Boronated phosphonium salts containing arylboronic acid, closo-carborane, or nido-carborane: Synthesis, X-ray diffraction, in vitro cytotoxicity, and cellular uptake

Article abstract of DOI:10.1007/s00775-010-0690-6

The preparation of boronated triaryl and tetraaryl phosphonium salts of the type [PPh₃CH₂R]Br [R is 4-boronophenyl (1), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-yl)phenyl (2), 3-boronophenyl (3), 3-(4,4,5,5-tetramethyl-1,3,2-dioxabo

Full text of DOI:10.1007/s00775-010-0690-6

J Biol Inorg Chem (2010) 15:1305–1318
DOI 10.1007/s00775-010-0690-6
ORIGINAL PAPER
Boronated phosphonium salts containing arylboronic acid,
closo-carborane, or nido-carborane: synthesis, X-ray diffraction,
in vitro cytotoxicity, and cellular uptake
•
Daniel E. Morrison Fatiah Issa Mohan Bhadbhade
•
•
•
•
Ludwig Groebler Paul K. Witting Michael Kassiou
•
•
Peter J. Rutledge Louis M. Rendina
Received: 23 February 2010 / Accepted: 8 July 2010 / Published online: 6 August 2010
Ó SBIC 2010
Abstract The preparation of boronated triaryl and tetra-
aryl phosphonium salts of the type [PPh3CH2R]Br [R is
4-boronophenyl (1), 4-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-yl)phenyl (2), 3-boronophenyl (3), 3-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-yl)phenyl (4), 2-boronophenyl
(5), 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-yl)phenyl (6),
and closo-1,2-carboran-1-yl (7)] is described. These
compounds were prepared by the reaction of triphenyl-
phosphine with benzylic bromides or 1-bromomethyl-closo-1,
2-carborane in acetonitrile solution at 85 °C. The zwitter-
ionic nido-7,8-carborane derivative PPh3CH2C2B9H11 (8)
was prepared by treatment of 7 with cesium fluoride in re-
fluxing ethanol. All compounds were fully characterized by
multinuclear (1H, 11B, 13C, and 31P) 1D- and 2D-NMR
spectroscopy, electrospray ionization mass spectrometry,
and elemental analysis, and single-crystal X-ray structures
were determined for compounds 1, 3, 7, and 8. The cyto-
toxicities and boron uptake of selected derivatives were
investigated in vitro using human glioblastoma (T98G) and
canine kidney tubule (MDCK II) cells. The zwitterionic
species 8 was found to be the least cytotoxic agent while also
delivering the greatest amount of boron to the T98G cells,
peaking at 9.15 2.65 lg B/mg protein.
Keywords Boron neutron capture therapy ꢀ Carborane ꢀ
Boronic acid ꢀ Phosphonium salt ꢀ Mitochondria
Introduction
Boron neutron capture therapy (BNCT) is an experimental
cancer treatment which is primarily used to treat tumors of
the CNS, most commonly the intractable and aggressive
brain malignancy known as glioblastoma multiforme [1–3].
The therapy is highly reliant upon 10B agents which can
D. E. Morrison and F. Issa contributed equally to this work.
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-010-0690-6) contains supplementary
material, which is available to authorized users.
D. E. Morrison ꢀ F. Issa ꢀ M. Kassiou ꢀ P. J. Rutledge ꢀ
L. M. Rendina (&)
School of Chemistry,
The University of Sydney,
Sydney, NSW 2006, Australia
e-mail: lou.rendina@sydney.edu.au
M. Kassiou
Discipline of Medical Radiation Sciences,
Faculty of Health Sciences,
The University of Sydney,
Cumberland Campus,
Lidcombe, NSW 2141, Australia
M. Bhadbhade
M. Kassiou
Solid State and Elemental Analysis Unit,
The University of New South Wales,
Kensington, NSW 2052, Australia
Brain and Mind Research Institute,
The University of Sydney,
Camperdown, NSW 2050, Australia
L. Groebler ꢀ P. K. Witting
Discipline of Pathology,
School of Medical Sciences,
The University of Sydney, Sydney,
NSW 2006, Australia
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Fig. 1 Structures of dequalini-
localize within tumor cells prior to irradiation with thermal
neutrons, resulting in neutron capture by a 10B nucleus and
the liberation of an a particle and a 7Li ion, a process which
is accompanied by a considerable amount of kinetic
energy. Selective uptake of boronated compounds into
tumor cells would significantly enhance the probability of a
neutron capture reaction with 10B, ultimately leading to an
enhanced tumor cell kill. The search for new agents that
can selectively target tumors and deliver sufficient quan-
tities of boron to critical tumor cell components such as
DNA and mitochondria is ongoing. Delocalized lipophilic
cations (DLCs) are known to accumulate selectively in the
mitochondria of tumor cells owing to a significant differ-
ence in the mitochondrial membrane potential between
tumor cells and normal, healthy cells [4–7]. Numerous
DLCs have been assessed for their anticancer properties
[4, 8–14], with selected examples entering human clinical
trials [4]. However, the study of DLCs as potential boron
carriers for BNCT is a more recent development. The first
reported instance of a boron-containing DLC was the
closo-1,12-carborane-containing analogue of dequalinium
chloride, which was found to accumulate selectively in
human epidermoid carcinoma of the oral cavity and rat
glioma in vitro, exhibiting uptake and retention properties
similar to those of rhodamine 123, MKT-077, and tetra-
phenylphosphonium (TPP) chloride (Fig. 1) [15]. More
recently, it has been shown that an anionic nido-7,8-car-
borane can be used as a counterion in selected DLC salts
that exhibit favorable tumor selectivity, a strategy that has
allowed selective boron uptake in human prostate epithelial
carcinoma in vitro [16].
um chloride (DECA), rhodamine
123 (Rh123), MKT-077, and
tetraphenylphosphonium chloride
(TPP)
Fig. 2 Structures of the triphe-
nylmethylphosphonium (TPMP)
cation, 4-boronophenylalanine
(BPA), and sodium mercaptoun-
decahydrododecaborate(-1)
(BSH)
Of the DLCs studied to date, phosphonium ions such as
TPP and triphenylmethylphosphonium (TPMP) have
demonstrated particularly high selectivity toward glioma
in vivo [17–19]. By comparison, 4-boronophenylalanine
and sodium mercaptoundecahydrododecaborate(-1)
(Na2[B12H11SH]; BSH) (Fig. 2), two boronated agents
used in clinical trials for BNCT, have shown at best a
tumor to healthy tissue selectivity of 6.4:1 when used in
tandem [20]. Thus, boronated phosphonium salts, in par-
ticular analogues of TPP and TPMP, may offer distinct
advantages as BNCT agents and may allow significantly
enhanced boron uptake by tumor cells, a key factor in
enhancing the efficacy of this cancer therapy.
and compounds incorporating a methylenecarborane spe-
cies in which the phosphorus is not directly linked to the
boron cluster. This structural change would necessarily
influence delocalization of the cationic charge associated
with the phosphonium center and may lead to significant
changes in reactivity (e.g., deboronation of the closo-car-
borane cage) [21], and changes in uptake by tumor cell
mitochondria. Modification of the boronic acid derivatives
by condensation with a diol such as pinacol, or complex
formation with sugars such as D-fructose, allows the
boronic acid to be masked with either a more lipophilic or a
more hydrophilic group, respectively, and thus provides
further scope for tailoring drug activity. We also report the
in vitro biological activity of selected derivatives toward
the T98G human glioblastoma multiforme cell line and
Previous work by Ioppolo et al. [21] led to the first
arylphosphonium salts containing a closo-carborane or
nido-carborane cage a to the phosphonium center. These
compounds demonstrated favorable in vitro cytotoxicity
during preliminary anticancer screening against the SF268
human glioblastoma line. Herein, we report the synthesis
and preliminary biological evaluation of two new classes of
boronated phosphonium derivatives (Fig. 3): salts in which
the boron moiety is an arylboronic acid or ester derivative,
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Scheme 1 Reagent: i PPh3
Fig. 3 The two classes of compounds investigated in this study:
arylboronic acid derivatives (i) and methylcarborane derivatives (ii)
assess the potential nephrotoxicity of these cations using
cultured kidney tubule epithelial cells (Madin–Darby
canine kidney type II; MDCK II). The selectivity of
phosphonium salts, determined as a ratio of IC50 values for
MDCK II and T98G cell lines, is also reported [22].
Finally, the results from a boron uptake study of selected
derivatives by T98G cells are provided.
Scheme 2 Reagents: i pinacol,
ii PPh3
Results and discussion
Syntheses
The synthesis of the benzylphosphonium salts (4-borono-
benzyl)triphenylphosphonium bromide (1), triphenyl(4-(4,
4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)phospho-
nium bromide (2), (3-boronobenzyl)triphenylphosphonium
bromide (3), triphenyl(3-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)benzyl)phosphonium bromide (4), (2-borono-
benzyl)triphenylphosphonium bromide (5), and triphenyl
(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)
phosphonium bromide (6) was achieved using a facile
nucleophilic substitution reaction of boronated alkyl bro-
mide precursors with triphenylphosphine [23, 24]. The
desired phosphonium salts containing either the free acid
(1, 3, and 5) or pinacol ester(2, 4, and 6) werereadily prepared
in high yield (83–97%) and purity (Schemes 1, 2). A similar
route proved ineffective for the preparation of the carborane-
containing compound (closo-1,2-carboran-1-ylmethyl)tri-
phenylphosphonium bromide (7). This is presumably due to
the strongly electron withdrawing character of the closo-
carborane cage in the 1-bromomethyl-closo-1,2-carborane
precursor, which would decrease the rate of the substitution
reaction. As a result, the reaction of 1-bromomethyl-closo-
1,2-carborane (15) and triphenylphosphine did not afford the
desired product, and undesired side reactions were found to
dominate. This diminished reactivity is typical of halom-
ethyl-closo-carboranes, and indeed a slow rate of halide
substitution has been observed previously in the preparation
of methyl-closo-carborane-derived Grignard reagents
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proton signal appeared at d 5.60 (2JH–P = 14.3 Hz), sig-
nificantly downfield from the signal for the corresponding
protons in the 1-bromomethyl-closo-1,2-carborane (15)
Scheme 3 Reagents: i PPh3,
ii CsF, EtOH
1
precursor at d 3.96. In the H NMR spectrum of 8, two
distinct doublet-of-doublet signals at d 4.18 and 3.62 can
be attributed to the diastereotopic methylene protons.
These protons were coupled to each other with a coupling
2
constant JH–H = 16.5 Hz (consistent with geminal cou-
pling), and to the phosphorus. Each signal was resolved
into a simple doublet in the 1H{31P} NMR spectrum. Both
7 and 8 gave typical 31P signals for arylphosphonium
compounds at d 22.8 and 23.4, respectively [29]. A clear
distinction between the closo-carborane and nido-carbo-
rane cages was apparent in the 11B{1H} NMR spectra. For
the monosubstituted closo-1,2-carborane 7, three signals
were observed in the 11B{1H} NMR spectrum between d
-2.7 and -12.0, typical for asymmetrically substituted
closo-carborane cages [30]. For the monosubstituted nido-
7,8-carborane 8, the resonances appeared further upfield in
the range d -8.5 to -35.3, characteristic of nido-carborane
cages [31]. The nido-carborane cage was further confirmed
by the presence of an upfield signal at d -3.60 in the
1H{11B} NMR spectrum, assigned to the endo-bridging
hydrogen atom [21, 32].
[25, 26]. Instead, the closo-1,2-carborane-containing phos-
phonium salt 7 was prepared by performing the reaction at
high temperature in the absence of solvent, an alternative
method to that previously reported by Kalinin et al. [27]. The
closo-1,2-carborane species was selectively deboronated
using cesium fluoride to afford the zwitterionic (7,8-dicarba-
nido-undecaborane-7-ylmethyl)triphenylphosphonium (8)
in high yield and purity (Scheme 3) [28].
X-ray diffraction studies
All compounds were characterized by a combination of
multinuclear (1H, 11B, 13C, and 31P) 1D- and 2D-NMR
spectroscopy, electrospray ionization (ESI) mass spec-
trometry (MS), and elemental analysis, and in the case of
compounds 1, 3, 7, and 8, X-ray structures were also
determined. The 1H NMR resonances due to the methylene
protons in both reagent and product gave a clear indication
of product formation owing to the appearance of 31P cou-
pling. These proton signals appear in the range d 4.89–5.63
for the free boronic acids 1, 3, and 5 and in the range d
5.38–5.63 for the corresponding pinacol esters 2, 4, and 6,
and display characteristic 31P coupling (2JH–P = 14.2–15.6
Hz). In addition, a singlet was observed in the 31P{1H}
NMR spectrum in the range d 24.2–28.4 for the boronic
acids 1, 3, and 5 and in the range d 24.2–24.9 for the
pinacol esters 2, 4, and 6, characteristic of a quaternary aryl
phosphonium species [29]. ESI-MS provided further evi-
dence for the formation of the desired salts: derivatives
containing boronic acid were observed as either singly or
doubly condensed esters of solvent MeOH, whereas the
pinacol ester derivatives were detected as clusters of parent
species with the loss of one bromide ion, i.e., [M–Br]? and
[2M–Br]?.
In addition to NMR and ESI-MS characterization, single-
crystal X-ray crystallography of the boronic acid contain-
ing phosphonium salts 1 (Fig. 4) and 3 (Fig. 5) and car-
borane-containing species 7 (Fig. 6) and 8 (Fig. 7)
confirmed their molecular structures. The boronate esters 2
and 4 underwent facile hydrolysis during crystallization to
afford boronic acids 1 and 3, respectively. Whereas the
hydrolysis of 4 led to crystals which were identical to those
obtained directly from 3, the hydrolysis of 2 resulted in a
polymorphic form of 1 (denoted as 10). The structure of 10
is presented in the electronic supplementary material.
The X-ray crystal structures of 1 and 3 closely resemble
that of the related [PPh3CH2Ph]? species with regard to
´
bond parameters about the phosphorus atom and torsion
angles about the P–CH2 bond [33]. Both 1 and 3 adopt a
similar distorted staggered conformation about this bond,
with torsion angles of -56.7° and -52.9° between the
benzyl and phenyl groups, respectively. The phosphorus
atom in both structures deviates marginally from an ideal
tetrahedral geometry, with bond angles from 107.7(1)° to
111.4(1)° and from 106.0(3)° to 112.6(3)° for 1 and 3,
respectively. The P–Caryl bond lengths range from 1.794(2)
The 1H, 1H{31P}, and 31P{1H} NMR spectra of the
carborane-containing species 7 and 8 showed features
similar to those reported above. For 7, the methylene
˚
to 1.803(2) A (1) and from 1.779(6) to 1.809(6) A (3),
˚
comparable to those found in the [PPh3CH2Ph]Br salt
˚
[from 1.789(4) to 1.797(4) A] [33]. The P–CH2 bond
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1309
Fig. 4 An ORTEP depiction and atomic numbering scheme of
(4-boronobenzyl)triphenylphosphonium bromide (1) with 50% dis-
˚
placement ellipsoids. Selected bond lengths (A): C(1)–P(1) =
Fig. 5 An ORTEP depiction and atomic numbering scheme of
(3-boronobenzyl)triphenylphosphonium bromide (3) with 50% displace-
˚
ment ellipsoids. Selected bond lengths (A): C(1)–P(1) = 1.779(6),
C(7)–P(1) = 1.809(6), C(13)–P(1) = 1.791(6), C(19)–P(1) = 1.822
(6), C(19)–C(20) = 1.503(8). Selected bond angles (deg): C(1)–P(1)–
C(7) = 112.6(3), C(1)–P(1)–C(13) = 107.7(3), C(1)–P(1)–C(19) =
111.1(3), C(7)–P(1)–C(13) = 108.7(3), C(7)–P(1)–C(19) = 106.0(3),
1.794(2), C(7)–P(1) = 1.803(2), C(13)–P(1) = 1.799(2), C(19)–
C(20) = 1.508(3), C(19)–P(1) = 1.821(2). Selected bond angles
(deg): C(1)–P(1)–C(7) = 109.6(1), C(1)–P(1)–C(13) = 109.2(1),
C(1)–P(1)–C(19) = 110.1(1), C(7)–P(1)–C(13) = 108.8(1), C(7)–P
(1)–C(19) = 107.7(1), C(13)–P(1)–C(19) = 111.4(1), C(20)–C(19)–
P(1) = 113.4(2). Selected torsion angles (deg): C(20)–C(19)–P(1)–
C(1) = 64.7, C(20)–C(19)–P(1)–C(7) = -175.9, C(20)–C(19)–P(1)–
C(13) = -56.7
C(13)–P(1)–C(19)
= 110.7(3), C(20)–C(19)–P(1) = 117.5(4).
Selected torsion angles (deg): C(20)–C(19)–P(1)–C(1) = –52.9,
C(20)–C(19)–P(1)–C(7) = –175.5, C(20)–C(19)–P(1)–C(13) = 66.8
˚
lengths are 1.821(2) and 1.822(6) A (1 and 3, respectively),
˚
compared with 1.802(4) A for [PPh3CH2Ph]Br [33].
The orientations of the phenyl rings in the structures
vary greatly between 1 and 3 and relative to the related
species [PPh3CH2Ph]Br [33]. The relative orientation of
the rings can be compared in two ways. The dihedral angle
between the plane of the benzyl ring and the plane formed
by carbon atoms C(1), C(7), and C(13) gives an indication
of the orientation of the benzyl ring relative to the rest of
the molecule. These dihedral angles are 24.3(0)° and
36.4(6)° for 1 and 3, respectively. The value for 3 shows
considerable deviation from the corresponding dihedral
angle observed in [PPh3CH2Ph]X (29.3°, 24.2°, and 24.8°
for the X = chloride, bromide, and iodide salts, respec-
tively) [33]. Alternatively, considering the dihedral angle
between the planes of the benzyl ring and the adjacent
phenyl rings gives an indication of the relative rotation of
aryl rings relative to each other. For 1, these angles are
71.1(1)°, 45.6(3)°, and 24.8(9)° and for 3 they are 47.0(8)°,
43.0(1)°, and 87.1(7)°.
In the carborane species 7 and 8, the P–CH2 bond lengths
˚
are 1.823(2) and 1.813(4) A, respectively, comparable to the
˚
P–CH2 bond length in [PPh3CH2Ph]Br [1.802(4) A] [33],
but both are considerably elongated relative to the P–CHPh
˚
bond of the ylide PPh3CHPh [1.696(3) A] [34]. The similar
P–CH2 bond lengths observed in 7 and 8 provides strong
support for a zwitterionic structure for the latter, in which
separate but delocalized positive and negative charges are
centered at the triphenylphosphonium moiety and the nido-
7,8-carborane cage, respectively (as has been previously
observed for the related species PPh2Me-7,8-C2B9H11) [21].
Indeed, the marginal shortening of the P–CH2 bond in 8
compared with 7 indicates that, in the former, this bond
possesses very little double-bond character. Compounds 7
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Fig. 7 An ORTEP depiction and atomic numbering scheme of (7,
8-dicarba-nido-undecaborane-7-ylmethyl)triphenylphosphonium (8)
˚
with 50% displacement ellipsoids. Selected bond lengths (A): C(1)–
Fig. 6 An ORTEP depiction and atomic numbering scheme of (closo-
1,2-carboran-1-ylmethyl)triphenylphosphonium bromide (7) with 50%
P(1) = 1.800(3), C(7)–P(1) = 1.806(3), C(13)–P(1) = 1.792(4), C(19)–
P(1) = 1.813(4), C(19)–C(20) = 1.531(5). Selected bond angles (deg):
C(1)–P(1)–C(7) = 108.6(2), C(1)–P(1)–C(13) = 110.1(2), C(1)–P(1)–
C(19) = 111.4(2), C(7)–P(1)–C(13) = 109.4(2), C(7)–P(1)–C(19)
= 108.3(2), C(13)–P(1)–C(19) = 108.9(2), C(20)–C(19)–P(1) =
114.5(3). Selected torsion angles (deg): C(20)–C(19)–P(1)–C(1) =
–77.3, C(20)–C(19)–P(1)–C(7) = 163.3, C(20)–C(19)–P(1)–C(13)
= 44.4
˚
displacement ellipsoids. Selected bond lengths (A): P(1)–
C(1) = 1.787(2), P(1)–C(7) = 1.791(2), P(1)–C(13) = 1.797(2),
P(1)–C(19) = 1.823(2), C(19)–C(20) = 1.523(3). Selected bond angles
(deg): C(1)–P(1)–C(7) = 108.3(1), C(1)–P(1)–C(13) = 108.7(1), C(1)–
P(1)–C(19) = 113.7(1), C(7)–P(1)–C(13) = 109.9(1), C(7)–P(1)–
C(19) = 111.9(1), C(13)–P(1)–C(19) = 104.2(1), P(1)–C(19)–C(20) =
122.7(2). Selected torsion angles (deg): C(1)–P(1)–C(19)–C(20) =
–78.0, C(7)–P(1)–C(19)–C(20) = 45.1, C(13)–P(1)–C(19)–C(20)
= 163.8
in the bond angles between the cage and adjacent phenyl
rings [C(1)–P(1)–C(19), C(7)–P(1)–C(19), P(1)–C(19)–
C(20)] and a decrease of the complementary C(13)–P(1)–
C(19) bond angle. This contrasts with the structure of 8,
where the reverse is observed: there is a relative decrease in
the bond angles between the nido-carborane and adjacent
phenyl rings [C(7)–P(1)–C(19), C(13)–P(1)–C(19), P(1)–
C(19)–C(20)], whereas the complementary C(1)–P(1)–
C(19) bond angle is increased. The overall distortion
resulting from these effects is smaller in 8 than in 7. This
reversal can be attributed to the reduction in overall steric
bulk of the cage owing to the presence of an open (nido)
rather than a closed (closo) face, plus electrostatic attraction
between the delocalized negative charge of the cage and the
positively charged phosphonium center in 8. This contrast
between 7 and 8 is also evident in the distance between the
substituted carbon atom of the cage and the ipso carbon of the
and 8 share a similar conformation about the P–CH2 bond
axis, in which the carborane cage is in an almost staggered
conformation relative to the phenyl rings at phosphorus. The
torsion angles are 45.1° and 44.4° between the carborane and
adjacent phenyl rings, which are orientated such that the
plane of each ring is almost perpendicular to the carborane
cage. The bond angles and lengths of both compounds are
comparable to those of typical [PPh3CH2R]? salts such as
[PPh3CH2Ph]Br [33, 35]. The central phosphorus atom is
close to tetrahedral in both compounds: in 7 the bond angles
range from 104.2(1)° [C(13)–P(1)–C(19)] to 113.7(1)°
[C(1)–P(1)–C(19)], whereas in 8 they range from 108.3(2)°
[C(7)–P(1)–C(19)] to 111.4(2)° [C(1)–P(1)–C(19)]. The
significant distortion exhibited by both compounds relative
to [PPh3CH2Ph]? is a direct result of the carborane cage [33].
The steric bulk of the closo-carborane in 7 causes an increase
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1311
adjacent phenyl ring. In 7 this distance [C(20)ꢀꢀꢀC(7)] is
Table 1 In vitro cytotoxicity of (4-boronobenzyl)triphenylphospho-
nium bromide (1), triphenyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)benzyl)phosphonium bromide (2), (closo-1,2-carboran-1-
ylmethyl)triphenylphosphonium bromide (7), (7,8-dicarba-nido-
undecaborane-7-ylmethyl)triphenylphosphonium (8), and sodium
mercaptoundecahydrododecaborate(-1) (BSH) toward the human
glioblastoma (T98G) cell line
˚
3.51(6) A, whereas the corresponding distance in 8
˚
[C(20)ꢀꢀꢀC(13)] is shortened to 3.25(9) A. The relative bulk
of the two carboranes is also apparent in the intramolecular
distances between the carborane hydrogen atoms and ring
centroids of adjacent aromatic rings. In 7, the intramolecular
B(3)Hꢀꢀꢀring centroid [C(7)-containing ring] distance is
Entry
Compound
IC50 (lM) ( SE, n)
˚
˚
2.60(3) A compared with 2.90(9) A in 8. This parameter
further demonstrates the reduced size of the nido-carborane
cage compared with that of the closo-carborane. Finally, the
carborane open-face centroidꢀꢀꢀring centroid [C(13)-con-
1
2
3
4
5
1
10.1 (1.1, n = 4)
15.8 (0.4, n = 3)
21.1 (5.4, n = 3)
60.6 (19, n = 3)
[800 (n = 3)
2
7
8
˚
taining ring] distance in 8 is 3.88(4) A, with an inclination of
BSH
19.5(3)° between these planes.
SE standard error
In vitro cytotoxicity and cell uptake studies with T98G
glioblastoma cells
Table 2 In vitro cytotoxicity of 1, 2, 7, 8, and BSH toward the
Madin–Darby canine kidney type II (MDCK II) cell line
Entry
Compound
IC50 (lM) ( SE, n)
The mean in vitro cytotoxicities of 1 and 2 were deter-
mined using the T98G human glioblastoma cell line and
were found to be similar, with IC50 values of
10.1 1.1 lM (n = 16 from four experiments) and
15.8 0.4 lM (n = 12 from three experiments), respec-
tively (Table 1, entries 1, 2). In vitro cytotoxicity screening
of 7 and 8 against the same cell line demonstrates that the
closo-carborane derivative 7 is approximately 3 times
more toxic than the corresponding nido derivative 8, as
determined by the respective mean IC50 values of
21.1 5.4 lM (n = 12 replicates from three independent
experiments) and 60.6 19.0 lM (n = 12 from three
independent experiments) (Table 1, entries 3, 4). By
comparison, the viability profile of T98G cells treated with
the clinically used agent BSH showed that this compound
was nontoxic to cells even at doses as high as 800 lM,
although its tumor selectivity is only marginal [3]. One-
way analysis of variance (nonparametric) using the New-
man–Keuls multiple-comparison test showed the difference
in IC50 values of the nido-carborane derivative 8 treatment
arm is statistically significant at a 95% confidence level
(p \ 0.05) to the treatment arms of 1, 2, and 7. In contrast,
compounds 1, 2, 7, and 8 displayed much lower cytotoxic
effects toward cultured MDCK II cells (Table 2), a result
which is consistent with the reported difference in the
mitochondrial membrane potentials of normal and can-
cerous cells [4–7]. Evidence for the interaction of cationic
arylphosphonium salts with DNA and its correlation with
cytotoxicity has recently been reported [36]. Indeed, sec-
ondary cytotoxic effects associated with the interaction of
boronated phosphonium compounds with chromosomal
DNA in the absence of neutrons may potentially prove
useful when coupled with BNCT in the clinic.
1
2
3
4
5
a
1
88.1 (19.5, n = 3)
136.6 (41.5, n = 3)
[125 (n = 3)
2
7a
8a
BSHa
[125 (n = 3)
[800 (n = 3)
Sigmoidal dose–response curves could not be determined owing to
the low water solubility of compounds 7 and 8 at high concentrations
and the nontoxic behavior of BSH
Table 3 In vitro selectivity of 1, 2, 7, 8, and BSH
Entry
Compound
Selectivity (IC50 MDCK II/IC50 T98G)
1
2
3
4
5
a
1
8.7 (2.2)
8.6 (2.6)
[5.9 (NRa)
[2.1 (NRa)
NDb
2
7
8
BSH
The error value is not reported (NR) because an accurate IC50 value
could not be determined owing to the low aqueous solubility of the
compound at higher concentrations
b
Could not be determined (ND) as MDCK II and T98G cells were
unaffected when treated with
compound
a 800 lM concentration of the
first reported by Rideout et al. [22] as a ratio of IC50 values.
Using this approach, the in vitro selectivity of phospho-
nium salts 1 and 2 were calculated to be 8.7 and 8.6,
respectively, whereas the selectivity of 7 was determined to
be more than 5.9 (Table 3). Owing to the low aqueous
solubilities of 7 and 8, doses above 125 lM were not
investigated and thus accurate IC50 values from a required
sigmoidal dose–response could not be determined for the
MDCK II cell line (Table 2). One should exercise some
degree of caution in interpreting the selectivity value (more
The in vitro selectivity of delocalized cations for cul-
tured cancer cells relative to kidney epithelial cells was
123

1312
J Biol Inorg Chem (2010) 15:1305–1318
than 2.1) for that of the zwitterionic salt 8 relative to those
of the other phosphonium salts because the compound
lacks an overall charge and thus it is unlikely to exhibit the
same mechanism of action as that of the cationic species.
Preliminary cell uptake studies were conducted to cor-
relate cytotoxicity with the extent of boron uptake into the
cultured T98G cells. Treatment of T98G cells with 7 or 8
(10 lM) over 72 h, followed by boron analysis by means
of inductively coupled plasma (ICP) MS, revealed that the
zwitterionic species 8 was taken up by the cells approxi-
mately twice as effectively as its charged closo-carborane
analogue 7 (Fig. 8). We postulate that 8 can traverse the
cell membrane more readily as a result of its neutral
charge, leading to a more effective accumulation within the
cell. Furthermore, there exist a series of organic cation and
anion transporters in cells and these may play an important
role in the differential uptake of 7 and 8 and their sub-
sequent detoxification [37–39]. Incubating cells with a
threefold higher concentration of 8 did not result in any
increase in boron uptake (Fig. 8). Although the exact
mechanism for the observed toxicity of these compounds is
unknown, the more cytotoxic, cationic species 7 is the
more likely of the two compounds to be taken up by the
mitochondria and ultimately result in apoptosis; further
investigation of the cellular organelle(s) targeted by these
drugs will be reported in due course. The addition of 7
(10 lM) to T98G cells delivered approximately 1 order of
magnitude more boron atoms to the cells than the boronic
acid 1 at an identical dose (Fig. 8). Although cellular boron
levels are much higher when a boron-rich carborane spe-
cies is used, the intracellular concentration of 1 must be
similar to that of 7 since the latter contains 10 times as
many boron atoms per molecule. Moreover, the uptake of 8
is significantly greater than that of either 1 or 7, in addition
to 8 being the least cytotoxic agent in vitro. Lipophilicity
appears to play only a minor role in cellular uptake of these
agents as the extent of accumulation of boronic acid 1 was
found to be similar to that of the closo-carborane species 7
(Fig. 8), with an uptake ratio of 1:1.1. A correlation of
lipophilicity involving the boronic acid 1 and pinacol ester
2 with regard to cellular uptake cannot be made as 2 was
found to readily hydrolyze during its crystallization to
afford 10 (vide supra) and so the boron uptake results
pertaining to 2 may actually be the result of its partial or
complete hydrolysis to 1 in aqueous solution.
Conclusions
A series of novel boronated phosphonium salts have been
synthesized and characterized. These compounds contain
either free benzylboronic acids (1, 3, and 5), the corre-
sponding boronate esters (2, 4, and 6), or a boron-rich
closo-1,2-carborane cage (7). The facile preparation of the
zwitterionic nido-7,8-carborane species 8 from 7 has also
been described. All compounds were fully characterized
using multinuclear (1H, 11B, 13C, and 31P) 1D- and
2D-NMR spectroscopy, ESI-MS, and elemental analysis,
and single-crystal X-ray diffraction studies were also per-
formed in the case of 1, 3, 7, and 8. In vitro cytotoxicity
and uptake studies demonstrated that the boronated phos-
phonium salts were taken up by cultured T98G cells to a
similar extent, regardless of the nature of the boron sub-
stituent(s), and all compounds were found to be signifi-
cantly less toxic toward cultured MDCK II cells. However,
the boron-rich carborane derivatives—the zwitterionic 8 in
particular—possess the distinct advantage of delivering
greater amounts of boron to the tumor cells on a per mole
basis when compared with the related boronic acids/boro-
nate esters. Furthermore, the accumulation of 8 was found
to be approximately two-fold higher than that of the anal-
ogous species 7 and it exhibited a lower cytotoxicity than
the corresponding closo-carborane species or the boronic
acid derivatives. An investigation of whether the boronated
phosphonium salts described in this work do indeed target
mitochondria and affect their function like other DLCs is
currently under way.
Materials and methods
nido-Decaborane(14) (B10H14) was purchased from Kat-
chem (Czech Republic), and 2-(bromomethyl)phenyl-
boronic acid, 3-(bromomethyl)phenylboronic acid, and
4-(bromomethyl)phenylboronic acid were purchased from
Boron Molecular (Australia). All other reagents were
Fig. 8 Inductively coupled plasma mass spectrometry determination
of boron uptake in human glioblastoma cells (T98G) treated with
compounds 1, 2, 7, and 8
123

J Biol Inorg Chem (2010) 15:1305–1318
1313
Triphenyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzyl)phosphonium bromide, 2
purchased from Aldrich. MeCN was freshly distilled from
CaH2 before use [40]. All other solvents were used without
prior purification.
1H, 13C{1H}, and 31P{1H} NMR spectra were recorded
at 27 °C using a Bruker Avance300 spectrometer (1H at
300 MHz, 13C at 75 MHz, 31P at 121 MHz). 11B{1H}
NMR spectra were recorded at 27 °C using a Bruker
DRX400 spectrometer at 128 MHz. NMR signals (d) are
reported in parts per million. 1H and 13C{1H} NMR spectra
were referenced to the solvent residual peaks. 11B{1H}
NMR spectra were referenced to external BF3ꢀOEt2
(0 ppm). 31P{1H} NMR spectra were referenced to external
P(OMe)3 (140.85 ppm). Low-resolution ESI-MS spectra
were recorded using a Finnigan LCQ mass spectrometer.
High-resolution ESI Fourier Transform ion cyclotron res-
onance MS data were recorded using a Bruker 7.0 T mass
spectrometer. Elemental analyses were performed by the
Campbell Microanalytical Laboratory, University of
Otago, New Zealand.
This compound was prepared using 4-(bromomethyl)
phenylboronic pinacol ester (12; 0.574 g, 1.93 mmol) to
give 2 (1.08 g, 97%) as a colorless powder. Melting point
1
259–261 °C. H NMR (CDCl3) d 7.75 (m, 9H, Ph), 7.64
(dd, 5H, Ph, JH–H = 7.17 Hz, JH–P = 3.9 Hz), 7.60
3
4
4
(d, 1H, Ph, JH–P = 3.6 Hz), 7.55 (d, 2H, Ph,
3
3JH–H = 7.59 Hz), 7.06 (dd, 2H, Ph, JH–H = 7.89 Hz,
4JH–P = 2.1 Hz), 5.41 (d, 2H, CH2, 2JH–P = 14.7 Hz), 1.32
(s, 12H, CH3). 13C{1H} NMR (CDCl3) d 135.2 (m, Ph),
134.5 (d, Ph, 3JC–P = 9.7 Hz), 130.9 (d, Ph,
4JC–P = 5.4 Hz), 130.3 (d, Ph, 2JC–P = 12.5 Hz), 117.9 (d,
1
Ph, JC–P = 85.1 Hz), 84.2 (s, COB), 31.4 (d, CH2,
1JC–P = 46.7 Hz), 25.0 (s, CH3). 31P{1H} NMR (CDCl3) d
24.2 (s). 11B{1H} NMR (CDCl3) d 30 (br, s). ESI-MS:
m/z 479.27 ([M–Br-]?), 1,039.07 ([2M–Br-]?), 1,071.07
([2M ? CH3OH–Br-]?). Found: (%) C 64.87, H 6.08.
Calc. for C31H33BO2PBrꢀH2O: (%) C 64.50, H 6.11.
General synthetic procedure for boronic acid
derivatives
(3-Boronobenzyl)triphenylphosphonium bromide, 3
This compound was prepared using 3-(bromomethyl)
phenylboronic acid (10; 0.50 g, 2.3 mmol) to give 3 as a
colorless, crystalline solid. Yield 0.90 g (83%). Melting
A solution containing 2-(bromomethyl)phenylboronic acid,
3-(bromomethyl)phenylboronic acid, or 4-(bromomethyl)
phenylboronic acid (or their respective pinacol esters) and
PPh3 (1.1 mol equiv) in MeCN (20 mL) was stirred at
85 °C for 12 h [41]. The solvent was removed in vacuo and
the residue was triturated with diethyl ether to yield a white
precipitate that was collected by filtration and recrystal-
lized from EtOH:Et2O (1:1 v/v) to afford the desired
product.
1
point 205–207 °C. H NMR (d6-DMSO) d 7.92 (m, 5H,
Ph/BOH), 7.74 (dt, 7H, Ph, 3JH–H = 7.80 Hz,
3
4JH–P = 3.6 Hz), 7.65 (dd, 6H, Ph, JH–H = 7.56 Hz,
3JH–P = 12.3 Hz), 7.47 (br, 1H, Ph), 7.18 (t, 1H, Ph,
3JH–H = 7.53 Hz), 6.96 (d, 1H, Ph, 3JH–H = 7.35 Hz), 5.14
(d, 2H, CH2, 2JH–P = 15.6 Hz). 13C{1H} NMR
(d6-DMSO) d 137.0 (d, Ph, 3JC–P = 5.6 Hz), 135.0 (s, Ph),
134.0 (d, Ph, 3JC–P = 9.8 Hz), 132.3 (d, Ph,
3JC–P = 5.0 Hz), 130.0 (d, Ph, 2JC–P = 12.3 Hz), 127.7 (d,
(4-Boronobenzyl)triphenylphosphonium bromide, 1
4
2
Ph, JC–P = 2.1 Hz), 126.8 (d, Ph, JC–P = 8.5 Hz), 117.8
This compound was prepared using 4-(bromomethyl)
phenylboronic acid (9; 0.29 g, 1.3 mmol) to give 1 (0.57 g,
1.2 mmol) as a colorless powder. Yield 0.57 g (89%).
1
1
(d, Ph, JC–P = 84.9 Hz), 28.3 (d, CH2, JC–P = 45.4 Hz).
31P{1H} NMR (d6-DMSO) d 24.3 (s). 11B{1H} NMR
(d6-DMSO) d 30 (br, s). ESI-MS: m/z 411.27 ([M ? CH3OH–
H2O–Br-]?), 425.20 ([M ? 2CH3OH–2H2O–Br-]?).
Found: (%) C 62.84, H 4.93. Calc. for C25H23BO2PBr: (%)
C 62.93, H 4.86.
1
Melting point 210–212 °C. H NMR [CDCl3/dimethly-d6
sulfoxide (d6-DMSO)] d 7.87 (t, 3H, Ph, 3JH–H = 7.41 Hz),
7.68 (m, 9H, Ph), 7.56 (dd, 5H, Ph, JH–H = 7.92 Hz,
3
3JH–P = 12.5 Hz), 7.39 (br, 2H, BOH), 6.93 (dd, 2H, Ph,
4
3JH–H = 7.89 Hz, JH–P = 2.0 Hz), 4.89 (d, 2H, CH2,
2JH–P = 14.9 Hz). 13C{1H} NMR (CDCl3/d6-DMSO) d
Triphenyl(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
benzyl)phosphonium bromide, 4
4
135.1 (d, Ph, JC–P = 2.3 Hz), 134.6 (d, Ph, JC–P
3
=
3
2.8 Hz), 134.0 (d, Ph, JC–P = 9.7 Hz), 130.0 (d, Ph,
2JC–P = 12.4 Hz), 128.8 (d, Ph, 2JC–P = 8.5 Hz), 117.6 (d,
This compound was prepared using 3-(bromomethyl)
phenylboronic pinacol ester (13; 0.652, 2.20 mmol) to give
4 as a colorless powder. Yield 1.21 g (97%). Melting point
1
1
Ph, JC–P = 85.3 Hz), 29.5 (d, CH2, JC–P = 46.6 Hz).
31P{1H} NMR (CDCl3/d6-DMSO) d 28.4 (s). 11B{1H}
NMR (d6-DMSO) d 27 (br, s). ESI-MS: m/z 411.33
([M ? CH3OH–H2O–Br-]?), 425.20 ([M ? 2CH3OH–
2H2O–Br-]?). Found: (%) C 62.69, H 4.90. Calc. for
C25H23BO2PBr: (%) C 62.93, H 4.86.
1
265–267 °C. H NMR (CDCl3) d 7.75 (m, 9H, Ph), 7.63
(m, 7H, Ph), 7.55 (d, 1H, Ph, JH–H = 7.68 Hz), 7.20
3
3
(t, 1H, Ph, JH–H = 7.50 Hz), 7.06 (br, 1H, Ph), 5.38 (d,
2
2H, CH2, JH–P = 14.2 Hz), 1.26 (s, 12H, CH3). 13C{1H}
123

1314
J Biol Inorg Chem (2010) 15:1305–1318
3
NMR (CDCl3) d 137.3 (d, Ph, JC–P = 5.2 Hz), 135.1 (d,
Ph, JC–P = 2.6 Hz), 134.8 (d, Ph, JC–P = 5.7 Hz), 134.7
1JC–P = 46.1 Hz), 24.7 (s, CH3). 31P{1H} NMR (CDCl3)
d 24.9 (s). 11B{1H} NMR (CDCl3) d 29.8 (br, s). ESI-MS:
m/z 479.27 ([M–Br-]?), 1,039.00 ([2M–Br-]?), 1,070.67
([2M ? CH3OH–Br-]?). Found: (%) C 64.83, H 5.77.
Calc. for C31H33BO2PBrꢀH2O: (%) C 64.50, H 6.11.
4
3
4
(d, Ph, JC–P = 3.7 Hz), 134.4 (d, Ph, JC–P = 9.7 Hz),
2
130.2 (d, Ph, JC–P = 12.5 Hz), 128.6 (d, Ph, JC–P
3
5
=
2
3.0 Hz), 126.2 (d, Ph, JC–P = 8.5 Hz), 117.7 (d, Ph,
1JC–P = 85.2 Hz), 83.9 (s, COB), 31.1 (d, CH2,
1JC–P = 46.8 Hz), 24.8 (s, CH3). 31P{1H} NMR (CDCl3) d
24.4 (s). 11B{1H} NMR (CDCl3) d 30 (br, s). ESI-MS:
m/z 479.33 ([M–Br-]?), 1,038.73 ([2M–Br-]?), 1,070.93
([2M ? CH3OH–Br-]?). Found: (%) C 65.88, H 6.04.
Calc. for C31H33BO2PBrꢀ0.5H2O: (%) C 65.52, H 6.03.
(Closo-1,2-carboran-1-ylmethyl)triphenylphosphonium
bromide, 7
A stirred mixture of 1-bromomethyl-closo-1,2-carborane
(15; 5.30 g, 22.3 mmol) and PPh3 (6.50 g, 24.8 mmol) was
heated to 100 °C in the absence of solvent for 4 days. The
mixture was then cooled and triturated with Et2O to yield 7
as a colorless solid. The unreacted starting materials were
isolated from the Et2O and recycled. Overall yield 1.77 g
(15.9%). Melting point above 350 °C (decomposed). 1H
(2-Boronobenzyl)triphenylphosphonium bromide, 5
This compound was prepared using 2-(bromomethyl)
phenylboronic acid (11; 0.44 g, 2.0 mmol) to give 5
(0.92 g, 1.9 mmol) as a pale-yellow residue, which was
recrystallized from EtOH to afford a colorless powder.
3
NMR (CDCl3) d 8.09 (dd, 6H, Ph, JH–H = 7.71 Hz,
3
3JH–P = 12.9 Hz), 7.83 (dt, 3H, Ph, JH–H = 7.50 Hz,
1
3
Yield 0.92 g (94%). Melting point 224–226 °C. H NMR
5JH–P = 2.0 Hz), 7.73 (dt, 6H, Ph, JH–H = 7.68 Hz,
(d6-DMSO) d 8.08 (s, 2H, BOH), 7.88 (t, 3H, Ph,
4JH–P = 3.6 Hz), 6.14 (br, 1H, C–H cage), 5.60 (d, 2H,
3JH–H = 7.37 Hz), 7.70 (dt, 7H, 3JH–H = 7.74 Hz,
2
CH2, JH–P = 14.3 Hz), 3.4–0.8 (v. br, 10H, B–H cage).
4JH–P = 3.3 Hz), 7.49 (dd, 6H, Ph, JH–H = 7.62 Hz,
13C{1H} NMR (CDCl3) d 135.7 (d, Ph, JC–P = 2.8 Hz),
134.3 (d, Ph, JC–P = 10.4 Hz), 130.7 (d, Ph, JC–P =
3
4
3JH–P = 12.4 Hz), 7.31 (m, 2H, Ph), 7.04 (m, 1H, Ph),
3
2
5.38 (d, 2H, CH2, JH–P = 15.6 Hz). 13C{1H} NMR
2
1
12.9 Hz), 117.6 (d, Ph, JC–P = 86.5 Hz), 66.7 (s, CH
4
1
(d6-DMSO) d 136.1 (d, Ph, JC–P = 2.4 Hz), 135.0 (d, Ph,
cage), 65.3 (s, C cage), 30.9 (d, CH2, JC–P = 53.2 Hz).
3
5JC–P = 2.2 Hz), 134.0 (d, Ph, JC–P = 9.6 Hz), 133.2 (d,
31P{1H} NMR (CDCl3) d 22.8 (s). 11B{1H} (CDCl3)
d -2.7 (br, s), -9.3 (br, s), -12.0 (br, s). ESI-MS:
m/z 419.40 ([M–Br-]?), 918.20 ([2M–Br-]?). Found: (%)
C 50.57, H 5.83. Calc. for C21H28B10PBr: (%) C 50.50, H
5.65.
3
Ph, JC–P = 9.0 Hz), 130.6 (d, Ph, JC–P = 5.0 Hz), 130.3
3
4
2
(d, Ph, JC–P = 3.0 Hz), 130.0 (d, Ph, JC–P = 12.2 Hz),
4
1
127.5 (d, Ph, JC–P = 3.6 Hz), 117.8 (d, Ph, JC–P
=
84.9 Hz), 28.4 (d, Ph, JC–P = 45.9 Hz). 31P{1H} NMR
(d6-DMSO) d 25.1 (s). 11B{1H} NMR (d6-DMSO) d 27 (br,
s). ESI-MS: m/z 411.27 ([M ? CH3OH–H2O–Br-]?),
425.20 ([M ? 2CH3OH–2H2O–Br-]?). Found: (%) C
62.90, H 5.12. Calc. for C25H23BO2PBr: (%) C 62.93,
H 4.86.
1
(7,8-Dicarba-nido-undecaborane-7-ylmethyl)
triphenylphosphonium zwitterion, 8
A solution of 7 (0.363 g, 0.727 mmol) and CsF (0.360 g,
2.37 mmol) in EtOH was stirred at reflux for 24 h. The
solvent was removed in vacuo to afford a colorless solid.
To this residue was added acetone (10 mL) and the insol-
uble borate products were filtered off. The acetone was
removed in vacuo and the crude material was recrystallized
in MeOH to afford 8 as a colorless solid (0.18 g, 61%).
Triphenyl(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
benzyl)phosphonium bromide, 6
This compound was prepared using 2-(bromomethyl)
phenylboronic pinacol ester (14; 0.44 g, 1.5 mmol) to
afford 6 as a colorless solid. Yield 0.81 g (97%). Melting
1
Melting point 297–299 °C. H NMR (d6-acetone) d 7.95
1
point 229–231 °C. H NMR (CDCl3) d 7.78 (t, 4H, Ph,
(dd, 6H, Ph, 3JH–H = 7.38 Hz, 3JH–P = 12.6 Hz), 7.90 (dt,
3JH–H = 6.89 Hz), 7.61 (dt, 8H, Ph, JH–H = 7.79 Hz,
3H, Ph, JH–H = 7.53 Hz, JH–P = 2.0 Hz), 7.77 (dt, 6H,
Ph, 3JH–H = 7.77 Hz, 4JH–P = 3.3 Hz), 4.18 (dd, 1H, CH2,
3
3
5
4JH–P = 3.4 Hz), 7.49 (dd, 5H, Ph, JH–H = 7.89 Hz,
3
3JH–P = 12.4 Hz), 7.30 (br, 1H, P), 7.29 (s, 1H, Ph), 5.63
2JH–H = 16.44 Hz, JH–P = 11.9 Hz), 3.62 (dd, 1H, CH2,
2
2
(d, 2H, CH2, JH–P = 15.0 Hz), 1.09 (s, 12H, CH3).
2
2JH–H = 16.50 Hz, JH–P = 10.7 Hz), 2.8 to -0.8 (v br,
9H, BH cage), 1.67 (br, 1H, CH cage), -3.60 (br, 1H, endo
13C{1H} NMR (CDCl3) d 137.2 (d, Ph, JC–P = 2.8 Hz),
4
4
135.0 (d, Ph, JC–P = 2.6 Hz), 134.2 (d, Ph, JC–P
3
=
H). 13C{1H} NMR (d6-acetone)
d
135.9 (d, Ph,
3
9.6 Hz), 133.7 (d, Ph, JC–P = 9.1 Hz), 132.1 (d, Ph,
3
4JC–P = 2.8 Hz), 135.4 (d, Ph, JC–P = 9.9 Hz), 131.2 (d,
3
4JC–P = 3.5 Hz), 130.9 (d, Ph, JC–P = 4.9 Hz), 130.0 (d,
Ph, 2JC–P = 12.3 Hz), 121.3 (d, Ph, 1JC–P = 84.7 Hz), 79.9
Ph, 2JC–P = 12.4 Hz), 127.9 (d, Ph, 4JC–P = 3.8 Hz), 117.6
(s, CH cage), 79.19 (d, C cage, JC–P = 32.7 Hz), 36.2 (d,
CH2, 1JC–P = 53.2 Hz). 31P{1H} NMR (d6-acetone) d 23.4
2
1
(d, Ph, JC–P = 85.3 Hz), 84.0 (s, COB), 30.3 (d, CH2,
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J Biol Inorg Chem (2010) 15:1305–1318
1315
(s). 11B{1H} NMR (d6-acetone) d –8.5 (br, s), –10.5 (br, s),
–12.3 (br, s), –18.9 (br, s), –20.1 (br, s), –31.4 (br, s), –35.3
(br, s). ESI-MS: m/z 408.13 ([M]ꢀ?). Found: (%) C 61.75,
H 6.65. Calc. for C21H28B9P: (%) C 61.71, H 6.91.
(100 units/mL), streptomycin (100 g/mL), and L-glutamine
(2.5 mM), at 37 °C in a humidified 5% CO2 atmosphere.
Cytotoxicity assays
X-ray crystal structure determinations
Cytotoxicity was assessed using the 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
[44, 45]. Briefly, cells were harvested with trypsin (0.1%
v/v), and cell pellets were isolated by centrifugation. Cells
were then resuspended to a single cell suspension, cell
numbers were counted using a hemocytometer (Weber),
and then cells were seeded (density 1 9 104 cells per well)
in growth medium (100 lL) using 96-well plates and were
allowed to adhere overnight at 37 °C. Cells were incubated
at 37 °C in a humidified atmosphere of 5% CO2 in the
presence of compounds 1, 2, 7, and 8, and the vehicle
(control). Serial dilutions of boron compound were added
to quadruplicate wells. After 72 h, MTT solution in phos-
phate-buffered saline (PBS; 20 lL, 0.25% w/v) was added
and the incubation was continued. After a further 4 h, the
culture medium and excess MTT solution were removed
and the resulting MTT–formazan crystals dissolved by
addition of 150 lL DMSO.
Cell viability was determined by measuring the absor-
bance at either 595 or 600 nm using an Ultramark multi-
wavelength plate reader (Bio-Rad, Australia) or a Victor3V
microplate reader (PerkinElmer), respectively. All readings
were corrected for absorbance from wells containing the
vehicle alone, and the level of MTT was expressed relative
to the corresponding vehicle-treated controls as percent
viability. Corresponding IC50 values for each of the com-
pounds tested were then determined at the dose required to
induce a 50% decrease in cell viability. All experiments
were conducted at least in triplicate and all IC50 values are
reported with standard errors where possible.
A suitable single crystal was selected under a polarizing
microscope (Leica M165Z) and was glued to a glass fiber
with a dot of silicon paste that fixed the crystal firmly upon
freezing at the temperature of data collection (-123 °C).
The intensities were measured with a Bruker kappa
APEXII CCD diffractometer equipped with graphite
˚
monochromated Mo Ka radiation (k = 0.71073 A) and
operating at a low temperature of -173(2) °C maintained
using an Oxford Cryosystems Cryostream 700 system.
Upon obtaining an initial refinement of unit-cell parame-
ters, the data collection strategy achieved a redundancy of
at least 4 throughout the resolution range (from infinity to
˚
0.80 A) at 10-s exposure time per frame making use of the
j offsets on the four-circle goniometer geometry. The data
integration and reduction with the multiscan absorption
correction method was carried out using the APEX2 [42]
software suite. The structure was solved by direct methods
using SHELXS-97 [43] and was refined by the full-matrix
least-squares refinement program SHELXL [43] to the final
R value. All non-hydrogen atoms were refined anisotropi-
cally. The hydrogen atoms were located in the difference
Fourier map, and some were fixed in their positions.
Molecular graphics were generated using ORTEP-3v2.
Key crystallographic data and refinement details are shown
in Table 4.
Crystallographic data (without structure factors) for the
structures reported in this paper have been deposited with
the Cambridge Crystallographic Data Centre (CCDC) as
supplementary publication nos. CCDC-768191 (compound
10), CCDC-768192 (compound 1), CCDC-768193 (com-
pound 3), CCDC-68194 (compound 7), and CCDC-768195
(compound 8). Copies of the data can be obtained free of
charge from the CCDC (12 Union Road, Cambridge CB2
1EZ, UK; Tel: ?44-1223-336408; Fax: ?44-1223-336003;
e-mail: deposit@ccdc.cam.ac.uk).
Cell uptake studies
Stock solutions of 1 and 2 (20 mM in DMSO), 7 (5 mM in
EtOH), and 8 (5 mM in DMSO) were prepared. Warm
(37 °C) culture medium was treated with the stock boron
solutions to final concentrations of 10, 30, and 40 lM. The
T98G cells were cultured as a monolayer in 75-cm2 flasks
to 70–80% confluence and then incubated with the boron-
containing culture medium at the respective concentrations
for 72 h at 37 °C in a humidified 5% CO2 atmosphere. The
medium was removed and the cells were washed once with
PBS (2 mL). PBS (4 mL) was added to the culture flask
and then cells were harvested with a rubber policeman,
rinsing with further PBS (2 mL). Harvested cells were
pipetted until a single cell suspension was achieved,
which was then divided into aliquots (100 lL) for pro-
tein analysis. The remaining cells were sedimented by
In vitro cytotoxicity and cell uptake studies
Human glioblastoma multiforme (T98G) cells were main-
tained as monolayers in minimum essential medium sup-
plemented with 10% fetal bovine serum, penicillin
(100 units/mL), streptomycin (100 g/mL) and L-glutamine
(2.5 mM), at 37 °C in a humidified 5% CO2 atmosphere.
MDCK II cells were maintained as monolayers in
Dulbecco’s modified Eagle’s medium/Ham’s F12 mixture
supplemented with 10% fetal bovine serum, penicillin
123

1316
J Biol Inorg Chem (2010) 15:1305–1318
Table 4 Crystal data for compounds 1, 3, 7, and 8
1
3
7
8
Crystal data
Chemical formula
Mr
C
25H23BBrO2P
C25H23BBrO2P
477.12
C21H28B10BrP
499.41
C21H28B9P
408.69
477.12
Cell setting, space group
Triclinic, P-1
Monoclinic, Pn
Monoclinic, P2(1)/n
Orthorhombic,
P2(1)2(1)2(1)
Temperature (°C)
-123(2)
-123(2)
-123(2)
-123(2)
˚
a, b, c (A)
9.3253(3), 10.0392(3),
12.7552(4)
9.8364(7), 9.4813(6), 11.2209(3), 11.7646(4), 8.9605(12), 14.892(2),
19.7629(6)
12.0475(7)
17.136(2)
a, b, c (°)
73.8260(10), 74.7010(10), 90, 94.761(2), 90
88.8710(10)
90, 103.8710(10), 90
90, 90, 90
3
˚
V (A )
1,104.35(6)
1,119.70 (12)
2,532.81(13)
2,286.7(5)
Z
2
2
4
4
Dx (Mg m-3
Radiation type
l (mm-1
)
1.435
1.415
1.31
1.187
Mo Ka
Mo Ka
Mo Ka
Mo Ka
)
1.95
1.93
1.7
0.13
Crystal form, color
Crystal size (mm3)
Data collection
Diffractometer
Data collection method
Absorption correction
Tmin
Cubic, colorless
0.14 9 0.10 9 0.07
Plates, colorless
0.18 9 0.08 9 0.04
Blocks, colorless
0.23 9 0.19 9 0.11
Blocks, colorless
0.20 9 0.16 9 0.13
Bruker kappa APEXII CCD area detector
u scans and x scans with j offsets
Multiscan
0.777
0.726
0.694
0.975
Tmax
0.882
0.936
0.83
0.983
No. of measured, independent and
observed reflections
15,727, 3,879, 3,606
7,737, 3,740, 3,179
17,896, 4,439, 4,003
15,417, 3,994, 3,269
Criterion for observed reflections
Rint
I [ 2r(I)
0.042
25
I [ 2r(I)
0.071
25
I [ 2r(I)
0.055
25
I [ 2r(I)
0.053
25
qmax (°)
Refinement
Refinement on
F2
F2
F2
F2
R[F2 [ 2r(F2)], wR(F2), S
No. of reflections
No. of parameters
H-atom treatment
Weighting scheme
(D/r)max
0.026, 0.089, 0.68
0.047, 0.129, 0.74
0.028, 0.097, 0.81
0.045, 0.130, 0.87
3,879
367
3,740
347
4,439
410
3,994
383
Mixture of independent and constrained refinement
Calculated w = 1/[r2(Fo2) ? (0.1P)2 ? 0.9602P], where P = (Fo2 ? 2F2c)/3
0.001
\0.0001
0.31, –0.32
0.001
\0.0001
0.30, –0.31
Dqmax, Dqmin (e-
A
)
0.79, –0.71
0.36, –0.43
-3
˚
centrifugation at 2,000 rpm for 3 min, then the supernatant
was removed and the cell pellet was analyzed for boron
content.
and 86 7% (n = 3) for 1, 2, 7, and 8, respectively. The
minor losses of boron can be attributed to volatilization
and/or surface adsorption.
The cell pellets were transferred to precleaned TFM
vessels with MQ water (2 9 0.25 mL) then dried for 1 h at
110 °C and cooled to room temperature. The cell pellets
were suspended in HNO3 (70%, 7.5 mL) and H3PO4 (85%,
2.5 mL) as described previously [46] and subjected to three
Microwave digestion and measurement of boron
concentration by ICP-MS
As certified boron reference materials are not available,
method control was performed by the determination of
boron using an accurate weight of 1, 2, 7, and 8. For
closed-system digestions, the determined recoveries were
103 14% (n = 3), 93 5% (n = 3), 94 6% (n = 3),
high-pressure microwave digestion cycles using
a
Milestone Ethos Plus microwave laboratory station. The
microwave digestion program involved heating to 200 °C
over 10 min, holding for 10 min then heating to 240 °C
123

J Biol Inorg Chem (2010) 15:1305–1318
1317
data, and Dorothy Yu (ICP Analytical Centre, The University of New
South Wales) for boron analysis. We are grateful to the Australian
Research Council (ARC), Cure Cancer Foundation of Australia, and
National Breast Cancer Foundation for financial support.
over 5 min, and sustaining this temperature for a further
20 min. Between program cycles, the closed sample
vessels were cooled to 150–100 °C. At the end of the
digestion process, the closed vessels were cooled to
room temperature to minimize volatile analyte losses,
and the resulting homogeneous solutions were diluted
with MQ water to a final volume of 30 mL. ICP-MS
analyses were performed using a PerkinElmer Elan
DRCII ICP-MS system. Beryllium (20 ng/mL) was used
as an internal standard by in-line injection and the 10B
and 11B lines were analyzed. Standard solutions of boric
acid were used to prepare a calibration plot. The intro-
duction of D-mannitol (0.25% w/v) and aqueous ammo-
nia (0.1 M) to all final samples/diluents and flush
solutions is known to minimize boron carryover and
memory effects [47]. However, it is also known to
reduce instrument sensitivity, so no additives were used
in ICP-MS analyses of these samples. Instead, all dilu-
tions of samples/standards and flushes between ICP-MS
runs were made with MQ water only. To account for any
variations in total cell number, the measured boron
content was normalized to cell protein measured in the
corresponding cell preparations and expressed in units of
micrograms of 11B per milligram of protein, analyzed as
described next.
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