tBu4octapa-alkyl-NHS for metalloradiopeptide preparation

Article abstract of DOI:10.1039/d0dt00845a

The peptide is an important class of biological targeting molecule; herein, a new bifunctional octadentate non-macrocyclic H₄octapa,^tBu₄octapa-alkyl-NHS, which is compatible with solid-phase peptide synthesis and thus usef

Full text of DOI:10.1039/d0dt00845a

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2020, DOI: 10.1039/D0DT00845A.
Volume 46
Number 10
14 March 2017
Pages 3073-3412
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DOI: 10.1039/D0DT00845A
tBu4octapa-alkyl-NHS for Metalloradiopeptide Preparation
Lily Lia,b, Hsiou-Ting Kuoc, Xiaozhu Wanga, Helen Merkensc, Nadine Colpoc, Valery
Radchenkob,d, Paul Schafferb, Kuo-Shyan Linc, François Bénardc, Chris Orviga*
a Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British
Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada
b Life Sciences Division, TRIUMF, 4004 Wesbrook Mall, Vancouver, British Columbia V6T
2A3, Canada
c Department of Molecular Oncology, BC Cancer, 675 West 10th Ave, Vancouver, British
Columbia V5Z 1L3, Canada
d Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British
Columbia V6T 1Z1, Canada
Abstract
The peptide is an important class of biological targeting molecule; herein, a new bifunctional
t
octadentate non-macrocyclic H4octapa, Bu4octapa-alkyl-NHS, which is compatible with solid-
phase peptide synthesis and thus useful for radiopeptide preparation, has been synthesized. To
preserve denticity, the alkyl-N-hydroxylsuccinimide linker was covalently attached to the
methylene-carbon on one of the acetate arms, yielding a chiral carbon center. According to density-
functional theory (DFT) calculations using [Lu(octapa-alkyl-benzyl-ester)]- as a simulation model,
the chirality has minimal effects on the complex geometry; regardless of the S-/R-stereochemistry,
DFT calculations revealed two possible geometric isomers, distorted bicapped trigonal antiprism
(DBTA) and distorted square antiprism (DSA), due to the asymmetry in the chelator. To evaluate
the biological behavior of the new bifunctionalization, two well-studied PSMA (prostate-specific
membrane antigen)-targeting peptidomimetics of varying hydrophobicity were chosen as proof-
of-principle targeting vector molecules. Radiolabeling both bioconjugates with lutetium-177 was
highly efficient at room temperature in 15 min at micromolar chelator concentration pH = 7. Both
the in vitro serum challenge and the lanthanum(III) challenge studies revealed complex lability,
and notably, progressive bone accumulation was only observed with the more hydrophobic linker
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(i.e. H4octapa-alkyl-PSMA617). This in vivo result informs potential alterations exerted by the
linker on the complex geometry and stability, with an appropriate biological targeting vector
adopted for such evaluations.
Introduction
Radiometalated bioconjugates are useful constructs in nuclear imaging and therapy. The primary
scaffold of a metallo-radiopharmaceutical consists of four synthetically exchangeable modules,
including the radiometal ion, the emission properties of which can be harnessed for diagnosis
and/or therapy, the chelator which stably sequesters the radiometal ion, and the linker which tethers
the chelator to the biological-targeting vector for disease targeting.1,2 Peptides, in particular, have
recently garnered considerable attentions in targeted drug delivery,3,4,5 due to the high affinity and
specificity, higher diffusibility, lower immunogenicity, and easier synthesis/modifications.6,7
Unlike monoclonal antibodies (mAb), which necessitate the use of long-lived radionuclides due to
the long blood circulation and clearance, peptide-conjugates are compatible with a wider range of
decay isotope half-life, from ~1 h (e.g. gallium-68),8 to >6 d (e.g. lutetium-177 and actinium-
225).9,10 Besides the favorable half-life, lutetium-177 is a highly potent theranostic radionuclide
due to the low energy -particles (maximum 498 keV),10 imageable -rays (208 and 113 keV),11
and efficient production routes (i.e. [176Lu(n,γ)177Lu] or [176Yb(n,)177Yb177Lu]) to meet the
large clinical demands.12,13,14
The essential component to join the otherwise incompatible radionuclide and targeting vector is a
bifunctional chelator that secures the radiometal ion in vivo. To date, the macrocycle DOTA
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) dominates radiometal chelation
(including with lutetium-177) in both preclinical and clinical studies, because its constrained
binding cavity often yields metal complexes with higher stability.15,16,17,18 However, prolonged
heating is often required in order to achieve quantitative radiochemical yield (RCY).1 Although
peptide-conjugates generally have higher tolerance to the elevated temperature, fast RT-
radiolabeling can facilitate radiotracer preparation in the clinical settings following “shake-and-
shoot” protocols, and avoid degradation of the products. In this regard, non-macrocyclic chelators
are preferable since, with enough rigidity, they can combine fast coordination kinetics with high
complex stability.19–21
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Multiple studies prove that the chemical properties (e.g. hydrophilicity, charges) of the radiometal
complex and the spacer can impact the drug biodistributions drastically, particularly when coupled
to small peptides.9,22,23,24,25,26,27,28 As a result, expanding the chelator-library beyond the
commercial chelating agents (e.g. DOTA or DTPA (diethylenetriamine-pentaacetic acid), Chart
1) is always worthwhile.
A family of non-macrocyclic chelators that target various radionuclides with fast coordination
kinetics at room temperature has been established.17,20,27,29,30,31,32,33 An octadentate member,
H4octapa (Chart 1), has demonstrated excellent affinity for a range of radiometal ions, including
that of lutetium-177. The complex was thermodynamically stable (log KLu(octapa) = 20.1 and pLu =
19.8).32 In addition, the biological stability of [177Lu][Lu(octapa)]- was demonstrated with the
conjugate of a monoclonal antibody, Trastuzumab, using the N-isothiocyanate functionalized
H4octapa-benzyl-NCS (Chart 1), in which the p-benzyl-N-isothiocyanate (p-Bn-NCS) linker was
covalently attached to the ethylenediamine backbone; the bioconjugate was radiolabeled in high
radiochemical yield (95%) with excellent in vitro serum and in vivo stability.32 Although the
antibody-conjugation was reported to be efficient, difficulties were encountered when conjugating
H4octapa-benzyl-NCS to a small glutamate-urea-lysine-based PSMA (prostate-specific membrane
antigen)-targeting peptidomimetic, perhaps due to the interferences from multiple free carboxylic
acid groups on both the chelator and the targeting pharmacophore. This prompted us to develop a
new bifunctional octapa-analogue in which all carboxylic acid groups are protected as tert-butyl
esters and the linker is attached to one of the acetate arms to maintain the overall denticity (i.e.
tBu4octapa-alkyl-NHS, Scheme 1). Additionally advantageous in the design is its compatibility
with solid-phase peptide synthesis (SPPS) which allows direct incorporation of the chelating agent
into the resin-bound peptide.33 In fact, most peptides are generated by SPPS;34 therefore,
tBu4octapa-alkyl-NHS is anticipated to have widespread application in metallo-radiopeptide
synthesis. Herein, the preparation and characterization of tBu4octapa-alkyl-NHS are detailed. The
bifunctional chelator was successfully coupled to two well-studied PSMA-targeting
pharmacophores with different hydrophobicity (named as PSMA-ureido and PSMA617 herein,
Chart 1) on solid supports,35,36 where the more hydrophobic conjugate (i.e. PSMA617) was shown
to have improved pharmacokinetics (i.e. longer biological half-life, higher tumor uptake and faster
kidney clearance).36 Both PSMA targeting motifs can be easily built via SPPS, and most
importantly, extensive research has proven its targeting-specificity to PSMA which is up-regulated
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in prostate cancer cells. As a result, they are the suitable candidates for proof-of-principle animal
studies with a new chelator scaffold.37 Radiolabeling, in vitro serum challenge and lanthanum(III)
challenge studies were performed with both peptide-conjugates to investigate their metal-
scavenging abilities under extremely dilute conditions and the kinetic inertness of the formed
complexes. After that, both radiolabeled tracers were injected into LNCaP-xenograft mice (n = 5)
for biodistribution studies to evaluate in vivo stabilities. As well, in order to study the effects from
the bifunctionalization on the metal coordination environment, density-functional theory
calculations (DFT) were performed to compare the geometry of different 177Lu-labeled H4octapa
complexes.
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OH
O
O
O
NH
OH
or
=
R = H (H4pypa)
O
NH
R
N
NH
O
O
HO
O
O
N
N
OH
HO
H4pypa-C7-PSMA-ureido
N
N
O
OH
HO
O
O
H
N
H
O
O
N
NH
or
=
O
OH
O
O
HO
OH
N
N
H
H
O
O
H4pypa-C7-PSMA617
NCS
O
O
O
O
O
O
O
O
O
O
HO
HO
HO
N
N
OH
N
N
OH
N
N
OH
N
N
N
HO
HO
O
OH
HO
O
N
N
N
OH
O
N
N
N
N
N
N
OH
O
N
O
OH
OH HO
H4octapa
OH HO
OH HO
H4octapa-benzyl-NCS
HO
OH
O
O
O
O
O
O
S-benzyl-H4octapa
CHX-A"-DTPA
DOTA
OH
O
O
H
O
OH
O
H
N
HO
N
OH
O
O
H
NH
O
N
HO
NH
O
HN
O
HO
O
HN
O
O
O
O
O
O
O
O
NH
N
O
O
HO
HO
N
N
OH
N
N
OH
HO
N
N
OH
N
N
N
N
N
OH HO
OH HO
O
O
O
O
OH HO
O
O
H4octapa-alkyl-PSMA617
H4octapa-alkyl-benzyl-ester
H4octapa-alkyl-PSMA-ureido
Chart 1. Chemical structures of discussed ligands.
Results and Discussion
Synthesis and Characterization
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NO2
NO2
i
O2
S
ii
O2
S
O
H2N
NH2
N
NH2
N
N
H
O
H
H
1
2
O
O
O
O
O
Br
O
iii
iv
v
N
N
N
N
HO
O
O
O
O
HO
OH
vi
5
4
3
O
NO2
O
O
O
O
O
O
O
O
SO2
NH
O
N
N
N
O
x
vii
N
N
O
N
N
N
O
N
N
N
O
O
O
O
O
O
O
O
O
7
6
10
ix
O
O
O
O
viii
O
O
O
OH
O
O
O
OH
Br
Br
NH2
(L)
xi
9
(both R and S)
8 (both R and S)
O
N
HO
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
N
O
xii
N
N
N
N
O
O
O
O
O
O
O
12
(both R and S)
tBu4octapa-alkyl-NHS
11 (both R and S)
Scheme 1. Reagents and conditions. i) THF, nosyl chloride, ambient temp, 18 h, 89%; ii) tert-butyl bromoacetate,
TEA, dry ACN, ambient temp, 18 h, 63%; iii) DCC, tert-butyl alcohol, DCM, ambient temp, overnight, 50%; iv)
NaBH4, dry MeOH, ambient temp, 3-4 h, 72%; v) PBr3, dry ACN, 0°C-ambient temp, 54%; vi) K2CO3, dry ACN,
60°C, 24 h, 70%; vii) PhSH, K2CO3, THF, 18 h, ambient temp, 63%; viii) NaBr, 2 N HBr, NaNO2, conc H2SO4, 18 h,
ambient temp, 45%; ix) TBTA/cyclohexane, DMA, BF3-etherate, CHCl3, ambient temp, 3d ; x) K2CO3, dry ACN,
60°C, 48 h, 30%; xi) Pd/C, H2, MeOH, ambient temp, 18 h, 87%; xii) NHS, EDCl, dry ACN, 69%.
The synthesis (Scheme 1) started with the asymmetric nosyl protection of the ethylenediamine
backbone. About 0.3 equivalent of 2-nitrobenzenesulfonyl chloride (NsCl) dissolved in
tetrahydrofuran (THF) was slowly added to the stirred ethylenediamine solution at 0°C to give
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compound 1 in high yield (89%). After selective protection, mono-alkylation of the primary amine
in compound 1 was achieved by dropwise addition of tert-butyl bromoacetate in THF at ambient
temperature in order to minimize over-alkylation (compound 2, 63%). After that, the secondary
amines in compound 2 were alkylated with the bromo-picolinate derivative (compound 5) to give
the nosyl-protected ligand scaffold (compound 6, 70%). To synthesize the bromo-picolinate 2,6-
substitute (compound 5), the carboxylic acid groups in the commercially available 2,6-picolinate
dicarboxylic acid were converted to tert-butyl esters using N,N’-dicyclohexylcarbodiimide (DCC)
(compound 3, 50%). The resulting ester-derivative was selectively reduced to tert-butyl 6-
(hydroxymethyl)picolinate (compound 4, 72%) using sodium borohydride (NaBH4). The first
equivalent of NaBH4 was added to the stirred mixture of compound 3 at 0˚C, and then the mixture
was stirred at room temperature for 1 h before adding another equivalent of NaBH4. The reduction
continued until the mono-reduced picolinate dominated and the dihydroxyl-byproduct started to
grow, as monitored by silica TLC every 30 min (5% MeOH in DCM). This step was followed by
bromination of the alcohol group with phosphorus tribromide (PBr3) in dry acetonitrile (ACN) to
give compound 5 (54%). To free the secondary amine in compound 6, the nosyl protecting group
was removed with thiophenol at ambient temperature overnight (compound 7, 63%). Due to the
strong affinity for silica, compound 7 was purified with the neutral alumina column eluted with
dichloromethane (DCM) and methanol (MeOH). After that, (2S/2R)-5-benzyl 1-(tert-butyl) 2-
bromopentanedioate (compound 9) derived from (L)-glutamic acid γ-benzyl ester was
incorporated into compound 7 by refluxing the mixture with potassium carbonate (K2CO3) in dry
ACN to give compound 10 in 30% yield. The unsatisfactory yield could be due to the steric
hindrance between the crowded secondary amine and the secondary alkyl bromide. The benzyl
ester in compound 10 was selectively removed through palladium/carbon (Pd/C, 10% w/w)-
catalyzed hydrogenation to give compound 11 (87%), while the resulting free carboxylic acid
group was activated with N-hydroxylsuccinimide and N-ethyl-N-(3-dimethylaminopropyl)-
carbodiimide hydrochloride (NHS/EDC·HCl) to give tBu4octapa-alkyl-NHS (compound 12, 69%).
t
The overall yield was ~1 %. For peptide-conjugation, Bu4octapa-alkyl-NHS was coupled to the
PSMA-targeting-peptide-bound resin (the peptide was either PSMA-ureido or PSMA617, Chart
1) by using N,N-diisopropylethylamine (DIPEA) in dry dimethylformamide (DMF) overnight.
After that, the peptide-bioconjugate was deprotected and simultaneously cleaved from the resin
using 95/5 trifluoroacetic acid (TFA)/triisopropylsilane (TIS) at ambient temperature to give either
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H4octapa-alkyl-PSMA-ureido or H4octapa-alkyl-PSMA617 (Chart 1). It is noted that the
compound 9 used for the reaction consisted of both R- and S-enantiomers because the intermediate
compound 8 was synthesized via the diazotization reaction, in which a carbocation derivative was
generated upon the release of N2(g) by the highly unstable diazonium cation. As a result, the final
bifunctional chelator is not enantiomerically pure. However, due to the difficulties in separating
the enantiomers and for the purpose of preliminary investigations of this new chelator scaffold,
the isomers were not isolated, but the effects of the stereochemistry were studies with DFT
calculations.
DFT Calculations
To study the effects of the bifunctionalization in tBu4octapa-alkyl-NHS on the metal coordination
environment, DFT calculations were performed to predict the structures of different Lu-octapa
conformations. [Lu(octapa-alkyl-benzyl-ester)] was used as the simulation model for the acetate-
arm-functionalized H4octapa (Chart 1, Figure 1 and Figure S27). Calculations for the non-
bifunctional [Lu(octapa)]- and [Lu(S-benzyl-octapa)]- (Chart 1, Figure S28) employed as the
model for the complex with the ethylenediamine-functionalized H4octapa-benzyl-NCS, were
performed for comparison. The selected bond lengths and bond angles are tabulated in Table 1.
The functionalization on the acetate arm obviates the 2-fold rotational symmetry about the
ethylenediamine bridge in the non-bifunctional H4octapa. Two acetate arms in H4octapa-alkyl-
benzyl-ester (and tBu4octapa-alkyl-benzyl-NHS) become inequivalent, and therefore, their relative
positions can alter the overall complex geometry, leading to two possible structural isomers as
shown in Figure 1 and Figure S27. Since the methylene-carbon on the functionalized acetate arm
contains both S- and R-configurations, while for each S-/R-configuration, the functional arm can
bind to the metal ion Lu3+ from the front or the back with respect to the five-membered chelate
ring of Lu-N1-C-C-N2. The front and the back positions are defined based on the presented figures
(Figures 1 and S25). When the functional acetate arm chelates from the back of the Lu-N1-C-C-
N2 plane (O1 into the page), the 8-coordinated complex adopts the geometry of distorted square
antiprism (DSA), where (N2, N4, O2, O4) and (N1, N3, O1, O3) form the top and the bottom faces
of the distorted square antiprism, respectively. When the arm is positioned at the front of the plane
(O1 out of the page), the structure is a distorted bicapped trigonal antiprism (DBTA) where N2,
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N3, N4, O1, O2 and O3 formed the distorted trigonal antiprism capped by N1 and O4. DBTA was
also the calculated geometry adopted by both [Lu(octapa)]- and [Lu(S-benzyl-octapa)]- complexes
with similar bond lengths (Figure S28 and Table 1), which means that the linker in H4octapa-
benzyl-NCS very possibly poses minimal disturbances in Lu3+ coordination. As for [Lu(octapa-
alkyl-benzyl-ester)]-, the adopted molecular shape avoids the S-/R-stereochemistry. Indeed, upon
comparing the Lu-X bond lengths between the S- and the R-geometries, except for the Lu-N2 bond
which was 0.1628 Å shorter in R-DBTA than S-DBTA, the S-/R-configurations on the arm have
minimal effects on the coordination bond distances considering the same geometry (i.e. difference
<0.1 Å). Within the same S-/R-configuration, most bond lengths are comparable between the two
complex geometries (DBTA vs. DSA), except for those with the tertiary amine nitrogen atoms (N1
and N2), particularly between the S-DBTA and the S-DSA conformations, where the Lu-N2 bond
distance in the latter was shorter by almost 0.3 Å. Furthermore, the Lu-N2 bonds in both DSA
conformations were also shorter than those in the [Lu(octapa)]- and [Lu(S-benzyl-octapa)]-
complexes, especially when compared with the [Lu(S-benzyl-octapa)]- complex (difference of
~0.3 Å). The shorter Lu-N1 and Lu-N2 bond distances in the S-/R-DSA geometries accompanied
larger N1-Lu-N2 bond angles (~68˚), which could be slightly more favorable considering the ideal
M-N bond length and N-M-N bond angle in a five-membered chelate ring with ethylenediamine
being ~2.5 Å and 69˚, respectively.38,39 This inverse relationship between the Lu-N1/N2 bond
lengths and the N1-Lu-N2 bond angle is consistent with the observation reported by Bazargan et
al.,40 who indicated that the variation of the N-M-N bond angle of the five-membered NCCNM
chelate ring is directly related to the M-N bond distance. Nonetheless, all four acetate-
functionalized Lu-octapa geometries (i.e. S-/R- DSA/DBTA) were calculated to be energetically
similar, with the energy differences < 7 kcal/mol. Therefore, it is possible that they co-exist in
solution. Importantly, the results revealed a significant difference between placing the linker on
the ethylenediamine and functionalizing via the acetate moiety, where the latter has altered the
bond geometry in the [Lu(octapa)]- coordination sphere, and the changes were regardless of the S-
/R-stereochemistry.
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41,42,43,44,45,46
Table 1. Selected DFT-calculated bond lengths and bond angles.
Bond length Å / bond angle˚
Acetate arm functionalization
[Lu(S-benzyl-
octapa)]-
[Lu(octapa)]-
R-
R-
S-
S-
DBTA
DSA
DBTA
DSA
N1
N2
N3
N4
O1
O2
Backbone 3o N*
Backbone 3o N
Picolinate-N*
Picolinate-N
Lu 2.8942 2.7442 2.8541 2.7731
Lu 2.7859 2.6712 2.9487 2.6604
Lu 2.4903 2.4849 2.5775 2.4659
Lu 2.4582 2.4895 2.5443 2.4958
Lu 2.2732 2.2211 2.2430 2.2227
Lu 2.2703 2.2388 2.2307 2.2372
2.8245
2.9616
2.4836
2.5626
2.2610
2.2445
2.2769
2.2492
62.3
2.8202
2.8219
2.4678
2.4662
2.2737
2.2721
2.2704
2.2704
63.4
Acetate-COO*
Acetate-COO
O3 Picolinate-COO* Lu 2.2592 2.3320 2.2599 2.3367
O4
Picolinate-COO
N1-Lu-N2 angle
O1-Lu-O2 angle
Lu 2.2675 2.3364 2.2777 2.3380
63.5
170
67.9
135
61.9
170
68.1
136
170
171
*The side of the chelator where the linker is located (no difference for H4octapa as it is symmetric).
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S-Distorted bicapped trigonal antiprism (S-DBTA)
S-Distorted square antiprism (S-DSA)
Figure 1. DFT-calculated structures for S-DBTA and S-DSA geometries of [Lu(octapa-alkyl-benzyl-ester)]- (Same
geometry adopted for the corresponding R-configuration, Figure S27).
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Radiolabeling, Human Serum Stability and Lanthanum Challenge Experiments
Rapid and quantitative radiolabeling under mild conditions (~RT, pH ~ 7) with low chelator
concentration provides flexibility in tuning the apparent molar activity of the radiotracer which
can considerably influence the biological profile. It was shown previously that high apparent molar
activity of [177Lu][Lu(PSMA-617)] correlated to higher tumor-to-background ratio, and therefore
enhanced tumoricidal effects.47 However, this trend is not generalizable because the opposite was
observed with [44Sc][Sc(pypa-C7-PSMA617)], with which lower apparent molar activity
significantly reduced the background organ uptakes, especially the kidney.48 For that reason, it is
important to be able to adjust the apparent molar activity while not compromising the
radiochemical yield (RCY). In this work, concentration-dependent radiolabeling of H4octapa-
alkyl-PSMA-ureido and H4octapa-alkyl-PSMA617 with lutetium-177 (2 MBq) and the serum
protein transchelation study were performed to determine the radiolabeling efficiency and the in
vitro stability of the 177Lu-complexes, respectively (Figures 2A and 2B). The results were analyzed
with instant thin-layer chromatography plate impregnated with silicic acid (iTLC-SA), developed
in ethylenediaminetetraacetic acid (EDTA) buffer (50 mM, pH = 5.2). Attempts to use HPLC to
identify the radiolabeled complexes failed, perhaps due to the decomplexation in the acidic HPLC
column (mobile phase: water/acetonitrile/0.1%TFA, pH ~ 2). To validate the human serum
challenge experiment, a control sample with only lutetium-177 in ammonium acetate (NH4OAc)
buffer (0.2 M, pH = 7) and serum was prepared, and analyzed with the same technique, which
confirmed that the EDTA buffer was able to carry both free and transchelated lutetium-177 to the
solvent front (i.e. only one radioactivity spot at the solvent front on the developed TLC plate).
Previously, H4octapa has demonstrated efficient radiolabeling with lutetium-177 resulting in a
reasonably stable complex upon challenged with serum proteins; while the biological stability of
the formed complex was confirmed with H4octapa-benzyl-Trastuzumab, a mAb-conjugate using
the ethylenediamine-functionalized H4octapa-benzyl-NCS.32,49 In the case of H4octapa-benzyl-
Trastuzumab, the conjugation did not hamper the radiolabeling efficiency (95%, RT, 15 min, 74-
111 MBq)32, nor did it significantly reduce the kinetic inertness (~89% intact in human serum on
day 5, 37°C).49 However, with the linker placed on one of the acetate arms instead, both 177Lu-
labeled H4octapa-alkyl-PSMA-ureido and H4octapa-alkyl-PSMA617 demonstrated transchelation
to the human serum proteins within one day, with the PSMA617 conjugate more labile (18±10%
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intact on day 1, 37°C) (Figure 2B and Table S1). The complex lability was disappointing since the
radiolabeling performance at room temperature was impervious to the bioconjugation with
quantitative radiochemical yields achieved using 10-6 M chelator concentration, in 15 min with
100
80
60
40
20
0
100
80
60
40
20
0
A
B
[
[
177Lu][Lu(octapa-alkyl-PSMA-ureido)]
177Lu][Lu(octapa-alkyl-PSMA617)]
[
[
177Lu][Lu(octapa-alkyl-PSMA617)]
177Lu][Lu(octapa-alkyl-PSMA-ureido)]
0
1
2
3
4
5
6
7
4
5
6
7
-log[M]
Day
100
100
80
60
40
20
0
C
80
60
40
20
0
D
177Lu][Lu(pypa)]- 0:1
177Lu][Lu(pypa)]- 3:1
177Lu][Lu(pypa-C7-PSMA-ureido)] 0:1
177Lu][Lu(pypa-C7-PSMA-ureido)] 3:1
177Lu][Lu(pypa-C7-PSMA617)] 0:1
177Lu][Lu(pypa-C7-PSMA617)] 3:1
[
177Lu][Lu(octapa)] 0:1
-
[
[
[
[
[
[
-
[
[
[
[
[
177Lu][Lu(octapa)] 3:1
177Lu][Lu(octapa-alkyl-PSMA-ureido)] 0:1
177Lu][Lu(octapa-alkyl-PSMA-ureido)] 3:1
177Lu][Lu(octapa-alkyl-PSMA617)] 0:1
177Lu][Lu(octapa-alkyl-PSMA617)] 3:1
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
Day
Day
Figure 2. (A) Concentration-dependent radiolabeling of H4octapa-alkyl-PSMA-ureido and H4octapa-alkyl-PSMA617
with lutetium-177 in 15 min at room temperature and pH = 7 (0.2 M NH4OAc). (B) Intact percentage of the
radiocomplexes upon human serum challenge over 7 days at 37oC. Lanthanum challenges on 177Lu-labeled (C)
H4octapa, H4octapa-alkyl-PSMA-ureido, H4octapa-alkyl-PSMA617, and (D) H4pypa, H4pypa-C7-PSMA-ureido,
H4pypa-C7-PSMA617, over 7 days.
2 MBq lutetium-177 for both conjugates (~80 GBq/mol) (Figure 2A and Table S2). To
understand such unexpected lability, the kinetic inertness of each of the 177Lu-labeled H4octapa,
H4octapa-alkyl-PSMA-ureido and H4octapa-alkyl-PSMA617 was challenged with excess La3+
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ions (Figure 2C and Tables S3-S5). To validate the challenge experiment and relate it to the in
vitro, and even in vivo kinetic inertness of the complex, comparative experiments should be
conducted on similar constructs that have shown promising stability results with lutetium-177. In
this work, H4pypa, H4pypa-C7-PSMA-ureido and H4pypa-C7-PSMA617 were adopted for direct
comparisons (Chart 1, Figure 2D and Tables S6-S8) since the corresponding 177Lu-labeled pypa-
complexes/conjugates have demonstrated excellent serum and biological stability (data with
H4pypa-C7-PSMA-ureido are unpublished).20 The challenge studies were conducted by adding
diluted natLa3+ AAS (atomic absorption spectroscopy) standard solution to the radiolabeled
samples with 3:1 La3+:ligand (mol/mol) ratio and then incubating at 37°C, pH = 7. Samples without
natLa3+ ion were prepared as the control. As seen in Figure 2C, substantial amount of lutetium-177
leached out of all the H4octapa complexes steadily in the presence of excess La3+ ions (intact %
on day 7: octapa = 51±14%, PSMA-ureido = 17±6%, PSMA617 = 39±11%), implying a high rate
of dissociation (off-rate) of the complexes. Surprisingly, even in the absence of La3+, both labeled
octapa-PSMA conjugates still decomplexed over time in the NH4OAc buffer, albeit much slower
(intact % on day 1 and 7: PSMA-ureido = 80% and 75%, PSMA617 = 88% and 57%, respectively)
(Figure 2C). As for [177Lu][Lu(octapa)]-, the dissociation was completely stimulated by the La3+
ions. In contrast, the 9-coordinated 177Lu-pypa-complexes were highly inert not only in the buffer
solution, but also under the La3+-competition (Figure 2D). The disturbances from the PSMA-
targeting vectors on the coordination spheres, albeit present, was much less severe compared to
the H4octapa-counterparts (intact % on day 7: PSMA-ureido = 92±2%, PSMA617 = 86±3%)
(Figure 2D). One explanation for these observations, particularly when comparing [177Lu][Lu(L)]-
(L = octapa or pypa), could be that a nine- rather than eight-coordinating environment might be
more favorable for the Lu3+ ion. The rigid pyridyl backbone in H4pypa might also contribute to a
more inert Lu-pypa complex compared to the ethylenediamine backbone in H4octapa, and
therefore, the metal-ion-exchange process could be much slower in the case of H4pypa. Another
possible reason is the destabilization resulting from the conjugation through the arm, which can
also explain the discrepancy in the kinetic inertness between the 177Lu-labeled octapa-PSMA
conjugates reported here and [177Lu][Lu(octapa-benzyl-Trastuzumab)] reported previously.32 In
fact, DFT calculations also predicted different complex conformations were adopted by the
ethylenediamine-functionalized and acetate-functionalized octapa ligands (Figures 1 and S25-S26).
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To validate the results from the competition and in vitro experiments, in vivo studies must be
conducted.
SPECT/CT Imaging and Ex Vivo Biodistribution Studies
Both 177Lu-labeled H4octapa-alkyl-PSMA-ureido and H4octapa-alkyl-PSMA617 were prepared at
an apparent molar activity of ~124 GBq/mol and then injected into the LNCaP-xenograft mice
(n = 5) at a dose of approximately 1.5-2 MBq per mouse (0.0121-0.0161 nmol, 12.0-16.0 ng for
PSMA-ureido, 16.1-21.4 ng for PSMA617, per mouse), except for the imaging mice (~48 MBq,
0.387 nmol, 514 ng, per mouse). Due to the fast clearance of [177Lu][Lu(octapa-alkyl-PSMA-
ureido)], the mice were monitored only up to 24 h post-injection (p.i.), but 72 h for those injected
with the PSMA617 analog which has a longer biological half-life. SPECT/CT MIP (maximum
intensity projection) images of [177Lu][Lu(octapa-alkyl-PSMA617)] are shown in Figure 3. Ex vivo
biodistribution studies (Figure 4 and Tables S9-S12) showed that both PSMA-targeting
radiotracers were cleared renally and collected in the bladder. In addition, they shared similar
biodistribution patterns where the major accumulations of the radioactivity were observed in the
PSMA-expressing organs, including spleen, adrenal glands, kidney, tumor and salivary glands.50,51
At 1 h p.i., for the PSMA-ureido conjugate, the uptakes of those organs were 9.60±5.15, 5.83±2.60,
75.0±9.3, 4.92±0.88 and 6.68±1.13% injected-dose-per-gram (% ID/g), respectively, while
25.0±13.0, 8.03±3.38, 143±17, 13.5±1.1 and 8.90±2.90 % ID/g, respectively, for the PSMA617
analog. The accumulations, except for the tumor and the kidney, were eliminated quickly over
time (≤1.53% ID/g at the last time point). Incorporated with the additional hydrophobic linker,
[177Lu][Lu(octapa-alkyl-PSMA617)] yielded significantly higher tumor uptake than
[177Lu][Lu(octapa-alkyl-PSMA-ureido)], peaked at 4 h p.i. (17.0±2.0 vs. 3.45±0.95% ID/g), and
retained at 12.3±4.6% ID/g on day 3, resulting in considerably higher tumor-to-muscle and tumor-
to-blood ratios (Tables S10 and S12). The day-3 tumor uptake was comparable to [177Lu][Lu(pypa-
C7-PSMA617)] (12.7±4.2% ID/g), although the uptake at 4 h p.i. was higher for the pypa-analog
(24.0±7.6% ID/g).20 Although the kidney uptake for [177Lu][Lu(octapa-alkyl-PSMA617)]
increased to 183±52% ID/g at 4 h p.i., while only 59.5±14.8% ID/g for [177Lu][Lu(octapa-alkyl-
PSMA-ureido)], the kidney clearance was much faster for [177Lu][Lu(octapa-alkyl-PSMA617)]
than [177Lu][Lu(octapa-alkyl-PSMA-ureido)], from 143±17% ID/g at 1 h p.i. to 26.0±10.9% ID/g
at 24 h p.i., and 7.22±3.43% ID/g at 72 h p.i., leading to a sharp increase in tumor-to-kidney ratio
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by 4.6-fold from 1 h to 24 h p.i. and 19-fold over the course of study, in contrast to only ~25%
drop in the kidney radioactivity over 24 h for the PSMA-ureido counterpart (49.5±4.4% ID/g, 24
h p.i.). Unfortunately, although the uptake of both octapa radiotracers were mostly in the PSMA-
expressing organs and the tumor accumulation for [177Lu][Lu(octapa-alkyl-PSMA617)] was
higher than the DOTA-based [177Lu][Lu(PSMA-617)] (12.3±4.6% vs. 7.80±3.69% ID/g on day
3),52 the bone radioactivity of the labeled octapa-alkyl-PSMA617 rose over time (Figure 5), from
0.643±0.285% ID/g (1 h p.i.) to 3.62±2.06% ID/g (24 h p.i.)), resulting in a dramatic drop in the
tumor-to-bone ratio (~21 to ~3). Of course, the tumor clearance also contributed to the reduced
contrast ratio. On the other hand, the growth in the bone uptake was significantly less appreciable
with the more hydrophilic [177Lu][Lu(octapa-alkyl-PSMA-ureido)], perhaps due to the rapid
excretion of the tracer. These in vivo biodistribution were due to the metal ion leaching out of the
chelator which was consistent with the in vitro serum instability, implying that the
bifunctionalization on the acetate arm in H4octapa was suboptimal for 177Lu-chelation – this is
significant for future chelator design. The lanthanum challenge experiment also predicted the
lability of the 177Lu-octapa PSMA-targeting conjugates seen in the biodistribution. As for the
177Lu-pypa-C7-PSMA analogs, the high inertness in the presence of excess La3+ ions was
consistent with the in vivo stability (<1% ID/g bone uptake on day 3 for [177Lu][Lu(pypa-C7-
PSMA617)]).20 Nonetheless, considering the observed differences in in vivo stability between
[177Lu][Lu(octapa-alkyl-PSMA-ureido)] and [177Lu][Lu(octapa-alkyl-PSMA617)], it should be
noted that the biological instability of the complex might not be revealed if the circulation half-
life of the radiotracer is very short, and therefore, a reasonably long-lived targeting vector should
be adopted for proof-of-principle biodistribution studies of a new chelator – another significant
fact for future chelator design.
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% ID/g
50
4 h
24 h
72 h
0
Figure 3. SPECT/CT MIP Images of [177Lu][Lu(octapa-alkyl-PSMA-617)] at different post-injection time points.
(LNCaP-xenograft male mice with tumor on the left shoulder)
90
80
70
60
50
240
A
B
1 h
4 h
24 h
72 h
1 h
4 h
24 h
230
180
170
160
150
140
40
30
20
10
0
10
0
Figure 4. Ex vivo biodistribution data of (A) [177Lu][Lu(octapa-alkyl-PSMA-ureido)] (B) [177Lu][Lu(octapa-alkyl-
PSMA-617)] at different post-injection time points. (LNCaP-xenograft male mice with tumor on the left shoulder)
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6
5
4
3
2
1
0
[
[
177Lu][Lu(octapa-alkyl-PSMA617)]
177Lu][Lu(octapa-alkyl-PSMA-ureido)]
0
20
40
60
Time (h)
Figure 5. Bone uptake (% ID/g) comparison between the 177Lu-labeled octapa-alkyl-PSMA-ureido and octapa-
alkyl-PSMA617 over time.
Conclusion
H4octapa has shown tremendous versatility in chelating a range of radiometal ions with high
t
stability,30,32,53 including lutetium-177. Herein, a new bifunctional analogue, Bu4octapa-alkyl-
NHS, was synthesized and completely characterized. The carboxylic acid moieties in the chelator
were protected as tert-butyl esters, facilitating the peptide conjugation via solid-phase peptide
t
synthesis, and therefore potentially valuable in metallo-radiopeptide preparation. In Bu4octapa-
alkyl-NHS, the linker is covalently attached to the methylene carbon on one of the acetate arms to
maintain the coordination integrity. This functionalization generates a chiral carbon center, which,
based on the DFT calculations, did not cause significant changes to the complex geometry.
Because the two acetate arms became inequivalent, depending on their relative position upon
complexation, there would be four isomers (i.e. S-/R-DBTA and S-/R-DSA). For proof-of-
principle biological evaluations of the new bifunctional 177Lu-octapa chelate, two easily accessible
and well-studied Glu-urea-Lys-based PSMA targeting motifs (i.e. PSMA-ureido and PSMA617)
t
were conjugated to Bu4octapa-alkyl-NHS. Both ligands coordinate lutetium-177 efficiently at
room temperature in 15 min at micromolar chelator concentration, despite the decomplexation in
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the presence of serum proteins and excess La3+ ions. To confirm the in vitro results, both
radiotracers were injected into a cohort of five LNCaP-xenografted mice for biodistribution studies.
The pharmacokinetics patterns of both tracers resembled with expected uptakes in the PSMA-
expressing organs. Similar to [177Lu][Lu(PSMA-617)],52 higher tumor uptake and faster kidney
clearance were seen with [177Lu][Lu(octapa-alkyl-PSMA617)], while the increasing bone
radioactivity concentration was also exclusively observed with the PSMA617-conjugate, perhaps
due to the enhanced blood circulation. Since the in vivo decomplexation was not seen in the
ethylenediamine-functionalized [177Lu][Lu(octapa-benzyl-Trastuzumab)],32 it is concluded that
the alkyl linker on the acetate arm perturbed the 177Lu-octapa coordination sphere. Considering
these results, it is important to understand the potential alterations exerted by the linker on the
metal coordination which is often overlooked; while the results reported in this work are not
conclusive to the analogous complexes with other radionuclides that H4octapa has demonstrated
high affinity for, such as 111In and 86/90Y, and therefore, future investigations on this new
bifunctional octapa with the corresponding radionuclides should be carried out to evaluate the
complex properties and stability.30,32,53
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. Deionized
water was filtered through the PURELAB Ultra Mk2 system. NMR spectra were recorded at
ambient temperature on Bruker AV400 instruments, as specified; NMR spectra are expressed on
the δ scale and referenced to residual solvent peaks. Low-resolution (LR) mass spectrometry was
performed using a Waters ZG spectrometer 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. The HPLC system used for 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)
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was used for purification of H4octapa-alkyl-PSMA-ureido. Phenomenex Luna 5 m C18 100 Å
LC column (250 mm × 10 mm) was used for purification of H4octapa-alkyl-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/neutral alumina columns (Teledyne Isco, Lincoln, NE). Analyses of
radiolabeled compounds were performed with thin layer chromatography (TLC) (i.e. iTLC-
impregnated with silicic acid (iTLC-SA) strips) purchased from Agilent Technologies). The TLC
scanner model was BIOSCAN (system 200 imaging scanner). 177LuCl3 was purchased from
Isotope Technologies Garching (ITG) as no-carrier added (n.c.a.).
N-(2-Aminoethyl)-2-nitrobenzenesulfonamide (1). A solution of 2-nitro-phenylsulfonyl
chloride (5.00 g, 22.6 mmol, 0.3 equiv) in tetrahydrofuran (THF) (25 mL) was added dropwise via
a dropping funnel over 2 h to a solution of ethylenediamine (5.00 mL, 67.9 mmol, 1 equiv) in THF
(12.5 mL) in a round-bottom flask at 0°C. At the end of the addition, the resulting mixture was
warmed up to room temperature and stirred for an additional 30 min. The crude mixture was then
concentrated, and the residue obtained was taken up in dichloromethane (DCM) (25 mL) and
washed with H2O (25 mL). The aqueous layer was further extracted with DCM (25 mL × 3). The
combined organic layers were dried over anhydrous sodium sulfate (Na2SO4), and then clarified
by filtration. The filtrate was concentrated in vacuo. The oily residue obtained was then treated
with concentrated hydrochloric acid (7.50 mL). The precipitate instantaneously formed was
separated by filtration to yield the by-product (N-[2-(2-nitrophenylsulfonyl)amino-ethyl]-2-nitro-
phenyl-sulfonamide) as a whitish solid. The resulting orange filtrate was rotary-evaporated to give
a bright yellow solid as the product (5.69 g, 89%). 1H NMR (400 MHz, 298 K, D2O): δ 8.10-8.05
(m, 1H), 8.01-7.96 (m, 1H), 7.94 – 7.81 (m, 2H), 3.39 – 3.31 (m, 2H), 3.18-3.14 (m, 2H). 13C NMR
(100 MHz, 298 K, D2O): δ 135.1, 133.8, 131.3, 130.7, 125.9, 40.4, 39.5. LR-ESI-MS: calcd for
[C8H11N3O4S+H]+ 246.0; found 246.2.
Tert-butyl-(2-((2-nitrophenyl)sulfonamido)ethyl)glycinate (2). A solution of tert-butyl
bromoacetate (2.98 mL, 20.2 mmol, 1 equiv) in THF (85 mL) was added dropwise via a dropping
funnel to a suspension of compound 1 (5.00 g, 17.7 mmol, 0.9 equiv) and triethylamine (TEA)
(8.45 mL, 60.6 mmol, 3 equiv) in THF (85 mL) in a round-bottom flask over 1 h at room
temperature. After that, another portion of TEA (1.41 mL, 10.1 mmol, 0.5 equiv) was added,
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followed by dropwise addition of tert-butyl bromoacetate (1.49 mL, 10.1 mmol, 0.5 equiv) in THF
(35 mL) over 30 min. The additional portions were necessary to achieve the complete conversion
of compound 1. Stirred overnight at room temperature, the crude mixture was then concentrated
in vacuo and the residue was taken up in DCM (100 mL) and washed with water (50 mL). The
aqueous layer was further extracted with DCM (100 × 2 mL). The combined organic layers were
dried over anhydrous Na2SO4, and then clarified by filtration. The filtrate was concentrated in
vacuo. The oily yellow residue obtained was purified with a silica column (A: hexanes B: ethyl
acetate, 80-100% B). The product fractions were rotary-evaporated to give a yellow oil (4.46 g,
63%). 1H NMR (400 MHz, 298 K, CDCl3): δ 8.18 – 8.11 (m, 1H), 7.89 – 7.82 (m, 1H), 7.75-7.71
(m, 2H), 3.19 (s, 2H), 3.17 – 3.10 (m, 2H), 2.80 – 2.73 (m, 2H), 1.44 (s, 9H). 13C NMR (100 MHz,
298 K, D2O): δ 171.5, 148.2, 133.7, 133.6, 132.8, 131.2, 125.4, 81.8, 51.0, 48.0, 43.2, 28.2. LR-
ESI-MS: calcd for [C14H21N3O6S+H]+ 360.1; found [M+H]+ 360.1.
Di-tert-butyl pyridine-2,6-dicarboxylate (3). To a stirred suspension of 2,6-pyridinedicarboxylic
acid (10.0 g, 59.8 mmol, 1 equiv) in 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 h. 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, A: DCM B: MeOH, 0-5% B). The product fractions were
1
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 (100 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.
Tert-butyl 6-(hydroxymethyl)picolinate (4). Compound 3 (1.40 g, 5.00 mmol, 1 equiv) was
dissolved in dry MeOH (150 mL) in a round-bottom flask. Sodium borohydride (NaBH4) (0.189
g, 5.00 mmol, 1 equiv) was added at 0˚C. The mixture was stirred at room temperature for 1 h and
then another equivalent 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 h. After that, the reaction mixture was diluted with DCM (100 mL) and then quenched
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with saturated sodium bicarbonate (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, A:
DCM B: MeOH, 0-5% B). 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 =
13
7.7 Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 4.82 (s, 2H), 1.59 (s, 9H). C NMR (100 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.
Tert-butyl 6-(bromomethyl)picolinate (5). Compound 4 (1.83 g, 8.73 mmol, 1 equiv) with a stir
bar in a three-neck round-bottom flask was purged with N2(g). Dry acetonitrile (ACN) (35 mL)
was added via a syringe to dissolve the starting material. Phosphorus tribromide (PBr3) (1.24 mL,
13.1 mmol, 1.5 equiv) was added in three portions via a syringe over 15 min to the stirred solution
of compound 5 at 0˚C. The mixture then continued to stir at room temperature for 4-5 h, followed
by dilution with DCM (50 mL) and saturated sodium carbonate (Na2CO3) in water (70 mL). The
organic layer was separated while the ACN in the aqueous layer was removed in vacuo. The
residual aqueous layer was further extracted with DCM (50 mL × 3). The combined organic layers
were dried over anhydrous magnesium sulfate (MgSO4), filtered and then concentrated in vacuo.
The crude product was further purified by a silica column (CombiFlash Rf automated column
system; 40 g HP silica; A: hexanes, B: ethyl acetate, 30-50% B). The product fractions were rotary-
evaporated to yield a waxy faint-yellow solid (1.36 g, 54%). 1H NMR (400 MHz, 298 K, CDCl3):
δ 7.93 (d, J = 8.4 Hz, 1H), 7.82 (t, J = 7.7 Hz, 1H), 7.65 (d, J = 7.7 Hz, 1H), 4.64 (s, 2H), 1.63 (s,
9H). 13C NMR (100 MHz, 298 K, CDCl3): δ 163.5, 157.4, 148.9, 138.0, 126.6, 123.9, 82.5, 33.3,
28.1. LR-ESI-MS: calcd for [C11H1481BrNO2+Na]+ 296.0; found [M(81Br)+Na]+ 296.1.
Tert-butyl-6-(((N-(2-((2-(tert-butoxy)-2-oxoethyl)((6-(tertbutoxycarbonyl)-pyridin-2-yl)-
methyl)amino)ethyl)-2- nitrophenyl)sulfonamido)methyl)picolinate (6). Compound 2 (1.08 g,
3.01 mmol, 1 equiv) was dissolved in anhydrous ACN in a round-bottom flask, followed by
addition of compound 5 (1.80 g, 6.63 mmol, 2.2 equiv) and potassium carbonate (K2CO3) (1.25 g,
9.04 mmol, 3 equiv). The reaction mixture was stirred at 60˚C for 48 h. Then, K2CO3 was separated
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by centrifugation and washed with DCM (5 mL). The combined supernatants were concentrated
in vacuo and purified by a silica column (CombiFlash Rf automated column system; 24 g HP silica;
A: DCM B: MeOH, 0-5% B). The product fractions were rotary-evaporated to yield a yellow oil
(1.56 g, 70%). 1H NMR (400 MHz, 298 K, CDCl3): δ 8.11-8.08 (m, 1H), 7.85 (d, J = 7.5 Hz, 2H),
7.76-7.70 (m, 2H), 7.67-7.66 (m, 1H), 7.64 – 7.59 (m, 3H), 7.53 (d, J = 7.7 Hz, 1H), 4.77 (s, 2H),
3.91 (s, 2H), 3.56-3.51 (m, 2H), 3.24 (s, 2H), 2.83-2.79 (m, 2H), 1.60-1.59 (m, 18H), 1.39 (s, 9H).
13C NMR (100 MHz, 298 K, CDCl3): δ 170.3, 164.1, 163.6, 160.0, 156.9, 148.9, 148.6, 148.1,
137.7, 137.3, 133.5, 133.3, 131.9, 130.9, 125.7, 125.1, 124.1, 123.6, 123.2, 82.1, 81.9, 81.1, 60.1,
55.8, 53.2, 52.6, 46.4, 28.1, 21.0, 14.2. LR-ESI-MS: calcd for [C36H47N5O10S+H]+ 741.3; found
[M+H]+ 742.3.
Tert-butyl-6-(((2-((2-(tert-butoxy)-2-oxoethyl)((6-(tert-butoxycarbonyl)-pyridin-2-yl)-
methyl)amino)ethyl)amino)methyl)picolinate (7). To a stirred solution of compound 6 (233 mg,
0.313 mmol, 1 equiv) in dry THF in a round-bottom flask was added thiophenol (80 L, 0.780
mmol, 2.5 equiv) and K2CO3 (0.130 g, 0.942 mmol, 3 equiv). The mixture was stirred at room
temperature overnight, followed by separation of K2CO3 through centrifugation. The K2CO3 salt
was washed with DCM (5 mL) and the combined supernatants were concentrated in vacuo. The
residue was purified through a neutral alumina column. (CombiFlash Rf automated column system;
12 g neutral alumina; A: DCM B: MeOH, 0-5% B). The product fractions were rotary-evaporated
1
to yield a yellow oil (0.11 g, 63%). H NMR (400 MHz, 298 K, CDCl3): δ 7.88 (d, J = 7.8 Hz,
2H), 7.85 – 7.71 (m, 3H), 7.54 (d, J = 7.6 Hz, 1H), 4.05 (s, 2H), 3.96 (s, 2H), 3.35 (s, 2H), 2.90 (t,
J = 5.7 Hz, 2H), 2.72 (t, J = 5.8 Hz, 2H), 1.63 (s, 18H), 1.46 (s, 9H). 13C NMR (100 MHz, 298 K,
CDCl3): δ 170.8, 164.0, 160.6, 148.8, 148.5, 137.4, 137.1, 129.8, 128.5, 125.6, 125.0, 123.1, 123.0,
81.9, 81.0, 60.6, 56.5, 54.9, 54.3, 53.5, 47.1, 28.2, 28.1. LR-ESI-MS: calcd for [C30H44N4O6 +H]+
557.3; found [M+H]+ 557.2.
5-Benzyl 1-(tert-butyl) glutamate (8). L-Glutamic acid γ-benzyl ester (4.66 g, 19.7 mmol, 1
equiv) was mixed with sodium bromide (6.01 g, 58.9 mmol, 3 equiv) in 1 N aqueous hydrobromic
acid (59 mL, 60 mmol, 3 equiv) at 0˚C in a round-bottom flask. Sodium nitrite (2.71 g, 39.3 mmol,
2 equiv) was slowly added over 30 min in three portions and the reaction was continuously stirred
for 10 h at ambient temperature. The reaction mixture was treated with concentrated sulfuric acid
(2.5 mL) and the acidified aqueous solution was extracted with diethyl ether (60 mL × 3). The
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combined organic phases were reduced in volume in vacuo before a wash with brine (20 mL × 2).
The ether layer was dried over anhydrous Na2SO4, and then clarified by filtration. The filtrate was
concentrated in vacuo and then purified by a silica column (CombiFlash Rf automated column
system; 40 g HP silica, A: hexanes B: ethyl acetate, 0-35% B). The product fractions were
combined, and rotary evaporated to yield a white solid (2.64 g, 45%). 1H NMR (400 MHz, 298 K,
CDCl3): δ 7.39-7.33 (m, 5H), 5.14 (s, 2H), 4.43-4.39 (m, 1H), 2.64-2.59 (m, 2H), 2.48-2.39 (m,
1H), 2.36-2.37 (m, 1H). LR-ESI-MS: calcd for [C12H13Br81O4+Na]+ 325.0; found [M(81Br)+Na]+
324.9.
5-Benzyl 1-(tert-butyl) 2-bromopentanedioate (9). Compound 8 (2.64 g, 8.76 mmol, 1 equiv) in
chloroform (CHCl3) (11 mL) was added dropwise over 20 min to tert-butyl-2,2,2-
trichloroacetimidate (TBTA) (4.79 g, 21.9 mmol, 2.5 equiv) in cyclohexane (13 mL) in a round-
bottom flask using a dropping funnel. Dimethylacetamide (2 mL) and boron trifluoride diethyl
etherate (175.2 µL) were then added to the reaction mixture sequentially. The mixture was stirred
for 3 d at room temperature, and then clarified by filtration. The filtrate was concentrated in vacuo
and then re-dissolved in hexanes (15 mL), followed by washing with water (15 mL × 3) in a
separating funnel. Following the evaporation of hexanes in vacuo, purification was performed with
a silica column (CombiFlash Rf automated column system; 40 g HP silica, A: hexanes B: ethyl
acetate, 0-30% B). The product fractions were rotary-evaporated to yield a colorless liquid (1.60
g, 51%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.36 (s, 5H), 5.13 (s, 2H), 4.25 (m, 1H), 2.59 –
2.52 (m, 2H), 2.43 – 2.18 (m, 2H), 1.47 (s, 9H). 13C NMR (100 MHz, 298 K, CDCl3) : δ 172.1,
168.4, 135.8, 128.8, 128.5, 128.4, 82.8, 66.7, 46.8, 31.8, 29.9, 27.9. LR-ESI-MS: calcd for
[C16H21Br81O4+Na]+ 381.1; found [M(81Br)+Na]+ 381.0.
5-Benzyl 1-(tert-butyl) N-(2-((2-(tert-butoxy)-2-oxoethyl)((6-(tert-butoxy-carbonyl)-pyridin-
2-yl)methyl)amino)ethyl)-N-((6-(tert-butoxycarbonyl)pyridin-2-yl)methyl)glutamate (10).
To a stirred solution of compound 7 (126 mg, 0.230 mmol, 1 equiv) in dry ACN(5 mL) in a round-
bottom flask was added compound 9 (161 mg, 0.423 mmol, 1.8 equiv) and K2CO3 (95 mg, 0.690
mmol, 3 equiv). The reaction mixture was stirred at 60˚C for 48 h. K2CO3 was separated through
centrifugation and then washed with DCM (10 mL). The combined supernatants were concentrated
in vacuo and then purified through a silica column. (CombiFlash Rf automated column system;
neutral alumina, A: diethyl ether B: ethyl acetate, 0-50% B). The product fractions were rotary-
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evaporated to a yellow oil (75.8 mg, 30%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.75 (d, J = 5.5
Hz, 2H), 7.61 (d, J = 4.4 Hz, 2H), 7.56-7.50 (m, 2H), 7.26 (s, 5H), 5.00 (s, 2H), 3.97 – 3.84 (m,
4H), 3.29 – 3.22 (m, 1H), 3.16 (s, 2H), 2.76-2.64 (m, 4H), 2.41-2.35 (m, 2H), 2.03-1.83 (m, 2H),
13
1.54 (s, 18H), 1.39-1.34 (2s, 18H). C NMR (100 MHz, 298 K, CDCl3): δ 173.0, 171.7, 170.5,
164.2, 161.4, 160.8, 148.6, 137.2, 136.0, 128.6, 128.3, 125.5, 125.1, 123.1, 123.0, 82.0, 81.4, 81.0,
66.3, 63.5, 60.6, 57.8, 56.3, 53.5, 50.2, 31.0, 28.4, 28.2, 28.2, 25.0. LR-ESI-MS: calcd for
[C46H64N4O10+H]+ 833.5 found [M+H]+ 833.6.
5-(Tert-butoxy)-4-((2-((2-(tert-butoxy)-2oxoethyl)((6(tertbutoxycarbonyl)-pyridin-2-yl)-
methyl)amino)ethyl)((6-(tertbutoxycarbonyl)pyridin-2-yl)-methyl)amino)-5-oxopentanoic
acid (11). Compound 10 (75.8 mg, 0.911 mol, 1 equiv) was dissolved in dry MeOH (7 mL) in a
three-neck round-bottom flask, saturated with N2(g). Pd/C (10 % w/w, 10.9 mg, 0.1 equiv) was
added under a stream of N2(g), and then the flask was saturated with H2(g) with a balloon. The
mixture was stirred vigorously for 6 h under H2(g) atmosphere, and then Pd/C was filtered off
through a pre-wet (MeOH) celite bed, washed with MeOH (10 mL). The filtrate was concentrated
to a yellow oil (58.0 mg, 87%) and used without purification. LR-ESI-MS: calcd for
[C39H58N4O10+H]+ 743.4; found [M+H]+ 743.5.
tBu4octapa-alkyl-NHS (12). To a two-neck round-bottom flask with a stirred solution of
compound 11 (58.0 mg, 78.0 mol) in dry ACN (2 mL) was added NHS (13.5 mg, 0.117 mmol,
1.5 equiv) and EDC·HCl (22.5 mg, 0.118 mmol, 1.5 equiv). The mixture was stirred at room
temperature under N2(g) overnight. After the reaction completed, the solvent was removed in
vacuo and the residue was redissolved in DCM (20 mL), and then washed with water (10 mL × 3)
and brine (10 mL × 2) using a separating funnel. The combined organic phases were dried over
anhydrous Na2SO4, and then clarified by filtration. The filtrate was rotary-evaporated to yield a
yellow oil (45.3 mg, 69%) and used directly without further purification. 1H NMR (400 MHz, 298
K, CDCl3): δ 7.70 (m, 6H), 4.03 – 3.90 (m, 4H), 3.44 – 3.40 (m, 1H), 3.20 (s, 2H), 2.80-2.69 (m,
10H), 2.15 – 1.96 (m, 2H), 1.58 (s, 18H), 1.44-1.38 (2s, 18H). 13C NMR (100 MHz, 298 K, CDCl3):
δ 171.3, 170.6, 169.1, 168.6, 164.2, 161.1, 160.8, 148.7, 148.6, 137.4, 137.3, 125.6, 125.2, 123.1,
82.0, 82.0, 81.7, 81.0, 62.9, 60.6, 57.7, 56.4, 53.5, 50.3, 28.4, 28.2, 28.2, 27.8, 25.7, 24.7. HR-ESI-
MS: calcd for [C43H61N5O12+H]+ 840.4395; found [M+H]+ 840.4398.
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DFT Calculations. All DFT simulations were performed as implemented in the Gaussian 09
revision D.01 suite of ab initio quantum chemistry programs (Gaussian Inc., Wallingford, CT) at
B3LYP/6-31G*41,42,43,44 level for first- and second-row elements, and the Stuttgart/Dresden and
associated effective core potentials’ basis set was applied for Lu.45,46 The structural geometry was
optimized in the presence of water solvent (IEF PCM as implemented in G09) without the use of
symmetry constraints. Normal self-consistent field (SCF) and geometry convergence criteria were
conducted for all the calculations. The calculated structures were visualized using Mercury 4.1.
Solid-Phase Peptide Coupling. Solid-phase synthetic procedures for H4octapa-alkyl-PSMA-
ureido and H4octapa-alkyl-PSMA617 were adapted from the literature.22 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 min).
The isocyanate derivative of di-tert-butyl ester of glutamate (0.138 mmol, 3 equiv) was prepared
according to literature procedures22 and added to the lysine-immobilized resin to react for 16 h.
After washing the resin with DMF, the ivDde-protecting group was removed with 2% hydrazine
in DMF (5 × 5 min). To synthesize the H4octapa-alkyl-PSMA617, that step was 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 tBu4octapa-alkyl-NHS (0.140 g, 0.167 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 h at room temperature. After filtration, the peptide was
precipitated by adding cold diethyl ether to the TFA solution. The crude conjugate was purified by
semi-preparative HPLC (H4octapa-alkyl-PSMA-ureido: 5-95% ACN in water containing 0.1%
TFA over 40 min at a flow rate of 10 mL/min, tR = 14.3 min; H4octapa-alkyl-PSMA617: 29%
ACN in water containing 0.1% TFA at a flow rate of 4.5 mL/min, tR = 10.5 min). The eluates
containing the desired peptide were collected, pooled, and lyophilized (H4octapa-alkyl-PSMA-
ureido: 12.0 mg, 14.6 mol, H4octapa-alkyl-PSMA617: 10.1 mg, 8.73 mol) HR-ESI-MS: calcd
for H4octapa-alkyl-PSMA-ureido [C35H45N7O16 + H+]+ 820.3001; found [M+H+]+ 820.2994; calcd
for H4octapa-alkyl-PSMA617 [C56H69N9O18 + H+]+ 1156.4839; found [M+H+]+ 1156.4800
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Radiolabeling Studies. An aliquot of ligand solution (H4octapa-alkyl-PSMA-ureido in MQ water
or H4octapa-alkyl-PSMA617 in MQ water with <5% DMSO, 10-3-10-6 M) (25 L) was mixed with
ammonium acetate (NH4OAc) solution (0.2 M, pH = 7, 223 L), followed by 177LuCl3 in HCl (aq,
0.04 M) (2 MBq, 2 L). The reaction was incubated at ambient temperature over 15 min. The
mixture (3 L) was spotted on an iTLC-SA plate and then developed in EDTA buffer (50 mM, pH
= 5.2). The TLC plate was read by a TLC reader, showing the free metal ion migrated to the solvent
front while the complex stayed at the baseline. The areas of both peaks were used to calculate
RCY%.
In vitro Human Serum Challenge. To the radiolabeled sample of H4octapa-alkyl-PSMA-ureido
or H4octapa-alkyl-PSMA617 solution (100 L) was added an equal volume (100 L) of human
serum. The mixture was incubated at 37˚C, and then an aliquot (5 L) was collected at desired
timepoints. The aliquot was spotted onto an iTLC-SA plate next to the control (lutetium-177 in
buffer with serum) and then developed in an EDTA solution (50 mM, pH = 5.2). The TLC plate
was read by a TLC reader. The free metal ion migrated to the solvent front while the complex
stayed at the baseline. The areas of both peaks were used to calculate % intact.
In vitro Lanthanum(III) Challenge. To the radiolabeled sample of H4octapa-alkyl-PSMA-ureido
or H4octapa-alkyl-PSMA617 solution (100 L, 0.25 nmol of conjugate) was added La3+ ions
(standard AAS solution) in 0.04 M HCl (aq) (7.5 L of 10-4 M, 0.75 nmol of La3+). The mixture
was incubated at 37˚C, pH = 7, and then 5 L aliquot was collected at desired timepoints. The
aliquot was spotted onto an iTLC-SA plate next to the control (without La3+ ions), and then
developed in an EDTA solution (50 mM, pH = 5.2). The TLC plate was read by a TLC reader.
The free metal migrated to the solvent front while the complex stayed at the baseline. The areas of
both peaks were used to calculate % intact.
Radiolabeling of Conjugates for in vivo Study. An aliquot of H4octapa-alkyl-PSMA-ureido in
MQ water or H4octapa-alkyl-PSMA617 solution in MQ water (<5% DMSO) (4.83 × 10-5 M, 33.4
L, 1.62 nmol) was added to NH4OAc solution (0.15 M, pH = 7, 170 L), followed by lutetium-
177 solution in ~0.04 M HCl (200 MBq, 10 L). The mixture was incubated at room temperature,
pH = 7 for 10 min before measuring the RCY % (>98%) with iTLC-SA plate as described above.
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SPECT/CT Imaging Studies. 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. Mice were anesthetized by
inhalation with 2% isoflurane in oxygen and implanted subcutaneously with LNCaP cells (1×107)
behind left shoulder. Mice were imaged or used in biodistribution studies when the tumor grew up
to reach 5-8 mm in diameter during 5-6 weeks. SPECT/CT imaging experiments were conducted
using the MILabs (Utrecht, The Netherlands) U-SPECT+/CT scanner. Each tumor-bearing mouse
was injected with 177Lu-labeled H4octapa-alkyl-PSMA617 (~48 MBq) 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 h after injection. At each time point, the mice were sedated
again with 2% isoflurane in oxygen and positioned in the micro-scanner. Body temperature was
maintained via a heating pad and vital signs were monitored throughout the scan. A baseline CT
scan was obtained for localization and attenuation correction with voltage setting at 60 kV and
current at 615 µA followed by a 60 min static emission scan acquired in list mode using an extra
ultra-high sensitivity big mouse (2mm multi-pinhole) collimator. Data was reconstructed using
MILabs reconstruction software centered on the 208 keV (177Lu) photopeaks. Reconstruction
parameters used similarity regulated ordered subset expectation maximization (128 subsets, 3
iterations) and a post-processing filter (Gaussian blurring) of 0.6 mm. Scatter correction was
performed using the automatic triple energy window setting and a calibration factor was applied
generating images in MBq/mL. Images were decay-corrected to injection time and divided by the
injected activity in PMOD (PMOD Technologies, Switzerland) to obtain quantitative images
expressed as the percentage of the injected dose per gram of tissue (% ID/g). Data were then
converted to DICOM for visualization using Inveon Research Workplace (Siemens Medical
Solutions USA, Inc.), a Gaussian filter of 0.4 mm was applied and maximum intensity projection
images were generated.
Biodistribution Studies. The LNCaP tumor-bearing mice were injected with the radiotracer (1.5-2
MBq) through the tail vein under anesthesia (2% isoflurane in oxygen). The mice were allowed to
recover and roam freely in their cages. At predetermined time points (1, 4, 24 and 72 hours p.i.),
the mice were euthanized by CO2 inhalation. Blood was withdrawn immediately from the heart,
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and the organs/tissues of interest were collected. The collected organs/tissues were weighed and
counted using a Perkin Elmer (Waltham, MA) Wizard2 2480 automatic gamma counter.
Acknowledgements
We thank the team of animal technicians and veterinarians for helpful assistance in the BC Cancer.
We gratefully acknowledge funding from NSERC CREATE IsoSiM at TRIUMF for PhD research
stipend (LL), both NSERC and CIHR for financial support via a Collaborative Health Research
Project (CHRP to CO, FB and PS) and NSERC Discovery (CO, PS, VR). TRIUMF receives
federal funding via a contribution agreement with the National Research Council of Canada.
Author Information Notes
*Corresponding Authors : orvig@chem.ubc.ca
ORCID
Lily Li : 0000-0002-8779-8689
Chris Orvig : 0000-0002-2830-5493
Notes: The authors declare no competing financial interest.
Associated Content (21 pages)
Supporting Information
t
NMR spectra of compounds 1-12; high-resolution mass spectra of Bu4octapa-alkyl-NHS,
H4octapa-alkyl-PSMA-ureido and H4octapa-alkyl-PSMA617; DFT-calculated structures of R-
DBTA, R-DSA geometries of [Lu(octapa-alkyl-benzyl-ester)]-, [Lu(S-benzyl-octapa)]- and
[Lu(octapa)]-; radiolabeling yield, % intact upon human serum challenge and lanthanum(III)
challenge experiments with H4octapa, H4octapa-alkyl-PSMA-ureido, H4octapa-alkyl-PSMA617,
H4pypa, H4pypa-C7-PSMA-ureido, H4pypa-C7-PSMA617; biodistribution studies data (% ID/g)
of 177Lu-labeled H4octapa-alkyl-PSMA-ureido and H4octapa-alkyl-PSMA617.
29