Enantiopure bifunctional chelators for copper radiopharmaceuticals - Does chirality matter in radiotracer design?

Article abstract of DOI:10.1016/j.ejmech.2014.04.071

It is well recognized that carbon chirality plays a critical role in the design of drug molecules. However, very little information is available regarding the effect of stereoisomerism of macrocyclic bifunctional chelators (BFC) on biological behaviors of

Full text of DOI:10.1016/j.ejmech.2014.04.071

European Journal of Medicinal Chemistry 80 (2014) 308e315
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Enantiopure bifunctional chelators for copper radiopharmaceuticals e
Does chirality matter in radiotracer design?q
,
b
,
,
Ajay N. Singh a 1, Marianna Dakanali a 1, Guiyang Hao a, Saleh Ramezani a, Amit Kumar a,
,c,
Xiankai Sun a
*
a Department of Radiology, The University of Texas Southwestern Medical Center, Dallas, TX, USA
b Department of Basic Science, Appalachian College of Pharmacy, Oakwood, VA, USA
c Department of Advanced Imaging Research Center, The University of Texas Southwestern Medical Center, Dallas, TX, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
It is well recognized that carbon chirality plays a critical role in the design of drug molecules. However,
very little information is available regarding the effect of stereoisomerism of macrocyclic bifunctional
chelators (BFC) on biological behaviors of the corresponding radiopharmaceuticals. To evaluate such
effects, three enantiopure stereoisomers of a copper radiopharmaceutical BFC bearing two chiral carbon
atoms were synthesized in forms of R,R-, S,S-, and R,S-. Their corresponding peptide conjugates were
prepared by coupling with a model peptide sequence, c(RGDyK), which targets the avb3 integrin for
in vitro and in vivo evaluation of their biological behaviors as compared to the racemic conjugate. Despite
the chirality differences, all the conjugates showed a similar in vitro binding affinity profile to the avb3
integrin (106, 108, 85 and 100 nM for rac-H2-1, RR-H2-1, SS-H2-1, and RS-H2-1 respectively with all p
values > 0.05) and a similar level of in vivo tumor uptake (2.72 ꢀ 0.45, 2.60 ꢀ 0.52, 2.45 ꢀ 0.48 and
2.88 ꢀ 0.59 for rac-64Cu-1, RR-64Cu-1, SS-64Cu-1, and RS-64Cu-1 at 1 h p.i. respectively). Furthermore,
they demonstrated a nearly identical biodistribution pattern in major organs (e.g. 2.07 ꢀ 0.21,
2.13 ꢀ 0.58, 1.70 ꢀ 0.20 and 1.90 ꢀ 0.46 %ID/g at 24 h p.i. in liver for rac-64Cu-1, RR-64Cu-1, SS-64Cu-1, and
RS-64Cu-1 respectively; 1.80 ꢀ 0.46, 2.30 ꢀ 1.49, 1.73 ꢀ 0.31 and 2.23 ꢀ 0.71 at 24 h p.i. in kidneys for
rac-64Cu-1, RR-64Cu-1, SS-64Cu-1, and RS-64Cu-1 respectively). Therefore we conclude that the chirality of
BFC plays a negligible role in avb3-targeted copper radiopharmaceuticals. However, we believe it is still
worthwhile to consider the chirality effects of BFCs on other targeted imaging or therapeutic agents.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Received 18 January 2014
Received in revised form
22 April 2014
Accepted 23 April 2014
Available online 25 April 2014
Keywords:
Molecular imaging
Positron emission tomography
Integrin avb3
Copper-64
Bifunctional chelator (BFC)
Imaging
Diagnosis
Prognosis
1. Introduction
detailed anatomical information for a better diagnosis. Currently,
18F (t1/2 ¼ 109 min) and 11C (t1/2 ¼ 20.3 min) are the most
Positron emission tomography (PET) is routinely used in clinical
and preclinical settings for diagnostic or prognostic imaging of
cancer or other diseases [1e5]. The success of PET lies in its high
sensitivity and specificity to detect physiological changes at cellular
or molecular level. Combined with computed tomography (CT),
dual-modal PET/CT imaging provides both molecular changes and
commonly used PET radionuclides for the development of PET
imaging probes. However, their short half-lives limit their appli-
cations mainly in small organic or biological molecules. Recently,
64Cu (t1/2 ¼ 12.7 h; Ebþ max ¼ 0.653 MeV, 17.4%), a non-standard PET
radionuclide, has drawn considerable attention in the community
of PET due to its low positron energy, commercial availability, and
reasonably long decay half-life [6,7]. More importantly, the well-
established coordination chemistry of copper potentially enables
a rapid translation from bench-top science to clinical practice of
copper radiopharmaceuticals with a variety of imaging or radio-
therapeutic applications involving peptides, antibodies or their
fragments, and nanoparticles [8e10].
q
Grant support: This work was partially supported by the Prostate Cancer
Research Program of the United States Army Medical Research and Materiel
Command (W81XWH-08-1-0305 and W81XWH-12-1-0336). The authors
acknowledge the generous support of a private donor that allowed the purchase of
the Siemens Inveon PET-CT Multi-modality System.
* Corresponding author. University of Texas Southwestern Medical Center, 5323
Harry Hines Blvd, Dallas, TX 75390-8542, USA.
The stability of the metal complex moiety is of critical impor-
tance in the design of a metal radiopharmaceutical. In the past
decade, we and other groups had research focused on a cross-
E-mail address: Xiankai.Sun@UTSouthwestern.edu (X. Sun).
Authors contributed to this work equally.
1
http://dx.doi.org/10.1016/j.ejmech.2014.04.071
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

A.N. Singh et al. / European Journal of Medicinal Chemistry 80 (2014) 308e315
309
bridged tetraazamacrocyclic chelator, CB-TE2A [4,11-bis-(carbo-
2. Results
tert-butoxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane] for
the development of novel copper radiopharmaceuticals because
CB-TE2A forms one of the most stable complexes with Cu(II), which
is more resistant to reductive metal loss than are other tetraaza-
macrocyclic complexes [11,12]. The high in vivo stability of Cu(II)-
CB-TE2A complex arises from the perfect match of Cu(II) and the
cavity size formed by the pre-organized CB-cyclam ligand [11,13]. In
this hexa coordinated complex, Cu(II) is completely encapsulated in
the CB-cyclam cavity by four coordinating nitrogen atoms and two
carboxylate oxygen atoms. However, once a pendent carboxylate
arm of the CB-TE2A is converted to amide during vector conjuga-
tion, the stability of the Cu(II)-CB-TE2A will be compromised and
the metal moiety become positively charged, which could be
detrimental to the biological behavior of the copper radiophar-
maceuticals. To avoid this problem, we have recently reported a
novel bifunctional chelating (BFC) scaffold based on the CB-TE2A
core [10]. The designed BFC scaffold, CB-TE2GA (Fig. 1), utilizes an
orthogonally protected glutaric heterodiester as the coordinating
side arm. The choice of this side arm allows selective deprotection
of the peripheral carboxylates for conjugation with targeting mol-
ecules, while the inner carboxylates can be preserved for 64Cu
complexation. We reported that the imaging probe so designed
(rac-64Cu-1) indeed showed high in vivo stability and enhanced
tumor uptake due to the multivalent effect [10].
However, our reported BFC scaffold, CB-TE2GA, bears two chiral
carbon atoms in the glutaric acid side arm, which can give rise to
three potential stereoisomers [14,15]. When targeting molecules
are attached to the side arms, the chirality difference may lead to
different spatial dispositions of the targeting moieties depending
on the chirality of the side arms, which can potentially impact their
binding affinities to the target receptors.
To evaluate the role of chiral BFC in the properties of copper
radiopharmaceuticals, we synthesized CB-TE2GA in three enan-
tiopure forms (RR-8, SS-8, RS-8) starting from the enantiopure side
arm precursors, namely R- and S-5-benzyl 1-(tert-butyl) 2-
((methylsulfonyl)oxy)pentadioate (R-6, S-6). The synthesized BFCs,
RR-8, SS-8, and RS-8, were conjugated with an avb3 integrin specific
peptide, c(RGDyK), to provide enantiopure peptide conjugates (RR-
H2-1, SS-H2-1, RS-H2-1), respectively. When labeled with 64Cu, the
peptide conjugates were evaluated by in vitro binding assay, PET/CT
imaging, and imaging-derived biodistribution profiles in tumor-
bearing mice. The racemic conjugate was used as the control for
comparative evaluation.
2.1. Synthesis
The synthesis of enantiopure RR-H2-1, SS-H2-1, and RS-H2-1 was
achieved in three steps; i) synthesis of the chiral side arms, S-6 and
R-6 ii) synthesis of the BFC scaffolds, RR-8, SS-8, and RS-8, and iii)
conjugation of two molecules of c(RGDyK) on to the BFCs (Schemes
1e3). The synthesis of the chiral side arms, S-6 and R-6, started
from
L- and
D
-glutamic acid, respectively. Following a reported
procedure [16,17],
respectively, via diazotization using sodium nitrite (NaNO2) in the
presence of excess HCl in 78% yield. The stereoselectivity of the
L
- or -glutamic acid was converted to S- or R-2
D
conversion of
L
-glutamic acid to S-2 depends critically on both the
rate of the addition of the aqueous NaNO2 solution to the acidic
L-
glutamic acid solution and on the reaction temperature that must
be maintained between 0 and 5 ꢁC during the addition [17]. The
synthesis of S-2 uses the stereoselectivity of the deamination re-
action, which is known to proceed with complete retention of the
configuration [16]. The tBu-protected ester S-3 was synthesized in
55% yield using a standard esterification procedure, namely 4-
dimethylaminopyridine
(DMAP)
and
N,
N0-dicyclohex-
ylcarbodiimide (DCC) in tetrahydrofuran (THF). Unlike the reported
procedure where S-3 was synthesized from the acid chloride of S-2,
the DMAP/DCC directed esterification was easy to handle and the
product, lactone S-3, could be purified and obtained at gram scale.
The obtained S-3 was then hydrolyzed using one equivalent of 1 N
KOH to provide the potassium salt of S-4. A small sample of the
above synthesized salt was treated with 3 N HCl to yield the cor-
responding acid in 60% yield for purification and characterization
purposes. The carboxylate salt of S-4 was alkylated with benzyl
bromide (BnBr) in dimethylformamide (DMF) to afford the crude S-
5, an orthogonally protected diester. Purification of crude S-5 using
column chromatography yielded the pure product in 52% yield. The
secondary alcohol of S-5 was then converted to the corresponding
mesylate as a leaving group to provide S-6 in quantitative yield.
Mesylate was chosen as the leaving group based on the fact that it is
stable in aqueous workup at room temperature and the controlled
alkylation of CB-cyclam by SN2 reaction affords the complete
inversion of stereochemistry of the product. Dialkylation of CB-
cyclam was carried out by adding the mesylated S-6 or R-6, to a
suspension of a half equivalent of CB-cyclam in acetonitrile pre-
heated to 50 ꢁC in the presence of potassium carbonate. The
orthogonally protected diester, RR-7 or SS-7, was obtained in 52%
Fig. 1. Structures of peptide conjugates: rac-64Cu-1, RR-64Cu-1, SS-64Cu-1 and RS-64Cu-1.

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A.N. Singh et al. / European Journal of Medicinal Chemistry 80 (2014) 308e315
Scheme 1. Synthesis of 5-benzyl 1-(tert-butyl) 2-((methylsulfonyl)oxy)pentadioate (S-6) and (R-6). Reagents and conditions: a) L-glutamic acid (1 equiv), HClconc, NaNO2 (1.5 equiv),
H2O/dioxane, 0 ꢁC then r.t., 20 h; b) t-BuOH (1.1 equiv), DMAP (0.4 equiv), DCC (1.1 equiv), CH2Cl2, r.t., 18 h; c) 1 N KOH (2 equiv), THF, 0 ꢁC then r.t., 4 h; d) BnBr (1 equiv), DMF, r.t.
8 h; e) MsCl (1.01 equiv), Et3N (1.4 equiv), CH2Cl2, 0 ꢁC then r.t., 2 h.
Scheme 2. Synthesis of RR-H2-1 and SS-H2-1. Reagents and conditions: a) K2CO3 (1.2 equiv), CH3CN, r.t. 24 h then 50 ꢁC 24 h; b) 10% Pd/C (catalytic), H2, 2-propanol, r.t. 12 h; c) NHS
(4 equiv), EDC HCl (4 equiv), CH3CN, r.t. 18 h; d) c(RGDyK) (4 equiv), DIPEA, DMF, r.t 24 h; e) TFA, r.t. 12 h.
Scheme 3. Synthesis of RS-H2-1. Reagents and conditions: a) K2CO3 (1.2 equiv), CH3CN, r.t. 24 h then 50 ꢁC 24 h; b) R-6 (1.5 equiv), K2CO3 (1.2 equiv), CH3CN, r.t. 24 h then 50 ꢁC
24 h; c) 10% Pd/C (catalytic), H2, 2-propanol, r.t. 12 h; d) NHS (4 equiv), EDC HCl (4 equiv), CH3CN, r.t. 18 h; e) c(RGDyK) (4 equiv), DIPEA, DMF, r.t. 24 h; f) TFA, r.t. 12 h.

A.N. Singh et al. / European Journal of Medicinal Chemistry 80 (2014) 308e315
311
yield. Addition of the chiral side arms, S-6 and R-6, provided the
BFC scaffolds RR-7 and SS-7, respectively, with the complete
inversion of configuration. The BFC scaffold RS-7 was synthesized
in two steps: reaction of CB-cylam with S-6, to yield scaffold R-9,
followed by alkylation with R-6 to provide RS-7 in 40% yield.
The synthesized BFC scaffolds RR-7, SS-7, and RS-7 contain two
purified in one step using a pre-conditioned C-18 Sep-Pak light
cartridge with a >90% recovery rate. The radiochemical purity of
the 64Cu-labeled conjugates after cartridge purification was >97%
as determined by radio-HPLC. The overall radiochemical procedure
including the synthesis and purification steps took less than
45 min.
protected carboxylate groups at
a and g positions of the side arms.
The benzyl protected -carboxylate groups were selectively
g
2.3. Binding assay
deprotected and the resulting acids were conjugated with the
c(RGDyK) peptide. Catalytic debenzylation of RR-7, SS-7, and RS-7
was achieved using 10% Pd/C in 2-propanol under hydrogen at-
mosphere to afford RR-8, SS-8, and RS-8 in quantitative yield. The
The avb3 binding affinities of rac-H2-1, RR-H2-1, SS-H2-1, and RS-
H2-1 were measured by a competitive cell-binding assay using
U87MG cells in which 125I-echistatin was employed as avb3-specific
radioligand for competitive displacement. The U87MG cell line was
chosen because the avb3 integrin density on the cell surface is the
highest among the solid tumor cell lines that have been assessed
[18]. The IC50 values of rac-H2-1, RR-H2-1, SS-H2-1, and RS-H2-1
which represent their concentrations required to displace 50% of
the 125I-echistation bound on the U87MG cells, were determined to
be 106, 108, 85 and 100 nM, respectively (n ¼ 4) with all p values
>0.05 (see Fig. 3).
obtained
g-carboxylic acids were activated by N-hydrox-
ysuccinimide (NHS) for acid-amine conjugation chemistry. The
conjugation of NHS-activated RR-8, SS-8, and RS-8 with two
equivalents of c(RGDyK) in the presence of N, N-diisopropylethyl-
amine (DIPEA) provided the t-butyl protected conjugates in quan-
titative yield. Finally, the a-carboxylate groups were deprotected
using 95% trifluoroacetic acid to provide RR-H2-1, SS-H2-1, and RS-
H2-1, each of which contains two free carboxylic acids for radio-
labeling with 64Cu.
Compounds, S- or R-2 to S- or R-6 were characterized by 1H and
13C NMR and found to be identical to those reported in the litera-
ture [17]. Compounds, S- or R-2 to S- or R-6, showed no differences
in 1H and 13C NMR spectra as expected for enantiomers. The pep-
tide conjugates were characterized by their molecular ion peak
shown on MALDI-mass spectra, and the purity of the conjugates
2.4. Small animal PET-CT imaging
To evaluate the effect of the chirality of BFC on the in vivo
properties of the avb3-targeted imaging agents, a comparative PET/
CT imaging study was performed in SCID mice bearing integrin
avb3-positive PC-3 prostate cancer xenografts on a Siemens Inveon
PET/CT Multimodality System. Representative trans-axial PET/CT
images at 1, 4, and 24 h p.i. are displayed in Fig. 4. The PC-3 tumors
were clearly visualized by all four probes up to 24 h p.i. The four
agents showed nearly identical tumor uptake at the three time
points, namely 2.72 ꢀ 0.45, 2.60 ꢀ 0.52, 2.45 ꢀ 0.48 and 2.88 ꢀ 0.59
for rac-64Cu-1, RR-64Cu-1, SS-64Cu-1, and RS-64Cu-1 respectively at
1 h p.i., 2.32 ꢀ 0.37, 2.13 ꢀ 0.49,1.58 ꢀ 0.32 and 1.73 ꢀ 0.36 at 4 h p.i.
and 1.77 ꢀ 0.32, 1.92 ꢀ 0.51, 1.16 ꢀ 0.20 and 1.22 ꢀ 0.31 at 24 h p.i.
with all p values > 0.05 except for SS-64Cu-1 that showed signifi-
cantly lower uptake than other conjugates at 4 and 24 h p.i. due to
its lower specific activity at the injection time. In addition, we
compared their in vivo distribution profiles in major organs and
was verified by HPLC. Optical rotation [a]D for compounds RR-7 and
SS-7 was recorded to be þ34.594 and ꢂ36.594 respectively, veri-
fying that these compounds are enantiomers. Furthermore, the
circular dichroism (CD) spectra measured in CH3CN:H2O (1:5) so-
lution, further confirm the enantiomeric nature of the optically
active BFC scaffolds, RR-8 and SS-8. The CD spectrum of RR-8 shows
a positive Cotton effect in the 230e280 nm region, while SS-8 ex-
hibits the opposite sign of the effect in the same range (Fig. 2).
2.2. Radiochemistry
All peptide conjugates (rac-H2-1, RR-H2-1, SS-H2-1, and RS-H2-1)
were successfully labeled (>90% RCY) with 64Cu within 30 min at
75 ꢁC in 0.4 mM NH4OAc buffer to provide rac-64Cu-1, RR-64Cu-1,
SS-64Cu-1, and RS-64Cu-1, respectively. A series of radiolabeling
conditions by decreasing the amount of peptide conjugates while
the radioactivity of 64Cu was fixed were tested to reach around
20 GBq/mmol specific activity. The 64Cu-labeled conjugates were
Fig. 3. The integrin avb3 binding affinities of cRGDyK peptide, RR-1, SS-1, RS-1 and rac-
1 measured by a competitive cell-binding assay using 125I-echistatin as the radioligand.
The IC50 values were calculated to be 396 nM (cRGDyK), 108 nM (RR-1), 85 nM (SS-1),
100 nM (RS-1) and 106 nM (rac-1) (R2: 0.91e0.94).
Fig. 2. Measured CD spectra of RR-8 and SS-8 in CH3CN:H2O 1:5.

312
A.N. Singh et al. / European Journal of Medicinal Chemistry 80 (2014) 308e315
Fig. 4. (A) Comparative transaxial PET/CT images of PC-3 tumors in SCID mice intravenously injected with rac-64Cu-1, RR-64Cu-1, SS-64Cu-1, and RS-64Cu-1 at 1 h, 4 h, and 24 h p.i.
(B) Quantitative tumor uptake analysis of the four 64Cu-labled peptide conjugates (n ¼ 6).
found that the stereoisomers shared a similar biodistribution
pattern in the mouse tumor model.
chirality of the side arms, which can potentially impact their
binding affinities to the target receptors. Indeed it was reported
that, when two binding motifs of gonadotropin-releasing hormone
(GnRH) peptide (intact peptide: pEHWSYGLRPG-NH2, binding
3. Discussion
motifs: pEHW and RPG) were connected by D-Lys or L-Lys, a dif-
ference of more than two orders of magnitude was observed in
their binding to GnRH receptor [25]. Therefore we reasoned that
the enantiopure peptide conjugates (RR-H2-1, SS-H2-1, RS-H2-1)
might yield different ligand-receptor affinity and hence in vivo
properties of the corresponding copper radiopharmaceuticals.
Synthesis of the enantiopure side arms was the key step towards
the success of this work. Enantiopure side arms, R-6 and S-6, were
Copper has several radioisotopes of interest for the develop-
ment of radiopharmaceuticals, which can be used for PET imaging
(
61Cu, 62Cu, and 64Cu) and radiotherapy (64Cu and 67Cu). Most
copper radiopharmaceuticals are designed and prepared by incor-
poration of copper radioisotope into a biological substrate through
the linkage often provided by a tetraazamacrocyclic BFC [6,7,19,20].
The BFCs usually consist of a Cu(II) chelating moiety and a pendent
linker for the attachment to a biomolecule of interest. As such, the
carbon atoms in the pendent linker may become chiral depending
on the chemical modification as in our reported design of CB-
TE2GA. In the case of drugs, these subtle stereo chemical differ-
ences may lead to one form of the chiral molecule being thera-
peutically beneficial, while the other form may be physiologically
neutral or even harmful [21,22]. However, very little information is
available on stereoisomerism of the radiolabeled macrocyclic BFC-
conjugate at tracer level [23e25].
The role of linker chirality on ligand-receptor interaction
partially depends upon the application of the linker. In case of
monovalent BFC, where linker is solely used as the attachment
point, the linker chirality is not supposed to play significant role in
ligand-receptor interaction. This is clearly exemplified by 99mTc-
(HYNIC-K(NIC)-3G-RGD2) (tricine) monovalent conjugates, where
no significant differences in biological behavior or properties were
observed for its separable enantiomers or diastereomers [23]. The
results for the monovalent conjugates are not surprising given the
fact that one radioligand binds to one receptor site, which perhaps
is the main reason that the commercial available BFCs, such as
DOTA-SCN and NOTA-SCN, are provided as racemates. In the case of
multivalent BFC, multiple linkers provide multiple attachment
points as well as different spatial orientations for the multi-
presentation of a biologically active molecule, where the linker
chirality may play a significant role in multi-valent or multiplexing
ligand-receptor interactions.
synthesized in gram quantity from D/L-glutaric acid, respectively
[16]. The enantio-selective synthesis relied on the use of an
enantio-specific deamination reaction to form the lactone (Scheme
1). Further functionalization of the lactone, as well as the ring
opening reaction does not affect the conformation of the molecule.
The synthesis of BFC scaffolds, RR-7, and SS-7, was achieved in one
step by dialkylation of CB-cyclam using enantiopure side arm, S-6,
and R-6, respectively. On the other hand, for the synthesis of RS-7,
stepwise alkylation was required using different enantiomer in
each step. The alkylation of CB-cyclam proceeded via an SN2 type
reaction to provide the product with inversion of configuration.
Finally, RR-H2-1, SS-H2-1, and RS-H2-1 were synthesized by
conjugating the c(RGDyK) peptide with the outer carboxylate
groups, followed by deprotection of the inner carboxylate groups
using 95% TFA for radiolabeling with copper radioisotopes.
The designed imaging agents, enantiopure (RR-64Cu-1, SS-64Cu-
1) and the meso (RS-64Cu-1) contain two c(RGDyK) units for
multivalent interactions. The distance between the two RGD motifs
in the conjugates is greater than 25 bonds (including the lysine
spacers), the minimum spacing length required to realize multi-
valent binding of RGD motifs to the avb3 integrin [26]. The spatial
orientations of the c(RGDyK) units are expected to be different in
each stereoisomeric conjugate. To evaluate the chirality effect of
BFC on the imaging properties of the agents, we performed PET/CT
imaging in PC-3 tumor bearing mice. To our surprise, all the agents
showed similar bio-distribution and tumor uptake at all-time
points. This observation suggests that the spatial orientation dif-
ference of the two c(RGDyK) units presented on the chiral BFC
scaffold has negligible effects on the bio-distribution profile of the
avb3-targeted imaging agents in the mouse tumor model. This may
indicate that the multivalent ligandereceptor interaction is likely a
stepwise mechanism. Since the multivalent binding can be a
concerted process, binding of one RGD unit to integrin avb3 enables
In our reported multivalent BFC scaffold design for 64Cu-based
PET imaging probe, the BFC scaffold provides multiple peripheral
functional points for multi-presentation of targeting vectors.
Bearing two chiral carbon atoms in the glutaric acid side arm, the
BFC scaffold can have three potential stereoisomers RR-8, SS-8, and
RS-8 [14,15]. Attachment of c(RGDyK) to the chiral side arms leads
to different spatial dispositions of the c(RGDyK) depending on the

A.N. Singh et al. / European Journal of Medicinal Chemistry 80 (2014) 308e315
313
the second RGD unit to reorient itself towards a second avb3
5.3. Integrin anb3 binding assay
binding site as long as the spacer between them including the
chelator moiety is flexible. However, given the fact that ligande
receptor interaction is receptor specific, what we have observed
with the chiral BFC scaffolds for integrin avb3 in this work may not
be necessarily conserved for other receptors. Judicious selection of
BFC scaffolds with regards to their chirality should still be consider
so as to achieve the optimal imaging properties for noninvasive
assessment of the expression status of specific biomarkers towards
personalized disease stratification.
The binding affinities of c(RGDyK), rac-H2-1, RR-H2-1, RS-H2-1,
and SS-H2-1 to integrin anb3 were determined by a competitive
cell-binding assay using 125I-echistatin (PerkinElmer) as the anb3
-
specific radioligand. The experiments were performed on U87MG
human glioblastoma cells following our previously reported
method [10]. Briefly, U87MG cells were grown in RPMI 1640 me-
dium supplemented with penicillin, streptomycin, and 10% (v/v)
fetal bovine serum (FBS) at 37 ꢁC under 5% CO2. Suspended U87MG
cells in binding buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM
CaCl2, 1 mM MgCl2, 1 mM MnCl2, 0.1% bovine serum albumin) were
seeded on multi-well DV plates (Millipore) with 5 ꢃ 104 cells per
well and then incubated with 125I-echistatin (10,000 cpm/well) in
the presence of increasing concentrations (0e5000 nM) of
c(RGDyK) peptide conjugates for 2 h. The final volume in each well
4. Conclusions
Three enantiopure BFC scaffolds for copper radiopharmaceuti-
cals were designed and synthesized. Using well-validated integrin
avb3 ligand, c(RGDyK), peptide conjugates of the enantiopure BFC
scaffolds were prepared to evaluate the effect of BFC chirality on
ligand-receptor binding and the implications on the in vivo
behavior of so-designed agents. Our work suggests that the
chirality of BFC scaffolds plays an insignificant role in integrin avb3
targeted copper radiopharmaceuticals. Although this observation
does not support our design rationale, the importance of BFC
chirality in radiopharmaceutical agents cannot be undervalued
without discretion when it is applied to other biological targets.
was maintained at 250 m
L. At the end of incubation, unbound 125I-
echistatin was removed by filtration followed by five rinses with
cold binding buffer. The retentive was collected and the radioac-
tivity was measured using a
g-counter. The best-fit IC50 values
(inhibitory concentration where 50% of the 125I-echistatin bound on
U87MG cells are displaced) of c(RGDyK), rac-H2-1, RR-H2-1, SS-H2-
1, and RS-H2-1 were calculated by fitting the data with nonlinear
regression using GraphPad Prism (GraphPadSoftware, Inc.). Ex-
periments were duplicated with quintuplicate samples.
5. Experimental section
5.4. Tissue culture and animal model
5.1. General methods and materials
All animal studies were performed in compliance with guide-
lines set by the UT Southwestern Institutional Animal Care and Use
Committee (IACUC). The PC-3 cell line was obtained from the
American Type Culture Collection (ATCC, Manassas, VA), and was
cultured in T-media (Invitrogen, Carlsbad, CA) at 37 ꢁC in an at-
mosphere of 5% CO2 and were passaged at 75% confluence in P150
plates. T-media was supplemented with 5% Fetal Bovine Serum
(FBS) and 1ꢃ Penicillin/Streptomycin. PC-3 cells were harvested
from monolayer using PBS and trypsin/EDTA, and suspended in T-
media with 5% FBS. The cell suspension was then injected subcu-
All reactions were carried out under N2 atmosphere in degassed
dried solvents. Commercially available starting materials were
purchased from vendors and used directly without further purifi-
cation unless otherwise stated. Milli-Q water (18 M
U cm) was ob-
tained from a Millipore Gradient Milli-Q water system (Billerica,
MA). All aqueous solutions were prepared with Milli-Q water. Silica
gel 60 (70e230 mesh, Merck) was used for column chromatog-
raphy. Analytical thin-layer chromatography (TLC) was performed
using F254 silica gel (precoated sheets, 0.2 mm thick) (Lawrence,
KS). 1H and 13C NMR spectra were recorded on a Varian 400 spec-
trometer; chemical shifts are expressed in ppm relative to TMS
(0.0 ppm). Matrix-assisted laser desorption/ionization (MALDI)
mass spectra were acquired on an Applied Biosystems Voyager-
6115 mass spectrometer. Optical rotation data were collected on a
APIV-6W Rudolph Research automatic polarimeter. Radiolabeled
conjugates were purified by Light C-18 Sep-Pak cartridges (Waters,
Milford, MA).
Bulk solvents were removed by rotary evaporator under
reduced pressure, and trace solvents were removed by vacuum
pump. 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-cyclam) was
synthesized according to a published procedure [27]. 64Cu(II) in
0.1 M HCl was purchased from either Washington University School
of Medicine in St. Louis or the University of Wisconsin at Madison.
taneously (5 ꢃ 105 cells in 100
mL media) into the front flanks of
male SCID (Severe combined immunodeficiency) mice. After in-
jection, animals were monitored three times a week by general
observations. The tumor was allowed to grow three weeks to reach
a palpable size (50e150 mm3) for microPET/CT imaging studies.
5.5. Mouse PET/CT imaging
The imaging studies were performed on a Siemens Inveon
Multimodality PET/CT system once the tumor size reached the
range of 50e150 mm3 (tumor volume ¼ ½ (length ꢃ width2)). One
hour prior to imaging, each mouse bearing PC-3 tumor was injected
with 100e125 m mL of saline via
Ci of a 64Cu labeled conjugate in 100
the tail vein. Ten minutes prior to imaging, the animals were
anesthetized using 3% isofluorane at room temperature until stable
vitals were established. Once the animal was sedated, it was placed
onto the imaging bed under 2% isofluorane anesthesia for the
duration of imaging data requisition. At each time point (1 h, 4 h,
and 24 h) post-injection (p.i.), a CT scan was performed (8 min),
followed immediately by a static PET scan (15 min). The CT imaging
5.2. High performance liquid chromatography (HPLC) methods
HPLC separation was performed on a Waters 600 Multisolvent
Delivery System equipped with a Waters 2996 Photodiode Array
detector. The mobile phase consisted of H2O with 0.1% TFA (solvent
A) and acetonitrile with 0.1% TFA (solvent B). The analytical HPLC
was performed on an XTerra RP18 column (150 ꢃ 4.6 mm) with a
gradient of 0% B to 100% B in 50 min at the flow rate of 1.0 mL/min.
The HPLC separation was performed on a semi-preparative XTerra
RP18 Column (250 ꢃ 10 mm) with a gradient of 0% B to 100% B in
50 min at the flow rate of 4.0 mL/min.
was acquired at 80 kV and 500 mA with a focal spot of 58 mm. The
total rotation of the gantry was 360ꢁ with 360 rotation steps ob-
tained at an exposure time of approximately 180 ms/frame. The
images were attained using CCD readout of 4096 ꢃ 3098 with a
binning factor of four and an average frame of one. Under low
magnification the effective pixel size was 103.03
mm. The CT images
were reconstructed with a down sample factor of two using Cobra

314
A.N. Singh et al. / European Journal of Medicinal Chemistry 80 (2014) 308e315
Reconstruction Software. PET images were reconstructed using
Fourier Rebinning and Ordered Subsets Expectation Maximization
3D (OSEM3D) algorithm. Reconstructed CT and PET images were
fused and analyzed using the manufacturer’s software. For quan-
tification, regions of interest were placed in the areas expressing
the highest 64Cu-labeled conjugate activity as determined by PET
and visually guided by CT images. The tissues examined include the
tumor, heart, liver, lung, kidney, and muscle. The resulting quanti-
tative data were expressed in percentage of the injected dose in per
gram of the tissue (%ID/g) on the assumption that the density of the
tissue is 1 g/cm3.
5.6.4. Synthesis of 5-benzyl 1-(tert-butyl) (S)-2-
hydroxypentanedioate (S-5)
S-4 (2.0 g, 9.8 mmol) was suspended in DMF (15 mL), to which
was added benzyl bromide (1.67 g, 9.8 mmol). After stirred for 8 h,
the mixture was poured into ice water (20 mL) and extracted with
EtOAc (3 ꢃ 25 mL). The combined organic layer was dried over
Na2SO4 and concentrated under reduced pressure. The residue was
purified by column chromatography (silica gel, gravity) using
hexane and EtOAc:hexanes (1:4) to give S-5 as a white solid (1.5 g,
52%) . 1H NMR (400 MHz, CDCl3)
1H), 2.88 (bs, 1H), 2.59e2.44 (m, 2H),2.17 (m, 2H), 1.91 (m, 1H), 1.48
(s, 9H). 13C NMR (100 MHz, CDCl3)
173.9, 173.0, 135.9, 128.5, 128.2,
d 7.35 (m, 5H), 5.13 (s, 2H), 4.08 (bs,
d
128.1, 82.8, 69.6, 66.3, 29.7, 29.4, 28.0.
5.6. Synthesis
5.6.5. Synthesis of 5-benzyl 1-(tert-butyl) (S)-2-((methylsulfonyl)
oxy)pentadioate (S-6)
5.6.1. Synthesis of (S)-5-oxotetrahydrofuran-2-carboxylic acid (S-2)
L
-glutamic acid (30.0 g, 200 mmol) was suspended in a water/
Methanesulfonyl chloride (0.42 g, 3.7 mmol) was added to a
mixture of S-5 (1.0 g, 3.4 mmol) and Et3N (0.47 g, 4.7 mmol) in
CH2Cl2 (25 mL) at 0e5 ꢁC. After the addition was completed, the
mixture was warmed to room temperature and stirred for 2 h. Upon
completion, water (10 mL) was added, the organic phase was
separated and washed with brine (3 ꢃ 10 mL), dried over Na2SO4
and concentrated to give S-6 (0.7 g, 60%). 1H NMR (400 MHz, CDCl3)
dioxane mixture (75/25 mL) and stirred at 0 ꢁC for 30 min. The
white slurry became clear after 40 mL of concentrated HCl (37%)
was added, followed by drop-wise addition of a solution of NaNO2
(21.0 g, 300 mmol) in 50 mL of water. The reaction temperature was
maintained around 0 ꢁC during the 4 h of addition. The reaction
mixture was then left stirring at room temperature for 20 h. Upon
completion, the solvent was evaporated under reduced pressure to
provide a white solid, which was then treated with EtOAc (300 mL)
and Na2SO4 for 30 min. The solution was filtered and the solvent
was evaporated to yield S-2 as a white solid (21.50 g, 78%). 1H NMR
d
7.36 (m, 5H), 5.14 (s, 2H), 4.98 (m, 1H), 3.11 (s, 3H), 2.55 (m, 2H),
2.30 (m, 1H), 1.49 (s, 9H). 13C NMR (100 MHz, CDCl3)
d
171.8, 167.4,
135.5, 128.5, 128.2, 128.2, 83.5, 76.6, 66.5, 38.9, 29.3, 27.8, 27.0.
The synthesis of 5-benzyl 1-(tert-butyl) (R)-2-((methylsulfonyl)
oxy)pentadioate (R-6) was accomplished in the same way as above
(400 MHz, CDCl3)
d 11.08 (bs, 1H), 5.01 (m, 1H), 2.71e2.55 (m, 3H),
2.45e2.37 (m, 1H); 13C NMR (100 MHz, CDCl3)
d
176.8, 174.5, 75.4,
from D-glutamic acid.
26.8, 25.7.
5.6.6. Synthesis of compound RR-7
Compound S-6 (500 mg,1.3 mmol) was added to a suspension of
cross-bridge cyclam (150 mg, 0.6 mmol) and K2CO3 (0.10 g) in
anhydrous acetonitrile (50 mL). The reaction was stirred at room
temperature for 24 h and then for another 24 h at 50 ꢁC. The re-
action mixture was filtered and the solid was washed twice with
chloroform (2 ꢃ 20 mL). The combined filtrates were concentrated
under reduced pressure and purified by column chromatography
(silica gel, 60e230 mesh) using 10:1 CHCl3/MeOH to 9:1 EtOAc/
isopropylamine to yield RR-7 as viscous oil (250 mg; Yield: 52%):
MALDI-TOF/MS [Mþ]: calc’: 778.49; found: 778.59.
5.6.2. Synthesis of tert-butyl (S)-5-oxotetrahydrofuran-2-
carboxylate (S-3)
In a solution of S-2 (10.0 g, 77 mmol) in CH2Cl2 (240 mL), t-
butanol (8 mL, 84 mmol) and 4-dimethylaminopyridine (DMAP,
3.75 g, 31 mmol) were added and the reaction mixture was cooled
to 0 ꢁC. To this solution, N, N-dicyclohexylcarbodiimide (DCC,16.2 g,
84.5 mmol) in CH2Cl2 (80 mL) was added dropwise. The reaction
was stirred at room temperature overnight and upon completion
the solvent was removed under reduced pressure. The residue was
purified by column chromatography (silica gel, gravity) using
hexane (250 mL) and EtOAc:hexane (1:4) to give S-3 as white solid
5.6.7. Synthesis of compound RR-H21
(7.0 g, 55%) : 1H NMR (400 MHz, CDCl3)
(m, 3H), 2.24 (m, 1H), 1.48 (s, 9H) ppm; 13C NMR (100 MHz, CDCl3)
176.2, 169.0, 83.1, 76.2, 27.9, 26.8, 25.8 ppm.
d
4.79 (m, 1H), 2.65e2.44
To a solution of RR-7 (13 mg, 16.7 mmol) in 0.5 mL of 2-propanol
was added portion wise 10 mg of 10% Pd/C. The suspension was
shaken in a hydrogenator (Parr, Moline, Illionis) at room tempera-
ture for 12 h under an H2 atmosphere (60 psi). After removal of the
solids, evaporation of the solvent afforded compound RR-8 as a
white foam in nearly quantitative yield. A mixture of compound RR-
d
5.6.3. Synthesis of potassium (S)-5-(tert-butoxy)-4-hydroxy-5-
oxopentanoate (S-4)
8 (10.0 mg, 16.7
mmol), N-hydroxysuccinimide (7.6 mg, 66.8
mmol)
Compound S-3 (5.0 g, 30 mmol) was dissolved in THF (60 mL),
and cooled to 0 ꢁC. To this mixture, 1 N KOH (aqueous, 66 mL) was
added dropwise. The resulting mixture was stirred at room tem-
perature over 4 h and upon completion the solvent was evaporated
to give S-4 as white solid. Compound S-4 was directly used for the
synthesis of S-5. However, for characterization of the compound, a
sample of S-4 was converted to its acid using 3 N hydrochloric acid.
The acidified aqueous layer was then extracted with EtOAc
(3 ꢃ 30 mL). The organic layers were combined and dried over
anhydrous MgSO4 and concentrated. The residue was purified by
column chromatography (silica gel, gravity) using hexane (100 mL)
and EtOAc:hexane (1:3) to yield the acid form of S-4 as a white solid
and EDC$HCl (12.8 mg, 66.8
m
mol) in 500 L of dry acetonitrile
m
(MeCN) was stirred under N2 for overnight. The solvent was
removed under reduced pressure and the residue was redissolved
in CHCl3 (1 mL) and then washed with water promptly three times
(3 ꢃ 2 mL). CHCl3 was evaporated under reduced pressure, the
residue was frozen by liquid nitrogen and the remaining water was
removed by a freeze dryer to give a pale yellow solid in quantitative
yield. The activated ester was used directly for the next reaction
without further purification. Cyclic Arg-Gly-Asp-D-Tyr-Lys
[c(RGDyK)] (10 mg, 16
(2.4 mg, 4 mol) in 200
m
mol) was mixed with the activated ester
m
mL of anhydrous DMF. To this solution, 30
mL
of N, N-diisopropylethylamine (DIPEA) were added. The mixture
was stirred at room temperature for 24 h under N2. Upon
completion, the solvent was evaporated under reduced pressure
and the crude product was purified by HPLC. The collected fractions
(3.0 g, 60%) : 1H NMR (400 MHz, CDCl3)
(m, 2H), 2.16e2.07 (m, 1H), 1.92e1.83 (m, 1H), 1.47 (s, 9H); 13C NMR
(100 MHz, CDCl3) 178.6, 173.9, 82.8, 69.5, 29.4, 29.0, 27.8.
d 4.10 (m, 1H), 2.57e2.41
d

A.N. Singh et al. / European Journal of Medicinal Chemistry 80 (2014) 308e315
315
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were combined and lyophilized to yield the t-butyl protected
product, which was then dissolved in 95% TFA and stirred at room
temperature for 12 h. After evaporation of the solvent, the residue
was purified by semi-preparative reverse-phase HPLC. The
collected fractions from multiple runs were collected and lyophi-
lized to afford RR-H21 as white solid at quantitative yield. MALDI-
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HCl were added. The reaction mixture was shaken and incubated at
75 ꢁC for 0.5 h. Then, 5
mL of 5 mM diethylenetriaminepentaacetic
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mm).
The gradient mobile phase started with 100% A (0.1% TFA in H2O) to
50% B (0.1% TFA in MeCN) and 50% A at 25 min with a flow rate of
1 mL/min.
5.8. Statistical analysis
Quantitative data were expressed as the mean ꢀ SD. Unpaired t
test (two-tailed, confidence intervals: 95%) was performed using
GraphPad Prism. P values of <0.05 were considered statistically
significant.
Conflict of interests
The authors declare no conflicts of interest.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.071.
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