Regioselective synthesis and photophysical and electrochemical studies of 20-substituted cyanine dye-purpurinimide conjugates: Incorporation of Ni II into the conjugate enhances its tumor-uptake and fluorescence-imaging ability

Article abstract of DOI:10.1002/chem.201203867

We report herein a simple and efficient approach to the synthesis of a variety of meso-substituted purpurinimides. The reaction of meso-substituted purpurinimide with N-bromosuccinimide regioselectively introduced a bromo functionality at the 20-position, which on further reaction with a variety of boronic acids under Suzuki reaction conditions yielded the corresponding meso-substituted analogues. Interestingly, the free base and the metalated analogues showed remarkable differences in photosensitizing efficacy (PDT) and tumor-imaging ability. For example, the free-base conjugate showed significant in vitro PDT efficacy, but limited tumor avidity in mice bearing tumors, whereas the corresponding Ni^II derivative did not produce any cell kill, but showed excellent tumor-imaging ability at a dose of 0.3 μmol kg^-1 at 24, 48, and 72 h post-injection. The limited PDT efficacy of the Ni ^II analogue could be due to its inability to produce singlet oxygen, a key cytotoxic agent required for cell kill in PDT. Based on electrochemical and spectroelectrochemical data in DMSO, the first one-electron oxidation (0.52 V vs. SCE) and the first one-electron reduction (-0.57-0.67 V vs. SCE) of both the free base and the corresponding Ni^II conjugates are centered on the cyanine dye, whereas the second one-electron reduction (-0.81 V vs. SCE) of the two conjugates is assigned to the purpurinimide part of the molecule. Reduction of the cyanine dye unit is facile and occurs prior to reduction of the purpurinimide group, which suggests that the cyanine dye unit as an oxidant could be the driving force for quenching of the excited triplet state of the molecules. An interaction between the cyanine dye and the purpurinimide group is clearly observed in the free-base conjugate, which compares with a negligible interaction between the two functional groups in the Ni^II conjugate. As a result, the larger HOMO-LUMO gap of the free-base conjugate and the corresponding smaller quenching constant is a reason to decrease the intramolecular quenching process and increase the production of singlet oxygen to some degree. Copyright

Full text of DOI:10.1002/chem.201203867

DOI: 10.1002/chem.201203867
Regioselective Synthesis and Photophysical and Electrochemical Studies of
20-Substituted Cyanine Dye–Purpurinimide Conjugates:
Incorporation of NiII into the Conjugate Enhances its Tumor-Uptake and
Fluorescence-Imaging Ability
Manivannan Ethirajan,[a] Ping Chen,[b] Tymish Y. Ohulchanskyy,[c] Lalit N. Goswami,[a]
Anurag Gupta,[a] Avinash Srivatsan,[a] Mahabeer P. Dobhal,[a] Joseph R. Missert,[a]
Paras N. Prasad,[c] Karl M. Kadish,*[b] and Ravindra K. Pandey*[a]
Abstract: We report herein a simple
and efficient approach to the synthesis
of a variety of meso-substituted purpur-
inimides. The reaction of meso-substi-
tuted purpurinimide with N-bromosuc-
cinimide regioselectively introduced a
bromo functionality at the 20-position,
which on further reaction with a varie-
ty of boronic acids under Suzuki reac-
tion conditions yielded the correspond-
ing meso-substituted analogues. Inter-
estingly, the free base and the metalat-
ed analogues showed remarkable dif-
ferences in photosensitizing efficacy
(PDT) and tumor-imaging ability. For
example, the free-base conjugate
showed significant in vitro PDT effica-
cy, but limited tumor avidity in mice
bearing tumors, whereas the corre-
sponding NiII derivative did not pro-
duce any cell kill, but showed excellent
0.3 mmolkgÀ1 at 24, 48, and 72 h post-
injection. The limited PDT efficacy of
the NiII analogue could be due to its in-
ability to produce singlet oxygen, a key
cytotoxic agent required for cell kill in
PDT. Based on electrochemical and
spectroelectrochemical data in DMSO,
the first one-electron oxidation (0.52 V
vs. SCE) and the first one-electron re-
duction (À0.57–0.67 V vs. SCE) of both
the free base and the corresponding
NiII conjugates are centered on the cya-
nine dye, whereas the second one-elec-
tron reduction (À0.81 V vs. SCE) of
the two conjugates is assigned to the
purpurinimide part of the molecule.
Reduction of the cyanine dye unit is
facile and occurs prior to reduction of
the purpurinimide group, which sug-
gests that the cyanine dye unit as an
oxidant could be the driving force for
quenching of the excited triplet state of
the molecules. An interaction between
the cyanine dye and the purpurinimide
group is clearly observed in the free-
base conjugate, which compares with a
negligible interaction between the two
functional groups in the NiII conjugate.
As a result, the larger HOMO–LUMO
gap of the free-base conjugate and the
corresponding smaller quenching con-
stant is a reason to decrease the intra-
molecular quenching process and in-
crease the production of singlet oxygen
to some degree.
À
Keywords: C C coupling · fluores-
cence
·
photodynamic therapy
·
photosensitizers · structure–activity
relationships · tumor imaging
tumor-imaging ability at
a dose of
Introduction
Nontoxic porphyrin-based compounds in combination with
harmless visible light, known as photodynamic therapy
(PDT), have shown great potential in several biological ap-
plications, including cancer therapy and noncancerous treat-
ments such as age-related macular degeneration (AMD)
and cardiovascular and infectious diseases.[1–6] Since the ap-
proval of Photofrinꢀ, a hematoporphyrin derivative (devel-
oped at the PDT Center, Roswell Park Cancer Institute,
Buffalo, NY), our laboratories have been investigating the
utility of naturally occurring chlorophyll and bacteriochloro-
phyll analogues for phototherapy and tumor imaging.[7]
Among the chlorophyll-based analogues, HPPH [3-(1’-hexy-
loxyethyl)-3-devinylpyropheophorbide-a], which has a long-
wavelength absorption at 665 nm (in vivo), has shown excel-
lent photosensitizing efficacy in mice with limited skin pho-
totoxicity, this being the main drawback with most other
[a] Dr. M. Ethirajan, Dr. L. N. Goswami, Dr. A. Gupta, Dr. A. Srivatsan,
Dr. M. P. Dobhal, J. R. Missert, Prof. R. K. Pandey
Chemistry Division, PDT Center, Cell Stress Biology
Roswell Park Cancer Institute, Buffalo, NY, 14263 (USA)
E-mail: ravindra.pandey@roswellpark.org
[b] Dr. P. Chen, Prof. K. M. Kadish
Department of Chemistry, University of Houston
Houston, TX, 77204-5003 (USA)
E-mail: kkdish@uh.edu
[c] Dr. T. Y. Ohulchanskyy, Prof. P. N. Prasad
Institute of Photonics and Biophotonics
University of Buffalo (SUNY)
Buffalo, NY 14221 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201203867.
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FULL PAPER
porphyrin-based compounds.[8,9] HPPH is currently in Pha-
se II human clinical trials for lung, esophagus, Barrettꢂs
esophagus, and head and neck cancers.[10–15] In our attempts
to synthesize longer-wavelength photosensitizers (>700 nm),
we observed that replacing the five-membered isocyclic ring
(e.g., HPPH, lmax =660 nm) with a six-membered fused
imide ring system leads to a significant redshift (40–45 nm)
in the electronic absorption spectra.[16] To investigate the
effect of substituents on PDT efficacy, we have introduced
the desired substituents at different positions of the purpuri-
nimide system[17–19] (but not at the 20-position), as indicated
in Figure 1. An in vivo biological investigation indicated
For quite some time, our laboratory has also been inter-
ested in developing an efficient method for the regioselec-
tive synthesis of 20-meso-substituted purpurinimides, which
could be utilized for developing a variety of photosensitizers
(cationic, anionic, and targeted analogues) and bifunctional
agents (Figure 2).[30] This report shows the utility of the
Figure 2. Modification of the purpurinimide skeleton.
Suzuki reaction[31] in synthesizing such candidates by intro-
ducing an additional functionality into the photosensitizer
and their application in developing efficient agents for fluo-
rescence imaging with and without PDT. The regioselective
introduction of a variety of substituents at the 20-meso-pos-
tion by the Suzuki reaction also provides an opportunity to
alter the overall lipophilicity and target specificity of the
molecule(s) by varying the nature of the substitution.[31,32] If
required, water-soluble analogues can be prepared by re-
placing the methyl ester functionality present at the 172-po-
sition with appropriate hydrophilic functionalities.
Figure 1. Various peripheral positions suitable for introducing the desired
substituents into the purpurinimide system for structure–activity relation-
ship studies.
that aside from the overall lipophilicity, the position of the
substituents in the tetrapyrrolic skeleton also plays a crucial
role in the long-term PDT response.[1,5]
Results and Discussion
Studies are underway in our laboratory (and others) to
improve the target specificity of the PDT agents by conju-
gating the effective candidates with peptide and non-pep-
tide-based compounds known to bind to receptors that are
over-expressed in certain tumors.[20–22] Photosensitizers with
high tumor avidity are also being used as vehicles to deliver
the desired imaging agents (radionuclides for nuclear imag-
ing, fluorophores for optical-imaging agents, GdIII chelates
for MR imaging) to the target site.[22–26] This approach has
shown great potential for the development of bifunctional
agent(s) for the “see and treatꢂ approach. Insertion of a
metal, for example, nickel, into the photosensitizer–cyanine
dye conjugate should eliminate its singlet oxygen yield and
the metalated analogue may be useful as a fluorescence-
imaging agent without any PDT capability. In recent years,
PDT has also been evaluated for non-oncological applica-
tions, in particular, cationic photosensitizers have shown
great promise for treating infectious diseases (bacterial,
fungal, parasitic, and viral infections).[27,28] The rapid killing
of bacteria by PDT and the unlikelihood of bacteria devel-
oping resistance to PDT suggests that this approach to treat-
ment should be at the forefront of new therapies for infec-
tious diseases.[29]
À
Among the growing number of palladium-catalyzed C C
coupling reactions, the Suzuki—Miyaura reaction[33,34] plays
a leading role. The major advantages of this reaction are
1) the stability of the boron reagents, 2) the easy access to a
broad variety of boronic acids, and 3) the tolerance for dif-
ferent functional groups. As a result of the simple experi-
mental conditions, this reaction has been widely used for the
preparation of a variety of biologically active molecules, in-
cluding various porphyrin-based analogues. We herein pres-
ent the utility of this synthetic approach in developing a va-
riety of purpurinimides as possible target-specific photosen-
sitizers and bifunctional agents for tumor imaging with and
without PDT.
In this study, N-hexylpurpurinimide (1) and the corre-
sponding N-[3,5-bis(trifluoromethyl)benzyl analogue 2 were
used as starting substrates. The NiII complexes were ob-
tained by treating these moieties with [NiACTHNURTGNENG(U acac)2] (acac=
acetylacetone) in toluene at reflux. Subsequent treatment of
the intermediates with Pd/C under hydrogen afforded the
NiII mesopurpurinimides 3 and 4, respectively (Scheme 1).
These metalated analogues, on stirring with N-bromosuc-
cinimide (NBS) at room temperature for 30 min, produced
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K. M. Kadish, R. K. Pandey et al.
Scheme 1. Regioselective bromination of purpurinimides.
the corresponding NiII 20-bromo-N-substituted-purpurini-
mides 5 and 6 in yields of 80 and 73%, respectively.
To prepare 20-iodopurpurinimide, compound 3 was initial-
ly treated with N-iodosuccini-
nimides, which provide an additional functionality for the
preparation of biologically active compounds with the de-
sired overall lipophilicity.
mide (NIS) and, as expected,
the desired iodo derivative 7
was isolated in a modest yield
with the deiodinated analogue
8
being isolated in minor
amounts (Scheme 2). However,
subjecting the NiII N-[3,5-bis-
ACHTUNGTRENNUNG(trifluoromethyl)benzyl]meso-
purpurinimide methyl ester 4 to
similar conditions did not pro-
duce the desired 20-meso-iodo
derivative, instead the purpuri-
nimide 9 with the oxidized D
ring was obtained as the major
product.
Scheme 2. Effect of N-substitution in the iodination with NIS.
To further investigate the
utility of the Suzuki reaction,
NiII
20-bromomesopurpurini-
mide 5 was treated with a series
of functionalized boronic acids
containing amino, formyl, car-
boxylic acid, or hydroxymethyl
functionalities (Scheme 3). This
approach was further extended
by introducing N,N-dimethyl-
A
a
furan
group at the 20-position. Thus,
meso-substituted analogues 10–
22 were synthesized, all in good
yield, except for the NiII 20-(p-
carboxyphenyl)-N-hexylpurpur-
inimide methyl ester 17a. This
compound was obtained in a
yield of only 5%, whereas the
corresponding m-carboxy deriv-
ative 17 was isolated in a yield
of 45% under similar reaction
conditions. This is the first
report of the regioselective syn-
Scheme 3. Regioselective synthesis of 20-substituted purpurinimides by Suzuki reaction. The purpurinimide
system allows the conjugation of these compounds to a series of DNA-intercalating and tumor-targeting moiet-
thesis of 20-substituted purpuri- ies. Boc=tert-butoxycarbonyl, dppf=1,1’-bis(diphenylphosphino)ferrocene, TBDMS=tert-butyldimethylsilyl.
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Cyanine Dye–Purpurinimide Conjugates
FULL PAPER
In recent years, the increasing occurrence of multi-antibi-
otic-resistant microbes has led to the search for alternative
methods of killing pathogens and treating infections. PDT
could be an alternate treatment for such infections. Howev-
er, there are certain limitations because the delivery of visi-
ble light is, almost, by definition, a localized process. There-
fore, PDT for infections is likely to be applied exclusively to
localized disease as opposed to systemic infections. Photo-
therapy has also been used to treat animal models for dental
infections.[35] In the first study, Porphyromonas gingivalis,
one of the major causative organisms of periodontitis, was
subjected to PDT on the buccal mucosa of the maxillary
molars of rats with no visible bacteria.[36] Certain cationic
photosensitizers have shown great promise in Mycobacteriun
tuberculosis, bacterial infections of Candida albicans, and
parasite and viral infections.[37] In collaboration with the
University of Rochester, certain cationic pyropheophorbide
analogues, synthesized at Roswell Park, were evaluated for
antimicrobial activity and a structure–activity relationship
(SAR) study revealed that the number of cationic groups
and their position in the molecule has a significant impact
upon microbial inactivation.[38] The Suzuki approach pre-
sented in this paper allows the facile incorporation of a vari-
able number of cationic groups into the purpurinimide
system by following the approach used for the synthesis of
compound 13.
fore a portion of the imparted energy may transfer from the
photosensitizer to the cyanine dye, thus decreasing the pho-
tosensitizerꢂs quantum yield of singlet oxygen.[39]
To develop an agent with decreased energy transfer and
an improved quantum yield of singlet oxygen, one of our
strategies was to combine the two moieties with a more
rigid linker, as shown in Scheme 4. We were then able to
prepare the desired purpurinimide–cyanine dye conjugates
27 and 28 by the Suzuki reaction. In brief, cyanine dye 25
was prepared by treating the commercially available 24 (IR-
820) with 4-mercaptobenzoic acid. The cyanine dye 25 was
treated with NiII 20-(p-aminomethylphenyl)purpurinImide
26a to produce the purpurinimide–cyanine dye conjugate 27
in a yield of 35%. Similarly, the free base 20-(p-aminome-
thylphenyl)purpurinimide 26b afforded the corresponding
free-base analogue 28. We have previously shown the utility
of 124I-pyropheophorbide-a analogues for positron emission
tomography (PET)/fluorescence imaging.[22] To develop
longer-wavelength
purpurinimide-based
“bifunctional
agents”, maleimide analogue 30 can be converted into the
corresponding 124I analogue by following the methodology
developed for the pyropheophorbide system.[40]
To investigate the utility of 20-(p-aminophenyl)mesopur-
purinimide 14 in preparing a target-specific molecule, we
were interested in introducing the maleimide moiety at the
20-position, which can be readily linked to various peptides
and other tumor-targeting groups known for their target
specificity towards receptors over-expressed in certain
In an attempt to prepare a “bifunctional agent” for tumor
imaging and phototherapy, we have previously shown that
conjugates of tumor-avid photo-
sensitizers (e.g., HPPH) and
NIR-absorbing cyanine dyes
can be used for tumor imaging
and PDT.[1] Most conjugates
synthesized to date consist of a
flexible linker joining the two
chromophores, which leads to
possible folding within the mol-
ecule thus resulting in signifi-
cant singlet oxygen quenching
(a key cytotoxic agent for
PDT). When folding occurs, the
molecules are brought closer to
each other, which could amplify
the phenomenon known as Fçr-
ster resonance energy transfer
(FRET). The mechanism for
FRET involves nonradiative
energy transfer between two
chromophores. For FRET to
occur there needs to be spectral
overlap between the fluores-
cence of the higher-energy
chromophore and the absorb-
ance of the lower-energy chro-
mophore. These photosensitiz-
er–cyanine dye conjugates ex-
Scheme 4. Simple approach for the preparation of tumor-targeting and bifunctional agents for tumor imaging.
hibit such an overlap and there- EDC=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide).
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K. M. Kadish, R. K. Pandey et al.
Figure 3. 1H NMR spectrum of the bifunctional agent 27 in a mixture of CD3OD and [D6]DMSO.
tumors.[41,42] To achieve our goal, bromopurpurinimide 5 was
treated with maleic anhydride at room temperature. The in-
termediate 29 thus obtained was not isolated but heated at
reflux with sodium acetate and
acetic anhydride to give the
maleimide 30 in an overall
yield of 48%. This approach is
quite versatile and provides an
easy access to a variety of de-
sired target molecules, including
certain linear or cyclic peptides
and monoclonal antibodies as
well as a variety of target-spe-
cific photosensitizers.
longer-wavelength absorption of the corresponding metalat-
ed analogue shows a blueshift (Figure 4A). This behavior
can also be observed in the absorption spectra of the pur-
The structure of each com-
pound was confirmed by NMR,
UV/Vis, and HRMS analyses
and their purity ascertained by
HPLC. As a representative ex-
ample, the NMR spectrum of
the photosensitizer–cyanine dye
conjugate 27 is shown in
Figure 3. The resonances of all
the protons were confirmed by
2D NMR techniques (see the
Supporting Information).
The absorption spectra for
26a and 26b are shown in Fig-
ure 4A, and those of the pur-
purinimide–cyanine dye conju-
gates 27 and 28, along with that
of 25, are shown in Figure 4B.
Figure 4. Absorption spectra of A) 26a and 26b and B) 25, 27, and 28, and the fluorescence emission spectra
for C) 26a and 26b when excited at 620 nm, D) 25, 27, and 28 when excited at 630 nm, and E) 25, 27, and 28
In comparison with the nonme-
talated
purpurinimide,
the when excited at 785 nm. The concentration of all compounds was 4 mm in methanol.
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Cyanine Dye–Purpurinimide Conjugates
FULL PAPER
purinimide–cyanine dye conjugates 27 and 28 (Fig-
ure 4B).The long-wavelength absorption bands for the free
base and nickel(II) purpurinimides are observed at 705 and
677 nm, respectively, and the cyanine dye moiety in both
conjugates has an intense absorption peak at around
830 nm. The fluorescence characteristics of 27 and 28 are
quite different. The fluorescence emission spectra displayed
in Figure 4C and D show that in 26a and 28, the fluores-
cence emission of the photosensitizer (PS) moiety is
quenched, which is associated with the presence of
The singlet oxygen decay curve of methanol was used as a
measure of the instrument response function (IRF) and
HPPH was used as a reference because of its known singlet
oxygen quantum yield (FD = 48%[8]). From the singlet
oxygen decay curves (Figure 5) it can be seen that 26b gives
the highest singlet oxygen yield and that conjugate 27 shows
limited singlet oxygen producing ability. Singlet oxygen lu-
minescence studies also confirmed that the presence of NiII
as the central metal significantly diminishes the singlet
oxygen producing ability of the photosensitizer.
ACHTUNGTRENNUNG
Photostability of 27 and 28: To a certain extent, the photo-
stability of the photosensitizer during light treatment is im-
portant for optimizing the PDT response. Therefore the
photostability of 27 and 28 was measured in 17% bovine
calf serum (BCS) by irradiating their solutions at 677 and
imaging and PDT, the existence of FRET between the PS
and NIR fluorophores results in a decrease in the availabili-
ty of singlet oxygen, which is directly correlated to PDT effi-
cacy. To evaluate FRET in the conjugates, their fluorescence
emissions were compared with those of the unconjugated
PS. When 28 was excited at 630 nm, the fluorescence emis-
sion intensity of the cyanine dye increased by a factor of 1.7
compared with those of 25 and 27, whereas the fluorescence
emission intensity of the PS showed an approximate four-
fold decrease compared with 26b (Figure 4D). This suggests
that energy is transferred from the PS to the cyanine dye.
To prove that the cyanine dye portion of the conjugates
could function independently of the PS, the conjugates were
excited at 785 nm. The fluorescence emission intensity of 25
is higher than those of 27 and 28 by around 300 a.u. Howev-
er, the difference in the fluorescence intensities of 27 and 28
is less than 70 a.u.. These results suggest that the cyanine
dye in the conjugate can indeed function independently of
the PS when excited at 785 nm.
705 nm, respectively, with
a light dose equivalent of
75 mWcmÀ2 at the center plane of the cuvette. The absorb-
ance intensity of the cyanine dye portion of the conjugates
27 and 28 was monitored at 852 and 849 nm, respectively,
and the percent-normalized absorbance was recorded as a
function of time. The photostability of 27 and 28 are shown
in Figure 6C. Based on the singlet oxygen luminescence
measurements, 27 should be more stable as negligible
amounts of singlet oxygen will be generated by irradiating
the metalated photosensitizer with light of an appropriate
wavelength. As expected, the absorbance of the cyanine dye
moiety of conjugate 28 had decreased to approximately 6%
of its initial value 21 min post-irradiation„ whereas the cya-
nine dye moiety of 27 (containing the free-base photosensi-
tizer) had photobleached to 72% of the initial value 78 min
post-irradiation. These data confirm that the presence of
NiII in the purpurinimide core enhances the photostability of
the photosensitizer and reduces its singlet oxygen producing
ability.
Singlet oxygen production for 25, 26a, 26b, 27, 28, and
HPPH: The production of singlet oxygen is one of the re-
quirements for effective PDT. The singlet oxygen producing
efficiencies of compounds 25, 26a, 26b, 27b, 28, and HPPH
were evaluated by measuring the phosphorescence of singlet
oxygen at 1270 nm upon excitation at 532 nm (Figure 5).
Electrochemistry and spectroelectrochemistry: The redox
properties of the cyanine dye (25) and its purpurinimide
nickel (27) and free-base (28) conjugates were examined in
DMSO containing 0.1m tetrabutylammonium perchlorate
(TBAP) to determine whether the central metal ion in the
purpurinimide of the conjugates influences the electron-ac-
cepting and -donating properties. Examples of the cyclic vol-
tammograms of these three compounds are shown in
Figure 7.
The cyanine dye 25 undergoes a reversible one-electron
oxidation at E1/2 =0.56 V and a reversible one-electron re-
duction at E1/2 =À0.56 V in DMSO containing 0.1m TBAP
with an additional irreversible reduction also seen at Ep =
À1.40 V at a scan rate of 0.1 VsÀ1. Under the same condi-
tions, the two conjugates undergo an oxidation at similar po-
tentials of E1/2 =0.52 V (27) and 0.53 V (28), two reversible
reductions at E1/2 =À0.57 and À0.81 V (27) and À0.67 and
À0.81 V (28), and an additional irreversible reduction at
Ep =À1.36 V (27) and À1.30 V (28) at a scan rate of
0.1 VsÀ1 (see Figure 7). A comparison of the potentials of
the three compounds suggests that the first oxidation and
Figure 5. Singlet oxygen production of 25, 26a, 26b, 27, 28, and HPPH in
methanol detected by measuring the phosphorescence of singlet oxygen
at 1270 nm upon excitation with a 532 nm laser. The instrument response
function (IRF) of methanol is also shown. The singlet oxygen production
of HPPH is shown as a reference (FD =48% for HPPH in methanol).
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K. M. Kadish, R. K. Pandey et al.
ing 0.1m TBAP is characterized
by an intense, split near-IR
band at 773 and 849 nm, where-
as 27 and 28 in their neutral
forms exhibit spectra that are a
simple sum of the absorptions
of the individual cyanine and
purpurinimide units. For exam-
ple, the 426 and 674 nm bands
of 27 in DMSO can be attribut-
ed to the NiII purpurinimide
unit,[42] whereas the intense,
split near-IR band at 773 and
850 nm of 27 has been attribut-
ed to contributions from the cy-
anine dye unit (see the top of
Figure 8b). Applying a reduc-
tion potential of À0.70 V to a
solution of 27 in DMSO con-
taining 0.1m TBAP led to the
disappearance of the split near-
IR band at 773 and 850 nm and
the appearance of a new visible
band at 531 nm (Figure 8b). No
change is observed in the NiII
purpurinimide bands of 27 (426
and 674 nm) during this one-
Figure 6. A) Absorption spectra of conjugate 28 in 17% BCS during the photobleaching process. B) Absorb-
ance spectra for conjugate 27 in 17% BCS during the photobleaching process. C) Comparison of photobleach-
ing rates for conjugates 27 and 28. The solutions were irradiated at 677 (27) and 705 nm (28) with a laser
power of 530 mW.
electron reduction whereas the cyanine dye bands undergo
exactly the same spectral changes as seen during the reduc-
tion of 25 at À0.70 V in the thin-layer cell (Figure 8a). This
unambiguously indicates that the first one-electron addition
to the conjugate 27 involves the cyanine portion of the mol-
ecule.
Shifting the applied potential from À0.70 to À1.00 V for a
DMSO solution of 27 led to a decrease in the intensity of
the 426 and 674 nm bands (Figure 8b), similar to the spectral
changes reported in the literature[42] for the first reduction
of NiII purpurinimide, which suggests that the second elec-
tron is added to the NiII purpurinimide part of the molecule.
The third reduction of 27 at À1.50 V is centered on the cya-
nine dye, based on the similar spectral changes observed
during controlled reduction of 25 at À1.60 V (Figure 8a).
The electron-transfer site of the oxidation can also be as-
signed on the basis of the data shown in Figure 9, which
shows almost identical spectral changes for 25 and 27 under
the same solution conditions, indicating that the first oxida-
tion of 27 occurs in the cyanine portion of the molecule. The
same initial sites for the redox processes are proposed for
conjugate 28, and the thin-layer spectral changes of 28
during each redox process for this compound are presented
in Figure S1 in the Supporting Information.
Figure 7. Cyclic voltammograms of 25, 27, and 28 in DMSO containing
0.1m TBAP. Scan rate=0.1 VsÀ1
.
the first and third reductions of both 27 and 28 all involve
the cyanine dye portion of the molecule and thus the second
one-electron reduction of the two conjugates at E1/2
=
Both the first oxidation and the first reduction of conju-
gates 27 and 28 are centered on the cyanine dyad with their
first oxidation potential negatively shifted by 30–40 mV in
comparison with that of 25 due to the influence of the
linked purpurinimide functional agent. Almost identical
half-wave potentials for the first reduction can be seen for
À0.81 V can confidently be assigned to the purpurinimide
part of the molecule.
More definitive evidence for these assignments is given by
the thin-layer spectroelectrochemical data presented in Fig-
ures 8 and 9. The spectrum of neutral 25 in DMSO contain-
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Chem. Eur. J. 2013, 19, 6670 – 6684

Cyanine Dye–Purpurinimide Conjugates
FULL PAPER
unit.[43,44] According to the
Rehm–Weller equation,[45] the
quenching constant would ap-
proximately depend on the po-
tential difference between the
first oxidation and the first re-
duction (the electrochemical
HOMO–LUMO gap) for mole-
cules with similar structures.
The HOMO–LUMO gaps of
1.09 V (27) and 1.20 V (28)
show that compound 28, which
has a larger HOMO–LUMO
gap, will have a smaller elec-
tron-transfer driving force and
avoid quenching of the excited
triplet state. In the case of 28,
this makes it possible to pro-
duce more singlet oxygen by
collision of an oxygen molecule
with the excited triplet state.
This is consistent with data for
the phosphorescence of singlet
oxygen for these compounds
provided earlier.
Figure 8. Thin-layer UV/Vis spectra of a) 25 and b) 27 during controlled reductions in DMSO containing 0.1m
TBAP.
Comparative in vitro photosensitizing efficacy of metalated
versus nonmetalated analogues: Conjugate 28 and the corre-
sponding NiII analogue 27 were evaluated for in vitro photo-
sensitizing efficacy in the murine colon carcinoma (Colon-
26) cell line by using the MTT-based phototoxicity assay.[46]
In brief, cells were incubated with increasing concentrations
of the photosensitizers for 24 h and then illuminated with
light at 706 nm for conjugate 28 and at 680 nm for conjugate
27. Neither compound exhibited dark (drug, no light) toxici-
ty (cell death), even at higher doses (6 mm). As expected,
the conjugate 27, which contains a NiII photosensitizer,
showed limited singlet oxygen producing efficiency, and
upon exposure to light it also showed minimal PDT efficacy
in comparison with the nonmetalated derivative 28. The re-
sults are summarized in Figure 10 and are represented in
two ways: A) At a fixed drug concentration (1.0 mm) with
variable light doses and B) at a fixed light dose with variable
concentrations of the drugs. As can be seen, the free-base
conjugate 28 showed significant PDT efficacy, whereas,
under similar treatment conditions, the corresponding NiII
derivative 27 did not exhibit any cell kill, which could be
due to its limited singlet oxygen producing ability.
Figure 9. Thin-layer UV/Vis spectra of a) 25 and b) 27 during the first ox-
idation in DMSO containing 0.1m TBAP.
cyanine dyad 25 and conjugate 27, but a potential difference
of 110 mV is seen for the first reduction of 28. The fact that
the cyanine dyad is reduced at a more negative potential
suggests electron communication between the cyanine dye
and the purpurinimide portion of the molecule. In other
words, the two functional agents are fairly interactive with
each other in the case of 28 whereas they are “almost inde-
pendent” of each other in the case for 27.
For the conjugates 27 and 28, reduction of the cyanine
dye unit is easier than reduction of the purpurinimide unit
(E1/2 =À0.57 to À0.67 V vs. SCE, À0.3 to À0.4 V vs. SHE).
Thus, the cyanine dye unit could be used as an oxidant to
quench the excited triplet state of the purpurinimide
Compound 27 with NiII as the central metal showed signifi-
cantly improved tumor contrast by fluorescence imaging:
The fluorescence imaging potentials of conjugates 27 and 28
were investigated in mice bearing Colon-26 tumors. Both
photosensitizers, at a dose of 0.3 mmolkgÀ1, were injected
(intravenously) and the mice were imaged at 24 h post-injec-
tion. The results depicted in Figure 11 reveal that compared
with the free base 28, the corresponding NiII complex 27
Chem. Eur. J. 2013, 19, 6670 – 6684
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6677

K. M. Kadish, R. K. Pandey et al.
ular position and also to alter the overall lipophilicity at
other position(s) of the molecule to gain the desired phar-
macokinetics. A molecule with multiple functionalities could
also help in developing “multifunctional” agents for imaging
and therapy. Thus, a single agent could be used for imaging
and therapy by following a “See and Treat” approach. This
study also illustrates the presence of metal in the photosen-
sitizer–cyanine dye conjugates has a significant effect not
only on its photophysical properties, but also in tumor local-
ization. Such properties may vary with the nature of the cen-
tral metal in the porphyrin core and studies along this line
are in progress. The higher in vivo uptake of the NiII purpur-
inimide–cyanine dye conjugate compared with the corre-
sponding free-base analogue reveals a remarkable difference
in the biodistribution between the two conjugates.
Figure 10. In vitro photosensitizing efficacy (MTT assay) of NiII purpuri-
nimide–cyanine dye conjugate 27 and the corresponding nonmetalated
analogue 28. A) PDT efficacy at a fixed concentration of the photosensi-
tizers with the cells exposed to variable light doses. B) PDT efficacy at a
fixed light dose and variable concentrations. On exposing the cells to
light without incubation with photosensitizers 27 and 28, no dark toxicity
(cell kill) was observed. The cells were incubated for 24 h before expo-
sure to light.
The regioselective synthesis of 20-bromopurpurinimide
(chlorin) and its further elaboration could also allow the
facile development of a wide variety of supramolecular
structures as models for photosynthetic reaction centers,
semiconductors for use in hybrid electronics, optoelectronic
applications, electrochemical sensors, and receptor models
with unique photophysical and electrochemical properties.
The investigated functional agents are electrochemically
active and systematic electrochemical and spectroelectro-
chemical studies on these photosensitizers with “multifunc-
tional” agents have been carried out. Their redox potentials,
electron-transfer sites, and UV/Vis spectra have been inves-
tigated in different oxidation states. Furthermore, analysis of
the electrochemical data shows the stability of the conju-
gates and the interaction that occurs between the two func-
tional agents in the donor–acceptor system. Detailed photo-
physical studies (fluorescence lifetime, quantum yield, com-
parative in vitro/in vivo photobleaching of the photosensitiz-
er and the cyanine dye) are in progress and will be publish-
ed separately.
Figure 11. Whole-body fluorescence imaging of A) a control mouse,
B) cyanine dye 25, C) conjugate 27, and D) conjugate 28 at a dose of
0.3 mmolkg at 24 h post intravenous injection.
showed improved tumor uptake and specificity. The best
tumor contrast was observed at 24 and 48 h post-injection
(see the Supporting Information). On the other hand, the
free-base analogue 28 showed a significantly higher uptake
in other organs (liver, kidney, and spleen) than that seen in
the tumor. Under similar imaging parameters, the cyanine
dye 25 alone did not show any tumor-imaging ability (data
not shown), which clearly indicates that conjugation of the
non-tumor-specific cyanine dye, which possesses the re-
quired photophysical properties, with tumor-avid purpurini-
mide-based photosensitizers enhances its tumor specificity.
This approach also provides a great platform for the devel-
opment of imaging agents with and without PDT capability.
To enhance the PDT capability of conjugate 28, it is impor-
tant to change the overall lipophilicity of the molecule,
which should make a significant difference to its tumor
uptake and pharmacokinetic and -dynamic characteristics.
These studies are in progress.
Experimental Section
All reactions were carried out in flame-dried glassware under an atmo-
AHCTUNGTREGsNNUN phere of nitrogen with magnetic stirring. TLC was carried out on
ANALTECH precoated silica gel GF PE sheets (Cat. 159017, layer thick-
ness 0.25 mm) and aluminum oxide NF PE sheets (Cat. 101016, layer
thickness 0.2 mm). Column chromatography was performed either on
silica gel 60 (70–230 mesh) or neutral alumina (Brockmann grade III,
50 mesh). In some cases preparative TLC plates were also used for the
purification (ANALTECH precoated silica gel GF glass plate, Cat.
02013, layer thickness 1.0 mm). Solvents were purified as follows: Trace
amounts of water and oxygen from THF were removed by heating at
reflux over sodium under an inert atmosphere and dichloromethane was
dried over P2O5. Anhydrous DMF, triethylamine, pyridine, and other
common chromatographic solvents were obtained from commercial sup-
pliers (J.T. Baker, EMD, and Aldrich) and used without further purifica-
tion. NMR spectra were recorded on a Bruker DRX 400 MHz spectrom-
eter. All chemical shifts are reported in parts per million (d). 1H NMR
(400 MHz) spectra were recorded at room temperature in CDCl3 or
CD3OD solutions and referenced to residual CHCl3 (7.26 ppm) or TMS
(0.00 ppm). EI-mass spectra were recorded on a Bruker Esquire ion-trap
mass spectrometer equipped with a pneumatically assisted electrospray
ionization source operating in positive mode. HRMS analyses were per-
Conclusion
We have shown the utility of the Suzuki reaction for the
synthesis of certain purpurinimide-based conjugates for
tumor imaging and therapy. The synthetic approach dis-
cussed herein is not only limited to the purpurinimide
system but can be extended to a wide variety of chlorin and
bacteriochlorin systems. The ability to introduce additional
functionality into a photosensitizer provides additional tools
for introducing desired tumor-targeting moieties at a partic-
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Chem. Eur. J. 2013, 19, 6670 – 6684

Cyanine Dye–Purpurinimide Conjugates
FULL PAPER
formed at the Mass Spectrometry Facility, Michigan State University.
UV/Vis spectra were recorded on a Varian Cary 50 Bio UV/Visible spec-
trophotometer using dichloromethane as solvent. All photophysical ex-
periments were carried out by using spectroscopic-grade solvents. Most
of the purpurinimides and the corresponding cyanine dye conjugates
tend to decompose at >1508C, which made it extremely difficult to
measure a precise melting point.
18-H), 3.58 (s, 3H; CO2Me), 3.46 (s, 3H; ring-CH3), 3.42–3.34 (m, 4H; 3-
CH2CH3, 8-CH2CH3), 2.94 (s, 3H; ring-CH3), 2.86 (s, 3H; ring-CH3),
2.62–2.55 (m, 2H; 172-CH2), 2.16–2.13 (m, 1H; 171-CH2), 1.93–1.89 (m,
1H; 171-CH2), 1.55–1.48 (m, 6H; 3-CH2CH3, 8-CH2CH3), 1.32 ppm (d,
J=6.8 Hz, 3H; 18-CH3); 13C NMR (100 MHz, CDCl3): d=189.6, 173.7,
166.8, 162.9, 162.5, 161.1, 150.9, 147.4, 145.0, 143.9, 141.1, 140.6, 138.4,
137.5, 135.2, 134.5, 134.1, 133.5, 133.1, 131.8, 131.3, 128.8, 118.7, 108.9,
102.5, 97.1, 94.8, 53.4, 51.5, 47.4, 42.1, 32.0, 29.6, 28.8, 20.8, 19.0, 17.0,
16.7, 12.3, 10.6 ppm; UV/Vis (CH2Cl2): lmax (e)=664 nm (3.2ꢃ
104 mÀ1 cmÀ1); MS (EI): m/z): 863 [M+H]+; HRMS: calcd for
C43H39F6N5NiO4: 861.2260; found: 861.2263.
NiII 20-Bromomesopurpurin-N-hexylimide methyl ester (5): In a 100 mL
dry round-bottomed flask, Ni mesopurpurin-N-hexylimide (3; 50.0 mg,
0.069 mmol) was dissolved in dry CH2Cl2 (30.0 mL). N-Bromosuccini-
mide (14.8 mg, 0.083 mmol) was added to this solution and the resultant
mixture was stirred for 30 min at room temperature under N2. The prog-
ress of the reaction was monitored by UV/Vis spectrophotometry and
TLC. After completion of the reaction, saturated sodium bicarbonate
(50.0 mL) was gradually added to the reaction mixture and then extract-
ed with dichloromethane (3ꢃ50.0 mL). The organic layers were collected,
combined, and washed with water (100.0 mL). The organic layer was sep-
arated, dried over anhydrous Na2SO4, and the solvent was evaporated.
The crude product obtained was purified through a column of silica by
using 1–2% acetone/dichloromethane as eluent. Yield: 45.0 mg (81.2%)
1H NMR (400 MHz, CDCl3): d=8.91 (s, 1H; meso-H), 8.61 (s, 1H; meso-
H), 4.55 (dd, J=3.6, 9.62 Hz, 1H; 17-H), 4.54 (q, J=7.2 Hz, 1H; 18-H),
4.23 (m, 2H; N-CH2-Hexyl), 3.64 (s, 3H; CO2Me), 3.44 (s, 3H; ring-
CH3), 3.42–3.35 (m, 4H; 3-CH2CH3, 8-CH2CH3), 3.08 (s, 3H; ring-CH3),
2.92 (s, 3H; ring-CH3), 2.66 (t, J=8.4 Hz, 2H; 172-CH2), 2.42 (m, 1H;
171-CH2), 2.04 (m, 1H; 171-CH2), 1.82 (m, 2H; CH2-Hexyl), 1.52–1.46 (m,
NiII mesopurpurin-18-N-hexylimide methyl ester (3): NiII purpurin-18-N-
hexylimide (1a; 350.0 mg, 0.48 mmol) was dissolved in distilled THF
(100 mL). Pd/C (10% w/w, 350.0 mg) and 5–6 drops of triethylamine
were added to the reaction mixture, which was degassed and flushed with
H2 and vigorously stirred for 12 h under H2. The progress of the reaction
was monitored by UV/Vis and 1H NMR spectroscopy. After completion
of the reaction, the mixture was degassed, flushed with N2, and filtered
over a bed of Celite. The filtrate was concentrated and the crude product
purified through a column of silica by using 0.5–1% acetone/dichloro-
ACHTUNGTRENNUNG
methane as eluent. Yield: 300.0 mg (85.4%). 1H NMR (400 MHz,
CDCl3): d=8.99 (s, 1H; meso-H), 8.61 (s, 1H; meso-H), 7.86 (s, 1H;
meso-H), 4.78 (dd, J=3.2, 9.2 Hz, 1H; 17-H), 4.27 (m, 2H; N-CH2-
Hexyl), 4.03 (q, J=7.2 Hz, 1H; 18-H), 3.60 (s, 3H; CO2Me), 3.48 (s, 3H;
ring-CH3), 3.44–3.36 (m, 4H; 3-CH2CH3, 8-CH2CH3), 2.95 (s, 3H; ring-
CH3), 2.87 (s, 3H; ring-CH3), 2.64–2.54 (m, 2H; 172-CH2), 2.20–2.18 (m,
1H; 171-CHH), 1.94–1.92 (m, 1H; 171-CHH), 1.87–1.82 (m, 2H; CH2-
Hexyl), 1.55–1.48 (m, 8H; 3-CH2CH3, 8-CH2CH3, CH2-Hexyl), 1.44–1.36
(m, 4H; 2 CH2-Hexyl), 1.33 (d, J=6.8 Hz, 3H; 18-CH3), 0.92 ppm (t, J=
7.2 Hz, 3H; CH3-Hexyl); 13C NMR (100 MHz, CDCl3): d=173.8, 166.8,
162.9, 162.3, 160.7, 150.4, 147.1, 144.8, 143.6, 141.5, 140.2, 138.3, 135.8,
133.3, 128.8, 128.4, 126.0, 119.6, 108.5, 102.3, 97.7, 94.5, 53.3, 51.5, 47.4,
41.9, 39.9, 32.0, 31.7, 28.8, 27.0, 22.7, 20.9, 19.1, 17.1, 16.8, 14.0, 12.2,
10.6 ppm; UV/Vis (CH2Cl2): lmax (e)=661 nm (3.2ꢃ104 mÀ1 cmÀ1); MS
(EI): m/z): 721 [M+H]+; HRMS: calcd for C40H47N5NiO4: 719.2982;
found: 719.2971.
8H; 3-CH2CH3, 8-CH2CH3, CH2-Hexyl), 1.41–1.35 (m, 4H;
2 CH2-
Hexyl), 1.18 (d, J=7.2 Hz, 3H; 18-CH3), 0.91 ppm (t, J=7.2 Hz, 3H;
CH3-Hexyl); 13C NMR (100 MHz, CDCl3): d=169.8, 162.4, 158.7, 153.0,
145.8, 143.5, 142.5, 139.7, 138.9, 137.0, 135.3, 134.4, 132.2, 131.2, 129.9,
124.9, 124.4, 122.1, 117.0, 104.6, 100.5, 93.2, 88.9, 49.5, 47.6, 46.1, 35.9,
28.2, 27.7, 25.7, 24.8, 23.9, 23.0, 18.7, 15.3, 15.1, 13.6, 13.0, 12.8, 12.2, 10.1,
8.3, 6.6 ppm; UV/Vis (CH2Cl2): lmax (e)=680 nm (3.0ꢃ104 mÀ1 cmÀ1); MS
(EI): m/z): 800.0 [M+H]+; HRMS: calcd for C40H46BrN5NiO4: 797.2087.
NiII N-Bis(3,5-trifluoromethyl)benzylpurpurin-18-N-hexylimide methyl
ester (2a): N-Bis(3,5-trifluromethyl)benzylpurpurin-18-N-hexylimide (2;
200.0 mg, 0.25 mmol) was dissolved in toluene (50 mL) and nickel acet
ACHTUNGERTNyNUNG l-
ACHTUNGTRENNUNGacetonate hydrate (640.0 mg, 2.45 mmol) was added. The reaction mix-
ture was heated at reflux vigorously for 2 h and the progress of the reac-
tion was monitored by TLC and UV/Vis spectrophotometry. After com-
pletion of the reaction, the mixture was cooled and directly loaded onto
a column of silica. The product 2a was eluted with 1% acetone/dichloro-
methane. Yield: 195.0 mg (91.1%). 1H NMR (400 MHz, CDCl3): d=9.00
(s, 1H; meso-H), 8.73 (s, 1H; meso-H), 8.09 (s, 2H; Ph-H), 7.91 (s, 1H;
meso-H), 7.78 (s, 1H; Ph-H), 7.55 (dd, J=11.6, 17.6 Hz, 1H; CH=CH2),
5.93 (d, J=10.4 Hz, 1H; CH=CH2), 5.91 (d, J=4.0 Hz, 1H; -CH=CH2),
5.59 (s, 2H; N-CH2-Ph), 4.72 (m, 1H; 17-H), 4.06 (q, J=7.2 Hz, 1H; 18-
H), 3.60 (s, 3H; CO2Me), 3.49 (s, 3H; ring-CH3), 3.40 (m, 2H; 8-
CH2CH3), 2.98 (s, 3H; ring-CH3), 2.93 (s, 3H; ring-CH3), 2.63–2.56 (m,
2H; 172-CH2), 2.19 (m, 1H; 171-CH2), 1.92 (m, 1H; 171-CH2), 1.50 (t, J=
7.2 Hz, 3H; 8-CH2CH3), 1.32 ppm (d, J=6.8 Hz, 3H; 18-CH3); 13C NMR
(100 MHz, CDCl3): d=173.6, 166.8, 162.9, 162.5, 160.5, 150.0, 145.6,
144.1, 143.8, 141.0, 140.0, 138.7, 135.6, 134.2, 134.0, 131.7, 131.3, 128.8,
128.5, 124.7, 121.1, 121.5, 119.1, 108.9, 104.4, 97.1, 95.1, 53.6, 51.5, 47.4,
42.1, 32.0, 29.6, 28.8, 20.8, 19.0, 17.0, 12.3, 11.6, 10.6 ppm; UV/Vis
(CH2Cl2): lmax (e)=675 nm (4.1ꢃ104 mÀ1 cmÀ1); MS (EI): m/z: 861
[M+H]+; HRMS: calcd for C43H37F6N5NiO4: 859.2103; found: 859.2107.
NiII 20-Bromo-N-bis(3,5-trifluoromethyl)benzylmesopurpurin-18-N-hexy-
limide methyl ester (6): In a 100 mL dry round-bottomed flask, Ni meso-
purpurin-N-bis(trifluoromethyl)benzyl-imide (50.0 mg, 0.058 mmol) was
dissolved in dry CH2Cl2 (30.0 mL). N-Bromosuccinimide (12.38 mg,
0.069 mmol) was added to this solution and the resultant mixture was
stirred for 30 min at room temperature under N2. The progress of the re-
action was monitored by UV/Vis spectrophotometry and TLC. After
completion of the reaction, saturated sodium bicarbonate (50.0 mL) was
gradually added to the reaction mixture and then extracted with di-
chloromethane (3ꢃ50.0 mL). The organic layers were collected, com-
bined, and washed with water (100.0 mL). The organic layer was separat-
ed, dried over anhydrous Na2SO4, and concentrated. The crude obtained
was purified through a column of silica by using 1–2% acetone/dichloro-
methane as eluent. Yield: 40.0 mg (73.3%). 1H NMR (400 MHz, CDCl3):
d=8.94 (s, 1H; meso-H), 8.61 (s, 1H; meso-H), 8.05 (s, 2H; Ph-H), 7.77
(s, 1H; Ph-H), 5.55 (s, 2H; Benzyl-CH2), 4.51–4.47 (m, 2H; 17-H, 18-H),
3.62 (s, 3H; CO2Me), 3.45 (s, 3H; ring-CH3), 3.42–3.36 (m, 4H; 3-
CH2CH3, 8-CH2CH3), 3.08 (s, 3H; ring-CH3), 2.96 (s, 3H; ring-CH3),
2.66–2.63 (m, 2H; 172-CH2), 2.22 (m, 1H; 171-CH2), 2.02 (m, 1H; 171-
CH2), 1.53–1.49 (m, 6H; 3-CH2CH3, 8-CH2CH3), 1.17 ppm (d, J=6.8 Hz,
3H; 18-CH3); 13C NMR (100 MHz, CDCl3): d=173.7, 166.4, 162.3, 162.0,
157.2, 150.0, 148.0, 146.8, 144.0, 143.4, 140.8, 139.3, 138.7, 136.0, 135.4,
134.0, 131.7, 131.4, 128.9, 128.3, 120.0, 109.1, 104.6, 96.5, 93.0, 53.6, 51.5,
50.1, 42.1, 32.1, 29.6, 27.9, 19.2, 19.0, 17.4, 16.9, 16.7, 16.1, 12.4, 10.5 ppm;
UV/Vis (CH2Cl2): lmax (e)=683 nm (3.3ꢃ104 mÀ1 cmÀ1);. MS (EI): m/z):
964 [M+Na]+; HRMS: calcd for C43H38BrF6N5NiO4: 939.1365; found:
939.1356.
NiII
N-Bis(3,5-trifluoromethyl)benzylmesopurpurin-18-N-hexylimide
methyl ester (4): Purpurinimide 2a (300.0 mg, 0.34 mmol) was dissolved
in distilled THF (100 mL). Pd/C (10% w/w, 600.0 mg) and 5–6 drops of
triethylamine were added to the reaction mixture, which was flushed with
H2 and stirred vigorously for 12 h under H2. The progress of the reaction
was monitored by UV/Vis and 1H NMR spectroscopy. After completion
of the reaction, the mixture was degassed, flushed with N2, and filtered
over a bed of Celite. The filtrate was concentrated and the crude product
was purified through a column of alumina (G-III) by using 0.5–1% ace-
tone/dichloromethane as eluent. Yield: 280.0 mg (93.1%). 1H NMR
(400 MHz, CDCl3): d=8.98 (s, 1H; meso-H), 8.59 (s, 1H; meso-H), 8.08
(s, 2H; Ph-H), 7.85 (s, 1H; meso-H), 7.77 (s, 1H; Ph-H), 5.58 (s, 2H; N-
CH2-Ph), 4.71 (dd, J=3.60, 8.80 Hz, 1H; 17-H), 4.04 (q, J=7.2 Hz, 1H;
NiII 20-iodopurpurin-N-hexylimide methyl ester (7): In a 100 mL dry
round-bottomed flask, NiII mesopurpurin-N-hexylimide (50.0 mg,
0.069 mmol) was dissolved in chloroform (30.0 mL). N-Iodosuccinimide
Chem. Eur. J. 2013, 19, 6670 – 6684
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6679

K. M. Kadish, R. K. Pandey et al.
(18.7 mg, 0.083 mmol) and 2 drops of trifluoroacetic acid (TFA) were
added and the resultant mixture was stirred for 3–4 min at room temper-
ature under N2. The progress of the reaction was monitored by UV/Vis
spectrophotometry and TLC. After completion of the reaction, saturated
sodium bicarbonate (50.0 mL) was gradually added to the reaction mix-
ture and then extracted with dichloromethane (3ꢃ50.0 mL). The organic
layers were collected, combined, and washed with water (100.0 mL). The
organic layer was separated, dried over anhydr. Na2SO4, and concentrat-
ed. The crude product obtained was purified through a column of silica
by using 1–2% acetone/dichloromethane as eluent to give compound 7.
Yield: 15.0 mg (25.5%). 1H NMR (400 MHz, CDCl3): d=8.88 (s, 1H;
meso-H), 8.56 (s, 1H; meso-H), 4.51 (dd, J=4.0, 9.6 Hz, 1H; 17-H), 4.47
(q, J=6.8 Hz, 1H; 18-H), 4.21 (m, 2H; N-CH2-Hexyl), 3.66 (s, 3H;
CO2Me), 3.43 (s, 3H; ring-CH3), 3.41–3.33 (m, 4H; 3-CH2CH3, 8-
CH2CH3), 3.08 (s, 3H; ring-CH3), 2.92 (s, 3H; ring-CH3), 2.70–2.65 (m,
2H; 172-CH2), 2.27 (m, 1H; 171-CH2), 2.09 (m, 1H; 171-CH2), 1.81 (m,
2H; CH2-Hexyl), 1.51–1.46 (m, 8H; 3-CH2CH3, 8-CH2CH3, CH2-Hexyl),
nimide (50.0 mg, 0.062 mmol), Cs2CO3 (20.16 mg, 0.062 mmol), methyl
boronic acid pinacol ester (88 mg, 0.62 mmol), and 1,1’-bis(diphenylphos-
phino)ferrocenepalladium(II)
chloride
([PdCl
G
9.06 mg,
0.0124 mmol) were dissolved in dry THF (35 mL). The resultant mixture
was heated at reflux for 12 h. The progress of the reaction was monitored
by UV/Vis spectrophotometry and TLC. After completion of the reac-
tion, the mixture was taken up in CH2Cl2 (100 mL) and washed with
brine. The organic layer was separated and dried over sodium sulfate.
The crude residue obtained was purified by chromatography through a
column of silica gel by using 0.5–1% acetone/CH2Cl2 to obtain product
10 in good yield. Yield: 38.0 mg (84.2%). 1H NMR (400 MHz, CDCl3):
d=8.91 (s, 1H; meso-H), 8.57 (s, 1H; meso-H), 4.58 (dd, J=4.0, 9.2 Hz,
1H; 17-H), 4.26–4.21 (m, 2H; N-CH2-Hexyl), 4.08 (q, J=6.8 Hz, 1H; 18-
H), 3.64 (s, 3H; CO2Me), 3.46 (s, 3H; ring-CH3), 3.43–3.35 (m, 4H; 8-
CH2CH3, 3-CH2CH3), 2.99 (s, 3H; ring-CH3), 2.97 (s, 3H; ring-CH3), 2.95
(s, 3H; ring-CH3), 2.66–2.61 (m, 2H; 172-CH2), 2.20 (m, 1H; 171-CHH),
2.00 (m, 1H; 171-CHH), 1.85–1.82 (m, 2H; CH2-Hexyl), 1.56–1.53 (m,
2H; CH2-Hexyl), 1.51–1.48 (m, 6H; 8-CH2CH3, 3-CH2CH3), 1.43–1.33
(m, 4H; 2 CH2-Hexyl), 1.13 (d, J=6.8 Hz, 3H; 18-CH3), 0.91 ppm (t, J=
6.8 Hz, 3H; CH3-Hexyl); 13C NMR (100 MHz, CDCl3): d=174.0, 166.7,
162.9, 160.0, 156.4, 150.2, 149.2, 145.3, 143.2, 142.9, 140.5, 138.8, 138.7,
135.7, 133.9, 133.0, 120.3, 108.1, 104.0, 103.0, 96.6, 54.0, 51.5, 45.6, 39.8,
32.2, 31.7, 28.8, 27.6, 27.0, 22.7, 19.0, 18.8, 17.9, 17.0, 16.8, 15.2, 14.0, 12.2,
10.5 ppm; UV/Vis (CH2Cl2): lmax (e)=677 nm (3.1ꢃ104 mÀ1 cmÀ1); MS
(EI): m/z): 757 [M+Na]+; HRMS: calcd for C41H49N5NiO4: 733.3138;
found: 733.3180.
1.41–1.34 (m, 4H;
2 CH2-Hexyl), 1.12 (d, J=6.8 Hz, 3H; 18-CH3),
0.90 ppm (t, J=7.2 Hz, 3H; CH3-Hexyl); 13C NMR (100 MHz, CDCl3):
d=173.8, 166.2, 162.2, 158.9, 150.1, 149.4, 146.7, 143.5, 143.1, 141.0, 139.4,
138.0, 136.3, 135.2, 133.7, 121.1, 108.8, 104.7, 62.3, 54.2, 53.7, 51.6, 39.8,
32.3, 31.6, 29.6, 29.3, 28.7, 27.3, 27.0, 22.6, 19.4, 19.0, 17.3, 16.9, 14.1, 12.2,
10.4 ppm; UV/Vis (CH2Cl2): lmax (e)=685 nm (3.4ꢃ104 mÀ1 cmÀ1). MS
(EI): m/z): 870 [M+Na]+; HRMS: calcd for C40H46IN5NiO4: 845.1948;
found: 845.1953.
NiII meso-Purpurin-N-hexylimide-emiraldine (8): Yield: 10.0 mg (20.0%).
1H NMR (400 MHz, CDCl3): d=9.24 (s, 1H; meso-H), 9.04 (s, 1H; meso-
H), 8.97 (s, 1H; meso-H), 4.37 (t, J=7.6 Hz, 2H; N-CH2-Hexyl), 3.90 (t,
J=7.6 Hz, 2H; 171-CH2), 3.72 (s, 3H; CO2Me), 3.67 (s, 3H; ring-CH3),
3.57 (m, 4H; 3-CH2CH3, 8-CH2CH3), 3.17 (t, J=8.4 Hz, 2H; 172-CH2),
3.16 (s, 3H; ring-CH3), 3.15 (s, 3H; ring-CH3), 3.14 (s, 3H; ring-CH3),
1.99 (m, 2H; CH2-Hexyl), 1.65–1.63 (m, 2H; CH2-Hexyl), 1.56 (m, 6H;
3-CH2CH3, 8-CH2CH3), 1.51–1.42 (m, 4H; 2 CH2-Hexyl), 0.96 ppm (t, J=
7.2 Hz, 3H; CH3-Hexyl); 13C NMR (100 MHz, CDCl3): d=173.9, 166.2,
163.1, 146.9, 145.4, 144.4, 143.2, 142.6, 142.4, 142.2, 141.1, 140.8, 140.4,
138.7, 136.9, 136.7, 136.5, 125.5, 101.7, 99.8, 98.7, 98.3, 51.5, 40.1, 36.3,
31.7, 29.6, 29.0, 27.1, 24.1, 22.7, 19.4, 19.3, 17.2, 14.1, 12.5, 11.7, 11.1,
11.0 ppm; UV/Vis (CH2Cl2): lmax (e)=647 nm (1.5ꢃ104 mÀ1 cmÀ1); MS
(EI): m/z): 719 [M+H]+; HRMS: calcd for C40H45N5NiO4: 717.2825;
found: 717.2300.
NiII 20-(tert-butyldimethylsilylacetylenyl)mesopurpurin-18-N-hexylimide
methyl ester (11): In a dry round-bottomed flask (50 mL), NiII 20-bromo-
N-hexylmesopurpurinimide (25.0 mg, 0.032 mmol), Cs2CO3 (10 mg,
0.032 mmol), tert-butyldimethylsilylacetylenylboronic acid (58 mg,
0.32 mmol), and [PdCl2ACTHNUTRGNE(UNG dppf)] (4.6 mg, 0.0064 mmol) were dissolved in
dry THF (15 mL). The resultant mixture was heated at reflux for 12 h.
The progress of the reaction was monitored by UV/Vis spectrophotome-
try and TLC. After completion of the reaction, the mixture was taken up
in CH2Cl2 (50 mL) and washed with brine. The organic layer was separat-
ed and dried over sodium sulfate. The crude residue obtained was puri-
fied by chromatography through a column of silica gel by using 0.5–1%
acetone/CH2Cl2 to obtain product 11 in good yield. Yield: 15.6 mg
(57%). 1H NMR (400 MHz, CDCl3): d=8.89 (s, 1H; meso-H), 8.55 (s,
1H; meso-H), 4.58 (dd, J=4.0, 9.2 Hz, 1H; 17-H), 4.50 (q, J=7.2 Hz,
1H; 18-H), 4.19–4.27 (m, 2H; N-CH2-Hexyl), 3.61 (s, 3H; CO2Me), 3.44
(s, 3H; ring-CH3), 3.33–3.40 (m, 4H; 8-CH2CH3, 3-CH2CH3), 3.12 (s, 3H;
ring-CH3), 2.92 (s, 3H; ring-CH3), 2.58 (t, J=7.2 Hz, 2H; 172-CH2), 2.16–
2.20 (m, 1H; 171-CHH), 1.90–1.96 (m, 1H; 171-CHH), 1.82 (m, 2H; CH2-
Hexyl), 1.49–1.52 (m, 8H; CH2-Hexyl, 8-CH2CH3, 3-CH2CH3), 1.42–1.46
(m, 4H; 2 CH2-Hexyl), 1.26 (d, J=6.8 Hz, 3H; 18-CH3), 1.07 (s, 9H; Si-
tert-Bu), 0.91 (t, J=6.8 Hz, 3H; CH3-Hexyl), 0.30 (s, 3H; Si-Me),
0.28 ppm (s, 3H; Si-Me); 13C NMR (100 MHz, CDCl3): d=173.7, 166.5,
164.4, 162.7, 161.4, 149.3, 148.7, 145.4, 143.8, 143.5, 141.0, 138.8, 138.7,
135.6, 135.2, 133.4, 126.4, 120.0, 109.1, 104.5, 103.5, 102.5, 98.4, 92.1, 53.0,
51.5, 48.4, 45.7, 39.9, 31.9, 31.7, 28.8, 28.4, 27.0, 26.2, 22.7, 19.1, 18.2, 17.1,
17.0, 16.8, 14.4, 14.1, 12.1, 10.6, À4.6 ppm; UV/Vis (CH2Cl2): lmax (e)=
670 nm (3.0ꢃ104 mÀ1 cmÀ1); MS (EI): m/z): 858 [M+H]+; HRMS: calcd
for C48H61N5NiO4Si+H: 858.3919; found: 858.3916.
NiII Bis(3,5-trifluoromethyl)benzylemeraldine methyl ester (9): In
a
100 mL dry round-bottomed flask, NiII mesopurpurin-N-hexylimide
(50.0 mg, 0.058 mmol) was dissolved in chloroform (30.0 mL). N-Iodosuc-
cinimide (15.6 mg, 0.069 mmol) and 2 drops of TFA were added and the
resultant mixture was stirred for 3–4 min at room temperature under N2.
The progress of the reaction was monitored by UV/Vis spectrophotome-
try and TLC. After completion of the reaction, saturated sodium bicar-
bonate (50.0 mL) was gradually added to the reaction mixture and then
extracted with dichloromethane (3ꢃ50.0 mL). The organic layers were
collected, combined, and washed with water (100.0 mL). The organic
layer was separated, dried over anhydr. Na2SO4, and concentrated. The
crude thus obtained was purified through a column of silica by using 1–
2% acetone/dichloromethane as eluent. Yield: 25.0 mg (50.2%).
1H NMR (400 MHz, CDCl3): d=9.38 (s, 1H; meso-H), 9.15 (s, 1H; meso-
H), 9.14 (s, 1H; meso-H), 8.19 (s, 2H; Ph-H), 7.81 (s, 1H; Ph-H), 5.65 (s,
2H; Benzylic-CH2), 3.83 (t, J=7.6 Hz, 2H; 171-CH2), 3.72 (s, 3H;
CO2Me), 3.65–3.61 (m, 4H; 3-CH2CH3, 8-CH2CH3), 3.57 (s, 3H; ring-
CH3), 3.21 (s, 3H; ring-CH3), 3.19 (s, 3H; ring-CH3), 3.17 (s, 3H; ring-
CH3), 2.98 (t, J=7.2 Hz, 2H; 172-CH2), 1.64–1.59 ppm (m, 6H; 3-
CH2CH3, 8-CH2CH3); 13C NMR (100 MHz, CDCl3): d=189.9, 173.5,
166.2, 162.8, 148.0, 146.1, 145.2, 145.0, 144.0, 143.4, 142.8, 141.7, 141.3,
140.9, 139.0, 137.6, 137.2, 131.8, 129.1, 125.1, 102.6, 100.6, 99.3, 97.5, 69.7,
51.4, 42.6, 36.0, 31.9, 29.7, 29.3, 24.3, 24.0, 22.7, 19.5, 17.3, 14.1, 12.8, 11.9,
11.2 ppm; UV/Vis (CH2Cl2): lmax (e)=655 nm (1.6ꢃ104 mÀ1 cmÀ1); MS
(EI): m/z): 861 [M+H]+; HRMS: calcd for C43H37F6N5NiO4: 859.2103;
found: 859.2101.
NiII
20-(6-dimethylaminopyridin-3-yl)mesopurpurin-18-N-hexylimide
methyl ester (12): Compound 12 was prepared by following the experi-
mental procedure for compound 10. Yield: 35.0 mg (67.8%). 1H NMR
(400 MHz, CDCl3): d=8.98 (s, 1H; meso-H), 8.64 (s, 1H; meso-H), 8.97,
8.26 (s, d, J=8.2 Hz, 1H; Py-H), 7.49, 6.88 (s, d, J=9.6 Hz, 1H; Py-H),
6.76, 6.48 (d, d, J=8.8, 8.8 Hz, 1H; Py-H), 4.52 (m, 1H; 17-H), 4.29–4.21
(m, 2H; N-CH2-Hexyl), 4.12, 3.74 (q, J=7.2 Hz, 1H; 18-H), 3.67 (s, 3H;
CO2Me), 3.48 (s, 3H; ring-CH3), 3.45–3.39 (m, 2H; 8-CH2CH3), 3.38–3.33
(m, 2H; 3-CH2CH3), 3.23 (s, 3H; Me-N-Me), 3.21 (s, 3H; Me-N-Me),
2.97 (s, 3H; ring-CH3), 2.69 (t, J=8.0 Hz, 2H; 172-CH2), 2.31 (m, 1H;
171-CHH), 2.15 (m, 1H; 171-CHH), 2.08, 2.07 (s, 3H; ring-CH3), 1.83 (m,
2H; -CH2-Hexyl), 1.50 (t, J=7.6 Hz, 3H; 8-CH2CH3), 1.47 (m, 2H;
-CH2-Hexyl), 1.45 (t, J=7.6 Hz, 3H; 3-CH2CH3), 1.38–1.36 (m, 4H; 2
CH2-Hexyl), 0.90 (t, J=6.8 Hz, 3H; CH3-Hexyl), 0.73 ppm (d, J=6.4 Hz,
3H; 18-CH3); 13C NMR (100 MHz, CDCl3, mixture of topoisomers): d=
NiII 20-methylmesopurpurinin-18-N-hexylimide methyl ester (10): In a
dry round-bottomed flask (100 mL), NiII 20-bromo-N-hexylmesopurpuri-
6680
www.chemeurj.org
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 6670 – 6684

Cyanine Dye–Purpurinimide Conjugates
FULL PAPER
174.0, 173.7, 166.8, 162.9, 161.5, 161.0, 158.8, 149.3, 149.0, 145.5, 143.6,
143.5, 143.2, 143.1, 140.7, 139.1, 138.6, 135.7, 135.2, 133.3, 127.8, 123.0,
122.8, 120.3, 114.6, 108.4, 103.2, 97.3, 53.5, 51.6, 45.1, 39.8, 32.5, 32.5, 32.4,
31.6, 29.6, 29.2, 28.8, 27.6, 27.0, 22.7, 19.0, 18.1, 17.0, 16.7, 14.6, 14.3, 14.0,
12.3, 10.6 ppm; UV/Vis (CH2Cl2): lmax (e)=677 nm (3.2ꢃ104 mÀ1 cmÀ1);
MS (EI): m/z: 840 [M]+, 862 [M+Na]+; HRMS: calcd for C47H55N7NiO4:
839.3669; found: 839.3672.
NiII 20-(3-carboxyphenyl)mesopurpurin-18-N-hexylimide methyl ester
(17): Compound 17 was prepared by following the experimental proce-
dure for compound 11. Yield: 12 mg (45%). 1H NMR (400 MHz, CDCl3,
mixture of both topoisomers) d=10.09, 9.84 (s, 1H; COOH), 8.93 (s, 1H;
meso-H), 8.63 (s, 1H; meso-H), 8.11, 7.66 (s, 1H; Ar-H), 8.06, 7.35 (d;
J=7.6, 1H, Ar-H), 7.88 (m, 1H; Ar-H), 7.51–7.68 (m, 1H; Ar-H) 4.56
(m, 1H; 17-H), 4.50 (m, 1H; 18-H), 4.23 (m, 2H; N-CH2-Hexyl), 3.64 (s,
3H; CO2Me), 3.44 (s, 3H; ring-CH3), 3.36–3.44 (m, 4H; 3-CH2CH3, 8-
CH2CH3), 3.09 (s, 3H; ring-CH3), 3.87 (s, 3H; ring-CH3), 2.67–2.69 (m,
2H; 172-CH2), 2.24 (m, 1H; 171-CHH), 2.04 (m, 1H; 171-CHH), 1.42–
1.53 (m, 10H; CH2-Hexyl, CH2-Hexyl, 8-CH2CH3, 3-CH2CH3), 1.34–1.40
(m, 4H; 2 CH2-Hexyl), 1.18 (d, J=6.8 Hz, 3H; 18-CH3), 0.88 ppm (t, J=
7.2 Hz, 3H; CH3-Hexyl); UV/Vis (CHCl3): lmax (e)=671 nm (3.3ꢃ
104 mÀ1 cmÀ1); MS (EI): m/z: 862 [M+Na]+; HRMS: calcd for
C50H51N5NiO6: 839.3193; found: 839.3277.
NiII 20-(4-aminophenyl)mesopurpurin-18-N-hexylimide methyl ester (14):
Compound 14 was prepared by following the experimental procedure for
compound 11. Yield: 15.7 mg (60%). 1H NMR (400 MHz, CDCl3): d=
8.99 (s, 1H; meso-H), 8.65 (s, 1H; meso-H), 7.97 (d, J=8.2 Hz, 1H; Ar-
H), 7.05 (d, J=9.6 Hz, 1H; Ar-H), 6.66 (d, J=8.8 Hz, 1H; Ar-H), 6.51
(d, J=8.8 Hz, 1H; Ar-H), 4.51 (m, 1H; 17-H), 4.22–4.29 (m, 3H; N-CH2-
Hexyl, 18-H), 3.47 (brs, 2H; NH2), 3.77 (s, 3H; CO2Me), 3.50 (s, 3H;
ring-CH3), 3.30–3.50 (m, 4H; 8-CH2CH3, 3-CH2CH3), 2.99 (s, 3H; ring-
CH3), 2.69 (t, J=8.0 Hz, 2H; 172-CH2), 2.32–2.35 (m, 1H; 171-CHH),
2.14–2.18 (m, 1H; 171-CHH), 2.04 (s, 3H; ring-CH3), 1.83–1.89 (m, 2H;
CH2-Hexyl), 1.27–1.55 (m, 12H; 8-CH2CH3, 3-CH2CH3, 3 CH2-Hexyl),
0.93 (t, J=6.8 Hz, 3H; CH3-Hexyl), 0.72 ppm (d, J=6.4 Hz, 3H; 18-
CH3); 13C NMR (100 MHz, CDCl3): d=173.7, 166.6, 164.9, 162.8, 162.3,
152.7, 149.2, 148.2, 147.6, 144.7, 141.7, 138.4, 138.8, 136.3, 135.0, 134.9,
134.6, 131.5, 119.6, 113.1, 109.3, 100.4, 100.1, 98.0, 95.0, 83.7, 52.8, 51.5,
48.0, 47.5, 40.1, 31.9, 31.6, 28.9, 28.7, 28.3, 27.0, 24.8, 22.6, 20.9, 19.0, 16.8,
16.7, 16.4, 14.0, 10.6 ppm; UV/Vis (CH2Cl2): lmax (e)=676 nm (3.2ꢃ
104 mÀ1 cmÀ1); MS (EI): m/z: 833.6 [M+Na]+; HRMS: calcd for
C46H52N6NiO4 +H: 811.3476; found: 811.3470.
NiII 20-(3-aminophenyl)mesopurpurin-18-N-hexylimide methyl ester (15):
Compound 15 was prepared by following the experimental procedure for
compound 11. Yield: 15.2 mg (59%). 1H NMR (400 MHz, CDCl3, mix-
ture of both topoisomers): d=8.97, 9.40 (s, 1H; meso-H), 8.64, 8.63 (s,
1H; meso-H), 7.58–7.60 (m, 0.95H; Ar-H), 7.48 (t, J=7.8 Hz, 0.40H; Ar-
H), 7.11 (t, J=7.2 Hz, 0.55H; Ar-H), 6.80–6.85 (m, 0.8H; Ar-H), 6.20 (d,
J=7.2 Hz, 0.40H; Ar-H), 6.07 (s, 0.35H; ArH), 4.42–4.62 (m, 1H; 17-H),
4.22–4.32 (m, 3H; N-CH2-Hexyl, 18-H), 3.71, 3.68 (s, 3H; CO2Me), 3.52
(s, 3H; ring-CH3), 3.31–3.57 (m, 4H; 8-CH2CH3, 3-CH2CH3), 2.97, 2.95
(s, 3H; ring-CH3), 2.63–2.70 (m, 2H; 172-CH2), 2.32–2.38 (m, 1H; 171-
CHH), 2.06–2.20 (m, 1H; 171-CHH, 3H; ring-CH3), 1.80–1.86 (m, 2H;
CH2-Hexyl), 1.22–1.61 (m, 12H; 8-CH2CH3, 3-CH2CH3, 3 CH2-Hexyl),
0.91 (t, J=6.8 Hz, 3H; CH3-Hexyl), 0.78, 0.76 ppm (d, J=6.6 Hz, 3H; 18-
CH3); 13C NMR (100 MHz, CDCl3, mixture of both topoisomers): d=
196.2, 173.4, 171.9, 156.1, 148.3, 144.4, 141.5, 141.1, 140.0, 139.6, 138.9,
136.7, 134.5, 134.3, 133.9, 133.6, 133.1, 133.0, 132.2, 132.1, 131.0, 127.2,
126.6, 115.4, 103.5, 98.7, 72.8, 69.6, 69.4, 52.0, 51.6, 48.6, 48.2, 44.6, 31.7,
31.6, 31.1, 30.2, 30.0, 29.8, 28.3, 26.0, 24.9, 24.5, 22.5, 21.0, 19.4, 17.3, 13.9,
13.8, 13.7, 13.4, 12.1, 11.3 ppm; UV/Vis (CH2Cl2): lmax (e)=675 nm (3.0ꢃ
104 mÀ1 cmÀ1); MS (EI): m/z: 833.6 [M+Na]+; HRMS: calcd for
C46H52N6NiO4 +H: 811.3476; found: 811.3487.
NiII 20-(4-carboxyphenyl)mesopurpurin-18-N-hexylimide methyl ester
(17a): Compound 17a was prepared by following the experimental pro-
1
cedure for compound 11. Yield: approx. 2 mg (5%). H NMR (400 MHz,
CDCl3): d=9.52 (s, 1H; meso-H), 9.30 (d, J=8.0 Hz, 1H; ArH), 8.98 (s,
1H; meso-H), 8.56 (d, J=8.6 Hz, 1H; Ar-H), 8.11 (d, J=8.0 Hz, 1H; Ar-
H), 7.68 (d, J=8.8 Hz, 1H; Ar-H), 4.60 (dd, J=4.3, 9.0 Hz, 1H; 17-H),
4.16–4.22 (m, 2H; N-CH2-Hexyl), 3.94–4.04 (m, 1H; 18-H), 3.55 (s, 3H;
CO2Me), 3.41 (s, 3H; ring CH3), 3.26–3.3.46 (m, 4H; 8-CH2CH3, 3-
CH2CH3), 2.94 (s, 3H; ring-CH3), 2.81 (s, 3H; ring-CH3), 2.43–2.61 (m,
2H; 172-CH2), 2.04–2.23 (m, 1H; 171-CHH), 1.69–1.88 (m, 1H; 171-
CHH), 1.22–1.51 (m, 17H; CH2-Hexyl, 18-CH3, 8-CH2CH3, 3 CH2-Hexyl,
3-CH2CH3), 0.82 ppm (t, J=6.8 Hz, 3H; CH3-Hexyl); 13C NMR
(100 MHz, CDCl3): d=189.8, 174.2, 166.1, 162.1, 161.7, 161.3, 150.2,
149.7, 148.3, 147.8, 142.8, 141.9, 137.7, 136.9, 135.1, 134.6, 132.3, 131.8,
129.8, 120.5, 113.2, 111.5, 105.0, 101.4, 99.8, 53.1, 51.6, 45.7, 40.0, 32.4,
31.8, 31.5, 29.6, 29.3, 28.6, 27.8, 26.9, 22.6, 22.5, 18.9, 18.5, 18.2, 16.7, 16.1,
14.0, 13.6, 10.4 ppm; UV/Vis (CHCl3): lmax (e)=672 nm (3.2ꢃ
104 mÀ1 cmÀ1); MS (EI): m/z: 862 [M+Na]+; HRMS: calcd for
C50H51N5NiO6: 839.3193; found: 839.3166.
NiII 20-(4-formylphenyl)- mesopurpurin-18-N-hexylimide methyl ester
(18): Compound 18 was prepared by following the experimental proce-
dure for compound 11. Yield: 20.3 mg (77%). 1H NMR (400 MHz,
CDCl3): d=10.19 (s, 1H; CHO), 9.00 (s, 1H; meso-H), 8.69 (s, 1H;
meso-H), 8.49 (d, J=8.0 Hz, 1H; Ph-H), 8.28 (d, J=7.6 Hz, 1H; Ph-H),
7.88 (d, J=7.2 Hz, 1H; Ph-H), 6.96 (d, J=7.2 Hz, 1H; Ph-H), 4.51 (dd,
J=3.6, 9.2 Hz, 1H; 17-H), 4.24 (m, 2H; N-CH2-Hexyl), 4.11 (q, J=
6.8 Hz, 1H; 18-H), 3.68 (s, 3H; CO2Me), 3.49 (s, 3H; ring-CH3), 3.42–
3.48 (m, 2H; 8-CH2CH3), 3.29–3.41 (m, 2H; 3-CH2CH3), 2.98 (s, 3H;
ring-CH3), 2.66–2.71 (m, 2H; 172-CH2), 2.33 (m, 1H; 171-CHH), 2.18 (m,
1H; 171-CHH), 1.90 (s, 3H; ring-CH3), 1.83 (m, 2H; CH2-Hexyl), 1.54–
1.56 (m, 2H; CH2-Hexyl), 1.52 (t, J=7.6 Hz, 3H; 8-CH2CH3), 1.45 (t, J=
6.8 Hz, 3H; 3-CH2CH3), 1.33–1.37 (m, 4H; 2 CH2-Hexyl), 0.91 (t, J=
7.2 Hz, 3H; CH3-Hexyl), 0.68 ppm (d, J=6.8 Hz, 3H; 18-CH3); 13C NMR
(100 MHz, CDCl3): d=191.8, 173.9, 166.6, 162.8, 161.2, 156.8, 149.3,
147.8, 146.3, 146.0, 143.7, 143.0, 141.1, 138.9, 138.8, 136.0, 135.6, 134.2,
133.6, 133.3, 132.7, 129.4, 120.6, 114.0, 110.4, 108.5, 103.9, 97.4, 53.7, 51.6,
45.0, 39.8, 32.5, 31.6, 28.8, 27.5, 27.0, 22.6, 19.0, 19.1, 18.1, 17.0, 16.7, 14.0,
13.6, 12.3, 10.6 ppm; UV/Vis (CH2Cl2): lmax (e)=676 nm (3.2ꢃ
104 mÀ1 cmÀ1); MS (EI): m/z: 824 [M]+; HRMS: calcd for C40H47N5NiO4:
823.3244; found: 823.9922.
NiII
20-(4-tert-butoxycarbonylphenyl)mesopurpurin-18-N-hexylimide
methyl ester (16): Compound 16 was prepared by following the experi-
mental procedure for compound 11. Yield: 18.6 mg (65%). 1H NMR
(400 MHz, CDCl3): d=9.00 (s, 1H; meso-H), 8.68 (s, 1H; meso-H), 8.48
(d, J=8.2 Hz, 1H; Ar-H), 8.39 (d, J=9.6 Hz, 1H; Ar-H), 7.90 (d, J=
8.8 Hz, 1H; Ar-H), 6.89 (d, J=8.8 Hz, 1H; Ar-H), 5.50 (dd, J=4.0,
9.2 Hz, 1H; 17-H), 4.20–4.25 (m, 2H; N-CH2-Hexyl), 4.10–4.14 (m, 1H;
18-H), 3.68 (s, 3H; CO2Me), 3.49 (s, 3H; ring CH3), 3.26–3.45 (m, 4H; 8-
CH2CH3, 3-CH2CH3), 2.99 (s, 3H; ring-CH3), 2.63–2.71 (m, 2H; 172-
CH2), 2.31–2.34 (m, 1H; 171-CHH), 2.13–2.18 (m, 1H; 171-CHH), 1.91 (s,
3H; ring-CH3), 1.82 (m, 2H; CH2-Hexyl), 1.20–1.67 (m, 21H; 8-CH2CH3,
3 CH2-Hexyl, 3-CH2CH3, t-Butyl), 0.90 (t, J=6.8 Hz, 3H; CH3-Hexyl),
0.67 ppm (d, J=6.4 Hz, 3H; 18-CH3); 13C NMR (100 MHz, CDCl3): d=
189.9, 173.8, 166.2, 165.7, 161.8, 161.6, 150.5, 150.0, 148.4, 147.8, 143.1,
141.9, 137.8, 137.2, 135.0, 134.5, 134.2, 132.3, 131.8, 129.3, 128.3, 120.6,
113.3, 111.4, 101.5, 99.8, 84.0, 81.5, 81.0, 53.3, 51.6, 45.7, 40.1, 32.3, 31.6,
28.7, 28.1, 27.8, 26.9, 24.8, 22.6, 19.0, 18.9, 18.2, 16.7, 16.4, 14.0, 13.9,
10.5 ppm; UV/Vis (CHCl3): lmax (e)=672 nm (3.2ꢃ104 mÀ1 cmÀ1); MS
(EI): m/z: 896 [M+H]+; HRMS: calcd for C51H59N5NiO6 +H: 896.3892;
found: 896.4222.
NiII 20-(3-formylphenyl)mesopurpurin-18-N-hexylimide methyl ester
(19): Compound 19 was prepared by following the experimental proce-
dure for compound 11. Yield: 15.3 mg (58%). 1H NMR (400 MHz,
CDCl3, mixture of both topoisomers): d=10.36, 9.94 (s, d, J=7.2 Hz, 1H;
CHO), 9.00 (s, 1H; meso-H), 8.80, 8.56 (s, d, J=7.6 Hz, 1H; Ph-H), 8.68
(s, 1H; meso-H), 8.08 (d, J=6.8 Hz, 1H; Ph-H), 7.95, 7.53 (t, t, J=8.0,
8.0 Hz, 1H; Ph-H), 7.28, 7.06 (s, d, J=6.8 Hz, 1H; Ph-H), 4.50 (m, 1H;
17-H), 4.24 (m, 2H; N-CH2-Hexyl), 4.09 (m, 1H; 18-H), 3.68 (s, 3H;
CO2Me), 3.49 (s, 3H; ring-CH3), 3.46–3.41 (m, 2H; 8-CH2CH3), 3.39–3.30
(m, 2H; 3-CH2CH3), 2.98 (s, 3H; ring-CH3), 2.71–2.66 (m, 2H; 172-CH2),
2.32 (m, 1H; 171-CHH), 2.19 (m, 1H; 171-CHH), 1.88 (s, 3H; ring-CH3),
1.82 (m, 2H; CH2-Hexyl), 1.53 (m, 2H; CH2-Hexyl), 1.52 (t, J=7.6 Hz,
3H; 8-CH2CH3), 1.44 (t, J=7.6 Hz, 3H; 3-CH2CH3), 1.38–1.33 (m, 4H; 2
Chem. Eur. J. 2013, 19, 6670 – 6684
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6681

K. M. Kadish, R. K. Pandey et al.
CH2-Hexyl), 0.90 (t, J=7.2 Hz, 3H; CH3-Hexyl), 0.67 ppm (d, J=6.8 Hz,
3H; 18-CH3); 13C NMR (100 MHz, CDCl3, mixture of both topoisomers):
d=192.4, 191.8, 174.0, 166.7, 162.8, 149.3, 148.1, 146.0, 143.7, 140.9, 138.9,
138.1, 137.7, 136.4, 134.2, 133.8, 133.5, 129.0, 128.8, 121.1, 127.8, 114.6,
110.2, 108.2, 103.8, 53.9, 53.7, 52.8, 51.6, 45.0, 39.8, 32.5, 31.6, 29.6, 28.8,
27.3, 27.0, 22.6, 19.0, 18.1, 17.0, 16.7, 14.0, 13.9, 12.3, 10.6 ppm; UV/Vis
(CH2Cl2): lmax (e)=675 nm (3.2ꢃ104 mÀ1 cmÀ1); MS (EI): m/z: 824 [M]+;
HRMS: calcd for C40H47N5NiO4: 823.3244; found: 823.1112.
procedure for compound 11. Yield: 18.3 mg (66%). 1H NMR (400 MHz,
CDCl3): d=8.98 (s, 1H; meso-H), 8.65 (s, 1H; meso-H), 8.15 (d, J=
8.2 H, 1H; Ar-H), 7.73 (d, J=9.6 Hz, 1H; Ar-H), 7.34 (d, J=8.8 Hz, 1H;
Ar-H), 6.66 (d, J=8.8 Hz, 1H; Ar-H), 4.50 (dd, J=4.0, 9.2 Hz, 1H; 17-
H), 4.16–4.24 (m, 3H; 18-H, N-CH2-Hexyl), 3.66 (s, 3H; CO2Me), 3.49 (s,
3H; ring CH3), 3.30–3.55 (m, 4H; 8-CH2CH3, 3-CH2CH3), 2.98 (s, 3H;
ring-CH3), 2.63–2.70 (m, 2H; 172-CH2), 2.28–2.36 (m, 1H; 171-CHH),
2.10–2.17 (m, 1H; 171-CHH), 1.95 (s, 3H; ring-CH3), 1.81–1.85 (m, 2H;
CH2-Hexyl), 1.37–1.53 (m, 19H; 8-CH2CH3, (CH3)2H-S, (CH3)2H-S, 3
CH2-Hexyl, 3-CH2CH3), 0.96 (t, J=6.8 Hz, 3H; CH3-Hexyl), 0.69 ppm
(d, J=6.4 Hz, 3H; 18-CH3); 13C NMR (100 MHz, CDCl3): d=174.0,
166.7, 162.9, 161.2, 157.7, 149.6, 148.5, 145.6, 143.6, 143.1, 140.9, 139.0,
138.7, 137.9, 135.8, 135.6, 134.9, 133.4, 132.9, 132.2, 131.1, 130.9, 120.4,
111.1, 108.3, 103.3, 97.3, 53.6, 51.6, 45.0, 39.8, 38.0, 32.2, 31.7, 28.8, 27.6,
27.0, 22.7, 19.0, 18.8, 17.9, 17.0, 16.8, 15.2, 14.0, 12.2, 10.5 ppm; UV/Vis
(CHCl3): lmax (e)=672 nm (3.2ꢃ104 mÀ1 cmÀ1); MS (EI): m/z: 870 [M]+;
HRMS: calcd for C49H57N5NiO4S+H: 870.3557; found: 870.3492.
NiII 20-(4-aminomethylphenyl)mesopurpurin-18-N-hexylimide methyl
ester (26a): Compound 20 (20 mg, 0.021 mmol) was treated with TFA/
CH2Cl2 (9:1, 5 mL) in an inert atmosphere for 6 h. The reaction mixture
was concentrated under vacuum and the resulting crude product was pu-
rified through a column of silica gel to provide pure 26a. Yield: 11.7 mg
(66%). 1H NMR (400 MHz, CDCl3): d=8.41 (s, 1H; meso-H), 8.40 (s,
1H; meso-H), 8.37 (d, J=8.2 Hz, 1H; Ar-H), 7.87 (d, J=9.6 Hz, 1H; Ar-
H), 7.43 (d, J=8.8 Hz, 1H; Ar-H), 6.87 (d, J=8.8 Hz, 1H; Ar-H), 4.28
(m, 3H; 17-H, Benzyl-CH2), 4.08 (q, J=6.8 Hz, 1H; 18-H), 3.98 (s, 2H;
NH2), 4.22–4.29 (m, 2H; N-CH2-Hexyl), 3.67 (s, 3H; CO2Me), 3.11–
3.3.30 (m, 2H; 8-CH2CH3), 2.86–3.00 (m, 5H; 3-CH2CH3, ring-CH3), 2.83
(s, 3H; ring-CH3), 2.56 (t, J=8.0 Hz, 2H; 172-CH2), 2.04–2.16 (m, 2H;
171-CHH, 171-CHH), 1.88 (s, 3H; ring-CH3), 1.61 (m, 2H; CH2-Hexyl),
1.27–1.55 (m, 9H; 8-CH2CH3, 3 CH2-Hexyl), 1.20 (t, J=7.6 Hz, 3H; 3-
CH2CH3), 0.88 (t, J=6.8 Hz, 3H; CH3-Hexyl), 0.59 ppm (d, J=6.4 Hz,
3H; 18-CH3); 13C NMR (100 MHz, CDCl3): d=175.5, 174.2, 167.5, 163.3,
154.0, 150.5, 145.2, 145.0, 142.1, 140.8, 139.5, 137.1, 136.4, 134.3, 133.9,
132.6, 132.4, 131.3, 127.5, 127.0, 116.2, 112.3, 106.5, 102.1, 97.7, 55.6, 51.5,
47.1, 40.4, 32.8, 31.7, 30.0, 29.6, 29.3, 28.9, 27.1, 22.7, 22.6, 20.7, 19.3, 19.1,
17.4, 16.7, 14.0, 13.2, 12.4, 11.0 ppm; UV/Vis (CHCl3): lmax (e)=672 nm
(3.1ꢃ104 mÀ1 cmÀ1); MS (EI): m/z: 825 [M+H]+; HRMS: calcd for
C47H54N6NiO4 +H: 825.3633; found: 825.3649.
NiII
20-(N-Boc-4-aminomethylphenyl)mesopurpurin-18-N-hexylimide
methyl ester (20): Compound 20 was prepared by following the experi-
mental procedure for compound 11. Yield: 20.1 mg (68%). 1H NMR
(400 MHz, CDCl3): d=8.97 (s, 1H; meso-H), 8.63 (s, 1H; meso-H), 8.16
(d, J=8.2 Hz, 1H; Ar-H), 7.60 (d, J=9.6 Hz, 1H; Ar-H), 7.22 (d, J=
8.8 Hz, 1H; Ar-H), 6.88 (d, J=8.8 Hz, 1H; Ar-H), 5.31 (brs, 1H; NH),
4.47 (m, 3H; 17-H, Benzyl-CH2), 4.22–4.29 (m, 3H; 18-H, N-CH2-Hexyl),
3.64 (s, 3H; CO2Me), 3.46 (s, 3H; ring-CH3), 3.26–3.44 (m, 4H; 8-
CH2CH3, 3-CH2CH3), 2.96 (s, 3H; ring-CH3), 2.61–2.68 (m, 2H; 172-
CH2), 2.26–2.31 (m, 1H; 171-CHH), 2.10–2.17 (m, 1H; 171-CHH), 1.87 (s,
3H; ring-CH3), 1.81 (m, 2H; -CH2-Hexyl), 1.35–1.44 (m, 21H; 8-
CH2CH3, 3 CH2-Hexyl, 3-CH2CH3, t-Butyl-Me3), 0.88 (t, J=6.8 Hz, 3H;
CH3-Hexyl), 0.65 ppm (d, J=6.4 Hz, 3H; 18-CH3); 13C NMR (100 MHz,
CDCl3): d=190, 173.8, 166.2, 163.0, 162.3, 161.7, 152.6, 150.1, 148.5,
147.7, 143.3, 141.9, 138.4, 137.9, 135.1, 134.4, 133.0, 132.9, 132.2, 120.4,
117.9, 117.7, 115.9, 114.6, 113.1, 111.8, 105.0, 101.0, 99.8, 53.0, 51.6, 50.5,
45.7, 40.1, 32.3, 31.8, 31.6, 29.6, 29.3, 28.7, 28.3, 27.8, 26.9, 22.6, 19.0, 18.2,
16.7, 16.3, 14.0, 13.8, 10.5 ppm; UV/Vis (CHCl3): lmax (e)=673 nm (3.1ꢃ
104 mÀ1 cmÀ1); MS (EI): m/z: 925 [M+H]+; HRMS: calcd for
C52H62N6NiO6 +H: 925.4157; found: 925.4136.
NiII 20-(4-hydroxyphenyl)mesopurpurin-18-N-hexylimide methyl ester
(21): Compound 21 was prepared by following the experimental proce-
dure for compound 11.Yield: 15.8 mg (61%). 1H NMR (400 MHz,
CDCl3): d=8.98 (s, 1H; meso-H), 8.64 (s, 1H; meso-H), 8.05 (d, J=
8.2 Hz, 1H; Ar-H), 7.19 (d, J=9.6 Hz, 1H; Ar-H), 6.80 (d, J=8.8 Hz,
1H; Ar-H), 6.58 (d, J=8.8 Hz, 1H; Ar-H), 5.44 (brs, 1H; OH), 4.49 (dd,
J=4.0, 9.2 Hz, 1H; 17-H), 4.14–4.29 (m, 3H; 18-H, N-CH2-Hexyl), 3.66
(s, 3H; CO2Me), 3.48 (s, 3H; ring CH3), 3.26–3.45 (m, 4H; 8-CH2CH3, 3-
CH2CH3), 2.97 (s, 3H; ring-CH3), 2.62–2.71 (m, 2H; 172-CH2), 2.28–2.34
(m, 1H; 171-CHH), 2.04–2.17 (m, 1H; 171-CHH), 1.96 (s, 3H; ring-CH3),
1.83 (m, 2H; CH2-Hexyl), 1.38–1.49 (m, 12H; 8-CH2CH3, 3 CH2-Hexyl,
3-CH2CH3), 0.89 (t, J=6.8 Hz, 3H; CH3-Hexyl), 0.69 ppm (d, J=6.4 Hz,
3H; 18-CH3); 13C NMR (100 MHz, CDCl3): d=174.0, 166.7, 162.9, 161.2,
158.4, 155.3, 149.0, 145.4, 143.6, 143.1, 140.8, 139.2, 138.6, 135.7, 135.3,
133.3, 133.0, 132.0, 127.8, 120.2, 114.7, 111.3, 108.3, 105.0, 103.1, 97.2,
83.5, 53.6, 51.6, 45.0, 39.8, 32.5, 31.6, 28.8, 27.6, 27.0, 24.8, 22.6, 19.0, 18.2,
17.0, 16.7, 14.0, 13.6, 12.3, 10.6 ppm; UV/Vis: lmax (e)=671 nm (3.6ꢃ
104 mÀ1 cmÀ1); MS (EI): m/z: 812 [M+H]+; HRMS: calcd for
C46H51N5NiO5 +H: 812.3316; found: 812.3313.
NiII 20-(4-hydroxymethylphenyl)mesopurpurin-18-N-hexylimide methyl
ester (22): Compound 22 was prepared by following the experimental
procedure for compound 11. Yield: 12.6 mg (48%). 1H NMR (400 MHz,
CDCl3): d=9.06 (s, 1H; meso-H), 8.68 (s, 1H; meso-H), 8.22 (d, J=
8.2 Hz, 1H; Ar-H), 7.84 (d, J=9.6 Hz, 1H; Ar-H), 7.38 (d, J=8.8 Hz,
1H; Ar-H), 6.78 (d, J=8.8 Hz, 1H; Ar-H), 4.90 (s, 2H; Benzyl-CH2),
4.50 (dd, J=4.0, 9.2 Hz, 1H; 17-H), 4.14–4.31 (m, 3H; 18-H, N-CH2-
Hexyl), 3.65 (s, 3H; CO2Me), 3.51 (s, 3H; ring CH3), 3.24–3.50 (m, 4H;
8-CH2CH3, 3-CH2CH3), 2.99 (s, 3H; ring-CH3), 2.60–2.74 (m, 2H; 172-
CH2), 2.30–2.38 (m, 1H; 171-CHH), 2.10–2.20 (m, 1H; 171-CHH), 1.98 (s,
3H; ring-CH3), 1.80–1.94 (m, 2H; CH2-Hexyl), 1.32–1.60 (m, 12H; 8-
CH2CH3, 3 CH2-Hexyl, 3-CH2CH3), 0.96 (t, J=6.8 Hz, 3H; CH3-Hexyl),
0.64 ppm (d, J=6.4 Hz, 3H; 18-CH3); 13C NMR (100 MHz, CDCl3): d=
173.9, 166.7, 161.2, 157.8, 149.0, 148.6, 145.5, 143.6, 143.1, 140.8, 140.5,
139.1, 139.0, 138.7, 135.8, 135.0, 133.3, 132.5, 132.0, 126.6, 120.3, 115.2,
111.5, 108.3, 103.3, 97.2, 65.0, 53.6, 51.5, 45.0, 39.8, 32.5, 31.6, 29.6, 28.8,
27.5, 27.0, 22.6, 19.0, 18.2, 17.0, 16.7, 14.0, 13.5, 12.3, 10.6 ppm; UV/Vis
(CHCl3): lmax (e)=671 nm (3.3ꢃ104 mÀ1 cmÀ1); MS (EI): m/z: 826
[M+H]+; HRMS: calcd for C47H53N5NiO5 +H: 826.3473; found: 826.3482.
20-(4-aminomethylphenyl) mesopurpurin-18-N-hexylimide methyl ester
(26b): Compound 26b was prepared by treating compound 26a (25 mg,
0.030 mmol) with conc. H2SO4 (4 mL) in an ice-cold bath and stirring at
room temperature for 30 min. The crude mixture was poured into ice-
cold water (25 mL) and extracted with dichloromethane (2ꢃ50 mL). The
resulting organic layer was washed with a sat. bicarbonate solution and
then water. The crude product was obtained by evaporating the organic
layer, which was purified by silica gel column chromatography. Yield:
9.7 mg (42%). 1H NMR (400 MHz, CDCl3): d=9.56 (s, 1H; meso-H),
9.28 (s, 1H; meso-H), 8.13 (d, J=8.2 Hz, 1H; Ar-H), 7.68 (d, J=9.6 Hz,
1H; Ar-H), 7.45 (d, J=8.8 Hz, 1H; Ar-H), 7.33 (d, J=8.8 Hz, 1H; Ar-
H), 5.16 (m, 1H; 17-H), 4.39–4.44 (m, 2H; N-CH2-Hexyl), 4.22 (q, J=
7.2 Hz, 1H; 18-H), 4.04 (brs, 2H; NH2), 3.81 (s, 2H; Benzyl-CH2), 3.61–
3.67 (m, 4H; 8-CH2CH3, 3-CH2CH3), 3.56 (s, 3H; CO2Me), 3.18 (s, 3H;
ring-CH3), 2.69–2.76 (m, 1H; 171-CHH), 2.25–2.45 (m, 3H; 172-CH2, 171-
CHH), 2.16 (s, 3H; ring-CH3), 2.13 (s, 3H; ring-CH3), 1.93–2.09 (m, 4H;
2 CH2-Hexyl), 1.66 (t, J=7.1 Hz, 3H; 3-CH2CH3), 1.54–1.58 (m, 5H; 8-
CH2CH3, CH2-Hexyl), 1.24–1.49 (m, 2H; CH2-Hexyl), 0.85–0.94 (m, 6H;
CH3-Hexyl, 18-CH3), 0.31 (brs, 1H), À0.33 ppm (brs, 1H); 13C NMR
(100 MHz, CDCl3): d=172.2, 166.7, 162.9, 157.8, 156.0, 150.5, 145.2,
145.0, 142.1, 140.8, 139.5, 137.1, 136.4, 134.3, 133.9, 132.6, 132.4, 131.3,
127.5, 127.0, 116.2, 112.3, 106.5, 104.2, 102.1, 97.7, 53.6, 51.5, 45.1, 39.8,
32.5, 31.7, 29.6, 28.8, 27.1, 22.7, 22.6, 20.7, 19.3, 19.1, 17.4, 16.7, 14.0, 13.2,
12.3, 10.6 ppm; UV/Vis (CH2Cl2): lmax (e)=705 nm (5.1ꢃ104 mÀ1 cmÀ1);
MS (EI): m/z: 769 [M+H]+; HRMS: calcd for C47H56N6O4 +H: 769.4436;
found: 769.4451.
NiII
aminomethylphen
20 mg, 0.1 equiv) was placed in a dry round-bottomed flask (50.0 mL)
purpurinimide–cyanine
dye
conjugate
27:
NiII
20-(4-
NiII 20-(4-isopropylthiophenyl)mesopurpurin-18-N-hexylimide methyl
ester (23): Compound 23 was prepared by following the experimental
ACHTUNGTRENNUNG
6682
www.chemeurj.org
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 6670 – 6684

Cyanine Dye–Purpurinimide Conjugates
FULL PAPER
and dissolved in anhydr. DMF (10 mL). Cyanine dye 25 (0.12 equiv),
Photostability measurements: In brief, conjugates 27 and 28 were formu-
lated in 1% Tween 80 in D5W and then diluted in 17% bovine calf
serum (BCS) to 5 mm. Changes in the concentration of the cyanine dye
portion of the conjugates were measured by a spectrophotometer at user-
specified intervals post-irradiation. The solutions were placed individually
in fluorescent cuvettes and exposed to radiation at 705 nm (28) and 677
(27) nm while being magnetically stirred to ensure the homogeneous dis-
tribution of singlet oxygen. An argon-pumped dye laser was used with a
power output of 530 mW for a 3 cm spot size. This yields 75 mWcmÀ2 at
the center plane of the cuvette. The decrease in the absorbance spectra
during irradiation was predominantly attributed to photodegradation
(photobleaching).
N-ethyl-N’-(3-di
G
N
hydrochloride
(0.15 equiv), and 4-di
N
ACHTUNGTRENNUNG
added and the resultant mixture was stirred for 12 h at room temperature
under N2. The reaction mixture was evaporated under vacuum and then
the crude product obtained was purified through a column of silica gel by
using 5–20% methanol/dichloromethane as eluent to yield 35%
(14.8 mg) of the desired conjugate. 1H NMR (400 MHz, 20% CD3OD/
5% [D6]DMSO/CDCl3): d=8.84 (s, 1H; meso-H), 8.68 (d, J=14 Hz,
2H), 8.58 (s, 1H; meso-H), 7.95–7.99 (m, 3H), 7.87 (d, J=9.6 Hz, 2H),
7.55–7.59 (m, 3H), 7.46 (d, J=8.8 Hz, 2H), 7.33–7.38 (6H), 7.01–7.05 (m,
3H), 6.30 (d, J=8.2 Hz, 2H), 6.05 (d, J=8.4 Hz, 2H), 4.60 (s, 2H;
Benzyl-CH2), 4.06–4.21 (m, 7H), 3.91 (q, J=7.1 Hz, 1H), 3.51 (s, 3H;
CO2Me), 3.32 (m, 8H), 2.81–2.84 (m, 12H), 2.61 (d, J=6.4 Hz, 3H), 2.44
(t, J=8.0 Hz, 3H), 2.15 (s, 3H), 2.13 (s, 3H), 2.04–2.16 (m, 2H), 1.86 (m,
12H), 1.73 (m, 2H), 1.64 (s, 3H), 1.42–1.58 (m, 10H), 0.89 (m, 6H),
0.26 ppm (d, J=6.4 Hz, 3H; 18-CH3); 13C NMR (150 MHz, 20%
CD3OD/5% [D6]DMSO/CDCl3): d=174.8, 174.3, 168.0, 167.3, 163.9,
161.9, 158.3, 149.8, 149.6, 145.8, 145.4, 144.4, 143.3, 142.2, 139.9, 139.75,
139.70, 139.4, 138.9, 138.7, 136.1, 135.7, 134.2, 134.1, 134.0, 132.8, 132.4,
132.2, 131.6, 131.2, 130.4, 128.8, 128.5, 128.0, 127.5, 127.2, 126.3, 125.6,
122.4, 120.3, 112.0, 110.9, 109.1, 103.8, 101.2, 54.1, 51.9, 51.4, 50.8, 45.4,
44.7, 43.5, 40.2, 32.8, 32.0, 29.1, 27.8, 27.6, 27.3, 26.8, 26.7, 23.0, 22.8, 21.1,
19.3, 19.2, 18.3, 17.2, 16.8, 14.1, 13.6, 12.4, 10.7 ppm; UV/Vis (MeOH):
lmax (e)=677 (3.2ꢃ104), 835 nm (19.2ꢃ104 mÀ1 cmÀ1); MS (EI): m/z: 1751
[MÀH]+; HRMS: calcd for C100H108N8NiO11S3: 1750.6653; found:
1750.6611.
In vitro PDT efficacy: The photosensitizing activities of the drugs 27 and
28 were determined in the Colon 26 tumor cell line as previously descri-
bed.[46] The Colon 26 tumor cells were grown in RPMI 1640 medium with
10% fetal calf serum, 1% l-glutamine, 1% penicillin, and 1% strepto-
mycin. Cells were maintained in 5% CO2/95% air and 100% humidity.
To determine the PDT efficacy, these cells were placed in 96-well plates
at a density of 5ꢃ103 cells per well in complete media. After overnight
incubation at 378C, the photosensitizers were added at variable concen-
trations (from 0.0193 to 6 mm) and incubated at 378C for 24 h in the dark.
Prior to light treatment, the cells were replaced with drug-free complete
media. The cells were then illuminated with an argon-pumped dye laser
set at 706 nm for drug 27 and at 680 nm for drug 28 at a dose rate of
3.2 mWcmÀ2 with variable light doses (0–2 JcmÀ2). After PDT, the cells
were incubated for 48 h at 378C in the dark. Following incubation, a
4.0 mgmLÀ1 solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide (MTT; 10 mL) dissolved in PBS (Sigma, St. Louis, MO) was
added to each well. After 4 h of further incubation at 378C, the MTT and
media were removed and DMSO (100 mL; Sigma, St Louis, MO) was
added to solubilize the formazine crystals. The 96-well plate was then an-
alyzed with a microtiter plate reader (BioTek Instruments, Inc., ELx800
Absorbance Microplate Reader) at an absorbance of 570 nm. The results
were plotted as percent survival of the corresponding dark (drug, no
light) control for each compound tested (see Figure 10). Each data point
represents the mean of three separate experiments, and the error bars
are the standard deviation. Each experiment was performed with six rep-
licate wells.
Purpurinimide–cyanine dye conjugate 28: Compound 28 was prepared by
following the experimental procedure for compound 27. Yield: 13.6 mg
1
(31%). H NMR (400 MHz, 20% CD3OD/CDCl3): d=8.80 (s, 1H; meso-
H), 8.68 (s, 1H; meso-H), 8.52 (d, J=14 Hz, 2H), 7.95–7.99 (m, 3H), 7.87
(d, J=9.6 Hz, 2H), 7.55–7.59 (m, 3H), 7.46 (d, J=8.8 Hz, 2H), 7.33–7.38
(6H), 7.01–7.05 (m, 3H), 6.30 (d, J=8.2 Hz, 2H), 6.05 (d, J=8.4 Hz,
2H), 4.60 (s, 2H; Benzyl-CH2), 4.06–4.21 (m, 7H), 3.91 (q, J=7.1 Hz,
1H), 3.51 (s, 3H; CO2Me), 3.32 (m, 8H), 2.81–2.84 (m, 12H), 2.61 (d, J=
7.0 Hz, 3H), 2.44 (t, J=8.0 Hz, 3H), 2.15 (s, 3H), 2.13 (s, 3H), 2.04–2.16
(m, 2H), 1.86 (m, 12H), 1.73 (m, 2H), 1.64 (s, 3H), 1.42–1.58 (m, 14H),
0.89 (m, 6H), 0.26 (d, J=6.4 Hz, 3H; 18-CH3), 0.22 (brs, 1H; NH),
À0.20 ppm (brs, 1H; NH) ppm; UV/Vis (MeOH): lmax (e)=705 (5.1ꢃ
104), 835 nm (19.3ꢃ104 mÀ1 cmÀ1); MS (EI): m/z: 1694 [MÀH]+; HRMS:
calcd for C100H110N8O11S3: 1694.7456; found: 1694.7511.
NiII 20-(4-maleimidophenyl)mesopurpurin-18-N-hexylimide methyl ester
(30): The aminophenylpurpurin 14 (25 mg, 0.030 mmol) was heated at
reflux with maleic anhydride (15 mg, 0.15 mmol) in acetic anhydride
(5 mL) and stirred at room temperature for 12 h. The resulting mixture
was treated with NaOAc in AcOH and then heated at reflux for 3 h. The
resulting crude product was diluted with dichloromethane and the mix-
ture was neutralized with a sat. bicarbonate solution. The organic layer
was concentrated and the crude obtained was purified on a silica gel
column by elution with 30% EtOAc/hexane as eluent to obtain the de-
sired maleimide derivative 30. Yield: 12.8 mg (48%). 1H NMR
(400 MHz, CDCl3): d=8.97 (s, 1H; meso-H), 8.65 (s, 1H; meso-H), 8.32
(d, J=8.2 Hz, 1H; Ar-H), 7.76 (d, J=9.6 Hz, 1H; Ar-H), 7.36 (d, J=
8.8 Hz, 1H; Ar-H), 6.91 (s, 2H; CO-CH=CH-CO), 6.82 (d, J=8.8 Hz,
1H; Ar-H), 4.50 (m, 1H; 17-H), 4.16–4.25 (m, 3H; N-CH2-Hexyl, 18-H),
3.65 (s, 3H; CO2Me), 3.48 (s, 3H; ring-CH3), 3.27–3.50 (m, 4H; 8-
CH2CH3, 3-CH2CH3), 2.96 (s, 3H; ring-CH3), 2.67 (t, J=8.0 Hz, 2H; 172-
CH2), 2.26–2.36 (m, 2H; 171-CHH, 171-CHH), 1.93 (s, 3H; ring-CH3),
1.77–1.85 (m, 2H; CH2-Hexyl), 1.23–1.53 (m, 10H; 8-CH2CH3, 3-
CH2CH3, 2 CH2-Hexyl), 0.82–0.92 (m, 5H; CH3-Hexyl, CH2-Hexyl),
0.69 ppm (d, J=6.4 Hz, 3H; 18-CH3); 13C NMR (100 MHz, CDCl3): d=
174.0, 172.0, 166.7, 162.8, 161.3, 157.4, 149.2, 145.8, 143.6, 143.0, 140.3,
139.0, 135.9, 134.7, 134.2, 133.5, 133.1, 132.5, 130.9, 127.9, 126.3, 120.5,
110.5, 108.4, 103.6, 97.3, 67.7, 53.8, 51.6, 45.0, 39.8, 35.8, 32.5, 31.6, 29.6,
28.8, 27.6, 27.0, 22.6, 20.5, 19.0, 18.2, 17.0, 16.7, 14.0, 13.6, 12.3, 10.6 ppm;
UV/Vis (CH2Cl2): lmax (e)=671 nm (3.8ꢃ104 mÀ1 cmÀ1); MS (EI): m/z:
891 [M+H]+; HRMS: calcd for C50H52N6NiO6 +H: 891.3375; found:
891.3369.
Optical imaging setup: Fluorescence planar optical imaging was per-
formed in accordance with the Guide for the Use of Laboratory Animals.
The mice (three per group) bearing subcutaneous Colon-26 tumors on
the shoulder were anesthetized with ketamine/xylazine by intraperitonial
injection. The conjugates 27 and 28 and compound 25 were injected in-
travenously at 0.3 mmolkgÀ1 and then imaged 24, 48, and 72 h post-injec-
tion.
Whole-body fluorescence imaging was carried out with a 12 Bit mono-
chrome Nuance CCD Camera (CRI, Woburn, MA). A 782 nm BWF
light source (B&W-Tek, Newark, DE), continuous wave laser was used to
excite the cyanine dye (CD) and the fluorescence emission was collected
beyond 830 nm with an 800 and 830 nm long-pass filter in series. To su-
perimpose the white-light image of the mouse on the fluorescence image
of compound 25, an image of the mouse with a tungsten halogen white-
light source was first taken, followed by an image as described above for
the CD. The white light and fluorescence images were superimposed by
using the NIH ImageJ software. The fluorescence images for 25, 27, and
28 were normalized to the same lookup table (LUT) and minimum and
maximum brightness in ImageJ.
Electrochemical measurements: Cyclic voltammetry was carried out with
an EG&G Model 173 potentiostat/galvanostat. A home-made three-elec-
trode cell was used and consisted of a platinum button or glassy carbon
working electrode, a platinum wire counter electrode, and a saturated
calomel reference electrode (SCE). The SCE was separated from the
bulk of the solution by a fritted-glass bridge of low porosity that con-
tained the solvent/supporting electrolyte mixture. All potentials are refer-
enced to the SCE.
UV/Vis spectroelectrochemical experiments were performed with an op-
tically transparent platinum thin-layer electrode of the type described in
the literature.[47] Potentials were applied with an EG&G Model 173 po-
Chem. Eur. J. 2013, 19, 6670 – 6684
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6683

K. M. Kadish, R. K. Pandey et al.
tentiostat/galvanostat. Time-resolved UV/Vis spectra were recorded with
a Hewlett-Packard Model 8453 diode-array rapid scanning spectropho-
tometer.
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from Fluka Chemical Co., recrystallized from ethyl alcohol, and dried
under vacuum at 408C for at least 1 week prior to use. DMSO (ꢀ99.9%)
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Acknowledgements
Financial support from the NIH (R.K.P., CA55791 and CA127369), the
Roswell Park Alliance Foundation (R.K.P.), the Robert A. Welch Foun-
dation (K.M.K., Grant E-680) and the shared resources of the Roswell
Park Cancer Support (Grant P30A15056) is gratefully acknowledged.
Mass spectrometry analyses were performed at the Biopolymer Facility,
Roswell Park Cancer Institute, Buffalo and the Michigan State Universi-
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Received: October 29, 2012
Revised: January 22, 2013
Published online: March 22, 2013
6684
www.chemeurj.org
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 6670 – 6684