Functionally Versatile and Highly Stable Chelator for 111In and 177Lu: Proof-of-Principle Prostate-Specific Membrane Antigen Targeting

Article abstract of DOI:10.1021/acs.bioconjchem.9b00225

Here, we present the synthesis and characterization of a new potentially nonadentate chelator H₄pypa and its bifunctional analogue ^tBu₄pypa-C7-NHS conjugated to prostate-specific membrane antigen (PSMA)-targeting peptidomimetic (Glu-urea-Lys). H₄pypa is very functionally versatile and biologically stable. Compared to the conventional chelators (e.g., DOTA, DTPA), H₄pypa has outstanding affinities for both ¹¹¹In (EC, t_1/2 ≈ 2.8 days) and ¹⁷⁷Lu (β^-,?, t_1/2 ≈ 6.64 days). Its radiolabeled complexes were achieved at >98% radiochemical yield, RT within 10 min, at a ligand concentration as low as 10^-6 M, with excellent stability in human serum over at least 5-7 days (<1% transchelation). The thermodynamic stabilities of the [M(pypa)]^- complexes (M³⁺ = In³⁺, Lu³⁺, La³⁺) were dependent on the ionic radii, where the smaller In³⁺ has the highest pM value (30.5), followed by Lu³⁺ (22.6) and La³⁺ (19.9). All pM values are remarkably higher than those with DOTA, DTPA, H₄octapa, H₄octox, and H₄neunpa. Moreover, the facile and versatile bifunctionalization enabled by the p-OH group in the central pyridyl bridge of the pypa scaffold (compound 14) allows incorporation of a variety of linkers for bioconjugation through easy nucleophilic substitution. In this work, an alkyl linker was selected to couple H₄pypa to a PSMA-targeting pharmacophore, proving that the bioconjugation sacrifices neither the tumor-targeting nor the chelation properties. The biodistribution profiles of ¹¹¹In- and ¹⁷⁷Lu-labeled tracers are different, but promising, with the ¹⁷⁷Lu analogue particularly outstanding.

Full text of DOI:10.1021/acs.bioconjchem.9b00225

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Article
Functionally Versatile and Highly Stable Chelator for 111In and 177Lu:
Proof-of-principle Prostate Specific Membrane Antigen Targeting
LILY LI, María de Guadalupe Jaraquemada-Peláez, Hsiou-Ting Kuo, Helen Merkens,
Neha Choudhary, Katrin Gitschalter, Una Jermilova, Nadine Colpo, Carlos Uribe-Munoz,
Valery Radchenko, Paul Schaffer, Kuo-Shyan Lin, Francois Benard, and Chris Orvig
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00225 • Publication Date (Web): 22 Apr 2019
Downloaded from http://pubs.acs.org on April 23, 2019
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Functionally Versatile and Highly Stable Chelator for 111In and 177Lu: Proof-of-principle Prostate
Specific Membrane Antigen Targeting
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Lily Lia,c, María de Guadalupe Jaraquemada-Peláeza, Hsiou-Ting Kuod, Helen Merkensd Neha
,
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Choudharya,c, Katrin Gitschtalerd, Una Jermilovaa, Nadine Colpod, Carlos Uribe-Munozd, Valery
Radchenkob,c, Paul Schafferc, Kuo-Shyan Lind, François Bénardd*, Chris Orviga*
a Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British
Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada
b Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British
Columbia V6T 1Z1, Canada
c Life Sciences Division, TRIUMF, 4004 Wesbrook Mall, Vancouver, British Columbia V6T
2A3, Canada
d Department of Molecular Oncology, BC Cancer, 675 West 10th Ave, Vancouver, British
Columbia V5Z 1L3, Canada
Abstract
Here, we present the synthesis and characterization of a new potentially nonadentate chelator,
t
H4pypa and its bifunctional analog Bu4pypa-C7-NHS conjugated to PSMA (prostate-specific
membrane antigen) - targeting peptidomimetic (Glu-urea-Lys). H4pypa is very functionally
versatile and biologically stable. Compared to the conventional chelators (e.g. DOTA, DTPA),
H4pypa has outstanding affinities for both 111In (EC, t1/2 ~2.8 d) and 177Lu (-, t1/2~6.64 d). Its
radiolabeled complexes were achieved at >98% radiochemical yield, RT within 10 minutes, at
ligand concentration as low as 10-6 M, with excellent stability in human serum over at least 5-7
days (<1% transchelation). The thermodynamic stabilities of the [M(pypa)]- complexes (M3+
=
In3+, Lu3+, La3+) were dependent on the ionic radii, where the smaller In3+ has the highest pM value
(30.5), followed by Lu3+ (22.6) and La3+ (19.9). All pM values are remarkably higher than those
with DOTA, DTPA, H4octapa, H4octox and H4neunpa. Moreover, the facile and versatile
bifunctionalization enabled by the p-OH group in the central pyridyl bridge of the pypa scaffold
(compound 14) allows incorporation of a variety of linkers for bioconjugation through easy
nucleophilic substitution. In this work, an alkyl linker was selected to couple H4pypa to a PSMA-
targeting pharmacophore, proving that the bioconjugation sacrifices neither the tumor-targeting
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nor the chelation properties. The biodistribution profiles of 111In- and 177Lu-labeled tracers are
different, but promising, with the 177Lu analog particularly outstanding.
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Introduction
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The potential of radionuclides in cancer diagnosis and therapy has been recognized for decades
since the discovery of radioactivity in 1901.1 The specificity and minimal invasiveness of targeted
radionuclide therapy compared to chemotherapy has poised the field for further growth, stimulated
by the technological advancements in production of both radionuclides and biological targeting
vectors (e.g. peptides and monoclonal antibodies). The biological safety of the radiometal is
ensured by a stably bound chelator coupled to a bio-targeting vector via a covalent linkage, which
also modulates the pharmacokinetics of the whole. 2-4
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An ideal chelator should possess rapid complexation kinetics and strong affinity for the radiometal
ion at mild conditions (RT, <15 minutes complexation), as well as high versatility of linker
incorporation (i.e. bifunctionalization) without sacrificing the coordination integrity. Although a
small peptidomimetic conjugate usually has higher tolerance to the harsh radiolabeling conditions
(e.g. 60-90 oC for 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA, Chart 1), fast
and quantitative radiolabeling at room temperature facilitate handling and avoid potential
degradation of the product. One of the benefits is that instead of relying on central manufacturing,
clinicians can conveniently prepare the radioactive tracer in a local hospital, which mimics the
existing practice for 99mTc kits.5 In terms of bifunctionalization, most of the commercial chelators
including DOTA and diethylenetriamine-pentaacetic acid (DTPA), lack a convenient spot for
adding a linker. The cumbersome functionalization on the polyamine backbone or the sacrifice of
a pendant arm can complicate the synthesis while restricting the linker variety, or even reduce the
complex stability.6-8 Additionally, a practically and clinically useful chelator should accommodate
(a pair of) theranostic isotopes (e.g. 177Lu, 111In, 86/90Y, 44/47Sc) for an accurate study of dosimetry
to achieve optimal therapeutic effects. In this regard, 177Lu (t1/2 ~ 6.64 days) is well-recognized for
treating small and metastatic tumors with its low-energy - particles (maximum 498 keV),9 along
with two useful  emissions (208 keV and 113 keV) for SPECT imaging, rendering it an excellent
theranostic isotope with a favorable half-life.10 The high thermal neutron capture cross section of
176Lu also allows direct production of 177Lu [176Lu(n,γ)177Lu] with high specific activity at multi-
Curie activity levels to meet the impending demand for targeted cancer therapy.11, 12 Of course,
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no-carrier-added (n.c.a.) 177Lu can also be produced through an indirect route (176Yb(n,)177Yb→
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177Lu).12, 13 Another potential theranostic radioisotope is the -ray and Auger-electron-emitting
111In (t1/2 ~ 2.8 days), which has proved induction of cytocidal double-strand break (DSBs) upon
internalization and translocation to the nucleus of the tumor cells, besides its widespread
applications in SPECT imaging. 2, 14-17
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To overcome the practical concerns on the radiolabeling conditions and the complex stability
encountered with the current commercial chelators, we report herein the synthesis and
characterization of a new potentially nonadentate pyridinecarboxylate-based ligand, H4pypa and a
bifunctional H4pypa conjugated to glutamate-urea-lysine-based PSMA (prostate-specific
membrane antigen)-targeting pharmacophore for prostate cancer (PCa) targeting. PCa is the most
common cancer in men in the United States accounting for around 26,730 deaths in 2017.18 PSMA
is primarily expressed in normal human prostate epithelium, but is up-regulated in prostate cancer
cells, even more in de-differentiated, metastatic and hormone-refractory carcinomas, rendering it
an attractive target in PCa.19 The current FDA-approved 111In-capromab pendetide scan
(ProstaScint® scan) has several drawbacks such as inducing the formation of human anti-mouse
antibody (HAMA) and targeting the intracellular epitope of PSMA, which somewhat limit its
applications.20, 21 The Glu-urea-Lys PSMA inhibitor adopted in this study was developed by
Maresca et al.,22 and the lipophilic spacer was suggested to improve the pharmacokinetics by
Benešová et al. who conjugated the whole moiety to DOTA (a.k.a. PSMA617) for 68Ga and 177Lu
radiolabels.23 In this paper, we show that both H4pypa and its PSMA-targeting counterpart
radiolabel 111In and 177Lu in excellent yield and stability with the LNCaP-tumor targeting ability
preserved. Moreover, the thermodynamic stabilities of M-pypa systems (M=In3+, Lu3+, La3+)
measured as pM are considerably higher than those with DOTA, DTPA, H4octapa, H4octox and
H4neunpa. In addition to chelation, a significant advantage of the pyridyl bridge is the facile and
versatile linker attachment via the central hydroxyl group. Undeniably, linkages play a pivotal role
in the biological behavior of the radiochelate, particularly the small peptide conjugates.23-27
Therefore, this versatility provides flexibility in not only the chemical properties of the whole (e.g.
hydrophilic, hydrophobic), but also the targeting vectors associated (e.g. peptide, antibody,
antibody fragment, nanoparticles).3 The combination renders H4pypa a promising theranostic
chelating agent for many different cancer treatments, despite the focus here on prostate cancer.
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R = H (H4pypa)
R
N
or
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OH
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H
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H4pypa-C7-PSMA617
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DTPA
CHX-A"-DTPA
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OH HO
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EDTA
p-NH2-Bn-CHX-A"-DTPA
TTHA
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OH
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OH HO
OH HO
O
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H4octapa
p-NO2-Bn-neunpa
H4octox
Chart 1. Chemical structures of selected chelators.
Result and Discussion
Synthesis and Characterization
Our group has established a significant library of non-macrocyclic chelators, including picolinate
and hydroxyquinoline derivatives, to target different radionuclides.28-30 H4pypa is a new member
of the picolinate-arm-based ligand family, designed to accommodate medium-to-large
radioisotopes (e.g. 177Lu, 111In, 44Sc, 86/89Y) in its potentially nine-coordinating cavity, securing the
metal ion with the rigid pyridyl cap on which a variety of linkers can be attached efficiently for
bioconjugation. As depicted in Scheme 1, the arm and pyridyl backbone of the non-bifunctional
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8, H4pypa
Scheme 1. Reagents and conditions: i) DCC, tert-butyl alcohol, DCM, RT, overnight, 50%; ii) NaBH4, dry MeOH,
RT, 3-4 h, 72%; iii) SeO2, 1,4-dioxane, 100oC, overnight, 56%; iv) 1. Dry MeOH, RT, 1 h; 2. NaBH3CN, dry
MeOH, 3 h, 70%; v) NaBH4, dry MeOH, RT, 12 h, 92%; vi) PBr3, dry CHCl3/ACN, 60 oC, 18 h, 70%; vii) K2CO3,
dry ACN, 60 oC, 24 h, 70%; viii) TFA/DCM, RT, overnight, 70%
H4pypa were strategically synthesized individually and then assembled in one convergence step
for higher synthetic efficiency. The backbone was synthesized by first reducing the starting
material dimethyl 2,6-pyridinedicarboxylate to pyridine-2,6-diyl-dimethanol (5, 92%), followed
by bromination with phosphorus tribromide, to give the dibromo-derivative (6, 70%) which was
coupled with 2 equivalents of the picolinate-acetate arm moiety (4). The tert-butyl esters on arm 4
were selected for the compatibility with solid-phase peptide coupling and it was synthesized
through one-pot Schiff base formation and reduction using the aldehyde (3) and tert-butyl glycinate
(70%). Compound 3 was prepared by converting 2,6-pyridinedicarboxylic acid to the
corresponding tert-butyl-ester analogue (1), followed by monoreduction with sodium borohydride
(NaBH4) to give compound 2 and subsequent oxidation to aldehyde (3) by selenium(IV) oxide
(SeO2) (cumulative yield ~20%). Finally, both pendant arm (4) and pyridyl bridge (6) were
connected through SN2 nucleophilic substitution (7, 70%) and then deprotection was followed to
give H4pypa (8, 70%). Purification with high-performance liquid chromatography (HPLC) yielded
the final product as a trifluoroacetic acid (TFA) salt (H2pypa·2TFA·1.7H2O as determined by
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elemental analysis). Regarding the bifunctional H4pypa (Scheme 2), commercially available
chelidamic acid monohydrate was selected as the backbone starting material. After converting the
carboxylic acids to methyl esters (9, >99%), the p-OH group on the pyridyl moiety was protected
with benzyl bromide (10, 64%).
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4 + 12
O
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ix
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O
O
O
O
O
O
O
O
O
17
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tBu4pypa-C7-NHS
Scheme 2. Reagents and conditions. i) SOCl2, MeOH, RT-60 oC, 26 h, >99%; ii) BnBr, ACN, K2CO3, 60 oC, overnight,
64%; iii) NaBH4, dry MeOH, RT, overnight, 82%; iv) PBr3, dry CHCl3/dry ACN, 60 oC, overnight, 70%; v) K2CO3,
dry ACN, 30 oC, 24 h, 73%; vi) Pd/C (10% w/w), H2 (g), MeOH, RT, overnight; vii) K2CO3, dry THF, RT-35 oC, 24
h, 90%; viii) Pd/C, MeOH, RT, overnight, 88%; ix) NHS, EDCI, dry ACN, RT, overnight, 86%.
Similar to the H4pypa preparation, the methyl esters in compound 10 were reduced to alcohols (11,
82%), and then brominated to give compound 12 (70%), which was coupled to arm 4 through the
aforementioned protocol to yield the protected pypa (13, 73%). Following debenzylation with
Pd/C-catalyzed hydrogenation, the alkyl linker was added through SN2 nucleophilic substitution
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to give compound 16 (90%). Deprotection and activation with 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride/ N-hydroxysuccinimide (EDCI/NHS)
generated the activated ester (18, 76% over 2 steps), which was coupled to the PSMA-targeting-
peptide-bound resin by using N,N-diisopropylethylamine (DIPEA) in dry DMF overnight. After
the coupling finished, the peptide-bioconjugate was deprotected and simultaneously cleaved from
the resin with 95/5 trifluoroacetic acid (TFA)/triisopropylsilane (TIS) for 2 hours at room
temperature to give H4pypa-C7-PSMA617 (Chart 1).
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Metal Complexation and Characterization
The complexation with non-radioactive In3+, Lu3+ and La3+ ions was studied and the complexes
were characterized with NMR spectroscopy (1H, 13C and HSQC) and high-resolution electrospray
ionization mass spectrometry (HR-ESI-MS). Being the largest lanthanide, La3+ is also of interest
in its complexation with H4pypa in comparison with that of Lu3+ which is the smallest in the
series.31 All three complexes appeared to be rigid coordination complexes with no observable
1
fluxionality, as confirmed by the sharply resolved H NMR peaks and the absence of extra
1
hydrogen and carbon signals in H and 13C NMR spectra (Figures S15-S22). In the case of
[In(pypa)]-, all the carbons in the complex were chemically distinct from one another, while there
were three pairs of visible diastereotopic methylene protons (2.94 and 3.22 ppm, 2J =17.6 Hz; 3.78
2
2
and 3.91 ppm, J =17.1 Hz; 4.10 and 4.44, J=17.4) (Figures 1A and S19), and each doublet
accounted for only one hydrogen atom; the rest were overlapping with the water signal (revealed
by the interaction with the adjacent carbon atom shown in 1H-13C HSQC, Figure S20B), indicating
the asymmetry in the complex in solution state. The asymmetry was also observed in [La(pypa)]-
which appeared in solution as a single isomer (Figures 1A and S21). On the other hand, [Lu(pypa)]-
presented in solution as a symmetric complex, marked by the unchanged number of carbon signals
(Figure S16), as well as the consistent 1H aromatic pattern (two triplets - 8.21 and 7.85 ppm, and
three doublets - 8.05, 7.80 and 7.44 ppm) compared with the uncomplexed chelator (Figures 1A
and S15). Furthermore, the complex only exhibited two pairs of diastereotopic protons (3.49 and
2
2
3.98 ppm, J=17.0 Hz; 4.07 and 4.39 ppm, J=14.7 Hz), while the last pair of methylene-H
appeared as a sharp singlet at pH = 1.5 but a clear doublet at pH = 11.5 (Figure 1B). However, the
much smaller splitting of the peak compared to the other diastereotopic pairs implied a much
weaker, or perhaps farther interaction with the Lu3+ ion. Additionally, this pair of methylene
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protons belonged to a carbon that has a very similar chemical shift with another methylene carbon
which suggested that both of them might be adjacent to the pyridyl group instead of the carboxylic
acid on the acetate arms (Figure S17). Interestingly, as determined by potentiometric titration, the
[Lu(Hpypa)] species deprotonates at pH = 3.35-3.60 which can be reasonably assigned to the
protonated central pyridine. In this case, it could be inferred that the interaction between the pyridyl
bridge and the Lu3+ ion is weaker when the central pyridine-N atom is protonated at low pH, and
is increased when is deprotonated at higher pH, which however, was not as strong as that with the
acetate and picolinate arms. This phenomenon could also be explained by the relatively small
atomic size and the hard nature of Lu3+.31
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Figure 1. (A) Partial 1H NMR spectra of [La(pypa)]-, [In(pypa)]-, [Lu(pypa)]-, H4pypa (top-bottom) (D2O, 400 MHz,
298 K). (B) Partial 1H NMR spectra of [Lu(pypa)]- at pH=11.5, 2.1 and 1.5 (top-bottom) (D2O, 400 MHz, 298 K).
X-ray Crystallography
X-ray quality single crystals of H[Lu(pypa)] were obtained by the slow evaporation of 1:1 LuCl3
and H4pypa solutions in water after adjustment of pH to 2. The ORTEP diagram of [Lu(pypa)]- is
shown in Figure 2 and its crystallographic data can be found in the Supporting Information. From
Figure 2, it can be seen that at pH 2 two of the carboxylic groups of the ligand are protonated. The
H atom attached to O2 (picolinic -COOH) is half occupied in one of the asymmetric units, as is
the case with Cl atom which is equally shared by two asymmetric units. While the H atom attached
to O6 (backbone -COOH) is fully occupied, hence the overall charge is neutral. The Lu(III) ion is
nine-coordinated by the N5O4 donor atoms of the ligand. Selected bond distances and bond angles
are provided in Table 1. The geometry is distorted but nevertheless, the structure provides the vital
visual insight to the coordination environment of the Lu(III) ion. It can be clearly seen from the
structure that Lu(III) sits in the cavity of the H4pypa ligand, capped by the pyridyl group. Also, the
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preference of Lu(III) for O-donor atoms can be seen from the shorter Lu-O bond distances
compared to longer Lu-N bonds.
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Figure 2. ORTEP diagrams of the anion in (A) H[Lu(pypa)] (B) C50H79ClLu2N10O33
Table 1. Selected bond lengths and bond angles in [Lu(pypa)]-.
Bond length/Å
Atom Atom Length/Å
Bond Angle/°
Atom Atom
Atom
N2
Angle/°
70.14(6)
129.28(6)
68.64(7)
67.31(6)
67.44(7)
66.17(6)
Lu
Lu
Lu
Lu
Lu
Lu
Lu
Lu
Lu
O1
O3
O5
O8
N1
N2
N3
N4
N5
2.3266(17)
2.3333(17)
2.3559(17)
2.2954(17)
2.4181(19)
2.537(2)
O3
O5
O8
N1
N3
N3
Lu
Lu
Lu
Lu
Lu
Lu
N5
N5
N2
N2
N4
2.451(2)
2.553(2)
2.3845(19)
Solution Thermodynamics
When evaluating ligands for metal complexation, knowledge of the basicity of the different
ionizable and non-ionizable protons is essential because the metal ion will compete for replacing
them at the basic sites. H4pypa possesses nine protonation sites, and in this work, we determined
the acidity constants for all of them. Combined potentiometric-spectrophotometric titrations were
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carried out by following spectral changes in the absorption band of the picolinate chromophore for
the first seven protonation equilibria and UV in batch spectrophotometric titrations for the last two
equilibria, as they deprotonate at a pH below the electrode threshold. The different absorption
features related to the above-mentioned equilibria as the pH is raised are presented in Figure S51.
Table 2 presents the protonation constants calculated from the experimental data using the
HypSpec201432 and Hyperquad201333 programs. Figure S52A presents one of the titration curves
of an acidified solution of H4pypa·2TFA·1.7H2O and it shows that nine equivalents of base (NaOH)
were consumed in the titration. The speciation plots of different species of H4pypa in Figure S52b
were calculated from the protonation constants in Table 2 with the Hyss software.34
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Table 2. Protonation constants of H4pypa at 25.0 °C, I = 0.16 M NaCl.
Equilibrium Reaction
L4- + H+⇆ HL3-
log β
log K
7.78 (1)
6.78 (1)
3.69 (1)
3.02 (1)
2.23 (2)
2.06 (6)
1.70 (2)
-0.37 (1) (a)
-0.58 (2) (a)
7.78 (1)
HL3- + H+ ⇆ H2L2-
H2L2- + H+ ⇆ H3L-
H3L- + H+ ⇆ H4L
H4L + H+ ⇆ H5L+
H5L+ + H+ ⇆ H6L2+
H6L2+ + H+ ⇆ H7L3+
H7L3+ + H+ ⇆ H8L4+
H8L4+ + H+ ⇆ H9L5+
14.56 (1)
18.25 (1)
21.27 (1)
23.50 (2)
25.56 (6)
27.26 (2)
26.89 (1) (a)
26.31 (2) (a)
(a) from in batch UV spectrophotometric titrations, not evaluated at constant ionic strength (0.16 M NaCl).
The structure of H4pypa resembles that of H4octapa,28, 35 with the difference in the backbone: in
H4pypa, a pyridyl ring bridges the two tertiary nitrogen atoms. Not surprisingly, also in this case,
the two most acidic protons (species H9L5+ and H8L4+, pK1 = -0.58 (2) and pK2 = -0.37 (1)) can be
attributed to the deprotonation of the pyridine nitrogen atoms in the picolinate moieties based on
the bigger spectral changes in the band of this chromophore. Following that, the species H7L3+ and
H6L2+ deprotonate with pK3 = 1.70 (2) and pK4 = 2.06 (6), respectively, and they can be attributed
to the deprotonation of the two acetate carboxylic acids. Species H5L+ and H4L deprotonate with
pK5 = 2.23 (2) and pK6 = 3.02 (1), respectively, and are attributed to the picolinate-COOH. The
last three dissociation steps are assigned to the tertiary nitrogen atoms and the central pyridine
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nitrogen (species H3L-, H2L2- and HL3-). Comparing the pKa values for the tertiary nitrogen atoms
of H4pypa (pK8 = 6.78 (1) and pK9 = 7.78 (1)) to those of H4octapa (pK7 = 5.43 (2) and pK8 = 8.58
(1))35, the ΔpKa for those equilibria is of 1 unit in H4pypa while it is 3.1 units in H4octapa. This
can be explained for the larger charge repulsion in H4octapa compared to H4pypa when the two
nitrogen atoms are protonated. The pK7 = 3.69 (1) can be allocated therefore to the central pyridine
nitrogen atom.
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Complex Formation Equilibria of H4pypa with In(III), Lu(III) and La(III)
Complex formation equilibria studies of H4pypa with metal ions of relevant interest in
radiopharmaceutical chemistry were carried out by different methods. The extent to which the
metal complexation occurred even at pH ~2 was too high to allow a direct determination of the
stability constants for the [ML]- species by simple potentiometric titrations of H4pypa with the
respective metal ions. Protonated species of the metal complexes, MHL, were found with H4pypa
and In3+, Lu3+ and La3+ ions by competition methods; ligand-ligand competition methods with the
competing ligand TTHA were used for In3+ and Lu3+ ions, while for La3+, EDTA was used as a
competitor. Additionally, acidic in batch UV spectrophotometric titrations were carried out for all
M3+-H4pypa systems (M3+ = In3+, Lu3+ and La3+) (Figures S53-S55). Once the stability constants
for the MHL species (log KMHL) were known, the direct potentiometric method was used to
determine the stability constants of the [ML]- and [M(OH)L]2- species for In3+ and Lu3+, and [ML]-
and [M2L2(OH)]3- species for La3+. Potentiometric and spectrophotometric experimental data
were refined using the HypSpec201432 and Hyperquad201333 programs and the stability constants
are presented in Table 3.
The metal complex stability of the [ML]- species formed (log KML) with H4pypa follows the order
In3+ > Lu3+ > La3+. The size and the acid character of the metal ion definitely play a role on its
metal complex stability, and from these results, the cavity of H4pypa seems best fitted for the
smaller In3+.
In order to compare the excellent stability of M3+-H4pypa complexes with the stability of other
metal complexes involving different chelators, it is necessary to use a parameter that takes into
account not just the stability of the metal complexes, but also the basicity and the denticity of the
different chelators to be compared. The parameter pM is widely used in medicinal inorganic
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chemistry for this purpose, and is a measurement of the metal sequestering ability of a determined
chelator towards an specific metal ion; it is defined as –log [Mn+]free at [ligand] = 10 M and [Mn+]
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Table 3. Stepwise stability constants (log K) of H4pypa complexes with In3+, Lu3+ and La3+.
9
Equilibrium reaction
M3+ + L ⇆ ML
In3+
29.99(4)a; 30.13(3)b 22.02(6)a; 22.20(2)b
4.06(5)a; 3.80(1)c 3.35(8)a; 3.60(6)c
10.59(7)a; 10.44(4)b 10.77(8)a; 10.86(3)b
Lu3+
La3+
19.74(3)d; 19.54(2)b
2.99(4)d; 3.24(5)c
-
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ML + H+ ⇆ MHL
M(OH)L + H+ ⇆ ML
M2L2(OH) + H+ ⇆ M2L2
pMe
-
-
33.88(7)d; 34.40(6)b
19.9
30.5
22.6
a) ligand-ligand potentiometric competition with H6ttha at I = 0.16 M (NaCl) and 25 °C; b) potentiometric titrations
at I = 0.16 M (NaCl) and 25 °C; c) in-batch acidic spectrophotometric competition at 25 °C, not evaluated at constant
I = 0.16 M (NaCl); d) ligand-ligand potentiometric competition with H4edta at I = 0.16 M (NaCl) and 25 °C; e) pM is
defined as -log [M]free at [L] = 10 M, [M] = 1 M and pH = 7.4. Charges are omitted for clarity.
= 1 M at pH = 7.4.36 In Figure 3A, the pM values for the most relevant chelators currently used
in the radiopharmaceutical field have been plotted for the metals of our interest and the H4pypa
performance exceeds that of all the other chelators. Additionally, Figure 3B shows the importance
of the basicity of different chelators on the metal sequestration ability or pM values; in particular,
it shows the metal sequestering ability in terms of pLu3+ as the pH is raised. For ligands with lower
overall basicities (Table S3), the protons will compete less with the metal ion and will show
complexation from lower pHs and in a broader pH range. This effect is more dramatic when
comparing the metal affinity of H4pypa and H4octox towards Lu3+. Although the overall stability
constant of [Lu(octox)]- (log K = 24.66(1))37 is higher than that of [Lu(pypa)]- (log K = 22.02(6)),
the higher basicity of H4octox prevents Lu3+ scavenging at lower pHs and more importantly, at
physiological pH = 7.4, the effectiveness of H4octox is 4.4 units lower than that of H4pypa. These
promising solution thermodynamics findings reveal H4pypa as an excellent chelator for further
studies in vitro and in vivo, particularly for the theranostic 177Lu isotope.
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In3+
H4octapa
DTPA
H4octox
H4neunpa
DOTA
Lu3+
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DTPA
H4octox
DOTA
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1.00
1.05
1.10
1.15
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0
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Ionic radius (CN = 8)
pH
Figure 3. (A) pM values vs ionic radius31 for M3+- ligand complexes (CN = 8); (B) Lu3+ scavenging ability of different
ligands as the pH is raised from 0-12, [Lu3+] = 1  10-6 M and [ligand] = 1  10-5 M. Solid line in B at indicates
physiological pH (7.4). Stability constants and acidity constants of ligands other than H4pypa used for the plotting are
reported in Tables S3-S4.
Radiolabeling and Human Serum Challenge Experiments
In most receptor targeting radiotracer formulations, the unlabeled receptor targeting vector is
preferably kept minimum to ensure that the receptor is not saturated by unlabeled targeting motif,
which in other words is to maximize the apparent molar activity.38 Quantitative radiolabeling at
mild conditions (RT, 10 minutes) with low ligand concentration and low ligand/radiometal
(mol/mol) (L/M) ratio obviates the need of post-labeling HPLC purification to attain high apparent
molar activity, which has significant impact on the biological profile. Fendler et al. reported that
high apparent molar activity of 177Lu-PSMA-617 was associated with higher tumor uptake, more
prominent DNA damage and more effective tumor growth inhibition.39 In our study,
concentration-dependent radiolabeling was applied to determine the lowest ligand (H4pypa and
H4pypa-C7-PSMA617) concentration required for quantitative radiometalation with both 177Lu
and 111In, while the corresponding L/M ratio was adjusted afterwards using a mixture of no-carrier-
added (n.c.a.) radioactive and non-radioactive isotopes from diluted AAS standards (i.e. 177/natLu
or 111/natIn). All radiolabeling studies were performed in triplicate. Firstly, both H4pypa and
H4pypa-C7-PSMA617 radiolabeled 177Lu and 111In quantitatively (>98% radiochemical yield,
RCY) in 10 minutes with 10-6 M ligand concentration at RT and pH=7 (Figure 4A), indicated by
a single sharp signal at the baseline of the iTLC-SA plate developed with EDTA solution (50 mM,
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pH = 5.5), which is consistent with the well-separated radiopeaks of the free metal and the complex
on the HPLC radiotraces (tR = 12.9 and 10.4 minutes for 177Lu- and 111In-pypa complexes; 15.0
and 14.1 minutes for 177Lu- and 111In-pypa-C7-PSMA617 complexes) (Figures S45-S50). The
corresponding complexes were highly kinetically inert with <1% transmetalation to the serum
proteins over at least 7 days for both 177Lu complexes and 5 days for [111In][In(pypa)]- (Figure 4B),
while that of [111In][In(pypa-C7-PSMA617)] was not determined due to unsuccessful separation
of the transchelated 111In and the radioactive complex with either PD10 column or iTLC-SA plate
(EDTA and DTPA solution as mobile phase). The results are in significant contrast with the
industrial “gold standard” macrocyclic chelator –DOTA – which required microwave heating at
80-100 oC over 20-30 minutes for quantitative radiolabeling with both 111In and 177Lu.28, 40 DTPA
and the cyclohexyl analogue (CHX-A”-DTPA) are two widely adopted non-macrocyclic ligands
in 111In radiopharmaceutical development in an effort to achieve high RCY at RT, but their inferior
in vitro stabilities unfortunately limit their clinical potential (88.3% and 89.9% for
[111In][In(DTPA)]2- and [111In][In(p-NH2−Bn−CHX-A″−DTPA)]2−, respectively, after 24
hours).28, 29 Encouraging radiolabeling results with 111In and 177Lu were reported with H4octapa,
an octadentate ligand previously reported by our group with reported in vitro stability 92.3% and
87.7%, respectively, after one day.2, 28 The nonadentate p-NO2−Bn−neunpa was developed for
111In with more favorable stability in human serum (97.8% over 5 days), but exhibited very low
affinity to 177Lu and therefore, further study was precluded.29 As mentioned above, the apparent
molar activity is a crucial parameter in preparing radiotracers. For [AE][E(pypa-C7-PSMA617)]
(AE=177Lu, 111In), at the optimal radiolabeling concentration (i.e. 10-6 M), radiolabeling yields with
different L/M ratios were tested using a mixture of no-carrier-added 177Lu or 111In, as well as non-
radioactive natLu or natIn in an attempt to achieve the lowest L/M ratio. For both radiotracers, L/M
ratio 2 has proved sufficient for ~98% RCY (RT), while equimolar gave only 76% (111/natIn) and
72% RCY (177/natLu). The results proved a cost-effective and comparable estimation for optimal
radiometal-to-chelator ratio without consuming a large amount of radioactivity which is very
expensive, while the resulting high apparent molar activity also conveniently obviates post-
labeling purification, and thus further enhances their potential in the practical applications.
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[
[
[
[
177Lu][Lu(pypa)]-
[
[
[
177Lu][Lu(pypa)]-
177Lu][Lu(pypa-C7-PSMA617)]
111In][In(pypa)]-
177Lu][Lu(pypa-C7-PSMA617)]
111In][In(pypa)]-
20
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111In][In(pypa-C7-PSMA617)]
0
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Time (h)
[-log(M)]
Figure 4. (A) Concentration dependent radiolabeling of H4pypa and H4pypa-C7-PSMA617 (10 minutes, room
temperature) in NH4OAc solution (0.15 M, pH = 7) with 177Lu/111In. (B) Human serum challenge of the radiolabeled
complexes over 5-7 days (37 oC).
SPECT/CT Imaging, Biodistribution Studies and Binding Affinity
[AE(pypa-C7-PSMA617)] (AE = natIn and natLu) inhibited the binding of 18F-DCFPyL to PSMA on
LNCaP cells in a dose-dependent manner (Figures S56-S57), and their calculated Ki values were
6.41 (1.80) nM and 7.88 (4.34) nM, respectively. For animal studies, 177Lu- and 111In-labeled
radiotracers with apparent molar activities of 207 GBq/mol and 459 GBq/mol, respectively,
were prepared and then injected into LNCaP-tumor-bearing mice (n=5 per time point). SPECT/CT
imaging (Figure 5) and ex vivo biodistribution studies (Figure 6 and Table S6-S7) showed that
both radioactive analogues excreted mainly via the renal pathway. The blood clearances for both
radiotracers were fast, measured at 0.89±0.42% injected dose per gram (ID/g) at 4-hour post-
injection (p.i.) and 1.04±0.34% ID/g at 1-hour p.i. for the 177Lu- and 111In-based tracers,
respectively. In both cases, uptake in non-specific organs and tissues were low (e.g. fat, intestine,
stomach, liver, pancreas, heart, muscle, bone), while high accumulations were observed in PSMA-
expressing tissues (e.g. kidney, spleen, adrenal glands, LNCaP tumor).41 For [177Lu][Lu(pypa-C7-
PSMA617)], the uptake in kidney, spleen, adrenal glands and tumors were 120±19% ID/g,
4.71±1.68% ID/g, 3.64±1.49% ID/g, 20.6±5.9% ID/g at 1-hour p.i., respectively. Except for tumor,
in which the accumulation grew to 24.0±7.6% ID/g at 4-hour p.i. and then gradually reduced to
12.7±4.2% ID/g after 3 days, the uptake in other organs was rapidly cleared within the first 4 hours,
resulting in a substantial increase in the tumor-to-background contrast ratio (Table S6). Moreover,
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the tumor uptake was more than 60% higher than that of 177Lu-PSMA-617 at both 4-hour and 72-
hour p.i., which was reported with 14.5±1.8% ID/g and 7.80 ±3.69% ID/g, respectively.42
Regarding [111In][In(pypa-C7-PSMA617)], similar to its 177Lu-counterpart, the background organs
and tissues uptakes were low and cleared rapidly except in the kidney and tumor. After 24 hours,
the kidney accumulation was 36.4±18.8% ID/g and the tumor uptake was 8.88±1.92% ID/g,
leading to a tumor-to-kidney ratio of 0.24±0.10 compared to 6.70±1.75 for the 177Lu-analogue (24-
hour post-injection) (Tables S6-S7). A DOTA-based construct containing the same PSMA-
targeting motif (Glu-urea-Lys) and IRDye800CW was synthesized and radiolabeled with 111In as
a dual-modality imaging agent.43 The PC3-PIP tumor uptake was 14.6±1.3% ID/g at 24-hour p.i.
and the calculated tumor-to-kidney ratio was around 0.3.43 Schottelius et al. published a DOTA-
based PSMA I&T which was metalated with 111In, 177Lu and 68Ga.44 In their case, [111In]PSMA-
I&T had the highest tumor uptake (~8% ID/g) at 1-hour p.i., along with the most kidney (~190%
ID/g) and spleen (~46% ID/g) accumulation which was claimed to be CB17 SCID-mice related.44
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%ID/g
1h
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%ID/g
Figure 5. Representative SPECT/CT images (MIP, coronal) of [AE][E(pypa-C7-PSMA617)] [AE=111In (left, 24.9
MBq), 177Lu (right, 44.1 MBq)] in LNCaP-tumor-bearing mice at different p.i. time points.
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Figure 6. Ex vivo biodistribution data [AE][E(pypa-C7-PSMA617)] [AE=177Lu (A), 111In (B)] in LNCaP-tumor-
bearing mice at selected p.i. time points (n=5 per time point).
Conclusion
H4pypa is a potentially nonadentate non-macrocyclic chelator with great affinity for 111In and 177Lu
which are excellent radionuclides in cancer diagnosis and therapy. In addition to the quantitative
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radiolabeling yields at fast complexation kinetics (10 minutes) under mild conditions (RT, pH =
7) with 111In and 177Lu, the corresponding H4pypa complexes are highly thermodynamically stable
and kinetically inert (<1% transmetalation to serum protein over 5-7 days), a benefit of the extra
rigidity exerted by the central pyridyl moiety that serves as a cap to further stabilize the whole
complex, as shown in the crystal structure of H[Lu(pypa)] where Lu is fully nine-coordinated by
H4pypa. Furthermore, the pM values of M3+-H4pypa systems (M=In3+ (30.5), Lu3+ (22.6) and La3+
(19.9)) are much higher than those with DOTA, DTPA, H4octapa, H4octox and H4neunpa.
Moreover, inclusion of a p-OH group on the central pyridyl ring also renders H4pypa a functionally
versatile chelator. One of the main advantages is that the precursor (compound 14) can be
synthesized in large scale with excellent stability while any linker of interest can be attached easily,
even in milligram scale, for fast screening. Here, an alkyl linker was selected to join the PSMA-
targeting pharmacophore and the [AE][E(pypa)] (AE=177Lu, 111In) complexes demonstrating vastly
different, but promising pharmacokinetics. In particular, the 177Lu-counterpart shows significantly
faster background clearance and higher tumor retention paving the way for potential theranostic
applications. Beyond the proof-of-principle PSMA targeting, H4pypa is also highly valuable for
other types of cancer treatments, particularly radioimmunotherapy, in which a mild radiolabeling-
condition is essential due to the temperature- and pH-sensitive antibody. Current efforts are
expanding the applications of H4pypa with other theranostic radionuclides (e.g. 44/47Sc, 86/90Y) and
225Ac. Different bifunctional derivatives have been developed and conjugated to other targeting
vectors, particularly to antibodies.
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Experimental Section
Materials and Methods
All solvents and reagents were purchased from commercial suppliers (TCI America, Alfa Aesar,
AK Scientific, Sigma-Aldrich, Fisher Scientific, Fluka) and were used as received. Human serum
was purchased frozen from Sigma-Aldrich. The analytical thin-layer chromatography (TLC) plates
used were aluminum-backed ultrapure silica gel 60 Å, 250 μm thickness; the flash column silica
gel (standard grade, 60 Å, 32−63 mm) was provided by Silicycle. 1H and 13C NMR spectra were
recorded at ambient temperature on Bruker AV300 and AV400 instruments, unless otherwise
specified; the NMR spectra are expressed on the δ scale and were referenced to residual solvent
peaks. Low-resolution (LR) mass spectrometry was performed using a Waters ZG spectrometer
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with an ESCI electrospray/chemical-ionization source, and high-resolution electrospray ionization
mass spectrometry (HR-ESI-MS) was performed on a Micromass LCT time-of-flight instrument
at the Department of Chemistry, University of British Columbia. Microanalyses for C, H, and N
were performed on a Carlo Erba Elemental Analyzer EA 1108. The HPLC system used for analysis
and purification of non-radioactive compounds consisted of a Waters 600 controller, Waters 2487
dual wavelength absorbance detector, and a Waters delta 600 pump. Phenomenex Synergi 4 
hydro-RP 80 Å column (250 mm × 21.2 mm semipreparative) was used for purification of
deprotected H4pypa and Phenomenex Luna 5 m C18 100 Å LC column (250 mm × 10 mm) was
used for purification of deprotected H4pypa-C7-PSMA617. Automated column chromatography
was performed using a Teledyne Isco (Lincoln, NE) Combiflash Rf automated system with solid
load cartridges packed with Celite and RediSep Rf gold reusable normal-phase silica columns
(Teledyne Isco, Lincoln, NE). Analyses of radiolabeled compounds were performed with both
Instant TLC (iTLC) plates, impregnated with silic acid (iTLC-SA) purchased from Agilent
Technologies and radio-HPLC. The TLC scanner model was BIOSCAN (system 200 imaging
scanner) and the HPLC system was from Agilent Technologies (1200 series). Phenomenex Synergi
4  hydro-RP 80 Å column (250 mm × 4.60 mm) was used for separation of free radioactivity and
radio-complex. 111InCl3 was cyclotron-produced and provided by BWX Technologies as a ∼0.05
M HCl solution; 177LuCl3 was purchased from Isotope Technologies Garching (ITG). All the
isotopes used were no-carrier added (n.c.a.). Deionized water was filtered through the PURELAB
Ultra Mk2 system.
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Di-tert-butyl pyridine-2,6-dicarboxylate (1). To a stirred suspension of 2,6-pyridinedicarboxylic
acid (10.0 g, 59.8 mmol, 1 equiv.) in dichloromethane (DCM) (30 mL) was added tert-butyl
alcohol (22.6 ml) and 4-dimethylaminopyridine (DMAP) (3.65 g, 29.9 mmol, 0.5 equiv.) at room
temperature. Then, N,N'-dicyclohexylcarbodiimide (DCC) (27.2 g, 0.132 mol, 2.2 equiv.) in DCM
(30 mL) was added dropwise using a dropping funnel over 1 hour. The mixture was left stirring at
room temperature overnight, and then the precipitate was filtered off by vacuum filtration. The
filtrate was concentrated in vacuo and then purified through a silica column (CombiFlash Rf
automated column system, 80 g gold silica column, DCM - methanol (MeOH), 0-5% MeOH). The
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product fractions were rotary-evaporated to give an off-white solid (8.36 g, 50%). H NMR (400
MHz, 298 K, CDCl3): δ 8.18 (d, J = 7.8 Hz, 2H), 7.95 – 7.90 (m, 1H), 1.64 (s, 18H). 13C NMR (75
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MHz, 298 K, CDCl3): δ 168.3, 150.0, 138.1, 127.3, 83.2, 27.9. LR-ESI-MS: calcd for [C15H21NO4
+ H]+ 280.1; found [M + H]+ 280.2
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Tert-butyl 6-(hydroxymethyl)picolinate (2). Compound 1 (1.40 g, 5.00 mmol, 1 equiv.) was
dissolved in dry MeOH (150 mL) in a round-bottom flask. NaBH4 (0.189 g, 5.00 mmol, 1 equiv.)
was added at 0 oC. The mixture was stirred at room temperature for 1 hour and then another equiv.
of NaBH4 was added. The reduction continued until the mono-reduced picolinate dominated, as
monitored by silica TLC (5% MeOH in DCM). The average reaction time was 3-4 hours. After
that, the reaction mixture was diluted with DCM (100 mL) and then quenched with saturated
NaHCO3 in water (100 mL). The organic phase was separated and the bulk of MeOH in the
aqueous phase was removed in vacuo to give an aqueous layer which was then extracted with
DCM (100 mL × 3). The combined organic phases were dried over anhydrous Na2SO4, and then
clarified by filtration. The filtrate was concentrated and then purified through a silica column
(CombiFlash Rf automated column system, 40 g gold silica column, DCM : MeOH, 0-5 % MeOH).
The product fractions were rotary evaporated to give an off-white powder (2.25 g, 72 %).1H NMR
(400 MHz, 298 K, CDCl3): δ 7.88 (d, J = 7.6 Hz, 2H), 7.77 (t, J = 7.7 Hz, 1H), 7.46 (d, J = 7.7 Hz,
1H), 4.82 (s, 2H), 1.59 (s, 9H). 13C NMR (75 MHz, 298 K, CDCl3): δ 164.1, 160.3, 148.3, 137.6,
123.6, 123.4, 82.4, 64.5, 28.2. LR-ESI-MS : calcd for [C11H15NO3 + Na]+ 232.1; found [M + Na]+
232.2
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Tert-butyl 6-formylpicolinate (3). To a round-bottom flask with a stirred solution of compound
2 (4.50 g, 21.5 mmol, 1 equiv.) in 1,4-dioxane (50 mL) was added SeO2 (1.19 g, 10.8 mmol, 0.5
o
equiv.). The mixture was refluxed at 100 C overnight. After the reaction completed, the hot
mixture was clarified by filtering through a Celite bed and the filtrate was concentrated in vacuo.
The crude mixture was purified through a silica column (CombiFlash Rf automated column system,
80 g gold silica column, hexanes : ethyl acetate (Hex : EtOAc), 0-60 % EtOAc) to give a pale
yellow solid (2.51 g, 56 %). 1H NMR (400 MHz, 298 K, CDCl3): δ 10.19 (s, 1H), 8.25 (d, J = 6.6
Hz, 1H), 8.11 (d, J = 8.8 Hz, 1H), 8.01 (t, J = 7.7 Hz, 1H), 1.67 (s, 9H). 13C NMR (75 MHz, 298
K, CDCl3): δ 193.2, 163.3, 152.9, 150.1, 138.3, 128.7, 123.9, 83.2, 28.2. LR-ESI-MS : calcd for
[C11H13NO3 - H]+ 206.1; found [M - H]+ 206.1
Tert-butyl 6-(((2-(tert-butoxy)-2-oxoethyl)amino)methyl)picolinate (4). To a round-bottom
flask with a stirred solution of compound 3 (0.500 g, 2.40 mmol, 1 equiv.) in dry MeOH (20 mL)
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was added tert-butyl glycinate (0.320 g, 2.40 mmol, 1 equiv.). The mixture was stirred for one
hour at room temperature and then sodium cyanoborohydride (NaBH3CN) (0.31 g, 4.87 mmol, 2
equiv.) was added. The reduction reaction was continued for three hours at room temperature
before quenching with saturated NaHCO3 in water (10 mL) and then extraction with DCM (20 mL
× 3). The combined organic phases were dried over anhydrous Na2SO4, and then clarified by
filtration. The filtrate was concentrated in vacuo and the residue was purified through a silica
column (CombiFlash Rf automated column system, 12 g gold silica column, DCM : MeOH, 0-5%
MeOH). The product fractions were combined and rotary-evaporated to give a pale yellow oil
(0.55 g, 70%).1H NMR (400 MHz, 298 K, CDCl3): δ 7.76 (d, J = 7.7 Hz, 1H), 7.65 (t, J = 7.7 Hz,
1H), 7.43 (d, J = 7.6 Hz, 1H), 3.91 (s, 2H), 3.26 (s, 2H), 1.48 (s, 9H), 1.32 (s, 9H).13C NMR (75
MHz, 298 K, CDCl3): δ 171.1, 163.9, 159.7, 148.6, 137.2, 124.9, 123.0, 81.9, 81.0, 54.2, 51.0,
27.9. LR-ESI-MS: calcd for [C17H26N2O4 + H]+ 323.2; found [M + H]+ 323.1
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2,6-Di(hydroxymethyl)pyridine (5). To a round-bottom flask with a stirred mixture of pyridine-
2,6-dicarboxylic acid dimethyl ester (3.00 g, 15.4 mmol, 1 equiv.) in dry MeOH (50 mL) at 0 oC
was slowly added NaBH4 (2.33 g, 61.5 mmol, 4 equiv.) in three portions over 15 minutes. The
solution was then stirred at room temperature for 12 hours. CHCl3 (25 mL) was added followed
by saturated Na2CO3 in water (50 mL) to quench the reaction. The organic phase was separated
and the MeOH in the aqueous phase was removed in vacuo to give a concentrated aqueous solution
which was then extracted with CHCl3 (100 mL × 10). Multiple extractions were required to recover
most of the product. The combined organic phases were dried over anhydrous Na2SO4, and then
clarified by filtration. The filtrate was concentrated to give a white solid (1.99 g, 92%). 1H NMR
(400 MHz, 298 K, CDCl3): δ 7.70 (t, J = 7.7 Hz, 1H), 7.20 (d, J = 7.7 Hz, 2H), 4.79 (s, 4H). 13C
NMR (75 MHz, 298 K, MeOD) δ 161.5, 139.2, 120.2, 65.5. LR-ESI-MS: calcd for [C7H9NO2 +
H]+ 140.1; found [M + H]+ 140.1
2,6-Bis(bromomethyl)pyridine (6). To a three-neck round-bottom flask with a stirred solution of
compound 5 (3.00 g, 21.2 mmol, 1 equiv.) in dry ACN (acetonitrile)/CHCl3 (30 mL, 50:50 v/v) at
0 °C was added PBr3 (6.02 mL, 63.4 mmol, 3 equiv.) dropwise using a dropping funnel over 15
minutes. The mixture was refluxed for 18 hours, and then cooled before water (20 mL) was added
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slowly at 0 C to quench the reaction. After extraction with CHCl3 (50 mL × 3), the combined
organic layers were dried over anhydrous MgSO4, and then clarified by filtration. The solvent was
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removed under reduced pressure and the product was obtained as a pure white solid (4.88 g, 87%).
1H NMR (400 MHz, 298 K, CDCl3): δ 7.76 (t, J = 7.8 Hz, 1H), 7.43 (d, J = 7.8 Hz, 2H), 4.59 (s,
4H). 13C NMR (75 MHz, 298 K, CDCl3) δ 156.8, 138.5, 123.1, 33.3. LR-ESI-MS: calcd for
[C7H7Br2N + H]+ 263.9; found [M(79Br) + H]+ 263.9
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Di-tert-butyl-6,6'-(((pyridine-2,6-diylbis(methylene))-bis((2-(tert-butoxy)-2-oxoethyl)aza-
nedi-yl))bis(methylene))dipicolinate (7). To a round-bottom flask with a stirred solution of
compound 6 (1.00 g, 3.80 mmol, 1 equiv) in dry ACN (15 mL) was added K2CO3 (1.38 g, 11.4
mmol, 3 equiv.), followed by compound 4 (2.45 g, 7.60 mmol, 2 equiv.) and KI (1.26 g, 7.60
mmol, 2 equiv.). The mixture was stirred at 40 oC for 24 hours, and then K2CO3 was separated by
centrifugation, followed by washing with DCM/ACN (10 mL × 3). The organic phase was
concentrated in vacuo and then purified through a silica column (CombiFlash Rf automated
column system, 24 g gold silica column, DCM : MeOH, 0-8% MeOH). The product fractions were
rotary-evaporated to give a yellow oil (1.99 g, 70%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.93 –
7.76 (m, 4H), 7.61 (t, J = 7.6 Hz, 1H), 7.49 (d, J = 7.4 Hz, 2H), 7.12 (d, J = 7.7 Hz, 2H), 4.23 (s,
4H), 3.97 (s, 4H), 3.18 (s, 4H), 1.32 (s, 18H), 1.24 (s, 18H). 13C NMR (75 MHz, 298 K, CDCl3):
δ 169.8, 164.7, 157.3, 157.0, 145.9, 138.1, 137.3, 126., 122.9, 121.8, 80.7, 58.5, 58.1, 53.6, 53.1,
51.9. LR-ESI-MS: calcd for [C41H57N5O8 + Na]+ 770.4; found [M + Na]+ 770.4
H4pypa (8). Compound 7 (37.6 mg, 5.03 × 10-5 mol) was dissolved in DCM (1 mL) in a round-
bottom flask and TFA (1 mL) was added dropwise to the stirred solution using a Pasteur pipette.
The mixture was stirred overnight at room temperature and then rotary-evaporated. The residue
was redissolved in H2O (2 mL) and then purified through reverse phase HPLC (A: ACN, B:
H2O/0.1% TFA, 5-50% A over 30 minutes, 10 mL/minute, tR = 16.8 minutes) (18.4 mg, 70%). 1H
NMR (400 MHz, 298 K, D2O): δ 8.16 – 8.03 (m, 4H), 7.92 (t, J = 7.8 Hz, 1H), 7.74 (d, J = 7.0 Hz,
2H), 7.49 (d, J = 7.9 Hz, 2H), 4.78 (s, 4H), 4.71 (s, 4H), 4.19 (s, 4H). 13C NMR (75 MHz, 298 K,
D2O): δ 169.8, 166.3, 150.3, 150.2, 146.3, 141.2, 140.9, 128.2, 125.5, 125.0, 58.1, 58.0, 55.2. HR-
ESI-MS: calcd for [C25H25N5O8 + H]+ 524.1781; found [M + H]+ 524.1783. Elemental analysis:
calcd % for H4pypa·2TFA·1.7H2O (C29H30.4F6N5O13.7 = 782.1739): C 44.53, H 3.92, N 8.95; found:
C 44.86, H 3.59, N 8.63.
Na[natIn(pypa)] Compound 8 (9.60 mg, 1.26 × 10-5 mol, 1 equiv.) was dissolved in H2O (0.5 mL)
in a scintillation vial and 0.1 M NaOH (aq) was added to adjust the pH to 7. In(NO3)3·6H2O (6.17
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mg, 1.51 × 10-5 mol, 1.2 equiv.) was added. The mixture was stirred at room temperature for 1
hour and the complexation was confirmed by LR-ESI-MS. 1H NMR (400 MHz, 298 K, D2O): δ
8.26 (t, J = 7.7 Hz, 1H), 8.20 – 8.15 (m, 2H), 8.02 – 7.94 (m, 2H), 7.88-7.83 (m, 2H), 7.50 (d, J =
7.7 Hz, 1H), 7.34 (d, J = 7.9 Hz, 1H), 4.44 (d, J = 17.4 Hz, 1H), 4.10 (d, J = 17.4 Hz, 1H), 3.91
(d, J = 17.1 Hz, 1H), 3.78 (d, J = 17.1 Hz, 1H), 3.22 (d, J = 17.5 Hz, 1H), 2.94 (d, J = 17.7 Hz,
1H). 13C NMR (100 MHz, 298 K, D2O): δ 176.7, 176.2, 168.3, 168.2, 154.0, 153.9, 153.2, 152.9,
145.1, 144.9, 142.5, 142.4, 142.2, 128.0, 127.7, 124.99, 123.48, 123.0, 117.8, 61.0, 60.7, 59.6,
57.4, 57.3, 54.8. HR-ESI-MS: calcd for [C25H21115InN5O8 + 2Na]+ 680.0224; found [M + 2Na]+
680.0223.
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Na[natLu(pypa)] Compound 8 (13.6 mg, 1.79 × 10-5 mol, 1 equiv.) was dissolved in H2O (0.5
mL) in a scintillation vial and 0.1 M NaOH (aq) was added to adjust the pH to 7. Lu(NO3)3·6H2O
(9.23 mg, 1.97 x 10-5 mol, 1.1 equiv.) was added. The mixture was stirred at room temperature for
1 hour and the complexation was confirmed by LR-ESI-MS. 1H NMR (400 MHz, 298 K, D2O): δ
8.21 (t, J = 7.8 Hz, 2H), 8.05 (d, J = 7.6 Hz, 2H), 7.85 (t, J = 7.8 Hz, 1H), 7.80 (d, J = 7.8 Hz, 2H),
7.44 (d, J = 7.8 Hz, 2H), 4.66 (d, J = 2.6 Hz, 4H), 4.39 (d, J = 14.7 Hz, 2H), 4.07 (d, 2J = 14.7 Hz,
2H), 3.98 (d, 2J = 16.6 Hz, 2H), 3.49 (d, 2J = 17.0 Hz, 2H). 13C NMR (100 MHz, 298 K, D2O): δ
179.1, 173.1, 156.5, 152.7, 150.5, 141.6, 140.0, 125.5, 124.0, 123.0, 64.6, 64.5, 63.4. HR-ESI-MS:
calcd for [C25H21175LuN5O8 + 2H]+ 696.0954; found [M + 2H]+ 696.0956.
Na[natLa(pypa)]Compound 8 (13.5 mg, 1.79 × 10-5 mol, 1 equiv.) was dissolved in H2O (0.5 mL)
in a scintillation vial and 0.1 M NaOH (aq) was added to adjust the pH to 6. La(ClO4)3·6H2O (10.7
mg, 1.97 x 10-5 mol, 1.1 equiv.) was added. The mixture was stirred at room temperature for 1
hour and the complexation was confirmed by LR-ESI-MS. 1H NMR (400 MHz, 298 K, D2O): δ
8.07 (t, J = 7.7 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.80 (s, 2H), 7.75 (t, J = 7.7 Hz, 1H), 7.67 (d, J
= 7.8 Hz, 1H), 7.44 (d, J = 7.7 Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 7.17 (s, 1H), 4.62 (d, J = 16.2
Hz, 1H), 4.50 (d, J = 14.0 Hz, 1H), 4.22 (d, J = 16.0 Hz, 1H), 4.06 (d, J = 18.6 Hz, 2H), 3.85 (d, J
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= 14.1 Hz, 4H), 3.64 (d, J = 16.3 Hz, 1H), 3.09 (s, 2H). C NMR (100 MHz, 298 K, D2O): δ
180.2, 173.4, 156.6, 156.1, 150.0, 145.3, 140.4, 126.5, 125.0, 123.7, 123.6, 123.3, 123.2, 63.5,
63.4, 62.5, 62.2. HR-ESI-MS: calcd for [C25H21139La N5O8 + 2Na]+ 704.0249; found [M + 2Na]+
= 704.0251.
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Dimethyl 4-hydroxypyridine-2,6-dicarboxylate (9). Thionyl chloride (SOCl2) (9.50 mL, 0.130
mol, 5 equiv.) was added slowly using a syringe to a stirred suspension of chelidamic acid
monohydrate (5.28 g, 26.2 mmol, 1 equiv.) in MeOH (60 mL) in a two-neck round-bottom flask
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at 0 C. The mixture was stirred at room temperature for 24 hours and then refluxed for an
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additional 2 hours. The solvent was removed under reduced pressure gently at room temperature
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and then D.I. water was added at 0 C. The mixture was neutralized with 1 M K2CO3 in water
solution and the precipitate was filtered by vacuum filtration, and then washed with 50% MeOH
in water solution (~10 mL). The white precipitate was dried under reduced pressure to give a white
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solid (5.54 g, >99 %). H NMR (400 MHz, 298 K, (CD3)2SO): δ 6.74 (s, 2H), 3.72 (s, 6H). C
NMR (75 MHz, 298 K, (CD3)2SO): δ 165.7, 149.2, 116.6, 52.7. LR-ESI-MS : calcd for [C9H9NO5
+ Na]+ 234.0; found [M + Na]+ 234.2
Dimethyl 4-(benzyloxy)pyridine-2,6-dicarboxylate (10). To a round-bottom flask with a stirred
solution of compound 9 (1.65 g, 7.82 mmol, 1 equiv.) in dry ACN was added anhydrous K2CO3
(2.19 g, 15.8 mmol, 2.02 equiv.) and benzyl bromide (1.02 mL, 8.60 mmol, 1.1 equiv.). The
reaction mixture was refluxed overnight at 60 oC. K2CO3 was filtered out by vacuum filtration and
then washed with DCM. The filtrate was concentrated in vacuo and then purified through a silica
column (CombiFlash Rf automated column system, 24 g gold silica column, DCM : MeOH, 0-5
% MeOH). The product fractions were rotary-evaporated to give a white powder (1.51 g, 64 %).
1H NMR (400 MHz, 298 K, CDCl3): δ 7.90 (s, 2H), 7.44-7.38 (m, 5H), 5.23 (s, 2H), 4.01 (s, 6H).
13C NMR (75 MHz, 298 K, CDCl3): δ 150.0, 129.0, 128.9, 127.9, 115.0, 71.0, 53.4. LR-ESI-MS :
calcd for [C16H15NO5 + Na]+ 324.1; found [M + Na]+ 324.1
(4-(Benzyloxy)pyridine-2,6-diyl)dimethanol (11). To a round-bottom flask with a stirred
solution of compound 10 (8.74 g, 29.0 mmol, 1 equiv.) in dry MeOH (90 mL) was added NaBH4
(3.29 g, 87.1 mmol, 3 equiv.) in three portions over 30 minutes at 0 oC. The reaction mixture was
stirred at room temperature. After 24 hours, the mixture was diluted with CHCl3 (50 mL) and then
quenched with saturated aqueous NaHCO3 (50 mL). The organic phase was separated and the bulk
of MeOH in the aqueous layer was removed in vacuo to give an aqueous solution which was
extracted with CHCl3 (50 mL × 4). The combined organic phases were dried over anhydrous
Na2CO3, and then clarified by filtration. The filtrate was rotary-evaporated to give a white solid
(5.86 g, 82 %).1H NMR (400 MHz, 298 K, CDCl3): δ 7.42-7.35 (m, 5H), 6.79 (s, 2H), 5.12 (s,
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2H), 4.70 (s, 4H). 13C NMR (75 MHz, 298 K, CDCl3): δ 184.4, 166.5, 162.7, 160.6, 149.6, 135.6,
128.9, 128.6, 127.6, 117.2, 111.8, 107.7, 106.5, 106.1, 105.2, 70.2, 64.5. LR-ESI-MS: calcd for
[C14H15NO3 + Na]+ 268.1; found [M + Na]+ 268.2
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4-(Benzyloxy)-2,6-bis(bromomethyl)pyridine (12). Compound 11 (1.76 g, 12.6 mmol, 1 equiv.)
was suspended in dry ACN/dry CHCl3 (40 mL, 50:50 v/v) in a three-neck round-bottom flask.
PBr3 (3.60 mL, 37.9 mmol, 3 equiv.) in CHCl3 (5 mL) was added dropwise using a dropping funnel
to the stirred solution of compound 11 at 0 oC over 15 minutes. The reaction mixture was stirred
at 60 oC for 18 hours and then saturated aqueous Na2CO3 was added slowly to quench the reaction
at 0 oC. The aqueous phase was extracted with CHCl3 (50 mL × 3). The combined organic phases
were dried over anhydrous Na2SO4, and then clarified by filtration. The filtrate was rotary-
evaporated to yield a colorless oil which later solidified to a white solid (3.28 g, 70 %). 1H NMR
(400 MHz, 298 K, CDCl3): δ 7.43 (m, 5H), 7.36 (s, 2H), 5.37 (s, 2H), 4.95 (s, 4H). 13C NMR (75
MHz, 298 K, CDCl3): δ 170.9, 154.5, 133.2, 129.5, 129.3, 128.3, 113.2, 73.0, 25.3. LR-ESI-MS:
calcd for [C14H1379Br2NO + H]+ 369.9; found [M(79Br) + H]+ 369.9
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Di-tert-butyl-6,6'-((((4-(benzyloxy)pyridine-2,6-diyl)bis(methylene))bis((2-(tert-butoxy)-2-
oxoethyl)azanediyl))bis(methylene))dipicolinate (13). Compound 12 (0.400 g, 1.30 mmol, 1
equiv.), K2CO3 (595 mg, 4.31 mmol, 3.3 equiv.) and KI (434 mg, 2.61 mmol, 2 equiv.) were added
sequentially to the stirred solution of compound 4 (0.837 g, 2.60 mmol, 2 equiv.) in dry ACN (15
mL) in a round-bottom flask. The mixture was stirred at 30 oC for 24 hours. K2CO3 was removed
by centrifugation and then washed with DCM/ACN (10 mL × 3). The combined supernatants were
concentrated in vacuo and then purified with a silica column (CombiFlash Rf automated column
system, 12 g gold silica column, DCM : MeOH, 0-5% MeOH). The product fractions were rotary-
evaporated to give a pale yellow oil (0.67 g, 73 %). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.92-
7.61 (m, 6H), 7.52-7.30 (m, 5H), 7.12 (s, 2H), 5.11 (s, 2H), 4.03 (s, 4H), 3.86 (s, 4H), 3.33 (s, 4H),
13
1.57 (s, 18H), 1.43 (s, 18H). C NMR (75 MHz, 298 K, CDCl3): δ 170.5, 166.2, 164.0, 160.2,
148.6, 137.2, 136.1, 128.5, 128.1, 127.7, 125.6, 123.3, 123.0, 107.8, 81.8, 80.9, 69.7, 64.4, 59.8,
56.1, 53.4, 28.0. LR-ESI-MS: calcd for [C48H63N5O9 + Na]+ 876.5; found [M + Na]+ 876.6
Di-tert-butyl-6,6'-((((4-hydroxypyridine-2,6-diyl)bis(methylene))bis((2-(tert-butoxy)-2-
oxoethyl)azanediyl))bis(methylene))dipicolinate (14). Compound 13 (0.170 g, 0.200 mmol) was
dissolved in dry MeOH (20 mL) in a three-neck round-bottom flask, saturated with N2(g). Pd/C
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(10 % w/w, 0.1 equiv.) was added under a stream of N2(g). The flask was purged with N2(g),
followed by H2(g) from a balloon. The mixture was stirred vigorously at room temperature
overnight under H2 atmosphere, and then Pd/C was filtered off through a Celite bed, washed with
MeOH (10 mL × 5). The filtrate was rotary-evaporated to a pale-yellow oil (0.150 g) and used
without purification. LR-ESI-MS: calcd for [C41H57N5O9 + H]+ 764.4; found [M + H]+ 764.6
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Benzyl 8-bromooctanoate (15). 8-Bromooctanoic acid (2.00 g, 8.96 mmol, 1 equiv.), benzyl
alcohol (0.930 ml, 8.96 mmol, 1 equiv.) and a catalytic amount of DMAP (0.1-0.2 equiv.) were
dissolved sequentially in dry DCM (20 mL) in a round-bottom flask. DCC (2.04 g, 9.86 mmol, 1.1
equiv.) in DCM (10 mL) was added dropwise using a dropping funnel over 1 hour. The mixture
was stirred at room temperature for 24 hours. The white precipitate was filtered off by filtration
and then the solvent was evaporated in vacuo. The residue was purified with a silica column
(CombiFlash Rf automated column system, 24 g gold silica column, DCM : MeOH, 0 - 5 %
MeOH). The product fractions were rotary-evaporated to yield a colorless oil (2.51 g, 89 %). 1H
NMR (400 MHz, 298 K, CDCl3): δ 7.35-7.32 (m, 5H), 5.12 (s, 2H), 3.39 (t, J = 6.8 Hz, 2H), 2.36
(t, J = 7.5 Hz, 2H), 1.83 (p, J = 6.9 Hz, 2H), 1.69 – 1.61 (m, 2H), 1.44-1.40 (m, 2H), 1.32 (dt, J =
13
7.3, 3.5 Hz, 4H). C NMR (75 MHz, 298 K, CDCl3): δ 173.7, 136.2, 128.7, 128.3, 66.3, 34.4,
34.0, 32.8, 29.0, 28.5, 28.1, 24.9. LR-ESI-MS: calcd for [C15H21BrO2 + Na]+ 335.1; found [M +
Na]+ 335.1
Di-tert-butyl-6,6'-((((4-((8-(benzyloxy)-8-oxooctyl)oxy)pyridine-2,6-diyl)bis-
(methylene))bis-((2-(tert-butoxy)-2-oxoethyl)azanediyl))-bis(methylene))-di-picolinate (16).
To a round-bottom flask with a stirred solution of compound 14 (152 mg, 0.200 mmol, 1 equiv.)
in dry tetrahydrofuran (THF) (4 mL) was added anhydrous K2CO3 (82.6 mg, 0.600 mmol, 3 equiv.).
The mixture was stirred for 1 hour before the addition of compound 15 (65.6 mg, 0.210 mmol,
1.05 equiv.). The mixture was stirred for 24 hours at 30 oC, followed by separation of K2CO3 by
centrifugation. The isolated salt was washed with DCM twice (~5 mL each) while the combined
organic phases were concentrated in vacuo to a yellow oil. The product crude was characterized
by MS and NMR, and then used directly in the next step (0.195 g, 90%). 1H NMR (400 MHz,
298 K, CDCl3): δ 7.85 – 7.80 (m, 4H), 7.72 (t, J = 7.7 Hz, 2H), 7.34-7.27 (m, 5H), 6.98 (s, 2H),
5.09 (s, 2H), 4.02 (s, 4H), 3.96 (t, J = 6.4 Hz, 2H), 3.84 (s, 4H), 3.32 (s, 4H), 2.34 (t, J = 7.5 Hz,
2H), 1.77 – 1.70 (m, 2H), 1.66-1.58 (m, 24H), 1.42 (s, 18H), 1.34 – 1.32 (m, 4H). 13C NMR (75
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MHz, 298 K, CDCl3): δ 173.6, 170.6, 166.7, 164.2, 160.4, 160.2, 148.7, 137.3, 136.1, 128.6, 128.2,
125.7, 123.1, 107.7, 82.0, 81.0, 67.9, 66.1, 64.3, 59.9, 56.2, 34.3, 29.1, 28.1, 25.9, 25.4, 24.9. LR-
ESI-MS: calcd for [C56H77N5O11 + H]+ 996.6; found [M + H]+ 996.7
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8-((2,6-Bis(((2-(tert-butoxy)-2-oxoethyl)((6-(tert-butoxycarbonyl)pyridin-2-yl)methyl)-
amino)methyl)pyridin-4-yl)oxy)octanoic acid (17). Compound 16 (94.8 mg, 0.0952 mmol) was
dissolved in dry MeOH (7 mL) in a three-neck round-bottom flask, saturated with N2(g). Pd/C (10
% w/w) was added under a stream of N2. The flask was purged with N2(g) again, followed by
H2(g) from a balloon. The mixture was stirred vigorously at room temperature overnight under H2
atmosphere, and then Pd/C was filtered off through a Celite bed, washed with MeOH(10 mL × 5).
The filtrate was concentrated in vacuo to a pale-yellow oil (75.70 mg, 88 %) and used without
purification. LR-ESI-MS: calcd for [C49H71N5O11 + Na]+ 928.5; found [M + Na]+ 928.7
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tBu4pypa-C7-NHS (18). To a two-neck round-bottom flask with a stirred solution of compound
17 (75.7 mg, 0.0837 mmol, 1 equiv.) in dry ACN (2 mL) was added N-hydroxysuccinimide (10.6
mg, 0.0922 mmol, 1.1 equiv.) and EDCI (19.2 mg, 0.101 mmol, 1.2 equiv.) under N2 (g). The
mixture was stirred at room temperature under an inert atmosphere overnight. Then, the solvent
was removed in vacuo and the residue was redissolved in DCM (10 mL), and then washed with
water (10 mL × 3) and brine (10 mL × 2). The combined organic phases were dried over anhydrous
Na2SO4, and then clarified by filtration. The filtrate was rotary-evaporated to yield a yellow oil
(71.70 mg, 86 %) which was used in next step without further purification. 1H NMR (400 MHz,
298 K, CDCl3): δ 7.92 – 7.77 (m, 4H), 7.77 – 7.67 (m, 2H), 6.98 (s, 2H), 4.11 – 3.92 (m, 6H), 3.83
(s, 4H), 3.30 (s, 4H), 2.79 (s, 4H), 2.59 – 2.54 (m, 2H), 1.76 – 1.69 (m, 4H), 1.56 (s, 18H), 1.40 (s,
24H). 13C NMR (75 MHz, 298 K, CDCl3): δ 173.7, 170.7, 169.3, 168.7, 164.2, 160.4, 148.8, 137.4,
128.7, 128.3, 125.8, 123.2, 82.1, 81.1, 77.4, 66.2, 60.0, 56.4, 31.0, 29.8, 29.0, 28.8, 28.3, 28.2,
25.9, 25.7, 24.6. HR-ESI-MS: calcd [C53H75N6O13 + H]+ 1003.5392 found [M + H]+ 1003.5358
Solid Phase Peptide Coupling. Solid-phase synthesis of H4pypa-C7-PSMA617 was modified
from literature procedures.26 Fmoc-Lys(ivDde)-Wang resin (0.046 mmol, 0.61 mmol/g loading)
was suspended in dimethylformamide (DMF) for 30 minutes. Fmoc was then removed by treating
the resin with 20% piperidine in DMF (3 × 8 minutes). The isocyanate derivative of di-t-butyl ester
of glutamate (0.138 mmol, 3 equiv.) was prepared according to literature procedures26 and added
to the lysine-immobilized resin to react for 16 hours. After washing the resin with DMF, the ivDde-
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protecting group was removed with 2% hydrazine in DMF (5 × 5 minutes), followed by coupling
of Fmoc-2-Nal-OH and Fmoc-tranexamic acid to the side chain of Lys using Fmoc-protected
amino acid (0.138 mmol, 3 equiv.), N,N,N’,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uronium
hexafluorophosphate (HBTU) (0.138 mmol, 3 equiv.), hydroxybenzotriazole (HOBt) (0.138 mmol,
3 equiv.) and N,N-diisopropylethylamine (DIPEA) (0.368 mmol, 8 equiv.). Afterwards, the
chelator tBu4pypa-C7-NHS (0.138 g, 0.138 mmol) was coupled to the peptide-bound resin by using
DIPEA (0.460 mmol, 10 equiv.) in DMF overnight. The peptide was then deprotected and
simultaneously cleaved from the resin by treating with 95/5 TFA/triisopropylsilane (TIS) for 2
hours at room temperature. After filtration, the peptide was precipitated by adding cold diethyl
ether to the TFA solution. The crude peptide was purified by semi-preparative HPLC (32%
acetonitrile in water containing 0.1% TFA at a flow rate of 4.5 mL/minute, tR = 8.8 minutes). The
eluates containing the desired peptide were collected, pooled, and lyophilized (5.16 mg, 3.91 mol,
8.5%) HR-ESI-MS: calcd [C66H82N10O19 + H]+ 1319.5836; found [M+H]+ 1319.7376.
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X-ray Crystallography. Single orange colored rhombic-shaped crystals of H[Lu(pypa)] were
obtained by the slow evaporation of 1:1 LuCl3 and H4pypa solutions in water after adjustment of
pH to 2. A suitable crystal 0.15×0.05×0.01 mm3 was selected and mounted on a suitable support
on a Bruker APEX-II CCD diffractometer. The crystal was kept at a steady T = 90(2) K during
data collection. The structure was solved with the ShelXT45structure solution program using the
dual solution method and by using Olex246 as the graphical interface. The model was refined with
version 2018/1 of ShelXL45 using Least Squares minimisation.
Solution Thermodynamics. All potentiometric titrations were carried out with a Metrohm
Titrando 809 and a Metrohm Dosino 800 with a Ross combined electrode. A 20 mL and 25 ºC
thermostated glass cell with an inlet-outlet tube for nitrogen gas (purified through a 10% NaOH
solution to exclude any CO2 prior to and during the course of the titration) was used as a titration
cell. The electrode was calibrated daily in hydrogen ion concentration by direct titration of HCl
with freshly prepared NaOH solution and the results were analyzed with Gran procedure47 in order
to obtain the standard potential Eº and the ionic product of water pKw at 25 °C and 0.16 M NaCl
as a supporting electrolyte. Solutions were titrated with carbonate-free NaOH (0.16 M) that was
standardized against freshly recrystallized potassium hydrogen phthalate. The experimental
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procedures for determination of the ligand protonation constants, complex formation constants and
pM values are described in the supporting information.
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Radiolabeling and Human Serum Challenge Experiments. Generally, for concentration-
dependent radiolabeling, an aliquot of a ligand solution (25 L) of desired concentration was
mixed with 177Lu/111In (~2 MBq) and diluted to a final volume (250 L) with ammonium acetate
solution (0.15 M, pH=7). The final mixture was incubated at room temperature for 10 minutes
before determination of radiochemical yield with iTLC-SA plate and radio-HPLC (Detailed
procedures of concentration- and ratio-dependent radiolabeling are described in the Supporting
Information). For the human serum challenge, to a quantitative radiolabeled complex solution was
added an equal volume of human serum. The mixture was incubated at 37 oC and an aliquot of the
mixture was spotted on iTLC-SA plate at desired time-point to determine the amount of intact
complex (%) (A detailed procedure is described in the Supporting Information).
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SPECT/CT Imaging, Biodistribution Studies and Binding Affinity.
Imaging and
biodistribution experiments were performed using NODSCID IL2RγKO male mice. The mice
were maintained, and the experiments were conducted according to the guidelines established by
the Canadian Council on Animal Care and approved by Animal Ethics Committee of the
University of British Columbia (A detailed procedure is described in the Supporting Information).
SPECT/CT imaging experiments were conducted using the MILabs (Utrecht, The Netherlands) U-
SPECT+/CT scanner. Each tumor bearing mouse was injected with 111Lu/111In labeled H4pypa-
C7-PSMA617 (44.1 MBq for 177Lu and 24.9 MBq for 111In) through the tail vein under anesthesia
of 2% isoflurane in oxygen. The mice were allowed to recover and roam freely in their cage and
imaged at 1, 4, 24, and 72 hours after injection. At each time point, the mice were sedated again
with 2% isoflurane in oxygen and positioned in the micro scanner (A detailed procedure is
described in the Supporting Information). For in vitro competition binding assays, non-specific
binding was determined in the presence of non-radiolabeled DCFPyL (10 µM). The assay mixtures
were further incubated for 1 hour at 37 °C with gentle agitation. Then, the buffer and hot ligand
were removed, and cells were washed twice with cold HEPES buffered saline. Trypsin solution
(400 µl, 0.25%) was then added to each well to harvest the cells. Radioactivity was measured on
the gamma counter. Data analyses of Ki were performed using the nonlinear regression algorithm
of GraphPad Prism 7 software (A detailed procedure is described in the Supporting Information).
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