Synthesis and evaluation of diastereoisomers of 1,4,7-triazacyclononane-1, 4,7-tris-(glutaric acid) (NOTGA) for multimeric radiopharmaceuticals of gallium

Article abstract of DOI:10.1021/bc300340g

In the conventional synthesis of 1,4,7-tris-(glutaric acid)-1,4,7- triazacyclononane (NOTGA), four isomeric species are usually generated by the alkylation of 1,4,7-triazacyclononane with α-bromoglutaric acid diester. To estimate their biological efficaci

Full text of DOI:10.1021/bc300340g

Article
pubs.acs.org/bc
Synthesis and Evaluation of Diastereoisomers of
1,4,7-Triazacyclononane-1,4,7-tris-(glutaric acid) (NOTGA) for
Multimeric Radiopharmaceuticals of Gallium
Francisco L. Guerra Gomez,† Tomoya Uehara,† Takemi Rokugawa,† Yusuke Higaki,†,‡ Hiroyuki Suzuki,†
Hirofumi Hanaoka,† Hiromichi Akizawa,§ and Yasushi Arano*,†
†Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan, 260-8675
‡School of Health Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Ishikawa, Japan, 920-0942
§Health Science University of Hokkaido, 1757 Kanazawa, Tobetsu-cho, Ishikari-gun, Hokkaido, Japan 061-0293
S
* Supporting Information
ABSTRACT: In the conventional synthesis of 1,4,7-tris-(glutaric acid)-1,4,7-
triazacyclononane (NOTGA), four isomeric species are usually generated by
the alkylation of 1,4,7-triazacyclononane with α-bromoglutaric acid diester. To
estimate their biological efficacies as well as their stability and radiochemistry,
the RRR/SSS and RRS/SSR NOTGA-tBu prochelators were isolated and the
corresponding cyclic RGDfK (RGD) conjugates with triethylene glycol linkages
were prepared. The RRR/SSS and RRS/SSR diastereomers were obtained in
69% and 17% yields, respectively. In the complexation reaction with 67GaCl3,
both diastereomers provided >98% radiochemical yields at pH 5 within 10 min
when the reaction was conducted at room temperature. However, the RRR/
SSS diastereomer exhibited more pH-sensitive radiochemical yields between
pH 3.5 to 4.5. Despite their diasteromeric nature, both 67Ga-labeled RGD-
NOTGA remained stable during the apo-transferrin challenge, exhibiting similar affinity for integrin αvβ3 and biodistribution with
predominant renal excretion. Similar tumor uptake was also observed in mice bearing U87MG tumor xenograft, which resulted in
impressively high contrast SPECT/CT images. These findings indicate that the RGD-NOTGA conjugates of both diastereomers
presented here possess equivalent biological efficacies and their combined usage would be feasible. It is worth noting that specific
properties of a given biomolecule, cell expression levels of the corresponding target molecule, and presence or absence of a
pharmacokinetic modifier would affect the structural differences between diastereomers on the ligand−receptor interactions and
pharmacokinetics. Thus, the preparation of corresponding conjugates and evaluation of their chemical and biological per-
formances still remains important for applying NOTGA to other biomolecules of interest using the diastereomerically pure
NOTGA-tBu prochelator.
NOTA-based bifunctional chelating agents (BCA) with dis-
similar functional groups in a pendant arm or on ethylene
bridge have been developed.6,8−10 Triazacyclononane (TACN)
has been selected as the core of the scaffold containing phosphinic
acid (triazacyclononane phosphinic acids; TRAP)11,12 or glutaric
acid (nonane triglutaric acid; NOTGA)13,14 for multiattachment of
biomolecules to the three pendant arms as a way to apply to the
multimeric concept (Figure 1).
INTRODUCTION
■
Gallium radioisotopes are of great interest for molecular imaging.
Gallium-68 (68Ga) is a PET radioisotope available from long-
lived 68Ge/68Ga generator systems allowing potentially cost-
effective production of 68Ga radiotracers far away from a cyclo-
tron facility. Its physical half-life of 67.7 min is attractive for
labeling low-molecular-weight probes with rapid pharmaco-
kinetics.1 Meanwhile, 67Ga is a γ-emitter (T1/2 = 3.3 d) currently
used in the diagnosis of infection and inflammatory processes as
well as tumor imaging by SPECT.2,3
Recently, Singh et al.14 exemplarily demonstrated that the
binding affinity and tumor accumulation of a trivalent cyclic
RGD peptide conjugated-NOTGA (68Ga-3) increased with
respect to its bivalent and monovalent counterparts. Similarly,
Notni et al. reported that the targeting ability of the trivalent
68Ga-TRAP(RGD)3 was superior to that of the monovalent
18F-Galacto-RGD.12 However, one aspect to consider when
For preparing 67/68Ga-based radiotracers, a macrocyclic chelator
1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA) is pref-
erably used due to the formation of a hexadentate gallium complex
of high thermodynamic (Log K = 30.98)4 and kinetic stabilities
arising from the good fit of the relatively small gallium cation in
the cyclic cavity.1 Moreover, this chelator has been efficiently
radiolabeled with 68Ga, even at room temperature.5−7 Since the
conjugation of targeting molecules to the carboxylic acids of
NOTA compromises its coordination ability with Ga, several
Received: June 26, 2012
Revised: October 7, 2012
Published: October 8, 2012
© 2012 American Chemical Society
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compound can be obtained without postlabeling purification by
selecting a diastereomer of preferable chemical and biological
performance. In the present study, pure diastereomers of NOTGA
using RGDfK as the targeting molecule, were synthesized, and
compared their performance in terms of radiochemical yields with
67GaCl3, kinetic stability, affinity for integrin αvβ3, and biological
behavior of each complex in normal and nude mice bearing
U87MG tumor xenografts.
EXPERIMENTAL SECTION
■
Figure 1. Structures of (A) NOTA and the NOTA-based BCAs,
(B) TRAP, and (C) NOTGA. TRAP contains phosphinic acids, whereas
NOTGA contains glutaric acids.
General. All commercially obtained chemicals were of ana-
lytical grade and used without further purification. 9-Fluoro-
phenylmethoxycarbonyl (Fmoc)-protected amino acids and
H-Gly-2-Cl-Trt Resin were purchased from Watanabe Chem-
ical Industries, Ltd. (Hiroshima, Japan). 67GaCl3 was supplied
by FUJIFILM RI Pharma Co., Ltd. (Tokyo, Japan). 1,4,7-
Triazacyclononane (TACN) was purchased from Sigma Aldrich
Chem. Co. (Milwaukee, WI, USA). Apo-transferrin (apo-Tf) Iron
free was purchased from Nacalai Tesque Inc. (Kyoto, Japan).
Reversed-phase HPLC was performed with a Cosmosil 5C18-
AR-300 column (4.6 mm i.d. × 150 mm, Nacalai Tesque Inc.) at
1 mL/min with a gradient mobile phase starting from 90% A
(0.1% aqueous trifluoroacetic acid (TFA) and 10% B (acetonitrile
with 0.1% TFA) to 70% A and 30% B at 30 min. The eluent was
monitored online with an UV−vis single-beam spectroscopy
detector (L-7405, Hitachi Co. Ltd., Tokyo) coupled to a NaI(Tl)
radioactivity detector (Gibi star, Raytest, Strubenhardt, Germany).
preparing NOTGA- and TRAP-based radiopharmaceuticals is
the presence of chiral centers in their pendant arms leading to
RRR, SSS, RRS, and SSR stereoisomers. In NOTGA, these
stereoisomers are formed as a result of the alkylation of TACN
with racemic (R/S) α-bromoglutaric acid diester (Figure 2),
TLC analyses was performed with silica plates (Silica gel 60 F254
,
Merck Ltd., Tokyo) developed with MeOH/0.1 M AcONH4
(1:1). Radioactivity was measured using a MiniGita Star Gamma
TLC Scanner (Raytest) and an autowell γ counter (ARC-380M,
Aloka, Tokyo). Mass spectrometry was carried out using an
Agilent 6130 Series Quadrupole LC/MS electrospray system
(Agilent Technologies, Tokyo) or MALDI TOF MS Kratos
Axima CFR Plus (Shimadzu Corporation, Kyoto). 1H, 13C NMR
spectra were recorded on a JEOL JNM-ECP-400 (400 MHz)
spectrometer (JEOL Ltd., Tokyo).
(2-{2-[2-(9H-Fluoren-9-ylmethoxycarbonylamino)-
ethoxy]-ethoxy}-ethoxy)-acetic acid (1) (Fmoc-TEG). 11-
Amino-3,6,9-trioxaundecanoic acid (0.79 g, 3.79 mmol) and
K2CO3 (1.04 g, 7.50 mmol) were dissolved in 5.8 mL of water
and stirred at room temperature for 15 min after which N-(9-
fluorenylmethoxycarbonyl) succinimide (1.28 g, 3.79 mmol)
was added and the mixture was stirred for 24 h, while the progress
of the reaction was monitored by TLC (CHCl3/MeOH/AcOH,
5:1:0.06). The salt was filtered and the filtrate was washed with
3 × 3 mL of Et2O, acidified to pH 1 using 3 N HCl, and the
desired compound extracted with 5 × 5 mL of CH2Cl2. After
removing the solvent in vacuo, the residue was purified with open
column chromatography using silica gel and subsequent elution
with CHCl3/MeOH/AcOH (40:1:0.1) to afford Fmoc-TEG
(1.19 g, 73%). ESI-MS, m/z: 430 [M+H]+: Found 430.
Synthesis of Fmoc-TEG-c(-Arg(Pbf)-Gly-Asp(OtBu)-
DPhe-Lys-) (2). Fmoc-TEG (0.16 g, 0.36 mmol), c(-Arg(Pbf)-
Gly-Asp(OtBu)-DPhe-Lys-) (0.3 g, 0.33 mmol)20 and 1-hydro-
xybenzotriazole monohydrate (HOBt, 0.049 g, 0.36 mmol)
were dissolved in 13 mL of dimethylformamide (DMF) and cooled
to −3 °C. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide·hydrochloride (EDC, 0.21 g, 1.08 mmol) in 12 mL
of DMF was added dropwise during 1 h under N2 pressure. The
reaction mixture was further stirred for 1 h and reacted overnight at
room temperature. The solvent was removed in vacuo, and then,
the residue dissolved in 100 mL of CH2Cl2, washed with 5% citric
Figure 2. Structures of the isomeric forms of NOTGA. The RRR and
SSS, as well as RRS and SSR, constitute enantiomers; while the pairs
RRR/SSS and RRS/SSR are diastereomers.
which was also observed in the alkylation of tetraazacyclodo-
decane,15,16 where the RRRR isomer of gadolinium complex
exhibited faster water exchange rate than the other isomers.16
In TRAP ligands, the phosphorpous atoms become chiral upon
coordination with the metal ion and four RRR, SSS, RRS, and
SSR isomers are formed depending on the substituent.11,17
A
mixture of diastereomers, with differences in spatial orientation
of the chelating unit or targeting molecules and physicochemical
properties, might influence the biodistribution of the molecular
probe.18,19 In cases where radiopharmaceuticals can form distinct
isomeric species, it is important to evaluate the individual product
separately to ensure that they both possess good biological
efficacy,18 as well as stability and radiochemistry.
In TRAP-based radiolabeled probes, separation of a potential
diastereomer can only be conducted after radiolabeling reaction.
On the other hand, the isolation of diastereomers in NOTGA is
feasible after alkylation of TACN, and diastereomerically pure
conjugates can be obtained. In NOTGA-based radiolabeled
probes, therefore, a diastereomerically pure 67/68Ga-labeled
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acid (2 × 40 mL). The organic layer collected and dried over
MgSO4. After removing the solvent in vacuo, the residue was
purified with open column chromatography using silica gel and
subsequent elution with CHCl3/MeOH (20:1) to obtained
compound 2 as a white solid (0.22 g, 51%). ESI-MS, m/z: 1345
[M+Na]+: Found 1345.
N−CH); 2.87−3.22 (br, 12H, N−CH2−CH2−N); 2.44−2.81
(br, 6H, CH2−COOH); 2.1.9−2.15 (br, 6H, N−CH−CH2);
1.51 (s, 27H, C(CH3)3). ESI-MS, m/z: 688 [M+H]+, Found
688.
NOTGA-(TEG-RGD)3 (7). NOTGA-tBu3 (RRR/SSS, 8.52 mg,
12.4 μmol; RRS/SSR, 10 mg, 14.5 μmol) was dissolved in dry
CH2Cl2 (0.5 mL) and 0.3 equiv of 4-dimethylaminopyridine
(DMAP), 3.5 equiv of TEG-c(-Arg(Pbf)-Gly-Asp(OtBu)-DPhe-
Lys-), 3.6 equiv of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimi-
de·hydrochloride (EDC) dissolved in 0.5 mL of CH2Cl2 were
added dropwise on an ice bath. After stirring at room temperature
for 12 h under N2, the solvent was evaporated in vacuo. The
residue was dissolved in CH2Cl2 (10 mL), washed with 5%
NaHCO3 (3 × 7 mL), and dried over MgSO4. After removing
the solvent, the residue was taken up by a small amount of
CHCl3. The precipitate formed upon addition of excess of Et2O
was filtered and dried in vacuum overnight to obtain a yellow
solid (RRR/SSS, 33.3 mg, 68.2%; RRS/SSR, 38.7 mg, 67.6%).
ESI-MS, m/z: 1313 [M+H]2+, Found 1313. After treatment
with TFA/triethylsilane/H2O (90:5:5) at room temperature for
3 h, the solvent was evaporated in vacuo and the precipitate
formed upon addition of excess of Et2O (RRR/SSS, 17.8 mg,
64.6%; RRS/SSR, 16 mg, 57.1%). ESI-MS, m/z: 1422 [M+2H]2+,
Found 1422.
Synthesis of TEG-c(-Arg(Pbf)-Gly-Asp(OtBu)-DPhe-
Lys-) (3). Fmoc-TEG-c(-Arg(Pbf)-Gly-Asp(OtBu)-DPhe-Lys-)
(0.47 g, 0.35 mmol) was dissolved in 23 mL of 10% piperidine
DMF solution and stirred for 2 h. After concentrating the
solvent, Et2O was added to the solution. The white precipitate
was filtrated, washed alternatively with Et2O and n-hexane, dried
in vacuum overnight to obtain compound 3 as a yellow solid
(0.30 g, 76%). ESI-MS, m/z: 1101 [M+H]+: Found 1101.
1,4,7-Tris-(1-tert-butoxycarbonyl-3-benzyloxycarb-
ony-propyl)-1,4,7-triazacyclononane (4) (NOTGA-tBu3-
Bz3). (R/S) α-Bromoglutaric acid 1-tert-butyl ester 5-benzyl
ester (1.3 g, 3.64 mmol)21 was dissolved in 3.5 mL of aceto-
nitrile and added dropwise to a solution of 1,4,7-triazacyclo-
nonane (0.142 g, 1.10 mmol) in 3.3 mL acetonitrile with
K2CO3 (0.913 g, 6.62 mmol) during 2 h at 0 °C under N2. The
mixture was stirred at room temperature for 24 h. The reaction
mixture was filtrated and the solvent was evaporated in vacuo.
The residue was dissolved in dichloromethane (15 mL) and
washed with 5% of NaHCO3 (3 × 10 mL), and the organic
layer was dried over MgSO4. After removing the solvent in
vacuo, the residue was purified with open column chromatog-
raphy using silica gel and subsequent elution with chloroform/
acetone (50:1) to afford compound 4 as a yellowish oil. RRR/
Ga-NOTGA-(TEG-RGD)3. NOTGA-(TEG-RGD)3 (RRR/
SSS, 0.8 mg, 0.28 μmol; RRS/SSR, 3 mg, 1.05 μmol) was dis-
solved in 500 μL of water, and 0.1 M Ga(NO3)3 solution (4.4
and 14.3 μL, respectively) was added. After 10 min heating at
70 °C, the pH was adjusted from 1.9 to 3.5 using 1 M sodium
acetate and heated for another 30 min. The complex was purified
by preparative HPLC and lyophilized to obtain Ga-NOTGA-
(TEG-RGD)3 (RRR/SSS, 0.7 mg, 85%; RRS/SSR, 2.67 mg,
87%). ESI-MS, m/z: 1455 [M+2H]2+, Found 1455.
1
SSS (0.637 g, 69%): H NMR (400 MHz, CDCl3), δ (ppm):
7.28−7.34 (m, 15H, Ph); 5.10 (s, 6 H, CH2−Ph); 3.09−3.13 (t,
3H, N−CH); 2.68, 2.95 (d, J = 0.031, 12H, N-CH2-CH2-N);
2.53−2.42 (m, 6H, CH2−COOBz); 1.82−2.03 (m-m, 6H,
N-CH-CH2); 1.43 (s, 27H, C(CH3)3). 13C NMR (100 MHz,
CDCl3), δ (ppm): 173.37 (COOBzl); 172.59 (COO-tBu);
128.44, 128.48, 128.81, 136.34 (Ph); 81.11 (C-Me3); 67.58
(C-Ph); 66.46 (N-C); 54.55 (N-C-C-N); 31.59 (C-COO-Bzl);
28.58 (tBu); 25.58 (C-COO-Bzl). RRS/SSR (0.165 g, 17%): 1H
NMR (400 MHz, CDCl3), δ (ppm): 7.26−7.34 (m, 15H, Ph);
5.09, 5.10 (d, 6 H, CH2−Ph); 3.09−3.13 (m, 3H, N−CH);
2.58, 2.93 (d, J = 0.031, 4H, N-CH2-CH2-N), 2.70−2.84 (m,
8H, N-CH2-CH2-N); 2.44−2.51 (m, 6H, CH2-COOBz); 1.82−
2.02 (m-m, 6H, N-CH-CH2); 1.43, 1.44 (d, 27H, C(CH3)3).
13C NMR (100 MHz, CDCl3), δ (ppm): 173.02, 173.13 (d,
COOBzl); 172.30, 172.42 (d, COO-tBu); 128.10, 128.14, 128.46,
135.99 (Ph); 80.77, 80.79 (C-Me3); 66.93 (C-Ph); 66.10, 66.27
(N−C); 54.29, 53.85, 53.54 (N−C−C−N); 31.02, 31.18 (C−
COO−Bzl); 28.24 (tBu); 25.17, 25.33 (C−COO−Bzl). ESI-MS,
m/z: 958 [M+H]+, Found 958.
67Ga-Radiolabeling. The complexation of 67Ga by both
diastereomeric pairs of ligands was studied regarding the reac-
tion pH and time at r.t. 67GaCl3 (10 μL, 1.5 MBq) in 0.05 M
HCl was mixed with 10 μL of 0.5 M sodium acetate buffer pH
4−5.5. The mixtures were added to Eppendorf tubes containing
0.4 nmol of ligand in 20 μL of 0.25 M acetate buffer pH 3−5.5
to give final solutions of 0.25 M A.B, pH 3.5−5.5, and 10 μM of
ligand concentration. The mixtures were incubated at 25 °C for
5, 10, and 15 min, and radiochemical yields were determined by
Radio-TLC.
In Vitro Stability. 67Ga-labeled complexes (RRR/SSS, RRS/
SSR) were purified by RP-HPLC to remove unlabeled ligands.
The radioactive peak was collected and the solvent was removed
in vacuo. The residue was reconstituted in apo-transferrin (apo-Tf)
solution (50 μM, 0.1 M carbonate buffer, pH 7.4) and incubated at
37 °C. Samples were withdrawn at 1, 3, and 6 h and analyzed by
Radio-TLC (n = 3).
Binding Affinity to Integrin αvβ3. To evaluate the binding
affinity of Ga-labeled compounds to integrin αvβ3, surface
plasmon resonance (SPR) technology-based ProteOn XPR36
protein interaction array system (Biorad Laboratories Japan,
Yokohama, Japan) was used. The SPR experiment was per-
formed according to the manufacturer’s instruction. In brief,
human purified integrin αvβ3 (20 μg/mL, Chemicon Interna-
tional, Temecula, CA, USA) dissolved in 10 mM acetate buffer
(pH 4.0) was immobilized on a ProteOn GLH sensor chip
(Biorad Laboratories Japan) by standard amine coupling
method. The Ga-NOTGA-(TEG-RGD)3 complexes (RRR/
SSS, RRS/SSR) at 10, 5, 2.5, and 1.25 μM concentrations and
c(RGDfk) at 200, 100, 50, 25, and 12.5 μM as a positive control
1,4,7-(α-Bromoglutaric Acid 1-tert-Butyl Ester 5-
Benzyl Ester)-1,4,7-triazacyclononane (5, 6) (NOT-
GA-tBu3). The RRR/SSS (0.57 g, 0.59 mmol) and RRS/SSR
(0.15 g, 0.16 mmol) fractions were dissolved in methanol/water
(5:1) and then 10% Pd/C (381 and 103 mg, respectively) was
added portionwise. The mixtures were stirred for 12 h under H2
atmosphere, then filtered over Celite, and evaporated to dry-
ness to obtain compounds 5 and 6 as white solids. The products
were used without further purification. RRR/SSS (408 mg,
1
96.4%). H NMR (400 MHz, CD3OD), δ(ppm): 3.69−3.70
(br, 3H, N−CH); 2.99−3.13 (br, 12H, N−CH2−CH2−N);
2.52−2.64 (br, 6H, CH2−COOH); 2.06−2.19 (br, 6H, N−
CH−CH2); 1.51 (s, 27H, C(CH3)3). RRS/SSR (104 mg, 95%).
1H NMR (400 MHz, CD3OD), δ(ppm): 3.62−3.72 (br, 3H,
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in 10 mM Tris-HCl buffer (50 mM NaCl, 1 mM MgCl2, 1 mM
MnCl2, pH 7.4) were injected simultaneously into the six
horizontal channels of the chip. Kinetic analysis was performed
by globally fitting curves describing a simple 1:1 biomolecular
model to the set of five sensorgrams.
Cell Line. Human glioblastoma U87MG cells were grown in
a 75 cm2 tissue culture flask with canted neck (Becton, Dickinson
and Company, Tokyo) in Dulbecco’s Modified Eagle Medium
(Sigma-Aldrich Japan K.K., Tokyo) supplemented with 10% fetal
calf serum (FCS, Nippon Biosupply Center, Tokyo) and GIBCO
BRL 1% penicillin−streptomycin (5000 unit-5000 μg/mL, In-
vitrogen, Life Technologies Japan Ltd., Tokyo), at 37 °C in a
humidified atmosphere containing 5% CO2.
Biodistribution Studies. Animal studies were conducted
in accordance with institutional guidelines approved by the
Chiba University Animal Care Committee. Male 6-week-old
ddY mice22 (Japan SLC, Inc., Shizuoka, Japan) were injected
via tail vein with either RRR/SSS or RRS/SSR diastereomers
of 67Ga-NOTGA-(TEG-RGD)3 (100 μL, 11.1 KBq, 10 μM
ligand concentration). The animal were sacrificed and dissected at
30 min, 1 h, 3 h, and 6 h after administration. Tissues of interest
were removed, weighed, and the radioactivity counts determined
with an autowell gamma counter. The urine and feces were
collected for 6 h and the radioactivity was also measured. Values
were expressed as mean SD for a group of 3−5 animals.
BALBc nu/nu male mice (Japan SLC, Inc., Shizuoka, Japan)
of 6 weeks old, weighing 18−20 g, were xenografted by sub-
cutaneous (s.c.) injection of U87MG human glioblastoma cells
(5 × 106 cells/80 μL of culture medium) into their right hind
legs. The mice were subjected to biodistribution studies as well
as SPECT/CT imaging studies when the tumor volume
reached 100−300 mm3.
Biodistribution studies were also conducted in male BALBc
nu/nu mice bearing U87MG xenografts 30 min after admin-
istration of each radiotracer (n = 4). The integrin αvβ3
specificity was estimated by coinjection of either RRR/SSS or
RRS/SSR diastereomers of 67Ga-NOTGA-(TEG-RGD)3 (100 μL,
11.1 KBq, 10 μM ligand concentration) and c(RGDyV) peptide
(3 mg/kg mouse body weight) into mice bearing U87MG tumors.
The animals were sacrificed and dissected at 2 h after admin-
istration (n = 4).
potassium carbonate at room temperature to obtain the fully
protected NOTGA-tBu3-Bz3 4 (Scheme 1). Purification was
carried out using silica-gel column chromatography and a
mixture of chloroform and acetone as the mobile phase. Two
fractions (a major fraction of 69% and the following of 17%)
were verified to be the target compound by mass spectrometry.
1
Structure assignment was possible by H NMR and 13C NMR,
which provides deeper insights into their structural differences
(Supporting Information). Focusing on the chiral carbon
resonance (label b in Figure 1S), a singlet was observed in
the 69% fraction Figure 1S A) indicating equivalent carbons,
and a doublet in the 17% fraction indicating the presence of
two chemically nonequivalent carbons (Figure 1SB). The 69%
fraction was assigned to the RRR/SSS diastereomeric pair and
the 17% to the RRS/SSR.
In order to evaluate the differences of both diastereomeric
pairs, each fraction was treated separately. After debenzylation
by palladium-catalyzed hydrogenolysis, the diastereomeric ortho-
gonally protected NOTGA-tBu3 prochelator were obtained in
quantitative yields.
The peptide substituent was prepared using an Fmoc chem-
istry to introduce the TEG spacer in the lysine side chain of
c(RGDfK) in 50% yield. The prochelators of NOTGA-tBu3 were
then conjugated to the partially protected TEG-c(-Arg(Pbf)-Gly-
Asp(OtBu)-DPhe-Lys-) by in situ activation using the standard
EDC-based coupling method followed by complete removal of all
protecting groups to obtain the desired ligands at moderate yields
after HPLC purification (>95% purity).
Radiochemistry. The RRR/SSS and RRS/SSR pairs of
NOTGA-(TEG-RGD)3 were radiolabeled with 67Ga and the
resulting complexes were analyzed by HPLC (Figure 3). In
both cases, a single peak was observed and the retention time of
the RRR/SSS was 24.5 min, slightly longer than that of RRS/
SSR (24.3 min). In both cases, their retention times were
identical to those of the corresponding nonradioactive gallium
complexes verified by mass spectrometry.
Formation kinetics as a function of pH is presented in Figure 4.
In both cases, radiochemical yields were low under acidic con-
ditions (pH 3.5) and quantitative at more basic pH 5 where the
time variation of the radiochemical yield was essentially the
same for both pairs (Figure 4B) and complete radiolabeling
with more than 98% yield was attained after 10 min of reaction.
Interestingly, while RRR/SSS was preferentially labeled at pH 5,
the RRS/SSR was pH-independent from pH 4 to 5.5 (Figure 4A).
Under the present condition, the specific activity of the
67Ga-labeled compounds was 4500 Mq/μmol.
Small Animal SPECT/CT Imaging Studies. SPECT/CT
images were taken 30 min after administration of either RRR/
SSS or RRS/SSR diastereomers of [67Ga]-NOTGA-(TEG-
RGD)3 (100 μL, 4.5 MBq, 10 μM ligand concentration) to
male BALBc nu/nu mice bearing U87MG xenografts from the
tail vein (n = 3). The mice were anaesthetized with 1−2% (v/v)
isoflurane (DS Pharma Animal Health, Osaka, Japan) and
positioned on the animal bed where anesthesia was continuously
delivered via a nose cone system. SPECT imaging and X-ray CT
imaging were performed by use of small animal SPECT/CT
system (FX-3200, Gamma Medica Inc., CA) equipped with a five
pinhole (0.5 mm) collimator. Data acquisition was performed for
64 min at 60 s per projection with stepwise rotation of 64
projections over 360°.
In Vitro Stability. The kinetic stability of [67Ga]-NOTGA-
(TEG-RGD)3 diastereomeric pairs purified by HPLC in order
to remove the excess ligand was estimated in an apo-transferrin
challenge (Table 1). After 6 h of incubation at 37 °C, more
than 98% of the radioactivity was still bound to the NOTGA
tripeptide conjugates of both pairs.
Binding Affinity. The binding kinetics of the two
diasteromeric gallium complexes of NOTGA-(TEG-RGD)3 to
αvβ3 integrin was estimated with the SPR technology using a
monovalent c(RGDfK) as a reference (Table 2). Both di-
astereomeric trivalent complexes exhibited higher association
and lower dissociation rates than that of a monovalent c(RGDfK)
with the rate constants similar each other. As a result, both tri-
valent complexes showed 10-fold higher dissociation constant
values (KD) than that of monovalent c(RGDfK).
Statistical Analysis. Quantitative data were expressed as
means SD. Means were compared using unpaired two-tailed
Student’s t test. P values <0.05 were considered statistically
significant.
RESULTS
■
Synthesis. TACN was alkylated with (R/S) α-bromogluta-
ric acid 1-tert-butyl ester 5-benzyl ester10,21,23 in acetonitrile and
In Vivo Experiments. The results of the biodistribution
studies of [67Ga]-NOTGA-(TEG-RGD)3 in normal mice at
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a
Scheme 1. Synthesis of NOTGA-(TEG-RGD)3
a
(a) Fmoc-O-Su, K2CO3, H2O; (b) c(-Arg(Pbf)-Gly-Asp(OtBu)-DPhe-Lys-), WSCI/HCl, HOBt, DMF; (c) 10% piperidine/DMF; (d) 1,4,7-triaza-
cyclononane, K2CO3, MeCN; (e) 10%Pd/C MeOH,H2O; (f) H2N-TEG-c(R(Pbf)GD(OtBu)fK), WSCI·HCl, DIMAP, CH2Cl2; (g) TFA/Water/Et3Si.
Figure 3. HPLC radiochromatograms of 67Ga-NOTGA-(TEG-RGD)3.
The RRR/SSS diastereomer (top) was eluted at 24.5 min, slightly longer
than that of the RRS/SSR counterpart (bottom, 24.3 min).
30 min, 1 h, 3 h, and 6 h after administration are shown in
Table 3 (RRR/SSS) and Table 4 (RRS/SSR). No significant
differences were observed in the uptake of both diastereomers
in almost all organs and tissues. These profiles were charac-
terized by rapid blood clearance with low accumulation in the
liver, and the majority of the radioactivity was localized in the
kidneys. Both radioligands were excreted in the urine with a
small amount in feces. Although in absolute terms the uptake in
Figure 4. Radiochemical yields of each diastereomer of 67Ga-NOTGA-
(TEG-RGD)3 (RRR/SSS, squares; and RRS/SSR, triangles) (A) at
different pH values for 10 min and (B) different reaction times at pH 5.0.
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Table 1. Stability of 67Ga-NOTGA-(TEG-RGD)3
Diastereomers against Apo-Transferrin
profiles of both diastereomeric pairs were characterized by rapid
blood clearance and high tumor uptake with comparatively low
accumulation in nontarget organs and renal excretion pathway. In
agreement with the studies in normal mice, the uptakes of the
diastereomeric pairs in organs of interest were comparable. Tumor
uptakes were 4.40 0.38 and 4.77 0.76%ID/g (p < 0.05) for
RRR/SSS and RRS/SSR, respectively. Tumor to organ ratios
(Figure 5B) were high for blood and muscle, moderate for the
liver, and much lower for the kidney. A combination of slightly
higher tumor and lower blood uptakes led to a statistically
significantly higher tumor to blood ratio for the RRS/SSR pair.
When the radiolabeled probes were coinjected with a high
amount of RGDyV, tumor accumulation was significantly dec-
reased (p < 0.05) to 0.82 0.17 and 0.68 0.2%ID/g at 2 h
p.i. (RRR/SSS and RRS/SSR, respectively), demonstrating in-
tegrin αvβ3 targeting specificity (Figure 6).
a
percent of intact radiolabeled complex
time (h)
RRR/SSS
RRS/SSR
1
3
6
98.6 0.2
98.9 0.3
98.0 0.4
98.9 0.4
98.3 0.9
98.4 0.2
a
Results are expressed as mean SD of three experiments.
the intestines was very low, a slight tendency of the RRR/SSS to
be excreted through the intestinal tract can be noted. Its
intestine accumulation was significantly higher at 1, 3, and 6 h
p.i. Likewise, relatively higher radioactivity in feces and lower
radioactivity in urine for the RRR/SSS were observed.
Figure 5 shows the biodistribution studies of [67Ga]-NOTGA-
(TEG-RGD)3 (RRR/SSS and RRS/SSS) at 30 min postinjection
to nude mice bearing U87MG xenografts. The biodistribution
SPECT/CT imaging studies were performed for both RRR/
SSS and RRS/SSS diastereomers of [67Ga]-NOTGA-(TEG-RGD)3
Table 2. Kinetic Binding Constants of 67Ga-NOTGA-(TEG-RGD)3 (RRR/SSS and RRS/SSR), c(RGDfK) to Integrin αvβ3
Determined Using the SPR Technology
analyte
Ka (1/Ms)
Kd (1/s)
KD (M)
RRR/SSS
RRS/SSR
c(RGDfK)
1.13 × 105 6.8 × 103
0.94 × 105 5.3 × 103
2.57 × 104 6.1 × 103
8.03 × 10−3 1.7 × 10−4
8.12 × 10−3 1.6 × 10−4
1.38 × 10−2 6.2 × 10−4
7.13 × 10−8 4.5 × 10−9
8.64 × 10−8 4.9 × 10−9
5.37 × 10−7 2.7 × 10−7
a
Table 3. Biodistribution of RRR/SSS Diastereomer of 67Ga-NOTGA-(TEG-RGD)3 in Normal Mice
RRR/SSS
organ
blood
30 min
1 h
3 h
6 h
0.61 0.13
1.27 0.22
2.09 0.35
5.82 1.22
1.06 0.19
1.00 0.07
2.52 0.33
0.84 0.14
0.63 0.06
2.98 0.54
0.16 0.02
1.32 0.15
1.26 0.12
5.04 1.15
0.72 0.09
0.69 0.05
1.41 0.11
0.55 0.12
0.74 0.16
3.67 0.27**
0.07 0.02
1.49 0.32
1.32 0.21
3.48 0.54
0.65 0.10
0.65 0.09
1.08 0.37
0.51 0.07
0.56 0.13
5.23 0.80*
0.09 0.02
1.50 0.30
1.77 0.56
3.00 0.43
0.66 0.13
0.73 0.11*
1.25 0.47
0.52 0.04
0.47 0.14
4.44 0.26**
63.22 6.83
4.25 1.30
liver
spleen
kidneys
pancreas
heart
lung
muscle
c
stomach
b,c
intestines
c
urine
c
feces
a
b
c
Data expressed as %ID/g SD (n = 5). (* p < 0.05, ** p < 0.01). Expressed as %ID.
a
Table 4. Biodistribution of RRS/SSR Diastereomer of 67Ga-NOTGA-(TEG-RGD)3 in normal mice
RRS/SSR
organ
blood
30 min
1 h
3 h
6 h
0.58 0.18
1.22 0.12
1.42 0.59
5.38 0.89
0.85 0.07
0.93 0.16
1.94 0.53
1.22 0.91
0.59 0.06
2.59 0.35
0.19 0.02
1.19 0.07
1.20 0.17
4.74 0.87
0.69 0.03
0.73 0.08
1.36 0.16
0.55 0.04
0.63 0.11
3.00 0.27**
0.09 0.03
1.56 0.38
1.40 0.06
3.58 0.39
0.71 0.09
0.73 0.09
1.33 0.35
0.55 0.06
0.61 0.11
4.10 0.62*
0.08 0.02
1.33 0.19
1.57 1.17
3.04 0.28
0.58 0.11
0.55 0.05*
1.00 0.04
0.62 0.17
0.40 0.02
2.90 0.38**
71.52 2.64
2.24 0.89
liver
spleen
kidneys
pancreas
heart
lung
muscle
c
stomach
b,c
intestines
c
urine
c
feces
a
b
c
Data expressed as %ID/g SD (n = 5). (* p < 0.05, ** p < 0.01). Expressed as %ID.
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Figure 6. Biodistribution data in tumor-bearing male nude mice at 2 h
after administration of 67Ga-NOTGA-(TEG-RGD)3 with (black) and
without (white) the presence of an excess of RGDyV. (A) RRR/SSS;
(B) RRS/SSR. Data expressed as %ID/g SD (n = 4).
Figure 5. (A) Biodistribution of 67Ga-NOTGA-(TEG-RGD)3 in
tumor-bearing male nude mice at 30 min after intravenous injection of
11.1 KBq of RRR/SSS (white) or RRS/SSR (black). (B) The tumor to
organ ratios of the 67Ga labeled conjugates. Data expressed as %ID/g
SD (n = 4, ** p < 0.01).
using nude mice bearing U87MG xenografts (Figure 7).
Tumors were clearly visualized as early as 30 min after admin-
istration. Radioactivity was concentrated in the kidneys and
bladder. Impressively, nonspecific accumulation in liver or
bowel was not observed, resulting in high-contrast images.
Figure 7. SPECT/CT images of U87MG tumor-bearing male nude
mice after 30 min of i.v. injection of 4.5 MBq of each diastereomer of
[67Ga]-NOTGA-(TEG-RGD)3. (A) RRR/SSS and (B) RRS/SSR.
Images were shown at the same signal intensity scale.
DISCUSSION
■
The cyclic RGD peptides have been widely study as a targeting
molecule due to its high affinity for integrin αvβ3 overexpressed
during tumor angiogenesis.24 It has also been well recognized
that in vitro binding affinity and in vivo tumor targeting ability
of multimeric RGD peptides are enhanced due to the multi-
valent effect and the enriched local RGD concentration.24−28
Moreover, it has been demonstrated that the spacer units of
appropriate length and hydrophilicity between a radiometal
chelate and each RGD motif increase avidity of the radiotracers
and simultaneously may act as pharmacokinetic modifiers to
improve pharmacokinetics of the radiotracer.24,29,30
enantiomers, as supported by similar MS and 13C NMR spectra.
Further isolation was not possible by the conventional methods. In
13C NMR, since the three chiral carbons in RRR/SSS are equi-
valent, this pair was assigned to the 69% fraction with a single
resonance. Similarly, two different carbons (R, S) are present in
RRS/SSR and were associated with the doublet observed in
the 17% fraction, as also observed in the tetraazacyclodecane
analogue.16
It should be mentioned that, besides the isomerism in
NOTGA ligands coming from the chiral carbon in the pendant
arms, NOTA-based complexes are chiral depending on the orien-
tation of the pendant arms around the metal center (Λ: clockwise
or Δ: anticlockwise) and the relative puckering of the ethylenedi-
amine subunit chelate rings (δδδ or λλλ conformers)31,32 in the
case of gallium, only the enantiomeric Δ(λλλ)/Λ(δδδ) com-
bination was observed.31 Therefore, each RRR/SSS and RRS/SSR
of Ga-NOTGA diastereomeric pair may further exist as
the enantiomeric combinations Δ(λλλ)-RRR/Λ(δδδ)-SSS or
Δ(λλλ)-SSS/Λ(δδδ)-RRR, as well as Δ(λλλ)-RRS/Λ(δδδ)-SSR
The preparation of NOTA-based radiopharmaceuticals was
conducted according to the procedure reported previously.13,14
In brief, the synthesis of the orthogonally protected prochelator
NOTGA-tBu3, followed by conjugation of the bioactive mol-
ecules to each carboxylic acid of the pendant arm and final
deprotection. Since racemic (R/S) α-bromoglutaric acid 1-tert-
butyl ester 5-benzyl ester was used in the alkylation of TACN,
intermediate 4 exists in different isomeric forms,15,16 RRR, SSS,
RRS, and SSR (Figure 2). The RRR and SSS isomers constitute
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Bioconjugate Chemistry
Article
or Δ(λλλ)-SSR/Λ(δδδ)-RRS. Nevertheless, both diastereomeric
ligands provided gallium complexes of high inertness, indicating
good retention of the metal inside the complex cavity.
10-folder higher than that of the monovalent cRGDfK. Thus,
both diastereomers might act as trivalent compounds and the
spatial arrangement of the RGD motifs in each complex would be
equivalent with respect to binding affinity to αvβ3 integrin.
No significant differences were also observed in the bio-
distribution of the 67Ga labeled diastereomers (Tables 3 and 4,
Figure 5). Both exhibited rapid blood clearance and renal ex-
cretion in normal and nude mice bearing U87MG tumors.
The high renal uptake was consistent with previous studies of
other multimeric RGD peptides.14,26,40−42 It was reported that
endothelial cells of the glomeruli vessels in the kidneys express
integrin αvβ3,43 which could partially explain the observation.
The presence of three guanidine groups in the trivalent RGD
conjugate would increase positive charge, which may have
facilitated reabsorption in proximal renal tubular cells.26,27,43,44
While both diastereomers exhibited similar biodistribution, the
elution order of the diastereomers from HPLC (Figure 3; RRS/
SSR followed by RRR/SSS) suggests that a small difference in
the lipophilicity between the two may be responsible for the
slight tendency of the RRR/SSS to be excreted through the
intestinal tract, though the excretion route is minimal. The
similar affinity for integrin αvβ3 along with similar pharmaco-
kinetics of the diastereomers resulted in similar tumor accu-
mulation of the two compounds (Figure 5), yielding SPECT/
CT images of high contrast as shown in Figure 7.
67Ga radiolabeling was performed at room temperature as
previously reported for NOTA and its derivatives.5−7 The reaction
condition allowed assessing the differences in the complexation
ability of the diastereomeric ligands that otherwise would be
suppressed by heating. Lower radiochemical yields under acidic
conditions were also found by Singh et al.14 when labeling
NOTGA and its disubstituted parent, in comparison to the
monosubstituted and the p-SCN-Bn-NOTA RGD conjugates,
possibly due to steric hindrance caused by the three
substituents. On the other hand, pH 5 was found optimal for
labeling NODAGA-RGD with 68Ga,33 and a study of the Ga-
NOTA formation in acetate solutions found faster reaction
rates at higher pH.34 Intriguingly, the RRS/SSR was almost pH-
independent from pH 4 to 5.5. A hypothesis to account for this
observation would be related to the formation mechanism of
the Ga-NOTA complex and the structural differences between
the diastereomers. It has been reported that complexation of
NOTA and other polyazamycrocycles containing pendant arms
proceed in two steps. The first step comprises a fast formation
of a monoprotonated intermediate species in which the metal
ion is outside of the macrocyclic cage and coordinated to the
three peripheric carboxylate oxygen atoms. The ring nitrogen
atoms remain unbound and the proton would be attached to
one of them. In the second step, deprotonation takes place
followed by migration of the metal to inside the cavity where it
also becomes coordinated to the nitrogen atoms.35 Due to elec-
trostatic repulsion between the nitrogen’s proton and the metal,
removal of the proton is considered to be the rate-determining
step.34,36,37 It has been proposed that this is an OH-catalyzed
deprotonation,34−37 explaining the faster complexation at higher
pH. It has also been proposed that the migration of the proton
from the NH+ group to a carboxylate facilitates the accessibility
with respect to OH− ions.37 In either case, the RRS/SSR con-
formation might facilitate access of the OH− ions to the nitrogen-
bound proton or the proton migration to the carboxylate, making
the complexation less pH-dependent.
The longer-lived 67Ga was used throughout the study. The
outcomes of this study would also be applicable to the synthesis
of 68Ga labeled NOTA-(TEG-RGD)3. Moreover, given the efforts
in processing generator eluates to reduce metal impurities5,45−47
and the sensitivity of PET imaging techniques, superior images of
in vivo integrin αvβ3 expression can be obtained.
CONCLUSIONS
■
The findings in this study show that the RGD conjugates of
both diastereomers presented here possess equivalent biological
efficacy, and the combined usage of the diastereomic mixture
would be feasible as far as the present compounds are con-
cerned. It is worth noting that the specific properties of a given
biomolecule, cell expression levels of the corresponding target
molecule, and presence or absence of pharmacokinetic modifier
might affect the structural differences between diastereomers on
the ligand−receptor interactions and biodistribution. Since the
synthesis of diastereomerically pure NOTGA-tBu prochelators
has been established, the preparation of corresponding con-
jugates and evaluation of their chemical and biological
performances still remains important for applying NOTGA to
other biomolecules of interest.
To further estimate the differences between the two di-
astereomers, the binding affinity of the two gallium complexes
of NOTGA-(TEG-RGD)3 to αvβ3 integrin were estimated by
SPR assay and found that the structural differences in the
NOTGA diastereomers presented here do not affect the inter-
action with the targets (Table 2). The binding to αvβ3 integrin
is strongly dependent on the molecular design of multimeric RGD
probes. There are two factors underneath the enhanced target
affinity: multivalency and enhanced local RGD concentration.
Although the local concentration factor is innate in all multimeric
probes, an appropriate distance between each of two sets of cyclic
RGD motifs is required to achieve multivalency.24,38,39 In this
design, the triethylene glycol spacer was inserted to provide a
distance between the three carboxylates in NOTGA and the lysine
side chains of the RGD motifs of 37 bonds. According to the
previous study,38 the distance between the two cyclic RGD motifs
of [67Ga]-NOTGA-(TEG-RGD)3 would be suitable for simulta-
neous αvβ3 integrin binding. In addition, Syngh et al. reported that
a trimeric RGD linked to NOTGA showed enhanced tumor
uptake and retention compared with monomeric RGD counter-
part, due to the multivalent effect partially.14 From these findings,
a combination of multivalency and enhanced local RGD con-
centration might be attributable to higher targeting capabilities of
[67Ga]-NOTGA-(TEG-RGD)3, reflected in a binding affinity
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR and LC-MS spectra of the diastereomeric pairs
of NOTGA-tBu3-Bz3. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +81-43-256-1782. Fax: +81-43-226-2897. E-mail: arano@
chiba-u.jp.
Notes
The authors declare no competing financial interest.
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Article
(16) Woods, M., Aime, S., Botta, M., Howard, J. A. K., Moloney, J.
M., Navet, M., Parker, D., Port, M., and Rousseaux, O. (2000)
Correlation of water exchange rate with isomeric composition in
diastereoisomeric gadolinium complexes of tetra(carboxyethyl)dota
and related macrocyclic ligands. J. Am. Chem. Soc. 122, 9781−9792.
ACKNOWLEDGMENTS
■
This work was partially supported by Special Funds for
Education and Research (Development of SPECT Probes for
Pharmaceutical Innovation) from the Ministry of Education,
Culture, Sports, Science and Technology, Japan. We would also
like to thank FUJIFILM RI Pharma Co., Ltd. for providing
[67Ga]Cl3.
̌
(17) Simecek, J., Schulz, M., Notni, J., Plutnar, J., Kubícek, V.,
̌
̌
̌ ́
Havlíckova, J., and Hermann, P. (2012) Complexation of metal ions
with TRAP (1,4,7-triazacyclononane phosphinic acid) ligands and
1,4,7-triazacyclononane-1,4,7-triacetic acid: Phosphinate-containing
ligands as unique chelators for trivalent gallium. Inorg. Chem. 51,
577−590.
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