Photo-control of cancer cell growth by benzodiazo N-substituted pyrrole derivatives

Article abstract of DOI:10.1016/j.jphotochem.2019.03.042

In order to expand the class of diazocompounds able to act as photo-activable microtubule inhibitors the potential of azo-heteroarenes has been explored. In this paper we focus on the synthesis, physical properties and biological effects of methyl rac-2-(2-((E)-(4-((R)-2,3-dihydroxypropoxy)phenyl) diazenyl)-1H-pyrrol-1-yl)-3-hydroxypropanoate (1a) and methyl rac-2-(2-((E)-(4-((S)-2,3-dihydroxypropoxy)phenyl) diazenyl)-1H-pyrrol-1-yl)-3-hydroxypropanoate (1b). Preliminary biological studies on the HCT-116 p53-/- cancer cell line have shown that the weak antiproliferative action of the trans isomers of these molecules, especially of 1a, is enhanced upon LED light irradiation at 435 nm. On A375 cells the molecules have not shown any effect on cell viability either in the dark or under irradiation. Moreover, the two diastereomeric components of 1a as pure stereomers have been synthesized and characterized for their chemical-physical properties. Interestingly, upon irradiation, 1a has shown an antiproliferative activity on the HCT-116 p53-/- cells greater than that of the pure stereomers, 1RR and 1RS. Tubulin polymerization assay has also demonstrated that 1a, 1RR and 1RS inhibit tubulin aggregation mostly after exposure of the samples to LED light irradiation.

Full text of DOI:10.1016/j.jphotochem.2019.03.042

Accepted Manuscript
Title: Photo-control of cancer cell growth by benzodiazo
N-substituted pyrrole derivatives
Authors: Concetta Imperatore, Maria Scuotto,
Mohammadhassan Valadan, Elisa Rivieccio, Assunta Saide,
Annapina Russo, Carlo Altucci, Marialuisa Menna, Anna
Ramunno, Luciano Mayol, Giulia Russo, Michela Varra
PII:
S1010-6030(18)31776-3
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2019.03.042
JPC 11779
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
5 December 2018
19 March 2019
25 March 2019
Please cite this article as: Imperatore C, Scuotto M, Valadan M, Rivieccio E,
Saide A, Russo A, Altucci C, Menna M, Ramunno A, Mayol L, Russo G,
Varra M, Photo-control of cancer cell growth by benzodiazo N-substituted pyrrole
derivatives, Journal of Photochemistry and amp; Photobiology, A: Chemistry (2019),
https://doi.org/10.1016/j.jphotochem.2019.03.042
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Photo-control of cancer cell growth by benzodiazo N-substituted pyrrole derivatives
Concetta Imperatore†a, Maria Scuotto†a, Mohammadhassan Valadanb, Elisa Rivieccioa, Assunta
Saidea, Annapina Russoa, Carlo Altuccib, Marialuisa Mennaa, Anna Ramunnoc, Luciano Mayola,
Giulia Russo*a and Michela Varra*a
a
Department of Pharmacy, University of Naples Federico II, Naples 80131, Italy. e-mail
varra@unina.it; correspondence can be also sent to giulia.russo@unina.it.
b Department of Physics “Ettore Pancini”, University of Naples Federico II, Naples 80126, Italy.
c Department of Pharmacy/DIFARMA, University of Salerno, 84084 Fisciano, Salerno, Italy.
†These authors contributed equally to the manuscript.
Graphical abstract
highlights


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

The new reported compounds easily switch from the trans to the cis form after LED irradiation;
the UV spectra reveal the reversibility of trans↔cis isomerisation;
after irradiation the trans-cis photo-conversions are detectable in 1H NMR spectra;
kinetics of the trans↔cis isomerisations were evaluated by UV and fast UV experiments;
the compounds differently affect the dynamic of MT polymerization after UV irradiation;
under irradiation the compounds affect HCT-116 p53-/- cell viability.
Abstract
In order to expand the class of diazocompounds able to act as photo-activable microtubule
inhibitors the potential of azo-heteroarenes has been explored. In this paper we focus on the
synthesis, physical properties and biological effects of methyl rac-2-(2-((E)-(4-((R)-2,3-
dihydroxypropoxy)phenyl) diazenyl)-1H-pyrrol-1-yl)-3-hydroxypropanoate (1a) and methyl rac-2-
(2-((E)-(4-((S)-2,3-dihydroxypropoxy)phenyl) diazenyl)-1H-pyrrol-1-yl)-3-hydroxypropanoate (1b).
Preliminary biological studies on the HCT-116 p53-/- cancer cell line have shown that the weak
antiproliferative action of the trans isomers of these molecules, especially of 1a, is enhanced upon
1

LED light irradiation at 435 nm. On A375 cells the molecules have not shown any effect on cell
viability either in the dark or under irradiation. Moreover, the two diastereomeric components of
1a as pure stereomers have been synthesized and characterized for their chemical-physical
properties. Interestingly, upon irradiation, 1a has shown an antiproliferative activity on the HCT-
116 p53-/- cells greater than that of the pure stereomers, 1RR and 1RS. Tubulin polymerization
assay has also demonstrated that 1a, 1RR and 1RS inhibit tubulin aggregation mostly after
exposure of the samples to LED light irradiation.
key words: N-substituted pyrrole, diazocompounds, tubulin inhibitors.
1. Introduction
Targeting a remote, non-invasive, spatiotemporal control of specific biological systems through
light induced stimuli has gained increasing attention in recent years [1-5]. Nowadays, the term
photopharmacology refers to these therapeutic approaches, in which the key role is played by
photo-responsive molecules or molecular portion(s), that, switching to different molecular states
upon specific irradiation, undergo changes in their pharmacokinetic or pharmacodynamic
properties [1-3].
Azobenzenes (AzBs) are one of the most interesting classes of photo-responsive molecules
because of their quantitative and reversible, light induced, trans-cis photoisomerization [4,5]. The
control of some ionotropic glutamate receptors [6,7], the folding-unfolding of the three-
dimensional structure of peptides [8] or nucleic acids [9], and the building of photosensitive supra-
molecular architectures [10], represent only a few examples of the potential applications of photo-
switchable AzBs.
Interestingly, the cis isomers of some AzBs, named photostatins (PSTs, Figure 1), are potential
tools for anti-cancer therapy in view of their ability to target microtubules [11-13] (MTs). MTs are
dynamic structures involved in the control of a great number of cellular events, including cell
signalling, cell division and mitosis [14]. Since cancer cells proliferate rapidly and uncontrollably,
MTs represent an attractive target in cancer therapy. However, since MTs are key components of
the cytoskeleton common to all eukaryotic cells, almost all microtubule targeting agents (MTAs)
are highly toxic. PSTs are analogues of combretastatin A-4 (CA4), a natural ligand of MTs; they
were designed by changing the exocyclic C=C into a N=N bond (Figure 1). The affinities of PSTs
towards the MT colchicine binding site were turned on by irradiation of their stable trans isomers
into the corresponding cis isomers. Thus, a local and specific activation through irradiation
prevents the side effects associated with the perturbation of the MT dynamics in normal cells,
revealing PSTs as new efficient MTAs [15-17]. Despite this, only a few examples of AzB analogues
as potential photo-activable MTAs have been reported until now [11-13,18]. In accordance with
their distinctive properties, the exploration of different typologies of AzBs is advisable.
Moreover, several CA4 analogues have been discovered in the last few years [17,19-21], modified
at the phenyl rings and/or at the double carbon bond. In particular, the replacement of the
exocyclic C=C with a heterocyclic aromatic moiety (i.e. pyrroles, imidazoles, thiazoles, thiophenes,
etc,) [19] not only allowed the desired cis-like conformational constraint but also introduced new
pharmacological properties, because of the incorporation of heteroatoms. It is noteworthy that
the etheroarene diazo derivatives show interesting optical properties, compared to those of the
unsubstituted AzBs [22-24]. Indeed, they switch from the trans to the cis form by irradiation with
low intensity light having red-shifted wavelengths, thus limiting the disadvantages of any possible
radiation treatment. In fact, it is known that radiation having a wavelength not significantly down-
shifted towards the UV side of the light spectrum (typically   400 nm) and intensities not
exceeding  10 mW/cm2, does not trigger any undesired damaging processes in the treated live
2

matter. Typically, such damaging processes occur with  < 400 nm and/or higher intensities [25
and references therein for a general presentation of the interaction mechanisms and triggered
processes between IR-Visible-UV radiation and live matter].
Furthermore, the regioselectivity at Cα of the pyrrole, that is retained in N-substituted pyrrole
derivatives, increases the yields of coupling in reactions with azonium salts, also enlarging the pool
of N-substituted diazo compounds to be investigated [17].
In order to explore the potential of diazo-heteroarenes as cancer cell growth inhibitors, we
synthesized methyl rac-2-(2-((E)-(4-((R)-2,3-dihydroxypropoxy)phenyl)diazenyl)-1H-pyrrol-1-yl)-3-
hydroxypropanoate 1a and methyl rac-2-(2-((E)-(4-((S)-2,3-dihydroxypropoxy)phenyl)diazenyl)-1H-
pyrrol-1-yl)-3-hydroxypropanoate 1b (Figure 1 and Scheme 1). The solvated samples 1a and 1b
were then subjected to in depth investigation to characterize their physical and biological
properties. In view of the increasing of the antiproliferative effect of 1a on the viability of the HCT-
116 p53-/- cells under irradiation compared to the darkness regimen, its pure stereomers 1RR and
1RS were also synthesized and tested (Scheme 1). The photo-activated 1RR and 1RS as well as
their effects on cancer cells were evaluated. In order to preliminarily investigate the mechanism of
action of 1a and of its pure components, a light-scattering-based tubulin polymerization assay was
run under both dark and irradiation conditions.
2. MATERIAL AND METHODS
2.1. General
Chemicals and anhydrous solvents were purchased from Sigma-Aldrich. TLCs were run on Merck
silica gel 60 F254 plates and the spots were visualized by means of an UV lamp (Vilber Lourmat VL-
4LC, 365 and 254 nm). Silica gel chromatography was performed using Merck silica gel 60 (0.063–
0.200 mm). HPLC purifications and analyses were carried out using a JASCO PU-2089 Plus HPLC
pump equipped with a JASCO BS-997-01 UV detector. CD experiments were performed on a JASCO
715 spectropolarimeter equipped with a PTC-348 temperature controller. UV experiments were
performed on a JASCO V-530 spectrophotometer, equipped with a PTC-348 temperature
controller. HRESIMS (high resolution electrospray ionization mass spectrometry) analyses were
performed on a Thermo LTQ Orbitrap XL mass spectrometer (Thermo-Fisher, San Josè, CA, USA).
The spectra were recorded by infusion into the ESI (Thermo-Fisher, San Josè, CA, USA) source
1
using MeOH as solvent. The [α] values were measured with JASCO polarimeter P-2000. H (500
MHz and 400 MHz) and 13C (125 MHz) NMR spectra were recorded on an Agilent INOVA
spectrometers (Agilent Technology, Cernusco sul Naviglio, Italy); chemical shifts were referenced
to the residual solvent signal (CD3OD: δH = 3.31, δC = 49.0 ppm; C6D5N: δH = 7.19, 7.55 and 8.71
1
ppm). For an accurate measurement of the coupling constants, the one-dimensional H NMR
1
spectra were transformed at 64 K points (digital resolution: 0.09 Hz). H connectivities were
determined by COSY and TOCSY experiments (mixing time 100 ms). Through-space 1H
connectivities were evidenced using a ROESY experiment with a mixing time of 500 ms. Two and
three bond 1H-13C connectivities were determined by gradient 2D HSQC experiments optimized for
a 2,3J of 8 Hz. 3JH-H values were extracted from 1D 1H NMR.
2.2. Synthesis
R or S 3-(4-nitrophenoxy)propane-1,2-diol (2R/2S). A mixture of 4-nitrophenol, (0.500 g, 3.6
mmol), KOH (1.0 mg, 1.8x10-2 mmol, dissolved in 50 μL of EtOH) and dry toluene (2 mL) was stirred
under reflux in a double neck flask. After 30 min, the mixture was cooled to r.t. and 287 µL of R or
S glycidol (0.320 g, 4.3 mmol) were slowly added by a syringe. The mixture was further stirred at
90 °C for 24 h. The solvent was then evaporated under vacuo and the residue dissolved in MeOH.
The obtained solution was embedded on silica and then evaporated to dryness. The resulting
3

powder was applied on a ethyl acetate:acetone 70:30 (v/v) packed silica gel column eluted using
isocratic conditions. The fractions containing the desired product were dried under vacuo to give R
or S stereomer of 2 as a yellow gel (yields 85% and 86%, respectively). TLC plate Rf= 0.73 (ethyl
acetate:acetone 70:30 (v/v)).
1H NMR (CD3OD) δH: 3.64–3.73 (m, 2H, CH2OH), 3.97-4.05 (m, 1H, CHOH), 4.09 (dd, J = 6.1, 9.8 Hz,
1H, OCH2), 4.18 (dd, J = 4.1, 9.8 Hz, 1H, OCH2), 7.05–7.10 (m, 2H, C2,6ArH), 8.14–8.20 (m, 2H,
C3,5ArH). 13C NMR (CD3OD) δC: 63.9 (CH2OH), 71.2 (CHO), 71.4 (CH2O), 115.8 (C2,6Ar), 126.8 (C3,5Ar),
142.8 (C4Ar), 165.5 (C1Ar). HRESIMS (positive ion mode, CH3OH) m/z 236.0533 [M+Na]+ (calcd for
C9H11NO5Na, 236.0529).
[α]25 R= -17.8 [c= 12.4 mg/mL; EtOH] S=+16.9 [c= 19.2 mg/mL; EtOH].
R or S 3-(4-aminophenoxy)propane-1,2-diol (3R/3S). A mixture of 2 (R or S, 0. 560 g, 2.6 mmol),
Pd/C (5% w/w, 28 mg) and 25 mL EtOH (90%, HPLC grade) was stirred in a Parr apparatus under
hydrogen atmosphere (P= 400 psi). After 1.5-2.0 h the reaction was monitored by disappearance
of UV spot of 2 on TLC plate (Rf= 0.73, ethyl acetate:acetone 70:30 (v/v) and the appearance of a
new UV spot strongly sensitive to ninhydrin (Rf= 0.46). The mixture was filtered, and the eluted
solution dried in vacuo, to obtain 3R or 3S as brick-red solid, that was further purified by
crystallization from DCM:MeOH 7:3 (0.440 g, yields 93%).
1H NMR (CD3OD) δH: 3.62 (dd, J = 5.4, 11.2 Hz, 1H, CH2OH), 3.67 (dd, J = 4.8, 11.2 Hz, 1H, CH2OH),
3.89–3.84 (m, 1H, CHOH), 3.91 (dd, J = 5.1, 9.8 Hz, 1H, OCH2), 3.95 (dd, J = 4.1, 9.8 Hz, 1H, OCH2),
6.70 (d, J = 8.8 Hz, 2H, C2,6ArH), 6.76 (d, J = 8.8 Hz, 2H, C3,5ArH). 13C NMR (CD3OD) δC: 64.3 (CH2OH),
71.1 (CHO), 71.9 (CH2O), 118.1 (C2,6Ar), 116.6 (C3,5Ar), 141.8 (C4Ar), 153.6 (C1Ar). HRESIMS (positive
ion mode, CH3OH) m/z 184.0980 [M+H]+ (calcd for C9H14NO3, 184.0974).
1a, 1b, 1RR and 1RS. A mixture of 3R or 3S (0.400 g, 2.2 mmol), 205 µL fluoroboric acid (water
solution, 48% v/v, 3.3 mmol) 4 mL of EtOH was cooled to -15 °C under argon and then 224 µL of
isoamyl nitrite (0.258 g, 2.2 mmol) were added. After 12 h under stirring, the produced diazonium
salt was first precipitated diluting the mixture with n-hexane, and then further washed with n-
hexane (three times). The solid residue was dried under vacuo to give 4R or 4S, which was used in
the next step without further treatment. The intermediate 4R or 4S (0.360 g, 1.8 mmol) was
dissolved in 10 mL of acetic acid (14 g, 232 mmol), under stirring. Sodium acetate (4.0 g, 511
mmol) and rac-5 or 5R or 5S (254 µL, 0.270 g, 2.2 mmol) were added, consecutively, to the
solution [26]. After 15 min at r.t., the formation of a gel-like mixture was observed. The reaction
time was further prolonged up to 1 h, under slow stirring. The final mixture was diluted with water
and extracted with DCM (4-5 times). The collected organic layers were dried under vacuo and the
residue dissolved in MeOH. The obtained solution was embedded on silica and then evaporated to
dryness. The resulting powder was applied on a DCM:propan-2-ol 90:10 (v/v) packed silica gel
column and then chromatographed using isocratic conditions. The collected fractions, dried under
vacuo (TLC 90:10 DCM:MeOH, v/v, Rf= 0.6) were further purified on HPLC RP-18 column (H2O:ACN,
80:20, v/v, tR= 12 min), to get 0.185 of 1b (yields 51%), 0.210 g of 1a (yields 58%), 0.214 g of 1RR
or 1RS (yields 59%). NMR spectra of each species are all reported in the SI.
1a. 1H NMR (CD3OD) δH: 3.66 (m, 2H, CH2OH), 3.67 (s, 3H, OCH3), 3.99 (m, 1H, CHOH), 4.13 (dd, J =
4.2, 9.6 Hz, 1H, OCH2), ), 4.19 (dd, J = 6.8, 11.7 Hz, 1H, CH2OH), 4.24 (dd, J = 4.7, 11.7 Hz, 1H,
CH2OH pyrrole 4.24 (dd, J = 6.0, 9.6 Hz, 1H, OCH2), 5.62 (dd, J = 4.8, 6.6 Hz, 1H, CHCOCH3), 6.32
(dd, J = 3.0, 3.38 Hz, 1H, C4pyrr), 6.67 (dd, J = 1.2, 3.9 Hz, 1H, C3pyrr), 7.04 (d, J = 8.9 Hz, 2H, C2,6ArH),
7.28 (s, m, 1H, C5pyrr), 7.70 (d, J = 8.9 Hz, 2H, C3,5ArH). 13C NMR (CD3OD) δC: 51.8 (OCH3), 60.2
(CHCOCH3), 62.8 (CH2OH), 70.5 (CHOH), 69.5 (OCH2), 69.4 (CH2OH), 99.9 (C3pyrr), 110.0 (C4 pyrr),
126.2 (C5 pyrr), 147.8 (C2pyrr), 114.7 (C2,6Ar), 123.4 (C3,5Ar), 145.5 (C4Ar), 160.7 (C1Ar), 170.2 (COCH3).
1b. 1H NMR (CD3OD) δH: 3.66 (m, 2H, CH2OH), 3.67 (s, 3H, OCH3), 3.99 (m, 1H, CHOH), 4.13 (dd, J =
4.2, 9.6 Hz, 1H, OCH2), ), 4.19 (dd, J = 6.8, 11.7 Hz, 1H, CH2OH), 4.24 (dd, J = 4.7, 11.7 Hz, 1H,
4

CH2OH pyrrole 4.24 (dd, J = 6.0, 9.6 Hz, 1H, OCH2), 5.62 (dd, J = 4.8, 6.6 Hz, 1H, CHCOCH3), 6.32
(dd, J = 3.0, 3.38 Hz, 1H, C4pyrr), 6.67 (dd, J = 1.2, 3.9 Hz, 1H, C3pyrr), 7.04 (d, J = 8.9 Hz, 2H, C2,6ArH),
7.29 (s m, 1H, C5pyrr), 7.70 (d, J = 8.9 Hz, 2H, C3,5ArH). 13C NMR (CD3OD) δC: 51.8 (OCH3), 60.2
(CHCOCH3), 62.8 (CH2OH), 70.5 (CHOH), 69.5 (OCH2), 69.4 (CH2OH), 99.9 (C3pyrr), 110.0 (C4 pyrr),
126.2 (C5 pyrr), 147.8 (C2pyrr), 114.7 (C2,6Ar), 123.4 (C3,5Ar), 145.5 (C4Ar), 160.7 (C1Ar), 170.2 (COCH3).
1RR. 1H NMR (CD3OD) δH: 3.66 (m, 2H, CH2OH), 3.67 (s, 3H, OCH3), 3.99 (m, 1H, CHOH), 4.13 (dd, J
= 4.2, 9.6 Hz, 1H, OCH2), ), 4.19 (dd, J = 6.8, 11.7 Hz, 1H, CH2OH), 4.24 (dd, J = 4.7, 11.7 Hz, 1H,
CH2OH pyrrole 4.24 (dd, J = 6.0, 9.6 Hz, 1H, OCH2), 5.62 (dd, J = 4.8, 6.6 Hz, 1H, CHCOCH3), 6.32
(dd, J = 3.0, 3.38 Hz, 1H, C4pyrr), 6.67 (dd, J = 1.2, 3.9 Hz, 1H, C3pyrr), 7.04 (d, J = 8.9 Hz, 2H, C2,6ArH),
7.29 (s m, 1H, C5pyrr), 7.70 (d, J = 8.9 Hz, 2H, C3,5ArH). 13C NMR (CD3OD) δC: 51.8 (OCH3), 60.2
(CHCOCH3), 62.8 (CH2OH), 70.5 (CHOH), 69.5 (OCH2), 69.4 (CH2OH), 99.9 (C3pyrr), 110.0 (C4 pyrr),
126.2 (C5 pyrr), 147.8 (C2pyrr), 114.7 (C2,6Ar), 123.4 (C3,5Ar), 145.5 (C4Ar), 160.7 (C1Ar), 170.2 (COCH3).
1RS. 1H NMR (CD3OD) δH: 3.66 (m, 2H, CH2OH), 3.67 (s, 3H, OCH3), 3.99 (m, 1H, CHOH), 4.13 (dd, J
= 4.2, 9.6 Hz, 1H, OCH2), ), 4.19 (dd, J = 6.8, 11.7 Hz, 1H, CH2OH), 4.24 (dd, J = 4.7, 11.7 Hz, 1H,
CH2OH pyrrole 4.24 (dd, J = 6.0, 9.6 Hz, 1H, OCH2), 5.62 (dd, J = 4.8, 6.6 Hz, 1H, CHCOCH3), 6.32
(dd, J = 3.0, 3.38 Hz, 1H, C4pyrr), 6.67 (dd, J = 1.2, 3.9 Hz, 1H, C3pyrr), 7.04 (d, J = 8.9 Hz, 2H, C2,6ArH),
7.29 (s m, 1H, C5pyrr), 7.70 (d, J = 8.9 Hz, 2H, C3,5ArH). 13C NMR (CD3OD) δC: 51.8 (OCH3), 60.2
(CHCOCH3), 62.8 (CH2OH), 70.5 (CHOH), 69.5 (OCH2), 69.4 (CH2OH), 99.9 (C3pyrr), 110.0 (C4 pyrr),
126.2 (C5 pyrr), 147.8 (C2pyrr), 114.7 (C2,6Ar), 123.4 (C3,5Ar), 145.5 (C4Ar), 160.7 (C1Ar), 170.2 (COCH3).
ε (CH3OH)= 1.05x104 M-1cm-1; ε(PBS)= 1.00x104 M-1cm-1.
HRESIMS (positive ion mode, CH3OH) 1a m/z 364.1510 [M+H]+; 1b m/z 364.1498 [M+H]+; 1RR m/z
364.1506 [M+H]+; 1RS m/z 364.1506 [M+H]+ (calcd for C17H22N3O6, 364.1503). [α]25 1RR = +12.0 [c=
26.6 mg/mL; MeOH] 1RS = -12.6 [c= 29.3 mg/mL; MeOH].
2.3. Chiral HPLC
2, as R or S stereomer and a mixture of both species were analyzed on ChiralpaK 1A column
(4.6x250 mm, particle size: 5micron, Figure S1) eluted with 90:10 n-hexane:propan-2-ol. tR = 33.85
min for R-3-(4-nitrophenoxy)propane-1,2-diol; tR = 38.55 min for S-3-(4-nitrophenoxy)propane-1,2-
diol. 1RR, 1RS and 1a were analyzed on cellulose-3 chiral column (4.6x100 mm, particle size: 3
micron, Figure S2) eluted with 80:20 n-hexane:propan-2-ol. tR of 1RR was 13.4 min and that of 1RS
was 19.8 min. 1RR and 1RS differed in their CD profiles, both showing a Cotton effect centred at
about 400 nm but having almost opposite signature (Figure S3).
2.4. UV spectroscopy
In order to obtain data related to the trans isomer, the cuvette (0.1 o.l. Vol. 400 μL) containing 40
or 80 μM of 1a or 1b in MeOH was taken in dark at r.t. for about 12 h. UV spectra of the cis
isomers were acquired after irradiation of the samples at 400, 435 or 505 nm (1 min, 1 LED 400,
435, or 505 nm Roithner Lasertechnik [11]). The cis thermal decay was monitored by further
sequential acquisition of spectra (every two minutes) until the UV profile became that of the initial
trans isomer. In order to obtain the t1/2 of cis isomer, after the sample irradiation at 435 nm, UV
time course measurement experiments were performed monitoring the absorbance values at 340,
390, 400, 425, and 505 nm, number of cycle 100-180, cycle time 60 s. The spectra showing the
cis→trans photo-conversion were obtained after irradiation with green light (1 min, 1 LED 505
nm). Samples for UV experiments in PBS were prepared mixing 63 or 126 μL of 1a or 1b (1.0 mM in
DMSO) and 1400 μL of PBS. Finally, the stability of 1a or 1b under the used light conditions, were
verified acquiring spectra after 24 h of irradiation using the same pulse-program employed for
biological assay. In particular, the UV cuvette containing 1a or 1b at 40 μM was exposed to pulsed
LED at 435 or 505 nm. LEDs were managed by an Arduino module (50 ms-ON/ 1.0 min-OFF, 50 ms
ON/1.0 s OFF; 50 ms ON /500 ms OFF; 500 ms ON/7.5 min OFF).
2.5. Fast UV spectroscopy
5

Samples were prepared by dissolving 1a and 1b molecules in different solvents: methanol, H2O,
and PBS. 6a and 6b were produced as a result of hydrolysis of the 1a/b in water-based solutions.
2.0 mL of the sample was hold in a quartz cuvette, stirred by a magnetic stirrer, and transmission
percentage at 404 nm in 10 mm optical path was monitored. A sketch of the optical setup for this
experiment is shown in scheme S1.
2.6. Cell cultures and treatments
The HCT-116 p53 -/- [27] and A375 cell lines were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM), supplemented with 10% fetal bovine serum (FBS) (Euroclone S.p.A), 2 mM L-glutamine
and 50 U/ml penicillin-streptomycin, under humidified atmosphere of 5% CO2 at 37 °C. Treatments
of cells were performed replacing the culture medium with those containing indicated compounds
at final concentration of 1 and 10 μM per well and exposed or not to light irradiation at 435 or 505
nm for 12 h, 24 h or 48 h. Cell viability after treatment was analysed by MTT assay [28].
2.7. MTT assay
HCT-116 p53-/- cells were seeded onto 96-well plates at density of 2 x 104 cells/well. Cell viability
was evaluated using the MTT assay procedure, which measures the level of mitochondrial
dehydrogenase activity of living cells employing the yellow 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) as substrate [29]. The absorbance was measured at 540
nm using a microplate reader (Labsystems Multiskan). Error bars represent mean ± SEM. Statistical
analysis was performed as previously described [30].
2.8. Tubulin polymerization assay
Standard tubulin and tubulin ligand polymerization reactions were carried out essentially using
manufacturer's instructions (tubulin polymerization assay Kit, Cytoskeleton). Briefly, in dark
regime, different wells of a microtiter plate were loaded with 10 μL of buffer solution, or 10 μL of
the 100 μM stock solutions of the tested reagents (taxol, 1a, 1RR or 1RS). Then 90 μL of a cooled
(4 °C) tubulin solution, (3.3 mg/mL) buffered at pH= 6.9 (80 mM PIPES, 0.5 mM EGTA, 2.0 mM
MgCl2, 1.0 mM GTP) was added to each well. Polymerization was started warming at r.t. the plate
and followed by absorbance reading at 340 nm every 60 s for 3 h. In the light regimen, a similar
loaded microtiter plate was kept 1 min under LED irradiation at 435 nm before starting the
acquisition of the absorption data. All data were reproduced in triplicate. For each treatment, the
absorption values were graphically reported vs time (min) as the means of the measured values.
3. RESULTS
3.1. Synthesis
Mixtures 1a and 1b were obtained in four synthetic steps (Scheme 1), starting from 4-nitrophenol,
which was coupled with R or S glycidol, slightly modifying a previously reported procedure [31].
Despite literature data indicating that very low yields in coupling products were obtained from the
reaction of R or S glycidol with diverse nitro-phenols [32], we observed that the use of catalytic
amounts of KOH (in ethanol solution) favoured the formation of 2R or 2S in good yields with a high
optical purity (Figure S1).
The reduction of 2, as the R or S stereomer, with H2/Pd(C) and the subsequent conversion of the
amino- to the azonium-group gave rise to 4R or 4S, that were coupled with racemic methyl 3-
hydroxy-2-(1H-pyrrol-1-yl)propanoate 5 [26] to give the mixtures 1a and 1b, respectively.
To obtain the pure diastereomers 1RS and 1RR, compound 4R was coupled with the R or S
stereomer of 5. The best yields of the final coupling step were obtained by treating the reactions
after 1 h from the mixture gelation. The final diastereomeric mixtures 1a and 1b, as well as the
pure 1RR and 1RS, and their intermediates, were fully characterized by 1D- and 2D-NMR,
HRESIMS, and UV. Furthermore, the species were analyzed on chiral HPLC (Figure S2).
6

3.2. UV experiments reveal the reversibility of the trans-cis conversion under different cycles of
irradiation at 435 nm
UV spectra of the thermodynamically stable trans 1a and 1b were obtained in the dark, using
three different solvents, MeOH, H2O and Phoshate Buffer Solution (PBS). In all examined
conditions the UV profiles of both species were similar (Figures 2A and S4). In H2O and PBS two
major broad bands with a comparable intensity, centred at 391 and 430 nm, were observed,
whereas in MeOH the relative intensity of the same two bands was appreciably different. Under
light exposure (400 or 435 nm), metastable cis isomers were obtained. Indeed, the UV profiles of
1a and 1b strongly changed, showing a significant decreasing of the band intensities in the range
350-460 nm, the occurrence of a new band centred at about 345 nm (Figure 2B, blue line), and a
broad absorption band that extended up to 475 nm. To check the cis→trans isomerization, the UV
spectra of 1a and 1b were sequentially collected after irradiation (Figures 2C and S3-C), and the
absorbance at 390 and 345 nm was monitored over time (Figures S5-S8). In both types of
experiments, the intensity of the UV band at 390 nm was almost restored in about 40 or 5 mins in
the case of 1a, and 80 or 5 mins for 1b depending on the solvent conditions (MeOH or PBS,
respectively) [33].
In the 475-550 nm range the cis isomers of 1a and 1b showed a UV absorption slightly higher than
that of the corresponding trans (Figure 2c'). However, after irradiating the samples with green
light the cis to trans conversions of 1a and 1b occurred faster than in darkness (Figures S9-S10).
Time-dependent UV experiments were performed also on 1RR and 1RS in PBS, and the resulting
t1/2s of the metastable isomers were 5.8 and 4.2 min, respectively (Figure S11).
3.3. 1H NMR spectra of samples in darkness or after irradiation at 435 nm confirm the occurrence
of the 1a/b trans-cis conversion
To gain further insights into the photo-conversion properties of 1a, we also analyzed the progress
of the cis to trans conversion by acquiring 1H NMR spectra in darkness and at different times after
the irradiation at 435 nm. In the dark the 1H NMR spectrum of 1a (CD3OD, 4 mg/mL) showed in the
downfield region five aromatic proton resonances at 7.70, 7.28, 7.04, 6.67, 6.32 ppm, as well as a
signal at 5.62 ppm due to the proton linked to the C-α of the methyl-ester (see Supplementary
data, p. 31). After light exposure (1 min, LED 435 nm) of the sample, in addition to the proton
resonances attributed to the trans 1a, a new set of five signals appeared in the same region of the
spectrum (Figure 3). Strikingly, the intensity of the new proton signals in the spectra was inversely
1
dependent on the sample concentration. Indeed, the H NMR spectra, acquired on samples
containing over 10 mg/mL and exposed at the same LED irradiation, did not show an appreciable
1
amount of new proton signals. Over time, the intensities of the new signals decreased, and the H
NMR spectrum evolved towards that obtained for 1a kept in the dark. Therefore, the transient
proton signals can be related to the metastable cis 1a, activated by LED light at 435 nm. The same
results were obtained for 1b (see supplementary material, p.36).
3.4. Kinetic parameters of the 1a/b and 6a/b trans→cis conversions and of the corresponding
reversed isomerisations were evaluated by fast UV experiments
We also tested the occurrence of the hydrolysis of the methyl ester groups of 1a and 1b dissolved
in PBS at r.t. During the first 2 days, neither hydrolyzed nor degraded products were observed on
the HRESIMS and TLC plates. After five days from the dissolution, mass spectra corresponding to
the hydrolyzed products (6a/b, Figure 1) were recovered (Figure S12). The UV spectra obtained
after the irradiation of 6a and 6b in PBS showed only slight differences with those measured in
darkness, thus suggesting a substantial decreasing of the life time of the corresponding metastable
cis forms.
In order to quantify the half life time of cis 6a/b, we performed kinetic studies by means of fast UV
spectroscopy. Samples were made by dissolving 1a/b in different solvents: methanol, H2O and
7

PBS. 6a and 6b were produced as a result of the hydrolysis of 1a/b in water-based solutions after
five days from the dissolution. An exponential decay model was used to fit the kinetic data (Figure
4), to obtain the half-life times (t1/2) for trans 1a, 1b, 6a and 6b to convert to their cis form, and for
the reverse cis to trans reaction. The resulting t1/2s are reported in Table S1. The obtained t1/2s of
cis 1a/b in methanol are longer than those measured in PBS by a factor of 2-4. This is ascribed
mostly to the different polarity of the two solvents, the more polar one, PBS, being characterized
by a faster transition, typically associated with a lower process quantum yield [1,34]. We also
notice that the two different apparatuses used to kinetically characterize the cis→trans
conversions, i.e. the UV experiments based on a spectrophotometer and fast UV spectroscopy, are
substantially consistent as shown by the third line in Table S1, where the t1/2 measured for the
sample of 1b in methanol is the same, within the error range. Additionally, we observed that the
measured value of the cis t1/2 for the hydrolyzed products, 6a/b, is 4-7 fold shorter than that
obtained for 1a/b, as seen in the case of the PBS (Table S1, Figures S6-S8).
3.5. The treatment with 1a, 1b, 1RR or 1RS in darkness and under irradiation affects in a
different manner the viability of the HCT-116 p53-/- and A375 cells
Both 1a and 1b were tested by MTT assay for their ability to inhibit the HCT-116 p53-/- and A 375
cell growth at 1 and 10 μM, in the dark and under irradiation.
In darkness (Figure 5A and Figure S13A and E), at a concentration of 1 μM, both 1a and 1b proved
to be almost ineffective on both cell lines. The viability of the HCT-116 p53-/- cells treated with 1b
at 10 μM was greater (50% at 24 h) than that of those treated with 1a (63% at 24 h). On the
contrary, at 10 µM neither 1a nor 1b showed any antiproliferative effect on A375 (Figure S13A and
E).
Furthermore, the HCT-116 p53-/- cells were treated for 12 h with 1a or 1b undergoing irradiation
at 435 or 505 nm. We used a pulse-program 50 ms-ON/ 1.0 min-OFF by means of an Arduino
module to manage the LED plates [11]. The LED irradiation at the wavelengths used proved to be
almost ineffective on the viability of the untreated cells (Figure S14).
Pulsed LED irradiation at 435 nm on HCT-116 p53-/- caused an enhancement of the
antiproliferative efficiency of 1a and 1b at both concentrations used. Indeed, the IC50 of 1a was
12.5 μM in the dark (Figure 5A) and < 1μM under irradiation (Figures 6A-B) while the IC50 of 1b was
10.0 μM in the dark (Figure 5B) and in the range of 1-5 μM under irradiation (Figures 6A-C).
In order to further explore the effects of 1a and 1b on HCT-116 p53-/- we also used the same
pulse program with LED light at 505 nm (Figure 6). Compared to the dark regimen, the viability of
the cells irradiated at 505 nm and treated with 1a or 1b at 1 and 10 μM was only weakly affected.
In view of the photo-responsive properties of 1a, also the pure stereomers 1RR and 1RS forming
1a were investigated for their antiproliferative action on HCT-116 p53-/-, in the dark and under
irradiation at 435 nm (Figure 7), following 24 h of treatment. The comparison between the
resulting MTT data showed that the increase of the incubation time produced a partial loss of the
antiproliferative effect of 1a (46% vs 60% of viability at 1 μM and 18% vs 35% of viability at 10 μM)
(Figures 6B and 7A). Remarkably, the mixture 1a showed, under irradiation, a higher
antiproliferative effect than each single diastereomer (Figures 6B and 7C-D) (60% cell viability of
1a vs 69% for 1RR and 78% for 1RS at 1 μM and 35% cell viability of 1a vs 65% of 1RR and 75% of
1RS at 10 μM)
The A375 cell line proved to be insensitive to 1a and 1b under irradiation, (Figure S15) also when
we adopted other light regimens (besides the pulse program above mentioned we also used 50 ms
ON/1.0 s OFF; 50 ms ON /500 ms OFF; and 500 ms ON/7.5 min OFF), and prolonged the incubation
time from 12 to 24 h.
3.6. 1a and 1RS affect the dynamics of MT polymerization after UV irradiation
8

In order to evaluate the effects of 1a, 1RR and 1RS on tubulin polymerization, we measured the
increasing in light scattering at 350 nm over time, caused by the polymer formation from
unassembled tubulin solutions. Indeed, as previously reported [35], there is almost a linear
relationship between the measured turbidity generated from the tubulin solutions and the total
amount of microtubules. In this assay taxol, a well known MT stabilizing agent, was used as the
reference compound.
The results of the tubulin polymerization assay mainly indicated that at 10 μM 1a and 1RS, but not
1RR, acted as MT destabilizing agents, hindering the tubulin polymerization [36] mostly after
irradiation of the plates with LED light at 435 nm for 1 min before the data acquisition (Figures S16
and S17). Indeed, the comparison of the data on the tubulin solutions treated with 10 μM of 1a, or
1RS, acquired in darkness or under irradiation, showed an increasing in the t1/2 values of the
polymerization (Table 1) of about 30 and 32.2 mins, whereas 10 μM of 1RR did not significantly
affect the t1/2 of tubulin.
compounds 10 μM t1/2 after irradiation (min)
t1/2 in the dark (min)
buffer
taxol
1a
1RR
1RS
61.0±0.1
10.2 ±0.4
97.0±1.0
66.9±0.5
104.0±1.0
66.5±0.2
62.3±0.2
71.8±0.5
Table 1. The t1/2 of the tubulin polymerization (≈66 μM) calculated from the fitted curves in the
presence of 10 μM of 1a, 1RR or 1RS in the dark and under LED irradiation at 435 nm.
4. Discussion
In this study some chiral benzodiazo N-substituted pyrrole derivatives as potential photo-activable
MTAs were synthesized and their biophysical properties were explored by means of UV, fast UV,
NMR, MTT and tubulin polymerization assay. All the collected UV and NMR data suggested that
irradiation of the samples at 435 nm induced the conversion of the thermodynamically stable
trans 1a and 1b to the corresponding metastable cis forms, and that a cis to trans reversion
occurred in darkness. Indeed, after LED light irradiation, the UV profiles significantly changed and
then evolved over time to become those obtained in the dark. Correspondingly, the comparison
1
between H NMR acquired in the dark and after LED exposure showed that the irradiation of the
samples caused the occurrence of new and transient proton signals, attributable to the presence
of the metastable cis isomers in the solution. The kinetics of the 1a/b cis-trans conversions was
evaluated by both UV and fast UV spectroscopy. The obtained data suggested that, in methanol,
but not in PBS, the chirality of the substituent at the oxygen atom of the benzene ring significantly
affects the half lives of the metastable cis forms (Figures S5-S8 and Table S1).
The occurrence of the hydrolysis of the methyl ester group in 1a/b in PBS at pH= 7.4 was also
evaluated. The hydrolysis of 1a/b in 6a/b also caused a significant decreasing of the half lives of
the metastable cis isomers, evaluated by means of UV spectroscopy. Our data are consistent with
the literature [37-38]. In particular, it appears that the solvation of diazocompounds by polar
solvents positively affects the rate of cis/trans isomerization. Accordingly, in PBS the free carboxyl
group in 6a/b should promote the solute-solvent interactions, thus producing a decreasing in the
half-lives of cis 6a/b with respect to 1a/b.
9

In order to explore the antiproliferative activity of 1a/b and 1RR/RS on cancer cell lines, a MTT
assay on HCT 116 p53 -/- and A375 was performed, both in the dark and under LED irradiation at
435 nm. The overall results showed that only HCT 116 p53-/- was responsive to the treatments.
It is noteworthy that both CA4 and the PSTs inhibit the A375 cell proliferation [11,39]. This finding
could be ascribed to a greater structural similarity between CA4 and the previously reported PSTs
than between CA4 and the herein described molecules. Considering that:
i) CA4 potently inhibits the A375 cell growth with an IC50 in the range of nanomolar
concentrations;
ii) A375 and melanoma cells, in general, are resistant to the induction of apoptosis when exposed
to chemotherapeutic agents via the activation of different protective mechanisms [40]; and
iii) the in vitro polymerization experiments showed an impact of 1a/b on tubulin, but the effect on
the t1/2 of tubulin is moderate (Table 1)
we suggest that CA4 and 1a/b could have a different mode of action. This hypothesis could be
supported also by the results obtained by Borowiak et al. [11]. Indeed, although PST-1 shares an
almost complete structural similarity with CA4, it inhibits cancer cell growth more significantly
than in vitro tubulin polymerization.
On the HCT 116 p53-/- cell line the cis stereomers forming 1a and 1b exhibited a greater
antiproliferative effect than the corresponding thermodynamically stable trans. Furthermore, the
photo-responsive antiproliferative profile of 1a appeared more promising than that of 1b.
On irradiating the HCT 116 p53 -/- cells with green light (505 nm) an in vitro reversion of the cis 1a
toxic effect only partially occurred. The UV spectra of 1a and 1b irradiated with LED at 435 nm or
505 nm also showed that the trans to cis conversion was efficiently induced only by LED at 435 nm
1
(Figures 2, S4 and the H NMR spectra after irradiation at 505 nm in the SI), while the green light
promoted, in part, the cis to trans conversion (Figures S9 and S10). In addition, the inability of the
green light to promote this reversion has also been observed for some PSTs by Borowiak et al. [11]
and should depend on the small difference in the absorption band between the trans/cis isomers.
The time-dependent UV experiments in PBS revealed that the t1/2s of cis 1RR and cis 1RS, the pure
stereomers forming 1a, were slightly different (5.8 and 4.2 min, respectively, Figure S11) whereas
the average of the two values corresponded to that of 1a (Figures S5 and S6). On prolonging the
incubation time from 12 to 24 h a partial loss of the toxic effect of 1a on HCT-116 p53-/- was also
observed both in the dark and under irradiation at 435 nm (Figures 5A, 6 and 7). This occurrence
has previously been described and could be attributed to the reduction of the diazo-compounds
by intracellular glutathione [13].
Remarkably, under irradiation, the antiproliferative effect of 1a on HCT 116 p53-/- was greater
than that of each single diastereomer (Figure 7B-C-D). Similar phenomena have already been
observed [41,42]; nevertheless, the apparent synergic behaviour of 1RR and 1RS is peculiar, since
it was amplified upon photo-activation (Figure 7A-B).
Finally, the t1/2 values obtained from the tubulin polymerization curves showed that the ability of
1a, 1RR and 1RS to interfere with the MT dynamics was activated by LED irradiation. Strikingly, the
chirality S of the substituent on the pyrrole portion in 1RS appeared essential to inhibit the tubulin
polymerization.
Despite the fact that more in-depth studies should be performed to clarify the reasons for these in
vitro properties, the described benzodiazo N-substituted pyrrole derivatives represent leads for
the discovery of a new class of photo-activable compounds useful for the local control of cancer
cell growth. Indeed, 1a contains four functional groups, that can be, in the future, easily employed
to produce a large number of 1a analogues, properly designed to improve both the
antiproliferative activity and the glutathione resistance [1-3, 43-44].
10

Figure 1. Natural ligands of MTs and AzB analogues.
Figure 2. A. The UV spectra of 1a in MeOH (black), PBS (blue) and H2O (violet). B. The UV spectra
of 1a in MeOH before (green) and after (blue) irradiation at 435 nm. C. The time-dependent UV
spectra of 1a (2 min) after irradiation at 400 nm in MeOH. The time gap between the two
consecutive spectra was 2 mins. c'. The enlargement of the spectra was in the range 475-550 nm.
11

C
B
A
Figure 3. The 1H NMR spectra (CD3OD) of 1a (7.9-5.5 ppm), sequentially acquired after 3 mins (A),
10 mins (B), and 25 mins (C) from the irradiation at 435 nm. The transient resonances indicated by
asterisks were produced by the protons of the metastable cis 1a.
12

Figure 4. An example of the 404-nm probe transmitted intensity changes: passing through 1b in
methanol solution as a function of time; (a) the trans to cis conversion [when the laser is switched
on and the sample is being irradiated with 10 mW, 435 nm laser beam], and (b) the cis to trans
conversion [in the dark].
13

120
100
80
60
40
20
0
A
12h
24h
NT
1µM
5µM
10µM
15µM
20µM
30µM
50µM
120
100
80
60
40
20
0
B
12h
24h
NT
1µM
5µM
10µM
15µM
20µM
30µM
50µM
Figure 5. Dose-response experiments on HCT-116 p53-/- treated in darkness with 1a (A) and 1b (B)
at different incubation times (12 and 24 h). The data are presented as a percentage (mean ± SEM)
of the viability of the untreated control cells (NT). The calculated IC50s at 12 h are 12.5 and 10 μM,
for 1a and 1b, respectively.
.
Figure 6. The HCT-116 p53-/- cell viability in the MTT assay conducted in different conditions. (A)
Cells treated for 12 h with 1a or 1b, in the dark. The results are presented as a percentage (mean ±
SEM) of the untreated control cells. (B and C) Cells treated with 1a or 1b and irradiated for 12 h
with LED light at 505 or 435 nm (12 h, 50 ms-ON/ 1.0 min-OFF). The results are presented as a
percentage (mean ± SEM) of the control cells treated in the dark with 1a or 1b.
14

(A)
No Treated
1μM
120
100
80
60
40
20
0
10μM
1a
1RR
1RS
(B)
(C)
(D)
No Led
435nm
No Led
435nm
No Led
435nm
120
120
100
80
60
40
20
0
120
100
80
60
40
20
0
100
80
60
40
20
0
1µM
10µM
1µM
10µM
1µM
10µM
1a
1RR
1RS
Figure 7. The HCT-116 p53-/- cell viability in the MTT assay conducted in different conditions. (A)
Cells treated for 24 h with 1a, 1RR or 1RS in the dark. The results are presented as a percentage
(mean ± SEM) of the untreated control cells. (B, C and D) Cells treated with 1a, 1RR and 1RS
respectively and irradiated for 24 h with LED at 435 nm (12 h, 50 ms-ON/ 1.0 min-OFF). The results
are presented as a percentage (mean ± SEM) of the control cells treated in the dark.
15

NO2
OH
NO2
NH2
N2 Cl
a
b
c
R
R'
R
R
O
R'
R'
O
OH
O
OH
OH
2R R= H, R'=OH
2S R= OH, R'=H
3R R= H, R'=OH
3S R= OH, R'=H
4R R= H, R'=OH
4S R= OH, R'=H
H3CO
HO
H3CO
HO
O
H3CO
O
HO
O
N
d
N
OH
N
N
4R or 4S
+
OH
N
O
N
O
N
or
1a
1b
(rac)
5
HO
HO
H3CO
HO
H3CO
HO
O
O
H3CO
HO
O
N
d
N
OH
N
OH
N
N
or
+
4R
O
N
O
N
HO
HO
5 (R or S)
1RS
1RR
Scheme 1. Preparation of compounds 1a, 1b, 1RS, and 1RR. Reagents and conditions: a) catalytic
KOH, dry toluene, 30 mins under reflux, then addition of R or S glycidol at r.t., and heating at 90 °C
for 24h; b) Pd/C, H2 (P= 400 psi) in EtOH, 2 h, r.t. c) fluoroboric acid (water solution, 48%), EtOH,-
15 °C, then isoamyl nitrite, r.t., 12 h; d) acetic acid, sodium acetate 1 h, r.t.
16

Conflicts of interest: There are no conflicts to declare.
Acknowledgements
This research was funded by Regione Campania under POR Campania FESR2007-2013 −O.O. 2.1
(FarmaBioNet). The authors are grateful to "Istituto Tecnico Industriale Statale Francesco Giordani
of Caserta" (I.T.I.S. Giordani) for their technical assistance on the LED System construction.
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