Multi-gram synthesis of a porphyrazine platform for cellular translocation, conjugation to Doxorubicin, and cellular uptake

Article abstract of DOI:10.1016/j.tetlet.2012.07.087

We report the synthesis of the near infrared (NIR) fluorescent porphyrazine (Pz) 285, with pendant hydroxyl groups, as a non-toxic platform for delivery of conjugated chemotherapeutic agents to tumor cells. Conjugation of Pz 285 to Doxorubicin via an acid

Full text of DOI:10.1016/j.tetlet.2012.07.087

Tetrahedron Letters 53 (2012) 5475–5478
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Multi-gram synthesis of a porphyrazine platform for cellular translocation,
conjugation to Doxorubicin, and cellular uptake
Evan R. Trivedi a, Carl M. Blumenfeld a, Todd Wielgos b, Sharon Pokropinski b, Prasad Dande b, Ton T. Hai b,
Anthony G. M. Barrett c, Brian M. Hoffman a,
⇑
a Departments of Chemistry and Molecular Biosciences, Northwestern University, 2145 Sheridan Rd, Evanston, IL 60208, USA
b Baxter Healthcare Corporation, One Baxter Parkway, Deerfield, IL 60015, USA
c Department of Chemistry, Imperial College of Science, Technology, and Medicine, London SW7 2AZ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis of the near infrared (NIR) fluorescent porphyrazine (Pz) 285, with pendant
hydroxyl groups, as a non-toxic platform for delivery of conjugated chemotherapeutic agents to tumor
cells. Conjugation of Pz 285 to Doxorubicin via an acid labile linker and initial biological studies are
reported.
Received 31 May 2012
Revised 13 July 2012
Accepted 19 July 2012
Available online 3 August 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Tetrapyrrole
Porphyrinoid
Chemotherapy
In recent years, cancer mortality rates have declined, in part be-
cause of improved early detection.1,2 One promising modality for
cancer detection is near infrared (NIR) optical imaging3,4 enabled
by use of a tumor specific contrast agent.5 Such an agent absorbs
and emits harmless NIR light that can penetrate soft tissues to con-
siderable depth, allowing non-invasive imaging of tumors, such as
those of the breast.6
Tetrapyrroles, including porphyrins and phthalocyanines, have
been studied as potential NIR contrast agents.7,8 They exhibit in-
tense absorption and emission at long wavelengths, and they have
been extensively studied as photosensitizers for photodynamic
therapy (PDT).9 Our work is focused on heteroatom substituted
porphyrazines (Pzs),10,11 a promising tetrapyrrole sub-class.
We have examined Pzs with peripheral heteroatom (S, O, N)
substituents for possible biomedical applications.12–15 These com-
pounds show promise as classic anti-tumor agents, PDT photosen-
sitizers, and NIR contrast agents. Most recently, we discovered a
chiral oxygen atom appended Pz that exhibits intense NIR emission
from cells in vitro,12 mediated by association with low-density
lipoprotein (LDL).16 Most importantly, this Pz exhibits highly pref-
erential tumor uptake in vivo, where its NIR fluorescence from
within the tumors can be visualized through the skin.
agents, which themselves are not taken up by either tumorigenic
or non-tumorigenic cells, were covalently attached to a Pz.17 Cellu-
lar uptake of the conjugates was observed when the MRI contrast
agent was conjugated to one side of the Pz, presumably allowing
the hydrophobic portion of the Pz to interact with serum proteins
while transporting the Gd(III) complex into the cell. Pandey and co-
workers have also recently shown that this strategy is viable for
producing tumor-selective tetrapyrrole/Gd(III) conjugates.18
The favorable properties of these initial Pz conjugates have led
us to examine the use of Pzs as translocation vectors for known
chemotherapeutics with the expectation that the drug would be
preferentially delivered to tumor tissue thereby lessening the dam-
age to healthy tissue. Herein we report the synthesis and cellular
uptake of a new Pz platform for both NIR-fluorescence optical
imaging on its own and for delivery of chemotherapeutics to tumor
cells. Pz 285 (Scheme 1) was designed to conjugate chemothera-
peutic agents on one side of the periphery so that they do not inter-
fere with the porphyrazine’s ability to interact with serum proteins
as the basis of cellular uptake with high tumor selectivity.19
The synthesis of 285 (Scheme 1) started with the preparation of
2-(2-chloroethoxy)tetrahydropyran (1) by a modified literature
procedure.20 Nucleophilic substitution of chloride 1 with commer-
cially available 1,4-dicyanohydroquinone gave dinitrile (2), which
was imidated to yield the iso-indoline (3). Compounds 2 and 3
were isolated as mixtures of diastereoisomers as confirmed by
their NMR spectra. Co-cyclization of iso-indoline 3 with (5R,6R)-
2,3-dicyano-5,6-dimethoxy-5,6-dimethyl-1,4-diox-2-ene21 gave a
mixture of Mg-Pzs with tetrahydropyran (THP) protected alcohol
As a first step toward use of the observed tumor selectivity in
generating a multimodal agent, paramagnetic Gd(III) MRI contrast
⇑
Corresponding author. Tel.: +1 847 491 3104; fax: +1 847 491 7713.
E-mail address: bmh@northwestern.edu (B.M. Hoffman).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.07.087

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E. R. Trivedi et al. / Tetrahedron Letters 53 (2012) 5475–5478
Scheme 1. Synthesis of 285 (i) THP, TsOH.H2O, neat, 0 °C, 2 h; (ii) (1), K2CO3, KI, DMF, 60 °C, 48 h; (iii) Na0, NH3, ethylene glycol, 140 °C, 6 h; (iv) Mg(O-n-Pr)2, (5R,6R)-2,3-
dicyano-5,6-dimethoxy-5,6-dimethyl-1,4-diox-2-ene, 120 °C, 18 h; (v) AcOH, THF, H2O (4:2:1), 80 °C, 18 h.
Figure 1. Electronic absorption (black) and emission (blue/red) of pz 285 in MeOH. Spectra were normalized to matching scales. Confocal fluorescence microscopic images of
(b) A549 human lung carcinoma cells and (c) WI38 VA13 human fibroblast cells. Fluorescence from pz 285 (red) is overlaid onto transmitted light images; 63Â.
groups. Attempts at macrocyclization without the THP protection
led to decomposition and very low yields. The Mg ion and THP
groups were removed from the Pz by reaction with acetic acid to
produce the enantiomerically pure, chiral 285 in 45% yield (2 steps)
after isolation by chromatography. Reaction with such yields was
found to be reproducible on a multi-gram scale (5–10 g), thus en-
abling extensive biological studies. It is important to note that the
chirality of 285 is merely a synthetic convenience and there is no
expectation or evidence that the chirality would have any effect
on biological properties.
rather hydrophobic, it can be administered in biological fluids
through use of DMSO (<1%) as solubilizing agent. Cellular uptake
of 285 was examined in vitro by fluorescence microscopy using
A549 human lung carcinoma cells (Fig. 1b) and WI38 VA13 human
fibroblast cells as a non-tumorigenic reference (Fig. 1c). Intracellu-
lar fluorescence from 285 is observed in both cell lines relative to
vehicle controls, but cellular uptake is much greater for the hyper-
proliferative tumor cells. Cellular uptake is also observed in the
MDA-MB-231 breast tumor cell line (See ESI; Supplementary Fig.
S1). Through a series of confocal microscopy experiments, not dis-
cussed in detail here, it was determined that 285 enters cells ac-
tively by endocytosis and is carried into cells by interactions
with the protein serum albumin (See ESI; Supplementary Figs.
S2–S6). Albumin binding has been shown with other porphyrinoid
systems22,23 and, in some cases enhances PDT efficacy in vivo.24
Intracellular endosomes are known to be slightly acidic,25 prompt-
ing the design of Pz – Doxorubicin conjugates connected through
Porphyrazine 285 exhibits typical Pz electronic absorption (kmax
=
594 nm, 662 nm) and S1 emission (kem = 740 nm, MeOH) (Fig. 1a).12
The emission falls within the wavelength range of maximum tissue
penetration for NIR optical imaging. Pzs with the core structure of
285 have negligible quantum yields of singlet oxygen generation
(U )12 so phototoxicity is not expected to interfere with optical
D
imaging or cellular translocation applications. Although 285 is
Scheme 2. Structures of Pz-Doxorubicin conjugates 285-Dox-1 and 285-Dox-2 linked with acid-labile oximes.

E. R. Trivedi et al. / Tetrahedron Letters 53 (2012) 5475–5478
5477
moeities and when allowed to react with 286 in excess, under basic
conditions, the corresponding Pzs 9 and 10 were obtained (See
ESI, Supplementary Scheme S3). Subsequent reaction of 9 and 10
with acid in the presence of Doxorubicin hydrochloride gave Pzs
285-Dox-1 and 285-Dox-2 in 70% and 29% yields, respectively
(Scheme 2).
Electronic absorption of Pz-Dox conjugates is as expected with a
combination of Pz and anthracycline absorption bands (Fig. 2a).
With the oxime linker intact, all fluorescence bands are almost
completely quenched, with the remaining green fluorescence from
Doxorubicin (k = 593 nm, H2O) being the strongest (See ESI; Sup-
plementary Fig. S7). This intramolecular quenching presents an
excellent qualitative method to track linker cleavage by monitor-
ing fluorescence intensity of the Doxorubicin fluorescence band.
For example, equimolar solutions of 285-Dox-1 (12.5 lM) were
prepared in buffer (KPi/Ac) of varying pH and incubated at 37 °C
for seven days. Fluorescence intensity of these solutions increased
with acidity (Fig. 2b) indicating, as expected, more extensive cleav-
age of the oxime linker at lower pH. Comparison of integrated fluo-
rescence intensity (Fig. 2b vs Supplementary Fig S7) indicated that
less than 5% oxime cleavage occurred at biologically relevant pH
(pH > 4.9). A more quantitative method to monitor oxime cleavage
was developed using capillary electrophoresis mass spectrometry
(CE-MS) and it was determined that >99% of 285-Dox-2 was intact
after 14 days at pH 4.0 (See ESI; Supplementary Fig. S8). This de-
gree of cleavage would not be expected to produce a beneficial
cytotoxic effect at biologically relevant pH.
Despite poor cleavage of the oxime linker in solution, cellular
uptake of Pz-Dox conjugates was examined. MDA-MB-231 breast
tumor cells were treated with both Pz-Dox conjugates and Doxoru-
bicin HCl alone as a positive control and imaged by confocal fluo-
rescence microscopy (Fig. 3).
Doxorubicin HCl treated cells display fluorescence from punc-
tate vesicles throughout the cell and in the nucleus, whereas cells
treated with 285-Dox-1 and 285-Dox-2 only display fluorescence
from intracellular vesicles. From this we can conclude that the
Pz-Dox conjugates successfully enter the cells but the linkers are
not cleaved enough to release Doxorubicin for its effective nuclear
localization.
The effect of Pz-Dox conjugates on cell viability was examined
using the MTS assay (Fig. 4). MDA-MB-231 cells were treated for
5 days with a range of concentrations of Doxorubicin HCl, 285-
Dox-1, and 285-Dox-2 with the maximum dosage corresponding
to the highest concentration of Pz-Dox conjugate that is stable in
aqueous solution over time. Comparison of cell viability versus
vehicle controls shows that, at these dosages, 285-Dox-1 has no ef-
fect on cell viability and 285-Dox-2 is ꢀ100-fold less toxic than
Figure 2. (a) Electronic absorption of Doxorubicin HCl (blue), 285 (black), and 285-
Dox-1 (red) in MeOH. (b) Anthracycline fluorescence of 285-Dox-1 increases with
decreasing pH indicating oxime cleavage.
an acid-labile oxime linker with the expectation that 285 would
carry Doxorubicin into the cell subsequently releasing the drug
once exposed to lower pH intracellular compartments.
The free hydroxyl groups of 285 were activated for nucleophilic
substitution by arene sulfonylation with 4-toluenesulfonyl and 4-
fluorobenzenesulfonyl chlorides to provide the corresponding Pzs
286 and 287 in near quantitative yields (See ESI; Supplementary
Scheme S1). Compounds 7 and 8 (See ESI; Supplementary Scheme
S2) were synthesized as linkers with terminal thiol and oxyamine
Figure 3. Confocal fluorescence microscopic images of MDA-MB-231 cells treated with (a) 10
vehicle; 488 nm ex., 560–610 nm em., 100Â magnification.
lM Doxorubicin HCl, (b) 5 lM 285-Dox-1, (c) 5 lM 285-Dox-2, and (d) DMSO

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E. R. Trivedi et al. / Tetrahedron Letters 53 (2012) 5475–5478
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.
07.087.
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Conclusions
In summary, the unsymmetrical Pz 285 was designed and syn-
thesized to exhibit NIR fluorescence while carrying two pendant
hydroxyl groups for conjugation to bioactive agents. A particular
advantage of 285 for this role is its vanishingly small quantum
yield for singlet oxygen formation, which should eliminate photo-
toxic side-effects. Cell studies show that Pz 285 undergoes active
endocytosis by hyperproliferative tumor cells to a greater extent
than by slower growing non-tumorigenic cells, with strong intra-
cellular NIR fluorescence upon uptake. Pz 285 was successfully
conjugated to Doxorubicin HCl through an acid-labile oxime linker.
Although cleavage of this linker is insufficiently rapid at biologi-
cally relevant pH for 285-Dox-1 and 285-Dox-2 to exhibit the de-
sired cytotoxicity, this report lays the foundation for future
experiments in which 285 is conjugated to Gd(III) MRI contrast
agents, or to Doxorubicin through more favorable linkers.
Acknowledgments
This work was supported by the Baxter Healthcare Corporation
through the Baxter-Northwestern Alliance. Imaging work was
performed at the Northwestern University Biological Imaging
Facility generously supported by the NU Office for Research.
25. Mellman, I. Annu. Rev. Cell Dev. Biol. 1996, 12, 575.