Synthesis and biological distribution study of a new carbon-11 labeled porphyrin for PET imaging. Photochemical and biological characterization of the non-labeled porphyrin

Article abstract of DOI:10.1142/S1088424615500728

Ideal reaction conditions were found to promote the "cold" monomethylation of 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin with CH₃I, at minute time scale, in the presence of base. The photophysical characterization, cellular uptake and dark cytotoxicity of the resulting monomethylated porphyrin were appraised. Moreover, the syntheses of the corresponding labeled porphyrin, using short-lived carbon-11, prepared in the automated radiolabeling system were efficiently performed. The purification of the labeled product was achieved via preparative HPLC with high radiochemical purity and specific radioactivity. Preliminary studies on the biodistribution of 5,10,15-tris(3-hydroxyphenyl)-20-(3-[¹1C]methoxyphenyl)porphyrin carried out in a BALB/C normal mouse, using PET imaging, showed that the liver is the main pathway for excretion.

Full text of DOI:10.1142/S1088424615500728

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2015; 19: 1–10
DOI: 10.1142/S1088424615500728
Published at http://www.worldscinet.com/jpp/
Synthesis and biological distribution study of a new
carbon-11 labeled porphyrin for PET imaging. Photochemical
and biological characterization of the non-labeled porphyrin
Nuno P. F. Gonçalvesa,b, Ana V. C. Simõesa, Artur R. Abreua, Antero J. Abrunhosac,
Janusz M. Da˛browskid and Mariette M. Pereira*a◊
a Department of Chemistry, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal
b Currently at Luzitin SA, Ed. Bluepharma, 3045-016 São Martinho do Bispo - Coimbra, Portugal
c Institute for Nuclear Sciences Applied to Health (ICNAS), 3000-548 Coimbra, Portugal
d Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland
Received 25 March 2015
Accepted 28 April 2015
ABSTRACT: Ideal reaction conditions were found to promote the “cold” monomethylation of
5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin with CH3I, at minute time scale, in the presence of base.
The photophysical characterization, cellular uptake and dark cytotoxicity of the resulting monomethylated
porphyrin were appraised. Moreover, the syntheses of the corresponding labeled porphyrin, using
short-lived carbon-11, prepared in the automated radiolabeling system were efficiently performed. The
purification of the labeled product was achieved via preparative HPLC with high radiochemical purity
and specific radioactivity. Preliminary studies on the biodistribution of 5,10,15-tris(3-hydroxyphenyl)-
20-(3-[11C]methoxyphenyl)porphyrin carried out in a BALB/C normal mouse, using PET imaging,
showed that the liver is the main pathway for excretion.
KEYWORDS: porphyrins, methylation, radiolabeling, carbon-11, PET imaging.
to allow good quality images without causing saturation
of the radio-detectors and, eventually, the organism’s
INTRODUCTION
Positron emission tomography (PET) is a powerful
non-invasive imaging technique, which allows the
detection of early stage tumors, in vivo brain functions
damage due to radiation exposure [1]. Fluorine-18 (t½ =
109.8 min), nitrogen-13 (t½ = 9.98 min), oxygen-15 (t½ =
2.05 min), and carbon-11 (t½ = 20.4 min) are short-
like Alzheimer and Parkinson diseases, blood flow and
lived PET radionuclides, which present high percentage
cardiacdisorders,suchascoronaryarterydisease,receptor
of decay by positron emission [3, 4]. The short life
binding and metabolism of drugs [1, 2]. PET imaging is
time of 13N and 15O make their clinical application very
based on the direct detection of a subpharmacological
limited, 18F being, until the last decade, the most studied
compound labeled with b-emitting radionuclides which
radionuclide for PET clinical imaging [5, 6]. However,
must have high specific activity. Ideally, the chemical
the widespread installation of cyclotrons nearby
structure of the labeled and unlabeled species must be
hospitals and research centers has greatly contributed to
very similar (although not equal); thus, both compounds
the increasing interest in the labeling of molecules with
will have similar biochemical properties. Additionally,
11C, which was previously considered difficult due to
in vivo imaging studies requires a careful choice of the
the required time for transportation from manufacturing
administered amount of radioactive compound in order
installations to hospitals. In this way, 11C is nowadays
considered one of the most exciting and challenging PET
isotopes, due to its easy preparation, purification, and
◊ SPP full member in good standing
also due to similar chemical and biological properties of
molecules to those of the unlabeled compounds [7].
*Correspondence to: Mariette M. Pereira, email: mmpereira@
qui.uc.pt, fax: +351 239-827-703, tel: +351 239-854-474
Copyright © 2015 World Scientific Publishing Company

2
N. P. F. GONÇALVES ET AL.
Unfortunately deaths by cancer will continue to rise,
based on projections, with an estimated population of
nine million people dying from cancer in 2015, and 11.4
million dying in 2030 [8]. Concerning the medicinal
research, the primary combat strategy against this
scourge consists in the early tumor detection, followed
by chemotherapy or Photodynamic Therapy (PDT)
techniques. Tetrapyrrolic macrocycles like porphyrin
and chlorins, in which Photofrin® [9] and Foscan®
[10] are included, have shown high accumulation rates
in carcinogenic cells and are used as photosensitizers
(PS) in several PDT treatments. Recently, our group
discovered a new bacteriochlorin PS [11, 12] (in clinical
trials — phase I), which 1 h after administration showed
remarkable activity and selectivity for tumors [13] and
high PDT effect upon NIR laser irradiation. So, the high
affinity of tetrapyrrolic macrocycles for accumulation
in tumor cells makes them promising compounds also
for development of contrast agents like radiolabeled
agents for cancer imaging. In this way, tetrapyrrolic
macrocycles are a class of agents with upmost interest
for the development of theranostic probes due to their
flexibility to synthetic modulation, low dark toxicity and
high biocompatibility [14–17]. The application of labeled
tetrapyrrolic macrocycles as PET probes has already
been described in recent literature, namely through
radionuclides with long semi-disintegration periods but
with lower positron emission decays, such as 64Cu [18,
19], 68Ga [20, 21], and 62Zn [22]. However, it should be
noted that all these processes required the introduction
of a foreign atom in the molecules, causing significant
changes in the pharmacokinetic and pharmacodynamic
characteristics of the radiotracer and even inducing
toxicity like the one observed with 68Ga-labeled
compounds [23, 24]. Other examples include the
radiolabeling of tetrapyrrolic macrocycles with 18F
radionuclide. For instance, Chi et al. [25] described
two different 18F-labeling methodologies: (i) acid
catalyzed condensation of pyrrole, m-anisaldehyde
and 4-[18F]fluorobenzaldehyde; (ii) the condensation
of the corresponding tetrapyrrane with 4-[18F]
fluorobenzaldehyde. A different approach was reported
by Lier et al. [26] who described a procedure for direct
18F-labeling using [18F]fluoride ion through nucleophilic
substitution reactions with tosylated Zn-porphyrins or
Zn-phthalocyanines.
Herein we report the laboratorial optimization of the
“cold” methylation reaction of 5,10,15,20-tetrakis(3-
hydroxyphenyl)porphyrin 1 (a precursor of Foscan®) with
CH3I, under a minute time scale, and describe the photo-
physical data, cellular uptake and dark cytotoxic properties
of the resulting monomethylated product. Moreover,
the pioneering synthesis of the corresponding labeled
5,10,15-tris(3-hydroxyphenyl)-20-(3-[11C]methoxy-
phenyl)porphyrin, using 11CH3I generated in ICNAS
cyclotron-University of Coimbra, is also described. The
biodistribution of the carbon-11 labeled 5,10,15-tris(3-
hydroxyphenyl)-20-(3-[11C]methoxyphenyl)porphyrin was
evaluated in vivo, in a BALB/C normal mouse, using PET
imaging, and its uptake on several organs is presented.
EXPERIMENTAL
General
All chemicals were of reagent grade and were used as
received. All solvents used in the synthesis of the meso-
substituted porphyrins were purified by standard methods
before use. The chloroform used in the preparative thin
layer chromatography was neutralized with neutral active
alumina and dried before use. Solutions were prepared
with doubly distilled water, either equilibrated with air or
bubbled with argon at room temperature. Reactions were
carried out under a N2 atmosphere and were monitored
by analytical (0.20 mm) silica TLC plates. Medium for
cell culture (RPMI, F10) and PBS (phosphate-buffered
saline) were purchased from Biomed, Lublin, Poland. All
reactants used in the synthesis of the meso-substituted
porphyrins, HPLC eluents, 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT), penicillin
and streptomycin were purchased from Sigma Chemical
Co. Trypsin/EDTA, cell culture media and supplements,
and animal serum were purchased from Biochrom,
Berlin, Germany. The disposable cell culture equipment
was from Technoplastics Products Ltd., Trasadingen,
Switzerland.
UV-visspectraofmeso-substitutedporphyrininethanol
were recorded with a UV-vis spectrophotometer CARY 3
(UV-2101 PC®, Shimadzu, Tokyo, Japan). Fluorescence
emission spectra of meso-substituted porphyrin were
measured in ethanol using a Luminescence Spectrometer
LS 50B (Perkin-Elmer). 1H NMR spectra were obtained
using a Brucker Avance III 400 MHz spectrometer.
Mass spectra (ESI-TOF) were acquired using a Brucker
MicroTof spectrometer. The MALDI-TOF MS data were
acquired using a Bruker Daltonics flexAnalysis apparatus.
Melting points were measured on an Electrothermal
capillary melting point non calibrated apparatus.
Despite all these advances, to the best of our knowledge
there are no examples in the literature describing the
synthesis of 11C-labeled tetrapyrrolic macrocycles,
although [11C]methyl iodide is one of the most
widespread methylation agent to promote the labeling of
hydroxylated aromatic compounds [27] like anisole or
raclopride. Furthermore, 11C is a promising radionuclide
to promote the labeling of hydroxylated tetrapyrrolic
macrocycles, in order to enhance the understanding of
early stage biodistribution and cancer cell accumulation
of this type of compounds.
Carbon-11 was produced using a Cyclone 18/9
cyclotron (IBA Cyclone 18/9, Louvaine-La-Neuve.
belgium). Preparative HPLC was made with a Bioscan
AutoLoop System (Eckert and Ziegler, Germany) with
Copyright © 2015 World Scientific Publishing Company
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SYNTHESIS AND BIOLOGICAL DISTRIBUTION STUDY OF A NEW CARBON-11 LABELED PORPHYRIN FOR PET IMAGING
3
KNAUER-200 UV detector (254 nm). Semi-preparative
column was a C18, 10 mm, 10 × 250 mm from Waters.
J3 = 8.5, 4H, HPh), 3.99 (s, 9H, OCH3), -2.80 (s, 2H, NH).
Analytical HPLC: tR = 1.90 min.
5,10,15,20-Tetrakis(3-methoxyphenyl)porphyrin
(5). Yield (12.7 mg, 30%); mp > 250°C. MS (MALDI-
TOF): m/z 757.2782 [M + Na]+ calcd. for C48H38N4NaO4+
Synthesis and “cold” methylation reaction
1
The synthesis of 5,10,15,20-tetrakis(3-hydroxyphenyl)
porphyrin (1) was carried out using the nitrobenzene
method [28] prepared by the condensation of the
3-hydroxybenzaldehyde (5.250 g, 43 mmol) with
pyrrole (43 mmol) in mixture of acetic acid (140 mL)
and nitrobenzene (70 mL) at 120°C (1 h). The solution
was cooled to room temperature and 50 mL of hexane
was added to promote precipitation. The crystals of
the porphyrin was filtered off, washed with hexane and
dried. The solid was purified by chromatography (2:1
chloroform/acetone). The data is in good agreement with
previously reported [29].
757.2790. H NMR (400 MHz, CDCl3): d, ppm 8.88 (s,
8H, Hb), 7.83–7.79 (m, 8H, HPh), 7.65 (t, J = 8.0 Hz, 4H,
HPh); 7.34 (dd, J2 = 1.8 hz; J3 = 8.5, 4H, HPh), 3.99 (s,
12H, OCH3), -2.79 (s, 2H, NH). Analytical HPLC: tR =
3.45 min.
UV-vis absorption spectra
UV-vis absorption spectra were recorded in quartz
cuvettes with Shimadzu 2100 spectrophotometer.
5,10,15,20-Tetrakis(3-hydroxyphenyl)porphyrin
(1). Yield (2.879 g, 10%); mp > 250°C. MS (ESI): m/z
679 [M + H]+. 1H NMR (400 MHz, CDCl3): d, ppm 8.92
(s, 8H, Hb), 7.68–7.65 (m, 8H, HPh), 7.60 (t, J = 7.8 Hz,
4H, HPh), 7.25 (dd, J2 = 1.2 Hz; J3 = 8.0 Hz, 4H, HPh), -2.80
(s, 2H, NH). For synthesis of methylated compounds
5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin1(30mg,
0.4 mmol) was dissolved in anhydrous DMF (1.5 mL)
and tetrabutylammonium hydroxide 1.0 M in methanol
(0.08 mL, 2.6 mmol, 6 equiv) was added. The mixture
was stirred at room temperature and iodomethane was
added (0.02 mL, 2.6 mmol, 6 equiv). The reaction was
monitored by TLC plate and stopped after 1 min, the
products were purified by column chromatography
(2:1 chloroform/acetone) to give selective methylated
porphyrins.
Fluorescence quantum yield measurements
The fluorescence spectra was measured in ethanol
with excitation wavelength at 415 nm (A415 = 0.025).
The fluorescence quantum yield of 2 was obtained from
the ratio of its fluorescence band in ethanol vs. that TPP.
In vitro studies
Cell culture and time dependent cellular accumu-
lation. The A549 cells were seeded into 96-well culture
plate at 1 × 104 cells per 0.2 mL of culture medium.
After attaching, the cells were incubated with different
concentrations (10-4–10-7 M) of photosensitize for 20 h
at 37°C. The solutions were prepared by diluting the
porphyrin stock (5 mg/mL DMSO) with the medium
to desired final concentrations (10-4–10-7 M). The
highest DMSO concentration did not exceed 0.5%. The
compound 2 solution was then replaced by 0.2 mL of
fresh medium. After 24 h, 20 mL (final concentration
0.5 mg/mL) of MTT was added and the MTT test was
performed using an ELISA plate reader (GENios Plus,
Tecan Trading AG, Switzerland). The cell survival was
expressed by the absorbance changes of the formazan
salt, and survival rate was given as the percent ratio of
viable treated cells vs. the number of viable untreated
cells. The number of cells was determined from linear
regression of a calibration curve.
Cellular uptake. Cells were prepared as described
above. After 24 h, the cells were incubated in 37°C with
different concentrations of porphyrin 2 for various time
intervals from 5 min to 24 h. The solutions of compounds
were prepared by diluting the porphyrin stock solution
in DMSO with the culture medium to the desired final
concentration. The highest concentration of DMSO in
medium did not exceed 0.5%. After incubation, the cells
were washed two times with warmed PBS and solubilized
in 30 mL of Triton X-100 and 70 mL of DMSO/ethanol
solution (1:3). The retention of cell-associated porphyrin
was detected by fluorescence measurement with the
5,10,15-Tris(3-hydroxyphenyl)-20-(3-methoxy-
phenyl)porphyrin (2). Yield (4.2 mg, 14%); mp >
250°C. MS (MALDI-TOF): m/z 693.2485 [M + H]+
1
calcd. for C45H33N4O4 693.2501. H NMR (400 MHz,
CD3OD): d, ppm 8.93 (s, 8H, Hb), 7.87 (s, 4H, HPh), 7.77–
7.75 (m, 2H, HPh), 7.64 (s, 6H, HPh), 7.57–7.54 (m, 3H,
OH), 7.39 (d, J = 8.4 Hz, H, HPh), 7.27 (d, J = 8.0 Hz, 2H,
HPh), 3.99 (s, 3H, OCH3), -2.8 (s, 2H, NH). Analytical
HPLC: tR = 0.55 min.
5,10-Bis(3-hydroxyphenyl)-15,20-bis(3-methoxy-
phenyl)porphyrin (3). Yield (8.3 mg, 25%); mp >
250°C. MS (MALDI-TOF): m/z 707.2637 [M + H]+
1
calcd. for C46H35N4O4 707.2658. H NMR (400 MHz,
CDCl3): d, ppm 8.88 (s, 8H, Hb), 7.82–7.78 (m, 8H, HPh),
7.69 (bs, 2H, OH), 7.64–7.58 (m, 4H, HPh), 7.34 (dd, J2 =
1,8 Hz; J3 = 8.5 Hz, 4H, HPh), 3.99 (s, 6H, OCH3), -2.80
(s, 2H, NH). Analytical HPLC: tR = 0.54 min. Analytical
HPLC: tR = 1.18 min.
5-(3-Hydroxyphenyl)-10,15,20-tris(3-methoxy-
phenyl)porphyrin (4). Yield (10.2 mg, 28%); mp >
250°C. MS (MALDI-TOF): m/z 743.2648 [M + Na]+
calcd. for C47H36N4NaO4 743.2634. 1H NMR (400 MHz,
CDCl3): d, ppm (s, 8H, Hb), 7.82–7.78 (m, 8H, HPh), 7.66–
7.62 (m, 4H, HPh), 7.59 (s, H, OH), 7.34 (dd, J2 = 1.8;
Copyright © 2015 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2015; 19: 3–10

4
N. P. F. GONÇALVES ET AL.
microplate reader (Tecan Infinite M200 Reader) using
excitation/emission wavelengths. After 24 h cell were
exposed to 20 mM 2 in PBS for various time intervals,
from 5 min up to 24 h to probe time-dependent drug
accumulation. At the end of the incubation interval, the
cells were washed three times with PBS and solubilized
in 100 mL of Triton X-100. Two mL of ethanol:DMSO
solution (75:25) was added and cell extract was
centrifuged at 2000 rpm for 10 min at room temperature.
The retention of cell-associated chlorin was detected
by fluorescence measurement of the accumulated mole-
cule using excitation/emission wavelengths of 413 and
≈660 nm, respectively.
Cells survival assay. The MTT assay for measuring
cell survival has been used successfully to quantitate
photosensitizer-mediated cytotoxicity. For each experi-
ment two identical 96-well plates with A549 cell culture
were used. After cell attachment, 2 solution in growth
medium (DMSO < 0.5%) at various concentrations.
Next, the 2 solution of each well was removed, cells
were washed in PBS and 200 mL fresh culture medium
supplemented with FBS and antibiotics was added to
each well and cells were returned to the incubator for
24 h. MTT was dissolved at 5 mg/mL in PBS. Briefly,
22 mL of MTT solution were added to each well (final
concentration 0.5 mg/mL) and the microplates were
furtherincubatedfor3h.Mediumwerethendiscardedand
100 mL of mixture of DMSO/methanol (1:1) were added
to the cultures and mixed thoroughly to dissolve the dark
blue crystals of formazan. Formazan quantification was
performed using an automatic microplate reader (Tecan
Infinite M200 Reader) by absorbance measurements
with a 565 nm test wavelength. Each experiment was
repeated three times. Data were expressed as mean
absorbance value of six samples and standard error of
the mean.
the cells with fresh medium, cells were incubated in
the dark with 5 mM sensitizers, diluted in cell medium,
for ≈6 h, at 37°C in a 95% atmospheric air and 5%
CO2 humidified atmosphere. After washing the cells
with HBSS/HEPES buffer, cells were incubated
with specific intracellular organelles probes: 100 nM
Mitotracker-Green, 1 mM ER-Tracker Green, 75 mM
Lysotracker Green (Molecular Probes, Invitrogen
Life Technologies), diluted in HBSS/HEPES buffer.
After 30 min incubation, at 37°C, in the dark, cells
were washed with HBSS/HEPES buffer and the slide
was transferred to the microscope stage and cells
were visualized under a confocal microscope (LSM
510 Meta, Carl Zeiss, Jena, Germany) with a 63x oil
immersion objective (Plan-Apochromat, 1.4 N.A., Carl
Zeiss, Jena, Germany). The porphyrin was excited at
488 nm, using an Argon/2 laser (45 mW); fluorescence
was collected after passage through a long-pass filter of
575 nm. A Helium-Neon laser (5 mW) was used as light
source at 633 nm for visualization of cell morphology.
General proceeding for 11C radiolabeling. 0.1 mg
(0.147 × 10-4 mmol) of 5,10,15,20-tetrakis(3-hydroxy-
phenyl)porphyrin 1 was dissolved in dry DMF (80 mL)
and 1.4 mL of TBAOH was added. The mixture was
introduced in 2 mL stainless loop of an automated
radiomethylation system (Bioscan AutoLoop, eckert
and Ziegler, Germany). The 11CH3I produced in the
automated methylation system is driven through the loop
and allowed to react with the precursor for 1 min at room
temperature. The reaction product was isolated using a
semi-preparative HPLC with a Waters C18 (10 mm, 10 ×
250 mm) column and CH3CN/0.1 M ammonium format
(buffer solution) 99/1 v/v as a mobile phase with a flow
of 4 mL/min. Detection was made by UV (254 nm)
and radioactivity. The solvent was removed by SPE
using
a Sep-Pak C18 Light cartridge (Waters®,
Toxicity in the dark. A549 cells were seeded into
96-well culture plate at 1 × 104 cells per 0.2 mL of culture
medium. After attaching, the cells were incubated with
compound 2 of different concentrations (0.02–30 mM)
for 6 h at 37°C. The photosensitizer solution was then
substituted by 0.2 mL of fresh medium. After 24 h 20 mL
(final concentration 0.5 mg/mL) of MTT was added
and MTT test was performed using an ELISA plate
reader (GENios Plus, Tecan Trading AG, Switzerland).
The cell survival was expressed by the absorbance
changes of the formazan salt, and survival rate was
given as the percent ratio of viable treated cells vs. the
number of viable untreated cells. The number of cells
was determined from linear regression of a calibration
curve.
USA). The analytical was perfomed using a HPLC
Agilent Technologies 1200 series with the Zorbax
Eclipse XDB-C18 (5 mm, 4.6 × 150 mm) column
using CH3CN/0.1
M
ammonium format (buffer
solution) 99/1 v/v as a mobile phase with a flow of
1 mL/min, controlled by UV-vis detector (420 nm).
PET imaging and biodistribution. For the in vivo
studies, a 30 g 12 months old female BALB/c mouse was
used. Anesthesia was induced by intraperitoneal injection
of tribromoethanol (Avertin®, 20 mg/mL) with a dosage
of 13 μL/g. PET imaging system was a ClearPEM
high resolution system (PETsysMedical PET Imaging
Systems, Portugal) with long LYSO crystals and depth-
of-interaction information. Image reconstruction was
performed with interactive reconstruction (5 iterations)
with gaussian smoothing at 1.2 mm spatial resolution.
Activity in the organs was counted on a Capintec CRC-
55tW well counter (Ramsey, NJ, USA). All animal
studies were performed by accredited researchers and
according to local and international guidelines on animal
experimentation.
Laser scanning confocal microscopy. The intra-
cellular distribution of porphyrin 2 was assessed in the
A549 cell line.A549 cells were plated at a density of 15 ×
103 cells per well in 8 wells slides (IBIDI, Germany)
and were kept at 37°C in a 95% atmospheric air and
5% CO2 humidified atmosphere, for 24 h. After washing
Copyright © 2015 World Scientific Publishing Company
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SYNTHESIS AND BIOLOGICAL DISTRIBUTION STUDY OF A NEW CARBON-11 LABELED PORPHYRIN FOR PET IMAGING
5
porphyrin 5 and the isolated yiels are presented inTable 1.
By other side, when 1.2 eq. of TBAOH was used
RESULTS AND DISCUSSION
(1 min reaction), 49% of 2, 14% of 3 and 3% of 4 were
isolated (Table 1, entry 2). Increasing the amount of
Synthesis and methylation reaction
base to 6 eq. led to the total consumption of the starting
material and 14% of 2, 25% of 3, 28% of 4 and 30% of
5 have been obtained (Table 1, entry 3). The increase
on the amount of base to 20 eq. led to the formation of
tetramethylated porphyrin 5 in 93% isolated yield, after
just 1 min reaction (Table 1, entry 5). Therefore, the use
of TBAOH as base in the “cold” methylation of meso-
tetra(hydroxyphenyl)porphyrins, at room temperature,
in a minute time scale has shown to significantly affect
the reaction selectivity.
These laboratorial optimization allowed the trans-
position of the methylation reaction to the radio-
synthesis of porphyrin 1, using 11CH3I generated in the
UC-ICNAS cyclotron. Considering that a low amount
of labeled 11CH3I is produced in the cyclotron, then the
monomethylated porphyrin 2 was expected to be the most
abudant labeled compound. Thus, the photophysical and
toxicological studies were exclusively performed for
compound 2.
The precursor 5,10,15,20-tetrakis(3-hydroxyphenyl)
porphyrin was prepared by condensation of equimolar
amounts of 3-hydroxybenzaldehyde with pyrrole in a
mixture of acetic acid and nitrobenzene at 120°C for 1 h.
After cooling to room temperature, hexane was added
to promote precipitation; the solid was filtered off and
washed with hexane. The solid was then purified by
chromatography (2:1 chloroform/acetone), yielding 10%
of 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin [29].
In order to promote the effective and fast methylation
of 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin with
11CH3I at room temperature, in the loop of the automated
radiolabeling apparatus, in a short period of time, a set
of “cold” experiments were performed using CH3I
(Scheme 1). The effect of the amount of base and the
concentration of the reagents on the reaction time and
yield were evaluated and the results are presente in
Table 1.
First, the methylation of 1 was tried at room
temperature in the absence of base under standard
conditions for radiolabeling (short period of time-1 min);
however the reaction did not occur (Table 1, entry 1).
Then, to evaluate the effect of the amount of base, a
set of experiments were made. In a typical experiment,
iodomethane (2.6 mmol) was added to a solution of 1
(0.4 mmol) and the desired amount of TBAOH in dry
DMF (1.5 mL). After 1 min, the reaction was quenched
by adding water to the solution (2 mL). After column
purification it was possible to quantify and characterize
5,10,15-tris(3-hydroxyphenyl)-20-(3-methoxyphenyl)
porphyrin 2, a mixture of 5,10-bis(3-hydroxyphenyl)-
15,20-bis(3-methoxyphenyl)porphyrin and 5,15-bis(3-
hydroxyphenyl)-10,20-bis(3-methoxyphenyl)porphyrin
3, 5-(3-hydroxyphenyl)-10,15,20-tris(3-methoxyphenyl)
porphyrin 4 and 5,10,15,20-tetrakis(3-methoxyphenyl)
Spectroscopy and photophysics characterization
The ground state absorption spectra of 2 recorded
at room temperature in ethanol are presented in Fig. 1.
The spectra show five bands characteristic for free based
porphyrin. The most intense Soret band is observed at
415 nm and four bands: 512 nm, 546 nm, 580 nm and
644 nm are assigned to the (0, 0) and (1, 0) levels of
the Qx and Qy bands. Using measured absorbance
for various concentrations of series of 2, the molar
absorption coefficients were determined for the highest
and the lowest absorption bands from Beer’s law and are
set to 2900000 M-1cm-1 and 2400 M-1 cm-1 for B (0. 0)
and Q (0. 0) respectively. Aggregation does not occur in
the mM concentration range, since the concentrations
R1
HO
R4
OH
NH
N
NH
N
N
N
CH3I
Base
dry DMF
HN
HN
R2
HO
(2) R1, R2, R3 = OH; R4 = OCH3
(3) R1, R2 = OH; R3, R4 = OCH3
and R1, R3 = OH; R2, R4 = OCH3
(4) R1 = OH; R2, R3, R4 = OCH3
(5) R1, R2, R3, R4 = OCH3
R3
OH
1
Scheme 1. Reagents and conditions for the synthesis of methylated hydroxyphenylporphyrins
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6
N. P. F. GONÇALVES ET AL.
Table 1. Optimization of the methylation of 5,10,15,20-
tetrakis(3-hydroxyphenyl)porphyrin
Entry
TBAOH
(eq)
Yield,
%
a
1
2
3
4
5
1
2
3
4
5
0
1.2
6
100
30
—
—
—
—
49
14
—
—
—
14
25
23
—
—
3
—
—
30
46
93
28
30
—
8
20
Reaction conditions: 5,10,15,20-tetrakis(3-hydroxyphenyl)
porphyrin 1 (0.4 mmol), anhydrous DMF (1.5 mL), tetrabutyl-
ammonium hydroxide (TBAOH), iodomethane (2.6 mmol),
room temperature, 1 min reaction. a Isolated yields.
Fig. 2. Time dependent cellular uptake of the compound 2
(20 mM) in A549 cells
Fig. 1. Electronic absorption and fluorescence spectra of 2,
measured in ethanol at room temperature. The fluorescence was
obtained with excitation at 415 nm
Fig. 3. Surviving fraction of A549 cells for various
concentrations of 2
after 18–24 h of incubation. Figure 2 presents time
dependent cellular uptake of the 2 in A549 cells.
were increased in ethanol, the absorption band of lowest
energy was not displaced to longer wavelengths.
Toxicity of 2 against A549 cells was tested in the dark
for various drug concentrations (0.02–30 mM) with a
6 h incubation time based on the cellular accumulation
studies and almost no dark toxicity was observed even at
the highest concentrations tested (Fig. 3).
The fluorescence spectrum of 2 in ethanol is shown in
Fig. 1. It shows the two typical porphyrin fluorescence
bands [30, 31] atributed to Q (0, 0) and Q (0, 1) at 647 nm
and 713 nm respectively. The fluorescence quantum
yield value determined for 2, using TPP (FF = 0.11) as
reference, [31] was FF = 0.0123. It should be noticed
that both, the absorption and fluorescent spectrum of the
new monomethylated porphyrin 2 are very similar to the
ones described for the precursor 5,10,15,20-tetrakis(3-
hydroxyphenyl)porphyrin which, as expected, shows
that the introduction of an extra methyl group does not
significantly affect the properties of the compound.
Subcellular localization. Porphyrin 2 has sufficient
detectable fluorescence so, confocal microscopy can be
used to determine its intracellular localization with the use
of fluorescent intracellular trackers for ER, mitochondria
and lysosomes. The overlaid images are shown in Fig. 4,
where the fluorescence of the porphyrin is shown in red
and the fluorescence of the organelle-specific probe is
shown in green. Confocal microscopy was coupled with
microspectrofluorimetry to define the spectral profiles of
photosensitizer and organelle probes in the same focal
plane. The topographical profiles of porphyrin and the
organelle marker were recorded along a strip within a
single living cell at the fluorescence emission wavelength
In vitro studies
Cellular uptake and dark toxicity. The uptake initially
increases very rapidly at relatively short incubation time
reaching maximum at 6 h and tends to saturation value
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J. Porphyrins Phthalocyanines 2015; 19: 6–10

SYNTHESIS AND BIOLOGICAL DISTRIBUTION STUDY OF A NEW CARBON-11 LABELED PORPHYRIN FOR PET IMAGING
7
Fig. 4. Fluorescence micrographs of A549 cells showing intracellular localization of 2 evaluated by confocal microscopy: cells were
marked with dyes for endoplasmic reticulum (ER-Tracker), lysosomes (Lyso-Tracker) and mitochondria (Mito-Tracker). For each
image, a profile of fluorescence intensity along the white arrow is shown. The green topographic profile corresponds to the emission
of the tracker and the red to the porphyrin emission
HO
HO
O11CH3
OH
NH
N
N
NH
N
N
11CH3I
TBAOH
dry DMF
rt, 1 min
HN
HN
HO
HO
OH
OH
6
Scheme 2. Synthesis of 5,10,15-tris(3-hydroxyphenyl)-20-(3-[11C]methoxyphenyl)porphyrin 6
of the porphyrin or of the organelle probe, as indicated
in the confocal microscopy images. The comparison
of the topographical profiles provides an indication of
the co-localization (or not) of the porphyrin 2 and the
specific organelle probe.
The data in Fig. 4 indicates a very high concentration
of 2 in the ER, a slightly lower extent in mitochondria, and
the lowest in lysosomes. Such desired localization can
be explained by the fact that more lipophilic molecules
goes more efficiently to ER and mitochondria than less
lipophilic ones.
These in vitro results show that the cellular uptake
of 2 is relatively fast, which is an important issue for its
potential applicationas PET imaging 11C radiotracer.
Carbon-11 radiolabeling. The precursor 1 was
dissolved in dry DMF and the previously optimized
ammount of base (TBAOH) to obtain the monomethylated
porphyrin was added. The mixture was introduced in 2 mL
stainless loop of an automated radiomethylation system
(Bioscan AutoLoop, eckert and Ziegler, Germany). The
11CH3I produced in the automated methylation system
is driven through the loop and allowed to react with the
precursor along 1 min, at room temperature, Scheme 2.
The reaction product was isolated using a semi-
preparative HPLC equiped with a C18 column and
CH3CN/0.1 M ammonium format (buffer solution) 99/1
v/v as a mobile phase with a flow of 4 mL/min. Detection
was made by UV (254 nm) and radioactivity. Both
detectors are monitored using the automated synthesizer
interface (Fig. 5).
The product fraction with activity signal (270 s) was
collected and the solvent was removed by solid phase
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N. P. F. GONÇALVES ET AL.
Fig. 5. Radio-chromatogram of the radiolabeled product of 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin 1. Ultraviolet absorbance
at 254 nm (green) and activity curve (pink). The retention times of DMF, precursor and 5,10,15-tris(3-hydroxyphenyl)-20-(3-[11C]
methoxyphenyl)porphyrin 6 are 190, 250 and 270 s, respectively
Fig. 6. Radio-HPLC chromatogram of isolated radiolabeled 5,10,15-tris(3-hydroxyphenyl)-20-(3-[11C]methoxyphenyl)porphyrin 6
(tR = 0.55 min). A: UV (420 nm) and B: g-detection (511KeV) recorded simultaneously
extraction (SPE). The radiolabeled product was injected
into an analytical HPLC system with the Zorbax Eclipse
XDB-C18 column using CH3CN/0.1 M ammonium
format (buffer solution) 99/1 v/v as a mobile phase with
a flow of 1 mL/min, controlled by UV detector (420 nm)
and radiation detector (Fig. 6).
From the analysis of Fig. 6 it is clearly observed
that only one product is formed (just one active peak)
(Fig. 6b). The desired product was identified by HPLC
retention time with comparison with a pure sample of a
non-radiolabeled product 2. The radiochemical purity and
Fig. 7. Whole body PET image of the biodistribution on a
specific radioactivity of 5,10,15-tris(3-hydroxyphenyl)-
normal mouse of the 11C-labeled porphyrin 6. Color scale
20-(3-[11C]methoxyphenyl)porphyrin were determined
saturates at 50% of the maximum intensity in the image. White
using the values obtained with the radioactivity and the
mass (UV) measurements from the chromatograms.
The results presented in Figs 5 and 6 show that we
developed a pioneering general synthetic methodology
for labeling 11C hydroxylated porphyrins in a very short
time, as required for this short-half live radionuclide.
arrow indicates the injection point in the tail vein
images were taken along one hour. Biodistribution was
quickly established and after 15 min no further changes
were observed. A summed image of the last 45 min is
presented in Fig. 7.
Uptake in the liver is clearly visible indicating this
as the main pathway for excretion of the compound.
Little uptake is visible on all the other organs. After the
PET imaging procedure, the mouse was sacrificed and
representative organs were harvested and counted on a
well counter. Values were normalized as percentage of
Exploratory in vivo PET imaging and biodistribution
In vivo biodistribution was performed on a normal
mouse. The animal was injected at the tail vein with
496 kBq of 5,10,15-tris(3-hydroxyphenyl)-20-(3-[11C]
methoxyphenyl)porphyrin and placed on
a high
resolution PET imaging system.Whole body PET planar
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J. Porphyrins Phthalocyanines 2015; 19: 8–10

SYNTHESIS AND BIOLOGICAL DISTRIBUTION STUDY OF A NEW CARBON-11 LABELED PORPHYRIN FOR PET IMAGING
9
Table 2. Key organ biodistribution in a normal mouse 1 h post
injection of compound 6
Fund in the framework of the Polish Innovation Economy
Operational Program (contract no. POIG.02.01.00-12-
023/08). J.M. Dabrowski thanks Ministry of Science
and Higher Education for the Iuventus Plus grant
nr IP2011009471 and NCN for grant nr 2013/11/D/
ST5/02995. We thank Luisa Cortes for the assistance
with the confocal microscopy.
Organ
Brain Heart Liver Lung Muscle
0.42 0.55 4.13 0.60 0.64
% injected dose
per gram
injected dose per gram of tissue and the data is collected
on Table 2.
Biodistribution profile confirms that the liver is the
main pathway for excretion and presents minimal uptake
on other organs including very low brain penetration
and low uptake on lung and muscle (heart and skeleton).
Motivated by this preliminary feasibility labeling, more
detailed investigations and validations of radiolabeled
porphyrin are therefore warranted to verify their potential
agent for PET imaging in a mouse with an induced tumor.
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