HeLa cells
respectively and subsequently exposed to P1, P2 MLT and 5-MeOT, at different concentrations
(ranging from 0 to 100 µM) for 24 or 48 h. After the treatments, 20 µl of MTS were added into each
well, and then incubated for 30 min at 37°C; the plates were read in a Benchmark microplate reader
(BioRad, Hercules, CA, USA) at 490 nm at a reference wavelength of 630 nm.
In some experiments, HeLa cells were seeded in 24 well plates at a density of 1.2 x 104 cells/well
and treated with 10, 50 and 100 µM P1 for 24 h; the number of live and dead cells was counted by
trypan blue staining: 20 μl of cell suspension was mixed with 20 μl of trypan blue 0.4% solution
(Sigma-Aldrich, UK) and the cells were counted using Countess II Automated Cell Counter
(Thermo Fisher Scientific, USA).
10
ACS Paragon Plus Environment

Page 11 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
Nrf2 activation. Nrf2 activation was assessed in NCTC-2544 cells pre-incubated overnight (o/n) in
serum-free medium (-FBS) in the presence or absence (NT) of the tested compounds P1, P2 or
MLT (100 µM, unless otherwise specified). The day after, the medium was removed and the cells
were maintained in culture with FBS-supplemented medium (+FBS) for up to 7 h. At time 0, 2, 4
and 7 h cells were harvested for nuclear extraction and western immunoblotting analysis.
In some experiments the medium containing FBS was supplemented with 10 mM GSH-C4 (kindly
provided by GLUOS S.r.l., Urbino) and cells were harvested after 4 h incubation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Nrf2 activation was also assayed in HeLa cells incubated with P1 or P2 (100 µM) under standard
growth conditions. At time 0, 1.5, 3, 6, 8 h, cells were washed in PBS and whole cell lysates were
submitted to western immunoblotting analysis.
Cell lysis and nuclear fractionation.
For nuclear protein extraction, cells were washed with PBS and lysed in hypotonic Buffer A [10
mM Hepes/KOH pH 7.9, 10 mM KCl, 0.1% (v/v) Nonidet-P40], supplemented with a cocktail of
protease inhibitors (Roche). After centrifugation at 14,000 x g at 4°C for 10 min., the pellet was
suspended in hypertonic Buffer B (20 mM Hepes/KOH pH 7.9, 25% glycerol, 0.42 M NaCl),
supplemented with protease inhibitors. Nuclear proteins were recovered by centrifugation at 14,000
x g in the supernatant.45 Protein concentration was determined by the method of Bradford, using
bovine serum albumin as standard.
Whole lysates were obtained by directly harvesting the cells in Sodium Dodecyl Sulfate (SDS)
buffer (50 mM Tris-HCl, pH 7.8, 0.25 M sucrose, 2% (w/v) SDS, 10 mM N-ethylmaleimide
supplemented with protease inhibitors as above). Lysates were boiled for 5 min, then sonicated at
100 Watts for 20 sec. Cell debris was removed by centrifugation at 14,000 x g at room temperature.
Protein content was determined by the Lowry assay.
Western immunoblotting analysis
11
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 12 of 38
1
2
3
4
5
6
7
8
9
Equal amounts of proteins were resolved by SDS-PAGE and gels were electroblotted onto a
nitrocellulose membrane (0.2 µm pore size) (BioRad laboratories Inc.). The blots were probed with
anti Nrf2 antibody (D1Z9C, Cell Signaling Technology). Anti Ying Yan 1 antibody (YY1, C-20,
sc-281) (Santa Cruz Biotechnology Inc.) was used as nuclear loading control. Anti actin (A 2066,
Sigma) was used to check equal protein loading for whole cell lysates. Bands were detected by
horseradish peroxidase (HRP)-conjugated secondary antibody (BioRad) and the enhanced
chemiluminescence detection kit WesternBright ECL (Advasta).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Flow cytometric analysis of ROS. Intracellular ROS levels were measured by flow cytometry
using 2’,7’-dichlorodihydrofluorescein diacetate (H2-DCF-DA, Sigma-Aldrich), which easily
diffuses into cells where it is transformed by esterases to 2’,7’-dichlorodihydrofluorescein (H2-
DCF). This membrane-impermeable product is then oxidized to the highly fluorescent 2’,7’-
dichlorofluorescein (DCF) by intracellular ROS, primarily by H2O2.46 NCTC-2544 and HeLa cells
were plated at a density of 8 x 104 and 2.6 x 104 cells per well in 24 well plates, respectively. The
day after the medium was removed and substituted with fresh medium containing the tested
compounds P1, P2 or MLT at a concentration of 100 µM for 6 h. The cells were harvested by
trypsin and suspended in 1 mL PBS supplemented with 3% (v/v) FBS. Then 2 x 105 cells were
labeled with 10 μM H2-DCF-DA for 30 min at RT in the dark and immediately analyzed for DCF
fluorescence using a FacScan flow cytometer (Becton Dickinson).
GSH determination. GSH levels in NCTC-2544 and HeLa cells treated with the tested compounds
P1, P2 or MLT (100 µM) o/n were determined following the previously described procedure.47
Briefly, the cells were washed twice in PBS and immediately lysed with 100 µL of lysis buffer
(0.1% Triton X-100, 0.1 M Na2HPO4, 5 mM EDTA, pH 7.5). Thereafter, 15 µL of 0.1 N HCl and
140 µL of precipitating solution (100 ml containing 1.67 g of glacial metaphosphoric acid, 0.2 g of
disodium EDTA, 30 g of NaCl), were added. After centrifugation at 12,000 x g for 10 min, the
12
ACS Paragon Plus Environment

Page 13 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
supernatant was collected and 25% (v/v) 0.3 M Na2HPO4 and immediately after 10% (v/v) 5,5'-
dithio-bis(2-nitrobenzoic acid) (DTNB) were added. The mixture was stirred for 1 min at RT, then
left at RT for another 5 min, and finally used for HPLC determination of thiols.48
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Statistical analysis. Statistical analysis was performed with GraphPad Instat. Data were analyzed
with the two-tail paired Student’s t test for pairwise comparisons or one-way analysis of variance
(ANOVA) for multiple comparisons. A P value of less than 0.05 was considered significant.
RESULTS and DISCUSSION
Design and synthesis of melatonin-arylboronate hybrids. Taking into account the properties of
MLT and p-QM we synthesized two new hybrid compounds (P1 and P2) by coupling MLT or 5-
MeOT with a p-boronate benzyl moiety through a carbamate linkage (Figure 1), that would be inert
under normal physiological conditions, but potentially able to release multiple active species in
response to high levels of ROS. A selective oxidative hydroxylation of the arylboronate trigger unit
by H2O2 should provide the corresponding phenol in equilibrium with phenolate (and the non toxic
boric acid or its pinacolate ester), which spontaneously could decompose to p-QM, CO2 and MLT
or 5-MeOT (see also Scheme 2 and 3).
The designed melatonin-arylboronate P1 was prepared by N1-carbamoylation of MLT with 4-
(4,4,5,5-tetramethyl-1,3,2-dioxoborolan-2-yl)benzyl 1H-imidazole-1-carboxylate which in turn was
obtained by treating 4-(hydroxymethyl)phenylboronic acid pinacol ester with carbonyldiimidazole
following a previously reported procedure41 (Scheme 1). To prepare the target hybrid compound P2,
MLT was first converted into 5-MeOT by microwave-assisted deacetylation using a combination of
ammonium bromide and ethylenediamine43, and then submitted to N-carbamoylation by treatment
with 4-nitrophenyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxoborolan-2-yl)benzyl) carbonate42 (Scheme 1).
13
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 14 of 38
1
2
3
4
5
6
7
8
9
NHCOCH3
O
B
H3CO
O
O
N
N
N
O
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
DBU/CH3CN
O
B
P1
NHCOCH3
O
H3CO
N
H
MLT
O
NH2
HN
O
H3CO
H3CO
NH4Br
TEA/CH2Cl2
O
B
O
N
H
N
H
NH2(CH2)2NH2
P2
O
O
5-MeOT
B
NO2
O
O
O
Scheme 1. Synthesis of pinacol arylboronate melatonin derivatives.
H2O2-induced transformation of hybrids P1 and P2. The in vitro stability of hybrids P1 and P2
in the absence of H2O2 (Figures 2 and 3) and their H2O2-induced (20 equiv) cleavage/activation
(Figures 4 and 5) were followed by HPLC-DAD-MS. Compounds P1 and P2 when incubated in
PBS partially converted to their corresponding acids, as highlighted by the appearance of the peak
at retention time (Rt) 6.65 min ([M+H]+ = 411, Figure 2) or the peak at Rt = 6.68 min ([M+H]+ =
369, Figure 3).
14
ACS Paragon Plus Environment

Page 15 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Stability of P1 (Rt = 8.95 min) and its corresponding boronic acid (Rt = 6.65 min) in the
absence of H2O2.
Figure 3. Stability of P2 (Rt = 8.85 min) and its corresponding boronic acid (Rt = 6.68 min) in the
absence of H2O2.
Upon treatment of P1 with H2O2 we observed complete disappearance of the compound, and of its
corresponding boronic acid, within 5-15 min, which is in accordance with previous reports
suggesting fast reactivity of arylboronic acids and their esters with H2O2.49 Formation of a new peak
after 15 min corresponding to p-hydroxybenzyl alcohol (p-HBA, resulting from the scavenging of
15
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 16 of 38
1
2
3
4
5
6
7
8
9
p-QM by water, Rt = 3.52 min), and of another peak corresponding to the intermediate 1-carboxy-
MLT (B, Rt =6.52 min, [M+H]+ = 277) which decomposed over a period of 7 h to generate MLT (Rt
= 5.52 min, [M+H]+ = 233), suggested the formation of the intermediate undetected phenolate A,
which spontaneously and irreversibly undergoes a 1,6-elimination to release p-QM and B (Scheme
2).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The formation of p-HBA is in good agreement with previous studies reporting that p-QM is
converted to p-hydroxybenzyl alcohol under aqueous conditions in the absence of nucleophiles such
as GSH .35
Indeed, p-HBA was not observed neither in positive nor in negative detection mode in the ESI mass
spectra of the mixture, as already reported in similar cases.50 However, its formation was proved in
HPLC-DAD (or TLC) by an authentic sample, the peak of which matched exactly with that of the
above cited p-HBA (Figures S1 and S2).
Figure 4. HPLC-DAD-MS chromatograms of the activation of P1 by H2O2 (20 equiv.) in a 1:1 v/v
CH3CN/PBS.
As expected, the intensity of the B peak (1-carboxy-MLT) decreases with increasing incubation
time, and simultaneously a consistent increase in MLT formation could be detected (Figure 4) in
agreement with the putative mechanism of P1 activation described in Scheme 2.
16
ACS Paragon Plus Environment

Page 17 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
OH
7
8
9
O
NHCOCH3
10
11
12
13
14
15
16
17
18
OH
O
O
N
NuH
O
O
OH
O
O
B
(H2O or GSH)
B
O
B
N
O
O
Nu
OCH3
H2O2
p-QM
Nu=OH, p-HBA
+
H+
H2O
NHCOCH3
-
O
OH
O
N
NHCOCH3
O
NHCOCH3
O
O
N
O
N
19
MLT
20
21
22
23
24
25
26
27
28
29
30
O
CO2
NHCOCH3
OCH3
P1
OCH3
OCH3
O
B
OCH3
A
Scheme 2. Proposed mechanism of MLT and p-QM formation from P1 and reaction of p-QM with
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
different nucleophiles (e.g. H2O or GSH).
HPLC-DAD-MS analysis showed that the H2O2-induced oxidative hydroxylation of the
arylboronate unit also rapidly occurred in the case of hybrid P2, leading to the formation of the
phenol C (Rt = 7.00 min, [M+H]+ = 341) (Figure 5) and of a small amount of 5-MeOT (Rt = 2.28
and 2.93 min) after 15 min of incubation with H2O2.
17
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 18 of 38
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. HPLC-DAD-MS chromatograms of the activation of P2 by H2O2 (20 equiv.) in a 1:1 v/v
CH3CN/PBS.
The mechanism of H2O2-induced activation of P2 remains substantially the same as that of P1.
HPLC-DAD-MS analysis showed that also the arylboronate unit of hybrid P2 was rapidly cleaved
to the corresponding phenol C (Figure 5), after 15 min of incubation with H2O2, but different from
P1, the expected subsequent 1,6-elimination with formation of p-QM and 5-MeOT proceeds much
more slowly.
In particular, the phenol intermediate C (Scheme 3) was fully decomposed only after 3 h of
incubation with H2O2 affording 5-MeOT, little amounts of p-HBA and some other products, such as
the adducts D (Rt = 3.70 min, [M+H]+ = 297) and E (Rt = 4.18 min, [M+H]+ = 403), deriving from
the reaction of 5-MeOT with one or two molecules of p-QM (Figure 5 and Scheme 3); the
formation of the latter products, probably due to the presence of the more nucleophilic 5-MeOT,
indirectly confirms the release of electrophilic p-QM also from P2.
In the chromatogram illustrated in Figure 5, 5-MeOT appeared as two splitted peaks (Rt = 2.28 and
2.93 min), sharing the same UV profile and m/z value [M+H]+ = 191; one possible cause of this
18
ACS Paragon Plus Environment

Page 19 of 38
Chemical Research in Toxicology
1
2
3
4
5
peak fragmentation is a difference in the elution strength between the diluent and the mobile phase
(see Figure S3 and references51-52 for similar peak splitting).
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
These observations suggest that both hybrids P1 and P2 quickly undergo a selective oxidative
hydroxylation of the arylboronate trigger unit by H2O2 resulting in the corresponding intermediates
A or C (Schemes 2 and 3 respectively). However, subsequent release of the electrophilic p-QM was
more rapid and consistent from hybrid P1 (in comparison to P2), due to additional thermodynamic
driving force from the release of the more stable leaving group 1-carboxy-MLT (compared to N-
carboxy-tryptamine) in the cascade reaction. These results are consistent with 1,6-elimination of the
leaving group being the rate determining step in this process; the relative stability of the leaving
group strongly affect p-QM release.
O
O
O
B
OH
NH
OH
OH
H2O2
p-QM
O
+
5-OMeT
O
+
N
NH
O
NH
O
OH
OH
NH2
O
B
CO2
O
NH
NH
NH
H3CO
H3CO
NH
NH
H3CO
H3CO
H3CO
D
E
C
5-OMeT
P2
Scheme 3. Proposed mechanism of H2O2-induced transformation of the hybrid P2.
Bioactivation of hybrids P1 and P2 within cells by endogenously produced H2O2. Having
established that the indoleboronate derivatives, P1 and P2, could be effectively activated in vitro by
H2O2, their bioactivation in cells by endogenously produced H2O2 was indirectly assessed by
measuring Nrf2 activation. In fact, p-QM rapidly reacts with –SH groups, including cysteine
19
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 20 of 38
1
2
3
4
5
6
7
8
9
residues of the Nrf2 inhibitor Keap1, leading to accumulation and nuclear translocation of the
transcription factor.53 To this end, NCTC-2544 cells were incubated o/n. with serum-free medium (-
FBS) in the absence (NT) or presence of MLT, P1 and P2 (100 µM). Then, the media were
substituted with complete medium (+FBS) to induce intracellular H2O2 production according to
Onumah et al.54 No toxicity was detected after o/n incubation of all compounds in serum-free
medium (data not shown). Western immunoblotting analysis of nuclear extracts demonstrates that
Nrf2 was almost undetectable after o/n incubation in all the experimental conditions (Figure 6,
panel A, lines 1-4). Upon FBS stimulation, the transcription factor was strongly activated by P1 and
to a lesser extent by P2 (Figure 6 panel A, lines 6 and 7), in agreement with the in vitro observation
of a slower release of p-QM from the P2 hybrid. A modest increase in Nrf2 nuclear levels was also
observed in MLT-treated and NT cells (Figure 6 panel A, lines 5 and 8), according to previous
observations that MLT can suppress degradation of Nrf2 and enhance its nuclear translocation.21
Thus, it cannot be excluded that the higher nuclear Nrf2 accumulation induced by P1 respect to P2
could also result from the additive effect of the simultaneous p-QM and MLT release. The ability of
P1 to induce Nrf2 activation was further investigated and demonstrated to be time- and dose-
dependent (Figure 6 panel B and C). Moreover, it was completely abrogated when cells were co-
treated with the highly nucleophile GSH-C4, a membrane soluble GSH derivative47 able to prevent
Keap1 oxidation and Nrf2 activation by trapping ROS and reacting with p-QM
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Altogether, these results demonstrate that the H2O2-induced activation of the hybrid molecules
occurs within cells, consistently with the activation of Nrf2 by the released p-QM.
20
ACS Paragon Plus Environment

Page 21 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
-FBS
+FBS
NT
P1
NT P1 P2 MLT NT P1 P2 MLT
0
2
4
7
0
2
4
7
h
Nrf2
YY1
Nrf2
A
B
8
9
YY1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
NT P1
NT P1
-
-
+
+
GSH-C4
Nrf2
0
10
50 100
µM P1
Nrf2
C
D
YY1
YY1
1
2
3
4
1
2
3
4
Figure 6. Western immunoblotting analysis to assess Nrf2 activation as a marker of p-QM release.
Five µg of nuclear extracts were loaded onto a 7% polyacrylamide gel, electroblotted and
immunoblotted with an antibody against Nrf2. YY1 was stained as nuclear loading control. (A)
Cells left untreated (NT) or treated with 100 µM molecule o/n before (lines 1-4) and after 4 h of
FBS-stimulation (lines 5-8). (B) Cells untreated (NT) or incubated o/n with 100 µM P1 in serum-
free medium at different times after FBS stimulation. (C) Cells treated o/n with different
concentrations of the P1 after 4 h of FBS stimulation. (D) Cells incubated o/n with 100 µM P1 after
4 h of FBS stimulation in the presence of 10 mM GSH-C4. Pictures of panel A, B and C are
representative of two independent experiments showing similar trends.
Potential protection by P1 against oxidative stress induced by H2O2. The ability of P1 to
efficiently activate Nrf2 prompted us to investigate its potential protective effects against ROS-
related oxidative stress.
To simulate a condition of oxidative stress, we treated the cells with cytotoxic doses of H2O2 (25
and 50 µM) for 2 h and observed a dose dependent reduction of cell viability (15 and 50%,
respectively) (Figure 7, left). Pre-treatment of NCTC-2544 cells with P1 (10, 50, 100 µM) did not
21
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 22 of 38
1
2
3
4
5
6
7
8
provide any protection against H2O2-mediated oxidative stress. At the highest doses (50 and 100
µM), cell viability was even further reduced with respect to untreated cells at both the H2O2
concentrations used.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
No H2O2
*
25 µM H2O2
50 µM H2O2
NT
NT
+
NT
-
P1 P1
1
0,5
0
-
-
+
-
-
25 µM H2O2
50 µM H2O2
**
**
-
+
+
Nrf2
YY1
**
1
2
3
4
5
NT
10 µM
50 µM
100 µM
P1 concentration
Figure 7. Cell viability and Nrf2 activation in NCTC-2544 cells pre-treated with P1 and then
exposed to H2O2. Left: Cells left untreated (NT) or pretreated with different concentrations of P1
o/n, were challenged with H2O2 for 2 h. At the end of the incubation time, the MTS reagent was
added into each well and incubated for 30 min at 37°C. The plates were read in a microplate reader
at 490 nm at a reference wavelength of 630 nm. Data are the mean ± SD of three separate
experiments each carried out on eight replicates. *p<0.05; **p<10-3 vs NT or NT challenged with
the same concentration of H2O2. Right: western immunoblotting analysis of Nrf2 activation. Cells
were pretreated with 100 µM P1 o/n and then exposed to 25 or 50 µM H2O2 for 2 h as described
above. Five µg of nuclear extracts were loaded onto a 7% polyacrylamide gel, electroblotted and
immunoblotted with an antibody against Nrf2. YY1 was stained as nuclear loading control.
The increased cytotoxicity observed at the highest doses of the hybrid P1 suggests that the products
released after H2O2 treatment could exert cytotoxic effects which may predominate over the
22
ACS Paragon Plus Environment

Page 23 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
beneficial effects expected from the Nrf2 regulated activation of the antioxidant response. Indeed,
analysis of Nrf2 activation revealed that the factor was only modestly activated in cells challenged
with H2O2 for 2 h, even in cells pretreated with 100 µM P1 (Figure 7, right). This result is
consistent with the evidence that after 2 h Nrf2 levels induced by P1, bioactivated by endogenously
generated H2O2, are still very low (Figure 6, lane 6). Hence, P1 is not effective in counteracting the
cytotoxic effects of H2O2 since the timing of Nrf2 activation may happen later than H2O2- and p-
QM-induced damage. Thus, the mounting of late defense mechanisms is inefficient in cell
protection.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cytotoxicity of hybrids P1 and P2 in NCTC-2544 and HeLa cells. Tumor cells produce high
levels of ROS, which make them distinctly different from normal cells.55 A putative strategy for
selective destruction of tumor cells involves the use of cytotoxic drugs, including QM-based
prodrugs, which can be activated by endogenously generated H2O2.56,57 Examples of arylboronate
analogues which can be activated by H2O2 to form QMs and directly alkylate DNA,58,59 or amplify
oxidative stress in cancer cells by alkylating GSH60 have been already reported.
Thus, we preliminarly tested whether the molecules were activated in HeLa cells, a very well-
known and broadly used human cancer cell line. Cells were cultured with 100 µM P1 or P2 for
different times and then harvested to assess Nrf2 activation as marker of molecule bioactivation.
Results shown in Figure 8 demonstrate that both molecules were able to stimulate Nrf2
accumulation with some differences in the kinetic of activation which occurred later in P2- (Figure
8, lanes 6-9) respect to P1-treated cells (Figure 8, lines 2-5).
23
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 24 of 38
1
2
3
4
5
P1
P2
6
7
8
0 1.5 3
6
8
1.5 3
6
8
h
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Nrf2
actin
1
2
3
4
5
6
7
8
9
Figure 8. Western immunoblotting analysis of Nrf2 activation in HeLa cells treated with P1 or P2.
Cells were incubated with 100 µM P1 or P2 for different times up to 8 h. Cells were lysed in SDS
buffer and 40 µg of whole cell lysates were loaded onto a 7% polyacrylamide gel, electroblotted
and immunoblotted with an antibody against Nrf2. Actin was stained as loading control. Picture is
representative of two independent experiments showing similar trends.
To evaluate the activity and selectivity of our hybrid compounds we measured cell viability in
normal NCTC-2544 and tumoral HeLa cell lines at concentrations ranging from 0 to 100 µM for 24
h. The highest concentration of P1 and P2 (100 µM) led to approximately 26% and 12% cell death
in NCTC-2544 cells, respectively, when compared to untreated cells (black and dark gray columns,
Figure 8). By contrast, MLT and 5-MeOT had no toxic effects at all the tested doses (light gray
columns, left and right respectively, Figure 9). No toxicity was observed in the cells grown in
medium containing only the vehicle (i.e. 0.1% v/v DMSO) (data not shown).
24
ACS Paragon Plus Environment

Page 25 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 9. Cell viability in NCTC-2544 cells exposed to P1, P2, MLT and 5-MeOT. The cells were
seeded in 96-well microtiter plates at a density of 5 x 103 cells/well and subsequently exposed to P1,
P2, MLT and 5-MeOT at increasing concentrations (1-100 µM) for 24 h. At this time, 20 µl of
MTS were added into each well, and then incubated for 30 min at 37°C. The plates were read in a
microplate reader at 490 nm at a reference wavelength of 630 nm. Data are the mean ± SD of three
separate experiments each carried out in eight replicates. * p<0.05 vs not treated cells (0 µM).
Based on these findings, the effects of our molecules were then studied in the tumoral HeLa cell
line at the concentrations of 10, 50, 100 µM for 24 h. All the compounds showed a dose-dependent
cytotoxicity when compared to untreated cells (Figure 9). Specifically, cell viability was almost
25
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 26 of 38
1
2
3
4
5
6
7
8
9
unaffected by 10 µM of all molecules, while a dose-related decrease of cell viability was observed
with concentrations of 50 and 100 µM of P1, P2 and MLT. In detail, hybrid P1 was the most
cytotoxic and statistically significant differences were found at 100 µM versus P2 and MLT (Figure
10). These results are compatible with the slower bioactivation of P2 to the p-QM, compared to P1
and, to a lesser extent, to the release of MLT from P1. Supporting this hypothesis, cytotoxicity tests
performed to determine the toxic effects of the combination of P2 with MLT (both 100 µM)
revealed an effect similar to that exerted by P1 (data not shown). At 100 µM, the cytotoxic action of
both hybrids was more evident in HeLa cells than in NCTC-2544 cells, while, at 50 µM, cell death
was only observed in HeLa cells (Figures 9 and 10). These data are consistent with an
overproduction of ROS in cancer cells, as demonstrated below (Figure 11), which can activate our
hybrid compounds more efficiently than in normal cells. However, even if Nrf2 levels persist for
long times (up to 8 h, Figure 8), they are not sufficient to defend cells from death. Normal and
tumoral cells seem to respond differently also to MLT treatment, i.e. cell viability of NCTC-2544
cells is not affected by MLT while the highest concentrations of MLT are able to display cytotoxic
action towards HeLa cells (Figures 9 and 10 dark gray, left). On the contrary 5-MeOT treatment did
not exert significant cytotoxic effect both in NCTC-2544 and HeLa cells (Figures 9 and 10 light
gray, right).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
26
ACS Paragon Plus Environment

Page 27 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0,7
0,6
0,5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
0,4
**
***
50
0,3
0,2
0,1
0
***
*** ‡
100
0
10
0
10
50
100
P1 (µM)
P2 (µM)
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
*
*
0
10
50
100
0
10
50
100
5-MeOT (µM)
MLT (µM)
Figure 10. Cell viability in HeLa cells exposed to P1, P2, MLT and 5-MeOT. The cells were
seeded in 96-well microtiter plates at a density of 2 x 103 cells/well and subsequently exposed to P1,
P2, MLT and 5-MeOT at 10, 50, 100 µM for 24 h. At this time, 20 µl of MTS were added into each
well, and then incubated for 30 min at 37°C. The plates were read in a microplate reader at 490 nm
at a reference wavelength of 630 nm. Data are the mean ± SD of three separate experiments each
carried out in eight replicates. Data were analyzed with one-way analysis of variance (ANOVA). *
p<0.05 vs not treated cells (0 µM); ‡ p<0.05 vs cells treated with P2 or MLT or 5-MeOT (100 µM);
** p<0.01, *** p<0.001 vs not treated cells (0 µM).
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To assess whether p-QM or MLT released from P1 could interfere with MTS assay, some data
points were confirmed by trypan blue assay. P1-treated HeLa cells, which provided more relevant
results, were used in the elucidation of cytotoxicity effects. The results confirmed those obtained by
MTS assay, in fact no toxicity was observed with 10 µM P1 while about 25% and 40% dead cells
were measured in HeLa cells treated with 50 µM and 100 µM P1 respectively (Figure S4).
27
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 28 of 38
1
2
3
4
5
6
7
8
Intracellular levels of GSH and ROS in NCTC-2544 and HeLa cells. To explain the differences
between the cytotoxic effects of hybrids P1 and P2 in HeLa compared to NCTC-2544 cells, the
GSH content and ROS levels in both cell lines were investigated. HeLa cells treated with P1 or P2
showed consistent reduction in the level of intracellular GSH in comparison with untreated cells
(Figure 11, right). Interestingly, treatment of HeLa cells with P1 and P2 led to dramatic decrease in
GSH content (from about 82 picomoles GSH/µg protein in not treated cells to about 40 and 64
picomoles GSH/µg protein in the cells treated with P1 and P2 respectively), while a moderate
reduction was observed in normal NCTC-2544 cells (from about 96 picomoles GSH/µg protein in
not treated cells to about 74 and 80 picomoles GSH/µg protein in the cells treated with P1 and P2
respectively). Regarding the content of ROS, as expected, untreated HeLa cells showed higher basal
intracellular ROS concentrations compared to NCTC-2544 cells (Figure 11, left). P2 treatment did
not affect ROS levels in NCTC-2544 cells and induced a moderate rightward shift of DCF
fluorescence in HeLa cells. On the contrary, P1 increased ROS level in both cell lines but only in
HeLa cells the fold increase vs untreated cells (NT) was statistically significant. All these results are
compatible to a preferential bioactivation of our hybrid compounds in cancer cells that overproduce
ROS, generating higher levels of the GSH-scavenging p-QM and a consequent further increase in
ROS levels above the threshold, not compatible with HeLa cells survival.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
28
ACS Paragon Plus Environment

Page 29 of 38
Chemical Research in Toxicology
1
2
3
4
NCTC-2544 cells
NCTC-2544 cells
5
6
7
8
120
100
80
60
40
20
0
6
5
4
3
2
1
0
*
*
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
NT
P1
P2
MLT
NT
P1
*
P2
MLT
6
5
4
3
2
1
0
120
100
80
60
40
20
0
*
NT
P1
P2
MLT
NT
P1
P2
MLT
HeLa cells
HeLa cells
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 11. Left: Production of ROS in NCTC-2544 (top panels) and HeLa cells (bottom panels).
Bars represent mean of fold increase of DCF fluorescence intensity (DCF MFI) in cells treated with
100 µM of P1, P2 and MLT o/n compared to the untreated sample (NT). In each sample, 10,000
events were counted and each experiment was conducted in duplicate and repeated at least three
times. *p<0.05 vs untreated cells (NT).
Right: GSH content in NCTC-2544 cells (top panel) and HeLa cells (bottom panel) either untreated
or treated with 100 µM of P1, P2 and MLT o/n. Briefly, after treatments, the cells were washed and
lysed with 100 µL of lysis buffer (0.1% Triton X-100, 0.1 M Na2HPO4, 5 mM EDTA, pH 7.5).
Thereafter, 15 µL of 0.1 N HCl and 140 µL of precipitating solution (100 ml containing 1.67 g of
glacial meta-phosphoric acid, 0.2 g of disodium EDTA, 30 g of NaCl), were added. After
centrifugation, the supernatant was collected and 25% (v/v) 0.3 M Na2HPO4 and immediately after
10% (v/v) DTNB were added. The mixture was stirred for 1 min at RT, then left at RT for another 5
min, and finally used for HPLC determination of GSH. Unpaired data were analyzed with the
Student’s t-test with Welch correction. *p<0.05 vs untreated cells (NT).
29
ACS Paragon Plus Environment