Synthesis and Evaluation of 68Ga- And 177Lu-Labeled (R)- vs (S)-DOTAGA Prostate-Specific Membrane Antigen-Targeting Derivatives

Article abstract of DOI:10.1021/acs.molpharmaceut.0c00777

Prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer cells and therefore is an attractive target for prostate cancer diagnosis and radionuclide therapy. Recently, published results from clinical studies using a new PSMA-targeting PET imaging agent, [68Ga]Ga-PSMA-093 ([68Ga]Ga-HBED-CC-O-carboxymethyl-Tyr-CO-NH-Glu), support the development of this agent for the diagnosis of prostate cancer. In this study, the HBED-CC chelating group in PSMA-093 was replaced by stereoselective (R)- or (S)-DOTAGA. This chelating group serves not only for chelating 68Ga but is also amendable for complexing other radioactive metals for radionuclide therapy. The corresponding optically pure (R)- and (S)-[68Ga/177Lu]-DOTAGA derivatives, (R)-[68Ga/177Lu]-13 and (S)-[68Ga/177Lu]-13, were successfully prepared. Comparison of radiolabeling, binding affinity, cell uptake, and biodistribution between the two isomers was performed. Radiolabeling of (R)-[177Lu]Lu-13 and (S)-[177Lu]Lu-13 at 50 °C suggested that rates of complex formation were time-dependent and the formation of (S)-[177Lu]Lu-13 was distinctly faster. The rates of complex formation for the corresponding 68Ga agents were comparable between structural isomers. The natGa and natLu equivalents showed high binding PSMA affinity (IC50 = 24-111 nM), comparable to that of the parent agent, [natGa]Ga-PSMA-093 (IC50 = 34.0 nM). Results of cell uptake and biodistribution studies in PSMA-expressing PC3-PIP tumor-bearing mice appeared to show no difference between the labeled (R)- and (S)-isomers. This is the first time that a pair of [68Ga/177Lu]-(R)- and (S)-DOTAGA isomers of PSMA agents were evaluated. Results of biological studies between the isomers showed no noticeable difference; however, the distinctions on the rate of Lu complex formation should be considered in the development of new 177Lu-DOTAGA-based radionuclide therapy agents in the future.

Full text of DOI:10.1021/acs.molpharmaceut.0c00777

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68
177
Synthesis and Evaluation of Ga- and Lu-Labeled (R)- vs
(S)‑DOTAGA Prostate-Specific Membrane Antigen-Targeting
Derivatives
Ruiyue Zhao, Karl Ploessl, Zhihao Zha, Seokrye Choi, David Alexoff, Lin Zhu, and Hank F. Kung*
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ABSTRACT: Prostate-specific membrane antigen (PSMA) is overexpressed in prostate
cancer cells and therefore is an attractive target for prostate cancer diagnosis and
radionuclide therapy. Recently, published results from clinical studies using a new PSMA-
targeting PET imaging agent, [68Ga]Ga-PSMA-093 ([68Ga]Ga-HBED-CC-O-carbox-
ymethyl-Tyr-CO-NH-Glu), support the development of this agent for the diagnosis of
prostate cancer. In this study, the HBED-CC chelating group in PSMA-093 was replaced
by stereoselective (R)- or (S)-DOTAGA. This chelating group serves not only for
chelating 68Ga but is also amendable for complexing other radioactive metals for
radionuclide therapy. The corresponding optically pure (R)- and (S)-[68Ga/177Lu]-
DOTAGA derivatives, (R)-[68Ga/177Lu]-13 and (S)-[68Ga/177Lu]-13, were successfully
prepared. Comparison of radiolabeling, binding affinity, cell uptake, and biodistribution
between the two isomers was performed. Radiolabeling of (R)-[177Lu]Lu-13 and (S)-
[
177Lu]Lu-13 at 50 °C suggested that rates of complex formation were time-dependent
and the formation of (S)-[177Lu]Lu-13 was distinctly faster. The rates of complex
formation for the corresponding 68Ga agents were comparable between structural isomers. The natGa and natLu equivalents showed
high binding PSMA affinity (IC50 = 24−111 nM), comparable to that of the parent agent, [natGa]Ga-PSMA-093 (IC50 = 34.0 nM).
Results of cell uptake and biodistribution studies in PSMA-expressing PC3-PIP tumor-bearing mice appeared to show no difference
between the labeled (R)- and (S)-isomers. This is the first time that a pair of [68Ga/177Lu]-(R)- and (S)-DOTAGA isomers of PSMA
agents were evaluated. Results of biological studies between the isomers showed no noticeable difference; however, the distinctions
on the rate of Lu complex formation should be considered in the development of new 177Lu-DOTAGA-based radionuclide therapy
agents in the future.
KEYWORDS: prostate cancer, gallium, lutetium, DOTAGA, chelating agents, optical isomers, PSMA, cell uptake, biodistribution
A large number of Ga and Lu complexes have been reported,
and they are usually based on macrocyclic or acyclic polyaza
carboxylic acids as chelating groups, including DTPA, TRAP,
AAZTA, DEDPA, DOTA, DOTAGA, and NOTAGA (see
Figure 1 and Abbreviations). Many of these ligands are
commonly employed to chelate radioactive metal ions including
68Ga(III) and 177Lu(III).7 Literature reports on DOTA and
related ligands7,8 suggest that they are the most versatile
chelating agents forming Ga(III) and Lu(III) complexes with
high thermodynamic stabilities. Nevertheless, the rate of
complexation of no-carrier-added (n.c.a.) 68Ga and 177Lu with
DOTA derivatives is relatively slow, thus requires heating at 80−
INTRODUCTION
■
In the past few years, 68Ge/68Ga generators have increasingly
played an important role in nuclear medicine clinics.1,2 It is
known that 68Ga generator-based PET imaging agents have
several unique advantages: (a) Germanium-68 (68Ge) (t1/2, 271
days), a long-lived isotope, facilitates a widespread generator
circulation; (b) physical properties of 68Ga (t1/2, 68 min; 89% β+;
1.92 MeV max energy) are appropriate for PET imaging; (c)
68Ge/68Ga generators provide an easy distribution for obtaining
a positron-emitting isotope without the need of a nearby
cyclotron.2,3 For radionuclide therapy, decay characteristics of
177Lu (t1/2 = 6.71 days, β−(max) = 497 keV) are very attractive
and they have been successfully applied in treatment of different
cancers. Recently, Lu-177 DOTATATE (Lutathera) was
approved by the FDA for the treatment of gastroenteropancre-
atic neuroendocrine tumors. This is the first time that a Lu-177
radiopharmaceutical has been approved for therapeutic
application.4 In addition, Lu-177 PSMA-617 is currently under
clinical trials as a PSMA-targeting radionuclide therapy agent.5,6
Received: July 26, 2020
Revised: October 13, 2020
Accepted: October 14, 2020
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© XXXX American Chemical Society
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Figure 1. Various chelating agents commonly used for developing 68Ga and177Lu imaging and radionuclide therapeutic agents. They include DTPA,
TRAP, AAZTA, DEDPA, DOTA, DOTAGA, DOTA, and NOTAGA.7
100 °C to complete the chelate formation. Generally, DOTA
derivatives are prepared with one of the chelating carboxylic acid
groups replaced by an amide substituted with additional groups;
therefore, most of the time, they were designated as the DO3AM
derivatives (Figure 1). It was reported that the kinetics and
thermodynamic equilibrium constant of Ga-DO3AMBu (log Kd
= 24.6) were less favorable than that of Ga-DOTA (log Kd =
26.1).9 As Ga(III) has a relatively small ionic radius (187 pm),
the complex formation between NOTA and Ga(III) is more
rapid than that of DOTA or DO3AM derivatives. It is likely due
to the fact that the smaller cavity created by NOTA derivatives is
better adapted to the ionic radius of Ga(III) (log Kd = 31.0). The
NOTAGA derivatives, containing an extra glutamic acid arm,
form Ga-NOTAGA complexes that exhibit higher thermody-
namic stability with faster complex formation kinetics. Complex
formation of Ga(III) generally requires an octahedral coordi-
nation sphere, and Ga-NOTAGA analogues provide optimal in
vitro and in vivo stability (Figure 1).10
Using a similar approach by adding one additional glutamic
arm to DOTA, an alternative strategy in improving the kinetics
of complex formation has been reported. Significantly,
[68Ga]Ga-DOTAGA-TATE and [68Ga]Ga-DOTAGA-TOC
showed improved 68Ga labeling and retained the biological
binding affinity to the somatostatin receptors for imaging
endocrine tumors.11 However, there was no information
provided on the optical purity of the complexing group
DOTAGA; presumably, it was a racemic mixture. The interest
of using DOTAGA for binding metal radionuclides does not
stop at 68Ga imaging agents. The chelating agent is also known
to complex many other useful radiometals, such as 177Lu, 44Sc,
111In, 89Zr, and 225Ac.7,12 Investigation on the effect of isomers
introduced by the extra glutamic acid arm of DOTAGA ((R)- vs
(S)-glutamic isomers) would be important for future develop-
ment of metal complexes for diagnosis and radionuclide therapy
in personalized medicine.13
It is well-known that the HBED chelating group is not suitable
for complexing Lu, and the cavity of DOTA derivatives is better
suited for formation of Lu complexes.7 By adding a glutamic acid
arm, several 68Ga- and 177Lu-labeled DOTAGA derivatives
designed for targeting PSMA binding were prepared and
evaluated previously. Notably, [68Ga]Ga-DOTAGA-Phe-Phe-
D-Lys(suberoyl)-Lys-urea-Glu and the related derivative PSMA-
I&T14 (see Figure 2) containing an extra glutamic acid arm
provided enhanced 68Ga and 177Lu complex stability, and
favorable pharmacokinetics and high specific binding to a PSMA
tumor were described. This PSMA-ligand, [68Ga/177Lu]Ga/Lu-
PSMA-I&T, displayed potential for the management of prostate
cancer.15,16 However, the optical center of the glutamic acid arm
for this agent was not specified, and it is assumed that racemic
mixtures were used. These previous studies have demonstrated
the “beneficial effect” of DOTAGA for DOTA substitution and
using a (D)-amino acid ((R)-isomer) peptide linker on PSMA to
improve affinity and metabolic stability for tumor uptake and
clearance of nonspecific binding.14,15,17
Recent reports on cancer statistics show that prostate cancer
(PCa) is the second most common cancer diagnosed in men and
fifth leading cause of cancer-related deaths worldwide.18,19
PSMA is overexpressed in hormone-refractory and metastatic
prostate cancer and is an excellent target for the diagnostic and
therapeutic applications.20,21 Many 68Ga-labeled PET imaging
agents targeting PSMA expression for diagnosis of prostate
cancer have been successfully tested clinically, including
[68Ga]Ga-PSMA-11,2 [68Ga]Ga-PSMA-THP,22 [68Ga]Ga-
PSMA-I&T,16 [68Ga]Ga-PSMA-617,23,24 and [68Ga]Ga-
PSMA-09325 (Figure 2). There are several advantages of using
HBED instead of commonly employed DOTA and NOTA for
chelating Ga(III). Stability constants (log Kd) for Ga-DOTA26
B
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Figure 2. Chemical structures of several 68Ga/177Lu-PSMA diagnostic imaging and radionuclide therapeutic agents. Each structure contains three parts
(as indicated by colored spheres): (1) Ga/Lu complexing group: either HBED-CC (in green) or DOTA (in blue) or DOTAGA (in red) with an (R)-
or (S)-optical center; (2) a linker (in black); and (3) a PSMA binding urea moiety, −NH-CO-NH-Glu, for PSMA binding (in purple). The new PSMA
agents, (R)- and (S)-DOTAGA-PSMA-093, are marked by the solid red box. It is noted that the extra glutamic acid arm attached to the DOTAGA
chelating group of rhPSMA-7.3,30−33 a radiohybrid PSMA-targeting agent, is reported to be the (S)-isomer. The derivatives of [177Lu]Lu-PSMA-617,
[
177Lu]Lu-PSMA-HTK03041, and [177Lu]Lu-PSMA-ALB-56, which improved the in vivo pharmacokinetics, were recently reported in animal and
clinical trials.34,35
and Ga-NOTA27 complexes were previously reported (log Kd =
21.3 and 31.0, respectively). Compared to DOTA and NOTA,
the HBED chelating group forms a stronger, more stable Ga(III)
complex: a log Kd value of 38.5 was reported for Ga-HBED.7,28 It
is important to note that [177Lu]Lu-PSMA-617, a DOTA
derivative, is currently the most well-studied PSMA-targeting
radionuclide therapy agent. Clinical studies have clearly
demonstrated its treatment potential in patients with metastatic
castrate-resistant prostate cancer.5
Based on [68Ga]Ga-PSMA-11, a new PSMA-targeting agent
[68Ga]Ga-PSMA-093 ([68Ga]Ga-P16-093), using a different
linker, was developed (see Figure 2). Similarly, it is composed of
three parts: [68Ga]Ga-HBED-CC complex (green circle), a
novel linker o-(carboxymethyl)-L-tyrosine, and a PSMA binding
group, −Lys-NH-CO-NH-Glu (purple circle) (see Figure 2).29
The unique o-(carboxymethyl)-Tyr linker (black square box) of
[68Ga]Ga-PSMA-093, as compared to the Ahx linker in
[68Ga]Ga-PSMA-11, led to improvements on biological proper-
ties of PSMA targeting and lowering renal excretion. In vitro
C
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a
Scheme 1. Synthesis of 2 and Diastereomers, (R)-(+)-α-Methylbenzylamide-DOTAGA(tBu)4 ((R,S)-10)
a
TBTA (tert-butyl trichloroacetimide) and TBBA (tert-butyl bromoacetate).
Scheme 2. Synthesis of (S)-6, (R)-6, and diastereomers, (R)-(+)-α-Methylbenzylamide-DOTAGA(tBu)4 ((R)-10 and (S)-10)
binding showed that [68Ga]Ga-PSMA-093 displayed fast
labeling, excellent affinity (IC50 = 33.5 nM), and specificity, a
value comparable to that of [68Ga]Ga-PSMA-11.29 Preliminary
human studies (IND 133,222)25 comparing PET images of
[68Ga]Ga-PSMA-093 and [68Ga]Ga-PSMA-11 at 60 min after iv
injection in the same prostate cancer patients showed similar
ability to detect PSMA-positive prostate cancer. Furthermore,
there are several notable advantages of [68Ga]Ga-PSMA-093 as
compared to that of [68Ga]Ga-PSMA-11, including a lower renal
clearance and minimum bladder activity while maintaining avid
tumor uptake as compared to background.25 Lower bladder
uptake reduced background in the pelvic region, which has
added advantage to visualize tumors in the prostate and regional
lymph nodes. [68Ga]Ga-PSMA-093 appeared to be a promising
candidate as a PET imaging radiotracer for detecting PSMA
expression in prostate cancer.
chelating group (see red solid line box in Figure 2). It was
hypothesized that using (R)- and (S)-DOTAGA derivatives,
containing an extra glutamic acid arm on DOTA, could lead to
development of new 68Ga/177Lu-PSMA theragnostic agents.
Both (R)- and (S)-DOTAGA isomers were expected to form
stable complexes with 177Lu.
Reported herein is the preparation, labeling, and biological
evaluation of (R)- and (S)-DOTAGA-PSMA-093, (solid red
box in Figure 2) as new PSMA-targeting agents for both
diagnostic imaging (68Ga) and radionuclide therapy (177Lu). In
particular, comparison of (R)- vs (S)-DOTAGA in 68Ga and
177Lu complex formation and potential effects of the optical
center of the glutamic acid arm on PSMA-targeting properties
were investigated.
MATERIALS AND METHODS
■
As mentioned above, HBED-CC is not suitable for complex-
ing 177Lu; in order to convert PSMA-093 for labeling with 177Lu,
the HBED-CC group was replaced by the (R)- or (S)-DOTAGA
General. All reagents were commercial products used
without further purification unless indicated. Solvents were
D
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a
Scheme 3. Synthesis of (R)-[natGa]Ga-13, (R)-[natLu]Lu-13, (S)-[natGa]Ga-13, and (S)-[natLu]Lu-13
a
HOBt (hydroxybenzotriazole), EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), and DIPEA (N,N-diisopropylethylamine).
dried through a molecular sieve system (Pure Solve Solvent
Purification System; Innovative Technology, Inc.). H NMR
purified by FC (hexane/EtOAc = 70/30) to give 2.0 g of (S)-4
(yield, 87%) as a white solid. 1H NMR (400 MHz, CDCl3): δ
4.81−4.78 (m, 1H), 2.65−2.44 (m, 3H), 2.28−2.23 (m, 1H),
1.48 (s, 9H).
1
spectra were recorded on a Bruker Avance II spectrometer at
400 MHz and referenced to NMR solvents as indicated. High-
resolution mass spectrometry (HRMS) data were obtained with
an Agilent (Santa Clara, CA) G3250AA LC/MSD TOF system.
Generally, crude compounds were purified by flash column
chromatography (FC) packed with silica gel (Aldrich). A
Wizard2 automatic gamma counter (PerkinElmer) measured
68Ga and 177Lu radioactivity. Reactions of nonradioactive
chemical compounds were monitored by TLC analysis with
precoated plates of silica gel 60 F254. Gallium-68 was obtained
from a 68Ge/68Ga generator (1.11 GBq (30 mCi)) produced by
ITG (Isotope Technologies Garching GmbH). Lutetium-177
used in this research was supplied by the U.S. Department of
Energy Isotope Program, managed by the Office of Science for
Nuclear Physics.
1-(tert-Butyl) 5-Methyl (S)-2-Hydroxypentanedioate ((S)-
5).37 To a solution of (S)-4 (2.0 g, 10.7 mmol) in methanol
(MeOH, 20 mL) was added sodium methoxide (0.6 g, 12.0
mmol) at 0 °C. After 30 min, the reaction was quenched with 1
M HCl and the mixture was concentrated under reduced
pressure. Then, the residue was extracted with EtOAc (30 mL ×
3). The combined organic layers were dried over MgSO4 and
filtered. The filtrate was purified by FC (hexane/EtOAc = 70/
30) to give 1.8 g of (S)-5 (yield, 77%) as a colorless oil. 1H NMR
(400 MHz, CDCl3): δ 4.10−4.07 (m, 1H), 3.68 (s, 3H), 2.54−
2.38 (m, 2H), 2.18−2.09 (m, 1H), 1.93−1.83 (m, 1H), 1.48 (s,
9H).
1-(tert-Butyl) 5-Methyl (S)-2-((Methylsulfonyl)oxy)-
pentanedioate ((S)-6).36 To a solution of (S)-5 (1.4 g, 6.4
mmol) in dichloromethane (CH2Cl2, 30 mL) were added
methanesulfonyl chloride (MsCl, 0.8 g, 7.0 mmol) and
triethylamine (Et3N, 1.0 g, 9.6 mmol) dropwise at 0 °C. After
completion of the addition, the reaction was warmed to rt and
stirred for 1 h. Then, the reaction mixture was washed with brine
(20 mL × 2) and water. The organic layers were dried over
MgSO4 and filtered. The filtrate was purified by FC (hexane/
EtOAc = 70/30) to give 1.7 g of (S)-6 (yield, 90%) as a colorless
oil. 1H NMR (400 MHz, CDCl3): δ 4.99−4.96 (m, 1H), 3.70 (s,
3H), 3.14 (s, 3H), 2.52−2.48 (m, 2H), 2.34−2.25 (m, 1H),
2.19−2.10 (m, 1H), 1.50 (s, 9H). [α]2D2 = −19.5 (MeOH, c = 2
mg/mL), [α]2D8 = −16.0 (MeOH, c = 2 mg/mL).
1-(tert-Butyl) 5-Methyl (R)-2-(1,4,7,10-Tetraazacyclodode-
can-1-yl)pentanedioate ((R)-7). To a solution of 1,4,7,10-
tetraazacyclododecane (344 mg, 2.0 mmol) in acetonitrile
(ACN, 10 mL), K2CO3 (137 mg, 1.0 mmol) was added at rt and
stirred for 10 min. Then, (S)-6 (296 mg, 1.0 mmol) was
dissolved in ACN (10 mL) and added dropwise. After
completion of the addition, the reaction was warmed to 50 °C
and stirred overnight. The reaction mixture was concentrated
under reduced pressure, and the residue was purified by FC
(CH2Cl2/MeOH/NH4OH = 80/20/2) to give 220 mg of (R)-7
(yield, 60%) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ
3.66 (s, 3H), 3.32−3.28 (m, 1H), 2.89−2.45 (m, 23H), 1.45 (s,
Chemistry. The synthesis of (R)-13 and (S)-13 is outlined in
Schemes 1−3. The details of the synthesis of 2 and (R)-6 are
included in the Supporting Information.
(S)-5-Oxotetrahydrofuran-2-carboxylic Acid ((S)-3).36 To a
solution of (S)-glutamic acid (3.0 g, 20 mmol) in water (20 mL)
was added hydrochloric acid (HCl, 37%, 5 mL) slowly at ice
bath temperature. After being stirred at 0 °C for 20 min, a
solution of sodium nitrite (2.1 g, 30 mmol) in water (5 mL) was
added dropwise over 2 h at 0 °C. After completion of the
addition, the reaction was warmed to rt and stirred overnight.
The reaction mixture was extracted with ethyl acetate (EtOAc,
30 mL × 3). The combined organic layers were dried over
magnesium sulfate (MgSO4) and filtered. The filtrate was
concentrated and without further purification yielded 2.1 g of
1
(S)-3 (yield, 80%) as a colorless oil. H NMR (400 MHz,
CDCl3): δ 5.01−4.98 (m, 1H), 2.72−2.52 (m, 3H), 2.46−2.36
(m, 1H).
tert-Butyl (S)-5-Oxotetrahydrofuran-2-carboxylate ((S)-
4).36 (S)-3 (1.6 g, 12.3 mmol) was dissolved in dichloromethane
(CH2Cl2, 10 mL) in a 50 mL round-bottom flask. To this
solution, tert-butyl 2,2,2-trichloroacetamidate (TBTA, 5.4 g,
25.0 mmol in 15 mL of cyclohexane) and boron trifluoride
diethyl etherate (BF3·Et2O, 0.2 mL, 1.2 mmol) were added at 0
°C. After the addition was complete, the reaction mixture was
warmed to rt and allowed to stir for 5 h. The reaction mixture
was concentrated under reduced pressure, and the residue was
E
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9H). HRMS: (M + H)+ calcd for C18H37N4O4, 373.2815; found,
373.2846.
3.55−3.53 (m, 1H), 3.43−3.30 (m, 3H), 3.06−2.98 (m, 3H),
2.85−2.79 (m, 4H), 2.74−2.71 (m, 3H), 2.65−2.50 (m, 5H),
2.31−2.25 (m, 4H), 2.11−1.98 (m, 4H), 1.45 (s, 9H), 1.43−
1.42 (m, 27H). HRMS: (M + H)+ calcd for C35H65N4O10,
701.4701; found, 701.4770.
(S)-5-(tert-Butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-
oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanoic
Acid ((S)-9). Compound (S)-9 (145 mg; yield, 70%) was
prepared from (S)-8 (212 mg, 0.29 mmol) following the same
procedure as for (R)-9 as a colorless oil. 1H NMR (400 MHz,
CDCl3): δ 3.63−3.61 (m, 1H), 3.51−3.47 (m, 1H), 3.41−3.28
(m, 2H), 2.94−2.73 (m, 7H), 2.66−2.57 (m, 8H), 2.45−2.34
(m, 2H), 2.25−2.17 (m, 3H), 2.08−2.04 (m, 3H), 1.44−1.46
(m, 36H). HRMS: (M + H)+ calcd for C35H65N4O10, 701.4701;
found, 701.4679.
1-(tert-Butyl) 5-Methyl (S)-2-(1,4,7,10-Tetraazacyclodode-
can-1-yl)pentanedioate ((S)-7). Compound (S)-7 (350 mg;
yield, 60%) was prepared from (R)-6 (466 mg, 1.57 mmol),
1,4,7,10-tetraazacyclododecane (516 mg, 3.1 mmol), and
K2CO3 (215 mg, 1.57 mmol) following the same procedure as
for (R)-7 as a colorless oil. 1H NMR (400 MHz, CDCl3): δ 3.68
(s, 3H), 3.32−3.22 (m, 1H), 2.92−2.44 (m, 23H), 1.46 (s, 9H).
HRMS: (M + H)+ calcd for C18H37N4O4, 373.2815; found,
373.2839.
1-(tert-Butyl) 5-Methyl 2-(1,4,7,10-Tetraazacyclododecan-
1-yl)pentanedioate ((R,S)-7). Compound (R,S)-7 (200 mg;
yield, 53%) was prepared from 2 (280 mg, 1.0 mmol), 1,4,7,10-
tetraazacyclododecane (344 mg, 2.0 mmol), and K2CO3 (137
mg, 1.0 mmol) following the same procedure as for (R)-7 as a
(R,S)-5-(tert-Butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-
2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanoic
Acid ((R,S)-9). Compound (R,S)-9 (156 mg; yield, 70%) was
prepared from (R,S)-8 (200 mg, 0.28 mmol) following the same
procedure as for (R)-9 as a colorless oil. 1H NMR (400 MHz,
CDCl3): δ 3.65−3.63 (m, 1H), 3.52−3.28 (m, 3H), 2.95−2.73
(m, 8H), 2.67−2.52 (m, 5H), 2.47−2.41 (m, 2H), 2.31−2.23
(m, 4H), 2.08−2.01 (m, 4H), 1.44−1.46 (m, 36H). HRMS: (M
+ H)+ calcd for C35H65N4O10, 701.4701; found, 701.4677.
Tri-tert-butyl 2,2′,2″-(10-((R)-1-(tert-Butoxy)-1,5-dioxo-5-
(((R)-1-phenylethyl)amino)pentan-2-yl)-1,4,7,10-tetraazacy-
clododecane-1,4,7-triyl)triacetate ((R)-10). To a solution of
(R)-9 (30 mg, 0.04 mmol) in CH2Cl2 (5 mL) were added (R)-
(+)-α-methylbenzylamine (8 mg, 0.06 mmol), hydroxybenzo-
triazole (HOBt, 8 mg, 0.06 mmol), and 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (EDC, 12 mg, 0.06
mmol) at 0 °C. The reaction mixture was stirred for 1 h before it
was concentrated under reduced pressure. Then, the residue was
purified by FC (CH2Cl2/MeOH/NH4OH = 90/9/1) to give 25
1
colorless oil. H NMR (400 MHz, CDCl3): δ 3.66 (s, 3H),
3.31−3.28 (m, 1H), 2.88−2.44 (m, 23H), 1.45 (s, 9H). HRMS:
(M + H)+ calcd for C18H37N4O4, 373.2815; found, 373.2842.
1-(tert-Butyl) 5-Methyl (R)-2-(4,7,10-Tris(2-(tert-butoxy)-2-
oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)-
pentanedioate ((R)-8). To a solution of (R)-7 (60 mg, 0.16
mmol) in ACN (10 mL), K2CO3 (137 mg, 1.0 mmol) and tert-
butyl bromoacetate (TBBA, 136 mg, 0.7 mmol) were added at 0
°C. The reaction mixture was warmed to rt and stirred overnight.
Then, the reaction mixture was concentrated under reduced
pressure and the residue was purified by FC (CH2Cl2/MeOH/
NH4OH = 90/9/1) to give 70 mg of (R)-8 (yield, 60%) as a
1
colorless oil. H NMR (400 MHz, CDCl3): δ 3.60 (s, 3H),
3.41−3.15 (m, 7H), 2.97−2.31 (m, 16H), 2.27−1.81 (m, 4H),
1.45−1.40 (m, 36H). HRMS: (M + H)+ C36H67N4O10,
715.4875; found, 715.4828.
1-(tert-Butyl) 5-Methyl (S)-2-(4,7,10-Tris(2-(tert-butoxy)-2-
oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)-
pentanedioate ((S)-8). Compound (S)-8 (212 mg; yield, 55%)
was prepared from (S)-7 (200 mg, 0.54 mmol), TBBA (421 mg,
2.16 mmol), and K2CO3 (444 mg, 3.24 mmol) following the
1
mg of (R)-10 (yield, 72%) as a colorless oil. H NMR (400
MHz, CDCl3): δ 8.07 (d, J = 8.0 Hz, 1H), 7.49−7.47 (m, 2H),
7.30−7.26 (m, 2H), 7.19−7.15 (m, 1H), 5.08−5.04 (m, 1H),
3.53−3.31 (m, 4H), 2.99−2.48 (m, 15H), 2.27−1.90 (m, 8H),
1.55 (d, J = 7.2 Hz, 3H), 1.46−1.43 (m, 36H). HRMS: (M + H)+
1
same procedure as for (R)-8 as a colorless oil. H NMR (400
MHz, CDCl3): δ 3.65 (s, 3H), 3.51−3.49 (m, 1H), 3.42−3.20
(m, 6H), 2.87−2.79 (m, 4H), 2.60−2.36 (m, 12H), 2.17−2.12
(m, 3H), 2.05−1.97 (m, 1H), 1.47 (s, 9H), 1.43−1.45 (m,
27H). HRMS: (M + H)+ calcd for C36H67N4O10, 715.4875;
found, 715.4793.
calcd for C43H74N5O9, 804.5487; found, 804.5510. [α]D25
=
−79.0 (MeOH, c = 1 mg/mL). The optical purity of compound
was analyzed by HPLC with a PRP-1 10 μm, 250 × 4.6 mm
column. The (R)-10 was eluted under isocratic conditions
(ACN/0.5% TFA in water (4/6), a flow rate of 2 mL/min,
temperature-controlled column compartment set at 10 °C), and
the retention time was 16.5 min.
1-(tert-Butyl) 5-Methyl 2-(4,7,10-Tris(2-(tert-butoxy)-2-ox-
oethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioate
((R,S)-8). Compound (R,S)-8 (200 mg; yield, 52%) was
prepared from (R,S)-7 (200 mg, 0.54 mmol), TBBA (409 mg,
2.10 mmol), and K2CO3 (438 mg, 3.20 mmol) following the
Tri-tert-butyl 2,2′,2″-(10-((S)-1-(tert-Butoxy)-1,5-dioxo-5-
(((R)-1-phenylethyl)amino)pentan-2-yl)-1,4,7,10-tetraazacy-
clododecane-1,4,7-triyl)triacetate ((S)-10). Compound (S)-10
(30 mg; yield, 52%) was prepared from (S)-9 (50 mg, 0.07
mmol), (R)-(+)-α-methylbenzylamine (12 mg, 0.1 mmol),
HOBt (13 mg, 0.1 mmol), and EDC (19 mg, 0.1 mmol)
following the same procedure as for (R)-10 as a colorless oil. 1H
NMR (400 MHz, CDCl3): δ 8.63 (d, J = 8.0 Hz, 1H), 7.50−7.49
(m, 2H), 7.28−7.24 (m, 2H), 7.17−7.13 (m, 1H), 5.05−5.01
(m, 1H), 3.55−3.29 (m, 4H), 2.97−2.43 (m, 15H), 2.25−1.92
(m, 8H), 1.54 (d, J = 6.8 Hz, 3H), 1.46−1.45 (m, 36H). HRMS:
(M + H)+ calcd for C43H74N5O9, 804.5487; found, 804.5507.
[α]2D5 = + 93.0 (MeOH, c = 1 mg/mL). (S)-10 was eluted under
isocratic conditions (ACN/0.5% TFA in water (4/6), a flow rate
of 2 mL/min, temperature-controlled column compartment set
at 10 °C), and the retention time was 18.8 min.
1
same procedure as for (R)-8 as a colorless oil. H NMR (400
MHz, CDCl3): δ 3.64−3.63 (m, 3H), 3.49−3.18 (m, 7H),
2.95−2.77 (m, 8H), 2.59−2.33 (m, 8H), 2.14−1.80 (m, 4H),
1.45−1.42 (m, 36H). HRMS: (M + H)+ calcd for C36H67N4O10,
715.4875; found, 715.4811.
(R)-5-(tert-Butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-
oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanoic
Acid ((R)-9). To a solution of (R)-8 (70 mg, 0.1 mmol) in
methanol (5 mL) was added sodium hydroxide (NaOH, 1 N, 2
mL) dropwise. After being stirred at rt for 2 h, HCl (1 N) was
added dropwise into the reaction mixture at 0 °C and adjusted
the pH to 5−6. Then, the reaction mixture was concentrated
under reduced pressure and the residue was purified by FC
(CH2Cl2/MeOH/NH4OH = 90/9/1) to give 52 mg of (R)-9
(yield, 75%) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ
F
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Tri-tert-butyl 2,2′,2″-(10-(1-(tert-Butoxy)-1,5-dioxo-5-(((R)-
1-phenylethyl)amino)pentan-2-yl)-1,4,7,10-tetraazacyclo-
dodecane-1,4,7-triyl)triacetate ((R,S)-10). Compound (R,S)-
10 (20 mg; yield, 59%) was prepared from (R,S)-9 (30 mg, 0.04
mmol), (R)-(+)-α-methylbenzylamine (8 mg, 0.06 mmol),
HOBt (8 mg, 0.06 mmol), and EDC (12 mg, 0.06 mmol)
following the same procedure as for (R)-10 as a colorless oil. 1H
NMR (400 MHz, CDCl3): δ 8.84−8.82 (m, 0.3H), 8.74−8.72
(m, 0.7H), 7.48−7.45 (m, 2H), 7.27−7.24 (m, 2H), 7.17−7.10
(m, 1H), 5.07−5.03 (m, 1H), 3.50−3.24 (m, 4H), 2.94−2.31
(m, 15H), 2.23−1.91 (m, 8H), 1.53−1.51 (m, 3H), 1.46−1.45
(m, 36H). HRMS: (M + H)+ calcd for C43H74N5O9, 804.5487;
found, 804.5493. (R,S)-10 was eluted under isocratic conditions
(ACN/0.5% TFA in water (4/6), a flow rate of 2 mL/min,
temperature-controlled column compartment set at 10 °C) with
a ratio of (R)-10/(S)-10 7/3.
Di-tert-butyl (((S)-1-(tert-Butoxy)-6-((S)-2-(2-(4-((S)-3-(tert-
butoxy)-2-(2-((R)-5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-
butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)-
pentanamido)acetamido)-3-oxopropyl)phenoxy)-
acetamido)-3-phenylpropanamido)-1-oxohexan-2-yl)-
carbamoyl)-L-glutamate ((R)-12). To a solution of (R)-9 (22
mg, 0.03 mmol) in N,N-dimethylformamide (DMF, 5 mL),
N,N-diisopropylethylamine (DIPEA, 13 mg, 0.1 mmol), HOBt
(7 mg, 0.05 mmol), EDC (9.5 mg, 0.05 mmol), and 11 (30 mg,
0.03 mmol) were added at 0 °C. The mixture was stirred at rt
overnight before 20 mL of EtOAc was added to the reaction
mixture. It was then washed with H2O (10 mL × 2) and brine
(10 mL), dried over MgSO4, and filtered. The filtrate was
concentrated, and the residue was purified by FC (DCM/
MeOH/NH4OH = 90/9/1) to give 40 mg of (R)-12 (yield,
78%) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ 8.52 (t, J
= 5.9 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.32−7.15 (m, 1H), 7.11
(d, J = 8.4 Hz, 2H), 6.77 (d, J = 8.4 Hz, 2H), 6.16 (d, J = 9.1 Hz,
1H), 5.99 (d, J = 7.9 Hz, 1H), 4.76 (d, J = 6.9 Hz, 1H), 4.61 (d, J
= 7.1 Hz, 1H), 4.35−4.19 (m, 2H), 4.03 (dd, J = 16.7, 6.6 Hz,
1H), 3.75 (dd, J = 16.8, 5.2 Hz, 1H), 3.68−3.54 (m, 2H), 3.51−
3.20 (m, 4H), 3.18−2.90 (m, 8H), 2.87−2.70 (m, 6H), 2.70−
2.46 (m, 6H), 2.46−2.14 (m, 6H), 2.09−2.06 (m, 4H), 1.88−
1.58 (m, 4H), 1.50−1.33 (m, 72H). HRMS: (M + H)+ calcd for
C85H139N10O22, 1652.0065; found, 1652.0026.
Di-tert-butyl (((S)-1-(tert-Butoxy)-6-((S)-2-(2-(4-((S)-3-(tert-
butoxy)-2-(2-((S)-5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-
butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)-
pentanamido)acetamido)-3-oxopropyl)phenoxy)-
acetamido)-3-phenylpropanamido)-1-oxohexan-2-yl)-
carbamoyl)-L-glutamate ((S)-12). Compound (S)-12 (60 mg;
yield, 51%) was prepared from (S)-9 (50 mg, 0.07 mmol), N,N-
diisopropylethylamine (DIPEA, 25 mg, 0.2 mmol), HOBt (14
mg, 0.09 mmol), EDC (20 mg, 0.1 mmol), and 11 (70 mg, 0.07
mmol) following the same procedure as for (R)-12 as a colorless
oil. 1H NMR (400 MHz, CDCl3): δ 8.64 (t, J = 5.6 Hz, 1H), 7.85
(d, J = 8.5 Hz, 1H), 7.56 (s, 1H), 7.37 (d, J = 7.4 Hz, 1H), 7.05
(d, J = 8.5 Hz, 2H), 6.67 (d, J = 8.5 Hz, 2H), 6.35 (d, J = 7.4 Hz,
1H), 6.12 (d, J = 7.8 Hz, 1H), 4.84−4.79 (m, 1H), 4.57−4.52
(m, 1H), 4.39−4.29 (m, 2H), 4.23−4.18 (m, 1H), 4.02−3.96
(m, 1H), 3.83−3.77 (m, 1H), 3.35−2.92 (m, 13H), 2.79−2.47
(m, 12H), 2.30−2.27 (m, 3H), 2.22−2.16 (m, 3H), 2.05−2.00
(m, 4H), 1.82−1.77 (m, 2H), 1.66−1.59 (m, 2H), 1.43−1.38
(m, 63H), 1.31 (s, 9H). HRMS: (M + H)+ calcd for
C85H139N10O22, 1652.0065; found, 1652.0078.
clododecan-1-yl)butanamido)acetamido)ethyl)phenoxy)-
acetamido)-3-phenylpropanamido)pentyl)carbamoyl)-L-
glutamic Acid ((R)-13). A solution of (R)-12 (40 mg, 0.024
mmol) in 1 mL of TFA was stirred at rt for 5 h. The reaction
mixture was evaporated in vacuo. The residue was dissolved in 1
mL of DMSO and purified by semipreparative HPLC (Luna 10u
C8(2) 250 × 10.00 mm, ACN/0.1% TFA in water = 65/35, 4
mL/min) to give 15.5 mg of (R)-13 (yield, 54%) as a colorless
oil. 1H NMR (400 MHz, DMSO): δ 8.18 (d, J = 6.9 Hz, 1H),
8.06 (d, J = 8.0 Hz, 1H), 7.21 (dd, J = 19.1, 9.0 Hz, 2H), 7.10 (d,
J = 5.5 Hz, 1H), 6.97 (s, 1H), 6.74 (d, J = 8.6 Hz, 1H), 6.36−6.26
(m, 1H), 4.56−4.51 (m, 1H), 4.41−4.37 (m, 2H), 4.11−4.03
(m, 1H), 3.77−3.58 (m, 4H), 3.34−2.86 (m, 25H), 2.33−2.22
(m, 3H), 1.92−1.15 (m, 10H). HRMS: (M + H)+ calcd for
C53H75N10O22, 1203.5057; found, 1203.5104. (The LC−MS
profile is shown in Figure S4.)
(((S)-1-Carboxy-5-((S)-2-(2-(4-((S)-2-carboxy-2-(2-((S)-4-
carboxy-4-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacy-
clododecan-1-yl)butanamido)acetamido)ethyl)phenoxy)-
acetamido)-3-phenylpropanamido)pentyl)carbamoyl)-L-
glutamic Acid ((S)-13). Compound (S)-13 (15.1 mg; yield,
41%) was prepared from (S)-12 (50 mg, 0.03 mmol) following
the same procedure as for (R)-13 described above as a colorless
oil. 1H NMR (400 MHz, DMSO): δ 8.18 (d, J = 6.9 Hz, 1H),
8.06 (d, J = 8.0 Hz, 1H), 7.21 (dd, J = 19.1, 9.0 Hz, 2H), 7.10 (d,
J = 5.5 Hz, 1H), 6.97−6.95 (m, 1H), 6.74 (d, J = 8.6 Hz, 1H),
6.33−6.28 (m, 1H), 4.56−4.51 (m, 1H), 4.40−4.42 (m, 2H),
4.13−4.03 (m, 1H), 3.77−3.58 (m, 4H), 3.37−2.82 (m, 25H),
2.67−2.21 (m, 3H), 1.93−1.23 (m, 10H). HRMS: (M + H)+
calcd for C53H75N10O22, 1203.5057; found, 1203.5094. (The
LC−MS profile is shown in Figure S5.)
(R)-[natGa]Ga-13. (R)-13 (1 mg, 0.83 μmol) was dissolved in
254 μL of dimethyl sulfoxide (DMSO) in a small vial, and 0.8 μL
of GaCl3 aqueous (1.14 M, 0.91 μmol) was added. The mixture
was carefully adjusted by dropwise addition of 5 μL of sodium
acetate (NaOAc, 1 N) to pH = 4 and diluted with water to a final
volume of 1 mL (1 mg/mL solution). The vial was closed, and
the reaction mixture was stirred overnight at rt. The completion
of the formation of (R)-[natGa]Ga-13 was verified by LC−MS
where no (R)-13 could be anymore detected. The resulting
aqueous solution (1 mg/mL (R)-[natGa]Ga-13) was diluted and
used in the in vitro binding affinity studies without further
processing. HRMS: (M + H)+ calcd for C53H72N10O22Ga,
1269.4078; found, 1269.4053. (The LC−MS profile is shown in
Figure S6.)
(S)-[natGa]Ga-13. (S)-[natGa]Ga-13 was prepared from (S)-
13 (1 mg, 0.83 μmol) following the same procedure as for (R)-
[
natGa]Ga-13. HRMS: (M)+ calcd for C53H72N10O22Ga,
1269.4078; found, 1269.4070. (The LC−MS profile is shown
in Figure S7.)
(R)-[natLu]Lu-13. (R)-13 (1 mg, 0.83 μmol) was dissolved in
254 μL of DMSO in a small vial, and 9 μL of LuCl3 aqueous (30
mg/mL, 0.91 μmol) was added. The mixture was carefully
adjusted by dropwise addition of 10 μL of NaOAc (1 N) to pH =
5 and diluted with water to a final volume of 1 mL (1 mg/mL
solution). The vial was closed, and the reaction mixture was
stirred for 2 h at 90 °C. The completion of the formation of (R)-
[natLu]Lu-13 was verified by LC−MS where no (R)-13 could be
anymore detected. The resulting aqueous solution (1 mg/mL
(R)-[natLu]Lu-13) was further diluted and used in in vitro
binding affinity studies without further processing. HRMS: (M +
H)+ calcd for C53H70N10O22Lu, 1375.4230; found, 1375.4173.
(The LC−MS profile is shown in Figure S8.)
(((S)-1-Carboxy-5-((S)-2-(2-(4-((S)-2-carboxy-2-(2-((R)-4-
carboxy-4-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacy-
G
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(S)-[natLu]Lu-13. (S)-[natLu]Lu-13 was prepared from (S)-13
(1 mg, 0.83 μmol) following the same procedure as for (R)-
[natLu]Lu-13. HRMS: (M + H)+ calcd for C53H70N10O22Lu,
1375.4230; found, 1375.4203. (The LC−MS profile is shown in
Figure S9.)
Tumor-bearing mice were used for studies when tumor reached
a diameter of approximately 5−10 mm.
In Vitro Competition Binding Assays for PSMA
Binding Affinity. Assays were carried out by incubating
PSMA-positive PC3-PIP cells with 0.2 nM, 3.7 MBq [125I]MIP-
109540 (structure shown in Figure S10) in the presence of 10
different concentrations (10−5−10−10 M) of competing “cold”
ligands. Nonspecific binding (NSB) was defined with 2 μM
PSMA-617. After incubation at 37 °C for 1 h, the bound and free
fractions were separated by vacuum filtration through a GF/B
filter paper using a Brandel M-24R cell harvester. The filters
were washed twice with cold Tris−HCl buffer (50 mM, pH 7.4),
and the radioactivity was counted in a gamma counter (Wizard2,
PerkinElmer). The IC50 values were calculated by GraphPad
Prism.
Comparison of Radiolabeling of (R)- and (S)-[68Ga]Ga-
13 and (R)- and (S)-[177Lu]Lu-13 to [68Ga]Ga-PSMA-617
and [177Lu]Lu-PSMA-617. Eluent 68GaCl3 (0.3 mL) in 0.05 M
HCl from a 68Ge/68Ga generator and 30 μL of 0.5 N NaOAc
were mixed and added to 5 μg of (R)- and (S)-13 or PSMA-617
(in DMSO). 177LuCl3 (10 μL, 1.85 MBq, ≤925 GBq/mg, in 0.05
M HCl (0.43 mL)) and 50 μL of 0.5 N NaOAc were mixed and
added to 2 μg of (R)- and (S)-13 or PSMA-617 (in DMSO).
The reaction mixtures were incubated at 50 °C, and aliquots
were removed at different time points (5, 10, 15, 20, 25, and 30
min). The radiochemical yields were determined by TLC
(iTLC-SG CH3OH, H2O, 30% NH4OH (2:2:0.1 v/v/v)).
Comparison of Radiolabeling of (R)- and (S)-[68Ga]Ga-
13, (R)- and (S)-[177Lu]Lu-13, and [177Lu]Lu-PSMA-617 for
In Vitro and In Vivo Studies. 68GaCl3 (0.5 mL, 55.5 MBq) in
0.05 M HCl of a 68Ge/68Ga generator and 50 μL of 0.5 N
NaOAc were mixed and added to 20 μg of (R)- and (S)-13 (in
DMSO). The reaction mixture was incubated at 80 °C for 10
min. [68Ga]Ga-PSMA-093 was synthesized following the
reported method.29 177LuCl3 (10 μL, 18.5 MBq, ≤925 GBq/
mg) was diluted with 0.05 M HCl (0.43 mL), and 50 μL of 0.5 N
NaOAc was added and mixed with 10 μg of (R)- and (S)-13 or
PSMA-617 (in DMSO). The reaction mixtures were incubated
at 80 °C for 20 min. Radiochemical yields were determined by
radio-HPLC (stationary phase: Supelco Ascentis C18, 5 μm,
150 × 4.6 mm) column; mobile phase: 0.1% TFA in water/ACN
with a gradient of 0−15 min, from 100% H2O with 0.1% TFA to
100% ACN; and a flow rate of 1 mL/min). Radiochemical
purities of all [68Ga]Ga-labeled and [177Lu]Lu-labeled agents,
(R)- and (S)-[68Ga]Ga-13 (30 μM, 1.6 GBq/μmol), (R)- and
(S)-[177Lu]Lu-13 (16 μM, 2.3 GBq/μmol), and [177Lu]Lu-
PSMA617, were >95%. The tracers were diluted and used in
vitro and in vivo experiments without purification.
Cell Binding and Internalization. PC3-PIP cells were
cultured in 12-well plates 24 h before cell uptake studies (5 × 105
cells/well). After washing, the cells were incubated with 37 kBq
of [68Ga]Ga-PSMA-093, (R)- and(S)-[68Ga]Ga-13, (R)- and
(S)-[177Lu]Lu-13, and [177Lu]Lu-PSMA-617 for 5, 15, 30, 60,
90, and 120 min at 37 °C. At the indicated time, the medium was
removed and the cells were washed twice with 1 mL of cold PBS.
Cells were subsequently incubated twice with 1 mL of glycine−
HCl in PBS (50 mM, pH 2.8) for 5 min to remove the surface-
bound fraction. The cells were lysed using 0.1 mL of 1 N NaOH.
The lysed cells were removed by filters and measured in a
gamma counter. The cell uptake was calculated as percent of the
initial dose bound to 106 cells [%ID/106 cells]. Experiments
were performed in triplicate.
Biodistribution Studies. [68Ga]Ga-PSMA-093, [68Ga]Ga-
PSMA-617, and (R)- and (S)-[68Ga]Ga-13 were injected (629
kBq, 0.16 nmol) via tail vein of PC-3 and PC3-PIP tumor-
bearing nude mice. Organs of interest were dissected and
weighed after the animals were sacrificed at 1 h. The
radioactivity was measured with a gamma counter, and the %
ID/g was calculated by comparison with samples of standard
dilution of the initial dose. All measurements were corrected for
decay.
In Vitro Stability in PBS and Plasma. (R)- and (S)-
RESULTS AND DISCUSSION
■
[
177Lu]Lu-13 were synthesized in 20 min at 80 °C as described
Syntheses of DOTAGA(tBu)4 ((R)-, (S)-, and (R,S)-9).
Syntheses of DOTAGA(tBu)4 ((R)-, (S)-, and (R,S)-9) were
successfully achieved in two steps: synthesis of the mixture and
chiral stereoselective side arms, 2 and (S)- and (R)-6, and
synthesis of DOTAGA(tBu)4 (Schemes 1 and 2). Compound 2,
containing a racemic side arm, was prepared from (S)-glutamic
acid 5-methyl ester using a synthesis method reported
previously.41 The chirality at the C2 position of 1 was partially
racemized during the displacement of bromide. Subsequently,
the acid group was protected with TBTA (tert-butyl
trichloroacetimide) to give (racemic)-2 in 80% yield. Com-
pounds (S)- and (R)-6, containing a C2 chiral −OMs group,
were prepared from the corresponding (S)- and (R)-glutamic
acids, respectively.36,37,42 Stereoselective preparation of (S)- and
(R)-3 was achieved by addition of NaNO2 solution to the excess
HCl reaction solution containing either (S)- or (R)-glutamic
acid at low temperature (between 0 and 5 °C). Under this
condition, the C2 optical center retained the chirality. The
above. These tracers (RCPs, >95%) were used for in vitro
stability without purification. (R)- and (S)-[177Lu]Lu-13 were
added to 0.1 M phosphate-buffered saline (PBS, pH 7.0) or
plasma (purchased from Innovative Research, MI), and the
solutions were incubated at rt for 7 days. The stabilities were
measured and analyzed by radio-HPLC to determine the
stability as a function of time. Corresponding stabilities of (R)-
and (S)-[68Ga]Ga-13 were also evaluated in PBS and similarly
analyzed by HPLC at 2 h at room temperature (rt) after the
preparation.
Cell Culture and Tumor Mouse Models. PC3-PIP38
(PSMA-positive, (generously provided by Dr. Sean Carlin,
University of Pennsylvania)) and PC3 (PSMA-negative,
purchased from ATCC, American Type Culture Collection)
cell lines39 were used for cell uptake studies and inoculation in
CD-1 nu/nu mice. The cells were cultured in the RPMI 1640
medium supplemented with 10% fetal bovine serum and 1%
antibiotics (penicillin/streptomycin) in a humidified incubator
equilibrated with 5% CO2 at 37 °C. After treatment with 0.25%
trypsin with 0.02% EDTA and being resuspended in HBSS, each
mouse was injected with 0.2 mL (5 × 106 cells) of the cell
suspension in the left flank (PC3-PIP) or right flank (PC3).
t
corresponding Bu-protected esters, (S)- and (R)-4, were
synthesized in 87% yield using the optimized procedure
described above. The reaction yields were better than those
reported previously using 4-dimethylaminopyridine (DMAP)
and N,N′-dicyclohexylcarbodiimide (DCC) as the catalyst.36
H
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Figure 3. HPLC profiles of (R)-(+)-α-methylbenzylamide derivatives, (R)-, (S)-, and (R,S)-10, under the following conditions: PRP-1 10 μm, 250 ×
4.6 mm column, ACN/0.5% TFA in water = 4/6, 2 mL/min, and 10 °C. (A, B) Two stereoisomers (R)-10 (A) and (S)-10 (B) separated under these
conditions with retention times of 16 and 19 min, respectively. (C) Prepared mixture of (R,S)-10 showed a 7 to 3 ratio of R to S. The corresponding
NMR spectroscopic data also support the conclusion. (NMR data of (R)-(+)-α-methylbenzylamide derivatives, (R)-, (S)-, and (R,S)-10, are included
in the Supporting Information; Figures S1−S3.)
(S)- and (R)-4 were subjected to the lactone ring opening by
sodium methoxide (NaOMe) in MeOH at 0 °C for 30 min to
give (S)- and (R)-5 in 77% yield. This one-step lactone ring
opening with NaOMe was faster and easier to handle than the
KOH hydrolysis and benzyl protection as reported earlier.36
Finally, the secondary alcohols, (S)- and (R)-5, were stereo-
selectively converted to the corresponding mesylates to provide
(S)- and (R)-6, respectively. Mesylate was chosen as the leaving
group because the controlled alkylation of cyclen by the SN2
reaction afforded a complete inversion of stereochemistry at the
C2 optical center of the glutamic acid side arm. The desired
products, DOTAGA(tBu)4 ((R)- and (S)-9), were readily
prepared in three steps in 30% yield.
and (S)-13 will be prepared as described above, and they will
show similar optical purity that described above for (R)-10 and
(S)-10, respectively (Scheme 3).
Synthesis of [natGa]Ga- and [natLu]Lu-DOTAGA-PSMA-
093, (R)-[natGa]Ga-13, (R)-[natLu]Lu-13, (S)-[natGa]Ga-13,
and (S)-[natLu]Lu-13. Synthetic schemes of unlabeled refer-
ence ligands, (R)-[natGa]Ga-13, (R)-[natLu]Lu-13, (S)-[natGa]-
Ga-13, and (S)-[natLu]Lu-13 complexes, are summarized in
Scheme 3. The key intermediate compound 11, containing the
linker and the PSMA binding group, −Glu-NH-CO-NH-Lys,
was prepared as previously reported.29 Compounds (R)- and
(S)-12 were prepared by a coupling reaction of DOTAGA-
(tBu)4, (R)- and (S)-9, in 78 and 51%, respectively. After
removing tert-butyl protection groups by TFA to give
compounds (R)- and (S)-13 in 54 and 41% yields, respectively,
(LC−MS profiles are shown in Figures S4 and S5, respectively.)
the desired precursors (R)- and (S)-13 were used for
radiolabeling of [68Ga]Ga-13 and [177Lu]Lu-13 and synthesis
of “cold” compounds, [natGa]Ga-13 and [natLu]Lu-13 (LC−MS
profiles are shown in Figures S6−S9). Unfortunately, attempts
in making the corresponding crystals for structural determi-
nation of the chelates, (R)- and (S)-13, and the corresponding
metal (natGa and natLu) complexes were not successful; this part
of the work remains to be accomplished in the future.
The optical purity of DOTAGA(tBu)4 ((R)-, (S)-, and (R,S)-
9), prepared above was not easily determined. The synthesis
employed optically defined precursors, 2, (S)-6, and (R)-6,
through which presumably optically specific (R)-, (S)-, and
(R,S)-9 were prepared (Schemes 1 and 2). However, the specific
optical purities of (R)-, (S)-, and (R,S)-9 cannot be easily
measured by HPLC as they are enantiomers and lack a
chromophore. To add a suitable chromophore and enhance
optical characteristics, small samples of (R)-, (S)-, and (R,S)-9
were converted to diastereomeric (R)-(+)-α-methylbenzyla-
mide derivatives, (R)-, (S)-, and (R,S)-10, respectively. The
HPLC profiles showed that the chiral purity ((R)-10 and (S)-
10) was >99%, and the racemic DOTAGA(tBu)4, (R,S)-10 was a
70%/30% (R/S) mixture of diastereomers (Figure 3). Addi-
tionally, the 1H NMR spectrum of (R,S)-10 also indicated that it
is consisted of a 70%/30% racemic mixture according to the
ratio of the NH proton on (R,S)-10 (Figures S1−S3). The same
starting materials were also employed for synthesis of optically
pure (R)- and (S)-13. It is expected that the optically pure (R)-
Radiosynthesis of [68Ga]Ga- and [177Lu]Lu-DOTAGA-
PSMA-093, (R)-[68Ga]Ga-13, (R)-[177Lu]Lu-13, (S)-
[68Ga]Ga-13, (S)-[177Lu]Lu-13, and [68Ga]Ga/[177Lu]Lu-
PSMA-617. Gallium-68 was obtained from a 68Ge/68Ga
generator (ITG, Germany), and lutetium-177 was obtained
from the National Isotope Development Center, Oak Ridge
National Laboratory (Oak Ridge, TN). Initially, it was
determined that complex formation of (R)- and (S)-[68Ga]Ga-
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Figure 4. (A, B) Kinetics of complex formation was measured at 50 °C for (R)- and (S)-[68Ga]Ga-13 and [68Ga]Ga-PSMA-617 (A) and (R)- and (S)-
[
177Lu]Lu-13 and [177Lu]Lu-PSMA-617 (B). Radiochemical yield (RCY) was determined by the TLC method (n = 3) (**statistically p < 0.01). The
formation of (S)-[177Lu]Lu-13 displayed a much faster rate than those of (R)-[177Lu]Lu-13 and [177Lu]Lu-PSMA-617. This suggests that the
stereochemistry of the extra glutamic acid arm on 177Lu-DOTAGA in combination with the stereochemistry of the linker and PSMA part played an
important role in complex formation. Results presented in this section clearly demonstrate that the speed of 177Lu-DOTAGA-PSMA-093 complex
formation is dependent on the stereochemistry of the extra glutamic acid arm.
13, (R)- and (S)-[177Lu]Lu-13, and [68Ga]Ga/[177Lu]Lu-
PSMA-617 were readily accomplished in a few minutes at
reaction temperature above 80 °C. To study the effect of
stereoselective isomers on the kinetics of complex formation,
labeling was performed at 50 °C, a temperature in which the
kinetics of complex formation was sufficiently slow and
measurable at different time points. As shown in Figure 4, the
labeling yields of (R)- and (S)-[68Ga]Ga-13, (R)- and (S)-
Gd-DOTAGA was amenable to conjugation to a variety of
chemical moieties. More importantly, the additional arm on
DOTAGA introduced by adding one or more glutamic acid
groups enhanced rotational immobilization and provided a large
enhancement of magnetic relaxivity and stability.43−45 Similarly,
lanthanide complexes of DOTA containing four glutamic arms,
resulting in adding four optical centers, have been reported. The
results of complex formation analysis and quantum mechanical
calculations for the Ln-DOTA(GA)4 complexes suggested that
there was a rapid formation of metastable species, consisting of
multiple isomeric forms, leading to a slow formation and
reorganization of the macrocycle. Specifically, greater stability of
these final Gd(III) complexes resulted from formation of
transitory bonds between the metal ion and pendant glutaric
arms, which contained four carboxylate groups. One major
improvement of adding glutamic arms on DOTA(GA)x (where
x = 1 to 4) for chelating Gd(III) was that the stability constants
of the final complexes had higher values than those for DOTA
without the added glutamic acid arms.46 It is reasonable to
conclude that there were at least two major advantages of
DOTAGA compared to DOTA: (1) faster kinetics of forming
Gd-DOTAGA complex(es) and (2) enhanced stability provided
by the additional glutamic arms.47 There were suggestions of
differences in the rate of formation and transition between
different stereoisomers; however, the extent of such effects was
not fully evaluated.46,47 We expect that these findings would
likely be consistent with the complex formation of Ga/Lu-
DOTAGA and would perhaps also be applicable to many other
3+ charged metals.8
[
177Lu]Lu-13, and [68Ga]Ga/[177Lu]Lu-PSMA-617 increased
over 30 min. The formation of (S)-[68Ga]Ga-13, the (S)-
DOTAGA chelator, displayed a small but noticeable faster
kinetics in comparison with (R)-[68Ga]Ga-13 and [68Ga]Ga-
PSMA-617, the (R)-DOTAGA and DOTA chelators, respec-
tively. Meanwhile, the formation of (S)-[177Lu]Lu-13 showed
significantly faster kinetics in comparison with (R)-[177Lu]Lu-
13 and [177Lu]Lu-PSMA-617, and the (R)-[177Lu]Lu-13 also
showed a noticeable faster complex rate compared with
[
177Lu]Lu-PSMA-617. It appeared that (S)-DOTAGA was the
preferred isomer showing a clearly faster rate of 177Lu complex
formation and a negligible faster rate of 68Ga complex formation
in comparison with (R)-DOTAGA and DOTA. Unlike the six
coordination of Ga(III)-DOTAGA, the coordination of Lu(III)
with DOTAGA prefer nine-coordinate covalent bonds, which
would be affected by the stereochemistry of an extra glutamic
acid arm.
For routine preparations, (R)- and (S)-[68Ga]Ga-13 were
successfully prepared at 80 °C by mixing [68Ga]GaCl3 in 0.05 M
HCl with 20 μg of (R)- and (S)-13 in a NaOAc buffer (pH = 5)
for 10 min. (R)- and (S)-[177Lu]Lu-13 and [177Lu]Lu-PSMA-
617 were successfully prepared at 90 °C by mixing [177Lu]LuCl3
in 0.05 M HCl with 10 μg of (R)- and (S)-13 in a NaOAc buffer
(pH = 5) for 20 min. Radiochemical purities were consistently
>95%, which were confirmed by radio-HPLC analysis.
Another example of adding (R)- or (S)-glutamic acid arms on
DOTA in developing MRI contrast agents has been
reported.43,44 The importance of the glutamic acid arm and its
optical isomers ((R)- vs (S)-glutamic isomers) on the stability
and in vivo kinetics of Gd-DOTAGA vs DOTA was not trivial,
and its significance has been identified and evaluated.43−45 It was
discovered that there were major differences between Gd-
DOTA and Gd-DOTAGA. The adjacent glutamic acid arm of
In Vitro Stability in PBS and Plasma. The stabilities of
(R)- and (S)-[177Lu]Lu-13, synthesized by routine preparations,
were investigated by incubation in PBS and in plasma at 37 °C.
After incubation for 7 days, the radiochemical purities remained
unchanged, maintaining a stable RCP of approximately >90%.
(Radio-HPLC profiles are shown in the Supporting Information,
Figures S10 and S11, respectively). Release of 177Lu from
complexes was not observed under respective experimental
conditions in the examined time. As expected, (R)- and (S)-
[
177Lu]Lu-13 were stable in PBS without any measurable
decomposition. HPLC profiles of (R)- and (S)-[68Ga]Ga-13
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suggested that they were stable in PBS at room temperature 2 h
after preparation.
Therefore, the (S)- and (R)-DOTAGA chelating groups showed
no significant influence in the PSMA binding affinity. This is
likely due to the fact that the binding pocket is far away from the
urea-binding moiety, which is the moiety responsible for PSMA
binding.
In Vitro Assay for PSMA Binding Affinity. The PSMA
binding affinities of natGa and natLu analogues were determined
in competitive binding assays using PC3-PIP cell homogenates
and [125I]MIP-1095 (structure shown in Figure S12), a known
PSMA radiotracer as the “hot” ligand for competition.40 The
relatively long half-life of 125I (t1/2, 60 days) makes it simple by
preparing one batch of [125I]MIP-1095 for multiple experi-
ments. The IC50 values (mean SD) are summarized in Table 1.
Cell Binding and Internalization Study. In vitro cell
binding and specific internalization (PC3-PIP cells over-
expressing PSMA) were determined using [68Ga]Ga-PSMA-
093 and [177Lu]Lu-PSMA-617 as known controls, respectively.
The cell binding and internalization of 68Ga-ligands and 177Lu-
ligands increased over incubation time. (Complete results are
presented in Figure S4.) The total cell uptake values of 68Ga-
ligands, (R)- and (S)-[68Ga]Ga-13 and [68Ga]Ga-PSMA-093,
were comparable (18.4, 14.5, and 17.4% ID/106 cells at 1 h;
25.3, 22.5, and 29.8% ID/106 cells at 2 h, respectively), while
[68Ga]Ga-PSMA-093 showed a significantly higher internal-
ization in comparison with (R)- and (S)-[68Ga]Ga-13 (12.0, 5.8,
and 5.5% ID/106 cells at 1 h; 21.9, 11.2, and 9.5% ID/106 cells at
2 h, respectively). This is likely due to the fact that the advantage
of HBED-CC aromatic stacking improved the internalization
(Figure 5A). (R)- and (S)-[177Lu]Lu-13 and [177Lu]Lu-PSMA-
617 displayed comparable total cell uptake (20.2, 20.6, and
20.0% ID/106 cells at 1 h; 24.2, 23.3, and 24.7% ID/106 cells at 2
h, respectively) and internalization (7.4, 7.2, and 7.3% ID/106
cells at 1 h; 10.9, 10.8, and 11.3% ID/106 cells at 2 h,
respectively) (Figure 5B). It is apparent that (R)- and (S)-
DOTAGA-based [68Ga/177Lu]-13 displayed negligible isomeric
effect on the cell uptake studies. Evidently, [68Ga]Ga-PSMA-093
was the only agent displaying a much higher internalization in
the PSMA-expressing cells under the same condition.
Table 1. Binding Affinities to PSMA (IC50 Values, Mean
a
SD, n = 4)
ligands
MIP-1095
PSMA-093
natGa]Ga-PSMA-093
(R)-13
(S)-13
(R)-[natGa]Ga-13
(S)-[natGa]Ga-13
(R)-[natLu]Lu-13
(S)-[natLu]Lu-13
IC50 (nM)
5.4 1.0
13.0 0.7
34.0 4.5
24.2 2.8
45.7 9.4
58.4 38.9
111.3 26.3
46.3 13.5
68.1 13.9
[
a
Binding assays were carried out using PC3-PIP cell homogenates and
125I]MIP-109540 as the “hot” ligand for competition.
[
(The detailed data are shown in Table S1.) All of (S)- and (R)-
13 and corresponding natGa and natLu equivalents displayed high
binding affinity (IC50 = 24−111 nM), values comparable or
slightly higher than that of the parent agent, [natGa]Ga-PSMA-
093 (IC50 = 34.0 4.5 nM). (S)-13, showed a slightly lower
affinity than (R)-13 (IC50 values of 45.7 9.4 vs 24.2 2.8 nM,
respectively). In addition, the inhibition potency for the
isomeric pair, (R)- and (S)-[natGa]Ga-13 and (R)- and (S)-
[natLu]Lu-13 demonstrated different IC50 values (58.4 38.9 vs
In Vivo Biodistribution Study in Tumor-Bearing Mice.
A comparison of the biodistribution (1 h p.i.) of [68Ga]Ga-
PSMA-093, [68Ga]Ga-PSMA-617, and (R)- and (S)-[68Ga]Ga-
13 in male nude mice bearing PSMA-positive PC3-PIP and
PSMA-negative PC-3 tumors is summarized in Table 2.
The biodistribution data between [68Ga]Ga-PSMA-093,
[68Ga]Ga-PSMA-617, and (R)- and (S)-[68Ga]Ga-13 displayed
comparable values. There was higher uptake of (R)- and (S)-
[68Ga]Ga-13 in the PSMA-expressing PC3-PIP xenograft
111.3
26.3 nM and 46.3
13.5 vs 68.1
13.9 nM,
respectively). It is apparent that the (R)-isomers displayed
slightly higher binding affinity in comparison with the (S)-
isomers, and all values were in the same order of magnitude.
tumors in mice (12.1
1.91 and 13.3
0.39% ID/g,
Figure 5. Cell uptake and internalization of 68Ga- and 177Lu-labeled compounds were evaluated in PC3-PIP cells for 1 and 2 h of incubation (n = 3).
Values are expressed as percent of the initial dose bound to 106 cells [% ID/106 cells] (**statistically p < 0.01). (A) (R)-[68Ga]Ga-13 (white), (S)-
[68Ga]Ga-13 (black), and [68Ga]Ga-PSMA-093 (gray) were evaluated in PC3-PIP cells for 1 and 2 h of incubation. (R)-[68Ga]Ga-13 (white) and (S)-
[68Ga]Ga-13 (black) showed very similar uptake values. Internalization values of [68Ga]Ga-PSMA-093 at 1 and 2 h were much higher than those of
(R)-[68Ga]Ga-13 (white) and (S)-[68Ga]Ga-13 (black). (B) (R)-[177Lu]Lu-13 (white), (S)-[177Lu]Lu-13 (black), and [177Lu]Lu-PSMA-617 (gray)
showed very similar cell uptake values at 1 and 2 h. Results of more detailed kinetics are shown in Figure S13 (Supporting Information).
K
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Table 2. Biodistribution at 1 h Post-Injection in Tumor-Bearing Mice of [68Ga]Ga-PSMA-093, [68Ga]Ga-PSMA-617, and (R)-
and (S)-[68Ga]Ga-13 (% ID/g, Avg SD, n = 3)
part
[68Ga]Ga-PSMA-093
[68Ga]Ga-PSMA-617
(R)-[68Ga]Ga-13
(S)-[68Ga]Ga-13
blood
heart
muscle
lung
kidney
spleen
pancreas
liver
stomach
skin
bone
PC3-PIP tumor
PC-3 tumor
0.64 0.10
0.63 0.09
0.68 0.39
2.37 0.70
165.1 50.1
20.0 13.2
1.32 0.58
0.94 0.34
0.60 0.54
1.56 0.35
0.31 0.06
19.2 5.66
1.77 0.63
0.44 0.11
0.15 0.04
0.11 0.03
0.55 0.15
25.6 31.5
0.86 0.32
0.18 0.12
1.94 0.57
0.06 0.01
0.36 0.19
0.09 0.03
10.1 1.84
0.50 0.30
0.86 0.09
0.45 0.16
0.29 0.04
1.18 0.13
144.6 16.2
2.52 0.70
0.76 0.18
0.34 0.03
0.33 0.15
0.85 0.29
0.50 0.23
12.1 1.91
0.96 0.05
1.54 0.07
0.57 0.12
0.35 0.04
1.57 0.04
131.4 1.43
2.80 0.23
1.13 0.62
0.56 0.04
0.62 0.14
1.23 0.20
0.43 0.02
13.3 0.39
1.41 0.03
a
a
a
PC-3 (PSMA−)/PC3-PIP (PSMA+) cells were implanted in nude mice.39
respectively) compared to [68Ga]Ga-PSMA-617 (uptake in
PC3-PIP tumor was 10.1 1.84 %ID/g), while there was very
low uptake in PC-3 tumor (PSMA negative). The new PSMA
agents, (R)- and (S)-[68Ga]Ga-13, clearly displayed a 30% lower
tumor uptake than [68Ga]Ga-PSMA-093 and 20% higher than
[68Ga]Ga-PSMA-617. All PSMA-093 ligands, [68Ga]Ga-PSMA-
093 and (R)- and (S)-[68Ga]Ga-13, showed higher kidney
noteworthy and should not be ignored in preparation of 177Lu-
DOTAGA derivatives for future applications.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.molpharma-
ceut.0c00777.
uptake compared with [68Ga]Ga-PSMA-617 (165.1
50.1,
31.5% ID/g,
144.6
16.2, 131.4
1.43, and 25.6
Synthesis of compounds 2 and (R)-6, 1H NMR of
compound (R,S)-10, 1H NMR of compound (R)-10, 1H
NMR of compound (S)-10, LC−MS of compound (R)-
13, LC−MS of compound (S)-13, LC−MS of compound
(R)-[natGa]Ga-13, LC−MS of compound (S)-[natGa]Ga-
13, LC−MS of compound (R)-[natLu]Lu-13, LC−MS of
compound (S)-[natLu]Lu-13, radio-HPLC profile of (R)-
respectively). However, 177Lu-ligands showed much lower
kidney uptake compared with 68Ga-ligands as previous
reported.48 It is likely due to the fact that the PSMA-targeting
properties of [177Lu]Lu-13 are comparable to those of
[
177Lu]Lu-PSMA-617, a theragnostic ligand currently in clinical
evaluation. The Ki of [177Lu]Lu-PSMA-617 was reported as
being 2.34 2.94 nM determined with LNCaP cells, while our
studies of [177Lu]Lu-13 were measured with PSMA-expressing
PC3-PIP cells.49 Apparently, the in vivo biodistribution was not
affected by the optical isomer formation for 68Ga/177Lu-
DOTAGA complexes. However, in vivo stability of these
isomers in humans needs to be evaluated further in the future.
More recently, a radiohybrid PSMA-targeting agent,
rhPSMA-7.3, was reported (see Figure 2).30−33 A phase 3
“SPOTLIGHT” trial, which examines the investigational PSMA-
targeting PET imaging agent, rhPSMA-7.3 F18, in men with
suspected prostate cancer recurrence, was recently announced
by Blue Earth Diagnostic Inc. It is noted that the glutamic acid
arm of the DOTAGA chelating group of rhPSMA-7.3 is reported
to be the (S)-isomer.
[
177Lu]Lu-13 and stability in PBS and plasma, radio-
HPLC profile of (S)-[177Lu]Lu-13 and stability in PBS
and plasma, structure of [125I]MIP-1095 and IC50 values,
and cell uptake and internalization of 68Ga- and 177Lu-
labeled compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Hank F. Kung − Department of Radiology, University of
Pennsylvania, Philadelphia, Pennsylvania 19104, United States;
Five Eleven Pharma Inc., Philadelphia, Pennsylvania 19104,
United States; Email: kunghf@gmail.com
Authors
Ruiyue Zhao − College of Chemistry, Beijing Normal University,
CONCLUSIONS
■
Beijing 100875, PR China
In summary, new PSMA-targeting agents were prepared by
combining stereoselective (R)- and (S)-DOTAGA chelators
and the PSMA-093 (without HBED) moiety. Resulting optically
pure isomers, (R)- and (S)-[68Ga/177Lu]-13, showed compara-
ble PSMA-targeting properties. However, the formation of (S)-
Karl Ploessl − Five Eleven Pharma Inc., Philadelphia,
Pennsylvania 19104, United States; orcid.org/0000-0001-
7896-5900
Zhihao Zha − Five Eleven Pharma Inc., Philadelphia,
Pennsylvania 19104, United States
Seokrye Choi − Five Eleven Pharma Inc., Philadelphia,
Pennsylvania 19104, United States
David Alexoff − Five Eleven Pharma Inc., Philadelphia,
Pennsylvania 19104, United States
[
177Lu]Lu-13 showed significantly faster labeling kinetics than
that of (R)-[177Lu]Lu-13, demonstrating a clear stereo effect of
(R)- and (S)-DOTAGA-PSMA-093 on 177Lu chelation. The
(S)-DOTAGA-PSMA ligand, (S)-13, may serve as a useful
candidate ligand for developing diagnostic and therapeutic
agents targeting PSMA binding in tumors. Although there are no
differences in PSMA targeting between (R)- and (S)-DOTAGA
isomers, their differences in forming metal complexes are
Lin Zhu − College of Chemistry, Beijing Normal University, Beijing
100875, PR China; orcid.org/0000-0001-9530-8531
Complete contact information is available at:
L
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Wang, J.; Rotile, N. J.; Schirmer, M. D.; Santos Ferreira, D. D.; Sui, J.;
Fuchs, B. C.; Lanuti, M.; Caravan, P. 68Ga-NODAGA-Indole: An
Allysine-Reactive Positron Emission Tomography Probe for Molecular
Imaging of Pulmonary Fibrogenesis. J. Am. Chem. Soc. 2019, 141,
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https://pubs.acs.org/10.1021/acs.molpharmaceut.0c00777
Notes
The authors declare the following competing financial
interest(s): This work was supported by Five Eleven Pharma
Inc. I am the founder of this company.
(11) Satpati, D.; Shinto, A.; Kamaleshwaran, K. K.; Sarma, H. D.;
Dash, A. Preliminary PET/CT Imaging with Somatostatin Analogs
[68Ga]DOTAGA-TATE and [68Ga]DOTAGA-TOC. Mol. Imaging
Biol. 2017, 19, 878−884.
ACKNOWLEDGMENTS
■
Five Eleven Pharma, Inc. was the sponsor of this study.
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Zarschler, K.; Wang, X.; Radchenko, V.; Kubeil, M.; Stephan, H.;
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McNeil, B. J.; Baumann, M.; Gambhir, S. S.; Hricak, H.; Weissleder, R.
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(14) Weineisen, M.; Schottelius, M.; Simecek, J.; Baum, R. P.; Yildiz,
A.; Beykan, S.; Kulkarni, H. R.; Lassmann, M.; Klette, I.; Eiber, M.;
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(15) Weineisen, M.; Simecek, J.; Schottelius, M.; Schwaiger, M.;
Wester, H. J. Synthesis and preclinical evaluation of DOTAGA-
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ABBREVIATIONS
■
PSMA, prostate-specific membrane antigen; DTPA, diethylene-
triaminepentaacetic acid; AAZTA, 6-[bis(hydroxycarbonyl-
methyl)amino]-1,4-bis(hydroxylcarbonylmethyl)-6-methylper-
hydro-1,4-diazepine; TRAP, 1,4,7-triazacyclononane-1,4,7-
triyltris(methylenephosphonic acid); DEDPA, 6,6′-[1,2-
ethanediylbis(iminomethylene)]bis[2-pyridinecarboxylic
acid]; NOTA, 1,4,7-triazacyclononane-1,4,7-triacetic acid;
DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid; DO3AM, 2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tet-
raazacyclododecan-1-yl)acetic acid; NOTAGA, 1,4,7-triazacy-
clononane-1-glutaric acid-4,7-acetic acid; DOTAGA, α-(2-
carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-
acetic acid; HBED-CC, N,N′-bis[2-hydroxy-5(carboxyethyl)-
benzyl]ethylenediamine-N,N′-diacetic acid
(16) Cytawa, W.; Seitz, A. K.; Kircher, S.; Fukushima, K.; Tran-Gia, J.;
Schirbel, A.; Bandurski, T.; Lass, P.; Krebs, M.; Połom, W.;
Matuszewski, M.; Wester, H.-J.; Buck, A. K.; Kubler, H.; Lapa, C.
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68Ga-PSMA I&T PET/CT for primary staging of prostate cancer. Eur. J.
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