A phthalocyanine-mestranol conjugate for photodynamic therapy prepared via click chemistry

Article abstract of DOI:10.1016/j.tetlet.2009.12.075

Introduction of an azide group onto the periphery of a photodynamically effective phthalocyanine (Pc) derivative gives a useful building block for the preparation of Pc conjugates via Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition ('click chemistry'). In this way, Pc-mestranol and Pc-hept-1-yne conjugates (for comparison purposes) are synthesized and their absorption, photochemical, and photophysical properties are studied. The conjugate retains the advantageous photophysical and photochemical properties of Pc. Conjugation with mestranol may lead to improved localization in tumors whereas the Pc unit is responsible for the photodynamic effect.

Full text of DOI:10.1016/j.tetlet.2009.12.075

Tetrahedron Letters 51 (2010) 1016–1018
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A phthalocyanine–mestranol conjugate for photodynamic therapy prepared
via click chemistry
*
Veronika Novakova, Petr Zimcik , Miroslav Miletin, Kamil Kopecky, Jana Ivincová
Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove 50005,
Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Introduction of an azide group onto the periphery of a photodynamically effective phthalocyanine (Pc)
derivative gives a useful building block for the preparation of Pc conjugates via Cu(I)-catalyzed azide-
alkyne 1,3-dipolar cycloaddition (‘click chemistry’). In this way, Pc–mestranol and Pc–hept-1-yne conju-
gates (for comparison purposes) are synthesized and their absorption, photochemical, and photophysical
properties are studied. The conjugate retains the advantageous photophysical and photochemical prop-
erties of Pc. Conjugation with mestranol may lead to improved localization in tumors whereas the Pc unit
is responsible for the photodynamic effect.
Received 13 November 2009
Revised 2 December 2009
Accepted 11 December 2009
Available online 21 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Phthalocyanines (Pc) are well-known organic dyes with appli-
cations in many areas. Medical applications include mainly photo-
dynamic therapy (PDT) which combines a photosensitizer (PS),
light, and oxygen to produce a cytotoxic effect.1 However, the
low targeting of the PS can often limit their use. This problem is
usually solved by the synthesis of PS conjugates with biomolecules
or other targeting moieties.2,3 Among them, alkynylestradiol–por-
phyrin conjugates have shown good promise for hormone-sensi-
tive tumors where the estrogen receptor (ER) is overexpressed.4
In another study, Pc–estradiol conjugates showed good receptor
binding affinities for the ER, particularly when the Pc was lipo-
philic.5 Nevertheless, the published syntheses of PS conjugates
are difficult and often suffer from low yields. The highly efficient,
selective and mild Cu(I)-catalyzed azide-alkyne 1,3-dipolar cyclo-
addition (‘click’ chemistry) is an attractive strategy for the prepara-
tion of bioconjugates.6 Although ‘click’ chemistry has been
thoroughly explored in recent years, only a few examples of Pc
postmodification by this interesting reaction can be found,7,8 most
likely due to the limited solubility of some Pcs.9
any molecule bearing a terminal alkyne. For example, conjugation
with mestranol and hept-1-yne is described.
First, 3-azidopropylamine (3) was prepared from the corre-
sponding bromide (Scheme 1). Amidation of 1 with 3 using HBTU
as the activator gave 2. However, the published synthesis of 1 is
relatively low-yielding (11%) due to problems with silica binding
during isolation.10 As compound 2 seemed to lack such problems,
we decided to synthesize it directly using a modified precursor
to obtain better overall yields (Scheme 1). Thus, amidation of
2,3-dicyanoquinoxaline-6-carboxylic acid with 3 using SOCl2 gave
4 in a reasonable 65% yield. Its mixed cyclotetramerization with
4,5-bis(tert-butylsulfanyl)phthalonitrile using magnesium butox-
ide as the initiator gave a statistical mixture composed of at least
six different Pcs (AAAA, AAAB, AABB, etc.). A 3:1 ratio of starting
material shifted the reaction toward the desired AAAB type. The
magnesium(II) complex of 2 was isolated in 33% yield and its
demetalation using p-toluenesulfonic acid gave metal-free 2 which
was subsequently chelated with zinc(II) to give Zn–2 in an overall
26% yield (based on starting dicarbonitriles). The higher overall
yield may be attributed to the significantly decreased silica binding
which seemed to be the limiting factor during purification of 1.
Compound 2 is a suitable active part of PS conjugates either
with improved physicochemical properties (e.g., water solubility)
or with targeting properties. Due to the above-mentioned reasons
we focused on PS–estrogen conjugates, but any other biomolecule
(e.g., saccharides, oligonucleotides) modified with a terminal al-
Recently, we described the synthesis of photosensitizer 1
(Scheme 1) which displayed advantageous photophysical and pho-
tochemical properties for application in PDT.10 It is highly soluble
in common organic solvents and absorbs light at wavelengths that
allow deeper penetration of activating light into human tissues
(726 nm). It possesses very good photosensitizing properties
(
(
UD = 0.80) and emits fluorescence for detection of PS localization
UF = 0.06).10 Herein, we describe a reliable synthesis of its deriva-
kyne can be used for conjugation. The 17a-ethynyl group of mes-
tive 2, which is a suitable building block for ‘click’ chemistry with
tranol represents a suitable reactive center for ‘click’ chemistry.
Various conditions were tested for the ‘click’ reaction. Refluxing 2
with mestranol in THF with CuI and DIPEA was revealed as the
most suitable procedure giving very high yields of conjugate 5.11
For example, DMF as the solvent and DBU as the base led to only
* Corresponding author. Tel.: +420 495067257; fax: +420 495067167.
E-mail address: petr.zimcik@faf.cuni.cz (P. Zimcik).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.12.075

V. Novakova et al. / Tetrahedron Letters 51 (2010) 1016–1018
1017
S
S
S
S
S
S
N
N
N
N
N
N
N
Zn
N
N
Zn
N
N
Zn
N
i)
v)
S
S
S
S
S
S
S
S
S
S
S
S
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
1
2
(26%)
N3
5, 6
NH
OH
NH
O
O
O
N
N
N
N
N
S
S
ii)
R
iv)
Br- +H3N
Br
H2N
N3
(55%)
3
(86%)
OCH3
(97%)
5
6
R =
R =
HO
O
O
N
N
N
iii)
N
N
N
OH
N
H
N3
N
N
4
(65%)
Scheme 1. Reaction conditions: (i) HBTU, anhyd. DMF, Et3N, rt, 18 h; (ii) NaN3, H2O, 90 °C, 23 h; (iii) SOCl2, anhyd toluene/THF, reflux, 6 h; (iv) Mg, anhyd butanol, reflux,
19 h?pTSA, THF/CHCl3, rt, 4 h?Zn(COOCH3)2, pyridine and DMF, 120 °C, 5 h; (v) 5-C„CH or 6-C„CH, CuI, THF, DIPEA, reflux, 19 h.
Table 1
a 29% yield and significant amounts of side products were detected
Absorption maxima of the Q-bands (kmax), singlet oxygen quantum yields (UD), and
fluorescence quantum yields (UF) of 1, 2, 5, and 6 in pyridine
on TLC. For comparative purposes, 2 was also reacted with hept-1-
yne to give 6 almost quantitatively. All the synthesized compounds
were fully characterized by NMR, IR, and UV–vis spectral methods
and the purity was confirmed by elemental analysis. MALDI-TOF
spectra showed clusters at expected m/z values with isotope pat-
terns corresponding with calculated distributions.
Compd
kmax (nm)/
e
(Â105 MÀ1 cmÀ1
)
UD
UF/kem (nm)
1a
2
5
726/1.390
727/1.503
727/1.045
727/1.335
0.80
0.78
0.84
0.69
0.060/734
0.040/738
0.042/738
0.040/738
6
An important question is whether the promising photophysical
and photochemical properties of 1 are retained in conjugates 5 and
6. No significant changes were observed in the absorption or fluo-
rescence emission spectra of 1, 5, and 6 (Fig. 1). The Q-band max-
ima were found at 727 nm allowing photosensitization using light
that penetrates deeper into human tissues. The broad Q-band
caused by splitting due to the unsymmetrical composition of the
macrocyclic system may be considered as another advantage. More
photons are expected to be absorbed during irradiation by conven-
tional lamps emitting full-spectrum light. The singlet oxygen quan-
tum yield (UD), the most important photochemical parameter in
PDT, was determined by a comparative method based on the
decomposition of 1,3-diphenylisobenzofuran. The results summa-
rized in Table 1 showed that neither introduction of an azide group
as in 2 nor conjugation with mestranol (5) or hept-1-yne (6) led to
significant reduction of UD values. All the tested compounds can
a
Data from Ref. 10.
therefore be considered as very efficient photosensitizers. The fluo-
rescence emission of 1, which can be a useful tool for tumor visu-
alization or photosensitizer distribution monitoring, is also
retained for 2 as well as for conjugates 5 and 6.
In conclusion, we have developed an efficient synthesis of a
photosensitizer substituted with an azide group 2 which is a versa-
tile substrate for ‘click’ chemistry with various molecules bearing a
terminal alkyne for both improved targeting (e.g., steroids, pro-
teins, oligonucleotides) and/or physicochemical properties (e.g., in-
crease of water-solubility after conjugation to oligosaccharides).
Formation of the stable linkage after Cu(I)-catalyzed azide-alkyne
1,3-dipolar cycloaddition does not affect the good photosensitizing
properties of the starting PS and no quenching of the excited states
occurs at the azide or final 1,2,3-triazolyl-modified Pc. Thus click
chemistry can be used for the preparation of targeting PS with
no decrease of the photosensitizing properties of the active part.
Conjugate 5 bearing mestranol should have improved targeting
properties for hormone-sensitive tumors where the ER is overex-
pressed and biological testing is under investigation.
Acknowledgments
Financial support from Research Project MSM0021620822 is
gratefully acknowledged.
Supplementary data
Supplementary material includes full synthetic details and char-
acterization of all compounds including 1H NMR spectra of final
Figure 1. Normalized absorption spectra of 1 (dotted line) and 5 (full line) in
pyridine. Normalized emission spectrum of 5 in pyridine (dashed line).

1018
V. Novakova et al. / Tetrahedron Letters 51 (2010) 1016–1018
11. Preparation of conjugate 5: Compound 2 (20 mg, 0.016 mmol), CuI (3.0 mg,
0.016 mmol) and mestranol (7.3 mg, 0.023 mmol) under argon were dissolved
in THF (10 mL). N,N-Diisopropylethylamine (DIPEA) (60.5 mg, 0.468 mmol)
was added dropwise. The solution was heated at reflux under argon for 7 h
after which furthur portions of CuI (3.0 mg, 0.016 mmol) and mestranol
(7.3 mg, 0.023 mmol) were added and reflux was continued for 10 h. The crude
product was purified by column chromatography on silica with toluene/
pyridine 10:2 as eluent and washed with methanol. Yield: 21.3 mg (86%), cyan-
blue solid. Anal. Calcd C83H96N14O3S6Zn+4H2O: C, 59.78; H, 6.29; N, 11.76.
Found: C, 59.50; H, 6.06; N, 11.34. m/z (MALDI-TOF) 1593 [M+H]+, 1615
[M+Na]+, 1631 [M+K]+; dH(300 MHz, pyridine-d5) 1.35 (3H, s, CH3Mestr.), 1.53
(9H, s, CCH3), 1.57 (9H, s, CCH3), 1.67 (18H, s, CCH3), 1.69 (18H, s, CCH3), 1.45–
1.75 (4H, m, aliphatic Mestr), 1.88–1.95 (4H, m, aliphatic Mestr), 2.14–2.29
(4H, m, aliphatic Mestr), 2.43–2.60 (3H, m, aliphatic Mestr and CH2CH2CH2),
2.70–2.83 (1H, m, aliphatic Mestr), 2.91–3.04 (1H, m, aliphatic Mestr), 3.56
(3H, s, OCH3), 3.89 (2H, q, J = 7.1 Hz, NHCH2), 4.81 (2H, t, J = 6.8 Hz, NCH2), 6.64
(1H, d, J = 2.5 Hz, Ar-Mestr.), 6.79 (1H, dd, J1 = 2.6 Hz, J2 = 8.8 Hz, Ar-Mestr.),
6.82 (1H, s, Ar-Mestr), 8.41 (1H, s, CH triazole), 8.89 (1H, d, J = 9.1 Hz, Ar-H),
9.01 (1H, d, J = 8.8 Hz), 9.81–9.86 (2H, m, Ar-H), 10.14 (2H, s, Ar-H), 10.16 ppm
(3H, s, Ar-H); dC(75 MHz, pyridine-d5) 15.07, 24.47, 26.97, 27.85, 30.15, 29.98,
31.38, 33.73, 37.98, 38.85, 39.98, 43.85, 47.84, 48.29, 48.74, 48.76, 48.90, 48.97,
54.94, 82.28, 111.94, 114.01, 126.72, 130.11, 130.73, 131.17, 131.42, 132.43,
132.59, 133.08, 134.83, 137.58, 138.07, 138.17, 138.28, 138.32, 141.67, 141.72,
142.63, 142.66, 143.16, 143.39, 143.44, 144.68, 154.56, 155.39, 155.66, 155.68,
155.93, 157.87 and 167.16 ppm. Additional analytical data can be found in the
Supplementary data.
compounds. Absorption, emission, and excitation spectra are also
presented. Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.2009.12.075.
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