Assessing the Dual Activity of a Chalcone–Phthalocyanine Conjugate: Design, Synthesis, and Antivascular and Photodynamic Properties

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Article

Assessing the dual activity of a chalcone-phthalocyanine conjugate:design, synthesis, antivascular and photodynamic propertiesSinem Tuncel, Aurélien Trivella, Devrim Atilla, Khalil Bennis, Huguette Savoie, Florian

Albrieux, Laetitia Delort, Hermine Billard, Virginie Dubois, Vefa Ahsen, FLORENCECALDEFIE-CHEZET, Claire Richard, Ross W. Boyle, Sylvie Ducki, and Fabienne Dumoulin

Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp400207v • Publication Date (Web): 12 Aug 2013

Downloaded from http://pubs.acs.org on August 30, 2013

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FOR TABLE OF CONTENTS USE ONLY

Assessing the dual activity of a chalcone-phthalocyanine

conjugate: design, synthesis, antivascular and photodynamic

properties

Sinem Tuncel, Aurélien Trivella, Devrim Atilla, Khalil Bennis, Huguette Savoie, Florian

Albrieux, Laetitia Delort, Hermine Billard, Virginie Dubois, Vefa Ahsen, Florence Caldefie-

Chézet, Claire Richard, Ross W. Boyle, Sylvie Ducki, Fabienne Dumoulin

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Molecular Pharmaceutics

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Assessing the dual activity of a chalcone-phthalocyanine

conjugate: design, synthesis, antivascular and photodynamic

properties

Sinem Tuncel1, Aurélien Trivella

2,4, Devrim Atilla

1, Khalil Bennis

3,4, Huguette Savoie

5,

Florian Albrieux6, Laetitia Delort

7,8, Hermine Billard

7,8, Virginie Dubois

7,8, Vefa Ahsen

1,

Florence Caldefie-Chézet7,8

, Claire Richard2,4

, Ross W. Boyle5, Sylvie Ducki

3,4*, Fabienne

Dumoulin1*

1 Department of Chemistry, Gebze Institute of Technology, P.O. Box 141, 41400 Gebze

Kocaeli, Turkey.

2 Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand,

Equipe Photochimie, BP 10448, F-63000 CLERMONT-FERRAND France

3 Clermont Université, ENSCCF, Institut de Chimie de Clermont-Ferrand, Equipe CESMA,

BP 10448, F-63000 CLERMONT-FERRAND France

4 CNRS, UMR 6296, ICCF, F-63171 AUBIERE France

5 Department of Chemistry, University of Hull, Kingston-upon-Hull, East Yorkshire, HU6

7RX, UK

6 Centre Commun de Spectrométrie de Masse UMR 5246, CNRS-Université Claude Bernard

Lyon 1, Université de Lyon, Bâtiment Curien, 43, bd du 11 Novembre, 69622 Villeurbanne

Cedex, France

7 Clermont Université, Université d'Auvergne, ECREIN-UNH, BP 10448, F-63000

CLERMONT-FERRAND France

8 INRA, UMR 1019, UNH, F-63009 Clermont-Ferrand France

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* Corresponding authors

Department of Chemistry, Gebze Institute of Technology, P.O. Box 141, 41400 Gebze

Kocaeli, Turkey. E-mail: [email protected]; Fax: +90 262 605 31 01; Tel: +90 262 605

31 06

Clermont Université, ENSCCF, Institut de Chimie de Clermont-Ferrand, BP 10187, F-63174

AUBIERE. E-mail: [email protected]; Fax: +33 4 73 40 70 08; Tel: +33 4 73 40 71 32

---------------------------------------------------------------------------------------------------------------

Abstract

Photodynamic therapy (PDT) and vascular-disrupting agents (VDA) each have their

advantages in the treatment of solid tumors, but also present drawbacks. In PDT, hypoxia at

the centre of the tumor limits convertion of molecular oxygen into singlet oxygen, while

VDAs are deficient at affecting the rim of the tumor. A phthalocyanine-chalcone conjugate

combining the VDA properties of chalcones with the PDT properties of phthalocyanines was

designed to address these deficiencies. Its vascular targeting, photophysical, photochemical,

photodynamic activities are reported herein.

Keywords

Phthalocyanine; chalcone; photodynamic therapy; antivascular; dual effect.

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Introduction

Photodynamic therapy (PDT) relies on the conversion of molecular oxygen into singlet

oxygen upon irradiation of a photosensitizer with light of a suitable wavelength. The

subsequent oxidative cellular damage leads to the destruction of the irradiated tissues/cells.

PDT is used to treat a wide range of medical conditions, including bacterial infections, acne,

and malignant cancers. In cancer treatment, PDT is often used as an alternative treatment to

surgery, chemotherapy or radiotherapy. Recently, PDT has been used for vascular targeting,

with the development of VEGFR-targeting photosensitizers1 or photosensitizing

nanoparticles2 targeting selectively the tumour neovasculature through the enhanced

permeation and retention (EPR) effect.3 This subfield of PDT is known as anti-vascular PDT.

Vascular disrupting agents (VDAs), such as chalcones,4 are also able to selectively destroy

tumor vasculature. They differ from anti-angiogenesis agents as they destroy, within minutes

to hours, newly formed blood vessels, rather than simply inhibiting growth of new vessels.

VDAs effectively starve the tumor to death, by depriving it of vital nutrients and oxygen,

leading to tumor necrosis. A majority of cancers are solid tumours that require neovasculature

to survive. VDAs are therefore emerging as a promising counterpart to conventional cytotoxic

chemotherapy.

Photodynamic therapy (PDT) and vascular-disrupting agents (VDA) each have their

advantages in the treatment of solid tumors, but also present drawbacks. In PDT, the light

penetrates and activates the photosensitizer mainly at the rim of the tumor, and hypoxia at the

center of the tumor limits conversion of molecular oxygen into singlet oxygen. On the other

hand, the presence of a viable rim of tumour cells at the periphery after VDA treatment, as

shown in pre-clinical studies5, reflects the inability of VDA to target the rim of the tumour,

The viable cells rapidly proliferate and recover their blood supply within 24 hours. It is hence

necessary to associate the VDAs with other therapies in order to counteract this deficiency.

Current approaches are therefore to combine VDAs with radiation therapy, anti-angiogenic

agents or conventional cytotoxic agents.6

In the present work, we covalently bound chalcones possessing VDA properties to

phthalocyanines possessing PDT properties to give a phthalocyanine-chalcone conjugate 1

(Figure 1). The conjugate was designed to overcome the poor hydrophilicity of the

tetrachalcone-phthalocyanine conjugate 2, recently reported7 (Figure 1), since it was too

hydrophobic to allow further biological investigations. In order to fully assess the relevance of

the conjugate 1, its vascular targeting, photophysical, photochemical, photodynamic activities

are reported.

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1

PEG-type spacerfor hydrophilicity

Non-peripheral substitutionAbsorption at 700 nm

O

O

N

N

N

N

N

N

N

N

Zn

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

OMe

OMe

OMe

O

MeO

HNC

O

Methylated triethyleneglycol moieties for

increased water-solubility

Cleavablebond

O

O

O

O

O

O

O

O O

O

O

O

O

O

O

O

O

O

O

N

N

N

N

N

N

N

N

Zn

OMe

OMeMeO

O

MeO

NHC

O

MeO

MeO OMe

O

OMe

HNC

O

MeO

MeO

MeO

O

OMe

HN

C

O

2

O

OMe

OMe

OMe

O

MeO

HNC

O

Figure 1. Structures of phthalocyanine-chalcone conjugates 1 and 2

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Results and discussion

Molecular design

The low hydrophilicity of conjugate 2 (Figure 1) prompted us to design the more

biocompatible phthalocyanine-chalcone mono-conjugate 1 (Figure 1) with the so-called AB3

type of phthalocyanine substitution pattern. Most of the structural features of the tetra-

conjugate 2 were retained to allow close comparison of results: thus both exhibit non-

peripheral substitution leading to bathochromic shifting of the maximum absorption into the

near infrared, and the chalcone grafted on to the phthalocyanine via a tetraethyleneglycol

spacer. Terminally methylated triethyleneglycol chains replace the chalcone substituents on

three isoindole subunits of the phthalocyanine to increase hydrophilicity. The water-

solubilizing effect of such polyethylene glycol moieties is currently often used to obtain water

soluble phthalocyanines for photodynamic therapy.8 As in conjugate 2, the chalcone moiety is

grafted at the extremity of a tetraethylene glycol chain through a carbamate link.

Synthesis and characterization

To prepare the monoconjugate 1, the monohydroxylated phthalocyanine 6 was prepared by a

mixed condensation of phthalonitriles 3 and 4. A ten-fold excess of 3 ensured the formation of

mainly two phthalocyanines: the symmetrically substituted 5 and the desired

monohydroxylated 6. Traces of A2B2 derivatives were nevertheless detected. The two

phthalocyanines, displaying different polarities, were readily separated on silica-gel column

chromatography, with the elution of the symmetric derivative 5 (eluent: dichloromethane-

ethanol 10/1) followed by the desired AB3 phthalocyanine 6 (eluent: dichloromethane-ethanol

4/1), obtained in a satisfactory yield of 24%. Next the aminochalcone 7, obtained by

condensation of the corresponding acetophenone and benzaldehyde, was converted into the

corresponding isocyanate in situ, and reacted with the monohydroxylated phthalocyanine 6 to

produce the carbamate 1 in 78% yield. Conjugate 1 was fully characterized by MALDI, ESI-

HRMS (Figure 2), NMR, IR and its HPLC profile confirmed a purity > 95%.

In vivo, the carbamate bond of conjugate 1 may be cleaved on the nitrogen side to give

aminochalcone 7 or on the oxygen side to yield chalcone 8. Hence as a reference, methyl

carbamate chalcone 8 was prepared, by reacting the isocyanate chalcone with methanol

(Scheme 2).

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3

6

O

O

N

N

N

N

N

N

N

N

Zn

O

O

O

O

O

O

O

O

O

O

O

O

O

O

OH

OOO

O

CN

CN

OOO

O

CN

CN

HO

+

4

Zn(OAc)2DMAE

1. Trisphosgene / Et3N2. 6, toluene /dichloromethane 48 h

Conjugate 1

5

O

O

N

N

N

N

N

N

N

N

Zn

O

O

O

O

O

O

O

O

O

O

O

O

O

O

+

O

O

O

O

7

NH2

O

Scheme 1. Preparation of conjugate 1 (only one of the possible regioisomers is shown)

Figure 2. ESI-HRMS isotopic pattern of conjugate 2: experimental (a) and theoretical (b)

peaks

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Scheme 2. Preparation of chalcone 8.

Vascular targeting properties

The ability of the phthalocyanine-chalcone conjugate 1 to target tumor vasculature was

investigated by assessing its effect on the motility of endothelial cells. Since this conjugate is

susceptible to metabolism in vivo, we also evaluated the properties of phthalocyanine 6,

chalcones 7 and 8, as potential metabolites. The resazurin viability assay was carried out to

determine the minimal concentrations required to inhibit cell migration for each compound.

Resazurin viability assay

As endothelial cells recruited for tumor angiogenesis undergo rapid cell proliferation, we first

evaluated the viability of Human Umbilical Vein Endothelial Cells (HUVEC) exposed to

compounds 1, 6-8 (25 nM - 50 µM range) after 24, 48 and 72 hours of treatment (Table 1,

Figure S1). The inhibitory effect of chalcones 7 and 8 on HUVEC was observed after 24

hours of treatment and increased until 72 hours. While chalcones 7 and 8 were able to

strongly inhibit the proliferation of HUVEC in a dose-dependent manner (IC50 = 25 and 156

nM respectively at 72 hours), phthalocyanine 6 displayed weak cytotoxic activity against

HUVEC (40% inhibition observed at 50 µM). A moderate inhibitory effect was observed for

the conjugate 1, demonstrating that this association conserved some cytotoxic activity (IC50 =

10.8 µM).

Table 1. Inhibitory concentrations IC10, IC25 and IC50 (determined by resazurin viability

assay after 24 h of treatment (n=3)) for compounds 1, 6-8 (from Figure S1).

Conjugate 1 Phthalocyanine 6 Chalcone 7 Chalcone 8

IC10 (µM) 2.5 5.0 0.012 0.176

IC25 (µM) 5.3 23.5 0.025 0.301

IC50 (µM) 16.8 >50 0.047 0.595

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Migration assay

Endothelial cell motility plays an essential role in supporting the survival and angiogenesis of

endothelial cells. Chalcones are vascular disrupting agents (VDAs) that also impair

endothelial cell motility, leading to diminished cell migration. We next challenged the

capacity of HUVEC to migrate into a denuded area to evaluate the vascular targeting

properties of compounds 1, 6-8. A "wound" was scraped across a confluent monolayer culture

of HUVEC and cell migration to “heal the wound” was measured in the presence of various

concentrations of compounds. We selected minimal inhibitory concentrations (IC10, IC25 and

IC50), (Table 1), as determined by the resazurin viability assay.

Twenty two hours after wounding, untreated HUVEC could be seen to have migrated into the

denuded area (Figure S2). As expected, chalcones 7 and 8, were able to inhibit the basal

migration of HUVEC in a dose-dependent manner after 22 hours of treatment (Figures 3B-D),

confirming that chalcones, at non-toxic doses, block endothelial cell migration. Although, no

significant inhibition of HUVEC migration could be observed for phthalocyanine 6, a slight

inhibition was observed when HUVEC were treated with the conjugate 1.

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1,1 1,1

9,8

44,3

0

10

20

30

40

50

% o

f m

igra

tio

n i

nh

ibit

ion

A a, b, c

a, b

Control 12 nM 25 nM 47 nM

3,95,9

13,1

41,2

0

10

20

30

40

50

% o

f m

igra

tio

n i

nh

ibit

ion

Ba, b, c

Control 176 nM 301 nM 595 nM

1,6

11,8 11,6

8,9

0

10

20

30

40

50

% o

f m

igra

tio

n i

nh

ibit

ion

C

a a

Control 5 µµµµM 23.5 µµµµM 50 µµµµM

0,0

11,1

15,4 15,9

0

10

20

30

40

50

% o

f m

igra

tio

n i

nh

ibit

ion

D

a a

Control 2.5 µµµµM 5.3 µµµµM 16.8 µµµµM

a

Figure 3. Migration assay for HUVEC treated with (A) chalcone 7, (B) chalcone 8, (C)

phthalocyanine 6 and (D) conjugate 1 after 22 hours of treatment at IC10, IC25 and IC50.

Results are expressed as the percentage of migration inhibition corresponding to the measure

of the scraped area recolonized by treated HUVEC compared to control (± SEM) (a : p<0.05

IC10 or IC25 or IC50 vs control; b: p<0.05 IC25 or IC50 vs IC10; c: p<0.05 IC50 vs IC25).

Photodynamic potential

Photophysics and photochemistry.

The effect of the presence of one or several chalcone units on the photophysical and

photochemical properties of phthalocyanines was investigated next. Properties of

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monoconjugate 1, tetraconjugate 2, phthalocyanine 5 and of chalcone 8 (used as the reference

chalcone as it bears a methyl carbamate in place of the phthalocyanine) were investigated, to

ascertain the importance of the covalent linkage, as well as the effect of chalcone, on singlet

oxygen generation.

Electronic absorption spectroscopy. The two phthalocyanine-chalcone conjugates 1 and 2,

and the reference phthalocyanine 5 are monomeric in acetonitrile (Figure 4). No aggregation

is observed in the concentration range (10-5

-10-6

M) in acetonitrile, as demonstrated by the

sharpness of the Q band.

250 300 350 400 450 500 550 600 650 700 750 8000,00

0,05

0,10

0,15

0,20

Absorbance

Wavelength (nm)

Figure 4. UV-Visible absorption spectra of conjugate 1 (dashed line), conjugate 2 (solid line),

reference phthalocyanine 5 (dotted line), and of carbamate chalcone 8 (dashed-dotted line) at

1 µM in acetonitrile.

A weak red-shift and an increase in the molar absorption coefficient of the Q bands are

observed when the number of chalcone per conjugate increases. The comparison in the UV

range of 8 and 1 spectra shows that the absorptions are only weakly modified when chalcones

are linked (Figure 4), thus suggesting that there is no significant electronic interaction

between the singlet oxygen production center and the chalcone.

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Singlet oxygen generation. Singlet oxygen quantum yields (Φ∆) were measured using 1,3-

diphenylisobenzofuran (DPBF) as a probe molecule. Values are reported in Table 2.

Phthalocyanine 5 shows a high Φ∆ value of 0.83±0.08; but when chalcone units are linked to

the phthalocyanine, Φ∆ is lower: 0.55±0.05 for monoconjugate 1 and 0.51±0.05 for

tetraconjugate 2. The presence of chalcone therefore reduces Φ∆ by approximately one third,

but the number of grafted chalcones units (one or four) seems to have little effect as no

significant difference was found between them. This led us to consider the importance of the

distance between the chalcone and the photosensitizing core, rather than the number of

chalcone units. To obtain a better insight into the effect of the chalcone (molecular ratio and

proximity to the phthalocyanine ring), its ability to trap singlet oxygen was measured.

Consequently, we investigated the effect of chalcone 8 on the DPBF phototransformation, by

assessing its capacity to quench singlet oxygen in the presence of two different molecular

photosensitizers, methylene blue (MB) or phthalocyanine 5. As shown by the Stern-Volmer

plot in Figure 5, the rate of DPBF consumption is higher in the absence than in the presence

of chalcone for both photosensitisers, providing experimental evidence of the quenching of

singlet oxygen by chalcone 8.

The bimolecular rate constant is estimated from the slope to be (1.9±0.5) ×108 M

-1s

-1. It can

be also seen that the scavenging effect of chalcone is low and hardly measurable for the

concentrations of 3.8 and 14.5 µM which correspond to the ratios 1:1 and 1:4 of

phthalocyanine:chalcone. This suggests that the scavenging effect of the chalcone when it is

linked to the phthalocyanines is related to its proximity. When chalcone and phthalocyanine

are covalently linked, the chalcone is maintained near the singlet oxygen generation centre

(the phthalocyanine core) allowing a more efficient scavenging than free chalcone units at

similar concentration, but randomly distributed in the medium. Consequently, the length of

the spacer between the phthalocyanine and the chalcone moieties may have a direct effect on

Φ∆.

Table 2. Singlet oxygen quantum yields (Φ∆) and photodegradation quantum yields (Φdeg)

determined in acetonitrile for conjugates 1 and 2, and reference phthalocyanine 5.

Compound Φ∆ (±0.05) Φdeg/10-5

1 0.55±0.05 8.7±0.09

2 0.51±0.05 2.4±0.05

5 0.83±0.08 0.62±0.10

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0 5 10 15 20 25 30 350,5

1,0

1,5

2,0

Ratio of DPBF consumption rate

[Chalcone] in µM

Figure 5. Ratio of DPBF consumption at various concentrations of chalcone 8, for MB

photosensitizer (black circles) and for photosensitizer 5 (white circles).

Phthalocyanine and conjugates photodegradation.

Prolonged exposures of phthalocyanines resulted in a decrease in Q-band intensity. The

photodegradation quantum yields (Φdeg) were estimated from these absorbance values. Table 2

displays the Φdeg values which are of (0.62±0.10)×10-5

, (2.4±0.5)×10-5

and (8.7±0.9)×10-5

for

phthalocyanines 5, 1, and 2, respectively. These values indicate good phthalocyanine

photostabilities.9

In vitro photodynamic activity

The in vitro photodynamic activity of the conjugate 1 was then investigated. The

phototoxicity was assessed in vitro using human colon adenocarcinoma (HT-29) cells, which

are routinely used to assess photodynamic toxicity of new photosensitizers.10

In order to

determine a possible influence of the chalcone unit on the phototoxicity of the conjugate 1,

phthalocyanine 6 was also examined.

Figure 6 shows that both the phthalocyanine 6 and conjugate 1 displayed no dark cytotoxicity

at concentrations < 10 µM. However, upon irradiation with red light (> 600 nm, at a dose of

3.6 J/cm2), the conjugate 1 displays significant cytotoxicity (LD50 = 0.51 µM, LD90 = 0.86

µM), and is 6-8 fold more phototoxic than the original photosensitizer 6 (LD50 = 3.32 µM,

LD90 = 6.90 µM) (Figure 6 and Table 3). The greater phototoxicity observed for the conjugate

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1, compared to the phthalocyanine alone 6, is statistically significant. The generation of

singlet oxygen in homogeneous solution does not, in this instance, correlate with

photodynamic activity in vitro, since previous experiments demonstrated lower singlet oxygen

generation in the presence of chalcone due to its scavenging effect.

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10

Concentration (µM)

% cell survival

Figure 6. Phototoxicity of conjugate 1 (circles) and reference phthalocyanine 6 (squares)

against HT-29 cells, after 1 hour incubation, in the dark (closed symbols) and after irradiation

(3.6 J/cm2) (open symbols).

Table 3. LD50 and LD90 phototoxocity values for conjugate 1 and reference phthalocyanine 6

Compound LD50 (µM) LD90 (µM)

1 0.51 0.86

6 3.32 6.90

Both Pcs are clearly internalized by HT-29 cells, but no well-defined differences in

intracellular distribution could be observed (Figure 7), suggesting that the modest increase in

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activity could be caused by a general increase in cellular uptake, rather than intracellular

distribution to sites more susceptible to oxidative damage. Since the differences in bulk

photosensitiser uptake are not likely to be large, based on the modest differences in LD90s,

these would not be expected to be detectable by qualitative fluorescence microscopy.

Figure 7. Fluorence microscopy subcellular images of conjugate 1 (a) and of reference

phthalocyanine 6 (b). Magnification 40x.

Since the increased phototoxicity is not related to an increased singlet oxygen generation,

several hypotheses can be formulated. The first being that the increased amphiphilic character

of the conjugate 1, compared to 6, contributes to enhanced photodynamic activity, an effect

already reported.11

The presence of the relatively hydrophobic chalcone unit in conjugate 1

increases its amphiphilicity (clog P = 0.29), compared to 6 (clogP = -3.14) or 5 (clogP = -

2.56), and may contribute to an improved cellular uptake of the conjugate and account for its

increased photocytotoxicity. Quantitative determination of cellular uptake is currently being

conducted to confirm or invalidate this hypothesis. Another hypothesis could be that the

chalcone is released upon irradiation of the conjugate 1 and contributes to cytotoxicity itself,

as demonstrated earlier in HUVEC cells.

Conclusion

In summary, we have designed, synthesised and characterized the phthalocyanine-chalcone

conjugate 1. The conjugate contains a photosensitizing phthalocyanine unit, which is

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covalently bound to an antivascular agent, the chalcone. As demonstrated by HUVEC

migration assay, a residual antivascular effect is conserved by the phthalocyanine-chalcone

conjugate 1 at minimal inhibitory concentrations. These results suggest that the vascular

targeting activity of 1 could be improved by designing a cleavable conjugate, which would

release the chalcone selectively at the tumor site. Photophysical and photochemical

measurements on the conjugate 1, compared with a reference phthalocyanine, confirmed that

the singlet oxygen generation capability of the conjugate was retained with limited adverse

effect of the chalcone, despite its singlet oxygen scavenging capacity. The presence of the

chalcone unit on the phthalocyanine resulted in a highly enhanced phototoxicity, presumably

due to the increased amphiphilic character of the photosensitizer favouring cellular uptake.

Alternatively, the chalcone may be released upon irradiation of the conjugate 1, contributing

to the cytotoxicity. Overall, we conclude that combining a photosensitizer and a VDA into

one molecule, represents a highly promising strategy for the treatment of solid tumors.

Acknowledgements

The Scientific and Technological Research Council of Turkey (TUBITAK) and the French

Embassy in Turkey are gratefully acknowledged for the bilateral Parteneriat Hubert Curien

between Turkey and France (project Bosphorus 109M356 / 26268ZM). The authors thank the

Regional Council of Auvergne (Conseil Régional d’Auvergne) and the European Fund for

Regional Economic Development (FEDER) for supporting the CA3D project.

Supporting information

Inhibition of proliferation of HUVEC (Figure S1) and representative migration pattern of

HUVEC (Figure S2) are available free of charge via the Internet at http://pubs.acs.org/.

Experimental section

Syntheses

Materials and methods.

Solvents and chemicals were purchased from Aldrich or Alfa Aesar and used as received. 3-

(4,7,10-trioxaundecane-1-oxanonyl)phthalonitrile (3)12

, 3-(2-{2-[2-(2 hydroxyethoxy) ethoxy]

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ethoxy}ethoxy)phthalonitrile (4)9 and chalcone 7

7 were prepared following described

procedures. All reaction solvents were dried and purified as described by Perin and

Armarego.13

Mass spectra were recorded on a MALDI (matrix assisted laser desorption

ionization) BRUKER Microflex LT using 2,5-dihydroxybenzoic acid as the matrix and on a

BRUKER MicroTOFQ-II with an ESI (electrospray ionization) ion source in positive mode.

In this later case, the sample was infused at 150 µL/h in 50:50 water and acetonitrile with

0.1% of formic acid. The gas flow of the sprayer is 0.6 bar and the spray voltage is 3.5 kV.

The capillary temperature is 200°C. The ions are transferred to the TOF by using mild

conditions on ion optics (the two ion funnels, the hexapole, the quadrupole and the collision

cell) to preserve the complex. The mass range of the TOF is 50-5000 m/z.

IR spectrum was recorded between 4000 and 650 cm-1

using a Perkin Elmer Spectrum 100

FT-IR spectrometer with an attenuated total reflection (ATR) accessory featuring a zinc

selenide (ZnSe) crystal. Electronic absorption spectra in the UV-visible region were recorded

with a Shimadzu 2001 UV spectrophotometer using a 1 cm path length cuvette at room

temperature. 1H and

13C NMR spectra were recorded in deuterated solvent solutions on a

Bruker 400 MHz or on a Varian 500 MHz spectrometer. The HPLC system is an Agilent 1100

series HPLC system (ChemStation software) equipped with a G1311A pump and G1315B

diode array detector monitoring the range 254–900 nm. A normal phase column Lichrosorb-

SI-60 (250 × 4.6 mm) from Alltech. Associates, Inc. was used. The mobile phase was

tetrahydrofuran. The flow-rate was set at 1 mL.min-1

. The column temperature was

maintained at 28 oC.

Preparation of phthalocyanine 6. Phthalonitrile 3 (725 mg, 2.50 mmol), phthalonitrile 4 (100

mg, 0.31 mmol) and anhydrous Zn(OAc)2 (257 mg, 1.40 mmol) were refluxed in dry N,N-

dimethylaminoethanol (8 mL) for 24 h under argon atmosphere. The crude product was

precipitated and N,N-dimethylaminoethanol was removed by adding hexane to the reaction

mixture. The solid was dissolved in DCM and filtered in order to remove inorganic impurities

then concentrated. Phthalocyanine 5 was obtained over a silica gel column firstly using 10:1

dichloromethane:ethanol mixture, then phthalocyanine 6 was obtained using 4:1

dichloromethane:ethanol mixture as eluent (101 mg, 26%). C61H74N8O17Zn, MW 1256.69. IR

(ATR, ν/cm-1

): 3426.57 (OH), 3066.43 (C-Har), 2871.70 (C-Hal), 1587.06 (C=Car), 1488.40,

1449.87, 1333.56, 1231.23, 1067.39, 884.72, 801.58, 744.01. 1H NMR (500 MHz, DMSO-

d6): δ 9.00-7.36 (bd; 12H; aromatics), 5.21-3.35 (m, 52H, CH2), 3.18-3.15 (t, 9H, CH3). 13

C

NMR (125 MHz, DMSO-d6): δ 155.89, 152.58, 141.54, 136.97, 132.18, 131.81, 131.20,

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130.75, 120.05, 117.33, 116.34, 115.39, 113.74, 72.80, 72.74, 71.74, 71.68, 70.96, 70.67,

70.53, 70.36, 70.16, 70.04, 69.11, 60.62, 58.48, 58.41. MS (MALDI-TOF): an isotopic pattern

peaking at m/z 1257.34 [100%, (M)+]. ESI-HRMS obs 1255.4491, calcd for C61H74N8O17Zn:

1255.4536. HPLC Rt : 31.36 min. UV-vis (DMSO) λmax (log ε): 704 (5.40), 376 (4.65), 320

(4.72).

Preparation of conjugate 1. Chalcone 7 (126 mg, 0.37 mmol) is stirred in toluene, triphosgene

(50 mg, 0.17 mmol) is added then triethylamine (100 µL). Upon the addition of triethylamine,

a white suspension appears in the yellow colored reaction mixture, which is heated to 65°C

overnight and has a brown color the next day. After cooling down, phthalocyanine 6 (46 mg,

0.036 mmol) dissolved in dichloromethane (5 mL) is added and the reaction mixture is heated

to 50°C during two days. After cooling, dilution with dichloromethane (100 mL), washing by

water 83 x 100 mL) and drying on magnesium sulfate, the desired conjugate is purified on a

silica gel column chromatography, using dichloromethane/ ethanol 100/1 as the eluent (45

mg, 78%). C81H93N9O23Zn, MW 1626.06. IR (ATR, ν/cm-1

): 3426.57 (NH), 3272.72, 3073.42

(C-Har), 2919.94-2870.62 (C-Hal), 1727.23 (C=O), 1657.69 (C=C), 1586.06, (C=Car),

1534.69, 1487.66, 1449.87, 1413.19, 1333.90, 1123.28, 1066.03, 932.92, 884.62, 847.34,

802.29, 744.53. 1H NMR (500 MHz, DMSO-d6): δ 9.10-6.94 (m; 20H; aromatics, CH=CH,

NH), 5.19-3.05 (m, 73H, CH2, CH2). 13

C NMR (125 MHz, DMSO-d6): δ 156.09, 152.87,

142.84, 141.50, 136.85, 135.50, 132.27, 131.84, 131.20, 130.75, 130.67, 126.65, 120.01,

117.34, 116.34, 115.39, 113.65, 72.80, 72.74, 71.74, 71.68, 70.96, 70.67, 70.53, 70.36, 70.16,

70.04, 69.11, 60.62, 58.48, 58.41. MS (MALDI-TOF): an isotopic pattern peaking at m/z

1627.14 [100%, (M+1)+]. ESI-HRMS obs 1626.5642, calcd for C81H93N9O23Zn: 1626.5725.

HPLC Rt : 7.95 min. UV-vis (DMSO) λmax (log ε): 704 (5.32), 362 (4.77), 320 (4.77).

Preparation of methyl N-{2-methoxy-5-[(1E)-3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-

yl]phenyl}carbamate 8. To a solution of the aminochalcone 7 (1 eq, 173 mg, 0.5 mmol) in dry

toluene (10 ml) in a previously dried flask, triphosgene (0.3 eq, 50 mg, 0.17 mmol) and

triethylamine (2 eq, 150 µL) was added. The solution was left to stir for 3 hours after which

methanol (1 eq, 119 mg) was added and continued to reflux over the night. The progress of

the reaction was monitored with TLC using (cyclohexane/ethylacetate 1/1) until no evolution

of the reaction. The mixture was extracted using dichloromethane, and the organic phase was

washed with saturated sodium bicarbonate solution and water, dried on magnesium sulfate

and concentrated. The resulting solid was purified using flash chromatography using

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cyclohexane/ethylacetate 1/1 as the eluent, to yield a pale yellow amorphous solid (128 mg,

64%). C21H23NO7, MW 401.41. 1H NMR (CDCl3, 400 MHz): δ 3.81 (s, 3 H), 3.93 (m, 12 H),

6.89 (d, 1 H, J 8.4 Hz), 7.27 (s, 2 H), 7.28 (d, 1 H,), 7.29 (d, 1 H, J 2 Hz), 7.39 (d, 1 H, J

15.5Hz), 7.76 (d, 1 H, J 15.5 Hz), 8.5 (br s, 1 H). 13

C NMR (CDCl3, 100 MHz): δ 52.4, 55.9,

56.4 (2xC), 61.0, 106.1(2xC), 110.0, 117.0, 120.2, 124.8, 128.2, 133.8, 142.3, 145.0, 149.5,

153.1(2xC), 153.8, 189.5. m.p. 79-81 °C. ESI-HRMS obs 402.1547, calcd 402.1553 [M+H]+.

Vascular targeting properties

Cell culture. Human Umbilical primary Vein Endothelial Cells (HUVEC) (pool of 3 donors)

from Millipore (Molsheim, France) were cultured in EndoGRO-VEGF complete media

containing 2% fetal calf serum (FCS), rh-VEGF (5 ng/ml), rh-EGF (5 ng/ml), rh-bFGF

(5 ng/ml), rh-IGF1 (15 ng/ml), ascorbic acid (50 µg/ml), hydrocortisone (1 µg/ml), heparin

sulfate (0.75 U/ml) and L-glutamine (10 mM). HUVEC subcultures were used from passages

2 to 8.

Resazurin viability assay.14,15,16

. Resazurin detects cell viability by converting from a

nonfluorescent dye to the highly red fluorescent dye (resorufin) in response to chemical

reduction of growth medium resulting from cell proliferation. Continued cell proliferation

maintains a reducing environment while inhibition of proliferation (or cytotoxicity) maintains

an oxidizing environment. HUVEC were plated (3 x 103 cells) in 96-well plates in complete

medium. After 24 h, cells were exposed to complete fresh medium, containing different

concentrations of compounds to test. After 24h, 48h and 72h, the medium was removed and

replaced by solution of resazurin (25 µg/ml PBS-10% FCS). The plates were incubated for 2 h

(37°C, 5% CO2, humidified atmosphere). Fluorescence was measured on an automated 96-

well plate reader (Fluoroskan Ascent FL, Thermo Fisher Scientific, Wilmington, USA;

excitation wavelength of 530 nm; emission wavelength of 590 nm). Cell proliferation assay

was performed 3 times (n=3, 6 wells for each assay). IC10, IC25 and IC50 were calculated at 24

and 72 h, and used for migration assays.

Migration assay. HUVEC migration was assayed by the wound healing method17

. Cells

(80,000 per well) were seeded into a 24-well plate and incubated with complete medium

(37°C, 5% CO2). At full confluence, cells in each well were scraped away horizontally using a

200 µl tip. At this moment, randomly selected views along the scraped line were measured

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and photographed using a microscope with a camera (40x magnification) and considered as

control. The medium was then replaced by fresh complete medium with different

concentrations of compounds corresponding to the previously calculated IC10, IC25 and IC50.

After 22 h of incubation, pictures of the selected views were taken in order to measure the

reduction in the scraped area, corresponding to cell migration (and not proliferation because

the doubling time population is superior to 24 hours) and the area modifications were

compared to these of the control. Data were expressed as mean normalized to the control ±

SEM (n=6), a: p<0.05 IC10 or IC25 or IC50 vs control; b: p<0.05 IC25 or IC50 vs IC10; c: p<0.05

IC50 vs IC25.

Photophysics and photochemistry.

Experimental setup. Acetonitrile (HPLC grade ≥ 99.9%), methylene blue (MB) and 1,3-

diphenylisobenzofuran (DPBF, 97% purity) were purchased from Sigma-Aldrich and used

without any further purification. Solutions of DPBF and photosensitizers in acetonitrile were

prepared freshly each day in a sodium lamp lighted room (589 nm). DPBF and the studied

photosensitizer (A = 0.04-0.17 at the irradiation wavelength) were mixed to get a 10-5

M

DPBF final concentration. Once made, the mixture was handled in the dark. At 10-5

M chain

reactions induced by DPBF can be neglected.18,19

A volume of 2.5 mL of the mixture was

filled in a cylindrical quartz cell of 1 cm pathlength. Irradiations were carried out at room

temperature using a xenon lamp (Oriel, 250 W) equipped with a monochromator Photomax

(Schoeffel). Samples were irradiated in the Q-band region at 630 nm (full width at half

maximum: 9 nm) all along the experiment The irradiation wavelength and the photon fluence

rate (6.7×1013

photons/cm²/s) were measured using a QE65000 UV-Visible-NIR spectrometer

(Ocean Optics). A 420 nm cut-off filter (Schott) was used to suppress residual ultraviolet

radiations. The initial DPBF absorbance at 410 nm and the rate of DPBF decay after

irradiation were monitored by UV-Visible spectroscopy. The measured absorbances were

corrected for the absorbance of the photosensitizer at the monitoring wavelength. Experiments

were made in triplicate.

The chemical probe DPBF not only reacts with singlet oxygen but can also react with other

oxidants. Control experiments were performed to confirm that DPBF only reacted with singlet

oxygen in our conditions20

.

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UV-Visible spectroscopy. Absorption spectra were recorded using a Cary 3 UV-Visible

spectrometer (Varian). Baselines and spectra were recorded at room temperature in the 800-

250 nm spectral range with a 1-nm resolution and a 600 nm/min scan rate.

Singlet oxygen quantum yield. Quantum yields of singlet oxygen photo-production of

phthalocyanines (Pc) (Φ∆) were determined in air-saturated acetonitrile using MB as a

reference sensitizer. The Φ∆ values were calculated from the following equation21,22

:

where is the singlet oxygen formation quantum yield for MB ( = 0.52 in

acetonitrile23

). and are the DPBF photo-bleaching rates, and

are the fractions of absorbed light, and and are the absorbances at

the irradiation wavelength of the phthalocyanines and MB, respectively. The DPBF photo-

bleaching was monitored at 410 nm. Quantum yields were calculated based on an absorbance

loss comprised between 5 and 10 %. The quenching of singlet oxygen by the chalcone was

evaluated using the Stern-Volmer plot:

Where and are the rates of DPBF loss in the absence and in the presence of

the chalcone, kr is the reaction rate constant of singlet oxygen with DPBF (9.8x108 L.mol

-1.s

-

1)24

, kd is the decay constant of singlet oxygen in acetonitrile (1.8x104 s

-1)19

, kQ is the reaction

rate constant of singlet oxygen with chalcone, and [DPBF] and [Chalc] are the initial

concentrations of DPBF and carbamate chalcone 8, respectively. An equivalent relationship

can be written when MB is used as a sensitizer instead of phthalocyanine. The singlet oxygen

quenching by the chalcone linked to the phthalocyanines was evaluated by comparing the Φ∆

values of 1 and 5.

Phthalocyanine photodegradation. The photodegradation of phthalocyanines 1, 2 and 5

dissolved in acetonitrile were monitored by UV-Vis spectroscopy at respectively 694, 696,

and 699 nm. Irradiations were performed in the same conditions than described above. The

quantum yields of photodegradation (Φdeg) were calculated from the following equation:

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where is the phthalocyanine photodegradation rate, NA is the Avogadro number, I0 is the

photon fluence rate expressed in Einstein L-1

.s-1

, is the fraction of absorbed light

by Pc, and is the absorbance at the irradiation wavelength of Pc.

In vitro photodynamic activity.

Phototoxicity. Each compound (1 and 6) was formulated in DMSO and diluted in medium

(McCoy’s 5A + 2 mM L-glutamine) to give concentrations ranging from zero to 5 x10-6

M.

The final concentration of DMSO for the highest concentration of photosensitiser was 4.1%

and we have previously determined that for this cell type no deleterious effects are observed

below 10%. The dilutions were added to HT-29 cells (Human Caucasian colon

adenocarcinoma) adjusted to a concentration of 1x106 cells /mL. Cells were then incubated in

the dark for an hour at 37 oC and 5% CO2 after which time they were washed in a 3x excess of

medium to eliminate any unbound phthalocyanine. The resulting pellets of cells were re-

suspended in 1ml medium and 4 x 100µl of each concentration was transferred in to two 96

wells plates. One plate was irradiated with red light (>600 nm) at a dose of 3.6 J/cm2 while

the other served as dark control. After irradiation, 5 µl of Fetal Bovine Serum was added to

each well and the plates were returned to the incubator overnight. After 18 to 24 hours, an

MTT cell viability assay was performed25

and the results expressed as percentage of cell

viability versus dye concentration; an LD90 (lethal dose where 90% of the cells are killed) was

determined from the resulting curves. Each experiment was repeated in triplicate. Materials:

McCoy’s 5A Medium Modified (Sigma M8403), L-Glutamine (Gibco 25030), Fetal Bovine

Serum Heat Inactivated (BioSera S1810), Dimethyl Suphoxide (DMSO) Hybri-Max (Sigma

D2650), HT-29 cells (ECACC 91072201). MTT (Thiazolyl blue; Sigma M5655). Light

Source: Oriel Instruments Quartz Tungsten-Halogen Lamp Housing (model 66188) powered

by 1KW Radiometric Power Supply (model 68835). Light delivered through water cooling

filter, Integrating Sphere and red Schott glass filter BP 580nm.

Protocol for fluorescence imaging on HT-29 cells + phthalocyanine 6 or conjugate 1. Each

compound formulated in DMSO was diluted in medium (McCoy’s 5A + 2 mM L-glutamine)

to give the LD90 concentration (8.3x10-7

M for conjugate 1 and 6.25x10-6

M for control

phthalocyanine 6). The diluted dyes were added to HT-29 cells which had been seeded the

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day before (at 1x106 cell/mL into ibidi 35 mm, glass bottomed dishes) after the cells had been

rinsed 3 times with PBS. The dishes were returned to incubate for 1 hour (37 ºC and 5% CO2)

after which they were again rinsed 3 times with PBS and 800 µl of HBSS was added.The

fluorescence image was produced by exciting via a Cairn Optoscan Monochromator which

delivers the light (produced by a 75W xenon/mercury arc lamp) to the microscope via a silica

fibre. The excitation wavelength was 405 nm. A bespoke filter cube with no excitation filter

but with a 600 nm dichroic filter and 610 nm longpass emission filter was used to further

modulate the wavelength. The images were acquired using a Leica DMIRB microscope,

40x/0.55 CORR Ph N PLAN Fluotar objective and Hamamatsu Orca AG deep-cooled digital

camera run by the MicroManager (1.4) plugin of ImageJ (1.44n9). The observed fluorescence

was weak and the acquisition times varied between 1 and 2 sec, because of these relatively

long excitation times, autofluorescence of cells only under the same condition was sought but

none could be detected. Materials: 35mm ibidi Glass Bottom Culture Dishes (Thistle

Scientific Ltd.), Leica DMIRB microscope (Leica Microsystems), Optoscan Monochromator,

Xenon arc lamp and power supply, light guide and silica fibre (Cairn Research Limited),

Hamamatsu ORCA AG deep-cooled camera and HCImage software (Hamamatsu Photonics

UK Limited), ImageJ program (Rasband, W.S., ImageJ, U. S. National Institutes of Health,

Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2012.)

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