Synthesis and toxicity of cobaltabisdicarbollide-containing porphyrins of high boron content

Article abstract of DOI:10.1142/S1088424611003902

Two porphyrins of high boron content (OCP and HCPPEG) were prepared in high yields from the reaction of the corresponding tri- and tetra-(dihydroxyphenyl) porphyrins with zwitterionic cobaltabisdicarbollide [3,3′-Co(8-C ₄H₈O₂

Full text of DOI:10.1142/S1088424611003902

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2011; 15: 973–983
DOI: 10.1142/S1088424611003902
Published at http://www.worldscinet.com/jpp/
Synthesis and toxicity of cobaltabisdicarbollide-containing
porphyrins of high boron content
N.V.S. Dinesh K. Bhupathirajua, Vijay Gottumukkalaa, Erhong Haob, Xiaoke Hua
,
Frank R. Fronczeka, David G. Bakerc, Nobuko Wakamatsuc and M. Graça H.Vicente*a
a Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA
b Anhui Key Laboratory of Functional Molecular Solids and Anhui Key Laboratory of Molecular Based Materials,
College of Chemistry and Material Science, Anhui Normal University, Wuhu, Anhui 241000, China
c Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge,
LA 70803, USA
Dedicated to Professor Karl M. Kadish on the occasion of his 65th birthday
Received 17 May 2011
Accepted 24 June 2011
ABSTRACT: Two porphyrins of high boron content (OCP and HCP–PEG) were prepared in high yields
from the reaction of the corresponding tri- and tetra-(dihydroxyphenyl)porphyrins with zwitterionic
cobaltabisdicarbollide [3,3′-Co(8-C4H8O2-1,2-C2B9H10)(1′,2′-C2B9H11)]. Both porphyrins were found
to have low cytotoxicity toward human HEp2 cells, and to localize subcellularly mainly in the cell
lysosomes.Animal toxicity investigations using male and female BALB/c mice also revealed low toxicity
for both compounds. The determined maximum tolerated dose (MTD) for these boronated porphyrins
administered intraperitoneally were 160 mg/kg for OCP and 320 mg/kg for HCP–PEG. Our studies
warrant further development of these porphyrins of high boron content, and in particular of HCP–PEG,
as boron delivery vehicles for BNCT.
KEYWORDS: cobaltabisdicarbollide, porphyrin, BNCT, PDT, toxicity.
BNCT has the potential to be a highly selective and local-
INTRODUCTION
ized cancer therapy, destroying 10B-containing tumor cells
Boronated porphyrins of high boron content and low
in the presence of normal boron-free cells. Therefore,
toxicity can find application as boron delivery agents for
research in the last two decades has centered on the dis-
boron neutron capture therapy (BNCT) of tumors [1].
covery of tumor-selective boronated agents able to deliver
BNCT is a binary therapy that involves the irradiation of
therapeutic boron concentrations (15–30 µg/g tumor) to
10B-rich tumors with low energy neutrons; this produces
targeted tumors with minimal normal tissue toxicity [2].
high linear energy transfer (high-LET) alpha-particles
Two compounds are currently in BNCT clinical trials for
and recoiling lithium-7 nuclei, and releases 2.4 MeV of
brain tumors, melanomas, and squamous cell carcino-
kinetic energy, according to the equation:
mas: sodium mercaptoundecahydro-closo-dodecaborate
(BSH) and (L)-4-dihydroxy-borylphenylalanine (BPA),
10B + 1n → 7Li3+ + 4He2+ + γ + 2.4 MeV
(1)
shown in Fig. 1 [3]. While BSH and BPA have shown
some effectiveness and low toxicity as boron delivery
agents for BNCT, both of these compounds have only
moderate selectivity and low retention times in tumors.
On the other hand, boronated porphyrin derivatives, in
particular those of high boron content, could potentially
deliver higher amounts of boron to tumors at the same
boron dose, with increased selectivity and retention time
The high-LET particles are highly cytotoxic and due
to their limited path lengths in tissue (less than 10 µm)
SPP full and student member in good standing
*Correspondence to: M. Graça H. Vicente, email: vicente@lsu.
edu, tel: +1 225-578-7442
Copyright © 2011 World Scientific Publishing Company

974
N.V.S.D.K. BhuPathIraJu et al.
SH
which is 34% boron by weight, bearing twelve (rather
than six) boron cages and one PEG chain, and evaluate
its cytotoxicity and cellular uptake in human HEp2 cells.
We also investigated and compared the toxicity of OCP
and HCP–PEG in BALB/c mice. The low toxicity found
for these boronated porphyrins of high boron content,
and in particular for HCP–PEG, is crucial in their further
development and investigation as boron delivery vehicles
for BNCT.
H
NH3
B
BH
Na2
CO2
(HO)2B
BPA
BSH
Fig. 1. Structures of BSH and BPA currently in BNCT clinical
trials
in tumor tissue [4]. Furthermore, boronated porphyrins
retain the absorption and emission properties characteris-
tic of this type of molecule, facilitating the quantification
of tumor-localized boron and treatment planning. How-
ever, the synthesis of boronated porphyrins of high boron
content often leads to low yields of the target products,
due to steric and/or electronic reasons. We have recently
developed an efficient methodology for the preparation
of cobaltabisdicarbollide-containing porphyrins, based
on the opening of the dioxane ring of zwiterionic [3,3′-
Co(8-C4H8O2-1,2-C2B9H10)(1′,2′-C2B9H11)] by pyridyl-
or phenoxy-containing porhyrin macrocycles [5]. Using
this strategy, we have prepared OCP (1), containing 39%
boron by weight, in one step from commercially avail-
able tetra(3,5-dihydroxyphenyl)porphyrin, in 95% yield
(Scheme 1) [5c]. Preliminary cellular investigations
showed that OCP had low cytotoxicity toward human HEp2
cells [5c] and that a pegylated cobaltabisdicarbollide-
porphyrin of comparatively low boron content (24%)
conjugated to a cell penetrating peptide (from the human
immunodeficiency virus I transcriptional activator, HIV-1
Tat 48–60), readily accumulated within cells, about
11-fold compared to the non-conjugated porphyrin [5d].
Herein we describe the synthesis of an unsymmetric high
boron-containing porphyrin designated HCP–PEG (2)
RESULTS AND DISCUSSION
Synthesis
While symmetrically substituted OCP (1) is read-
ily prepared in a one-step reaction from commercially
available tetra(3,5-dihydroxyphenyl)porphyrin, as shown
in Scheme 1, the unsymmetric HCP–PEG (2) required
initial synthesis of a porphyrin containing two differ-
ent phenyl substituents, i.e., porphyrins 3a–c, as shown
in Scheme 2. A mixed-aldehyde condensation using 3
equiv. of 3,5-dimethoxybenzaldehyde, 1.5 equiv. of an
appropriately para-functionalized benzaldehyde and
4equiv. ofpyrroleunderLindsey’sreactionconditions[6],
produced the corresponding A3B-type porphyrin as the
major product, in yields ranging from 9–12%. Crystals of
porphyrins 3a–c suitable for X-ray analysis were grown
from slow evaporation of dichloromethane and hexane.
The molecular structures for these three porphyrins are
shown in Fig. 2. In all porphyrins, the 24-atom porphyrin
core is fairly planar. In both 3a and 3c, there is a slight
saddle distortion, with maximum and mean deviations
0.254(2) and 0.091 Å for 3a; 0.279(2) and 0.104 Å for 3c.
K
K
Co
Co
O
O
O
O
K
K
O
Co
O
Co
OH
OH
O
O
O
O
O
O
O
O
O
O
O
Co
OH
OH
N
H
H
N
HO
HO
O
N
H
H
N
N
N
N
N
O
O
K2CO3, acetone, 60 °C
(95%)
Co
Co
O
O
K
OH
HO
K
O
O
CH
B
BH
O
O
Co
Co
K
K
1: OCP
Scheme 1. One-step synthesis of octa-cobaltacarboranylporphyrin (OCP)
Copyright © 2011 World Scientific Publishing Company
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975
CHO
CHO
R = CO2CH3, OCH2Ph, OCOCH3
N
H
H3CO
OCH3
1. BF3•OEt2
R
2. DDQ (9-12%)
OCH3
OH
OCH3
OH
OCH3
OH
H3CO
H3CO
HO
HO
N
N
H
H
N
1. BBr3 (90%)
H
H
N
N
N
N
N
2. Boc-Gly, DIEA
HOBt, TBTU, DMF
(80%)
O
R
OCH3
OH
HN
4
3a: R = CO2CH3
3b: R = OCH2Ph
3c: R = OCOCH3
CO2tBu
1. [3,3’-Co(8-C4H8O2-1,2-C2B9H10)(1’,2’-C2B9H11)]
K2CO3, acetone (85%)
2. TFA, acetone (95%)
3. NH2(CH2CH2O)3CH2CH2CO2tBu, DMF
DIEA, HOBt, HATU (20%)
4. TFA, acetone (95%)
K
K
Co
Co
O
O
O
O
K
K
O
Co
Co
O
O
O
O
O
O
O
N
H
H
N
N
N
B
BH
CH
O
O
O
H
O
N
Co
O
N
O
O
H
K
O
O
O
OH
O
O
2: HCP-PEG
Co
K
Scheme 2. Synthesis of hexa-cobaltacarboranylporphyrin-PEG (HCP–PEG)
For 3b, the distortion from planarity is smaller, with
maximum and mean deviations 0.111(3) and 0.036 Å.
The conformations of the methoxy groups in 3a and 3c
are also similar, five pointing away from the center of the
molecule and one inward in both, while in 3b, five point
inward and only one outward.
of porphyrin 3a, and the benzyl and acetyl ethers of por-
phyrins 3b and 3c, respectively. The free carboxylic acid
group resulting from deprotection of porphyrin 3a reacted
with Boc-protected glycine in the presence of HOBt and
TBTU as coupling agents [7] to afford porphyrin 4 in
80% yield. The glycine linker decreases steric interac-
tions and increases the yield of the conjugation reaction
to the PEG group [8]. Reaction of porphyrin 4 with
Deprotection of the methoxyl groups of porphyrins
3a–c using BBr3 [5d] also cleaved the methyl ester group
Copyright © 2011 World Scientific Publishing Company
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N.V.S.D.K. BhuPathIraJu et al.
Fig. 2. Molecular structures of porphyrins 3a, 3b, and 3c with 50% ellipsoids
zwiterionic cobaltabisdicarbollide [3,3′-Co(8-C4H8O2–1,2-
C2B9H10)(1′,2′-C2B9H11)], prepared from reaction of com-
mercially available Cs[Co(C2B9H11)2] with 1,4-dioxane in
the presence of BF3·Et2O [9], gave the corresponding hexa-
cobaltacarboranylporphyrin in 85% yield. Deprotection
of the tert-butyl ester group using TFA proceeded nearly
quantitatively, and the resulting carboxylated porphyrin
was characterized by NMR, MS and UV-vis. Subsequent
conjugation of this porphyrin to commercially available
tert-butyl-12-amino-4,7,10-trioxadodecanoate followed by
deprotection of the tert-butyl group using TFA, gave HCP–
PEG (2) in 15% overall yield from porphyrin 4. HCP–PEG
is a valuable precursor for the conjugation of various biomol-
ecules, such as the HIV-1 Tat 48–60 peptide [5d], that might
enhance tumor cell uptake and overall biological efficacy of
the boronated porphyrin as BNCT delivery agent [2b].
to 50 µM for OCP (higher concentrations were not evalu-
ated to avoid precipitation due to compound aggregation
[5c]) and up to 400 µM for HCP–PEG in the dark, the
compounds were found non-toxic to the cells, as evalu-
ated using the Cell Titer Blue assay [10] (Fig. 3a). Upon
exposure to approx. 1 J/cm2 light dose, neither compound
was found to be toxic, as shown in Fig. 3b for HCP–PEG.
Fluorescence microscopy was used to investigate cel-
lular uptake and subcellular localization, as previously
reported for OCP [5c] and shown in Fig. 4 for HCP–PEG.
As observed for OCP [5c], HCP–PEG showed a punctu-
ate pattern that correlated mostly with the cell lysosomes,
as seen in Fig. 4j. A minor/partial site of localization was
the Golgi apparatus. Preferential lysosomal localization
of cobaltabisdicarbollide-containing porphyrins [5b–d]
and other boronated porphyrins [11, 12] might result
from endocytosis of small porphyrin aggregates and their
subsequent localization in endosomal vesicles, some of
which might fuse with the cell lysosomes.
Cell studies
The subcellular distribution and cytotoxicity of OCP
(1) and HCP–PEG (2) were evaluated in human carci-
noma HEp2 cells and the results obtained using HCP–
PEG are shown in Figs 3 and 4 (we have previously
published the results for OCP [5c]). At concentrations up
Animal toxicity
Both male and female BALB/c mice were used in
these studies, as previously reported for other boronated
Fig. 3. Cytotoxicity of HCP–PEG (2) toward HEp2 cells using the Cell Titer Blue assay, (a) in the dark and (b) after exposure to 1
J/cm2 light dose
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Fig. 4. Subcellular localization of HCP–PEG (2) in HEp2 cells at 10 µM for 6 h; (a) phase contrast, (b) overlay of HCP–PEG fluo-
rescence and phase contrast, (c) ER tracker Blue/White fluorescence, (e) MitoTracker Green fluorescence, (g) BODIPY Ceramide,
(i) LysoSensor Green fluorescence, and (d, f, h, j) overlays of organelle tracers with HCP–PEG fluorescence. Scale bar: 10 µm
Copyright © 2011 World Scientific Publishing Company
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N.V.S.D.K. BhuPathIraJu et al.
Table 2. Serum chemistry values for 12 BALB/c mice admin-
istered HCP–PEG (2) in 3% DMSO in PBS (groups 8, 9, 10,
11, 12) or only 3% DMSO in PBS (vehicle: group 7), by ip
injection
porphyrins [11c]. OCP or HCP–PEG were dissolved in
3% DMSO in PBS and all mice received intraperito-
neal (ip) injections of 0.15–0.74 mL. Group 6 received
a maximum dose of 160 mg/kg of compound OCP (1),
representing maximum saturation of 4 mg/mL. Group
12 received maximum dose of 320 mg/kg of HCP–PEG
(2), representing maximum saturation of 20 mg/mL.
No clinical chemistry changes occurred corresponding
to either vehicle or compound injected. Tables 1 and 2
show the clinical data obtained for OCP and HCP–PEG,
respectively.
Group
7
8
9
10
227.5 140.5 137.0 151.5
(19.0) (37.5) (0.5) (6.0) (3.5)
11
12
Glucose
(mg/dl)
AST
150.0 166.0
(6.0)
1049.0 207.7 2248.5 147.5 146.5 589.5
(947.0) (201.35) (1006.5) (80.5) (8.5) (294.5)
(U/L)
ALT
159.5 244.0
337.0 28.5 26.5 125.5
No signs of toxicity were observed in any of the
experiments using HCP–PEG or OCP. For OCP the
slightly elevated glucose levels in Table 1 might indicate
a diabetic condition, or may simply occur due to excite-
ment and increased corticosteroid release. The latter is
considered responsible for increases in serum glucose
levels observed in mice of group 3. On the other hand,
the trends toward decreases in total protein, albumin,
and globulin may be explained by decreased produc-
tion or increased loss. With HCP–PEG (Table 2) plasma
glucose levels generally increased, likely due to excite-
ment and increase corticosteroid release. One mouse
in group 7 (control) and both mice in group 9 (40 mg/
kg) had highly elevated plasma AST levels, most likely
due to liver trauma during compound injection. Values
for both mice in group 12 (320 mg/kg) were moderately
(U/L)
(135.5) (175.0) (142.0) (6.5) (1.5) (74.5)
AP
116.5 112.0
130.0 125.5 108.0 152.0
(10.0) (17.5) (12.0) (49.0)
(U/L)
(17.5)
0.30
(7.0)
0.20
(0.0)
5.0
Bilirubin
(mg/dl)
Total Prot.
(g/dl)
0.40
(0.0) (0.05) (0.0) (0.05)
5.5 5.9 5.9 5.4
0.20 0.20 0.30
(0.05)
5.4
(0.45) (0.05) (0.05) (0.15) (0.45) (0.20)
Albumin
(g/dl)
3.1
(0.30)
2.3
2.8
(0.0)
2.2
3.2
(0.0) (0.05) (0.15) (0.15)
2.3 2.6 2.5 2.5
3.3
3.4
3.0
Globulin
(g/dl)
(0.15) (0.05) (0.05) (0.10) (0.30) (0.05)
Values represent mean serum chemistry levels and the standard
error of the mean (SEM) values are given in parenthesis.
elevated possibly suggestive of mild hepatocyte dam-
age though this was not observed histologically. A more
likely explanation for the moderate elevations in mice of
group 12 was muscular overexertion. One mouse in each
of groups 8 and 9 (20 and 40 mg/kg, respectively) had
mildly elevated plasma ALT levels. One of these mice
(from group 9) also showed elevated AST levels. These
mild elevations were likely due to trauma to the liver dur-
ing compound injection. Overall, there did not appear
to be a treatment effect on ALT. Plasma alkaline phos-
phatase levels were within normal limits suggestive of
a lack of toxicity for several of the major organ systems
including the renal, gastrohepatic, immune, and skeletal
systems. Plasma bilirubin levels were within normal lim-
its. Plasma total protein levels plateaued in mice of group
11 (160 mg/kg) and declined slightly in mice of group 12
(320 mg/kg) most likely because of reductions in albu-
min levels. However, levels of total protein in all mice
remained within normal limits. Plasma albumin levels
declined slightly in mice of group 12 (320 mg/kg) but
remained within normal limits. No treatment effects were
noted in plasma globulin levels. The values for all mice
were within normal limits. Histopathologic examination
revealed no lesions attributable to compound administra-
tion. Extramedullary hematopoiesis was observed in all
mice, and may represent mild levels of stress, normal for
these animals. Moderate thickening of Bowman’s cap-
sule (periglomerular fibrosis) was also observed in one
renal glomerulus of one mouse in group 2 and likely rep-
resented an incidental finding.
Table 1. Serum chemistry values for 11 BALB/c mice adminis-
tered OCP (1) in 3% DMSO in PBS (groups 2, 3, 4, 5, 6) or only
3% DMSO in PBS (vehicle: group 1), by ip injection
Group
1
2
3
4
5
6
Glucose 190.0 212.0a 258.0b 217.5a 211.0a 219.0a
(mg/dl)
AST
(0)
(8.0)
(5.0)
(0.5)
(6.0) (7.0)
244.5 224.5
92.0 577.0 150.5
282.5
(U/L)
ALT
(0) (468.0) (87.5) (178.5) (156.5) (61.5)
26.0 319.0 68.5 289.0 199.0 185.5
(0) (260.0) (21.5) (257.0) (130.0) (20.5)
114.0 117.5 158.5 127.0 170.5 138.5
(0) (11.5) (31.5) (3.0) (11.5) (8.5)
0.10 0.15 0.15 0.15 0.1 0.2
(0) (0.05) (0.05) (0.05) (0) (0)
4.4
(0.05) (0.1) (0.2)
(U/L)
AP
(U/L)
Bilirubin
(mg/dl)
Total Prot. 5.0
4.6
(0)
4.7
4.75
2.7
4.6
(g/dl)
(0)
2.9
(0)
2.1
(0.1)
2.75
Albumin
(g/dl)
2.6
(0.1)
2.0
2.7
(0)
1.9
2.55
(0.05)
1.85
(0.05) (0.1)
1.95 2.05
Globulin
(g/dl)
(0)
20.0
(0)
(0.1)
18.5
(1.5)
(0.05) (0.05) (0.1) (0.15)
BUN
19.5
(0.5)
15.5
(0.5)
22.5 18.0
(3.5) (4.0)
(mg/dl)
Values represent mean serum chemistry levels and the standard
error of the mean (SEM) values are given in parenthesis. For
individual analytes measured, rows with superscripts in com-
mon (a or b) are not different from one another (p > 0.05).
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Compound OCP was found to be non-toxic up to a
dose of 160 mg/kg, while HCP–PEG was non-toxic up to
a dose of 320 mg/kg, which is in aggrement with the low
toxicity observed for other boronated porphyrins of lower
boron content [11c, 13–16]. Indeed, these compounds are
comparatively less toxic than other previously reported
boronated porphyrins, including BTPP, BOPP, ZnDPE
and NiNTCP-H [13, 15]. Therefore, porphyrins of high
boron content such as OCP and HCP–PEG might be able
to deliver high and therapeutic boron concentrations to
targeted tumors, which warrants their further evaluation
as boron delivery agents for BNCT. Furthermore, con-
jugation of peptides and other tumor cell-targeting mol-
ecules to HCP–PEG might increase its tumor cell uptake
and specificity.
under argon for 20 min. BF3·OEt2 (0.16 mL of a 2.5 M
solution in CH2Cl2) was added at once. The reaction mix-
ture was stirred for 2 h under argon in the dark. DDQ
(0.492 g, 2.16 mmol) was added and the stirring contin-
ued for 45 min. Then the resulting mixture was neutral-
ized using aqueous NaHCO3 and filtered through a silica
gel pad using CH2Cl2 as the solvent. The resulting residue
was further purified by silica gel column chromatography
using CH2Cl2/hexanes (9.8:0.2) for elution. The second
and main fraction was collected and dried under vac-
uum (0.09 g, 11%), mp > 300 °C. 1H NMR (CDCl3, 400
MHz): δ, ppm 8.96 (d, J = 7.08 Hz, 6H, β-H), 8.78 (d,
J = 4.64 Hz, 2H, β-H), 8.45 (d, J = 8.04 Hz, 2H, o-PhH),
8.31 (d, J = 8.00 Hz, 2H, m-PhH), 7.40 (s, 6H, o-PhH),
6.91 (s, 3H, p-PhH), 4.12 (s, 3H, COOCH3), 3.97 (s, 18H,
OCH3), -2.81 (s, 2H, NH). 13C NMR (CDCl3, 100 MHz):
δ, ppm 167.4, 158.9, 147.0, 143.9, 134.5, 131.3, 129.6,
127.9, 120.1, 120.0, 118.6, 113.9, 100.2, 55.6, 52.5. MS
(MALDI-TOF): m/z 852.779, calcd. for C52H44N4O8
852.316. UV-vis (acetone): λmax, nm (ε) 422 (466600),
519 (14500), 553 (1330), 596 (880).
EXPERIMENTAL
General
Commercially available reagents and solvents (HPLC
grade) were purchased from Sigma-Aldrich and used
without further purification. COSAN was purchased from
Katchem, Inc. Silica gel 60 (70–230 mesh, Merk) used
for column chromatography and silica gel TLC plates
(0.2 mm thickness) were purchased from Sorbent Tech-
5,10,15-tri(3,5-dimethoxyphenyl)-20-(4-benzyl-
oxyphenyl)porphyrin (porphyrin 3b). 3,5-dimethoxy-
benzaldehyde (0.498 g, 3.0 mmol), 4-benzyloxybenzaldehyde
(0.318 g, 1.5 mmol), and freshly distilled pyrrole (0.278 mL,
4 mmol) were mixed in a 1 L flask. Dry CH2Cl2 (500 mL)
was added and the solution stirred under argon for 20
min. BF3·OEt2 (0.16 mL of 2.5 M in CH2Cl2) was added
at once. The reaction mixture was stirred for 2 h under
argon and in the dark. DDQ (0.492 g, 2.16 mmol) was
added and the stirring continued for 45 min. Then the
resulting mixture was neutralized using aqueous NaHCO3
and filtered through a silica gel pad using CH2Cl2 as the
solvent. The resulting residue was further purified by
silica gel column chromatography using CH2Cl2/hexanes
(9.8:0.2). The main product was isolated and dried (0.09
1
nologies. H NMR and 13C NMR spectra were obtained
using a Bruker AV-400 MHZ spectrometer; chemical
shifts are expressed in ppm. Electronic absorption spec-
tra were measured on a Perkin Elmer Lambda 35 UV-vis
spectrophotometer. Mass analysis was conducted at the
LSU Mass Spectrometry Facility on a Bruker Omniflex
MALDI-TOF mass spectrometer and exact mass were
obtained from HRMS-ESI under negative mode on an
Applied Biosystems QSTAR XL. Melting points were
measured on a Thomas hoover melting point appara-
tus. Analytical reverse phase HPLC was performed on a
Waters system including a 2545 quaternary gradient mod-
ule pump with a 2489 UV-vis detector and a fraction col-
lector III. Analytical column (4.6X250 mm-XBridgeTM
BED300 C18 5 µm) and acetonitrile/water solvent sys-
tem, varying gradient from 100% water (2 min) to 75%
acetonitrile/water (5 min) to 100% acetonitrile (10 min).
[3,3′-Co(8-C4H8O2-1,2-C2B9H10)(1′,2′-C2B9H11)] was pre-
pared as previously described [9]. Tetra(3,5-dihydroxy-
phenyl)porphyrin was purchased from Frontier Scientific
and used without further purification in the preparation of
OCP, as previously described [5c].
1
g, 10%), mp > 300 °C. H NMR (CDCl3, 400 MHz): δ,
ppm 8.96 (d, J = 7.08 Hz, 6H, β-H), 8.78 (d, J = 4.64
Hz, 2H, β-H), 8.45 (d, J = 8.04 Hz, 2H, o-PhH), 8.31
(d, J = 8.00 Hz, 2H, m-PhH), 7.40 (s, 6H, o-PhH), 6.91
(s, 3H, p-PhH), 4.12 (s, 3H, COOCH3), 3.97 (s, 18H,
OCH3), -2.81 (s, 2H, NH). 13C NMR (CDCl3, 100 MHz):
δ, ppm 158.9, 147.2, 142.9, 135.1, 131.2, 129.5, 127.8,
127.2, 120.3, 120.2, 118.6, 113.8, 100.9, 72.0, 55.8. MS
(MALDI-TOF): m/z 901.358, calcd. for C52H44N4O8
901.355. UV-vis (acetone): λmax, nm (ε) 418 (414400),
513 (16100), 553 (4400), 596 (2600).
5,10,15-tri(3,5-dimethoxyphenyl)-20-(4-acetoxy-
phenyl)porphyrin (porphyrin 3c). 3,5-dimethoxyben-
zaldehyde (0.498 g, 3.0 mmol), 4-acetoxybenzaldehyde
(0.246 g, 1.5 mmol), and freshly distilled pyrrole (0.278
mL, 4.0 mmol) were mixed in a 1 L flask. Dry CH2Cl2
(500 mL) was added and the mixture stirred under argon
for 20 min. BF3·OEt2 (0.16 mL of 2.5 M in CH2Cl2) was
added at once. The reaction mixture was stirred for 2 h
under argon and in the dark. DDQ (0.492 g, 2.16 mmol)
was added and the stirring continued for 45 min. Then the
Synthesis
5,10,15-tri(3,5-dimethoxyphenyl)-20-(4-methyl-
esterphenyl)porphyrin (porphyrin 3a). 3,5-dimethoxy-
benzaldehyde (0.498 g, 3.0 mmol), methyl-4-formyl
benzoate (0.246 g, 1.5 mmol), and freshly distilled pyr-
role (0.278 mL, 4.0 mmol) were mixed in a 1 L flask.
Dry CH2Cl2 (500 mL) was added and the mixture stirred
Copyright © 2011 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2011; 15: 979–983

980
N.V.S.D.K. BhuPathIraJu et al.
resulting mixture was neutralized using aqueous NaHCO3
and filtered through a silica gel pad using CH2Cl2 as the
solvent. The resulting residue was further purified by
silica gel column chromatography using CH2Cl2/hexanes
(9.8:0.2). The main product was isolated and dried (0.102
(d-acetone, 100 MHz): δ, ppm 169.0, 167.0, 156.7, 145.2,
143.7, 142.9, 134.4, 133.8, 128.3, 127.1, 125.8, 124.5,
120.4, 120.3, 118.9, 118.7, 114.5, 102.3, 80.8, 42.2, 27.4.
MS (MALDI-TOF): m/z 867.913, calcd. for C51H41N5O9
867.290. UV-vis (acetone): λmax, nm (ε) 423 (364000),
519 (18300), 555 (7400), 598 (6000), 654 (3500).
1
g, 12%), mp > 300 °C. H NMR (CDCl3, 400 MHz): δ,
ppm 8.96 (d, J = 7.08 Hz, 6H, β-H), 8.78 (d, J = 4.64
Hz, 2H, β-H), 8.45 (d, J = 8.04 Hz, 2H, o-PhH), 8.31
(d, J = 8.00 Hz, 2H, m-PhH), 7.40 (s, 6H, o-PhH), 6.91
(s, 3H, p-PhH), 4.12 (s, 3H, COOCH3), 3.97 (s, 18H,
OCH3), -2.81 (s, 2H, NH). 13C NMR (CDCl3, 100 MHz):
δ, ppm 170.0, 158.7, 147.1, 143.7, 134.8, 131.2, 129.0,
127.9, 120.1, 120.0, 118.6, 113.9, 100.2, 55.5, 20.1. MS
(MALDI-TOF): m/z 852.322, calcd. for C52H44N4O8
852.316. UV-vis (acetone): λmax, nm (ε) 419 (426700),
515 (15200), 549 (4200), 588 (2700).
HCP–PEG (porphyrin 2). Porphyrin 4 (14.7 mg,
0.017 mmol) and K2CO3 (28.0 mg, 0.20 mmol) were
refluxed in 10 mL acetone in a 50 mL round bottom flask
under argon for 15 min. The reaction mixture was cooled
toroomtemperatureand[3,3′-Co(8-C4H8O2–1,2-C2B9H10)
(1′,2′-C2B9H11)] (42.0 mg, 0.10 mmol) was added. The
resulting mixture was stirred at room temperature for
2 h under argon, then refluxed overnight. The second por-
tion of [3,3′-Co(8-C4H8O2-1,2-C2B9H10)(1′,2′-C2B9H11)]
(21.0 mg, 0.05 mmol) was added and reflux continued for
24 h. The last portion of [3,3′-Co(8-C4H8O2–1,2-C2B9H10)
(1′,2′-C2B9H11)] (21.0 mg, 0.05 mmol) was added and
reflux continued for an additional 24 h. The reaction was
stopped, acetone was evaporated and the resulting solid
was washed with diethyl ether several times to remove
excess cobaltabisdicarbollide. The resulting orange-red
color solid of the corresponding tert-butyl-protected
porphyrin was kept under vacuum to remove traces of
diethyl ether; this porphyrin was obtained (49 mg) in
85% yield. 1H NMR (d-acetone, 400 MHz): δ, ppm 9.01
(s, 6H, β-H), 8.86 (s, 2H, β-H), 8.37 (s, 4H, o,m-PhH),
7.48 (s, 6H, o-PhH), 7.08 (s, 3H, p-PhH), 4.40 (s, 12H,
OCH2), 4.17 (s, 24H, OCH2), 3.93 (s, 12H, OCH2), 3.67
(s, 24H, Carb-H), 1.6–3.0 (br, 104, BH, CH2), 1.54 (s, 9H,
COOC(CH3)3), -2.79 (s, 2H, NH). 13C NMR (d-acetone,
100 MHz): δ, ppm 170.1, 162.0, 158.3, 158.0, 149.9,
145.1, 143.7, 114.5, 101.1, 100.7, 72.2, 69.4, 68.4, 68.0,
59.7, 54.5, 46.6, 28.8. HRMS-ESI: m/z 670.4627 [M - 6K
+ Na]5-, calcd. for [C99H209B108Co6NaN5O21]5- 670.0437.
UV-vis (acetone): λmax, nm (ε) 424 (367702), 519 (7041).
To this porphyrin (49 mg, 15 mmol) dissolved in 5 mL
of CH2Cl2/acetone 1:1, TFA (5 mL) was added and the
final mixture was stirred at room temperature for 4 h. The
solvents were removed under vacuum and the resulting
residue was washed several times with diethyl ether and
dried under vacuum. The carboxylated porphyrin was
5,10,15-tri(3,5-dihydroxyphenyl)-20-(4-carboxy-
phenyl)porphyrin. To porphyrin 3a (0.095 g, 0.1 mmol)
in dry CH2Cl2 (20 mL) at -20 °C was added dropwise
a solution of BBr3 (0.52 mL, 5.3 mmol) in CH2Cl2
(1.5 mL) with vigorous stirring under argon over a period
of 30 min. The reaction mixture was stirred at room tem-
perature for 24 h and then poured into water and extracted
with ethyl acetate (3 × 50 mL). The combined organic
layers were washed successively with brine and aqueous
NaHCO3. The organic layer was dried over Na2SO4 and
evaporated to dryness to give the title porphyrin in 90%
1
yield, mp > 300 °C. H NMR (d-acetone, 400 MHz): δ,
ppm 9.05 (s, 6H, β-H), 8.86 (s, 2H, β-H), 8.73 (broad
s, 6H, OH), 8.50 (d, J = 7.88 Hz, 2H, o-PhH), 8.39 (d,
J = 7.88 Hz, 2H, m-PhH), 7.25 (s, 6H, o-PhH), 6.84 (s,
3H, p-PhH), -2.79 (s, 2H, NH). 13C NMR (d-acetone, 100
MHz): δ, ppm 166.9, 156.8, 146.8, 143.7, 134.5, 130.1,
128.1, 120.4, 120.3, 118.5, 114.5, 102.3. MS (MALDI-
TOF): m/z 755.432, calcd. for C45H30N4O8 755.209. UV-
vis (acetone): λmax, nm (ε) 422 (360600), 519 (16700),
553 (5900), 596 (5400), 654 (2800).
5,10,15-tri(3,5-dihydroxyphenyl)-20-[4-carboxy-
Gly(Boc)]phenyl)porphyrin (porphyrin 4). To 5,10,
15-tri(3,5-dihydroxyphenyl)-20-(4-carboxyphenyl)por-
phyrin (0.05 g, 0.066 mmol) was added N,N-diisopropyl-
ethylamine (0.0854 g, 0.66 mmol) and 1.5 mL DMF and
the resulting mixture was stirred for 5 min. HOBt (0.009
g, 0.066 mmol) and TBTU (0.021 g, 0.066 mmol) were
added and the stirring continued at room temperature for
15 min. Glycine tert-butyl ester hydrochloride (0.101 g,
0.1 mmol) was added and the final reaction mixture was
stirred at room temperature under nitrogen for 48 h. Ethyl
acetate was added and the organic solution was washed
with brine. The resulting residue was purified by silica
gel column chromatography using ethyl acetate for elu-
tion. The title porphyrin was obtained in 80% yield, mp
1
obtained (45 mg) as a green powder in 95% yield. H
NMR (d-acetone, 400 MHz): δ, ppm 9.01 (s, 6H, β-H),
8.86 (s, 2H, β-H), 8.37 (s, 4H, o,m-PhH), 7.47 (s, 6H,
o-PhH), 7.08 (s, 3H, p-PhH), 4.39 (s, 12H, OCH2), 4.19
(s, 24H, OCH2), 3.93 (s, 12H, OCH2), 3.67 (s, 24H,
Carb-H), 1.50–2.51 (br, 104, BH, CH2), -2.79 (s, 2H, NH).
HRMS-ESI m/z 818.3561 [M - 6K + 2H]4-, calcd. for
[C95H203B108Co6N5O21]4- 818.2954. UV-vis (acetone):
λ
max, nm (ε) 424 (584300), 520 (28500), 556 (8000), 599
(7700), 655 (2900). This porphyrin (45 mg, 0.013 mmol)
was dissolved in DMF (200 µL) and DIEA (13 mg,
0.1 mmol) and the solution stirred at room tempera-
ture for 30 min. HOBt (1.84 mg, 0.014 mmol) and
HATU (5.18 mg, 0.014 mmol) were added and the mix-
ture was stirred for 1 h. Tert-butyl-12-amino-4,7,10-
1
> 300 °C. H NMR (d-acetone, 400 MHz): δ, ppm 9.05
(s, 6H, β-H), 8.86 (s, 2H, β-H), 8.37 (broad s, 6H, OH),
7.90 (d, J = 8.44 Hz, 2H, o-PhH), 7.69 (d, J = 8.32 Hz,
2H, m-PhH), 7.26 (s, 6H, o-PhH), 6.85 (s, 3H, p-PhH),
1.54 (s, 9H, C(CH3)3), -2.79 (s, 2H, NH). 13C NMR
Copyright © 2011 World Scientific Publishing Company
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SyNtheSIS aND tOxICIty Of COBaltaBISDICarBOllIDe-CONtaININg POrPhyrINS Of hIgh BOrON CONteNt
981
trioxadodecanoate (4.7 mg, 0.014 mmol) was added to
the reaction solution at once and the final mixture was
stirred for 72 h at room temperature, under nitrogen. The
solvents were evaporated under vacuum and the resulting
residue dissolved in ethyl acetate. The organic solution
was washed with saturated aqueous NaCl and water. The
solvent was evaporated to give a dry residue, which was
purified by silica gel column chromatography using ace-
tonitrile/CH2Cl2 7:3 as the eluant. The resulting tert-butyl-
protected porphyrin-PEG was obtained in 20% yield
(9.7 mg).1H NMR (d-acetone, 400 MHz): δ, ppm 9.03 (s,
6H, β-H), 8.88 (s, 2H, β-H), 8.39 (s, 4H, o,m-PhH), 7.47
(s, 6H, o-PhH), 7.07 (s, 3H, p-PhH), 4.38 (s, 12H, OCH2),
4.24 (s, 24H, OCH2), 3.93 (s, 12H, OCH2), 3.66–3.57
(m, 36H, Carb-H, OCH2), 1.50–2.51 (br, 108, BH, CH2),
-2.79 (s, 2H, NH). HRMS-ESI: m/z 714.5167 [M - 5K]5-,
calcd. for [C108H226B108Co6KN6O25]5- 714.0612. UV-vis
(acetone): λmax, nm (ε) 424 (455900), 519 (5740), 555
(2500), 598 (2050), 654 (1400). To a solution of the tert-
butyl-protected porphyrin-PEG (9.7 mg, 0.0025 mmol)
in acetone (1 mL), TFA (1 mL) was added and the reac-
tion mixture was stirred at room temperature for 4 h.
The solvents were evaporated under vacuum and the
resulting solid was washed several times with diethyl
ether. After drying, porphyrin 5 was obtained in 95% (9
mg) yield. 1H NMR (d-acetone, 400 MHz): δ, ppm 9.15
(s, 6H, β-H), 9.01 (s, 2H, β-H), 8.61 (s, 4H, o,m-PhH),
7.91 (s, 3H, p-PhH), 7.31 (s, 6H, o-PhH), 4.51 (s, 12H,
OCH2), 4.30 (s, 24H, OCH2), 4.02 (s, 12H, OCH2), 3.69
(s, 24H, Carb-H), 3.60 (s, 12H, OCH2), 1.55–2.85 (br,
108, BH, CH2). HRMS-ESI: m/z 707.1274 [M - 5K - H +
Na]5-, calcd. for [C104H217B108Co6KNaN6O25]5- 707.0457.
UV-vis (acetone): λmax, nm (ε) 424 (493600), 519
(25400), 555 (10250), 598 (8350), 654 (5300). HPLC
tR = 8.76.
β = 78.429(6), γ = 89.623(5)°, V = 2331.0(3) Å3, T =
90.0(5) K, Z = 2, ρcalcd = 1.300 g.cm-3, µ(CuKα) = 1.10
mm-1. A total of 23,985 data was collected at to θ =
69.0°. R = 0.047 for 7789 data with Fo2 > 2σ(Fo2) of
8270 unique data and 628 refined parameters, CCDC
824813. For 3b: C57H48N4O7, triclinic space group P1, a =
9.420(2), b = 9.786(2), c = 13.609(3) Å, α = 102.099(14),
β = 102.101(15), γ = 97.645(15)°, V = 1178.7(4) Å3,
T = 90.0(5) K, Z = 1, ρcalcd = 1.269 g.cm-3, µ(CuKα) =
0.68 mm-1. A total of 14,545 data was collected at to θ =
68.4°. R = 0.065 for 4739 data with Fo2 > 2σ(Fo2) of 6619
unique data and 619 refined parameters, CCDC 824814.
.
For 3c: C52H44N4O8 0.85 CH2Cl2, triclinic space group
P-1, a = 9.7004(5), b = 13.3343(9), c = 18.2186(10) Å,
α=86.830(5), β=78.328(5), γ=89.435(5)°,V=2304.3(2)
Å3, T = 90.0(5) K, Z = 2, ρcalcd = 1.333 g.cm-3, µ(CuKα) =
1.61 mm-1. A total of 22,993 data was collected at to θ =
68.9°. R = 0.039 for 7440 data with Fo2 > 2σ(Fo2) of 8188
unique data and 585 refined parameters, CCDC 824815.
Cell studies
All tissue culture media and reagents were obtained
from Invitrogen. Human HEp2 cells were obtained
from ATCC and maintained in a 50:50 mixture of
DMEM:Advanced MEM containing 5% FBS. The cells
were sub-cultured biweekly to maintain sub-confluent
stocks.
Cytotoxicity. The HEp2 cells were plated at 10 000
per well in a Costar 96 well plate and allowed to grow
36 h. Conjugate stocks 1–5 were prepared in DMSO at
a concentration of 10 mM and then diluted into medium
to final working concentrations. The cells were exposed
to increasing concentrations of HCP–PEG up to 400 µM
and incubated overnight. The loading medium was then
removed and the cells fed with medium containing Cell
Titer Blue (Promega) as per manufacturer’s instructions.
Cell viability was then measured by reading the fluores-
cence at 520/584 nm using a BMG FLUOstar plate reader.
The signal was normalized to 100% viable (untreated)
cells and 0% viable (treated with 0.2% saponin from
Sigma) cells. For the phototoxicity experiment the cells
were prepared as described above and exposed to HCP–
PEG concentrations up to 100 µM.The cells were exposed
to a 100 W halogen lamp for 20 min, filtered through a
610 nm long pass filter to provide approximately 1 J/cm2
light dose. The cells were kept cool by filtering the IR
radiation through 10 mm of water and placing the culture
in an ice-water bath. After exposure to light, the plate was
incubated overnight and the cell viability was measured
as described above.
X-ray crystallographic data
Crystals of porphyrins 3a, 3b and 3c for X-ray crys-
tallographic analysis were grown by slow evaporation
of their hexane/dichloromethane solutions. Diffrac-
tion data were collected at low temperature on a Bruker
Kappa Apex-II CCD diffractometer equipped with CuKα
radiation (λ = 1.54178 Å) and an Oxford Cryosystems
Cryostream chiller. Refinement was by full-matrix least
squares using SHELXL [17], with H atoms in idealized
positions, guided by difference maps. For porphyrins 3a
and 3c, the NH hydrogen atoms are disordered, and were
placed into four half-populated sites. In porphyrin 3a,
dichloromethane and hexane solvent molecules shared
a disordered site on an inversion center. For porphyrin
3c, electron density amounting to 0.85 molecules of dis-
ordered dichloromethane per porphyrin molecule was
removed using the SQUEEZE [18] procedure.
Microscopy. The HEp2 cells were incubated in a glass
bottom 6-well plate (MatTek) and allowed to grow for
48 h, before being exposed to 10 µM of HCP–PEG for
6 h. For the co-localization experiments the cells were
incubated for 24 h concurrently with porphyrin and
one of the following organelle tracers (Invitrogen), for
.
.
Crystallographic data. For 3a: C52H44N4O8 0.307 C6H14
0.386 CH2Cl2, triclinic space group P-1, a = 9.7437(5),
b = 13.4281(10), c = 18.2079(15) Å, α = 87.136(6),
Copyright © 2011 World Scientific Publishing Company
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982
N.V.S.D.K. BhuPathIraJu et al.
30 min: ER Tracker Blue/White 100 nM (endoplasmic
reticulum), MitoTracker Green at 250 nM (mitochon-
dria), LysoSensor Green at 50 nM (lysosomes), BODIPY
FL C5-ceramide at 50 nM (Golgi network). The slides
were washed three times with growth medium and new
medium containing 50 mM HEPES pH 7.4 was added.
The images were acquired using a Leica DMRXA micro-
scope with 40× NA 0.8 dip objective lens and DAPI, GFP
and Texas Red filter cubes (Chroma Technologies).
Kaysville, UT). Variables of interest were statistically
evaluated for group effect, using One-Way ANOVA.
When the overall F statistic was significant (p < 0.05),
the Fisher’s Least Significant Differences test was per-
formed to compare the groups. Significant differences
existed when p > 0.05.
CONCLUSION
Two porphyrins of high boron content (34 and 39%
boron by weight) were synthesized in high yields, via
reaction of the corresponding hydrophyphenyl porphy-
rins with cobaltabisdicarbollide [3,3′-Co(8-C4H8O2-
1,2-C2B9H10)(1′,2′-C2B9H11)]. One of the porphyrins
was conjugated to a low molecular weight PEG group
to increase its solubility and to provide a spacer for
future conjugations to peptides and other molecules.
OCP showed no cytotoxicity towards human HEp2 cells
at concentration up to 50 µM; at higher concentrations
compound precipitation was observed. On the other hand
HCP–PEG was found to be non-toxic up to 400 µM in
the dark, and up to 100 µM after exposure to 1 J/cm2
light dose. The preferential sites of subcellular localiza-
tion were found to be the lysosomes, maybe as a result
from an endocytic internatization mechanism. No signs
of toxicity were observed in BALB/c mice upon sin-
gle ip injection of high doses of these porphyrins. The
determined MTD values for OCD and HCP–PEG were
160 mg/kg and 320 mg/kg, respectively. Such low toxic-
ity warrants further evaluation of these porphyrins of high
boron content as boron delivery vehycles for BNCT, and
these studies are currently underway in our laboratories.
Animal toxicity
The mice which we used in this project were obtained
from the breeding colony operated by the Division of
Laboratory Animal Medicine, School of Veterinary
Medicine, Louisiana State University. The experiments
were conducted according to the guidelines in the proto-
col approved by Louisiana State University Institutional
Animal Care and Use Committee, fully accredited by the
Association of Laboratory Animal Care, International.
Twelve groups of two BALB/c mice, 4–11 weeks of age
and weighing 12–24 g, were used; among these 23 were
females and 1 male. Mice in groups 2,3,4,5, and 6 were
administrated OCP in 3% DMSO diluted in PBS once via
ip injection, at increasing dosages; group 2 (20 mg/kg of a
2 mg/mL solution), group 3 (40 mg/kg of a 2 mg/mL solu-
tion), group 4 (80 mg/kg of a 4 mg/mL solution), group 5
(120 mg/kg of a 4 mg/mL solution), group 6 (160 mg/kg
of a 4 mg/mL solution). Mice in groups 8,9,10,11, and 12
were administrated HCP–PEG in 3% DMSO diluted in
PBS, once via ip injection, at increasing dosages; group 8
(20 mg/kg of a 2 mg/mL solution); group 9 (40 mg/kg of
a 2 mg/mL solution); group 10 (80 mg/kg of a 2 mg/mL
solution); group 11 (160 mg/kg of a 20 mg/mL solution);
and group 12 (320 mg/kg of a 20 mg/mL solution). Mice
in groups 1 and 7 served as vehicle controls and were
administered 1.0 mL/25 gms body weight of 3% DMSO,
which corresponded to the volume of DMSO injected
into mice in group 3. This represented the maximum
volume of 3% DMSO injected into any group of mice.
Groups of mice were dosed sequentially and each group
evaluated daily for signs of toxicity, including hunched
posture, rough hair coat, and decreased responsiveness.
Mice were anesthetized with CO2 48 h after compound
administration, and blood collected by cardiocentesis for
plasma clinical chemistry evaluation. Plasma chemistries
performed included glucose, aspartic acid aminotransam-
inase (AST), alanine aminotransaminase (ALT), alkaline
phosphatase (AP), bilirubin, total protein (TP), albumin,
and globulin. Mice were exsanguinated and a necropsy
performed. Tissues, including lung, kidney, thymus,
heart, Harderian gland, spleen, stomach, small intestine
and colon were fixed in 10% neutral buffered formalin.
Fixed tissues were processed routinely and examined by
a board-certified pathologist. For the statistical analysis
the clinical chemistry values were compared using the
Number Cruncher Statistical System software (NCSS,
Acknowledgements
The work described was supported by the US National
Institutes of Health, grant number R01 CA098902.
Supporting information
Crystallographic data have been deposited at the Cam-
bridge Crystallographic Data Centre (CCDC) under num-
bers CCDC 824813–824815. Copies can be obtained on
request, free of charge, via www.ccdc.cam.ac.uk/conts/
retrieving.html or from the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK
(fax: +44 1223-336-033 or email: deposit@ccdc.cam.
ac.uk).
REFERENCES
1. a) Hawthorne MF. Angew. Chem. Int. Ed. Eng. 1993;
32: 950. b) Soloway AH, Tjarks W, Barnum BA,
Rong FG, Barth RF, Codogni IM and Wilson JG.
Chem. Rev. 1998; 98: 1515. c) Barth RF, Soloway
AH, Goodman JH, Gahbauer RA, Gupta N, Blue TE,
Yang WL and Tjarks W. Neurosurg. 1999; 44: 433.
Copyright © 2011 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2011; 15: 982–983

SyNtheSIS aND tOxICIty Of COBaltaBISDICarBOllIDe-CONtaININg POrPhyrINS Of hIgh BOrON CONteNt
983
2. a) Barth RF, Coderre JA, Vicente MGH and Blue TE.
Clinical Cancer Research 2005; 11: 3987. b) Sib-
rian-Vazquez M and Vicente MGH. In Boron Sci-
ence: New Technologies & Applications, Hosmane
NS. (Ed.) CRC Press: 2011; pp 203.
b) Gottumukkala V, Luguya R, Fronczek FR and
Vicente MGH. Bioorg. Med. Chem. 2005; 13: 1633.
c) Gottumukkala V, Ongayi O, Baker DG, Lomax
LG and Vicente MGH. Bioorg. Med. Chem. 2006;
14: 1871.
3. Barth RF and Joensuu H. Rad. Oncol. 2007; 82:
119.
4. Vicente MGH and Sibrian-Vazquez M. In The
Handbook of Porphyrin Science, Vol 4, Kadish KM,
Smith KM and Guilard R. (Eds.) World Scientific
Publishers: Singapore, 2010; pp 191.
5. a) Hao E and Vicente MGH. Chem. Commun. 2005:
1306. b) Hao E, JensenTJ, Courtney BH andVicente
MGH. Bioconjugate Chem. 2005; 16: 1495. c) Hao
E, Sibrian-Vazquez M, Serem W, Garno JC, Fronc-
zek FR and Vicente MGH. Chem. Eur. J. 2007; 13:
9035. d) Sibrian-Vazquez M, Hao E, Jensen TJ and
Vicente MGH. Bioconjugate Chem. 2006; 17: 928.
e) Hao E, Zhang M, E WB, Kadish KM, Fronczek
FR, Courtney BH and Vicente MGH. Bioconjugate
Chem. 2008; 19: 2171.
6. a) Lindsey JS, Hsu HC and Schreiman IC. Tetrahe-
dron Lett. 1986; 27: 4969. b) Lindsey JS and Schre-
iman IC. J. Org. Chem. 1987; 52: 827.
7. Sibrian-Vazquez M, Jensen TJ and Vicente MGH. J.
Med. Chem. 2008; 51: 2915.
8. Sibrian-Vazquez M, Jensen TJ, Fronczek FR, Ham-
mer RP and Vicente MGH. Bioconjugate Chem.
2005; 16: 852.
12. a) Woodburn K, Phadke AS and Morgan AR.
Bioorg. Med. Chem. Lett. 1993; 3: 2017. b) Nguyen
T, Brownell GL, Holden SA and Teicher BA. Bio-
chem. Pharm. 1993; 45: 147. c) Callahan DE, Forte
TM, Afzal SMJ, Deen DF, Kahl SB, Bjornstad KA,
Bauer WF and Blakely EA. Int. J. Rad. Oncol. Biol.
Phys. 1999; 45: 761.
13. a) Miura M, Micca PL, Fisher CD, Heinrichs JC,
Donaldson JA, Finkel GC and Slatkin DN. Int. J.
Cancer. 1996; 68: 114. b) Miura M, Micca PL,
Fisher CD, Gordon CR, Heinrichs JC and Slat-
kin DN. Br. J. Radiol. 1998; 71: 773. c) Miura M,
Micca PL, Heinrichs JC, Gabel D, Fairchild RG and
Slatkin DN. Biochem. Pharmacol. 1992; 43: 467.
14. a) Vicente MGH, Wickramasighe A, Nurco DJ,
Wang HJH, Nawrocky MM, Makar MS and Miura
M. Bioorg. Med. Chem. 2003; 11: 3101.
15. a) Kahl SB, Joel DD, Nawrocky MM, Micca PL,
Tran KP, Finkel GC and Slatkin DN. Proc. Natl.
Acad. Sci. U.S.A. 1990; 87: 7265. b) Zhou R,
Balasubramanian SV, Kahl SB and Straubinger RM.
J. Pharm. Sci. 1999; 88: 912. c) Tibbitts J, Fike JR,
Lamborn KR, Bollen AW and Kahl SB. Photochem.
Photobiol. 1999; 69: 587.
9. Teixidor F, Pedrajas J, Rojo I, Vinas C, Kivekas R,
Sillanpaa R, Sivaev I, Bregadze V and Sjoberg S.
Organometallics 2003; 22: 3414.
16. Hao E, Friso E, Miotto G, Jori G, Soncin M, Fab-
ris C, Sibrian-Vazquez M and Vicente MGH. Org.
Biomol. Chem. 2008; 20: 3732.
10. Zhang JH, Chung TD and Oldenburg KR. J. Biomol.
Screen. 1999; 4: 67.
17. Sheldrick GM. Acta Crystallogr. Sect. A 2008; 64:
112.
11. a) Vicente MGH, Edwards BF, Shetty SJ, Hou Y
and Boggan JE. Bioorg. Med. Chem. 2002; 10: 481.
18. Spek AL. Acta Crystallogr. Sect. D-Biol. Crystal-
logr. 2009; 65: 148.
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