Synthesis, biological evaluation, and radioiodination of halogenated closo -carboranylthymidine analogues(1)

Article abstract of DOI:10.1021/ic202150b

The synthesis and initial biological evaluation of 3-carboranylthymidine analogues (3CTAs) that are (radio)halogenated at the closo-carborane cluster are described. Radiohalogenated 3CTAs have the potential to be used in the radiotherapy and imaging of ca

Full text of DOI:10.1021/ic202150b

Article
pubs.acs.org/IC
Synthesis, Biological Evaluation, and Radioiodination of Halogenated
closo-Carboranylthymidine Analogues1
Rohit Tiwari,† Antonio Toppino,†,‡ Hitesh K. Agarwal,† Tianyao Huo,§ Youngjoo Byun,†,¶ Judith Gallucci,∥
Sherifa Hasabelnaby,†,⊥ Ahmed Khalil,†,▽ Ayman Goudah,†,# Robert A. Baiocchi,× Michael V. Darby,†
Rolf F. Barth,§ and Werner Tjarks*,†
†Division of Medicinal Chemistry & Pharmacognosy, The Ohio State University, 500 West 12th Avenue, Columbus, Ohio 43210,
United States
‡Dipartimento di Chimica Generale e Chimica Organica, Universita
̀
degli Studi di Torino, Torino, Italy
§Department of Pathology, The Ohio State University, Columbus, Ohio 43210, United States
¶College of Pharmacy, Korea University, Chungnam, South Korea
∥Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United States
⊥Division of Pharmaceutical Organic Chemistry, College of Pharmacy, Helwan University, Cairo, Egypt
▽Chemistry Department, Faculty of Science, Zagazig University, Zagazig, Egypt
#Division of Pharmacology, College of Veterinary Medicine, Cairo University, Giza, Egypt
×Division of Hematology and Oncology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio 43210,
United States
S
* Supporting Information
ABSTRACT: The synthesis and initial biological evaluation of 3-carboranylthymidine
analogues (3CTAs) that are (radio)halogenated at the closo-carborane cluster are described.
Radiohalogenated 3CTAs have the potential to be used in the radiotherapy and imaging of
cancer because they may be selectively entrapped in tumor cells through monophosphorylation
by human thymidine kinase 1 (hTK1). Two strategies for the synthesis of a 127I-labeled form of
a specific 3CTA, previously designated as N5, are described: (1) direct iodination of N5 with
iodine monochloride and aluminum chloride to obtain N5-127I and (2) initial monoiodination
of o-carborane to 9-iodo-o-carborane followed by its functionalization to N5-127I. The former
strategy produced N5-127I in low yields along with di-, tri-, and tetraiodinated N5 as well as
decomposition products, whereas the latter method produced only N5-127I in high yields.
N5-127I was subjected to nucleophilic halogen- and isotope-exchange reactions using Na79/81Br
and Na125I, respectively, in the presence of Herrmann’s catalyst to obtain N5-79/81Br and
N5-125I, respectively. Two intermediate products formed using the second strategy, 1-(tert-
butyldimethylsilyl)-9-iodo-o-carborane and 1-(tert-butyldimethylsilyl)-12-iodo-o-carborane, were
subjected to X-ray diffraction studies to confirm that substitution at a single carbon atom of 9-iodo-o-carborane resulted in the
formation of two structural isomers. To the best of our knowledge, this is the first report of halogen- and isotope-exchange reactions of
B-halocarboranes that have been conjugated to a complex biomolecule. Human TK1 phosphorylation rates of N5, N5-127I, and
N5-79/81Br ranged from 38.0% to 29.6% relative to that of thymidine, the endogenous hTK1 substrate. The in vitro uptake of N5,
N5-127I, and N5-79/81Br in L929 TK1(+) cells was 2.0, 1.8, and 1.4 times greater than that in L929 TK1(−) cells.
decaborate(2) (B10H102−), or dodecaborate (B12H122−).3 By
INTRODUCTION
■
analogy to electrophilic radiohalogenation of tyrosine residues in
proteins,2 labeling of these negatively charged boron clusters mainly
was carried out with electrophilic halogen species produced in situ
by the action of Chloramine T or Iodogen.3 Nucleophilic labeling
is another widely used strategy for the introduction of carbon-
bound radiohalogens in biomolecules.2,4,5 Methods employing
iron-, copper-, and palladium-catalyzed nucleophilic halogen
Radiopharmaceuticals containing carbon-bound radiohalogens play
an important role in a variety of therapy and imaging applications.2
There is, however, a convincing body of evidence indicating that
most radiopharmaceutical biomolecules (e.g., antibodies, carbohy-
drates, growth factors) that contain radiolabeled boron clusters are
less susceptible to dehalogenation under physiological conditions
than their counterparts containing conventional radiohalogen−
carbon bonds.3 The vast majority of these radiopharmaceuticals
possess negatively charged boron clusters, such as nido-carbo-
rane(1−) (C2B9H12−), closo-monocarborane(1−) (C1B11H12−),
Received: October 4, 2011
Published: December 16, 2011
© 2011 American Chemical Society
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Inorganic Chemistry
Article
exchange at simple boron cluster structures were developed,6−11
but they have not as yet been applied to the radiohalogenation of
more complex biologically active carborane-containing compounds.
Nucleophilic halogen exchange can be carried out at hydrophobic
neutral boron clusters and thus could be a useful alternative in the
radiohalogenation of low-molecular-weight boron-cluster-containing
bioconjugates that may rely on hydrophobic properties for biological
activity.12
flight spectrometer. Electron impact (EI) MS spectra were obtained at
the University of Illinois Mass Spectrometry Laboratory (Urbana−
Champaign, Illinois) on a Micromass 70-VSE double-focusing-sector
spectrometer on TSSPro3.0 system. For all carborane-containing
compounds, the mass of the most intensive peak of the isotopic
pattern was reported for a 80% 11B to 20% 10B distribution. Measured
patterns agreed with calculated patterns. Melting points were
measured in sealed capillaries on a Thomas−Hoover capillary melting
point apparatus and are uncorrected. Silica gel 60 (0.063−0.200 mm)
from Merck was used for gravity column chromatography, whereas
silica gel 60 (0.015−0.049 mm) from EM Science was used for flash
column chromatography. Precoated glass-backed thin-layer chroma-
tography (TLC) plates with silica gel 60 F254 (0.25-mm layer
thickness) from Dynamic Adsorbents (Norcross, GA) were used for
TLC studies. For all nonradioactive materials, (semi)preparative high-
performance liquid chromatography (HPLC) purification was
performed either with a Gemini 5μ C18 column (21.20 mm ×250
mm, 5 μm particle size), supplied by Phenomenex Inc. (Torrance,
CA), or with a Supelco Discovery HS C18 column (10 mm ×250 mm,
10 μm particle size), supplied by Sigma-Aldrich (St. Louis, MO). A
LiChrocart 250-4 HPLC cartridge packed with a LiChrospher RP-18
stationary phase (4 mm ×250 mm, 5 μm particle size), supplied by
EM Science (Gibbstown, NJ), was used for analytical reversed-phase
chromatography. For all radioactive materials, semipreparative HPLC
purification was performed with a Supelco Discovery HS C18 column
(10 mm × 250 mm, 10 μm particle size). A Beckman Ultrasphere
column (4.6 mm × 250 mm, 5 μm particle size), supplied by
Beckmann Coulter Inc. (Brea, CA), was used for analytical reversed-
phase chromatography. Further general experimental conditions are
provided in the Supporting Information.
5-(o-Carboran-1-yl)pentyl 4-Methylbenzenesulfonate
(2).28 6-Heptynyl 4-methylbenzenesulfonate (1;29 1.0 g, 3.7 mmol),
decaborane (240 mg, 2 mmol), and 1-butyl-3-methylimidazolium
chloride (bmim+Cl−; 300 mg, 1.72 mmol) were vigorously stirred in
anhydrous toluene at 120 °C for 10 min. Subsequently, the reaction
mixture was diluted with CH2Cl2 and filtered through silica gel. The
filtrate was evaporated, and the residue was purified by column
chromatography using hexanes/EtOAc (2:1) to afford 2 (420 mg,
55%) as a white solid. 1H NMR (CDCl3) data were the same as those
obtained previously for the same compound using a different
procedure.28 The measured melting point was slightly lower than
the one previously reported (105−106 °C vs 109−110 °C).
Thymidine (dThd) analogues with a hydrophobic boron cluster
(closo-carborane) attached through spacers to the N3 position
(3-carboranylthymidine analogues or 3CTAs) may be good
examples for such compounds. Both boronated and nonboronated
N3-substituted dThd analogues have attracted considerable
attention in a variety of biomedical applications in recent
years.13−21 3CTAs have been evaluated as boron delivery agents
for boron neutron capture therapy (BNCT) of cancer.19,22 One of
the lead 3CTAs that was identified is N5 (3; Figure 1 and
Scheme 1).14,23 This agent appears to selectively accumulate
within cancer cells following monophosphorylation by human
thymidine kinase 1 (hTK1), which is overexpressed in cancer cells.
This form of selective entrapment of nucleoside analogues in
tumor cells has been referred to as “kinase mediated trapping”
(KMT).13 Furthermore, 3CTAs did not appear to be substrates of
nucleoside-catabolizing enzymes such as 5′-nucleotidases
(5′-NTs) and nucleoside phosphorylases, and preliminary data
indicated that these compounds might pass cell membranes by
passive diffusion.12,14,23 It is conceivable that cage-monoradioiodi-
nated forms of 3 (e.g., N5-125I [13a or 13b, Scheme 3]) could be
used in the radiotherapy and imaging of cancer, as well as for
BNCT-related biodistribution/pharmacokinetic experiments. They
may be superior to conventional radioiodinated nucleosides, such
5-[125I]iodo-2′-deoxyuridine.24−27 Under physiological conditions,
the latter is cleaved within a few minutes by nucleoside phos-
phorylases to 5-[125I]iodoracil. This is followed by rapid
dehalogenation, which can lead to the accumulation of significant
quantities of radioiodine in the thyroid and the stomach.24−27
In this paper, we describe strategies for the controlled intro-
duction of a single 127I atom to the closo-o-carborane cluster of
3, the halogen and isotope exchange of iodine-127 to bromine-
79/81 and iodine-125, respectively, via palladium-catalyzed
nucleophilic halogen/isotope exchange, and the initial bio-
logical evaluation of cage-halogenated forms of 3 in phosphoryl
transfer assays (PTAs) with hTK1 and in cell uptake
experiments with L929 TK1(+) and L929 TK1(−) cells. To
the best of our knowledge, this is the first description of this
type of nucleophilic halogen/isotope-exchange reaction at a
neutral boron cluster in a complex biomolecule. The method-
ology that we have developed could be applicable for the
synthesis of a wide range of low-molecular-weight bioconju-
gates containing neutral radioiodinated boron clusters for
bioimaging and the treatment of cancer.
3-[5-(o-Carboran-1-yl)pentyl]thymidine (3).28 To a solution of
2 (650 mg, 1.7 mmol) in N,N-dimethylformamide (DMF)/acetone
(1:1, 15 mL) were added dThd (500 mg, 2.06 mmol) and potassium
carbonate (700 mg, 5.07 mmol). The solution was stirred for 24 h at
50 °C. The reaction mixture was filtered, and the filtrate was concentrated
in vacuo. The residue was purified by column chromatography using
CH2Cl2/CH3COCH3 (4:1) as the solvent system to give compound 3
(490 mg, 64%) as a waxlike solid. Spectroscopic data were consistent with
those previously reported for the same compound.28
3-[5-(9-Iodo-o-carboran-1-yl)pentyl]thymidine and 3-[5-(12-
Iodo-o-carboran-1-yl)pentyl]thymidine (4a/4b, [N5-I]) [Strategy 1].
To a stirred solution of 328 (25 mg, 0.055 mmol) and AlCl3
(73 mg, 0.55 mmol) in anhydrous CH2Cl2 was added dropwise ICl
(1 M in CH2Cl2; 55 μL, 0.055 mmol) at 0 °C. The reaction mixture
was stirred for 2 h at 0 °C, quenched with a saturated solution of
Na2S2O3, and extracted with CH2Cl2. The organic layer was separated
and the aqueous layer washed two times with 10 mL of CH2Cl2. The
combined organic layers were washed with brine, dried over
magnesium sulfate, and evaporated in vacuo. The product was crudely
separated from the decomposition products by column chromatog-
raphy using CH2Cl2/MeOH (10:1) or, alternatively, PhMe/CH3CN
(3:2) as the solvent system. Rf = 0.34 (CH2Cl2/MeOH, 10:1).
Final purification, primarily by separation from the diiodinated
product, was accomplished by preparative RP-18 HPLC (acetoni-
trile/water, 60:40) to furnish 4a/4b (5.0 mg, 16%) as a white foam.
1H NMR (CD3COCD3): δ 0.6−4.0 (m, 9H, BH), 1.32 (m, 4H, −CH2),
1.57 (m, 8H, CH2CH2), 1.83 (s, 6H, −CH3), 2.25−2.39 (m,
8H, −CH2Ccarborane and H-2′), 3.78 (d, 4H, H-5′, J = 2.7 Hz), 3.86
EXPERIMENTAL SECTION
■
NMR spectra were obtained on a Bruker Avance 400 at The Ohio
State University College of Pharmacy (400 MHz for 1H, 100 MHz for
13C, and 128 MHz for 11B). Chemical shifts (δ) are reported in parts
per million from internal deuterated chloroform, deuterated acetone,
or an external BF3·Et2O standard. Coupling constants are reported in
hertz. High-resolution electron spray ionization (HR-ESI) mass
spectrometry (MS) spectra were obtained at The Ohio State
University Campus Chemical Instrumentation Center on a Micromass
Q-TOF II and a Micromass LCT spectrometer and at the University
of Illinois Mass Spectrometry Laboratory (Urbana−Champaign,
Illinois) on a Waters Q-TOF Ultima tandem quadrupole/time-of-
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Inorganic Chemistry
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(m, 4H, −CH2N), 3.93 (m, 2H, H-3′), 4.26 (m, 2H, −OH), 4.41 (d, 2H,
H-4′, J = 3.2 Hz), 4.49 (s, 2H, −OH), 4.90 (s, 1H, HCcarborane), 5.10 (s, 1H,
HCcarborane), 6.34 (t, 2H, H-1′, J = 6.8 Hz), 7.83 (s, 2H, H-6). 11B{1H}
NMR (CD3COCD3): δ −17.79 (s, 1B, B12−I, B9−I), −16.15 (s, 1B, B9−
I/B12−I), −11.04 (m, 12B, B3, B4, B5, B6, B7, B11), −7.19 (s, 4B, B8,
B10), −3.96 (s, 1B, B12−H/B9−H), −0.58 (s, 1B, B9−H/B12−H). 11B
NMR (CD3COCD3): δ −17.93 (s, 1B), −16.26 (s, 1B), −11.17 (m, 12B),
−7.33 (d, 4B, J = 156.2 Hz), −4.10 (d, 1B, J = 145.7 Hz), −0.70
(d, 1B, J = 153.5 Hz). HPLC retention time = 9.91 min (RP-18 analytical
HPLC, 1 mL flow rate, solvent system CH3CN/H2O, 60:40, isocratic
elution). MS (HR-ESI) for C17H33B10IN2O5 [(M + Na)+]. Calcd: m/z
603.2324. Found: m/z 603.2344. N5-I2: HPLC retention time = 11.21 min
(RP-18 analytical HPLC, 1 mL flow rate, solvent system CH3CN/H2O,
60:40, isocratic elution). MS (HR-ESI) for C17H32B10I2N2O5
[(M + Na)+]. Calcd: m/z 729.1259. Found: m/z 729.1310.
3-[5-(8,9,10,12-Tetraiodo-o-carboran-1-yl)pentyl]thymidine
(5, N5-I4). To a stirred solution of 328 (100 mg, 0.22 mmol) and
AlCl3 (440 mg, 3.3 mmol) in anhydrous CH2Cl2 was added dropwise a
1 M ICl solution in CH2Cl2 (1.1 mL, 1.1 mmol) at 0 °C. The reaction
mixture was stirred for 2 h at 0 °C and then refluxed for 2 days. The
reaction mixture was quenched with a saturated solution of Na2S2O3
and extracted with CH2Cl2. The organic layer was separated, and the
aqueous layer was washed with 2 × 10 mL of CH2Cl2.. The combined
organic layers were washed with brine, dried over anhydrous MgSO4,
and evaporated in vacuo. The product was crudely separated from the
decomposition products by column chromatography using CH2Cl2/
MeOH (10:1) or, alternatively, PhMe/CH3CN (3:2) as the solvent
system. Rf = 0.34 (CH2Cl2/MeOH, 10:1). Final purification, primarily by
separation from the triiodinated product, was accomplished by preparative
RP-18 HPLC (CH3CN/H2O, 60:40, isocratic elution) to furnish 5
(27 mg, 13%) as a yellow solid. HPLC retention time = 13.81 min (RP-
18 analytical HPLC, 1 mL flow rate, solvent system CH3CN/H2O, 60:40,
isocratic elution). 1H NMR (CD3COCD3): δ 0.6−4.0 (m, 6H, BH), 1.33
(m, 2H, −CH2), 1.61 (m, 4H, −CH2CH2), 1.83 (s, 3H, −CH3), 2.23
(dd, 2H, H-2′, J = 4.7 and 6.7 Hz), 2.45 (m, 2H, −CH2Ccarborane), 3.78
(m, 2H, H-5′), 3.88 (m, 2H, −CH2N), 3.94 (dd, 1H, H-3′, J = 3.1 and 6.1
Hz), 4.27 (s, 1H,−OH), 4.42 (s, 1H, −OH), 4.49 (s, 1H, H-4′), 5.72 (s,
1H, HCcarborane), 6.34 (t, 1H, H-1′, J = 6.8 Hz), 7.84 (s, 1H, H-6). 11B{1H}
NMR (CD3COCD3): δ −17.8 (s, 4B, B8−I, B9−I, B10−I, and B12−I),
−12.1 (s, 1B), −10.2, −8.4 (m, 5B). 11B NMR (CD3COCD3): δ −17.9
(s, 4B, B8, B9, B10, and B12), −12.1 (d, 1B, J = 205.2 Hz), −10.1, −8.3
(m, 5B). MS (HR-ESI). C17H30B10I4N2O5 [(M + H)+]. Calcd: m/z
960.9371. Found: m/z 960.9373. N5-I3: HPLC retention time = 13.60
min (RP-18 analytical HPLC, 1 mL flow rate, solvent system CH3CN/
H2O, 60:40, isocratic elution). MS (HR-ESI) for C17H31B10I3N2O5 [(M +
Na)+]. Calcd: m/z 855.0226. Found: m/z 855.0277.
layer was extracted with 2 × 30 mL of diethyl ether. The combined
organic layers were dried (MgSO4) and concentrated in vacuo. The crude
residue was purified by column chromatography using pentane to afford
8a/8b as colorless oil (2.0 g, 66%). Rf = 0.24−0.31. 1H NMR
(CD3COCD3): δ 0.30 (s, 6H, −Si(CH3)2), 0.33 (s, 6H, −Si(CH3)2),
1.04 (s, 9H, −C(CH3)3), 1.07 (s, 9H, −C(CH3)3), 1.47−2.23 (m, 9H,
BH), 4.52 (s, 1H, HCcarborane), 4.74 (s, 1H, HCcarborane). MS (HR-EI) for
C8H25B10ISi (M+). Calcd: m/z 384.1774. Found: m/z 384.1774.
A quantity of 100 mg of the mixture of 8a and 8b was separated by
column chromatography using pentane as the solvent system to give
58 mg of 8a (Rf = 0.31; mp 105−106 °C) and 31 mg of 8b (Rf = 0.24;
mp 125−126 °C). 8a. 1H NMR (CD3COCD3): δ 0.33 (s, 6H,
−Si(CH3)2), 1.09 (s, 9H, −C(CH3)3), 1.47−2.23 (m, 9H, BH), 4.77
(s, 1H, HCcarborane). 11B{1H} NMR (CD3COCD3): δ −15.0 (s, 1B,
B9−I), −11.7 (m, 4B, B3, B4, B5, B6), −8.9 (s, 2B, B7, B11), −4.6
(s, 2B, B8, B10), 2.3 (s, 1B, B12). 11B NMR (CD3COCD3): δ −15.0
(s, 1B), −10.4 (m, 6B), −4.6 (d, 2B, J = 152.6 Hz), 2.3 (d, 1B, J =
157.9 Hz, B12). MS (HR-EI) for C8H25B10ISi (M+). Calcd: m/z
1
384.1774. Found: m/z 384.1758. 8b. H NMR (CD3COCD3): δ 0.31
(s, 6H, −Si(CH3)2), 1.04 (s, 9H, −C(CH3)3), 1.47−2.23 (m, 9H,
BH), 4.55 (s, 1H, HCcarborane). 11B{1H} NMR (CD3COCD3): δ −14.4
(s, 1B, B12−I), −12.3 (m, 4B, B3, B4, B5, B6), −11.0 (m, 2B, B7,
B11), −5.2 (s, 2B, B7, B11), −0.2 (s, 1B, B9). 11B NMR
(CD3COCD3): δ −14.3 (s, 1B), −13.0 (s, 1B), −10.4 (m, 1B, J =
76.7 and 168.5 Hz), −5.2 (d, 1B, J = 154.3 Hz), −0.3 (d, 1B, J = 152.1
Hz). MS (HR-EI) for C8H25B10ISi (M+). Calcd: m/z 384.1774.
Found: m/z 384.1791.
1-(tert-Butyldimethylsilyl)-2-[5-(tert-butyldimethylsilyloxy)-
pentyl]-9-iodo-o-carborane and 1-(tert-Butyldimethylsilyl)-2-
[5-(tert-butyldimethylsilyloxy)pentyl]-12-iodo-o-carborane
(9a/9b). To a stirred solution of a mixture of 8a and 8b (6.52 g, 17
mmol) in 35 mL of anhydrous THF at −78 °C was added dropwise a
2.5 M solution of n-BuLi in hexanes (8.48 mL, 21.2 mmol). The reaction
mixture was stirred for 30 min at room temperature and then cooled to
0 °C, and TBDMSCl (8.21 g, 22 mmol) dissolved in THF (20 mL) was
added dropwise. The solution was stirred for 24 h, quenched with 20 mL
of water, and extracted with 60 mL of diethyl ether. The separated
aqueous layer was extracted with 2 × 30 mL of diethyl ether, and the
combined organic layers were dried (MgSO4) and concentrated in vacuo.
The crude residue was purified by flash chromatography to afford 9a/9b
(6.5 g, 66%). Rf = 0.72 (pentane/diethyl ether, 30:1). 1H NMR
(CD3COCD3): δ 0.05 (s, 6H, −Si(CH3)2), 0.05 (s, 6H, −Si(CH3)2), 0.39
(s, 6H, −Si(CH3)2), 0.43 (s, 6H, −Si(CH3)2), 0.89 (s, 9H, −C(CH3)3),
0.90 (s, 9H, −C(CH3)3), 1.09 (s, 9H, −C(CH3)3), 1.12 (s, 9H,
−C(CH3)3), 1.34−1.67 (m, 12H, CH2CH2CH2), 1.3−3.27 (m, 18H,
BH), 2.22 (m, 2H, CH2Ccarborane), 2.34 (m, 2H, CH2Ccarborane), 3.63 (dd,
4H, −CH2OTBDMS, J = 6.2 and 13.2 Hz). 11B{1H} NMR
(CD3COCD3): δ −15.9 (s, 1B, B12−I/B9−I), −14.2 (s, 1B, B9−I/
B12−I), −9.0 (m, 12B, B3, B4, B5, B6, B7, B11), −5.3 (m, 4B, B8, B10),
−2.5 (s, 1B, B12−H/B9−H), 2.0 (s, 1B, B9−H/B12−H). 11B NMR
(CD3COCD3): δ −15.9 (s, 1B), −14.2 (s, 1B), −9.2 (m, 12B), −5.3 (m,
4B), −2.4 (d, 1B, J = 148.6 Hz), 2.1 (d, 1B, J = 148.8 Hz). MS (HR-EI)
for C19H49B10IOSi2 [(M − 56)−]. Calcd: m/z 528.2744. Found: m/z
528.2760.
9-Iodo-o-carborane (7).30,31 To a solution of o-carborane (6; 2.0 g,
14 mmol) in anhydrous CH2Cl2 was added dropwise ICl (27.7 mmol,
27.7 mL of a 1 M solution in CH2Cl2) at 25 °C. The reaction mixture was
stirred for 5 h at 40 °C, quenched with a saturated aqueous solution of
Na2S2O3, and extracted with CH2Cl2. The organic layer was washed with
brine, dried (MgSO4), and evaporated in vacuo. The residue was purified
by column chromatography to afford 7 (2.7 g, 72%) as a off-white solid.
Rf = 0.38 (hexanes/ethyl acetate, 10:4). 1H NMR (CD3COCD3): δ 1.53−
3.21 (m, 9H, BH), 4.74 (s, 1H, HCcarborane), 4.95 (s, 1H, HCcarborane).
11B{1H} NMR (CD3COCD3): δ −16.5 (s, 1B, B9−I), −13.7 (s, 2B, B3,
B6), −12.8 (s, 2B, B4, B5), −12.1 (s, 2B, B7, B11), −7.3 (s, 2B, B8, B10),
−1.1 (s, 1B, B12). 11B NMR (CD3COCD3): δ −16.5 (s, 1B), −13.1 (m,
6B), −7.3 (d, 2B, J = 155.0 Hz), −1.2 (d, 1B, J = 151.2 Hz). MS (HR-EI)
for C2B10H9I (M+). Calcd: m/z 270.0909. Found: m/z 270.0912.
1-(tert-Butyldimethylsilyl)-9-iodo-o-carborane (8a) and 1-
(tert-Butyldimethylsilyl)-12-iodo-o-carborane (8b). To a stirred
solution of 7 (2.1 g, 7.8 mmol) in anhydrous tetrahydrofuran (THF)
at −78 °C was added dropwise a 2.5 M solution of n-BuLi in hexanes
(3.26 mL, 8.15 mmol). The reaction mixture was allowed to stir for
30 min at room temperature and cooled to 0 °C, and tert-
butyldimethylsilyl chloride (TBDMSCl; 1.29 g, 8.55 mmol) in
15 mL of anhydrous THF was added dropwise. The solution was
refluxed overnight, carefully quenched with 10 mL of water, and extracted
with 60 mL of diethyl ether. The layers were separated, and the aqueous
5-(9-Iodo-o-carboran-1-yl)pentan-1-ol and 5-(12-Iodo-o-car-
boran-1-yl)pentan-1-ol (10a/10b). To a solution of 9a/9b (2.37 g,
4.05 mmol) in 40 mL of THF was added dropwise at −78 °C a 1.0 M
solution of tetrabutylammonium fluoride (TBAF) in THF (6.1 mL,
6.1 mmol). The reaction mixture was stirred at 4 °C for 30 min,
acidified with 10% methanolic/HCl (20 mL), and then stirred for an
additional 30 min at 4 °C. The reaction mixture was extracted with
ethyl acetate (3 × 35 mL), and the combined organic layers were
washed with water, then saturated aqueous NaHCO3, dried over
anhydrous magnesium sulfate, and concentrated in vacuo. The residue
was purified by column chromatography (hexanes/ethyl acetate, 3:7)
to furnish 10a/10b (1.1 g, 76%). Rf = 0.54 (hexanes/ethyl acetate,
1
3:7). H NMR (CD3COCD3): δ 1.28−1.59 (m, 12H, −CH2CH2CH2),
1.3−3.27 (m, 18H, BH), 2.25 (m, 2H, −CH2Ccarborane), 2.38 (m, 2H,
−CH2Ccarborane), 3.50 (dd, 4H, −CH2OH, J = 5.3 and 10.2 Hz), 4.88 (br,
1H, HCcarborane), 5.09 (br, 1H, HCcarborane). 11B{1H} NMR (CD3COCD3):
631
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Inorganic Chemistry
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δ −18.4 (s, 1B, B12−I/B9−I), −16.7 (s, 1B, B9−I/B12−I), −11.7 (m, 12B,
B3, B4, B5, B6, B7, B11), −7.7 (m, 4B, B8, B10), −4.4 (s, 1B, B12−H/B9−
H), −1.15 (s, 1B, B9−H/B12−H). 11B NMR (CD3COCD3): δ −18.1 (s,
1B), −16.4 (s, 1B), −11.3 (m, 14B), −7.3 (m, 2B, J = 152.5 Hz), −4.1 (d,
1B, J = 153.3 Hz), −0.7 (d, 1B, J = 150.4 Hz). MS (HR-EI) for
C7 H2 1 B1 0 IO (M+ ). Calcd: m /z 356.1641. Found:
m/z 356.1647.
DMF (10 mol %) was added to the reaction vial. To this reaction
mixture was added dropwise 10 μL (100 μCi) of the diluted alkaline
Na125I solution. The reaction mixture was then heated at 110 °C for
1 h, cooled to room temperature, and filtered using a GHP Acrodisc
13-mm syringe filter to separate the catalyst from the reaction mixture.
The filtrate was evaporated under an argon flow at 30 °C, and the
residue was purified by a semipreparative RP-18 HPLC to yield 13a/
13b (N5-125I; 3.2 mg, radiochemical yield = 8%, specific activity =
2.52 μCi/mg, radio chemical purity: > 98%). HPLC retention time =
25.50 min (RP-18 analytical HPLC, 1 mL flow rate, solvent system
CH3CN/H2O, 50:50, isocratic elution).
X-ray Diffraction Studies. Crystals of 8a and 8b were obtained by
diffusion crystallization using diethyl ether/pentane as the solvent
system. Both data collections were carried out at −123 °C on a Nonius
Kappa CCD diffractometer with Mo Kα radiation and a graphite
monochromator. ϕ and ω scans were used for data collection with a
frame width of 1.0°. Data integration was carried out with Denzo,32 and
an absorption correction and merging of the data were carried out with
Sortav.33,34 The structures were solved by the direct methods procedure
in SHELXS-97.35 Full-matrix least-squares refinements based on F2 were
performed in SHELXL-97,35 as incorporated in the WinGX package.36
For structure 8b, there are four independent molecules in the
asymmetric unit (Z′ = 4), and these are labeled as A−D (Table 3 and
Figure S2 in the Supporting Information). For each methyl group, the
hydrogen atoms were added at calculated positions using a riding model
with U(H) = 1.5Ueq (bonded carbon atom). The torsion angle, which
defines the orientation of the methyl group about the C−C or Si−C
bond, was refined. The remaining hydrogen atoms in each cluster were
refined isotropically. Neutral-atom scattering factors were used and
include terms for anomalous dispersion.37 The values of the Flack
parameters38,39 for 8a and 8b are −0.05(1) and −0.042(7), respectively,
indicating that the models for these structures are of the correct chirality.
Additional crystallographic details are shown in Table 2.
Docking Studies. The homology model of hTK1 (PDB ID #
1W4R) was developed from the crystal structure of Thermotoga
maritima Thymidine Kinase (TmTK, PDB ID # 2QPO) using the
alignment mode of SWISS-MODEL.40 The sequence identity and
similarity between hTK1 and TmTK are 36% and 55%, respectively.41
The obtained homology model was saved in pdb format, and hydrogen
atoms were added using Accelrys Discovery Studio Visualizer v2.5.
After the addition of hydrogen atoms, the pdb file was imported into
Surflex, version 2.1142 (Biopharmics LLC, San Francisco, CA),
installed on a Dell optimplex GX 270 desktop computer with a
Windows operating system environment, for the protomol generation.
The default mode was chosen for docking. The obtained docking
poses were visualized with Accelrys Discovery Studio Visualizer v2.5.
Surflex does not have parameters for boron atoms. By default, Surflex
treats such unrecognizable atoms as “funky atoms”. However, this does
not seem to affect significantly the accuracy of the docking studies with
compounds containing carborane clusters.43
PTAs. Recombinant hTK1 was expressed and purified from the
bacterial expression system BL21(DE3) pLysS with transformed vector
pET-14b + hTK1), kindly provided by Dr. Staffan Eriksson, Biomedical
Centre, Uppsala, Sweden, according to a procedures described pre-
viously.28,44 dThd and the carboranylthymidine conjugates were dissolved
in dimethyl sufloxide (DMSO; 5 mM concentration) and further diluted
with water to produce stock solutions of concentrations (0.4 mM). The
PTAs were carried out as described previously28,44 with minor
modifications. The reaction mixture contained 100 μM nucleoside and
100 μM ATP [with a small fraction of 0.13 μM [γ-32P]ATP (Perkin-
Elmer)], 25 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 125 mM KCl, 1 mM
DTT, and 0.5 mg/mL bovine serum albumin. In all reactions, the final
concentration of DMSO was set to 2%. The reaction mixture was
incubated at 37 °C for 20 min in the presence of 135 ng of enzyme.
Somewhat higher concentrations of enzyme were used than in previous
studies45 because this specific enzyme preparation appeared to be less
active (441 nmol of thymidine monophosphate formed per minute and
mg of hTK1). Following the incubation period, the enzyme was
inactivated by heating for 2 min at 99 °C. The reaction mixture was
centrifuged, and 2 μL sample portions were spotted on PEI−cellulose
5-(9-Iodo-o-carboran-1-yl)pentyl 4-Methylbenzenesulfo-
nate and 5-(12-Iodo-o-carboran-1-yl)pentyl 4-Methylbenzene-
sulfonate (11a/11b). To a solution of 10a/10b (1.65 g, 4.63 mmol)
in CH2Cl2 (12 mL) was added triethylamine (0.84 mL, 6 mmol) and a
catalytic amount of 4-(dimethylamino)pyridine (DMAP; 113 mg, 0.93
mmol). A solution of p-toluenesulfonyl chloride (1.33 g, 6.95 mmol) in
13 mL of CH2Cl2 was added at 0 °C, and the reaction mixture was stirred
at room temperature for 7 h. The reaction was quenched with a saturated
NH4Cl aqueous solution; the organic phase was washed with water and
brine, dried (MgSO4), and concentrated in vacuo. The residue was
purified by column chromatography to afford 11a/11b (2.2 g, 93%) as an
1
oil. Rf = 0.65 (hexanes/ethyl acetate, 1:1). H NMR (CD3COCD3): δ
1.27−1.69 (m, 12H, −CH2CH2CH2), 1.3−3.27 (m, 18H, BH), 2.23
(m, 2H, −CH2Ccarborane), 2.36 (m, 2H, −CH2Ccarborane), 2.47 (s, 6H,
−CH3), 4.02 (m, 4H, −CH2OTs), 4.84 (br, 1H, HCcarborane), 5.05
(br, 1H, HCcarborane), 7.49 (d, 4H, ArH, J = 7.3 Hz), 7.79 (dd, 4H, ArH,
J = 3.0 and 8.4 Hz). 11B{1H} NMR (CD3COCD3): δ −18.0 (s, 1B, B12−
I/B9−I), −16.3 (s, 1B, B9−I/B12−I), −11.5 (m, 12B, B3, B4, B5, B6, B7,
B11), −7.3 (s, 4B, B8, B10), −4.1 (s, 1B, B12−H/B9−H), −0.66 (s, 1B,
B9−H/B12−H). 11B NMR (CD3COCD3): δ −17.9 (s, 1B), −16.3 (s,
1B), −11.4 (m, 12B), −7.3 (d, 2B, J = 155.0 Hz), −4.0 (d, 1B, J = 149.8
Hz), −0.66 (d, 1B, J = 152.7 Hz). MS (HR-EI) for C14H27O3SIB10 (M+).
Calcd: m/z 510.1729. Found: m/z 510.1724.
Synthesis of 4a/4b (N5-I) [Strategy 2]. To a solution of 11a/
11b (1.18 g, 2.31 mmol) in DMF/acetone (1:1, 40 mL) was added
dThd (1.68 g, 6.93 mmol) and potassium carbonate (960.0 mg, 6.94
mmol). The solution was stirred for 2.5 h at 40 °C and filtered. The
filtrate was concentrated in vacuo, and the residue was added to water
and extracted with ethyl acetate (3 × 50 mL). The combined organic
layers were washed with brine, dried over anhydrous MgSO4, and
concentrated in vacuo. The residue was purified by column
chromatography using CH2Cl2/MeOH (10:1, Rf = 0.34) as the eluent
to afford 4a/4b (800 mg, 60%).
3-[5-(9-Bromo-o-carboran-1-yl)pentyl]thymidine and 3-[5-
(12-Bromo-o-carboran-1-yl) pentyl]thymidine (12a/12b, N5-Br). To
a solution of 4a/4b (100 mg, 0.175 mmol) in 3 mL of CH2Cl2
was added a solution of NaBr (180 mg, 1.75 mmol) dissolved in
5 mL of water. The solvents were evaporated in vacuo to obtain an
anhydrous residue. A solution of Herrmann’s catalyst (41 mg,
25 mol %) in 1 mL of anhydrous DMF was added. The reaction mixture
was stirred at 110 °C for 1 h. Following evaporation, the residue was
added to water and extracted with CH2Cl2. The organic phase was
dried (MgSO4) and concentrated in vacuo, and the residue was
purified by column chromatography to yield 12a/12b (48 mg, 52%).
1
Rf = 0.34 (CH2Cl2/MeOH, 10:1). H NMR (CD3COCD3): δ 1.3−
3.22 (m, 18H, BH), 1.31 (m, 4H, −CH2), 1.56 (m, 8H, −CH2CH2),
1.83 (s, 6H, −CH3), 2.23 (m, 4H, H-2′), 2.36 (m, 4H, −CH2Ccarborane),
3.77 (m, 4H, H-5′), 3.86 (m, 4H, −CH2N), 3.92 (m, 2H, H-3′), 4.27
(s, 2H, −OH), 4.42 (s, 2H, −OH), 4.49 (d, 2H, H-4′, J = 2.9 Hz), 4.84
(s, 2H, HCcarborane), 6.34 (t, 2H, H-1′, J = 6.7 Hz), 7.83 (s, 1H, H-6).
11B{1H} NMR (CD3COCD3): δ −11.9 (m, 12B, B3, B4, B5, B6, B7,
B11), −8.1 (s, 4B, B8, B10), −4.9 (s, 1B, B12−H/B9−H), −1.3 (s, 1B,
B9−H/B12−H), 0.4 (s, 2B, B9−Br and B12−Br). 11B NMR
(CD3COCD3): δ −12.7 (m, 12B), −8.1 (d, 4B, J = 151.9 Hz), −4.9
(m, 2B), −0.72 (m, 2B). MS (HR-ESI) for C17H33B10BrN2O5 [(M +
Na)+]. Calcd: m/z 557.2401. Found: m/z 557.2431.
3-[5-(9-[125I]Iodo-o-carboran-1-yl)pentyl]thymidine and 3-
[5-(12-[125I]Iodo-o-carboran-1-yl)pentyl]thymidine (13a/13b,
N5-125I). An alkaline Na125I solution (10 μL, pH 8−11, 1 mCi,
specific activity of 17 Ci [629 GBq]/mg) was diluted to 100 μL using
deionized water. Compound 4a/4b (10 mg, 0.017 mmol), dissolved in
200 μL of DMF, was transferred to a 3 mL conical reaction vial
(MINUM-WARE). A solution of Herrmann’s catalyst in 100 μL of
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TLC plates (EMD Chemicals Inc.). The TLC plates were developed in a
solvent system containing isobutyric acid/ammonium hydroxide/water
(66:1:33) over a period of 6 h. The radiolabeled spots were visualized by
placing the TLC plates on a BioMax XAR film (Kodak) overnight and
developing the films using an X-ray film developer (Tiba M6B, series VI B
Rapid processor; Commonwealth X-ray Inc., Cincinnati, OH). The spot
intensities of the phosphorylated compounds were calculated by using
Photoshop, and the values were compared with those of dThd.
to calculations). Therefore, we hypothesize that the introduc-
tion of a single iodine to 3 may not significantly affect its
phosphorylation by hTK1, and more importantly, its tumor-
localizing properties. Docking studies of the triphosphate forms
of 3 and 4b with a homology model of hTK1 seem to confirm
our hypothesis (Figure 1). The extensive conformational changes
Cellular Uptake Studies. The L929 (#CCL1, American Type
Culture Collection, Manassas, VA) cell line was one of the first to be
established in a continuous culture, and clone 929 was the first cloned
strain that was developed. The parent L strain was derived from
normal subcutaneous areolar and adipose tissue from a C3H/An
mouse, and clone 929, which is TK1(+), was from the 95th subculture
generation of the parent strain. Its TK1(−) counterpart (#CCL1.3
L-M ATCC) was derived from the wild-type cell line L929. The L929
TK1(+) cells were grown in Dulbecco’s Modified Eagle’s Medium
(DMEM), supplemented with 10% fetal bovine serum (FBS), 1%
L-glutamine (#25030, Invitrogen, Carlsbad, CA), and a 1% penicillin
and streptomycin mixture (Penstrep, #15140, Invitrogen). The L929
TK1(−) cells were grown in media with 10% FBS, 1% L-glutamine, 1%
Penstrep, and 10 mM 5-bromo-2′-deoxyuridine in order to inhibit
reversion to the TK1(+) phenotype. Cells were grown in T-75 flasks
(#430641, Corning, Corning, NY) and split after reaching 70−80%
confluency for propagating the cell line.
Uptake experiments were carried out as follows: 48 h prior to the
addition of the test compounds, L929 TK1(−) or TK1(+) cells were
seeded at a density of 1 × 106 or 2.5 × 106 cells per T-75 flask,
respectively, with six flasks for each cell type. When the cells reached
approximately 90% confluency, the test compounds were added to the
culture medium. Cells were seeded at different densities because
TK1(−) cells grew faster than TK1(+) cells. Before treatment, the cell
culture medium was removed and the cells were washed once with
DMEM. After that, 12.5 mL of media containing 17.5 μM N5 (3),
N5-79/81Br (12a/12b), or N5-127I (4a/4b) was added to the cultures. A
total of 24 h following incubation at 37 °C, the medium containing the
compound was removed and the cells were washed twice with cold
phosphate-buffered saline at pH 7.4. Cells were then trypsinized with
5 mL of 0.5% trypsin/ethylenediaminetetraacetic acid (#15400,
Invitrogen) for 5 min, followed by the addition of 5 mL of DMEM
with 10% FBS to neutralize the trypsin. Cells with media from two T-75
flasks were pooled into one 50 mL centrifuge tube, following which they
were centrifuged (600g). After centrifugation, the trypsin-containing
medium was decanted, the cell pellets were resuspended in 2 mL of
serum-free DMEM, and the number of cells per tube was counted
by means of a hemocytometer. Cells with media then were transferred
to borosilicate free glass test tubes, heated to dryness in an oil
bath at 105 °C, and then 1 mL of concentrated sulfuric acid was added
to digest them for 2 h. Following this, 1 mL of 50% hydrogen
peroxide was added to each tube to decolorize the digested
material. The contents were transferred to 15 mL plastic centrifuge
tubes, and the volume was adjusted to 4 mL by the addition of
deionized water. Boron concentrations were determined by means of
inductively coupled plasma optical emission spectroscopy (ICP-OES)
using a spectrometer located at the Nanotech West Facility of The Ohio
State University.
Figure 1. Docking poses of the 5′-triphosphate forms of 3 (A) and 4b
(B) in a homology model of hTK1 (PDB ID # 1W4R) that was
developed from the crystal structure of TmTK (PDB ID # 2QPO).
that TK1-like enzymes undergo as a result of ATP and/or dThd
binding have been discussed in detail by Lavie et al.46 The
homology model of hTK1 was developed from an apo form of
Thermotoga maritima Thymidine Kinase (TmTK, PDB ID #
2QPO) and, thus, should represent an apo form of hTK1. The
triphosphate forms of 3 and 4b were chosen for docking because
we have found that this modification produces realistic docking
poses of the dThd scaffold within the hTK1 active site. As can be
seen from parts A and B of Figure 1, the carborane clusters of
both compounds are located outside the substrate binding
pocket and monoiodination in 4b does not generate significant
differences in the dThd binding patterns between 3 and 4b.
Similar results have been observed previously in docking studies
of other 3CTAs with varying substitution patterns at the
carborane cluster in a hTK1 homology model.47
Chemistry. Theoretically, radioiodination of the closo-o-
carborane cage can be achieved in three ways. The first method
employs a “closo-cluster reconstruction” from nido-o-carborane
via treatment with n-butyllithium (n-BuLi) followed by boron
triiodide (BI3) to generate iodinated closo-o-carborane.48,49 The
B-iodocarborane synthesized in this way has iodine either at
B3 or at B6 (Figure 2). The second method is the direct
introduction of iodine to the closo-o-carborane by means of a
RESULTS AND DISCUSSION
■
Computational Studies. Human TK1 tolerates extensive
modification at the N3 position of dThd,19 and the differences
in the surface areas and the molecular volumes between 3 and
13a/13b, or their nonradioactive counterparts (N5-127I,
[4a/4b, Scheme 1], were relatively small. The molecular
surface area and volume for 3 were 575.53 Å2 and 1297.05 Å3,
respectively. The corresponding values for 4a and 4b were
630.77 and 630.83 Å2, respectively, for molecular surface areas
and 1379.36 and 1379.75 Å3, respectively, for molecular
volumes (see the Supporting Information for details related
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a Lewis acid in a Friedel−Crafts halogenation strategy already
discussed above and (2) initial monoiodination of unsubstituted
closo-o-carborane with ICl followed by its functionalization to 4a/
4b using conventional synthetic organic methods.28
Direct Iodination of Preformed 3 [Strategy 1]. A new
strategy for the synthesis of 3 was applied (Scheme 1). The
reaction of 6-heptynyl tosylate (1)29 with decaborane (B10H14)
in a biphasic mixture of toluene and the ionic liquid 1-butyl-3-
methylimidazolium chloride (bmim+ Cl−)52 yielded the o-
carboranyl tosylate 228 in 55% yield. The reaction of compound
2 with dThd in DMF/acetone in the presence of K2CO3
produced compound 3 in 56% yield, as described previously by
Lunato et al.28 Overall, the yields for both compounds 2 and 3
described here are comparable with those reported previously
for the same compounds.28 However, the synthesis of 3 is much
shorter, far more convenient, and also safer than the procedure
described previously involving the synthesis and isolation of an
intermediate decaborane/acetonitrile complex.28
Figure 2. Atom numbering and electron density distribution in closo-o-
carborane. Boron atoms shown in red are the most electron-rich, whereas
the boron atoms shown in blue are the most electron-deficient.
Friedel−Crafts-type (electrophilic) halogenation using I2 or ICl
as iodine sources.48,50,51 The electrophilic attack of positively
charged species on o-carborane is regiospecific based on the
charge distribution in the cluster.48,50,51 Electrophilic halogen-
ation occurs first at the most electron-rich boron atoms 9 and
12 (Figure 2), which are furthest away from both carbon atoms
followed by the boron atoms 8 and 10.48,50,51 Thus, reaction of
closo-o-carboranes with halogenating species can result in
mono-, di-, tri-, or tetraiodination depending on the applied
reaction conditions and stoichiometry of halogenating
species.48,49 A disadvantage of both methods discussed above
is that 125I2, 125ICl, or B125I3, which would be necessary for the
synthesis of 125I-labeled 3 (N5-125I, 13a/13b, Scheme 3), are
not readily available from commercial sources.
The third method is a halogen/isotope-exchange reaction
using a nonradioactive B-iodocarborane as the starting material
that is treated with a nucleophilic radiohalogen source in the
presence of a suitable catalyst.6,8−10 The advantage of this
method is that the radiohalogen source required for this
strategy (Na125I) is commercially available. This strategy was
therefore chosen for the synthesis of 13a/13b.
Several different reaction conditions were explored to
optimize the synthesis of 4a/4b via direct iodination of 3, as
shown in Scheme 1 and Table 1. The treatment of 3 with 5
Table 1. Reaction Conditions To Control the Degree of
Iodination by Strategy 1
a
% in reaction mixtures
reactions
(Scheme 1)
dec
prod
3
(recov)
4
N5-I2 N5-I3
5
c
d
e
∼10
∼35
100
∼45
0
∼40
0
∼5
0
0
∼10
0
0
∼55
0
0
0
0
a
Estimations based on TLC and 1H NMR analyses of reaction
mixtures.
equiv of ICl in the absence of Lewis acid (AlCl3) at room
temperature for 1 h in CH2Cl2 proved to be detrimental to
the nucleoside scaffold, resulting in complete decomposition.
When 3 was treated with 1 equiv of ICl in the presence of
For the synthesis of 13a/13b by means of isotope exchange,
the nonradioactive precursor, 4a/4b, must be synthesized first.
Two methods were explored for its synthesis: (1) direct iodination
of the carborane cage of 3 using either ICl or I2 in the presence of
a
Scheme 1
a
Reagents and conditions: (a) B10H14, (bmim)+Cl−, toluene, 120 °C, 10 min; (b) dThd, K2CO3, DMF/acetone (1:1), 40 °C, 24 h; (c) ICl, AlCl3,
DCM, 0 °C, 2 h; (d) ICl, AlCl3, DCM, 40 °C, 48 h; (e) ICl, DCM, rt, 1 h.
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a
Scheme 2
a
Reagents and conditions: (a) ICl, DCM, 40 °C, 5 h; (b) n-BuLi, TBDMSCl, THF, 66 °C, 12 h; (c) n-BuLi, 5-(tert-butyldimethylsilyloxy)pentyl 4-
methylbenzenesulfonate; THF, 66 °C, 12 h; (d) (i) TBAF, THF, −78 to +4 °C, 30 min; (ii) 10% methanolic HCl, 4 °C, 30 min; (e) TsCl, Et3N,
DMAP, 0 °C to rt, 7 h; (f) dThd, K2CO3, DMF/acetone (1:1), 40 °C, 2.5 h.
10 equiv of AlCl3 in CH2Cl2 at 0 °C for 2 h, the reaction
mixture contained 40% of 4a/4b, ∼45% of unreacted 3, and
minor quantities of diiodinated 3 (N5-I2) and decomposition
products (Table 1). The reaction of 3 with 5 equiv of ICl and 15
equiv of AlCl3 at 40 °C in CH2Cl2 for 48 h resulted in the
formation of ∼55% of compound 5 (N5-I4) along with smaller
quantities of triiodinated 3 (N5-I3) and decomposition products
in the reaction mixture (Table 1). Preliminary analytical data
indicated that the decomposition products may include
nucleobases and pyranosyl nucleosides (data not shown). In
general, compounds 4a/4b, 5, N5-I2, and N5-I3 were crudely
separated from decomposition products (primarily nucleobases)
and inorganic impurities by column chromatography followed by
complete purification via semipreparative HPLC. When AlCl3/I2
instead of AlCl3/ICl was used in these reactions, overall similar
product ratios were observed (data not shown).
Initial Monoiodination of o-Carborane followed by
Functionalization to Compounds 4a/4b [Strategy 2]. Scheme
2 describes the synthesis of 4a/4b according to strategy 2. The
reaction of o-carborane with ICl in the absence of any Lewis
acids under reflux conditions in CH2Cl2 furnished 9-iodo-o-
carborane (7)30,31,53 in 70% yield without producing detectable
amounts of di-, tri-, or tetraiodinated o-carboranes. This
monoiodination of o-carborane was operationally very con-
venient, and 7 was the only product obtained even when an
excess of ICl was used. It should be noted that, in the absence
of Lewis acid, I2 did not cause any iodination of o-carborane
(data not shown), whereas both ICl and I2 were far more
reactive in the presence of AlCl3, causing even tetraiodination
of 3, as discussed above.
(see the Experimental Section). The same was true for all other
compound mixtures described in Schemes 1−3 because of very
close Rf values (see the Experimental Section). Therefore, we
decided to carry out all followup reactions, optimization of the
halogen/isotope reactions, and all biological evaluations with
isomeric mixtures because this would not affect most of the
major objectives of this study. As already discussed, the
carborane cage of 3CTAs may be located outside the hTK1
substrate-binding site. Therefore, the regioselectivity of
monoiodination (B9−I vs B12−I) should not affect the
phosphorylation by hTK1 significantly (Figure 1). It was,
however, important to confirm the formation of structural
isomers with iodine and the TBDMS group in meta and para
orientations, respectively, and to ensure that nucleophilic
displacement/exchange of iodine did not occur after the
treatment of 9-iodo-o-carborane with n-BuLi (Figure 4).
Therefore, a small quantity of 8a/8b was separated by silica
gel chromatography for a thorough analysis by 1H, 13C, and 11
NMR, X-ray crystallography (Figure 4), and HRMS.
B
In the next reaction step, the carboranyl C−Hs of isomeric
8a/8b were deprotonated by n-BuLi followed by the reaction
with 5-(tert-butyldimethylsilyloxy)pentyl 4-methylbenzenesulfonate
to afford 9a/9b in 66% yield. Complete desilylation of 9a/9b
was accomplished by the reaction with TBAF/THF for 30 min
at 0 °C followed by the addition of 10% methanolic HCl56 and
continued stirring for 30 min at room temperature to obtain
10a/10b in 76% yield. The reaction with TBAF alone resulted
in complete deprotection of the C-TBDMS group of 9a/9b but
only in the partial removal of the O-TBDMS group. The
complete cleavage of the O-TBDMS group by TBAF using
longer reaction times, higher temperatures, and/or higher
equivalents (2.2−2.5) of TBAF was not explored because there
was a possibility of degradation of the closo-o-carborane cage to
nido-carborane.48,57 On the other hand, the reaction of 9a/9b
with 10% methanolic HCl56 alone for 30 min at room
temperature led to the complete removal of the O-TBDMS
group, whereas the C-TBDMS remained unaffected. The free
hydroxyl groups of 10a/10b were functionalized to the corre-
sponding tosylates (11a/11b) in 70% yield, which were then
subjected to treatment with dThd in the presence of K2CO3 in
Deprotonation of the carboranyl C−H of 7 by n-BuLi,
followed by the reaction with TBDMSCl, resulted in the
formation of a mixture of structural isomers 8a and 8b in 66%
overall yield with iodine at either B9 or B12 (Scheme 2 and
Figure 2). This protection of one of the carboranyl carbon
atoms of 7 with the TBDMS group was carried out to
guarantee the subsequent monofunctionalization of carboranyl
carbon.54,55 Unfortunately, the column chromatographic
separation of larger quantities of 8a and 8b from their mixture
was very tedious even though they have distinct Rf values
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DMF/acetone (1:1) to afford 4a/4b in 60% yield after purification
by column chromatography. All compounds described in Schemes
1
1 and 2 were analyzed by H, 13C, and 11B NMR and HRMS.
Halogen/Isotope-Exchange Reactions with 4a/4b. In order
to optimize the reaction conditions for the final isotope-
exchange reaction (Scheme 3, reaction b), compounds 4a/4b
a
Scheme 3
a
Reagents and conditions: (a) Na79/81Br, Herrmann's catalyst, DMF,
110 °C, 1 h; (b) Na125I, Herrmann's catalyst, DMF, 110 °C, 1 h.
Figure 3. HPLC analysis of N5-125I (13a/13b): (A) radioanalytical
trace; (B) UV chromatogram. Split peaks are likely due to the presence
of two structural isomers.
were initially subjected to a halogen-exchange reaction using
10 equiv of NaBr in the presence of Herrmann’s catalyst
(25 mol %) in DMF at 110 °C for 1 h (Scheme 3, reaction a).
This resulted in 100% halogen exchange to afford N5-79/81Br
(12a/12b) in 52% yield following column chromatographic
purification, as indicated by 1H and 11B NMR and mass
spectrometry. Commercial Na125I is available in the form of an
aqueous alkaline solution (pH 8−11). Strong bases such as the
methoxide ion, ammonia, alkylamines, and piperidine are
known to degrade closo-o-carboranes into the corresponding
nido-carboranes.48,57 In order to measure the possible extent of
degradation of 4a/4b during the isotope-exchange reaction
using an alkaline solution of Na125I, 4a and 4b were also treated
with 0.5 equiv of NaBr and 5 mol % NaOH in DMF at 110 °C
for 1 h. 1H and 11B NMR spectroscopy did not indicate the for-
mation of measurable quantities of the corresponding nido species
during this pilot reaction. Additional pilot reactions for optimization
of halogen/isotope exchange with different iodo-o-carboranyl com-
pounds are described in the Supporting Information.
The isotope-exchange reaction of 4a/4b was carried out with
Na125I in the presence of 10 mol % Herrmann’s catalyst in
DMF at 110 °C for 1 h followed by semipreparative HPLC
purification. The reaction was also monitored by radio-TLC
(Supporting Information). Both the analytical HPLC UV
chromatogram and the radioanalytical trace of the purified
product (13a/13b) displayed split peaks, which is consistent
with the presence of two structural isomers (Figure 3). The specific
activity, radiochemical yield, and radiochemical purity of 13a/13b
were 2.55 μCi/mg, 8%, and 98%, respectively. The specific activity
obtained may be sufficient for preliminary in vitro uptake studies of
13a/13b.58 The primary intention of this isotope-exchange
reaction was proof-of-concept, and no attempts were made to
improve the specific activity and radiochemical yield. This could be
accomplished by varying the reaction conditions and stoichiome-
tries of the reagents. Furthermore, the specific activity of the final
product could be improved significantly by employing a “reversed”
halogen exchange (B−79/81Br to B−125I) followed by the chro-
matographic separation of the halogenated species. Preliminary
HPLC studies indicated that it may be possible to separate N5-127
I
(4a/4b) from N5-79/81Br (12a/12b) (see the Supporting
Information).
X-ray Crystallography. X-ray crystallography confirmed
the formation of the structural isomers 8a and 8b with iodine and
the TBDMS group in meta and para orientations, respectively
(Figure 4). Compound 8a crystallized with one molecule in the
asymmetric unit (Z′ = 1), whereas compound 8b was obtained as
a collection of four slightly different rotamers (Z′ = 4 and labeled
as A−D; see Table 2 for crystallographic details and Figure S2 in
the Supporting Information for structures). The four rotamers of
8b have different torsion angles for C5−Si1−C1−C2, which are
−131.8(2)° (A), −140.6(2)° (B), 138.5(2)° (C), and 142.1(2)°
(D). Interestingly, the C5−Si1−C1−C2 torsion angle found for
8a was 6.3(2)°. Structure 8a does not show any significant I···I
halogen bond-type interactions, whereas structure 8b does form
two of these. Both structures show significant Ccarb−H···I−B
interactions (see Table 3). These types of intermolecular
interactions are similar to those observed by Puga et al. in the
crystal lattices of iodinated o-carboranes.53 Table 3 lists selected
bond lengths in 8a and in all four rotamers of 8b along with the
Ccarb−H···I−B and I···I interactions. The bond lengths found for
both crystal structures were in agreement with those reported by
Puga et al. for similar compounds.53
Biological Studies. PTAs. In order to evaluate their hTK1
substrate characteristics, PTAs28,44 with recombinant hTK1 were
carried with N5 (3), N5-127I (4a/4b), N5-79/81Br (12a/12b),
and N5-I4 (5). The results are shown in Figure 5 and Table 4. The
phosphorylation rate of dThd, being a natural substrate of hTK1, is
set to 100%, and the phosphorylation rates of the test compounds
are expressed relative to that of dThd. They indicate that
substitution of hydrogen in the carborane cluster of 3 by either
iodine (4a/4b) or bromine (12a/12b) only had minimal effects
on the phosphorylation rates (38.0% for 3 vs 36.9% for 12a/12b
vs 29.6% for 4a/4b). The observed phosphorylation rate for 3 was
comparable to that reported previously for the same compound.28
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Table 2. Crystallographic Data for 8a and 8b
8a
8b
C8H25B10ISi
formula
fw
C8H25B10ISi
384.37
384.37
space group
a, Å
Cc (No. 9)
6.8264(1)
20.6488(3)
12.5390(2)
90.346(1)
1767.43(5)
4
Pca21 (No. 29)
13.6530(1)
19.5285(1)
27.7225(2)
b, Å
c, Å
β, deg
volume, Å3
Z
density (calcd), g/cm3 1.444
μ, cm−1
cryst size, mm3
7391.45(8)
16
1.382
18.59
17.78
0.04 × 0.12 × 0.38
0.19 × 0.19 × 0.27
final R indices
R1 = 0.0238, wR2 =
0.0421
R1 = 0.0303, wR2 =
0.0460
a
[I > 2σ(I)]
a
R indices (all data)
R1 = 0.0316, wR2 =
0.0440
R1 = 0.0538, wR2 =
0.0489
a
2
2
R1 = ∑||Fo| − |Fc||/∑|Fo|. wR2 = [∑[w(Fo − Fc )2]/
2
∑[w(Fo )2]]1/2
.
Table 3. Selected Bond Lengths (Å) and Selected
Intermolecular Interactions for 8a and the Four Rotamers of
8b (A−D)
selected
a
a
b
bonds
8a
8b (A)
8b (B)
8b (C)
8b (D)
B−I
C1−Si
C1−C2
2.182(3)
1.941(3)
1.664(4)
2.175(4) 2.178(4)
1.937(3) 1.943(3)
1.667(4) 1.664(4)
2.177(4)
1.930(3)
1.664(4)
2.175(4)
1.940(3)
1.661(4)
selected intermolecular
interactions
a
b
8a
8b (A)
8b (B) 8b (C) 8b (D)
Ccarb−H·····I−B
I·····I
3.37 3.17−3.38
NA 3.70 and 4.10
c
The Mercury program, version 2.4,59 of the Cambridge Crystallographic
Data Centre was used to calculate these intermolecular distances. See
a
b
Figure 4 for the numbering of 8a and 8b (A). Numbers for 8b (B), 8b
c
(C), and 8b (D) are the same as that for 8b (A). NA: not applicable.
may be somewhat more susceptible to nido formation than non-
halogenated carboranes.48
Cellular Uptake Studies. Uptake of 3, 12a/12b, and 4a/4b
by L929 TK1(+) cells was quantified by determining the boron
concentrations by means of ICP-OES (Table 4). These were 2,
1.4, and 1.8 times greater than those of L929 TK1(−) cells,
indicating that TK1 activity may play a major role in the in vitro
uptake of these compounds. However, there was no obvious
correlation with the hTK1 phosphorylation rates of these
compounds. We do not have an explanation for the uptake data
of 4a/4b in both L929 TK1(+) and TK1(−) cells, which were
higher than those of 3 and 12a/12b in the same cell lines. Com-
pound 5 was not evaluated in the cellular uptake studies because of
lack of material. However, cellular uptake of boronophenylalanine
(BPA), a drug used clinically for BNCT,60 did not differ significantly
(P value = 0.563) between L929 TK1(+) and L929 TK1(−), as
determined by a two-sample t test. This was expected because BPA
is a boron-containing amino acid that is not a substrate of TK1.
Uptake of 3, 12a/12b, and 4a/4b in L929 TK1(−) cells was also
very high, indicating that other factors, such as compound
lipophilicity, causing nonspecific retention in cellular membranes,
or nucleoside/nucleotide influx and/or efflux, may have played
important roles in the uptake and retention of these compounds.
Figure 4. ORTEP plots of 8a and 8b drawn with 50% probability
displacement ellipsoids. The hydrogen atoms are omitted for clarity.
Only one of the four molecules in the asymmetric unit, molecule A, is
shown for structure 8b.
The moderate decrease in phosphorylation of 4a/4b and 12a/12b
compared with 3 substantiates our hypothesis that the carborane
cage of 3CTAs is located far away from the site of phosphorylation
and that the introduction of one iodine or bromine at the
carborane cage did not significantly affect hTK1 substrate
characteristics. On the other hand, the introduction of
four iodine atoms at the carborane cage, as in compound 5,
affected binding to hTK1, resulting in markedly decreased
phosphorylation, which could not be accurately determined
(Figure 5). The chromatogram shown in Figure 5 displays
additional spots for two unidentified 32P-containing
compounds (b) that were formed during phosphoryl
transfer or the following workup. It is conceivable that
these spots were the monophosphates of the nido-carboranyl
forms of 4a/4b and 12a/12b because halogenated carboranes
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Inorganic Chemistry
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result in improved specific activity and radiochemical yield of
13a/13b. To the best of our knowledge, this is the first report of
halogen- and isotope-exchange reactions at a B-halocarborane,
which is conjugated to a complex biomolecule such as dThd.
Noteworthy is the observed stability of the B−I bond under a
variety of reaction conditions. In general, the synthetic technology
developed at the examples of 12a/12b and 13a/13b may pave
the way for the synthesis of a wide range of carborane cage
radiohalogenated therapeutics and diagnostics.
Only small differences in hTK1 phosphorylation rates were
found between 3, 12a/12b, and 4a/4b. This supports our original
assumption that monohalogenation of 3 will not drastically change
its hTK1 substrate characteristics. In contrast, tetraiodination of 3,
as in compound 5, notably decreased the hTK1 phosphorylation
rate, indicating that there is a limit for size increase at the carborane
cage. These findings also reinforce our initial hypothesis that
mono(radio)iodination at the carborane cage of 3 is at least an
appropriate tool to study the real-time pharmacokinetics of this
3CTA. The phosphoryl transfer rates obtained for 3, 12a/12b, and
4a/4b were supported by a higher uptake in L929 TK1(+) than in
L929 TK1(−) cells. However, uptake of all three compounds in
L929 TK1(−) cells was also substantial, indicating that mechanisms
other than KMT, such as passive diffusion, may have contributed to
their accumulation.
Figure 5. Phosphorylation of dThd, N5 (3), N5-79/81Br (12a/12b),
N5-127I (4a/4b), and N5-127I4 (5) by recombinant TK1. Assay
products were separated by PEI−cellulose TLC. (a) Monophosphates
of N5 (3) N5-127I (4a/4b), and N5-79/81Br (12a/12b). (b)
Unidentified phosphorylation products with relative phosphorylation
rates of 14 0.7% (N5-127I lane) and 25 1.1% (N5-79/81Br lane).
Table 4. Human TK1 Phosphorylation Rates of N5 (3),
N5-127I (4a/4b), and N5-79/81Br (12a/12b), Relative to That
of dThd, and in Vitro Uptake Data of N5 (3), N5-127
I
a
(4a/4b), N5-79/81Br (12a/12b), and BPA
uptake (μg of B/109 cells)
ASSOCIATED CONTENT
* Supporting Information
■
phosphorylation
rate (%)
S
compounds
dThd
L929 TK1(+) L929 TK1(−)
Additional data, including additional general experimental
information, 13C NMR chemical shifts, supporting synthetic,
computational, chromatographic, and radioanalytical studies, as
well as detailed X-ray crystallographic and spectroscopic
information are available. This material is available free of
charge via the Internet at http://pubs.acs.org.
100
nd
nd
d
b
N5 (3)
38.0 2.2
36.9 0.4
29.6 0.5
77.6 6.2
76.0 9.0
38.6 3.3
54.0 5.3
65.5 5.3
42.1 13.6
N5-79/81Br (12a/12b)
N5-127I (4a/4b)
c
118.2 24.3
48.0 8.9
e
e
BPA
nd
a
Data represent the means of four replicates SD for phosphorylation
rates, three replicates SD for cellular uptake studies with 3, 12a/12b,
AUTHOR INFORMATION
Corresponding Author
*E-mail: tjarks.1@osu.edu.
■
5, and BPA, and six replicates SD for cellular uptake studies with
b
c
d
4a/4b. P < 0.05. P < 0.01. P < 0.001, compared with L929 TK1(−).
e
BPA: boronophenylalanine. nd: not determined. BPA is not a
nucleoside analogue. Hence, phosphorylation rate determination was
not applicable.
ACKNOWLEDGMENTS
■
The present studies were supported by funds from The Ohio
State University College of Pharmacy, the Dipartimento di
Chimica Generale e Chimica Organica Studi di Torino, Torino,
Italy, NIH Grant 1 R01 CA127935-01A2, and the National
Center for Research Resources (Award UL1RR025755). The
content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Center
for Research Resources or the National Institutes of Health.
The authors thank Drs. John Byrd and Fengting Yang from the
Division of Hematology and Oncology, Department of Internal
Medicine, The Ohio State University, for generously availing
their laboratory facilities and assistance for work related to
hTK1 expression, purification, and PTAs. The authors thank
George Hinkle, RPh, MS, BCNP, FASHP, FAPhA, Director of
Nuclear Pharmacy Services at The Ohio State University
Medical Center, as well as Drs. Staffan Eriksson and Elena
Sjuvarsson from the Biomedical Centre in Uppsala, Sweden, for
helpful discussions.
SUMMARY AND CONCLUSIONS
■
Both synthetic strategies explored for the synthesis of 4a/4b
were successful. However, even under optimized reaction
conditions, strategy 1 generated a complex reaction mixture con-
taining decomposition products, diiodinated material, and
substantial quantities of unreacted 3. This was a major dis-
advantage of strategy 1 because it necessitated the purification of
4a/4b not only by conventional column chromatography but
also by semipreparative HPLC. On the other hand, strategy 1
also generated the tetraiodinated compound 5. PTAs indicated
that this compound might not be a good substrate of hTK1,
presumably because of increased bulkiness at the carborane cage.
However, tetraradioiodination potentially may be an important
approach for other closo-carboranyl radiopharmaceuticals,
especially those that might be used for cancer treatment. From
this perspective, it is important to develop a general strategy for
the tetraiodination of closo-carboranyl biomolecules, as reported
in this study. In general, strategy 2 allowed for a more controlled
synthesis of 4a/4b. Potentially, separation of the isomers 4a and
4b is also possible using this strategy on the level of compounds
8a and 8b. Radiohalogenation of 4a/4b to obtain 13a/13b was
successful. Further optimization of the reaction conditions should
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