Methyl pyropheophorbide-a analogues: Potential fluorescent probes for the peripheral-type benzodiazepine receptor. Effect of central metal in photosensitizing efficacy

Article abstract of DOI:10.1021/jm050039k

Pyropheophorbides and their metal complexes were synthesized to investigate their applications as nonradioactive peripheral benzodiazepine receptor (PBR) binding probes and photosensitizers for use in photodynamic therapy. They were found to be localized in mitochondria and showed significant binding to PBR. In some cases, the PBR binding values were similar to that for 17 (PK11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)isoquinoline-3-carboxamide). However, no direct correlation between 17 displacement ability and photosensitizing efficacy of photosensitizers was observed.

Full text of DOI:10.1021/jm050039k

3692
J. Med. Chem. 2005, 48, 3692-3695
ligand) for the active derivatives of the alkyl ether
derivatives of pyropheophorbide-a, but no effect was
found for the less active analogues. Kessel et al.5 studied
three very similar analogues (protoporphyrins III, IX,
and XIII) to understand the role of the peripheral
benzodiazepine receptor in the apoptotic response to
PDT. Only protoporphyrin IX had significant affinity
for the PBR. These data suggest that the relationship
between PDT efficacy and PBR affinity may hold only
for sensitizers with PpIX-like configurations. A recent
report from Oleinick6 on the importance of PBR receptor
in phthalocyanine photosensitizer Pc-4 concluded that
the binding of Pc-4 to PBR is less relevant to the
phototoxicity of Pc-4-PDT than other mitochondrial
events, such as photodamage to Bcl-2, and that the
observed inhibition of Pc-4-PDT induced apoptosis by
17 likely occurs through a mechanism independent of
PBR.7 Therefore, the role of PBR in PDT is still a subject
of discussion.
It has been shown that mitochondria are important
intracellular targets for PDT and play a critical role in
energy production and involvement in apoptosis.8,9 For
porphyrins and porphyrin-based structures, accumula-
tion of photosensitizers can occur in the membranes of
organelles such as the mitochondria or lysosomes. In
our studies with a series of photosensitizers related to
chlorins and bacteriochlorins, we observed that most of
the effective photosensitizers are localized in mitochon-
dria.10 Interestingly, in a series of alkyl ether analogues
of pyropheophorbide-a analogues, the overall lipophi-
licity was found to play an important role in the site of
intracellular localization. For example, the lower alkyl
ether analogues (C1-C8) that are localized in mito-
chondria were found to be more effective in vivo than
higher alkyl analogues (C9-C12) with a preferrential
localization in lysosomes. Among the alkyl ether ana-
logues of pyropheophorbide-a, the corresponding hexyl
ether derivative (HPPH), which produced the best in
vivo photosensitizing activity, also showed affinity
toward the PBR. However, the PBR binding data
obtained from HPPH was significantly lower than the
data observed for 17.
The present work describes the synthesis of a series
of pyropheophorbides (free-base and the corresponding
metalated analogues), their PBR binding affinity, in-
tracellular localization pattern, and photosensitizing
efficacy. The main objectives of this study were (i) to
develop nonradioactive, efficient fluorescent probes for
PBR binding and (ii) to investigate any correlation
between the PBR binding affinity of these compounds
and photosensitizing efficacy.
The compounds synthesized for our study are shown
in Schemes 1 and 2. For our initial study, to determine
the effect of the central metal, pyropheophorbide-a 1
was converted into the corresponding Zn(II), In(III), and
Ni(II) complexes. Among these analogues the In(III)
complex showed the best PDT efficacy and significant
PBR binding affinity. The Ni(II) complexes, because of
its inability to produce singlet oxygen, did not show any
PDT efficacy.11 However, a strong PBR binding affinity
with this compound was observed. Because our objective
Methyl Pyropheophorbide-a Analogues:
Potential Fluorescent Probes for the
Peripheral-Type Benzodiazepine
Receptor. Effect of Central Metal in
Photosensitizing Efficacy
Yihui Chen,† Xiang Zheng,† Mahabeer P. Dobhal,†,‡
Amy Gryshuk,†,§ Janet Morgan,§
Thomas J. Dougherty,† Allan Oseroff,§ and
Ravindra K. Pandey*,†,‡
PDT Center, Department of Dermatology, and
Department of Nuclear Medicine and Radiology,
Roswell Park Cancer Institute, Buffalo, New York 14263
Received January 14, 2005
Abstract: Pyropheophorbides and their metal complexes were
synthesized to investigate their applications as nonradioactive
peripheral benzodiazepine receptor (PBR) binding probes and
photosensitizers for use in photodynamic therapy. They were
found to be localized in mitochondria and showed significant
binding to PBR. In some cases, the PBR binding values were
similar to that for 17 (PK11195, 1-(2-chlorophenyl)-N-methyl-
N-(1-methylpropyl)isoquinoline-3-carboxamide). However, no
direct correlation between 17 displacement ability and pho-
tosensitizing efficacy of photosensitizers was observed.
Peripheral benzodiazepine receptor (PBR) is an 18
kDa protein of the outer mitochondrial membrane.1 It
is involved in numerous functions including steroid
biosynthesis, mitochondrial respiration, heme biosyn-
thesis, cell proliferation, and calcium channel modula-
tion.1 In addition, PBR plays an important role in the
regulation of stress response, anxiety disorders, mood
disorders, and neurodegenerative disorders (Parkinson’s
disease and Alzheimer’s disease). Elevated PBR expres-
sion is also related to certain cancers, such as those of
breast, colon-rectum, and prostate tissue, and has been
proposed as a novel prognostic indicator of an aggressive
phenotype for these indications.2 Therefore, the PBR
provides an attractive target molecule for the develop-
ment of compounds that may be used for the regulation
of the above-mentioned PBR-dependent functions.
Another intriguing aspect of the PBR is its association
with photodynamic therapy (PDT). PDT is a relatively
new modality for cancer treatment; it refers to the
photosensitization of tumor-avid photosensitizers by
light, resulting in singlet-oxygen-mediated cytotoxicity
to tumor cells. Most photosensitizers for PDT are
porphyrin-based compounds. Verma et al.3 were the first
to study the binding affinity of various porphyrins to
the PBR with prominent physiological functions. A
previous report from our laboratory has shown the
importance of the PBR as a binding site for a series of
chlorin-type photosensitizers such as pyropheophor-
bide-a ethers.4 It was found that the PDT effect was
inhibited by 17 (PK11195, 1-(2-chlorophenyl)-N-methyl-
N-(1-methylpropyl)isoquinoline-3-carboxamide) (PBR
* To whom correspondence should be addressed. Phone: 716-845-
3203. Fax: 716-845-8920. E-mail: ravindra.pandey@roswellpark.org.
† PDT Center.
‡ Department of Nuclear Medicine and Radiology.
§ Department of Dermatology.
10.1021/jm050039k CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/10/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3693
Scheme 1. Structures of 1-10
Figure 1. Comparative intracellular localization of 7 and 8
with MitoTracker Green: (A) 7; (B) MitoTracker Green; (C)
overlap of 7 with MitoTracker Green; (D) 8; (E) MitoTracker
Green; (F) overlap of 8 with MitoTracker Green.
Scheme 2. Structures of 11-16a
4 > 2 > 1 > 3, i.e., nickel complex > indium complex >
free base > zinc complex. Because of the limited singlet-
oxygen-producing ability of the Ni(II) complex 4, we
focused our attention on In(III) analogues, and py-
ropheophorbide-a derivatives 7-10 and 14 with variable
lipophilicity were used for our studies.
For investigation of the effect of lipophilicity in PBR
binding, compounds with hydrophilic substituents con-
taining carboxylic groups 5 and 6 or mono-/di-PEG
substituents 9 and 10 were synthesized. Despite a
similar lipophilicity between 5 and 6, 6 showed higher
PBR binding affinity than 5. These results suggest that
lipophilicity alone may not be an indicator of PBR
binding affinity.
In recent years, several indole analogues have also
been investigated for PBR binding. Among these ana-
logues, an indole-NBD conjugate 16 has been reported
as an excellent PBR binding agent. The use of the NBD
moiety in the conjugate was based on its fluorescence
properties, but its presence also enhanced the PBR
binding affinity. However, in contrast to 16, the pres-
ence of the NBD moiety in 13 and 14 diminished the
PBR binding affinity (Figure 2C). The IC50 values for
these pyropheophorbide-a analogues are summarized in
Table 1 and show the following order of PBR binding
affinities: 4 > 2 > 1 > 6 > 5 ∼ 3 > 10 > 9 > 14 > 8 >
7 > 13. Because of their strong fluorescence in the range
650-720 nm, these compounds could be promising
candidates as fluorescent probes and may have a
significant advantage (cost effective, easy to handle)
over the radioactive probes (e.g., tritiated 17) currently
being used.
a Prepared by reacting 15 with NBD-chloride 11, following the
literature procedure.1
was to investigate the PBR binding affinity with pho-
tosensitizing ability, this study was extended to a series
of In(III) analogues with variable lipophilicity in which
the vinyl group at position 3 was replaced with hexyl
ether 8 and mono- and di-PEG substituents 9 and 10.
Kozikowski1 showed that incorporation of NBD chloride
11 to PBR binding indole 15 enhances the PBR binding
affinity. Therefore, to investigate the effect of such
introduction in pyropheophorbide-a, a corresponding
NBD conjugate 13 and the related In(III) complex 14
were synthesized (Scheme 2). For a comparative study,
16 (Kozikowski’s compound) was prepared by reacting
15 with NBD chloride 11, following the literature
procedure.1
Porphyrin-based compounds are reported to be ligands
for the mitochondrial PBR. The first report on correla-
tion between the photodynamic activities of several
porphyrin photosensitizers and their binding affinities
for the PBR was from Verma et al.3 Therefore, before
evaluation of the methylpheophorbide-a analogues for
PBR binding, their intracellular localization character-
istic was determined. Interestingly, they all were found
to be localized in mitochondria. A representative local-
ization pattern of the most effective photosensitizers 7
and the corresponding In(III) complex 8 is shown in
Figure 1.
The in vitro PDT efficacy of photosensitizers was
investigated in RIF tumor cells by following the MTT
assay.12 After careful comparison, the in vitro PDT
efficacy (LD50 light dose either at 0.06 or 1 micromolar
concentration) was in the order of 2 ) 9 > 8 > 7 > 10
> 14 > 6 ∼ 1 > 3 > 5 > 13 (Table 2).
Compared to the free-base analogues, the correspond-
ing indium complexes generally produced higher PBR
binding affinities and also were found to have more
effective in vitro photosensitizing ability. For example,
2 at 0.06 µM and a light dose of 0.625 J/cm2 gave 100%
cell kill, whereas 1 at the same concentration required
a light dose of 2 J/cm2 and only 60% tumor cell-kill was
observed (Figure 3A). At 1 µM, although the zinc
complex 3 showed some PDT efficacy, it was less
effective than the corresponding free-base analogue 1.
Among the methyl pyropheophorbide-a 1 and the
related metal complexes 2-4, the In(III) and Ni(II)
analogues 2 and 4, respectively, produced a PBR binding
affinity similar to that of 17. Among the compounds
investigated, the binding affinities was in the order of

3694 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Letters
Table 2. Light Dose (J/cm2) for LD50 (See Figure 3)
compd:
1
2
3
4
5
6
light dose at 0.06 µM
light dose at 1.00 µM
a
b
0.02
a
0.70
a
a
a
0.87
a
0.10
compd:
7
8
9
10
13
14
light dose at 0.06 µM 0.43 0.05 0.02 0.96
light dose 1.00 µM
a
a
1.64
0.01
a Greater than 2.00 J/cm2 light dose. b Dark toxicity.
Figure 2. Displacement of titrated 17 (PBR probe) with
pyropheophorbide-a analogues.
Table 1. IC50 Displacement of Titrated 17 from the PBR (See
Figure 2)
compd:
17
1
2
3
4
IC50 (×10-7 M):
3.9
18.2
11.2
28.9
6.6
compd:
5
6
7
8
9
IC50 (×10-7 M):
28.8
20.4
169.8
147.9
104.7
Figure 3. In vivo PDT efficacy studies of pyropheophorbide-a
analogues (RIF tumor).
compd:
IC50 (×10-7 M):
10
89.1
13
371.5
14
16
107.1
3.2
possesses an ability to replace the radioactive probes
currently being used. Interestingly, as can be seen in
Figures 2 and 3, compared to free-base analogues, the
related indium complexes were found to have higher
PDT efficacy with enhanced PBR binding ability (8 >
7, 14 > 13). Although the overall lipophilicity of 5 and
6 was similar, a significant difference in their in vitro
photosensitizing efficacy was observed (Figure 3C), with
6 being more effective than 5, which correlates with the
PBR binding values (Figure 2B). On the other hand, 1
has higher PBR binding affinity than 7 but was less
In summary, a correlation between the in vitro PDT
efficacy and the PBR binding affinity of these com-
pounds was observed and it was in the order of 2 > 1 >
3 (Figure 2A and 3A).
The Ni(II) complex 4, which exhibited limited 1O2
efficiency,11 showed insignificant photosensitizing ability
in RIF tumor cells (MTT assay) (see Figure 3A). Since
the PBR binding affinity of the Ni(II) complex 4 is
similar to that of 17, this compound has the potential
to be an effective fluorescent PBR binding probe and

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3695
Acknowledgment. Financial support from NIH
Grant CA 55792 and the shared resources of the RPCI
Support Grant P30CA16056 is highly appreciated. We
also thank the National Science Foundation under the
Integrative Graduate Education and Research Train-
eeship (Grant DGE0114330) for providing a graduate
fellowship to A.G. The help rendered by Adam Sumlin
in in vivo experiments is highly appreciated.
Supporting Information Available: Synthesis, NMR
spectra, and HRMS analyses of new compounds, detailed PBR
binding, and in vitro PDT efficacy data. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
Figure 4. In vivo photosensitizing efficacy of 7 and 8 in C3H
mice bearing RIF tumors (10 mice/group). The mice were
treated with laser light (135 J/cm2, 75 mW/cm2, 665 nm for 7
and 655 nm for 8) at 24 h postinjection of the drug. The control
has no photosensitizer and no light exposure.
(1) Kozikowski, A. P.; Kotoula, M.; Ma, D.; Boujrad, N.; Tuckmantel,
W.; Papadopoulos, V. Synthesis and Biology of a 7-Nitro-2,1,3-
benzoxadiazol-4-yl Derivative of 2-Phenylindole-3-acetamide: A
Fluorescent Probe for the Peripheral-Type Benzodiazepine
Receptor. J. Med. Chem, 1997, 40 (16), 2435-2439 and refer-
ences therein.
(2) Han, Z.; Slack, R. S.; Li, W. P.; Papadopoulos, V. Expression of
Peripheral Benzodiazepine Receptor (PBR) in Human Tumors:
Relationship to Breast, Colorectal, and Prostate Tumor Progres-
sion. J. Recept. Signal Transduction Res. 2003, 23, 225-238.
(3) Verma, A.; Facchina, S. L.; Hirsch, D. J.; Song, S. Y.; Dillahey,
L. F.; Williams, J. R.; Snyder, S. H. Photodynamic Tumor
Therapy: Mitochondrial Benzodiazepine Receptors as a Thera-
peutic Target. Mol. Med. 1998, 4, 40-50.
(4) Dougherty, T. J.; Sumlin, A. B.; Greco, W. R.; Weishaupt, K. R.;
Vaughan, L. A.; Pandey, R. K. The Role of the Peripheral
Benzodiazepine Receptor in Photodynamic Activity of Certain
Pyropheophorbide Ether Photosensitizers: Albumin Site II as
a Surrogate Marker for Activity. Photochem. Photobiol. 2002,
76 (1), 91-97.
(5) Kessel, D.; Antolovich, M.; Smith, K. M. The Role of the
Peripheral Benzodiazepine Receptor in the Apoptotic Response
to Photodynamic Therapy. Photochem. Photobiol. 2001, 74 (2),
346-349.
(6) Morris, R. L.; Varnes, M. E.; Kenney, M. E.; Li, Y. S.; Azizuddin,
K.; McEnery, M. W.; Oleinick N. L. The Peripheral Benzodiaz-
epine Receptor in Photodynamic Therapy with the Phthalocya-
nine Photosensitizer Pc 4. Photochem. Photobiol. 2002, 75 (6),
652-661.
(7) Oleinick, N. L.; Morris, R. L.; Belichenko, T. The Role of
Apoptosis in Response to Photodynamic Therapy: What, Where,
Why, and How. Photochem. Photobiol. Sci. 2002, 1 (1), 1-21.
(8) Kessel, D.; Luo, Y. Intracellular Sites of Photodamage as a Factor
in aPoptotic Cell Death. J. Porphyrins Phthalocyanines 2001, 5
(2), 181-184.
(9) Zhang, Z.; Blesington, D.; Hui, L.; Busch, T. M.; Glickson, J.;
Luo, Q.; Chance, B.; Zheng, G. Redox Ratio of Mitochondria as
an Indicator for the Response of Photodynamic Therapy. J.
Biomed. Opt. 2004, 9 (4), 772-778.
(10) Chen, Y.; Graham, A.; Potter, W.; Morgan, J.; Vaughan, L.;
Bellnier, D. A.; Henderson, B. W.; Oseroff, A.; Dougherty, T. J.;
Pandey, R. K. Bacteriopurpurinimides: Highly Stable and
Potent Photosensitizers for Photodynamic Therapy. J. Med.
Chem. 2002, 45 (2), 255-258.
(11) Mosinger, J.; Micka, Z. Quantum Yields of Singlet Oxygen of
Metal Complexes of meso-Tetrakis(sulphonatophenyl)porphine.
J. Photochem. Photobiol. 1997, 107, 77-82.
efficacious. In summary, in the pyropheophorbide-a
series, no direct correlation between PBR binding af-
finity and in vitro PDT efficacy was observed.
On the basis of in vitro screening, the indium com-
plexes in general were found to be more effective than
the corresponding free-base counterparts. Among the
free-base and metalated analogues investigated so far,
7 and the related In(III) analogue 8 produced the best
photosensitizing activity. Therefore, these compounds
were tested for in vivo PDT efficacy in C3H mice bearing
RIF tumors at variable drug doses. The results sum-
marized in Figure 4 show that at 0.2 µmol/kg, compared
to 7, the corresponding In(III) complex 8 was more
effective with 8/10 mice tumor-free on day 90. The
therapeutic response of 7 at 0.4 µmol/kg was similar to
that of the In(III) analogue at 0.05 µmol/kg, which was
8-fold more than the parent compound 7.
In summary, our results with a series of pyropheophor-
bide-a analogues suggest that the presence of a core
metal makes a significant difference in PBR binding and
photosensitizing efficacy. Because of their strong fluo-
rescence, these compounds have great potential as
fluorophores to replace radioactive PBR probes (e.g.,
tritiated 17). The presence of metals also had a signifi-
cant impact on photosensitizing efficacy, with the In-
(III) complexes proving to be the most efficacious, which
could be a result of their higher singlet oxygen produc-
tion. The detailed photophysical characterization of
these compounds is currently underway. Replacing cold
indium with radioactive indium (In-III, half-life 72 h)
may also provide an opportunity to develop bifunctional
agents for tumor-imaging (nuclear imaging) and pho-
todynamic therapy. Recently, the In(III) complex of
pyropheophorbide was reported as a potential candidate
for the treatment of age-related macular degeneration
(AMD),13,14 a leading cause of blindness that is most
prevalent in people over the age of 60. Therefore, the
compounds discussed herein with variable lipophilicity
could be ideal candidates for investigating structure-
activity relationships within a particular series of
compounds for such indication, and these studies are
currently in progress.
(12) Chen, Y.; Sumlin, A.; Morgan, J.; Dougherty, T. J.; Gryshuk,
A.; Oseroff, A.; Henderson, B. W.; Dougherty, T. J.; Pandey, R.
K. Synthesis and Photosensitizing Efficacy of Isomerically Pure
Bacteriopurpurinimide. J. Med. Chem. 2004, 47 (20), 4814-
4817.
(13) Dolmans, D. E. J. G. J.; Kadambi, A.; Hill, J. S.; Waters, C. A.;
Robinson, B. C.; Walker, J. P.; Fukumura, D.; Jain, R. K.
Vascular Accumulation of a Novel Photosensitizer, MV6401,
Causes Selective Thrombosis in Tumor Vessels after Photody-
namic Therapy. Cancer Res. 2002, 62, 2151-2156.
(14) Ciulla, T. A.; Criswell, M. H.; Snyder, W. J. Small, W., IV.
Photodynamic Therapy with PhotoPoint Photosensitizer MV6401,
Indium Chloride Methyl Pyropheophorbide, Achieves Selective
Closure of Rat Corneal Neovacularisation and Rabbit Chorio-
capillaris. Br. J. Ophthalmol. 2005, 89, 113-119.
JM050039K