Inorganic Chemistry
Article
1
oil (3.30 g, 60%). H NMR (400 MHz, CDCl ): δ 3.93 (d, J = 2.3 Hz,
2
(Varian Inc./Agilent Technologies) and Bruker Avance 400 and 500
MHz spectrometers at room temperature. The solutions of closomer
contrast agent CA-9 were prepared in serum and in PBS buffer at
varied Gd(III) ion concentrations of 1, 0.5, and 0.25 mM. For
3
H), 3.44−3.39 (m, 4H), 3.38−3.32 (m, 4H), 3.27 (t, J = 7.1 Hz, 2H),
.17 (s, 8H), 2.54 (t, J = 7.8 Hz, 4H), 2.45 (t, J = 6.1 Hz, 2H), 2.40 (t,
J = 6.3 Hz, 4H), 2.26 (t, J = 2.3 Hz, 1H). C NMR (100 MHz,
3
13
CDCl ): δ 170.27 (4C), 80.28 (4C), 79.41, 74.46, 70.25, 70.09, 70.07,
comparison, T relaxation rates of 1 mM solution of the clinical MRI
1
3
6
9.44, 68.74, 57.98, 55.89 (2C), 53.72, 53.42, 51.87, 27.93 (12C).
CA Omniscan in PBS buffer and in serum as well as serum blank
samples and PBS buffer blank samples were measured in the same
+
HRMS (m/z) Calcd for C H N O [M + H] : 730.4854. Found:
37
67
3
11
7
30.5893.
Synthesis of 6-(2-(Bis(carboxymethyl)amino)ethyl)-3-(car-
boxymethyl)-9,12,15-trioxa-3,6-diazaoctadec-17-yn-1-oic acid
5). A solution of 4 (1.30 g, 1.80 mmol) in formic acid (50 mL, 90%)
experiment. A T -weighted MRI pulse sequence was applied with TE =
1
15 ms, and TR = 500 ms, slice thickness =1 mm, matrix = 256 × 256,
and FOV = 30 × 30 mm. A series of inversion recovery (IR) spin−
echo images were acquired using TR = 3 s, TE = 15 ms, and the
following inversion delays: 0.082, 0.1, 0.12, 0.16, 0.24, 0.32, 0.64, 1.28,
2.56, 3.6 s. The water signal intensities were measured using the
VnmrJ software (Varian Inc./Agilent Technologies). The relaxation
rates were calculated using a three-parameter experiential recovery
fitting in Origin8 (OriginLab Corporation).
(
was stirred for 15 h at 65 °C and then concentrated under reduced
pressure. The residue was dissolved in a minimal amount of MeOH,
and the crude product was precipitated by adding ether. Yield: 0.90 g
1
(
89%). H NMR (400 MHz, CD OD): δ 4.21 (d, 2H, J = 2.4 Hz),
3
3
.87 (m, 2H), 3.70−3.66 (m, 16H), 3.50 (m, 3H), 3.42 (m, 4H), 3.27
(
m, 4H), 2.91 (t, 1H, J = 2.4 Hz). HRMS (m/z) Calcd for
In Vivo MRI Studies. In vivo MRI studies of closomer CA-9 were
performed in SCID (severe combined immunodeficient) mice bearing
human PC-3 prostate cancer xenografts. ICR SCID outbred mice (4−
5 week old) were obtained from Taconic (Germantown, NY). Mice
were housed four animals per cage in sterile microisolator cages in a
temperature- and humidity-controlled room with a 12-h light/12-h
dark schedule. The animals were fed autoclaved rodent chow (Ralston
Purina Company, St. Louis, MO) and water ad libitum. All animal
studies were conducted in accordance with the highest standards of
care as outlined in the NIH guide for “Care and Use of Laboratory
Animals and the Policy and Procedures for Animal Research” at the
Harry S. Truman Memorial Veterans’ Hospital. Mice were inoculated
with human prostate cancer PC-3 tumor cells on the right flank;
average body weight was between 25 and 30 g at the time of MRI.
All MRI experiments were performed on a 7 T Varian Unity Inova
MRI equipped with a Millipede quadrature RF coil (40 mm i.d.). A T1
weighted multislice spin echo sequence was performed to record
sequential images pre- and post-injection of CAs. The animals were
injected with 120 μL (10 mM Gd) of CA (in 2% Tween-80 in PBS)
via the tail vein and imaged immediately. The mice were again imaged
at 2, 4, and 24 h post-injection time points. Vitals were continually
monitored, and mice were released to their cage after each imaging
session in order to fully recover. Mice were then euthanized for tissue
collection. Organs (including tail, tumor, blood, heart, lungs, liver,
spleen, stomach, large intestine, small intestine, kidney, brain, muscle)
were collected for ICP-OES analysis of gadolinium and boron
concentrations.
+
:
C H N O [M + H] 506.2350. Found: 506.2003.
21
35
3
11
Synthesis of 6-(2-(Bis(carboxymethyl)amino)ethyl)-3-(car-
boxymethyl)-9,12,15-trioxa-3,6-diazaoctadec-17-yn-1-oic
acid-dysprosium(III) Complex (6). A mixture of 5 (0.35 g, 0.69
mmol) and dysprosium chloride (0.25 g, 0.69 mmol) in pyridine (25
mL) was stirred for 15 h at 70 °C. The solvent was removed under
vacuum, and the residue was dissolved in a minimum amount of
ethanol and passed through a Celite pad to remove any solid present.
Ether was added to precipitate an off-white powder which was then
filtered, washed with ether, and dried to obtain the product as a white
solid. Yield: 0.40 g (87%). Mp: 215 °C (decomposed). HRMS (ESI)
−
m/z for C H DyN O [M] Calcd 665.1250. Found: 665.1483.
21
31
3
11
Synthesis of Closomer 8. A mixture of 12-fold azidoacetate
closomer 7 (0.10 g, 0.06 mmol) and DTTA ligand 4 (2.40 g, 3.30
mmol) was dissolved in acetonitrile−tetrahydrofuran (ACN−THF)
(
30 mL, 50:50 mixture). To this mixture, DIPEA (N,N-diisopropy-
lethylamine) (0.85 g, 6.60 mmol) and copper(I) iodide (0.13 g, 0.66
mmol) were added, and the resulting mixture was stirred for 3 days at
RT under an argon atmosphere. The reaction mixture was then
concentrated to dryness, and the residue was redissolved in DCM and
filtered. The filtrate was washed with an aqueous 2% ethyl-
enediaminetetraacetic acid disodium salt (EDTA·2Na) solution, and
the organic layer was separated, dried, and concentrated. The closomer
8
was purified via size-exclusion chromatography over Lipophilic
Sephadex (LH-20) using ACN as eluent. Yield: 0.42 g (76%), obtained
as viscous oil. H NMR (400 MHz, CD CN): δ 7.58 (s, 12H), 4.84 (s,
1
3
2
(
4H), 4.52 (s, 24H), 3.54−3.43 (m, 125H), 3.32 (m, 98H), 2.68−2.55
Image Analysis. Image analysis and processing were performed
with VnmrJ software (Varian/Agilent Technologies). A water tube
placed under the animal was treated as a reference signal. Regions of
interest (ROIs) were manually drawn on the tumor tissue, kidney
cortex, liver, and the muscle tissue near the tumor for each time point.
Signal intensity (SI) was measured as the mean of the intensity over
the segmented ROI. SIs were normalized according to the signal
intensity of the muscle assuming the enhancement of signal of the
muscle tissue is zero 2 h post-injection of CAs. The contrast
enhancement ratio (CER) was calculated according to:
m, 117H), 1.34 (m, 444H). 13C NMR (100 MHz, CD CN): δ 171−
3
6
5
4, 166.55, 145.32, 125.82, 81.44, 70.97, 70.23, 64.70, 56.88, 54.64,
4.21, 52.79, and 28.43. 11B NMR (128 MHz, CD CN): δ 18.07.
3
+
HRMS (ESI) m/z for C468H828B N O [M] Calcd: 10083.6210.
12
72 156
Found: 2522.2649 [M]4
+
Synthesis of CA-9. Closomer 8 (0.20 g, 0.02 mmol) was treated
with 80% TFA/DCM (10 mL) (TFA = trifluoroacetic acid) for 6 h at
RT under an argon atmosphere. The reaction mixture was
concentrated under reduced pressure, and the resulting residue was
dissolved in a minimum amount of methanol and precipitated by
adding ether. The solid was filtered, washed with ether, and dried
under high vacuum overnight. It was then dissolved in 1 M citrate
buffer (15 mL, pH-7) and added to to a solution of GdCl ·6H O (0.44
g, 1.19 mmol) in 15 mL of 1 M citrate buffer over 5 h at RT with
vigorous stirring. The pH of the reaction mixture was maintained at
approximately 7 using 0.3 N NaOH. The reaction mixture was stirred
for additional 24 h at RT and was sonicated a few times during the
course of the reaction. The reaction mixture was then dialyzed in
deionized water for 2 days using 1000 MWCO (MWCO = molecular
weight cutoff) membrane tubes (Spectra/Por). The product CA-9 was
obtained as an off-white solid after lyophilization. Yield: 0.15 g (82%).
Mp: 265 °C (decomposed). HRMS (ESI) m/z for
CER = (SIpost − SIpre)/SIpre × 100%
3
2
Long-Term Toxicity Studies. Six CF-1 female mice (Charles
River), 6 weeks old, were randomly assigned to two groups. Group
one, as a placebo control group consisting of three mice, was injected
with 120 μL saline (bacteriostatic 0.9% sodium chloride) via the tail
vein. Group two, also consisting of three mice, was injected with 120
μL of CA-9 at a gadolinium dose of 0.04 mmol/kg body weight in
saline solution via the tail vein. Mice were provided water and food ad
lib. The mice were observed for health status and behavior three times
a week. Body weight was measured once every week. At the end of 6
weeks of observation, all mice were euthanized and tissues were
immediately collected. Organs (including tail, blood, heart, lungs, liver,
spleen, stomach, large and small intestines, kidneys, brain, muscle)
were collected, and weighed for ICP-OES analysis of gadolinium
concentration.
3
‑
C276H408B Gd N O [M + Na + K] Calcd: 3146.5606. Found:
3
12
12 72 156
146.6066. ICP-OES analysis for Gd calcd 12, found 11.3.
Relaxivity Studies. Relaxivity measurements in serum and in PBS
buffer were performed using a 7 T Varian Unity Inova MRI system
1
699
dx.doi.org/10.1021/ic3017613 | Inorg. Chem. 2013, 52, 1694−1700