Design, synthesis and evaluation of novel bifunctional tetrahydroxamate chelators for PET imaging of89Zr-labeled antibodies

Article abstract of DOI:10.1016/j.bmcl.2017.01.052

Two compact and symmetrical bifunctional tetrahydroxamate chelators, 1 and 2, were synthesized and evaluated for labeling antibodies with⁸⁹Zr for imaging with positron emission tomography. Using 2,2′-iminodiacetamide as the backbone, four hydro

Full text of DOI:10.1016/j.bmcl.2017.01.052

Bioorganic & Medicinal Chemistry Letters 27 (2017) 708–712
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of novel bifunctional tetrahydroxamate
chelators for PET imaging of 89Zr-labeled antibodies
Julie Rousseau a, Zhengxing Zhang a, Gemma M. Dias a, Chengcheng Zhang a, Nadine Colpo a,
François Bénard a,b,c, , Kuo-Shyan Lin a,b,c,
⇑
⇑
a Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC V5Z 1L3, Canada
b Department of Functional Imaging, BC Cancer Agency, Vancouver, BC V5Z 4E6, Canada
c Department of Radiology, University of British Columbia, Vancouver, BC V5Z 4E3, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Two compact and symmetrical bifunctional tetrahydroxamate chelators, 1 and 2, were synthesized and
89
evaluated for labeling antibodies with Zr for imaging with positron emission tomography. Using 2,20-
Received 3 December 2016
Revised 15 January 2017
Accepted 16 January 2017
Available online 18 January 2017
iminodiacetamide as the backbone, four hydroxamate-containing moieties coupled to the diacetamide
nitrogen were used for 89Zr labeling, while a pendant connected to the amino group provided an isoth-
iocyanate group for coupling to the antibody. Both 1- and 2-conjugated Trastuzumab were labeled with
89Zr efficiently (>90% radiolabeling yield), and their 89Zr-labeled products maintained comparable
immunoreactivity to Trastuzumab. Compared to 89Zr-labeled deferoxamine-conjugated Trastuzumab,
89Zr-1- and 89Zr-2-Trastuzumab showed faster demetalation in mouse plasma, and also displayed higher
bone uptake in mice. Despite suboptimal stability of 89Zr complexes of 1 and 2, our design strategy led to
tetrahydroxamate chelators for efficient 89Zr labeling, and could be potentially modified to provide novel
chelators with improved stability.
Keywords:
Zirconium-89
Bifunctional chelators
Antibody
Trastuzumab
Molecular imaging
Positron emission tomography
Ó 2017 Elsevier Ltd. All rights reserved.
Positron Emission Tomography (PET) is a powerful non-invasive
molecular imaging modality with high sensitivity, spatial resolu-
tion and quantification capability, and is routinely used in the
clinic for cancer imaging.1 With high affinity and specificity for
their targets, monoclonal antibodies (mAb) have been successfully
used in the clinic as molecularly-targeted therapeutics.2 The devel-
opment of innovative PET isotopes production and radiolabeling
techniques further stimulates the application of immunoPET
imaging to provide information on target expression and the
pharmacokinetics of mAb.3 Zirconium-89 (89Zr) is an appealing
radionuclide for immunoPET imaging with mAb.4,5 Its half-life
(78.4 h) is well suited to the slow pharmacokinetics of mAb, and
its low positron energy (Emax = 908 keV) results in high spatial-
resolution PET images. Moreover, efficient procedures for radiola-
beling mAb with 89Zr have been established6 and several
89Zr-labeled mAb are currently under investigation in the clinic.7–10
Despite great potential of 89Zr for labeling mAb for PET imaging,
the derivatives (maleimide and isothiocyanate) of the bacterial side-
rophore deferoxamine (DFO) are currently the only commercially
available bifunctional chelators for 89Zr labeling. Significant bone
uptake (up to 15.1%ID/g, 5 days after injection) resulting from
demetalation of 89Zr-labeled DFO-conjugated mAb has been con-
stantly observed in mice in preclinical studies, especially in high
bone remodeling regions.11 This has been suggested due to the fact
that DFO can provide only three hydroxamate groups (hexadentate)
for chelation but octadentate chelators are required to stably chelate
Zr4+ 12,13
The high bone uptake reduces the overall tumor uptake and
.
the ability to detect smaller bone metastatic lesions. Therefore,
development of novel bifunctional chelators that lead to higher
in vivo stability of 89Zr-labeled mAb is needed to facilitate the appli-
cation of 89Zr for immunoPET imaging.
As shown in Fig. 1, in the past 10 years, several bifunctional
chelators have been synthesized and evaluated for labeling
antibodies with 89Zr. Perk et al.14 reported that labeling
MX-DTPA-conjugated Zevalin with 89Zr resulted in <0.1% labeling
yield. Price et al.15 showed that labeling H6phospa-conjugated
Trastuzumab with 89Zr resulted in up to 12% radiochemical yield,
whereas no 89Zr chelation was observed using H4octapa-
conjugated Trastuzumab. The tripodal tris(hydroxypyridinone)
ligand YM-103 was reported by Ma et al.16 and showed >98%
radiochemical yield for labeling Trastuzumab with 89Zr. However,
massive demetalation was observed in mice as uptake in bone
⇑
Corresponding authors at: 675 West 10th Avenue, Rm 14-111, Vancouver, BC
V5Z 1L3, Canada (F. Bénard). 675 West 10th Avenue, Rm 4-123, Vancouver, BC V5Z
1L3, Canada (K.-S. Lin).
E-mail addresses: fbenard@bccrc.ca (F. Bénard), klin@bccrc.ca (K.-S. Lin).
http://dx.doi.org/10.1016/j.bmcl.2017.01.052
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.

J. Rousseau et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 708–712
709
Fig. 1. Reported bifunctional chelators for labeling antibodies with 89Zr.
was 25.9 0.58% ID/g at seven days post-injection. Evaluation
of a 3-hydroxypyridin-2-one based macrocyclic chelator BPDE-
TLysH22-2,3-HOPO for labeling mAb with 89Zr was reported by
Tinianow et al.17 Despite good radiolabeling yield (60 – 69%),
89Zr-labeled BPDETLysH22-2,3-HOPO-conjugated Trastuzumab
showed higher bone uptake compared to that of 89Zr-DFO-
Trastuzumab (15.1 2.7 and 10.6 1.0% ID/g, respectively at Day
6. Deri et al.18 reported the evaluation of p-SCN-Bn-HOPO for
labeling mAb with 89Zr. Compared to 89Zr-DFO-Trastuzumab,
89Zr-HOPO-Trastuzumab showed much less bone uptake in mice
at Day 14 (17.0 4.1 and 2.4 0.3% ID/g, respectively), demonstrat-
ing its superior in vivo stability. A cyclam-based trihydroxamate
chelator L5 was reported by Boros et al.19 L5-conjugated Tras-
tuzumab was radiolabeled with 89Zr in quantitative radiochemical
yield, but 89Zr-L5-Trastuzumab suffered significant in vivo demet-
alation in mice (18.9 1.1% uptake in bone at Day 4). Recently,
Vugts et al.20 reported adding an additional hydroxamate group
to the DFO chelator, so the resulting DFO⁄ chelator could fulfill
the octadentate requirement to stably complex Zr4+. Labeling DFO⁄
with 89Zr was achieved in >95% radiochemical yield. Furthermore,
89Zr-DFO⁄-Trastuzumab was demonstrated to be more stable than
89Zr-DFO-Trastuzumab as their uptake values in knees in mice
were 1.38 0.23 and 8.20 2.94% ID/g, respectively at Day 7.
Despite superior in vivo stability of 89Zr-HOPO and 89Zr-DFO⁄
complexes, the synthesis of these two bifunctional chelators is
not trivial.18,20 The reported synthesis methods involved multiple
steps and HPLC purification, and only a few mg batch production
of both chelators was demonstrated.18,20 To develop novel
bifunctional chelators for labeling mAb with 89Zr, we designed
and synthesized two compact and symmetrical bifunctional
tetrahydroxamate chelators 1 and 2 (Fig. 1). 2,20-Iminodiacetamide
was used as the backbone for both chelators. A pendant arm
stretching out from the central amino group was functionalized
to provide an isothiocyanate group for coupling to antibodies. Four
hydroxamate groups to fulfill the octadentate requirement were
connected to the two amide nitrogen with different length of linker

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J. Rousseau et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 708–712
(Fig. 1, 1: X = CH2; 2: X = CH2CH2). The chelators were designed to
be compact and symmetrical, so they could be facilely synthesized
and characterized.
The synthetic scheme for the preparation of bifunctional
chelators 1 and 2 is depicted in Fig. 2. To construct the hydroxam-
ate-containing moieties, the N-Boc protected 2,20-iminodiacetic
acid 3 was acquired commercially, whereas the N-Boc protected
3,30-iminodipropionic acid 4 was synthesized in 79% yield by
treating 3,30-iminodipropionic acid with di-tert-butyl dicarbonate.
quantitatively. Compound 12 was subsequently converted to the
activated di-(2,3,5,6-tetrafluoro)phenyl diacetate 13 in 84% yield.
Coupling 13 with amines 9 or 10 provided tetrahydroxamate-
conjugated 2,20-iminodiacetamides 14 and 15 in 76 and 58% yields,
respectively. The N-Boc protecting group was removed by treating
14 and 15 with trifluoroacetic acid, and free amines 16 and 17 were
obtained in 99 and 87% yields, respectively. The four O-benzyl pro-
tecting groups of 16 and 17 were removed by catalytic hydrogena-
tion to afford 18 and 19 in 94 and 89% yields, respectively.
Coupling 18 and 19 with 1,4-phenylene diisothiocyanate gave
the desired bifunctional tetrahydroxamate chelators 1 and 2 in
66 and 72% yields, respectively.
Compounds
3
and
4
were converted to their activated
di-(2,3,5,6-tetrafluoro)phenyl diesters 5 and 6 in 57 and 78% yields,
respectively. Di-O-benzyl protected dihydroxamates 7 and 8 were
obtained in 78 and 53% yields, respectively, by coupling 5 and 6
with O-benzyl-N-methylhydroxylamine. The free amines 9 and
10 were prepared in 80 and 98% yields, respectively, after
removing the N-Boc protecting group with trifluoroacetic acid.
To construct the 2,20-iminodiacetamide backbone with a pen-
dant arm for mAb coupling, N-Boc-1,4-butanediamine was coupled
with 2 equivalents of benzyl 2-bromoacetate to form 11 in 96%
yield. The O-benzyl protecting group was removed by Pd-catalyzed
hydrogenation to give N-substituted 2,20-iminodiacetic acid 12
To assess the efficiency of bifunctional chelators 1 and 2 for
labeling mAb with 89Zr, Trastuzumab was selected as the model
mAb as it was used previously by others to test new 89Zr
chelators.15–20 Conjugation of Trastuzumab with 1,
2 and
p-SCN-DFO, and radiolabeling of the resulting conjugates
with 89Zr followed the protocol reported by Vosjan et al.6 On
average, 0.55 0.04, 0.91 0.02 and 0.94 0.04 of DFO, 1 and 2,
respectively were conjugated to Trastuzumab. The radiolabeling
yields of 89Zr-DFO-Trastuzumab, 89Zr-1-Trastuzumab and
Fig. 2. Synthesis of bifunctional tetrahydroxamate chelators 1 and 2 for labeling antibodies with 89Zr.

J. Rousseau et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 708–712
711
89Zr-2-Trastuzumab were very efficient (>90%) as confirmed by
iTLC-SG. After purification by PD-10 column, the radiochemical
purities were >99% for all three radioimmunoconjugates and their
specific activities were in the range of 0.3 – 0.6 MBq/lg. Compared
to unmodified Trastuzumab, no significant change in immunoreac-
tivity of all three radioimmunoconjugates was observed
(96, 104 and 101% immunoreactivity for 89Zr-DFO-Trastuzumab,
89Zr-1-Trastuzumab, and 89Zr-2-Trastuzumab, respectively). This
indicates that chelator conjugation and 89Zr labeling did not reduce
the binding capability of Trastuzumab to the HER2 antigen.
To assess the stability of 89Zr complexes of 1 and 2, in vitro
stability assays were conducted for 89Zr-1-Trastuzumab,
89Zr-2-Trastuzumab and 89Zr-DFO-Trastuzumab. These three
radioimmunoconjugates were incubated in mouse plasma at
37 °C, and the extent of demetalation was monitored over time
and quantified by iTLC. As shown in Table 1, progressive demet-
alation was observed for all three radioimmunoconjugates in
mouse plasma. 89Zr-DFO-Trastuzumab displayed the best plasma
stability. While 77% of 89Zr-DFO-Trastuzumab remained intact
after one day, only 25% of 89Zr-1-Trastuzumab and 46%
89Zr-2-Trastuzumab were still intact. On Day 3, there were 3,
16 and 55% of 89Zr-1-Trastuzumab, 89Zr-2-Trastuzumab and
89Zr-DFO-Trastuzumab, respectively, remained intact.
In addition to in vitro stability assays, PET imaging was also
conducted to assess the in vivo behaviour of these three radioim-
munoconjugates. Mice were injected with 8.4 2.0 lg of radioim-
Fig. 3. Representative maximum intensity projection PET images of 89Zr-labeled
Trastuzumab conjugates obtained at 1 and 3 days post-injection. Spleen is indicated
by arrows.
munoconjugates and PET/CT images were acquired at one and
three days post-injection. As shown in Fig. 3, spleen was clearly
visualized for all three radioimmunoconjugates on both images
acquired on Day 1 and 3. Spleen is the organ that is typically
observed in antibody imaging, and was used here as a surrogate
target. Clear spleen visualization indicated that significant por-
tions of radioimmunoconjugates remained intact and reached this
target organ. Bone uptake (especially in the knees) was also
observed and increased over time, suggesting there was progres-
sive demetalation for all three radioimmunoconjugates. The bone
uptake of 89Zr-1-Trastuzumab and 89Zr-2-Trastuzumab was higher
than that of 89Zr-DFO-Trastuzumab as observed from both images
taken on Day 1 and 3. The observed inferior in vivo stability of
complexes of 89Zr-1 and 89Zr-2 was consistent with the observa-
tions from the in vitro plasma stability assays.
At Day 3, all mice were euthanized and tissues/organs of inter-
est were harvested and counted to obtain comprehensive biodistri-
bution data of all three radioimmunoconjugates. Compressive
tissue uptake values were provided in Supplementary Table 1. As
shown in Fig. 4, significantly higher spleen uptake (p < 0.001)
was observed using 89Zr-DFO-Trastuzumab (48.9 7.92% ID/g)
than 89Zr-1-Trastuzumab (24.6 4.08% ID/g) and 89Zr-2-Tras-
tuzumab (27.64 4.22% ID/g). In addition, bone uptake was also
significantly lower (p < 0.001) for 89Zr-DFO-Trastuzumab
(7.58 0.36% ID/g) than 89Zr-1-Trastuzumab (19.5 3.59% ID/g)
and 89Zr-2-Trastuzumab (18.3 2.91% ID/g). These biodistribution
data were consistent with observations from PET images, and con-
firmed that 89Zr-1-Trastuzumab and 89Zr-2-Trastuzumab had a
higher extent of in vivo demetalation leading to higher bone uptake
and lower spleen uptake.
Fig. 4. Biodistribution of 89Zr-labeled Trastuzumab conjugates at three days post-
injection. Data are expressed as mean SD (n = 6). For statistics analysis, * (p < 0.05)
and *** (p < 0.001) indicate the difference is significant when compared to the
uptake of 89Zr-DFO-Trastuzumab.
Both in vitro and in vivo studies of 89Zr-1-Trastuzumab and 89Zr-
2-Trastuzumab suggested that chelators 1 and 2 did not form a
stable complex with 89Zr. This could be due to for example (1) the
Zr complexes of 1 and 2 were not the expected eight-coordinate
complexes despite the presence of four hydroxamate groups per
chelator; or (2) the eight-coordinate Zr complexes of 1 and 2 were
not in an optimized coordination arrangement due to the steric con-
straints imposed by the 2,20-iminodiacetamide backbone and the
shorter arm (X = CH2 for 1 and X = CH2CH2 for 2, Fig. 1) of hydroxa-
mate moieties. A similar finding was reported by Boros et al.19 on
the comparison of stability between 89Zr complexes of two
hydroxamate chelators L3 and L4 (Fig. 1) that shared a same cyclam
backbone. L3 was an octadentate tetrahydroxamate chelator with
an (N-methyl-N-hydroxy)carboxamidomethyl group connected to
all four amino groups of the cyclam. For L4, it was a hexadentate tri-
hydroxamate chelator with substitution of a hydroxamate group
with a longer arm ((N-methyl-N-hydroxy)carboxamidoethylcar-
Table 1
In vitro plasma stability of 89Zr-DFO-Trastuzumab, 89Zr-1-Trastuzumab and
89Zr-2-Trastuzumab. Data are presented as the percentage of the total activity
corresponding to the intact radioimmunoconjugates.
89Zr-DFO-Trastuzumab 89Zr-1-Trastuzumab 89Zr-2-Trastuzumab
Day 0 >99
Day 1 77
Day 2 69
Day 3 55
>99
25
13
3
>99
46
18
16

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J. Rousseau et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 708–712
bonyl) at three of the four amino groups of the cyclam. Despite being
a hexadentate chelator, the complex of L4 with 89Zr was more stable
against EDTA (ethylenediaminetetracetic acid) challenge than the
complex of the tetrahydroxamate chelator L3 with 89Zr. Computer
calculations revealed that L3-Zr complex might not form the
expected eight-coordinate complex, but a seven-coordinate species
due to steric constraints. For L4 with longer hydroxamate arms, it
could adopt a more ideal geometry with decreased ligand strain
and form a more stable Zr complex. Therefore, based on these
observations, it might be possible to improve the design of our
tetrahydroxamate chelators by reducing steric constraints. This
could be achieved for example by increasing the arm length of
hydroxamate moieties to propylene (CH2CH2CH2) or longer
(Fig. 1) and/or replacing the 2,20-iminodiaetamide backbone with
3,30-iminodipropinamide.
Acknowledgements
This work was supported by the Terry Fox Research Institute.
The authors would like to thank Milan Vuckovic, Wade English
and Baljit Singh for their technical assistance for 89Zr production.
Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.01.
052.
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