Boron-containing protoporphyrin ix derivatives and their modification for boron neutron capture therapy: Synthesis, characterization, and comparative in vitro toxicity evaluation

Article abstract of DOI:10.1002/chem.200901532

A novel series of boronated porphyrins for potential use in boron neutron capture therapy (BNCT) and photodynamic therapy (PDT) for tumor suppression is described. Protoporphyrin IX {i.e., bis(α-methyl-β-pen-tylethylether)protoporphyrin IX, and bis(α-methyl-β-dodecanylethylether)protoporphyrin IX} bearing polyhedral borane anions (B₁₂H₁₁SH^2-, B₁₂H₁₁NH₃^-, or B₁₂H₁₁OH^2-) were synthesized with reasonable yields. Modification of the protoporphyrin IX structure was achieved by variation of the lengths of the alkyl chains (pentyl and dodecanyl) attached through ether linkages to the former vinyl groups. The goal of this modification was to develop boronated porphyrins with chemical and physical properties that differed from those of protoporphyrin IX. Performance of an MTT assay with each derivative revealed that the synthesized boronated porphyrins showed low cytotoxicities in a variety of cancer cells. Of these compounds, B₁₂H₁₁NH₂^2--conjugated porphyrin induced a significant increase in the level of boron accumulation and PDT efficacy against HeLa cells.

Full text of DOI:10.1002/chem.200901532

FULL PAPER
DOI: 10.1002/chem.200901532
Boron-Containing Protoporphyrin IX Derivatives and Their Modification for
Boron Neutron Capture Therapy: Synthesis, Characterization, and
Comparative In Vitro Toxicity Evaluation
Mohamed E. El-Zaria,[a, b] Hyun Seung Ban,[a] and Hiroyuki Nakamura*[a]
Abstract: A novel series of boronated
porphyrins for potential use in boron
neutron capture therapy (BNCT) and
photodynamic therapy (PDT) for
tumor suppression is described. Proto-
porphyrin IX {i.e., bis(a-methyl-b-pen-
tylethylether)protoporphyrin IX, and
bis(a-methyl-b-dodecanylethylether)-
protoporphyrin IX} bearing polyhedral
cation of the protoporphyrin IX struc-
ture was achieved by variation of the
lengths of the alkyl chains (pentyl and
dodecanyl) attached through ether
linkages to the former vinyl groups.
The goal of this modification was to
develop boronated porphyrins with
chemical and physical properties that
differed from those of protoporphy-
AHCTUNGTRENNUNG
showed low cytotoxicities in a variety
of cancer cells. Of these compounds,
B12H11NH22À-conjugated porphyrin in-
duced a significant increase in the level
of boron accumulation and PDT effica-
cy against HeLa cells.
Keywords: BNCT · boron · cluster
borane
anions
(B12H11SH2À,
compounds
·
photodynamic
B12H11NH3 , or B12H11OH2À) were syn-
thesized with reasonable yields. Modifi-
À
therapy · porphyrinoids
Introduction
can palliate or ablate 10B-rich tumors. The quantity of sensi-
tizer required for successful therapy has been estimated to
be around 20–30 ppm boron.[1c,4]
Boron neutron capture therapy (BNCT)[1] and photodynam-
ic therapy (PDT)[2] are two bimodal therapies with the po-
tential to control local recurrences of malignant tumors.
Both rely on the selective uptake and/or retention of a sen-
sitizer molecule by the target cells and activation of that
sensitizer by application of an external radiation source.
PDT depends upon photosensitization of porphyrins and
use of red laser light, which catalyzes the conversion of mo-
lecular oxygen into rapidly reactive cell toxins.[3] BNCT acts
on similar principles, but makes use of the high capture
cross-section of the boron-10 nucleus for thermal neutrons.
The family of dyes most extensively studied with respect
to PDT and BNCT are the porphyrins and related macrocy-
cles (e.g., chlorines, phthalocyanines, and porphyrazines).[1c,5]
In many aspects, these drugs appear to act similarly. For a
porphyrin to be useful in either of these therapies, it should
be accumulated or retained in the tumor tissue in preference
to the surrounding healthy tissue. There are several uptake
mechanisms for porphyrins into tumor cells,[6] but there
exists no general rule for the nature of the structure/uptake
correlation. The compounds currently used for clinical trials
of BNCT applied to malignant brain tumors are the sodium
mercaptoundecahydrododecaborate Na2B12H11SH (BSH)
and the amino acid p-boronophenylalanine (BPA), which
yield tumor/blood boron concentration ratios of 1:1 and 3:1,
respectively.[7,8] It would be useful if combinations of these
and/or new boron carriers could achieve higher tumor-selec-
tive ratios without undesirable chemotoxicity.
An excited 11B nucleus decays to yield highly energetic He
and Li with a microscopic range of 5–9 mm. These particles
4
7
[a] Prof. Dr. M. E. El-Zaria, Dr. H. S. Ban, Prof. Dr. H. Nakamura
Department of Chemistry, Faculty of Science, Gakushuin University
1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588 (Japan)
Fax : +81-3-5992-1029
Strategies for achieving this goal focus on obtaining
higher boron concentrations in the tumor and better selec-
tivity of the boron carrier for tumor cells than for blood and
normal tissues. A variety of boronated porphyrins designed
to have a higher weight proportion of boron per molecule
than BPA have been synthesized.[9–12] Previous studies have
E-mail: hiroyuki.nakamura@gakushuin.ac.jp
[b] Prof. Dr. M. E. El-Zaria
Permanent address:
Department of Chemistry, Faculty of Science, University of Tanta
31527 Tanta (Egypt)
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shown that the tetrakis-carborane carboxylate ester of 2,4-
bis-(a,b-dihydroxyethyl)deuteroporphyrin IX (BOPP) is one
such porphyrin. It contains ~30% boron by weight and ap-
pears to be a good boron carrier in different glioma animal
models.[13] The pharmacokinetic behavior of BOPP, in con-
trast both with that of BPA and with that of BSH, in both
small and large animals, as well as in humans, is character-
ized by a relatively slow rise in tumor boron levels over a
period of several hours or even days.[13,14]
The disadvantages of BOPP are as follows: i) it has toxic
side effects, most notably thrombocytopenia, which limits
the dose that can be tolerated by humans, ii) relative boron
levels reached in human tumors are insufficient to permit
clinically effective BNCT, and iii) the plasma pharmacoki-
netic behavior of BOPP in humans is characterized by a
prolonged clearance phase, giving rise to potentially toxic
metabolites and cutaneous photosensitivity.[15]
Figure 1. Schematic structures of protoporphyrin IX (1) and sodium salts
of boronated protoporphyrin derivatives.
Another strategy tested in an attempt to optimize this
treatment was through the investigation of a boronated por-
phyrin (BOPP) in combination with BPA (a cocktail of two
BNCT drugs). The results showed that the combination of
BOPP and BPA gave much higher tumor boron concentra-
tions and better tumor selectivity than either drug adminis-
tered alone.[16] Recently, tetraphenylporphyrins containing
polyhedral borane anions were synthesized, and their toxico-
logical profiles in rats were determined.[17] The results
showed that thrombocytopenia toxicity limited the dose of
boronated porphyrins in mammals, which suggests that the
toxicity may arise from the porphyrin moiety rather than
from the borane or carborane moiety.
produces virtually no side products. Treatment of 3 with
pentan-1-ol and potassium carbonate (K2CO3) for 2 h at
room temperature (RT) gave the corresponding ether com-
pound {bis(a-methyl-b-pentylethylether)protoporphyrin IX
dimethyl ester (4)} in 95% isolated yield. Incubation of 4 in
a THF/MeOH/H2O (1:1:1) mixture with LiOH·H2O for 6 h
resulted in cleavage of the dimethyl esters to give bis(a-
methyl-b-pentylethylether)protoporphyrin IX
5
in 87%
yield with no evidence of ether bond cleavage (Scheme 1).
Similarly, bis(a-methyl-b-dodecanylethylether)protoporphy-
AHCTUNGTRENNUNG
Here we report a series of protoporphyrin IX derivatives
with systematically altered properties. These compounds
differ from one another not only in the natures of the cova-
lent bonds between the porphyrin and the dodecaborate
anion components, but also in the lengths of the alkyl chains
that chemically link the dodecaborate, through ether bonds,
to the former vinyl groups of protoporphyrin IX (1, Fig-
ACHTUNGTRENNUNG
and 90% yields, respectively, with dodecan-1-ol as the alco-
hol source (Scheme 1).
An alternative route for direct conversion of 1 into the
corresponding ether compound (5 or 7) without esterifica-
tion of the free carboxylic acid groups is also possible, but
this method, although it saves time, results in only a 55%
isolated yield.
ACHTUNGTRENNUNGure 1).Variation of the alkyl chain lengths of the linkages, as
well as testing with different borate moieties, are important
for the creation of molecules that exhibit a wide spectrum
of solubility.
One of the goals of our synthetic work was the successful
preparation and characterization of a structurally diverse
series of water-soluble dodecaborate anions derived from
compounds 1, 5, and 7. Polyhedral borane anions (such as
À
B12H11SH2À, B12H11NH3 , or B12H11OH2À)[19,20] are often the
Results and Discussion
clusters of choice for binding to porphyrins because of their
known chemistry, hydrophilic properties, high boron con-
tents, and chemical stabilities. Moreover, the carboxylic acid
group was chosen for the linkage chemistry largely as a
result of the breadth of stable derivatives that could poten-
tially be formed from known polyhedral dodecaborate
anions. As a result, amide, thioester, and oxoester deriva-
tives were successfully prepared and characterized
(Figure 1).
Activation of the carboxylic acid groups of the porphyrins
1, 5, or 7 with oxalyl chloride allowed the attachment of
[(CH3)4N]2B12H11SH (BSH), to give surprisingly stable thio-
esters. This reaction was utilized to prepare bis(tetramethyl-
Synthesis: Naturally occurring porphyrin systems have occa-
sionally been used for conjugation with boron clusters.[5c]
Here we report the synthesis of a series of compounds with
systematically varying properties. Esterification of commer-
cially available 1 with H2SO4/MeOH (5%) for 18 h at
À108C in the dark gave protoporphyrin IX dimethyl ester
(PP-IX dimethyl ester, 2).[18] Even on a scale of 1 g, yields of
2 exceeding 95% are routinely obtained. The vinyl groups
in 2 were hydrobrominated with HBr/AcOH (25%) to yield
the corresponding bis(a-methyl-b-bromo)protoporphyrin IX
dimethyl ester 3 in >92% yield (Scheme 1). This very mild,
clean method does not hydrolyze the ester side chains and
AHCTUNGTREGaNNNU mmonium)protoporphyrin IX monothioester undecahydro-
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Boron-Containing Protoporphyrin IX Derivatives
FULL PAPER
their sodium salts (compounds 9, 11, and 13, respectively)
by ion-exchange chromatography.
In a similar approach, the oxoester (bis(methyltriphenyl-
phosphenium)protoporphyrin IX monooxoester undecahy-
dro-closo-dodecaborate, 14) was prepared by coupling
(MePh3P)2B12H11OH (BOH) to the acid chloride of com-
pound 1 (Scheme 2). The water-soluble sodium salt (15) was
obtained by ion exchange. Synthesis of 14 by the procedures
of Kahl et al.[17] is also possible, but the yield of the product
was very low. The amide (bis(tetramethylammonium)proto-
AHCTUNGTRENNUNG
1
(Scheme 2). Ion exchange afforded the sodium salt 17,
which was water-soluble.
Similarly to 9, tetrakis(tetramethylammonium)protopor-
AHCTUNGTREGpNNUN hyrin IX bis(thioester) undecahydro-closo-dodecaborate
(18) was prepared in 74% yield from the acid chloride of 1
and BSH (Scheme 3). As in the cases of the lipophilic
tetramethylACTHUNTGRNEUNGammonium salts, this compound was also con-
verted into the water-soluble sodium salt (19) by ion ex-
change prior to administration. The advantage of a com-
Scheme 1. Synthesis of long-chain PP-IX derivatives 5 and 7 from 1.
a) H2SO4/MeOH (5%), À108C, 18 h. b) HBr/ACOH (25%), RT, 2 h.
c) ROH, CH2Cl2, K2CO3, RT, 1 h. d) LiOH, MeOH/H2O/THF, RT, 6 h.
closo-dodecaborate (8), bis(tetramethylACTHNUGRTENUNGammonium)bis(a-
methyl-b-pentylethylether)protoporphyrin IX monothioester
undecahydro-closo-dodecaborate (10), or bis(tetramethyl-
ACHTUNGTRENNUNGammonium)bis(a-methyl-b-dodecanylethylether)protopor-
phyrin IX monothioester undecahydro-closo-dodecaborate
(12) in reasonable yields (Scheme 2). One of the most im-
portant requirements of BNCT agents is water solubility.
The transformation of the tetramethylACTHNUTRGNEaNUG mmonium salt into
the sodium salt is the simplest way to increase the water sol-
ubility of a BSH-containing porphyrin. The compounds 8,
10, and 12 were highly water-soluble when converted from
Scheme 3. Synthesis of compound 19: a) CH2Cl2, (COCl)2, RT, 1 h.
b) [(CH3)4N]2B12H11SH, CH3CN, C5H5N, RT, 72 h. c) Ion-exchange with
Amberlite 120R (Na+ form).
the tetramethylACHTUNGTRENNUNGammonium salts used in the synthesis into
Scheme 2. General pathway for the synthesis of anionic boronated porphyrins 8, 10, 12, 14, and 16: a) CH2Cl2, (COCl)2, RT, 1 h. b) B12H11XH2À (X=S or
O), CH3CN, C5H5N, RT, 72 h. c) CH2Cl2, (COCl)2, RT, 1 h. d) [(CH3)4N]B12H11NH3, DMF, NaH, C5H5N, RT, 72 h. e) Ion-exchange with Amberlite 120R
(Na+ form).
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1545

H. Nakamura et al.
pound such as 19, bearing such a high percentage of boron
by weight (~27%), is that it can potentially deliver high
(therapeutic) quantities of boron to tumor targets with toler-
able toxicity.
(Ff) of compounds 1, 9, 11, 13, 15, 17, and BOPP were cal-
culated relative to rhodamine 101 in ethanol as a standard
(Ff =1.00)[21a] by the comparative method.[21b,c] Free proto-
porphyrin IX (1) showed a Ff value of 0.1, whereas BOPP,
11, and 13 showed Ff values of 0.091, 0.095, and 0.093, re-
spectively. Conjugation of the propanoic acid group of 1
with dodecaborate anions enhanced the fluorescence of 1
only slightly more than BOPP (Ff =ca. 0.12). Presumably,
the binding of the carborane ester or alkoxy moieties at the
former vinyl groups of 1 locked the rotational mobility of 1
in a rigid structure, whereas compounds 9, 15, 17, and 19 did
not bind significantly to 1.
These results are interesting because these compounds
differ in the natures of the functional groups linking the por-
phyrin carboxylic group and the dodecaborate anion, the re-
sulting overall charges of the porphyrin-borane conjugates,
and the lengths of the ether chains. The structures and puri-
1
ties of the new compounds were confirmed by 11B, H, and
13C NMR spectroscopy, IR, UV/Vis, and fluorescence spec-
troscopy, mass spectrometry, and elemental analysis (see Ex-
perimental Section).
The IR spectra of compounds 5 and 7 showed absorption
bands in the 3585–3565 cmÀ1 and 1745 cmÀ1 regions, charac-
teristic of the free OH and C=O groups, respectively, of free
diacids. However, the boronated porphyrins (8–17) each
showed two different vibrational frequencies for the C=O
Spectroscopic characterization: The 1H NMR spectra of
compounds 3–17 were consistent with their proposed struc-
tures, showing the expected features and correct integration
ratios. We observed high-field shifts of the CH components
of the former vinyl groups in compounds 3–7 from 7.58 ppm
to ca. 6.1 ppm. Large deviations in the positions of these sig-
nals were observed in compounds 3–7, as would be expect-
ed. Moreover, the chemical shifts of the CH2 components of
the former vinyl groups were also changed, from 7.25 ppm
to ca. 2.2 ppm, indicating the presence of the saturated form
(CH3 groups). In addition, the 13C NMR spectra showed
high-field shifts of ca. 18 ppm for the CH carbon atoms of
bonds {nACHTUNGTRNEUNG
(C=O)}, at ca. 1725 and 1685 cmÀ1, indicating sub-
stantial electron donation from the borane anion into the
linkage group. Moreover, the vibrational frequencies of the
À
À
À
À
B H bonds {nACTHNUTRGNENG(U B H)} or the B B bonds {nACHTUNGTNER(NUGN B B)} were not
found to be sensitive to conjugation with the boron cluster.
À1
À
For compounds 8–19, n
range, whereas n
(B H) lay in the 2495–2500 cm
À1
À
(B B) varied from 1050 to 1080 cm . Of
2À
the frequencies associated with the B12H12
moiety
{nACTHNUTRGENNGU ACHTUNGTERN(NUGN B B) 1073 to 1057 cm },
(B H) 2486 to 2462 cmÀ1; n
À1 [22]
À
À
compounds 4–7 (74.2 ppm) relative to compound
1
only slight differences between the compounds were found,
indicating that intracluster bonding was not perturbed by
the substitution of the icosahedron.
(56.2 ppm). Other NMR spectroscopic data for the series of
compounds (5, 7, and 10–13) were similar, although there
were some minor variations in the proton shielding as the
alkoxy groups changed (see Experimental Section).
Biology: Boronated porphyrins are an important class of
tumor-localizing agents in two bimodal therapies for cancer:
BNCT and PDT. The boronated porphyrins are character-
ized by several desirable properties: i) they are easily syn-
thesized, pure, and well-characterized drugs, ii) they are
stable in vivo and are tumor-specific, iii) they yield high
tumor/blood and tumor/normal tissue boron concentration
ratios, and iv) they are minimally toxic.
Polyhedral borane anions of the types B12H11SH2À,
À
B12H11OH2À, and B12H11NH3 were easily identified by
NMR spectroscopy, because their 11B NMR spectra present-
ed a characteristic shielding pattern over the quite remark-
able range of ca. À22 to +7 ppm, showing only minor differ-
ences in the overall 11B cluster shielding patterns. The spec-
tra of all boronated porphyrins (8–19) showed 1:5:5:1 pat-
terns typical of monosubstituted B12 derivatives.
In view of the above desirable characteristics, we pre-
pared and characterized a series of porphyrins bearing poly-
hedral borane anions. We first examined the cell toxicities
of the synthesized boronated porphryins toward a variety of
cancer cells by means of an MTT—3’-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide—assay. Of the boronat-
ed porphyrins, the BSH-conjugated porphyrin 9 showed low
cytotoxicity in relation to its alkylated compounds 11 and 13
and BOPP (Table 1). Furthermore, the BOH- and BNH2-
conjugated porphyrins (15 and 17) also exhibited lower
levels of cytotoxicity than BOPP toward various cancer
cells, with the exception of C6 cells (Table 1). We also exam-
ined the cytotoxicity of porphyrin 19, with two polyhedral
borane anions in the molecule: conversely, the more hydro-
philic porphyrin 19 (IC50 =182.9 mm) was found to be more
cytotoxic toward C6 cells than porphyrin 9 (IC50 =382.2 mm).
These results indicate that conjugation of only one poly-
hedral borane anion to porphyrin resulted in reduced cytox-
icity.
Evidence in support of structures 2–7 was also obtained
by mass spectrometry. The ESI-MS spectra of these com-
pounds in MeOH showed molecular ion peaks—[M]+ or
[M+1]+—consistent with the formula of each compound.
The boronated porphyrins 8–15, 18, 19, and 16–17 showed
the molecular ion peaks—([M]À/2) and ([M]À)—attributable
to the typical pattern of boron isotopes (10B and 11B, respec-
tively).
The UV/Vis absorption spectra of all porphyrins in
CH3CN or H2O (10 mm) each contained a high-energy Soret
band (peak position=ca. 410 nm) and lower-energy Q-
bands (peak positions=ca. 515, 535, 585, 635 nm) arising
from p–p* transitions, typical for the protoporphyrin IX (1)
framework. However, emission spectral studies in ethanol
each showed an emission fluorescence band at ca. 635 nm
for compounds 1, 9, 15, 17, and 19, caused by the bathochro-
mic shift of 10 nm in relation to the BOPP, 11, and 13 emis-
sion bands at ca. 625 nm. The fluorescence quantum yields
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Boron-Containing Protoporphyrin IX Derivatives
FULL PAPER
Table 1. Cytotoxicities in IC50 [mm] of boronated porphyrins.
synthesis, which explains the
current interest in finding effi-
cient synthetic methods for the
preparation of these species.
Importantly, these new structur-
ally varied boronated porphyr-
ins displayed a wide spectrum
of water solubilities and surpris-
ing spectroscopic properties
with extremely stable and non-
Porphyrin HeLa
C6
B16
CT26
HepG2
HCT15
HCT116
9
11
13
15
17
BOPP
306.7Æ13.9 382.2Æ35.4 533.3Æ1.4
382.6Æ6.5
811.1Æ6.0
404.4Æ4.3
436.2Æ20.2
88.3Æ6.6
94.7Æ2.2
161.8Æ15.9
51.9Æ2.2
75.8Æ2.8
69.1Æ0.6
69.2Æ6.7
138.9Æ3.5
124.3Æ10.6 214.7Æ15.9 142.2Æ1.4
81.0Æ12.5 107.7Æ7.2
182.9Æ9.7
248.2Æ2.7
658.7Æ4.1
175.1Æ1.3
169.3Æ12.8
196.4Æ25.7
241.2Æ9.2
301.3Æ4.7
167.6Æ7.1
359.1Æ9.0
374.4Æ5.5
182.2Æ1.3
235.7Æ18.9 212.8Æ4.6
227.2Æ14.9 246.8Æ9.8
95.9Æ15.1 114.1Æ5.1
233.5Æ1.3
175.7Æ8.4
285.1Æ5.1
128.3Æ14.4
The various cancer cells were incubated for 72 h in the presence of each boronated porphyrin, and the ratios
of viable cells were determined by MTT assay. The drug concentration required to inhibit cell growth by 50%
(IC50) was determined from semilogarithmic dose-response plots, and results represent the means ÆSDs of
triplicate samples.
toxic
closo-borane
anions.
These improvements indicate
the potential to decrease the
We next examined the levels of intracellular accumulation
of the boronated porphyrins through determination of
boron concentrations in HeLa cells by ICP-AES (induc-
tively coupled plasma atomic emission spectroscopy). As
shown in Figure 2, the accumulations of boron increased
with increasing concentration of boronated porphyrins. The
accumulated boron concentrations obtained with com-
pounds 9 and 11 were similar to that seen with BOPP. Inter-
estingly, compounds 13, 15, and 17 each showed a fourfold
increase in selective accumulation relative to other com-
pounds. The assays with compounds 9, 11, and 13 showed
that cell uptake increased with increasing porphyrin lipophi-
licity. Furthermore, fluorescence imaging after treatment for
3 h revealed that compound 17 and BOPP accumulated
mainly in the cytosol (Figure 3).
compoundsꢁ plasma half-lives relative to those of previously
investigated boronated porphyrins such as BOPP. Further-
more, we found that the boronated porphyrins exhibited low
cytotoxicities and high levels of accumulation and induced
potent PDT effects with a variety of cancer cell lines. These
encouraging results suggest the possible application of these
compounds for combined BNCT and PDT tumor suppres-
sion.
Table 2 shows the PDT response driven by boronated por-
phyrins and BOPP in HeLa cells. Concentrations of 14.4,
2.71, 4.97, 0.6, 0.21, and 4.83 mm for compounds 9, 11, 13, 15,
17 and BOPP, respectively, did not induce cytotoxicity,
whereas the boronated porphyrins reduced cell viability. Of
the compounds, BOH- and BNH2-conjugated porphyrins (15
and 17) showed activities more potent than that of BOPP,
with IC50 values of 0.60 and 0.21 mm, respectively. However,
the BSH-conjugated porphyrin (compound 9) displayed a
threefold reduction in PDT efficacy relative to BOPP. These
results suggest that the PDT ef-
Figure 2. Intracellular uptake of boronated porphyrins. Various cancer
cell types were incubated for 3 h in the presence of each boronated por-
phyrin. After incubation, the cells were digested and their boron contents
were determined by inductively coupled plasma atomic emission spec-
troscopy (ICP-AES).
ficacies of boronated com-
pounds, determined by cell via-
bility, were correlated with the
levels of cell accumulation in
the cases of compounds 15 and
17.
Conclusions
We have demonstrated a very
simple, efficient, and practical
strategy for the synthesis of
novel water-soluble boronated
porphyrins in good to high
yields. Functionalized porphyr-
ins are very useful precursors in
advanced boronated porphyrin
Figure 3. Fluorescence imaging of boronated porphyrins in HeLa cells. The cells were incubated for 3 h either
with compound 17 or with BOPP (100 mm). After incubation, the cells were washed three times with PBS, and
the boronated porphyrins were detected by fluorescence microscopy. The cell nuclei were stained with DAPI,
shown in blue.
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H. Nakamura et al.
Table 2. PDT effects of boronated porphyrins.
2989 s, 2927 s (NH), 1755 s (C=O), 1710 s (C=C), 1605 s (NH), 1415 s
(CH3), 11495 s (CN), 2965 m, 2895 m, 1475 m, 1442 m, 1409 m, 1169 m,
1098 s, 752 m cmÀ1 (d, g of CH2 groups); UV/Vis (CH3CN): lmax =415,
523, 537, 594, 635 nm; MS (ESI, positive): m/z (%): 767 (100) [M]+; ele-
mental analysis calcd (%) for C46H62N4O6 (767.01):C 72.03, H 8.15, N
7.30; found: C 71.96, H 8.12, N 7.27.
Porphyrin
IC50 [mm]
Porphyrin
IC50 [mm]
9
11
13
14.4Æ0.30
2.71Æ0.14
4.97Æ0.24
15
17
BOPP
0.60Æ0.06
0.21Æ0.04
4.83Æ0.75
HeLa cells were incubated for 3 h in the presence of each boronated por-
phyrin and were then irradiated for 5 min with a xenon lamp (400–
800 nm, average 2.4 mWcmÀ2). After further incubation for 12 h, the
ratios of viable cells were determined by MTT assay. The drug concentra-
tions required to inhibit cell growth by 50% (IC50) were determined
from semilogarithmic dose-response plots, and results represent the
means ÆSDs of triplicate samples.
Bis(a-methyl-b-pentylethylether)protoporphyrin IX (5): Compound
4
(1.0 mmol, 767 mg) was dissolved in a THF/MeOH/H2O mixture (15 mL,
1:1:1). LiOH·H2O (2.2 mmol, 92.3 mg) was added and the reaction mix-
ture was stirred for 6 h at room temperature. The reaction mixture was
filtered and the volume of the filtrate was reduced under vacuum (5 mL).
HCl (1n, 2 mL) was slowly added to this solution with stirring to precipi-
tate the dark violet solid, which was filtered off and then washed with
water (5 mL) to give 5. Yield (642 mg, 87%) as a dark violet solid;
1H NMR (300 MHz, [D6]DMSO, 258C, TMS): d=10.65–10.43 (s, 4H;
meso-H), 6.14 (m, 2H; a-CH), 4.33 (m, 4H; porphyrin-CH2), 3.69, 3.67,
3.65, 3.63 (s, 12H; b-CH3), 3.54 (t, J=12.6 Hz, 4H; CH2COOH), 3.08 (m,
4H; OCH2), 2.17 (d, J=8.0 Hz, 6H; CH3), 1.73, 1.35, 1.16 (m, 12H;
CH2), 0.7 (t, J=16.2 Hz, 6H; CH3), À3.96 ppm (s, 2H; NH); 13C NMR
(75 MHz, [D6]DMSO, 258C, TMS): d=175.11 (2C; CO), 147.2, 146.51,
145.79, 145.78, 140.02, 136.92, 136.28, 124.88 (16C; C pyrrole), 98.39,
Experimental Section
Materials and methods: 1H NMR and 13C NMR spectra were measured
with Jeol JNM-AL 300 (300 MHz) and Varian Unity-Inova 400
(400 MHz) spectrometers. 1H NMR and 13C NMR chemical shifts are ex-
pressed in parts per million (ppm, d units), and coupling constants are ex-
pressed in hertz (Hz). 11B NMR spectra were recorded with a JEOL
JNM-AL 300 spectrometer (96.3 MHz) and the chemical shifts are re-
ported in d units relative to external BF3·Et2O in CDCl3. IR spectra were
measured with a Shimadzu FTIR-8200 A spectrometer. UV/Vis spectra
were measured with a Shimadzu 2450 PC spectrophotometer over the
300–700 nm wavelength range. Elemental analyses were performed with
a Perkin–Elmer 2400 automatic elemental analyzer. The fluorescence
(excitation and emission) spectra were determined with a Jasco FP-
6500 PC spectrophotometer. Electron spray ionization (ESI) mass spectra
were recorded with a Shimadzu LCMS-2010 eV spectrometer. Analytical
thin-layer chromatography (TLC) was performed on glass plates (Merck
Kieselgel 60 F254, layer thickness 0.2 mm). Samples were visualized with
the aid of UV light (254 nm), I2, or KMnO4. Column chromatography
was performed on silica gel (Merck Kieselgel 70–230 mesh). Preparative
thin layer chromatography (TLC) was carried out with 0.75 mm layers of
silica gel G (Merck, GF254) made from water slurries on glass plates of di-
mensions 20ꢂ20 cm2, followed by drying in air at 1008C. All reactions
were performed in darkness in dry solvents and under argon with use of
standard Schlenk techniques. Most chemicals were of analytical grade
and were used without further purification. BOPP, PP-IX dimethyl ester
(2), [(CH3)4N]2B12H11SH, [(CH3)4N]B12H11NH3, and (MePh3P)2B12H11OH
were prepared as described in the literature[9d,18–20] and stored at À208C.
À
97.97, 97.72, 97.36 (4C; meso-C), 74.05, 72.47 (2C; O CH), 60.67, 56.61
(2C; OCH2), 32.18, 30.4 (2C; CH2COOH), 29.4, 29.21, 28.12, 27.7, 25.25,
À
À
25.08 (6C; CH2 ), 22.32, 22.22 (2C; CH2CH2COOH), 21.99, 21.88,
13.92, 13.73, 11.2, 11.56 ppm (8C; CH3); IR (KBr disc): n˜ =3309 w (CH),
2987 s, 2927 s, 2860 s (OH/NH), 1745 s (C=O), 1706 s (C=C), 1600 s
(NH), 1410 s (CH3), 1145 s (CN), 2961 m, 2889 m, 1471 m, 1439 m,
1405 m, 1165 m, 1093 s, 749 m cmÀ1 (d,
g of CH2 groups); UV/Vis
(CH3CN): lmax =415, 520, 539, 595, 639 nm; MS (ESI, positive): m/z (%):
739 (100) [M]+; elemental analysis calcd (%) for C44H58N4O6 (738.95):C
71.52, H 7.91, N 7.58; found: C 71.33, H 7.81, N 7.49.
Bis(a-methyl-b-dodecanylethylether)protoporphyrin IX dimethyl ester
(6): This compound was prepared from 2 (1.0 mmol, 590 mg) and dodeca-
nol (2.2 mmol, 410 mg) by the procedure described for 4, to give 6
(934 mg, 97%) as a dark violet solid; Rf =0.52 (CH2Cl2/MeOH 17:3);
1H NMR (300 MHz, [D6]DMSO, 258C, TMS): d=10.65, 10.57, 10.35,
10.12 (s, 4H; meso-H), 6.08 (m, 2H; a-CH), 4.34 (m, 4H; porphyrin-
CH2), 3.85, 3.79 (s, 6H; COOMe), 3.67, 3.64, 3.59, 3.50 (s, 12H; b-CH3),
3.01 (m, 4H; CH2COOMe), 2.56 (m, 4H; OCH2), 2.13 (d, J=6.0 Hz, 6H;
CH3), 1.71–1.17(m, 20H; CH2), 0.83 (t, J=13.2 Hz, 6H; CH3), À3.95 ppm
(s, 2H; NH); 13C NMR (75 MHz, [D6]DMSO, 258C, TMS): d=179.02
(2C; CO), 147.26, 147.05, 146.23, 145.14, 141.15, 134.65, 133.92, 124.95
À
(16C; C pyrrole), 99.21, 98.25, 97.65, 97.47 (4C; meso-C), 75.03 (2C; O
CH), 57.07 (2C; OCH2), 48.35 (2C; COOMe), 31.47, 30.13 (2C;
Bis(a-methyl-b-pentylethylether)protoporphyrin IX dimethyl ester (4):
Compound 2 (1.0 mmol, 590 mg) was dissolved in HBr/acetic acid (25%,
30 mL) under argon and the mixture was stirred for 2 h. All volatile sol-
vents were evaporated under vacuum to yield the green colored bis(a-
methyl-b-bromo)PP-IX dimethyl ester (3). The resulting substance was
directly dissolved in dichloromethane (50 mL). Pentan-1-ol (2.2 mmol,
194 mg) and K2CO3 (2.2 mmol, 304 mg) were added to the dichlorome-
thane solution. The reaction mixture was again stirred under argon for
2 h and filtered, and the resulting filtrate was concentrated to dryness.
The residue, consisting of the crude product, was then purified by repeat-
ed thin layer chromatography (TLC) on silica gel with CH2Cl2 and
MeOH (17:3) as liquid phase to yield compound 4 as dark violet crystals.
Yield (728 mg, 95%) as a dark violet solid; Rf =0.45 (CH2Cl2/MeOH
17:3); 1H NMR (300 MHz, [D6]DMSO, 258C, TMS): d=10.58–10.36 (s,
4H; meso-H), 6.12 (m, 2H; a-CH), 4.28 (m, 4H; porphyrin-CH2), 3.89,
3.82 (s, 6H; COOMe), 3.71, 3.69, 3.68, 3.62 (s, 12H; b-CH3), 3.48 (t, J=
13.2 Hz, 4H; CH2COOMe), 3.12 (4H; m, OCH2), 2.22 (d, J=7.0 Hz, 6H;
CH3), 1.75, 1.41, 1.21 (12H; m, CH2), 0.78 (t, J=12.9 Hz, 6H; CH3),
À3.95 ppm (s, 2H; NH); 13C NMR (75 MHz, [D6]DMSO, 258C, TMS):
d=177.08 (2C; CO), 145.2, 144.98, 144.05, 143.87, 139.15, 136.85, 136.18,
125.03 (16C; C pyrrole), 98.54, 98.02, 97.98, 97.74 (4C; meso-C), 75.14,
À
À
CH2COOH), 29.01–25.36 (20C;
CH2 ), 24.99, 24.12 (2C;
CH2CH2COOMe), 23.16, 22.06, 14.95, 13.14, 11.36, 11.43 ppm (8C; CH3);
IR (KBr disc): n˜ =3310 w (CH), 3300 s, 2950 s (NH), 1755 s (C=O),
1681 s (C=C), 1609 s (NH), 1417 s (CH3), 1152 s (CN), 2965 m, 2866 m,
1479 s, 1454 s, 1412 m, 1316 m, 1168 m, 1100 s, 885 m, 715 m cmÀ1 (CH2
groups); UV/Vis (CH3CN): lmax =419, 526, 538, 595, 639 nm; MS (ESI,
positive): m/z (%): 963 (100) [M]+; elemental analysis calcd (%) for
C60H90N4O6 (963.38): C 74.8, H 9.42, N 5.82; found: C 74.69, H 9.38, N
5.77.
Bis(a-methyl-b-dodecanylethylether)protoporphyrin IX (7): This com-
pound was prepared from 6 (1.0 mmol, 963 mg) by the procedure de-
scribed for 5 to give 7 (842 mg, 90%) as a dark violet solid; 1H NMR
(300 MHz, [D6]DMSO, 258C, TMS): d=10.59, 10.5, 10.47, 10.15 (s, 4H;
meso-H), 6.07 (m, 2H; a-CH), 4.37 (m, 4H; porphyrin-CH2), 3.71, 3.68,
3.65, 3.63 (s, 12H; b-CH3), 2.95 (m, 4H; CH2COOH), 2.61 (m, 4H;
OCH2), 2.18 (d, J=6.0 Hz, 6H; CH3), 1.85–0.81 (m, 20H; CH2), 0.72 (t,
J=13.2 Hz, 6H; CH3), À3.92 ppm (s, 2H; NH); 13C NMR (75 MHz,
[D6]DMSO, 258C, TMS): d=177.82 (2C; CO), 148.78, 147.35, 146.33,
145.6, 141.12, 134.76, 133.73, 125.14 (16C; C pyrrole), 98.39, 97.97, 97.72,
97.36 (4C; meso-C), 74.72 (2C; O CH), 56.17 (2C; OCH2), 31.27, 30.56
(2C; CH2COOH), 29.5–25.95 (20C;
CH2CH2COOH), 24.26, 22.06, 16.65, 13.92, 11.65, 11.54 ppm (8C; CH3);
IR (KBr disc): n˜ =3310 w (CH), 3197 s, 2927 s, 2856 s (OH/NH), 1745 s
(C=O), 1675 s (C=C), 1605 s (NH), 1415 s (CH3), 1150 s (CN), 2965 m,
À
À
À
À
73.16 (2C; O CH), 61.27, 57.19 (2C; OCH2), 47.86 (2C; COOMe),
CH2 ), 25.57, 24.29 (2C;
31.98, 30.79 (2C; CH2COOMe), 28.56, 28.06, 27.47, 27.17, 26.25, 25.74
À
À
(6C; CH2 ), 23.21, 23.25 (2C; CH2CH2COOMe), 21.57, 21.65, 14.02,
13.63, 11.36, 11.86 ppm (8C; CH3); IR (KBr disc): n˜ =3310 w (CH),
1548
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 1543 – 1552

Boron-Containing Protoporphyrin IX Derivatives
FULL PAPER
2856 m, 1475 s, 1452 s, 1405 m, 1313 m, 1165 m, 1100 s, 883 m, 705 m cmÀ1
(CH2 groups); UV/Vis (CH3CN): lmax =415, 523, 537, 593, 638 nm; MS
(ESI, positive): m/z (%): 936 (100) [M+1]+; elemental analysis calcd (%)
for C58H86N4O6 (935.33): C 74.48, H 9.27, N 5.99; found: C 74.37, H 9.01,
N 5.82.
(2C; CO), 149.19, 148.67, 147.45, 146.77, 142.06, 141.08, 138.11, 118.2
(16C; C pyrrole), 100.17, 99.38, 98.29, 97.61 (4C; meso-C), 74.12, 73.85
À
(2C; O CH), 60.67 (2C; OCH2), 50.7 (8C; N
G
À
CH2COOH), 29.37, 29.33, 25.68, 25.46 (6C; CH2 ), 23.95, 23.71 (2C;
CH2CH2COOH), 22.31, 23.05, 14.13, 14.1, 11.98, 11.92, 11.8, 11.65 ppm
(8C; CH3); 11B NMR (96.3 MHz, CD3CN, 258C, BF3 Et2O): d=À14.39,
Bis(tetramethylACHTUNGTRENNUNGammonium)protoporphyrin IX monothioester undecahy-
À10.91, À9.58, À5.89 ppm; IR (KBr disc): n˜ =3568 s, 3421 s (OH/NH),
3311 w (CH), 2500 s (BH), 1722, 1685 s (C=O), 1620 s (C=C), 1608 s
dro-closo-dodecaborate (8): Protoporphyrin IX (1, 0.16 mmol, 90 mg)
was dissolved in anhydrous dichloromethane (10 mL) under argon in a
25 mL round-bottomed flask. Freshly distilled oxalyl chloride (8.2 mmol,
0.7 mL) was added to this solution, and the reaction mixture was stirred
at room temperature for 1 h. The dichloromethane and unreacted oxalyl
chloride were removed under vacuum. The resulting green solid of the
porphyrin acid chloride was dissolved in anhydrous acetonitrile (15 mL).
[(CH3)4N]2B12H11SH (0.25 mmol, 80 mg) and anhydrous pyridine
(3.7 mmol, 0.3 mL) were added. This reaction mixture was stirred for
72 h and the volatile components were removed under vacuum. The
crude product was purified by column chromatography (CH2Cl2/MeOH
17:3) to give 8. Yield (71 mg, 51%) as a violet colored solid; Rf =0.31
(CH2Cl2/MeOH 17:3); 1H NMR (300 MHz, CD3CN, 258C, TMS): d=
10.29, 10.22, 10.2, 10.15 (m, 4H; meso-H), 8.49 (m, 2H; CH=CH2), 6.45
(d, J=16.0 Hz, 2H; CH=CH2), 6.17 (d, J=12.4 Hz, 2H; CH=CH2), 4.32
(m, 4H; porphyrin-CH2), 3.77, 3.73, 3.66, 3.64 (s, 12H; b-CH3), 3.24 (m,
À
(NH), 1386 s (CH3), 1157 s (CN), 1080 s (B B), 2986 m, 2864 m, 1471 m,
1440 m, 1400 m, 1185 m, 1093 s, 721 m cmÀ1 (d, g of CH2 groups); UV/Vis
(CH3CN): lmax =414, 522, 538, 592, 635 nm; elemental analysis calcd (%)
for C52H92N6B12O3S (1011.12): C 61.77, H 9.17, N 8.31; found: C 61.49, H
8.96, N 8.19.
Bis
G
mono-
thioester undecahydro-closo-dodecaborate (11): This compound was pre-
pared from 10 (0.087 mmol, 88 mg) by the procedure described for 9, to
give 11 (29 mg, 37%) as
a
deep red solid; 1H NMR (300 MHz,
[D6]DMSO, 258C, TMS): d=12.05 (s, 1H; COOH), 10.73, 10.64, 10.52,
10.24 (m, 4H; meso-H), 6.17 (m, 2H; a-CH), 4.32 (m, 4H; porphyrin-
CH2), 3.72, 3.66, 3.64, 3.62 (s, 12H; b-CH3), 3.35 (t, J=13.8 Hz, 4H;
CH2COOH), 2.97 (t, 4H; OCH2), 2.35 (s, 6H; CH3), 1.7, 1.35, 1.25 (m,
12H; CH2), 0.7 (t, J=12.5 Hz, 6H; CH3), À3.91 ppm (s, 2H; NH);
13C NMR (75 MHz, [D6]DMSO, 258C, TMS): d=208.93, 177.47 (2C;
CO), 148.15, 147.14, 146.21, 146.77, 142.06, 141.08, 138.11, 118.45 (16C; C
4H; CH2COOH), 3.08 (s, 24H; NACHTUNGTRNEUNG(CH3)4), 0.55–1.82 (brs, 11H; B12H11),
À3.85 ppm (s, 2H; NH); 13C NMR (75 MHz, CD3CN, 258C, TMS): d=
176.43, 176.69 (2C; CO), 145.39, 143.54, 142.37, 137.03, 136.85, 136.74,
130.61, 126.77 (16C; C pyrrole), 97.92–97.14 (4C; meso-C), 56.73, 56.6
À
pyrrole), 98.29, 97.61 (4C; meso-C), 73.78, 72.46 (2C; O CH), 68.47
(2C; OCH2), 30.82 (2C; CH2COOH), 29.32, 29.27, 25.46, 25.19 (6C;
À
À
(4C; CH=CH2), 54.59 (8C; N
G
CH2 ), 22.98, 22.64 (2C; CH2CH2COOH), 22.05, 21.97, 14.17, 14.09,
23.28, 22.67 (2C; CH2CH2COOH), 13.45, 12.57, 11.56, 11.28 ppm (4C;
11.96, 11.91, 11.76, 11.68 ppm (8C; CH3); 11B NMR (96.3 MHz,
.
CH3); 11B NMR (96.3 MHz, CD3CN, 258C, BF3 Et2O): d=À15.37,
[D6]DMSO, 258C, BF3 Et2O): d=À14.39, À10.91, À9.58, À5.89 ppm; UV/
À11.97, À9.78, À5.49 ppm; IR (KBr disc): n˜ =3587 s, 3375 s, 3228 s (OH/
NH), 3025 w (CH), 2495 s (BH), 1725, 1685 s (C=O), 1618 s (C=C),
Vis (H2O): lmax =410, 517, 536, 585, 635 nm.
Bis(tetramethyl
porphyrin IX monothioester undecahydro-closo-dodecaborate (12): This
compound was prepared from (0.16 mmol, 150 mg) and
[(CH3)4N]2B12H11SH (0.25 mmol, 80 mg) by the procedure described for
8, to give 12 (110 mg, 57%) as a deep red solid; Rf =0.39; 1H NMR
(300 MHz, CD3CN, 258C, TMS): d=10.55, 10.42, 10.12, 10.0 (s, 4H;
meso-H), 5.94 (m, 2H; a-CH), 4.25 (m, 4H; porphyrin-CH2), 3.69, 3.67,
3.63, 3.6 (s, 12H; b-CH3), 3.37 (m, 4H; CH2COOH), 3.15 (s, 24H; N-
ACHTUNGTRENaNUGN mmonium)bis(a-methyl-b-dodecanylethylether)proto-
1605 s (NH), 1410 s (CH3), 1152 s (CN), 1050 s cmÀ1 (B B); UV/Vis
(CH3CN): lmax =417, 526, 535, 592, 637 nm; MS (ESI, negative): m/z
(%): 359.22 (100) [M]À/2; elemental analysis calcd (%) for
C42H68N6B12O3S (868.62): C 58.20, H 7.91, N 9.70; found: C 58.05, H 7.73,
N 9.49.
7
Ion exchange to form bis
ACHTUNGTRENNUNG
decahydro-closo-dodecaborate (9): The bis(tetramethylAHCTUNGTRENNUNG
of compound 8 (0.081 mmol, 71 mg) was dissolved in CH3CN/H2O (1:1 v/
v; 50 mL) and then stirred overnight with Amberlite 120R previously
generated in the Na+ form (25 g). The dark red solution was filtered, and
the resin was washed with CH3CN/H2O (1:1 v/v) until the filtrate was
very pale pink. Acetonitrile was evaporated under reduced pressure and
the water was then completely removed by freeze-drying to provide 9 as
ACHTUNGNERT(NGNU CH3)4), 2.61 (m, 4H; OCH2), 2.2 (d, J=6.0 Hz, 6H; CH3), 1.74–0.75 (m,
20H; CH2), 0.71 (t, J=13.2 Hz, 6H; CH3), À0.69 ppm (s, 2H; NH);
13C NMR (75 MHz, CD3CN, 258C, TMS): d=213.41, 213.08 (2C; CO),
149.19, 148.60, 147.51, 146.76, 142.06, 141.95, 138.73, 138.19 (16C; C pyr-
À
role), 99.78, 99.61, 98.36, 97.41 (4C; meso-C), 73.99, 69.61 (2C; O CH),
À
50.73 (2C; OCH2), 34.96, 32.33 (2C; CH2COOH), 29.94–26.99 (20C;
À
a
violet crystalline powder. Yield (20 mg, 32%) as
a
violet solid;
CH2 ), 25.66, 24.26 (2C; CH2CH2COOH), 24.26, 23.11, 16.65, 14.21,
1H NMR (300 MHz, [D6]DMSO, 258C, TMS): d=10.44, 10.34, 10.28,
10.25 (m, 4H; meso-H), 8.52 (m, 2H; CH=CH2), 6.44 (d, J=18.1 Hz,
2H; CH=CH2), 6.21 (d, J=9.6 Hz, 2H; CH=CH2), 4.25 (m, 4H; porphy-
rin-CH2), 3.74, 3.71, 3.65, 3.62 (s, 12H; b-CH3), 3.13 (m, 4H;
CH2COOH), 0.55–1.81 (brs, 11H; B12H11), À3.84 ppm (s, 2H; NH);
13C NMR (75 MHz, [D6]DMSO, 258C, TMS): d=176.43, 176.69 (2C;
CO), 145.39, 143.54, 142.37, 137.03, 136.85, 136.74, 130.61, 126.77 (16C; C
pyrrole), 97.92–97.14 (4C; meso-C), 56.73, 56.6 (4C; CH=CH2), 31.27,
30.56 (2C; CH2COOH), 23.28, 22.67 (2C; CH2CH2COOH), 13.45, 12.57,
11.93, 11.65 ppm (8C; CH3); 11B NMR (96.3 MHz, CD3CN, 258C,
BF3·Et2O): d=À15.35, À11.98, À9.75, À5.45 ppm; IR (KBr disc): n˜ =
3568 s, 3423 s (OH/NH), 3305 w (CH), 1735 s, 1689 (C=O), 1635 s (C=C),
À
1611 s (NH), 1458 s (CH3), 1125 s (CN), 1080 s (B B), 2995 m, 2854 m,
1475 s, 1458 s, 1410 m, 1282 m, 1165 m, 1058 s, 885 m, 721 m cmÀ1 (CH2
groups); UV/Vis (CH3CN): lmax =412, 520, 535, 591, 634 nm; MS (ESI,
negative): m/z (%): 936 (100) [M+2]+; elemental analysis calcd (%) for
C66H120N6B12O3S (1207.49): C 65.65, H 10.02, N 6.96; found: C 65.34, H
9.83, N 6.7.
11.56, 11.28 ppm (4C; CH3); 11B NMR (96.3 MHz, [D6]DMSO, 258C,
Bis(sodium)bis(a-methyl-b-dodecanylethylether)protoporphyrin-IX-mono-
AHCTUNGTRENNUNG
.
BF3 Et2O): d=À15.24, À11.85, À9.95, À5.65 ppm; UV/Vis (H2O): lmax
=
411, 515, 535, 583, 637 nm.
Bis(tetramethyl
phyrin IX monothioester undecahydro-closo-dodecaborate (10): This
compound was prepared from (0.16 mmol, 118 mg) and
[(CH3)4N]2B12H11SH (0.25 mmol, 80 mg) by the procedure described for
8, to give 10 (88 mg, 55%) as a deep red solid; Rf =0.35; 1H NMR
(300 MHz, CD3CN, 258C, TMS): d=10.65, 10.56, 10.49, 10.12 (s, 4H;
meso-H), 6.16 (m, 2H; a-CH), 4.37 (m, 4H; porphyrin-CH2), 3.74, 3.69,
3.66, 3.65 (s, 12H; b-CH3), 3.3 (t, J=12.4 Hz, 4H; CH2COOH), 3.0 (t,
G
a
[D6]DMSO, 258C, TMS): d=10.57, 10.45, 10.21, 10.12 (s, 4H; meso-H),
6.11 (m, 2H; a-CH), 4.34 (m, 4H; porphyrin-CH2), 3.68, 3.66, 3.64, 3.61
(s, 12H; b-CH3), 3.35 (m, 4H; CH2COOH), 2.85 (m, 4H; OCH2), 2.18
(d, J=6.0 Hz, 6H; CH3), 1.85–0.81 (m, 20H; CH2), 0.76 (t, J=13.2 Hz,
6H; CH3), À3.92 ppm (s, 2H; NH); 13C NMR (75 MHz, [D6]DMSO,
258C, TMS): d=207.41, 168.42 (2C; CO), 149.19, 148.60, 147.51, 146.76,
142.06, 141.95, 138.73, 118.25 (16C; C pyrrole), 98.56, 98.05, 96.85, 96.54
À
J=13.5 Hz, 4H; OCH2), 2.98 (s, 24H; N
(CH3)4), 2.42 (s, 6H; CH3), 1.75,
(4C; meso-C), 72.85, 71.65 (2C; O CH), 52.65 (2C; OCH2), 31.86, 28.89
À
À
1.39, 1.21 (m, 12H; CH2), 0.75 (t, J=12.7 Hz, 6H; CH3), À0.48 ppm (s,
(2C; CH2COOH), 27.86–22.74 (20C;
CH2 ), 25.66, 24.26 (2C;
2H; NH); 13C NMR (75 MHz, CD3CN, 258C, TMS): d=210.86, 178.47
CH2CH2COOH), 22.54, 21.19, 15.75, 12.41, 11.52 ppm (8C; CH3);
Chem. Eur. J. 2010, 16, 1543 – 1552
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H. Nakamura et al.
11B NMR (96.3 MHz, CD3CN, 258C, BF3·Et2O): d=À15.40, À11.95,
1065 s cmÀ1 (B B); UV/Vis (CH3CN): lmax =415, 524, 536, 595, 639 nm;
MS (ESI, negative): m/z (%): 701.6 (100) [M]À; elemental analysis calcd
(%) for C38H56N6B12O3 (776.64): C 58.77, H 7.53, N 10.82; found: C
58.47, H 7.26, N 10.67.
À
À9.85, À5.42 ppm; UV/Vis
ACHTNUGTRNEUN(GN H2O): lmax =412, 520, 535, 591, 634 nm.
Bis(methyltriphenylphosphenium)protoporphyrin IX monooxoester un-
decahydro-closo-dodecaborate (14): This compound was prepared from 1
(0.16 mmol, 90 mg) and (MePh3P)2B12H11OH (0.25 mmol, 178 mg) by the
procedure described for 8, to give 14 (140 mg, 70%) as a dark violet
solid; Rf =0.31; 1H NMR (300 MHz, CD3CN, 258C, TMS): d=9.48, 9.26,
8.84 (s, 4H; meso-CH), 7.63 (m, 2H; CH=CH2), 7.83–7.53 (m, 30H;
Ph3P), 6.1 (m, 4H; CH=CH2), 4.12 (m, 4H; porphyrin-CH2), 3.74, 3.65,
3.64, 3.61 (s, 12H; b-CH3),3.35 (m, 4H; CH2COOH), 3.1 (s, 6H; CH3P),
0.4–1.69 (brs, 11H; B12H11), À2.05 ppm (s, 2H; NH); 13C NMR (75 MHz,
CD3CN, 258C, TMS): d=177.26, 175.08 (2C; CO), 148.33, 148.45, 140.9,
137.12, 136.52, 133.21, 133.07, 130.18 (16C; C pyrrole), 130.13, 129.96,
121.29, 121.23, 121.20, 119.24 (36C; Ph3P), 97.92–97.14 (4C; meso-C),
54.78 (2C; CH=CH2), 36.62 (2C; CH3P), 31.35, 30.76 (2C; CH2COOH),
23.15, 22.97 (2C; CH2CH2COOH), 13.05, 12.31, 12.01, 11.74 ppm (4C;
Sodium-protoporphyrin IX monoamide undecahydro-closo-dodecaborate
(17): This compound was prepared from 16 (0.1 mmol, 80 mg) by the pro-
cedure described for 9, to give 17 (36 mg, 49%) as a violet solid;
1H NMR (300 MHz, CD3CN, 258C, TMS): d=10.39, 10.3, 10.26, 10.22 (s,
4H; meso-H), 8.47 (m, 2H; CH=CH2), 6.46 (d, J=13.2 Hz, 2H; CH=
CH2), 6.22 (d, J=11.1 Hz, 2H; CH=CH2), 5.57 (s, 2H; NH2), 4.28 (m,
4H; porphyrin-CH2), 3.71–3.61 (s, 12H; b-CH3), 2.93 (m, 4H;
CH2COOH), 0.27–1.89 (brs, 11H; B12H11), À3.82 ppm (s, 2H; NH);
13C NMR (75 MHz, [D6]DMSO, 258C, TMS): d=176.55, 175.04 (2C;
CO), 146.38, 145.41, 143.38, 140.12, 137.65, 136.96, 133.14 (16C; C pyr-
role), 97.99–97.41 (4C; meso-C), 56.64, 56.23 (2C; CH=CH2), 33.17,
30.87 (2C; CH2COOH), 22.98, 22.05 (2C; CH2CH2COOH), 14.12, 13.57,
11.36, 11.05 ppm (4C; CH3); 11B NMR (96.3 MHz, CD3CN, 258C,
.
CH3); 11B NMR (96.3 MHz, CD3CN, 258C, BF3 Et2O): d=À23.46,
BF3·Et2O): d=À15.25, À11.80, À9.95, À5.65 ppm; UV/Vis (H2O): lmax
=
À17.55, À14.78, 6.41; IR (KBr disc): n˜ =3567 s, 3431 s (OH/NH), 2998 w
415, 525, 535, 595, 639 nm; MS (ESI, negative): m/z (%): 701.6 (100)
[M]À.
(CH), 2484 s (BH), 1725 s, 1690 s (C=O), 1675 s (C=C), 1615 s (NH),
1377 s (CH3), 1153 s (CN), 1065 s cmÀ1 (B B); UV/Vis (CH3CN): lmax
=
À
415, 522, 536, 595, 639 nm; MS (ESI, negative): m/z (%): 358 (65) [M]À/
2; elemental analysis calcd (%) for C72H80N4B12O4P2 (1257.11): C 68.79,
H 6.41, N 4.46; found: C 68.52, H 6.19, N 4.16.
Tetrakis(tetramethylACTHNUTRGNEaUNG mmonium)protoporphyrin IX bisthioester undeca-
hydro-closo-dodecaborate (18): Protoporphyrin IX (1, 0.16 mmol, 90 mg)
was dissolved in anhydrous dichloromethane (10 mL) under argon in a
25 mL round-bottomed flask. Freshly distilled oxalyl chloride
(17.5 mmol, 1.5 mL) was added, and the reaction mixture was stirred at
room temperature for 1 h. The dichloromethane and unreacted oxalyl
chloride were removed under vacuum. The resulting green solid of por-
phyrin acid chloride was dissolved in anhydrous acetonitrile (15 mL), and
[(CH3)4N]2B12H11SH (0.5 mmol, 161 mg) and anhydrous pyridine
(6.2 mmol, 0.5 mL) were added. The mixture was stirred for 72 h and the
volatile components were removed under vacuum. The crude product
was purified by column chromatography on silica gel with CH2Cl2/MeOH
BisACHTUNGTRENNUNG(sodium)protoporphyrin IX monooxoester undecahydro-closo-dodeca-
borate (15): This compound was prepared from 14 (0.11 mmol, 140 mg)
by the procedure described for 9, to give 15 (48 mg, 58%) as a dark
violet solid; 1H NMR (300 MHz, CD3CN, 258C, TMS): d=10.3, 10.25,
10.19, 10.13 (s, 4H; meso-CH), 8.5 (m, 2H; CH=CH2), 6.46 (d, J=
16.4 Hz, 2H; CH=CH2), 6.14 (d, J=12.2 Hz, 2H; CH=CH2), 4.34 (m,
4H; porphyrin-CH2), 3.73–3.60 (s, 12H, b-CH3), 3.21 (m, 4H;
CH2COOH), 0.21–1.55 (brs, 11H; B12H11), À3.81 ppm (s, 2H; NH);
13C NMR (75 MHz, CD3CN, 258C, TMS): d=176.43, 176.69 (2C; CO),
145.39, 143.54, 142.37, 137.03, 136.85, 136.74, 130.61, 126.77 (16C; C pyr-
role), 97.92–97.14 (4C; meso-C), 56.73, 56.6 (2C; CH=CH2), 31.27, 30.56
(2C; CH2COOH), 23.28, 22.67 (2C; CH2CH2COOH), 13.45, 12.57, 11.56,
(7:3) to give 18. Yield (139 mg, 74%) as
a violet solid; Rf =0.38;
1H NMR (300 MHz, CD3CN, 258C, TMS): d=10.45, 10.39, 10.26, 10.35
(m, 4H; meso-H), 8.59 (m, 2H; CH=CH2), 6.39 (d, J=14.0 Hz, 2H; CH=
CH2), 6.21 (d, J=9.0 Hz, 2H; CH=CH2), 4.35 (m, 4H; porphyrin-CH2),
3.75, 3.70, 3.65, 3.60 (s, 12H; b-CH3), 3.26 (m, 4H; CH2COOH), 3.07 (s,
.
11.28 ppm (4C; CH3); 11B NMR (96.3 MHz, CD3CN, 258C, BF3 Et2O):
d=À23.73, À17.63, À14.9, 6.51 ppm; UV/Vis (H2O): lmax =415, 525, 535,
24H; N
A
595, 639 nm.
13C NMR (75 MHz, CD3CN, 258C, TMS): d=202.13 (2C; CO), 146.56,
145.17, 143.25, 137.14, 136.92, 135.56, 131.66, 127.72 (16C; C pyrrole),
98.19–97.05 (4C; meso-C), 56.96, 56.62 (4C; CH=CH2), 55.07 (8C; N-
Synthesis of bis(tetramethylACTHNURTGNEUaGN mmonium)protoporphyrin IX monoamide
undecahydro-closo-dodecaborate (16): Protoporphyrin IX (1, 0.16 mmol,
90 mg) was dissolved in anhydrous dichloromethane (10 mL) under
argon in a 25 mL round-bottomed flask. Freshly distilled oxalyl chloride
(8.2 mmol, 0.7 mL) was added, and the reaction mixture was stirred at
room temperature for 1 h. The dichloromethane and the unreacted oxalyl
chloride were removed under vacuum. The resulting green solid was dis-
AHCTNUGTERNG(NNU CH3)4), 31.45, 29.99 (2C; CH2COOH), 23.65, 23.02 (2C;
CH2CH2COOH), 13.59, 12.59, 11.49, 11.74 ppm (4C; CH3); 11B NMR
(96.3 MHz, CD3CN, 258C, BF3·Et2O): d=À15.35, À11.94, À9.71,
À5.53 ppm; IR (KBr disc): n˜ =3254 s (NH), 3026 w (CH), 2499 s (BH),
1682 s (C=O), 1620 s (C=C), 1609 s (NH), 1416 s (CH3), 1160 s (CN),
solved in anhydrous DMF (10 mL). The tetramethylACTHNURTGNEUaGN mmonium salt of
1053 s cmÀ1 (B B); UV/Vis (CH3CN): lmax =415, 523, 536, 595, 639 nm;
À
B12H11NH3 (0.5 mmol, 110 mg) was dissolved in anhydrous DMF (10 mL)
in a 30 mL round-bottomed flask and the solution was cooled to 08C in
an ice bath. NaH (70 mg, 1 mmol, 60% suspension in oil) was added and
the mixture was stirred for 30 min. This reaction mixture was added
dropwise to the porphyrin acid chloride solution over the course of
10 min. Anhydrous pyridine (3.7 mmol, 0.3 mL) was added and the reac-
tion mixture was stirred for 72 h. The volatile components were removed
under vacuum and the crude product was purified twice by preparative
TLC with MeOH/CH2Cl2 (1:4) as eluent to give 16. Yield (80 mg, 65%)
MS (ESI, negative): m/z (%): 436.65 (100) [M]À/2; elemental analysis
calcd (%) for C50H102N8B24O2S2 (1170.99): C 51.28, H 8.78, N 9.57; found:
C 51.12, H 8.75, N 9.38.
TetrakisACTHUNRTGNEUNG(sodium)protoporphyrin IX bis(thioester) undecahydro-closo-do-
decaborate (19): This compound was prepared from 18 (0.12 mmol,
139 mg) by the procedure described for 9, to give 19 (100 mg, 65%) as a
violet solid; 1H NMR (300 MHz, [D6]DMSO, 258C, TMS): d=10.37,
10.27, 10.15, 10.01 (m, 4H; meso-H), 8.52 (m, 2H; CH=CH2), 6.46 (d, J=
18.0 Hz, 2H; CH=CH2), 6.14 (d, J=18.0 Hz, 2H; CH=CH2), 4.34 (m,
4H; porphyrin-CH2), 3.73, 3.71, 3.60, 3.57 (s, 12H; b-CH3), 3.15 (m, 4H;
CH2COOH), 0.19–1.65 (brs, 11H; B12H11), À3.81 ppm (s, 2H; NH);
13C NMR (75 MHz, [D6]DMSO, 258C, TMS): d=179.79 (2C; CO),
146.41, 144.64, 142.47, 136.98, 136.52, 136.12, 130.3, 127.07 (16C; C pyr-
role), 97.95–97.33 (4C; meso-C), 56.79, 56.65 (4C; CH=CH2), 32.25,
31.02 (2C; CH2COOH), 23.19, 22.87 (2C; CH2CH2COOH), 14.05, 12.97,
11.59, 11.56 ppm (4C; CH3); 11B NMR (96.3 MHz, [D6]DMSO, 258C,
1
as a violet solid; Rf =0.33; H NMR (300 MHz, CD3CN, 258C, TMS): d=
9.59, 9.28, 10.12 (m, 4H; meso-H), 8.02 (m, 2H; CH=CH2), 6.11 (m, 4H;
CH=CH2), 4.74 (S, 2H; NH2), 4.28 (m, 4H; porphyrin-CH2), 4.07, 3.88
(m, 12H; b-CH3), 3.48, 3.27 (m, 4H; CH2COOH), 2.88 (s, 12H; N-
A
(75 MHz, CD3CN, 258C, TMS): d=176.89, 176.21 (2C; CO), 145.42,
143.93, 141.73, 140.34, 136.63, 136.33, 135.85, 130.14 (16C; C pyrrole),
97.46–97.04 (4C; meso-C), 54.35, 56.6 (2C; CH=CH2), 54.31 (4C; N-
BF3·Et2O): d=À15.21, À11.89, À10.05, À5.72; UV/Vis
ACHTUGNERTN(NUNG H2O): lmax =415,
ACHTUNGTRENNUNG(CH3)4), 33.0, 30.56 (2C; CH2COOH), 23.28, 23.0 (2C; CH2CH2COOH),
522, 536, 591, 639 nm.
13.45, 12.57, 11.56, 11.28 ppm (4C; CH3); 11B NMR (96.3 MHz, CD3CN,
258C, BF3·Et2O): d=À15.37, À11.97, À9.78, À5.49 ppm; IR (KBr disc):
n˜ =3565 s, 3382 s, 3246 s (OH/NH), 2985 w (CH), 2493 s (BH), 1710 s,
1675 s (C=O), 1616 s (C=C), 1605 s (NH), 1406 s (CH3), 1165 s (CN),
Biological studies
Cell viability (MTT) assay: Human cervical carcinoma HeLa cells, rat
glioma C6 cells, mouse melanoma B16 cells, mouse colon cancer CT26
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 1543 – 1552

Boron-Containing Protoporphyrin IX Derivatives
FULL PAPER
cells, human hepatocellular carcinoma HepG2 cells, and human colon
cancer HCT15 and HCT116 cells were used for the cell viability assay.
The cells (5ꢂ103 cells per well of a 96-well plate) were incubated at 378C
for 72 h in RPMI-1640 medium (100 mL) containing various concentra-
tions of the boronated porphyrins. After the incubation, 3’-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) in PBS
(5 mgmLÀ1, 10 mL) was added to each well, and the cells were further in-
cubated at 378C for 4 h. After the removal of the medium, DMSO
(100 mL) was added and the absorbance at 570 nm was determined with a
microplate reader. The drug concentration required to reduce cell viabili-
ty by 50% (IC50) was determined from semilogarithmic dose-response
plots.
Boron accumulation study: HeLa cells (1ꢂ106 cells) were incubated at
378C for 3 h in medium containing each boronated porphyrin. After the
incubation, the cells were washed three times with PBS and digested with
perchloric acid/hydrogen peroxide (2 mL) at 708C for 6 h. Boron con-
tents in the solution were determined by an inductively coupled plasma
atomic emission spectroscopy (ICP-AES).
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Acknowledgements
This work was supported in part by a Grant-in Aid from the Ministry of
Education, Science and Culture, Japan (20390379), and from the Ministry
of Health, Labour and Welfare (20100201). ME thanks the Japan Society
for the Promotion of Science (ID No. P 08363) for the financial support.
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