Preparation and Evaluation of Radiolabeled Antibody Recruiting Small Molecules That Target Prostate-Specific Membrane Antigen for Combined Radiotherapy and Immunotherapy

Article abstract of DOI:10.1021/acs.jmedchem.5b01881

The feasibility of developing a single agent that can deliver radioactive iodine and also direct cellular immune function by engaging endogenous antibodies as an antibody-recruiting small molecule (ARM) was determined. A library of new prostate-specific membrane antigen (PSMA)-binding ligands that contained antibody-recruiting 2,4-dinitrophenyl (DNP) groups and iodine were synthesized and screened in vitro and in vivo. A lead compound (9b) showed high affinity for PSMA and the ability to bind anti-DNP antibodies. Biodistribution studies of the iodine-125 analogue showed 3% ID/g in LNCaP xenograft tumors at 1 h postinjection with tumor-to-blood and tumor-to-muscle ratios of 10:1 and 44:1, respectively. The radiolabeled analogue was bound and internalized by LNCaP cells, with both functions blocked using a known PSMA inhibitor. A second candidate showed high tumor uptake (>10% ID/g) but had minimal binding to anti-DNP antibodies. The compounds reported represent the first examples of small molecules developed specifically for combination immunotherapy and radiotherapy for prostate cancer.

Full text of DOI:10.1021/acs.jmedchem.5b01881

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Article
Preparation and Evaluation of Radiolabeled Antibody Recruiting
Small Molecules that Target Prostate Specific Membrane
Antigen (PSMA) for Combined Radiotherapy and Immunotherapy
Afaf R Genady, Nancy Janzen, Laura Banevicius, Mahmoud
El-Gamal, Mohamed E. El-Zaria, and John F Valliant
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01881 • Publication Date (Web): 19 Feb 2016
Downloaded from http://pubs.acs.org on February 21, 2016
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Preparation and Evaluation of Radiolabeled Antibody Recruiting Small
Molecules that Target Prostate Specific Membrane Antigen (PSMA) for
Combined Radiotherapy and Immunotherapy
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Afaf R. Genady,†,‡ Nancy Janzen,† Laura Banevicius,† Mahmoud ElꢀGamal,†
Mohamed E. ElꢀZaria,† and John F. Valliant†,#,*
†Department of Chemistry and Chemical Biology, McMaster University, 1280 Main Street
West, Hamilton, Ontario L8S 4M1, Canada.
‡Department of Chemistry, Faculty of Science, Tanta University, 31527 Tanta, Egypt.
#Centre for Probe Development and Commercialization, 1280 Main Street West, Hamilton,
Ontario Canada, L8S 4K1.
*email: valliant@mcmaster.ca; telephone: 905ꢀ525ꢀ9140 ext. 20182; fax: 905ꢀ522ꢀ2509.
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Abstract
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The feasibility of developing a single agent that can deliver radioactive iodine and also direct
cellular immune function by engaging endogenous antibodies, as an antibody recruiting small
molecule (ARM), was determined. A library of new prostateꢀspecific membrane antigen
(PSMA)ꢀbinding ligands that contained antibody recruiting, 2,4ꢀdinitrophenyl (DNP) groups
and iodine, were synthesized and screened in vitro and in vivo. A lead compound (9b) showed
high affinity for PSMA and the ability to bind antiꢀDNP antibodies. Biodistribution studies of
the iodineꢀ125 analogue showed 3% ID/g in LNCaP xenograft tumors at 1 h postꢀinjection,
with tumorꢀtoꢀblood and tumorꢀtoꢀmuscle ratios of 10:1 and 44:1. The radiolabeled analogue
was bound and internalized by LNCaP cells, with both functions blocked using a known
PSMA inhibitor. A second candidate showed high tumor uptake (>10% ID/g), but had
minimal binding to antiꢀDNP antibodies. The compounds reported represent the first
examples of small molecules developed specifically for combination immunotherapy and
radiotherapy for prostate cancer.
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Introduction
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Immunotherapies that activate, focus, or release immune mechanisms from aberrant
regulation within tumors are changing cancer therapy paradigms, and introducing the
prospect of immunization against recurrence. Agents such as antiꢀCTLAꢀ4 and antiꢀPDꢀ1 /
PDꢀL1 antibodies are improving the prognosis of metastatic melanoma patients,1 whose five
year survival rate was previously estimated as only 16%.2 For example, a pivotal trial of the
antiꢀCTLAꢀ4 antibody ipilumumab in metastatic melanoma patients demonstrated overall
survival of 10 months versus 6.4 months in the control group.3 As a result, there has been a
subsequent surge in efforts to find new targets and molecules to stimulate antiꢀtumor immune
response. One such method is the antibody recruiting small molecule (ARM) strategy. ARMs
are designed to direct antibodies to bind specific cells and mark them for destruction by
immune effector cells.4
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One type of ARM used to promote immune response utilized 2,4ꢀdinitrophenyl (DNP)
groups, considering that antiꢀDNP antibodies exist in a large proportion of the North
American population,5 potentially as a result of environmental exposure to related hapten
structures.4 The ARM strategy has been employed to label the surface of prostate cancer
cells with DNP groups, using prostate specific membrane antigen (PSMA) as the target
(Figure 1).6 PSMA is a cell surface protein that has significantly higher expression on
prostate cancer cells compared to normal tissue.7 The agents, referred to as ARMꢀP (ARMꢀ
targeting prostate cancer), were based on DNP linked to a potent PSMA inhibitor through a
PEGꢀ8 spacer (ARMꢀP8), which promoted antibody dependent killing of PSMA expressing
LNCaP cells, in the presence of peripheral blood mononuclear cells and antiꢀDNP
antibodies.6 PSMA negative, DU145 cells were not susceptible to antibody dependent cell
cytotoxicity (ADCC) under the same conditions. Subsequent, 14 day in vivo studies, using
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human xenografts and an analogous DNP containing ARM, demonstrated target dependent
ADCC, inhibiting the growth of PSMA positive tumors.8
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The same construct that was used to prepare the PSMA targeting ARMs, has been
used to develop radiotherapeutic agents for treating metastatic prostate cancer.9ꢀ12 Babich,
Haberkorn and coworkers showed that a 131IꢀPSMA inhibitor (MIPꢀ1095) significantly
decreased prostate specific antigen levels and pain in men with metastatic, castration resistant
prostate cancer.12,13 We have noted that the amount of ARM needed to induce ADCC in vivo
was comparable to the molar quantity of the radiopharmaceutical used during PSMA targeted
radionuclide therapy. This suggested that a PSMA binding ARM containing a therapeutic
medical isotope could offer a means of combining immunotherapy and radionuclide therapy
within a single construct. There are an increasing number of reports that show both external
beam radiotherapy and therapeutic radiopharmaceuticals induce an antiꢀtumor immune
response. This “radiation vaccination” strategy offers not only the opportunity to kill the
tumor, but to provide sustained immunity against tumor recurrence as well.14ꢀ17 In addition,
there are significant practical advantages to using a single construct with two therapeutic
functions as opposed to two separate agents, in that it simplifies determining the optimal
dosing schedule and the order of administration, and would decrease overall development
costs.
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To test the feasibility of developing a combination agent (a radiolabeled ARMꢀP), we
prepared a series of PSMAꢀbinding DNP derivatives, using a glutamineꢀureaꢀlysine targeting
construct that contained iodineꢀ125 within the backbone. The goal was to identify compounds
that had high affinity for PSMA, would retain binding to antiꢀDNP antibodies, and would
localize in PSMA expressing tumors.
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Results and Discussion
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Synthesis
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The target library prepared was based on the ARMꢀP constructs reported previously6
in which iodine is added at the 4 or 5 position of the triazole ring. Iodine was selected
because iodineꢀ131, a βꢀemitting isotope, is widely used clinically for the treatment of thyroid
cancer and hyperthyroidism and because previous SAR and modeling data suggested that the
addition of the halogen to the triazole would not have a detrimental impact on binding to
PSMA.18 For the present study, iodineꢀ125 was used for cell uptake and biodistribution
studies, after in vitro screening was performed using the nonꢀradioactive iodinated analogues.
The initial class of compounds synthesized, contained the iodine atom at the 5ꢀ
position of the triazole. Iodination and formation of the triazole was achieved through a
concomitant reaction between a DNP alkyne, having a PEGꢀ4 (4a) or a PEGꢀ8 spacer (4b),
and an azide derivative of the PSMA binding ligand (5), in the presence of copper iodide and
Nꢀiodosuccinimide (NIS, Scheme 1). The spacer group length was selected based on the
PSMA binding data for the ARMꢀP4 and ARMꢀP8 reported previously.6 The 5ꢀiodoꢀtriazoles
6a and 6b were obtained in 75% and 83% yields, respectively.
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The identity of iodoꢀtriazoles 6a and 6b were confirmed by a variety of
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characterization methods (see Supporting Information). The H NMR indicated the NH
proton linked to the DNP moiety appeared at 6.97 ppm, while the NH protons of urea linkage
were at 5.07 ppm. In the 13C NMR spectrum, the triazole carbon atom signals were at 148.5
and 156.8 ppm, and were shifted by 3 ppm compared to the corresponding desꢀiodo
analogues. The presence of iodine at C5 was verified by the absence of triazole CH proton in
1H NMR. Moreover, HRMS were consistent with the formation of both the IꢀPEGꢀ4 and Iꢀ
PEGꢀ8 derivatives, as they showed the expected masses, matching the molecular formula of
6a (m/z 1059.3464 [M + Na]+) and 6b (m/z 1213.4752 [M + H]+).
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The IR spectra where similarly characteristic with intense bands appearing at 3358, 2931
and 1730 cmꢀ1 for 6a, and 3358, 2867 and 1735 cmꢀ1 for 6b, corresponding to the NH and
carbonyl stretches, respectively. Deprotection of 6a and 6b to the corresponding free acids 7a
and 7b was achieved using a mixture of TFA and DCM, with microwave heating.
Compounds 7a and 7b were isolated in 92% and 94% yield after HPLC purification, where
the loss of the signals associated with the tBu protecting groups was evident in the 1H and 13C
NMR spectra.
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A second family of 5ꢀiodoꢀDNP derivatives were prepared as high affinity PSMA
ligands using 2ꢀazidoacetic acid as a spacer group, based on the work of Darwish et al.19
(Scheme 2). The azide 8 was prepared in 90% yield by treatment of glutamateꢀureaꢀlysine20
with methyl 2ꢀazidoacetate in methanol at 60 oC for 48 h. A oneꢀpot click reaction between 8
and the DNPꢀalkynes 4a or 4b in the presence of Nꢀiodosuccinimide, followed by acidic
deprotection of tBu esters, produced two additional 5ꢀiodoꢀtriazoles 9a and 9b in 63% and 70%
yield respectively. The 1H and 13C NMR spectra of 9a and 9b were similar to those of 7a and
7b respectively, with notable differences being the additional signals associated with the extra
methylene protons that appeared at 5.10 ppm, and the carbonyl resonance at 176.2 ppm.
HRMS data provided additional evidence to support the formation of the desired products 9a
and 9b.
The 4ꢀiodotriazole derivatives were prepared from the corresponding stannanes
(Scheme 3). Here, the alkynes used to prepare the 5ꢀiodo derivatives were converted to the
alkynyl tin derivatives 10a and 10b, by treating 4a/4b with Bu3SnOMe in the presence of
zinc bromide.21 The PEGꢀ4 and PEGꢀ8 alkynes were isolated in 50% and 62% yield
respectively. These were coupled to the azide 8 using a thermal click reaction to give 11a and
11b in 30% and 28 % yield respectively. It should also be noted that this reaction also led to
the formation of approximately 10% of the desꢀiodotriazoles that can readily be separated
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from the desired product by preparative TLC. 119Sn NMR confirmed the presence of tin in the
product with the 119Sn chemical shifts of alkynes 10a and 10b observed at ꢀ64.7 ppm, and
then shifted downfield to ꢀ58.7 ppm, upon formation of the triazoles 11a and 11b. The two
sets of isomers could be distinguished by analytical HPLC, where the 5ꢀiodo derivatives have
shorter retention times compared to the 4ꢀiodo analogues. The two desired compounds for in
vitro screening, 12a and 12b, were prepared by iodination of the tin precursors, which
proceeded in high yield, and deprotection of the tꢀbutyl groups using TFA.
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As controls, we prepared the 4ꢀiodo derivatives without a DNP group and with DNP
linked to the PSMA binding ligand via a short spacer group, such that binding to antiꢀDNP
antibodies would be prevented. To accomplish this, an amino triazole was prepared using the
pthalimide 13, which was coupled to the azide 8, and the product isolated in 50% yield as a
single isomer (Scheme 4). For preparation of the iodine derivative 16, compound 14 was
treated with iodine, prior to deprotection with hydrazine. HRMS data for 14 was consistent
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with the expected mass (m/z 752.2842 [M + H]+), and the H NMR showed the three
characteristic NH protons at 7.65, 5.94 and 5.61 ppm. Moreover, the triazole carbon atoms
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were evident in the C NMR spectrum appearing at 157.6 and 141.0 ppm, and key carbonyl
resonances at 172.1, 171.9, 171.8 and 171.6 ppm.
To prepare the DNP derivative, 16 was combined with 1ꢀchloroꢀ2,4ꢀdinitrobenzene
to produce 18 in 38% yield (Scheme 5). The appearance of signals associated with DNP (9.13,
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8.32 and 7.05 ppm) and amide groups (8.93 and 7.77 ppm) in the H NMR spectrum of 18
provided evidence of the successful coupling. Deprotection was accomplished by treatment
of 18 with TFA over 10 h, where 19 was isolated in 87% yield.
In Vitro Screening
The affinity (IC50) of the reported compounds was assessed using an established
competitive binding assay with 125I-23; a known high affinity PSMA ligand (Figure 2).19 All
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compounds were assayed using PSMA expressing LNCaP cells under the same conditions.
Experiments were performed in triplicate using 2ꢀ(phosphonomethyl)ꢀpentanedioic acid
(PMPA), a known PSMA inhibitor, as a positive control. The previously reported desꢀiodo
ligands 1 and 2 were also tested to assess the impact of the addition of iodine to the triazoles,
on the binding to PSMA.
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The IC50 values of the 5ꢀiodo derivatives ranged from 14 nM to 30 nM, with the most
potent ligand being the acetamide linked, PEGꢀ8 derivative 9b (Table 1). The PEGꢀ4
derivative 9a was comparable with an IC50 of 15 nM. For compounds containing only the
triazole linker (7a, 7b), the products were still potent ligands, however the measured IC50
values nearly doubled compared to 9a/9b. For the 4ꢀiodo series, the PEGꢀ4 derivative 12a
showed an IC50 of 19 nM, while the PEGꢀ8 derivative 12b was nearly 100 nM. Interestingly,
the short chain derivative bearing no DNP was the least potent compound tested (123 nM),
and its DNP derivative showed the lowest IC50 (3 nM), indicating the presence of an arene
binding region.
The IC50 values measured for the parent ARMꢀPs was 9 nM for the PEGꢀ4 derivative
versus 23 nM for the longer chain PEGꢀ8 form. Although the affinities were higher in our
assay than those reported using a different screening method,6 the data are consistent with the
literature in that they showed both constructs had good affinity for PSMA and that ARMꢀP4
was more potent than ARMꢀP8. In the case of the iodinated compounds, there was evidence
of impact on affinity when the triazole alone was used as the linker between the PEG group
and the PSMA binding portion of the molecule. When the acetamide linker was added, the
affinities were similar or slightly better than that observed for ARMꢀP8 (2).6
Compound 9b was selected as the lead compound and its ability to bind to both
PSMA and antiꢀDNP antibodies was measured, using a previously reported in vitro
fluorescence assay.6 PSMA expressing LNCaP cells were incubated with 9b or 2 (the desꢀ
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iodo analogue reported previously), with and without the PSMA inhibitor PMPA. This was
followed by AlexaFluor488 conjugated, rabbit antiꢀDNP antibody, to detect the amount of
DNP bound to the LNCaP cells. Antibody binding was evident for 9b, which was reduced by
approximately 70% when the assay was performed in the presence of PMPA (Figure 3A).
Similar results were obtained with the positive control 2.6 A second test compared 9b with 19,
a potent ligand that has no spacer group between the PSMA binding portion of the molecule
and the DNP group. As expected, background levels fluorescence were obtained with 19,
indicating negligible antiꢀDNP binding, with no effect observed with PMPA blocking (Figure
3B).
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Radiochemistry
A method for producing 125Iꢀ9b was developed in order to assess the ability of the
labeled construct to target PSMA in vivo, using PMSAꢀexpressing xenograft tumors. Given
its unusually high affinity for PSMA, the iodineꢀ125 analogue of 19 was also prepared for
comparison. The synthesis of 125I-9b was performed using a methodology that is similar to
the synthesis of the nonꢀradioactive compound, in that the alkyne 4b was treated with cupric
chloride, followed by the azide 8 and Na125I, in place of Nꢀiodosuccinimide (Scheme 6).
These conditions resulted in concomitant formation of the triazole and the addition of the
iodine at the 5ꢀposition of the ring. The desired product was isolated by HPLC in 70% yield,
and the retention time of the product matched that of the reference standard used to determine
the IC50 value, with a measured radiochemical purity of 97% (Figure 4). The product was
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stable in saline up to 48 h, and for three weeks when stored as a solid at ꢀ10 C. The log P
value for 125I-9b measured using the “shakeꢀflask” method22 was ꢀ2.54 ± 0.03, which was
higher than the iodoꢀtriazole 23,19 which had a log P of ꢀ3.23 ± 0.05. The log P value for 125I-
9b was however less than those for pꢀiodoꢀbenzyl and iodoꢀphenylurea derivatives of
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glutamateꢀureaꢀlysine (0.26 and 0.53 respectively), which have been evaluated in patients as
PSMA targeting radiopharmaceuticals.23
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For 19, the corresponding stannane was prepared in order to introduce iodineꢀ125 at
the 4ꢀposition of the triazole. This was accomplished by treatment of 14 with hydrazine
hydrate, followed by coupling with 1ꢀchloroꢀ2,4ꢀdinitrobenzene to produce the stannane
precursor 21 (Scheme 7). The radiolabeling of 21 to form 125I-19 was performed at in 5%
AcOH/MeCN containing Na[125I] and Iodogen® as an oxidant. After 15 min, the reaction
was quenched with Na2S2O5 and deprotected with TFA for 1 h and the product purified by
HPLC to obtain 125I-19 in 72% yield and high (>98%) radiochemical purity (Figure 5). As
expected the log P value of 125I-19 (ꢀ1.4 ± 0.05) was higher than 125I-9b that contains a PEG
spacer.
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Biodistribution Studies in Mice with LNCaP Xenografts
Biodistribution studies on 125I-9b and 125I-19 were performed using NCr nu/nu mice
bearing LNCaP xenografts, at 4ꢀ7 weeks post inoculation. Mice were administered between
0.19 and 0.36 MBq of either 125I-9b or 125I-19 formulated in 0.9% saline, via tail vein
injection. Animals were sacrificed and tissues collected and counted at t = 1 and 6 h post
injection. 125I-9b showed tumor uptake of 3.01 ± 0.17 %ID/g at 1 h, and remained near that
level (3.24 ± 0.06 %ID/g) at 6 h (Figure 6). Tumorꢀtoꢀblood ratios were approximately 10:1
at 1 h but decreased to 3.4:1 at 6 h. Tumorꢀtoꢀmuscle ratios were 44:1 at 1 h and 24:1 at 6 h.
High uptake at 1 h was observed in the gallbladder, which declined significantly by 6 h.
There was also greatly increasing levels in the thyroid at the later time point, indicating
deiodination of the product.
For 125I-19, high tumor uptake (12.21 ± 1.85 %ID/g) was observed at 1 h, with only
a small decrease (9.80 ± 1.55 %ID/g) by 6 h (Figure 7). The tumorꢀtoꢀblood ratios were 35:1
at 1 h and increased to 59:1 at 6 h. There was a tumorꢀtoꢀmuscle ratio of 37:1 at 1 h and 73:1
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at 6 h. Rapid blood clearance was observed by 1 h (0.36 ± 0.07 %ID/g), and similar to that
observed for 125I-9b, there was high gall bladder uptake at 1 h (92.46 ± 20.7 %ID/g) with a
decrease to 7.89 ± 1.67 %ID/g at 6 h. High and sustained kidney uptake was also observed
(68.35 ± 16.56 %ID/g at 1 h, and 66.46 ± 45.10 %ID/g, at 6 h), which is typical of
radiolabeled, small molecule PSMA probes.19,23 The small intestine showed high uptake
(47.37 ± 7.86 %ID/g) at 1 h, but had decreased substantially to 2.18 ± 0.19 %ID/g at 6 h.
Cell Binding and Internalization
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Despite the IC50 value, the extent of tumor uptake for 125I-9b was modest when
compared to 125I-19 and 123I-23.19 This could be due to nonꢀspecific binding in vivo, deꢀ
iodination, or possibly due to reduced internalization following binding to PSMA. To gain
further insight, cell binding assays were performed for 125I-9b and the known PSMA ligand
125I-23 (Figure 8). The total binding of 125I-23 to LNCaP cells was almost 9 fold higher than
that of 125I-9b. When the binding was performed in the presence of the cold analogues of
each compound or PMPA as blocking agents, the measured binding of either radiolabeled
compound to LNCaP cells was reduced to the same degree. This indicated that both agents
bound PSMA specifically and had minimal nonꢀspecific binding to LNCaP cells. The extent
of internalization of by LNCaP cells was also assessed for both compounds, over 2 h of
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incubation at 37 C or 4 C. Both radiolabeled compounds were continuously internalized
over 2 h at 37 oC, with larger quantities of 125I-23 internalized compared to 125I-9b (Figure 9).
After 2 h approximately one half of the cell associated radioactivity was internalized for both
compounds. Low amounts of internalization for both compounds occurred at 4 oC, indicating
that uptake at 37 oC was an active process.
To assess the impact of internalization on the extent of DNP antigen available on the
cell surface, flow cytometry experiments were performed using a fluorophore labeled, antiꢀ
DNP antibody. Compound 9b was incubated with LNCaP cells for 0, 0.5, 1, 2 and 4 h at 37
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oC followed by the fluorescent antiꢀDNP antibody. Flow cytometry showed appreciable
binding of the antibody with modest decrease between 2 and 4 h of incubation (Figure 10).
This was likely due to internalization, as was observed for the radiolabeled analogue (Figure
9B). As a control, the studies were repeated in the presence of a known PSMA inhibitor
(PMPA), which blocked all observed binding to background levels. This data indicates that
despite internalization observed with the radiotracer there is sufficient antigen (DNP)
available over time to allow for antibody recruitment, and that the binding is due to PSMA
expression.
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It is clear that the presence of the DNPꢀPEG spacer in 9b reduces the extent of tumor
binding and uptake, but is nonetheless necessary for binding to antiꢀPSMA antibodies.
Spiegel and coworkers demonstrated that while their ARM compound with a PEGꢀ4 linker
(ARMꢀP4) had higher affinity for PSMA, the extent of antiꢀDNP binding compared to ARMꢀ
P8 was greatly diminished.6 Notwithstanding, one therapeutic advantage of the ARMꢀP
strategy is that it requires binding to PSMA and the antiꢀDNP antibody.8 Consequently, the
impact of lower than optimal target binding on the tumor may be reduced through the need to
form a ternary complex (PSMAꢀDNPꢀantibody). Given the positive in vitro and in vivo data
reported for DNPꢀderived PSMA ARMs,8 Sufficient amounts of ligand must be getting to the
tumors to induce an ADCC response. Unfortunately, the same benefit is not imparted to
radiotherapy, where high off target binding can cause unwanted dose being delivered to
normal tissue. Potential explanations for the modest cell and tumor uptake of 125I-9b versus
other radiolabeled PSMA derivatives are that the construct has inadequate affinity, the
presence of the DNPꢀPEG group and/or addition of the iodine label decreases uptake, or that
rapid deiodination occurs in vivo, prior to PSMA binding. To provide further insight, the
tritiated analogue of 2 is being prepared and uptake and distribution in PSMA expressing
cells and tumor models assessed. One of the immediate benefits of the reported approach is
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that creation of radiolabeled ARMs provides a convenient means to determine the extent of
tumor binding, internalization and overall biodistribution of novel constructs. This data can
subsequently be used to fine tune pharmacokinetics and enhance the efficacy of the next
generation of ARMs and analogues containing therapeutic radionuclides.
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Conclusions
A series of radiolabeled antibody recruiting molecules were prepared and their
binding to PSMA assessed, with several potent compounds identified. Two lead constructs
were evaluated in vivo using LNCaP tumor models. One DNP derivative (9b) was capable of
recruiting antiꢀDNP antibodies in vitro, and when iodinated, showed modest tumor uptake in
vivo, with good tumorꢀtoꢀblood ratios. In addition, a novel iodinated PSMA binding ligand
(19) was also discovered, and showed high tumor uptake and good tumorꢀtoꢀblood and
tumorꢀtoꢀmuscle ratios. While combining radiotherapy and ARM in one construct will require
additional work to find compounds with greater tumor uptake ratios, the availability of
radioiodinated compounds 19 and 23, along with other known PSMAꢀtargeted
radiotherapeutics, provides an opportunity to combine radiotherapy and immunotherapy
using 2 and related analogues. In addition, access to radiolabeled ARMs provides the means
to use in vivo imaging to support the development of new immune stimulating constructs in
addition to seeking potent dual function therapeutics.
Experimental Section
General Procedures, Materials and Instrumentation. All chemicals and reagents for
synthesis were purchased from SigmaꢀAldrich. Solvents were purchased from Caledon and
dried using a PureꢀSolv drying apparatus (Innovative Technology). Compounds 1ꢀ5 were
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prepared according to literature procedures.6 Preparative TLC was carried out using 0.75 mm
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layers of silica gel 60 GF254 made from water slurries on glass plates of dimensions 20 × 20
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cm2, followed by drying in air at 100 C. Iodineꢀ125 was obtained from the McMaster
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Nuclear Reactor as Na[125I] in 0.1 N NaOH. For labeling experiments, radioactivity was
measured using a dose calibrator (Capintec, Ramsey, NJ, USA). Caution: Iodineꢀ125 is
radioactive and should only be handled in an appropriately licensed and equipped laboratory.
1H and 13C NMR spectra were measured on a Bruker Avance AVꢀ600 spectrometer
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(1H = 600.13 MHz, 13C = 150.90 MHz, 119Sn = 225 MHz). H NMR, 119Sn NMR and 13C
NMR chemical shifts are expressed in parts ppm (δ units), and coupling constants are
expressed in Hz. SnMe4 was used as an external reference in 119Sn NMR. IR spectra were
recorded on a Nicolet 6700 FTꢀIR spectrometer (KBr disc). Lowꢀresolution mass spectra
were obtained on an Agilent 630 ion trap ESI instrument, using a 1200 series LC system and
an eluent consisting of H2O:MeOH (1:1). HRMS were obtained using a Waters Micromass
Global Ultima QꢀTOF in ESI mode. HPLC (analytical and semiꢀpreparative) was performed
on a Varian ProStar HPLC system fitted with an IN/US γꢀRAM Model 3 detector or a Waters
1525 Binary HPLC system connected to a Bioscan γꢀdetector and a 2998 photodiode array
detector monitoring at 254 nm. For analysis of compounds, a Phenomenex Gemini column (5
µm, 4.6 × 250 mm, C18) was used, eluting at a flow rate of 1.0 mL/min. For semiꢀpreparative
HPLC, a Phenomenex Luna column (5 µm, 10.0 × 250 mm, C18) was used, eluting at a flow
rate of 4.0 mL/min. HPLC protocols were as follows: Analytical and semiꢀpreparative HPLC:
Solvent A = 0.1% trifluoroacetic acid (TFA) in water; Solvent B = 0.1% TFA in acetonitrile:
gradient elution, 5% B (0ꢀ8 min), 39% B (8ꢀ20 min), 5% B (21ꢀ22 min).
Cell Culture. LNCaP cells were purchased from ATCC (CRL 1740). Cells were
propagated using RPMI 1640 with 2mM glutamine, supplemented with 10% fetal bovine
serum, 2mM Lꢀglutamine, 1mM sodium pyruvate, 10mM HEPES, 1% Penicillin
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Streptomycin and 0.25% Dꢀglucose, and grown at 37˚C and 5% CO2. Cells were used
between passage numbers 7 to 28.
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Animal Studies. Animal studies were approved by the Animal Research Ethics Board at
McMaster University in accordance with Canadian Council on Animal Care (CCAC)
guidelines. Male, 4ꢀ5 week old NCr nu//nu mice were purchased from Taconic (Germantown,
NY) and were maintained under SPF conditions in an established animal facility, with 12 h
light/dark cycles, and given food and water ad libitum. Mice were injected with 2 × 106
LNCaP cells, in Matrigel:DPBS (1:1 v/v) subcutaneously in the right flank, to provide tumor
xenografts.
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PSMA Binding Studies
Binding studies were done as described by Hillier et al.23 with some modifications. Briefly,
equal numbers (3 x 105) of LNCaP cells in RPMI 1640 with 2 mM glutamine and 0.5% BSA,
where incubated for 1 h at rt with either 10 nM 125I-9b or 3 nM 125I-23 alone, or in the
presence of 10 µM of the cold analogues (9b or 23) or 10 µM PMPA. Cells and media were
transferred to microcentrifuge tubes and were washed 3 times with centrifugation for 30 s, at
21,130 x g. Final pellets were counted for 10 min, using a Perkin Elmer Wizard 1470
Automatic Gamma Counter. The resulting CPM values were used to calculate fmol bound per
sample.
Fluorescence Assay for Binding to PSMA Expressing Cells and anti-DNPAntibodies
Fluorescence based assessment of 2, 9b, and 19 binding to both PSMA and antiꢀDNP
antibodies was performed as described,6 with minor changes to the procedure. LNCaP cells
were detached from the flask and reꢀsuspended in TBS, having a density of 6.25 × 105
cells/mL. Aliquots of 0.2 mL of the cell solution were prepared in Eppendorf tubes. Three
compound solutions were prepared: 220 nM solutions of 2, 9b, or 19, containing either 887
µM PMPA (block) or no PMPA. AlexaFluor488 conjugated rabbit antiꢀDNP IgG (Invitrogen)
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was added (final concentration, 580 nM) immediately before the mixture was added to
LNCaP cells. A 57.3 ꢁL volume of each solution was added to designated Eppendorf tubes
containing the LNCaP cells. Aliquots were incubated at 37 °C for 1 h on a shaker. To each
tube, 750 ꢁL TBS was added, and the cells were washed an additional time with TBS. PBS
was used to wash the cells once more. The aliquots were reꢀsuspended in 0.5 mL lysing
buffer and incubated at 37 °C. Three 150 ꢁL aliquots of each 500 ꢁL lysed cell sample was
added to a 96ꢀwell plate, and fluorescence intensity was analyzed with the Tecan Infinite
M1000 plate reader by exciting at 494 nm and reading the fluorescence intensity at 520 nm.
Internalization Studies of Radiolabeled Compounds
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Internalization studies were done as described by Hillier et al.23 with some modifications.
Briefly, aliquots (3 x 105) of LNCaP cells were incubated in HEPES buffered saline with 10
nM 125Iꢀ9b or 5 nM 125Iꢀ23 and 90 nM 9b or 95 nM 23 respectively, at 4 °C or 37 °C for 0 to
2 h. At designated times, cells in media were transferred to microcentrifuge tubes and
washed twice with PBS using centrifugation for 30 s, at 21,130 x g, to remove unbound
radioactivity. The cells were then washed (50 mM Glycine, 150 mM NaCl, pH 3.0) at 4 °C
for 5 min, to release surfaceꢀbound radioactivity. Cell aliquots were then centrifuged at
20,000 × g for 5 min. Both the supernatant (released surface bound radioactivity) and the cell
pellet (internalized radioactivity) were counted for 10 min with a Perkin Elmer Wizard 1470
Automatic Gamma Counter. The resulting cpm values were used to calculate surface bound
and internalized fmol of each radiolabeled compound.
Flow Cytometry
Flow cytometry studies were conducted as described in Murelli et al.6 with minor
modifications. Briefly, LNCaP cells were detached, counted, washed, resuspended in TBS (25
mM TrisꢀHCl, 150 mM NaCl, 1.5% BSA, 5mM Glucose, 1.5 mM MgCl2, pH 7.2) at a
density of 5×105 cells/mL and 0.25 mL aliquots were added to Eppendorf tubes. Compound
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9b in water (2.5 µL of 5 µM stock) was added to the aliquots followed by either vehicle (2.3
µL of saline) or the blocking PMPA solution (2.3 µL of 22.1 mM PMPA solution in saline).
Samples were incubated with shaking at 37 °C prior to the addition of 2 mg/mL (2.5 µL)
AlexaFluor488 conjugated rabbit antiꢀdinitrophenyl IgG (Invitrogen), at t=0, 0.5, 1, 2, and 4
h. Samples were protected from light and incubated at 37 °C with shaking for a further 1 h.
Cells were diluted and washed with 0.75 mL of TBS and centrifuged for 30 s at max speed.
The wash was repeated once more with 1 mL of TBS followed by a final wash with 1 mL of
PBS. Cells were resuspended in 0.5 mL PBS and kept on ice until analyzed on a FACS Aria
III (BectonꢀDickinson). AlexaFluor488 (Flꢀ1 channel) was determined, after dead cells
were excluded based on propidium iodide (500 µg/mL, 2 µL) staining (Flꢀ2). Data was
analyzed using FlowJo software (Tree Star Inc.) and all experiments performed in triplicate.
Synthetic Procedures
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For all compounds evaluated purity was >95% as determined by HPLC, HRMS and H and
13C NMR.
General Procedure for Synthesis of 6a and 6b. To a solution of copper(I) iodide (63.0 mg,
0.33 mmol) and triethylamine (19.7 mg, 0.33 mmol) in dry DMF (1.0 mL) were added alkyne
(4a or 4b, 0.33 mmol), azide 5 (190 mg, 0.33 mmol) and Nꢀiodosuccinimide (82 mg, 0.37
mmol). The resulting mixture was stirred at rt under an atmosphere of argon for 4ꢀ8 h. After
the specified reaction time, water (10 mL) was added and the product mixture extracted with
DCM (3 × 10 mL). The organic layers were combined, extracted with 20 mL of brine and
dried over MgSO4. After filtration, the solvent was removed under reduced pressure and the
desired product was purified by preparative TLC (5% MeOH/DCM).
6a: Yellow waxy solid (75% yield, 256 mg, Rf = 0.45). 1H NMR (CDCl3): δ 9.13 (d, J = 3 Hz,
1H, CH), 8.80 (bs, 1H, CH), 8.26 (d, J = 12 Hz, 1H, CH), 6.97 (d, J = 6 Hz, 1H, CH), 5.07
(bs, 2H, NH), 4.59 (s, 2H, CH2), 4.34 (m, 4H, CH2), 3.84 (d, J = 6 Hz, 2H, CH2), 3.68 (m,
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12H, CH2), 3.60 (m, 2H, CH2), 2.30 (m, 2H, CH2), 2.07 (m, 1H, CH), 1.86 (m, 4H, CH2),
1.65 (m, 1H, CH), 1.46, 1.44, 1.42 (s, 27H, CH3), 1.30 (m, 2H, CH2) ppm. 13C NMR (CDCl3):
δ 172.4, 172.2, 172.0, 156.8, 148.5, 148.1, 136.0, 130.4, 130.0, 124.4, 114.2, 81.9, 80.6, 80.2,
70.8, 70.7, 70.6, 70.4, 69.7, 68.6, 64.2, 53.2, 53.0, 50.4, 43.3, 32.6, 31.6, 29.5, 28.4, 28.1,
28.0, 22.1 ppm. IR (KBr): 3358, 2976, 1730, 1621 cmꢀ1. HRMS (ESI+) m/z for
C41H65O15N8INa: calcd 1059.3512, obsd 1059.3464 [M + Na]+.
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6b: Yellow waxy solid (83% yield, 332 mg, Rf = 0.38). 1H NMR (CDCl3): δ 9.13 (d, J = 3 Hz,
1H, CH), 8.80 (s, 1H, CH), 8.26 (dd, J = 3, 6 Hz 1H, CH), 6.97 (d, J = 6 Hz, 1H, CH), 5.07
(m, 2H, NH), 4.59 (s, 2H, CH2), 4.31 (m, 4H, CH2), 3.83 (t, J = 6 Hz, 2H, CH2), 3.65 (m,
32H, CH2), 2.29 (m, 2H, CH2), 2.09 (m, 1H, CH), 1.83 (m, 4H, CH2), 1.62 (m, 1H, CH), 1.46,
1.45, 1.42 (s, 27H, CH3), 1.34 (m, 2H, CH2). 13C NMR (CDCl3): δ 172.5, 172.2, 172.0, 156.8,
148.6, 136.2, 130.5, 130.2, 124.4, 114.2, 82.1, 82.0, 80.6, 70.8, 70.7, 70.6, 70.4, 69.6, 68.6,
64.3, 53.2, 53.1, 50.4, 43.3, 32.6, 31.6, 29.4, 28.4, 28.1, 28.0, 22.0 ppm. IR (KBr): 3359,
2868, 1736 cmꢀ1. HRMS (ESI+) m/z for C49H82O19N8I: calcd 1213.4741, obsd 1213.4752 [M
+ H]+.
Synthesis of 7a. Bocꢀprotected 6a (50.0 mg, 0.048 mmol) was dissolved in a mixture of
TFA (2 mL) and CH2Cl2 (1 mL) in a 5 ml microwave vial that was capped and subjected to
microwave heating for 5 min at 70 oC. The resulting reaction mixture was concentrated under
reduced pressure. The desired product was purified by semiꢀpreparative HPLC and
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lyophilized to give 7a. Yellow waxy solid (92% yield, 38 mg, tr = 14.1 min). H NMR
(CD3OD): δ 8.91 (d, J = 3 Hz, 1H, CH), 8.17 (m, 1H, CH), 7.11 (d, J = 6 Hz, 1H, CH), 4.45
(s, 2H, CH2), 4.33 (t, J = 6 Hz, 2H, CH2), 4.19 (m, 2H, CH2), 3.70 (t, J = 6 Hz, 2H, CH2),
3.56 (m, 16H, CH2), 3.21 (s, 4H, CH2), 2.30 (m, 2H, CH2), 2.04 (m, 1H, CH), 1.85‒1.75 (m,
4H, CH2), 1.60 (m, 1H, CH), 1.34 (m, 2H, CH2) ppm. 13C NMR (CD3OD): δ 176.3, 160.1,
149.8, 137.0, 131.7, 131.0, 124.7, 116.2, 71.7, 71.6, 71.5, 70.9, 70.0, 65.0, 53.8, 51.7, 44.2,
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32.9, 31.1, 30.5, 29.0, 23.5 ppm. IR (KBr): 3356, 2924, 2868, 1734 cmꢀ1. HRMS (ESI+) m/z
for C29H42O15N8I: calcd 869.1815, obsd 869.1805 [M + H]+.
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Synthesis of 7b. Compound 7b was prepared from 6b (12.0 mg, 0.01 mmol) following the
procedure used to synthesize 7a, to give 7b. Yellow waxy solid (93% yield, 9.0 mg, tr = 12.3
min). 1H NMR (CD3OD): δ 8.93 (d, J = 3 Hz, 1H, CH), 8.19 (dd, J = 6, 8 Hz, 1H, CH), 7.13
(d, J = 12 Hz, 1H, CH), 4.48 (s, 2H, CH2), 4.34 (t, J = 6 Hz, 2H, CH2), 4.22 (m, 1H, CH),
4.18 (m, 1H, CH), 3.71 (t, J = 6 Hz, 2H, CH2), 3.55 (m, 34H, CH2), 2.30 (m, 2H, CH2), 2.04
(m, 1H, CH), 1.80 (m, 4H, CH2), 1.59 (m, 1H, CH), 1.35 (m, 2H, CH2) ppm. 13C NMR
(CD3OD): δ 176.4, 176.2, 175.8, 160.1, 149.9, 149.5, 137.1, 131.6, 131.2, 124.8, 116.2, 83.4,
71.6, 71.5, 71.4, 71.3, 70.8, 70.0, 65.1, 53.8, 53.4, 51.6, 44.0, 32.9, 31.1, 30.4, 29.0, 23.5 ppm.
IR (KBr): 3360, 2919, 2868, 1717, 1684 cmꢀ1. HRMS (ESI+) m/z for C37H58O19N8I: calcd
1045.2863, obsd 1045.2814 [M + H]+.
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General Procedure for Synthesis of 9a and 9b. To a mixture of alkyne 4a or 4b (0.066
mmol), and azide 8 (31.0 mg, 0.054 mmol) in dry DMF (0.5 mL) was added copper(I) iodide
(13.3 mg, 0.070 mmol), triethylamine (7.24 mg, 0.070 mmol) and Nꢀiodosuccinimide (15.0
mg, 0.073 mmol). The resulting mixture was allowed to stir at rt under an atmosphere of
argon for 3 h. The reaction was deemed complete by TLC (and mass spectrometry), filtered
through a plug of celite, and concentrated to give a yellow oil, which was redissolved in
CH2Cl2 (2 mL) and TFA (4 mL) in a microwave vial. The vial was capped and subjected to
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microwave irradiation for 5 min at 70 C. The resulting reaction mixture was concentrated
under reduced pressure, and chromatographed using semiꢀpreparative HPLC and lyophilized.
9a: Yellow waxy solid (63% yield, 37 mg, tr = 11.97 min). 1H NMR (CD3OD): δ 8.93 (d, J =
6 Hz, 1H, CH), 8.17 (dd, J = 6, 12 Hz, 1H, CH), 7.11 (d, J = 12 Hz, 1H, CH), 5.06 (m, 2H,
CH2) 4.48 (s, 2H, CH2), 4.22 (m, 1H, CH), 4.17 (m, 1H, CH), 3.71 (t, J = 6 Hz, 2H, CH2),
3.55 (m, 16H, CH2), 3.16 (t, J = 6 Hz, 2H, CH2), 2.31 (m, 2H, CH2), 2.05 (m, 1H, CH), 1.78
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(m, 2H, CH2), 1.52 (m, 1H, CH), 1.42 (m, 2H, CH2), 1.34 (m, 2H, CH2) ppm. 13C NMR
(CD3OD): δ 176.4, 176.3, 175.9, 167.1, 160.1, 150.0, 149.8, 137.1, 131.1, 124.8, 116.3, 85.2,
71.7, 71.6, 71.5, 70.9, 70.0, 65.1, 54.0, 53.7, 53.6, 44.1, 40.5, 33.2, 31.1, 30.0, 28.9, 24.0,
22.0 ppm. IR (KBr): 3356, 2925, 1676, 1622 cmꢀ1. HRMS (ESI+) m/z for C31H45O16N9I: calcd
926.2029, obsd 926.2056 [M + H]+.
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9b: Yellow viscous oil (70% yield, 50 mg, tr = 10.95 min). 1H NMR (CD3OD): δ 8.93 (d, J =
3 Hz, 1H, CH), 8.20 (dd, J = 6, 8 Hz, 1H, CH), 7.13 (d, J = 6 Hz, 1H, CH), 5.08 (m, 2H,
CH2), 4.50 (s, 2H, CH2), 4.22 (m, 1H, CH), 4.17 (m, 1H, CH), 3.71 (t, J = 6 Hz, 2H, CH2),
3.55 (m, 32H, CH2), 3.16 (t, J = 6 Hz, 2H, CH2), 2.31 (m, 2H, CH2), 2.05 (m, 1H, CH), 1.84ꢀ
1.73 (m, 2H, CH2), 1.57 (m, 1H, CH), 1.49 (m, 2H, CH2), 1.38 (m, 2H, CH2) ppm. 13C NMR
(CD3OD): δ 176.4, 176.4, 175.9, 167.1, 161.9, 161.7, 160.2, 149.9, 149.8, 137.1, 131.0, 124.7,
116.2, 85.3, 71.6, 71.5, 71.4, 71.3, 70.8, 70.0, 65.1, 54.0, 53.7, 53.6, 44.2, 40.4, 33.3, 31.2,
29.9, 28.9, 24.1 ppm. IR (KBr) 3359, 2919, 2870, 1726, 1683 cmꢀ1. HRMS (ESI+) m/z for
C39H61O20N9I: calcd 1102.3077, obsd 1102.3107 [M + H]+.
Synthesis of 10a. To a solution of ZnBr2 in THF (0.05 M, 2.0 mg/0.3 mL) and DNPꢀPEG4ꢀ
alkyne6 (50 mg, 0.12 mmol) was added dropwise Bu3SnOMe (43 uL, 0.15 mmol). The
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mixture was stirred for 4 h at 60 C, and then quenched by H2O (5 mL). The mixture was
extracted with diethyl ether (3 × 10 mL). The collected organic layers were dried (MgSO4).
Evaporation of volatiles gave a waxy residue which was diluted with EtOAc (20 mL) and
washed by NH4F (aq) (10%, 20 mL). The obtained white precipitate was filtered off, and the
filtrate was dried (MgSO4). Evaporation of volatiles gave an oily yellow product. Purification
by prepꢀTLC using hexane/EtOAc (1:1) gave compound 10a. Yellow oil (41% yield, 36 mg,
Rf = 0.35). It is noteworthy that alkyne 10a is unstable and must be kept in the refrigerator to
prevent fast decomposition. Typically a freshly prepared sample of 10a was used in all
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subsequent reactions. H NMR (CD3CN): δ 8.77 (d, J = 6 Hz, 1H, CH), 8.51 (bs, 1H, CH),
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8.06 (dd, J = 6, 8 Hz, 1H, CH), 6.94 (d, J = 12 Hz, 1H, CH), 3.91 (m, 2H, CH2), 3.57 (t, J = 6
Hz, 2H, CH2), 3.38 (m, 16H, CH2), 1.75 (m, 2H, CH2), 1.36 (m, 4H, CH2), 1.13 (m, 6H, CH2),
0.79 (m, 4H, CH2), 0.69 (t, J = 6 Hz, 9H, CH3) ppm. 13C NMR (CD3CN): δ 148.6, 123.5,
114.8, 105.9, 89.4, 70.0, 69.9, 69.7, 69.6, 68.6, 68.2, 68.1, 58.4, 42.8, 28.4, 26.4, 12.6, 10.4
ppm. 119Sn NMR: (225 MHz, CD3CN) ꢀ65.8. IR (KBr): 3326, 2798, 1731 cmꢀ1. HRMS (ESI+)
m/z for C29H49O8N3SnH: calcd 688.2626, obsd 688.2623 [M + H]+.
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Synthesis of 10b. Compound 10b was prepared as described for 10a using ZnBr2 (0.12 M,
5.0 mg/0.5 mL THF), DNPꢀPEG8ꢀalkyne6 (50 mg, 0.09 mmol) and Bu3SnOMe (50 µL, 0.17
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mmol). The reaction mixture was stirred for 3 days at 60 C, and then quenched by H2O (5
mL). Purification by preparativeꢀTLC using 4% MeOH/DCM gave compound 10b. Yellow
oil (62% yield, 46 mg, Rf = 0.65). 1H NMR (CD3CN): δ 9.00 (d, J = 3 Hz, 1H, CH), 8.75 (bs,
1H, CH), 8.28 (dd, J = 3, 8 Hz, 1H, CH), 7.17 (d, J = 12 Hz, 1H, CH), 4.17 (m, 2H, CH2),
3.80 (t, J = 6 Hz, 2H, CH2), 3.62 (m, 32H, CH2), 1.58 (m, 4H, CH2), 1.37 (m, 6H, CH2), 1.04
(m, 6H, CH2), 0.92 (t, J = 6 Hz, 9H, CH3) ppm. 13C NMR (CD3CN): δ 148.6, 129.8, 123.4,
115.1, 105.8, 89.2, 74.3, 70.0, 69.8, 69.5, 68.7, 68.2, 68.1, 58.3, 57.4, 42.7, 28.4, 26.3, 12.7,
10.4 ppm. 119Sn NMR (CD3CN): δ ꢀ65.7 ppm. IR (KBr): 3112, 2960, 1734 cmꢀ1. HRMS (ESI+)
m/z for C37H65O12N3SnNH4: calcd 881.3943, obsd 881.3953 [M + NH4]+.
General Procedure for Synthesis of 11a and 11b. Alkyne 10a or 10b (0.04 mmol) was
dissolved in toluene (2 mL) followed by the addition of the azide 8 (24 mg, 0.04 mmol). The
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reaction mixture was stirred at 100 C for 16ꢀ24 h. After cooling to rt, the reaction mixture
was concentrated under reduced pressure and purified by prepꢀTLC.
11a. Ethyl acetate/hexane (1:1) was used as prepꢀTLC eluent and the product isolated. Yellow
waxy solid (30% yield, 15 mg, Rf = 0.47). 1H NMR (CD3CN): δ 8.94 (d, J = 3 Hz, 1H, CH),
8.70 (bs, 1H, NH), 8.23 (dd, J = 3, 6 Hz, 1H, CH), 7.12 (d, J = 6 Hz, 1H, CH), 6.86 (t, J = 6
Hz, 1H, NH), 5.48 (d, J = 12 Hz, 1H, NH), 5.36 (d, J = 12 Hz, 1H, NH), 5.07 (s, 2H, CH2),
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4.52 (s, 2H, CH2), 4.13 (m, 1H, CH), 4.03 (m, 1H, CH), 3.75 (t, J = 3 Hz, 2H, CH2), 3.58 (m,
16H, CH2), 3.16 (m, 2H, CH2), 2.23 (m, 2H, CH2), 1.93 (m, 1H, CH), 1.75 (m, 1H, CH), 1.65
(m, 1H, CH), 1.53 (m, 1H, CH), 1.46 (m, 6H, CH2), 1.40 (m, 27H, CH3), 1.30 (m, 9H, CH2),
1.11 (m, 6H, CH2), 0.86 (t, J = 6 Hz, 9H, CH3) ppm. 13C NMR (CD3CN): δ 172.1, 171.8,
171.6, 165.7, 157.0, 148.4, 144.1, 140.1, 140.6, 129.7, 123.4, 114.8, 80.8, 80.4, 79.6, 70.0,
69.9, 69.8, 69.5, 69.3, 68.1, 61.9, 53.3, 52.6, 50.4, 42.7, 38.4, 31.4, 30.9, 28.4, 28.3, 27.5,
27.0, 26.9, 26.6, 22.1, 12.7, 9.3 ppm. 119Sn NMR (CD3CN): δ ꢀ58.6 ppm. IR (KBr): 3334,
2978, 1730 cmꢀ1. HRMS (ESI+) m/z for C55H95N9O16SnH: calcd 1258.6011, obsd 1258.6044
[M + H]+.
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11b. 5 % MeOH/DCM was used as prepꢀTLC eluent and the product isolated. Yellow viscous
oil (28% yield, 16 mg, Rf = 0.32). 1H NMR (CD3CN): δ 9.00 (s, 1H, CH), 8.75 (bs, 1H, CH),
8.28 (dd, J = 6,6 Hz, 1H, CH), 7.17 (d, J = 12 Hz, 1H, CH), 6.95 (t, J = 6 Hz, 1H, CH), 5.53
(d, J = 12 Hz, 1H, CH), 5.42 (d, J = 12 Hz, 1H, CH), 5.13 (s, 2H, CH2), 4.59 (s, 2H, CH2),
4.18 (m, 1H, CH), 4.08 (m, 1H, CH), 3.80 (t, J = 6 Hz, 2H, CH2), 3.66 (m, 4H, CH2), 3.60 (m,
28H, CH2), 3.21 (m, 2H, CH2), 2.27 (m, 2H, CH2), 1.80 (m, 1H, CH), 1.71 (m, 1H, CH), 1.57
(m, 8H, CH2), 1.45 (m, 29H, CH3, CH2), 1.36 (m, 10H, CH2), 1.17 (m, 2H, CH2), 1.15 (m,
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6H, CH2), 0.90 (m, 9H, CH3) ppm. C NMR (CD3CN): δ 172.2, 171.9, 171.6, 165.7, 157.1,
148.5, 144.1, 140.7, 129.7, 123.4, 114.8, 80.8, 80.4, 79.6, 69.9, 69.8, 69.5, 69.3, 68.1, 61.8,
53.3, 52.6, 50.4, 42.7, 38.4, 31.4, 30.9, 28.5, 28.3, 27.5, 27.0, 26.9, 26.6, 22.1, 12.7, 9.3 ppm.
119Sn NMR (CD3CN): δ ꢀ58.7 ppm. IR (KBr): 3326, 2930, 1731 cmꢀ1. HRMS (ESI+) m/z for
C63H112O20N9Sn: calcd 1434.7063, obsd 1434.7029 [M + H]+
General Procedure for Synthesis of 12a and 12b. To a solution of 11a or 11b (0.006 mmol)
in DCM (3 mL), I2 (3 mg, 0.012 mmol) was added at rt and the mixture was stirred for 30
min. The reaction was passed through a silica plug (2 g, 10 wt% KF mixed in silica) and
concentrated under vacuum to dryness. The resulting brown oily crude was redissolved in a
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mixture of TFA and CH2Cl2 (2 mL, 1:1) in a 5 ml microwave vial. The vial was capped and
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subjected to microwave irradiation for 4 min at 70 C. The resulting reaction mixture was
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preparative HPLC and lyophilized.
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12a: Yellow waxy solid, (97% yield, 5 mg, tr = 12.45 min). 1H NMR (CD3OD): δ 9.02 (d, J =
3 Hz, 1H, CH), 8.28 (dd, J = 3, 6 Hz, 1H, CH), 7.18 (d, J = 12 Hz, 1H, CH), 5.29 (s, 2H, CH2)
4.61 (s, 2H, CH2), 4.31 (m, 1H, CH), 4.26 (m, 1H, CH), 3.82 (t, J = 6 Hz, 2H, CH2), 3.65 (m,
16H, CH2), 3.24 (m, 2H, CH2), 2.42 (m, 2H, CH2), 2.15 (m, 1H, CH), 1.87 (m, 2H, CH2),
1.66 (m, 1H, CH), 1.57 (m, 1H, CH), 1.45 (m, 1H, CH), 1.32 (m, 4H, CH2) ppm. 13C NMR
(CD3OD): δ 176.4, 176.3, 175.9, 167.6, 162.3, 160.2, 149.8, 138.7, 137.3, 131.2, 124.7, 116.2,
91.1, 71.6, 71.5, 71.2, 71.1, 69.9, 62.7, 54.0, 53.6, 52.7, 44.1, 40.5, 33.2, 31.1, 29.9, 28.9,
24.0 ppm. IR (KBr): 3362, 2926, 1686 cmꢀ1. HRMS (ESI+) m/z for C31H45O16N9I: calcd
926.2029, obsd 926.2039 [M + H]+.
12b: Yellow waxy solid (93% yield, 6 mg, tr = 12.92 min). 1H NMR (CD3CN): δ 8.99 (t, J =
6 Hz, 1H, CH), 8.72 (t, J = 6Hz, 1H, NH), 8.27 (dd, J = 3, 6 Hz, 1H, CH), 7.15 (m, 2H, CH,
NH), 6.12 (m, 2H, NH), 5.11 (s, 2H, CH2) 4.59 (s, 2H, CH2), 4.27 (bs, 1H, CH), 4.19 (bs, 1H,
CH), 3.80 (t, J = 6 Hz, 2H, CH2), 3.64 (m, 34H, CH2), 3.24 (d, J = 6 Hz, 2H, CH2), 2.42 (m,
2H, CH2), 2.11 (m, 1H, CH), 1.91 (m, 1H, CH), 1.80 (m, 1H, CH), 1.67 (m, 1H, CH), 1.58 (m,
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2H, CH2), 1.44 (m, 2H, CH2) ppm. C NMR (CD3CN): δ 173.8, 173.7, 173.4, 164.6, 158.4,
148.4, 148.0, 135.5, 129.7, 123.4, 114.8, 82.9, 69.7, 69.5, 69.4, 69.1, 68.8, 68.6, 68.0, 63.5,
53.0, 52.4, 42.5, 38.5, 30.8, 29.7, 28.0, 26.7, 22.2 ppm. IR (KBr): 3327, 3058, 1693 cmꢀ1.
HRMS (ESI+) m/z for C39H60O20N9INH4: calcd 1119.3344, obsd 1119.3333 [M + NH4]+.
Synthesis of 13. Method A: (3ꢀBromopropꢀ1ꢀynꢀ1ꢀyl)tributylstannane was prepared
according to a literature method21 and treated with potassium phthalimide as described
previously24 to give 13 in 33% yield. Method B: To a 0.5 M solution of ZnBr2 in THF (0.5
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mL) containing Nꢀproparglphthalimide (185 mg, 1 mmol) was added dropwise Bu3SnOMe
(345 µL, 1.2 mmol). The mixture was stirred for 3 h at 60 oC, and then quenched by H2O (10
mL). The mixture was extracted with diethyl ether (3 × 10 mL). The collected organic layers
were dried (MgSO4), and evaporated. The resulting residue was diluted with EtOAc (30 mL)
and extracted with 10% m/v NH4F (aq) (20 mL). The solution was filtered and the filtrate
dried (MgSO4) and evaporated producing an oily white product. Purification by preparative
TLC using DCM gave compound 13. Colorless oil (49% yield, 235 mg, Rf = 0.71). The
alkyne 13 is unstable and must be kept in the refrigerator to prevent decomposition. It is
advantageous to use a freshly prepared sample for further chemical transformations. 1H NMR
(CDCl3): δ 7.89 (m, 2H, CH), 7.74 (m, 2H, CH) 4.48 (s, 2H, CH2), 1.53 (m, 6H, CH2), 1.31
(m, 6H, CH2), 0.97 (m, 6H, CH2), 0.87 (m, 9H, CH3) ppm. 13C NMR (CDCl3): δ 167.1,
134.0, 132.2, 123.4, 103.1, 86.7, 28.8, 28.2, 26.9, 13.6, 11.0 ppm. 119Sn NMR (CDCl3): δ ꢀ
64.5 ppm. IR (KBr): 3245, 2917, 2113, 1734 cmꢀ1. HRMS (ESI+) m/z for C23H33O2NSnH:
calcd 476.1616, obsd 474.1614 [M + H]+.
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Synthesis of 14. Azide 8 (300 mg, 0.53 mmol) was added to a solution of 13 (362 mg, 0.76
mmol) in toluene (5 mL) and the reaction mixture stirred at 80 oC for 16 h. After cooling to rt
the reaction mixture was concentrated and the desired product was purified twice by
preparative TLC, using ethyl acetate/hexane (2:1) as eluents and the product isolated.
Colorless oil (50% yield, 275 mg, Rf = 0.50). 1H NMR (CDCl3): δ 7.83 (m, 2H, CH), 7.72 (m,
2H, CH), 6.98 (t, J = 6 Hz, 1H, NH), 5.86 (d, J = 12 Hz, 1H, NH), 5.56 (d, J = 6 Hz, 1H, NH),
5.35 (s, 2H, CH2), 5.00 (m, 2H, CH2), 4.36 (m, H, CH), 4.31 (m, H, CH), 3.22 (m, 1H, CH),
3.01 (m, 1H, CH), 2.34 (m, 2H, CH2), 2.07 (m, H, CH), 1.84 (m, H, CH), 1.74 (m, 1H, CH),
1.48 (m, 29H, CH3, CH2), 1.31 (m, 6H, CH2), 1.24 (m, 4H, CH2), 0.86 (t, J = 6 Hz, 9H, CH3)
ppm. 13C NMR (CD3CN): δ 172.1, 171.9, 171.8, 171.6, 167.5, 166.9, 165.4, 157.0, 145.9,
139.2, 134.1, 131.6, 122.8, 80.9, 80.6, 79.7, 53.2, 52.5, 51.3, 49.8, 38.3, 38.2, 31.5, 31.4, 30.9,
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30.6, 28.6, 28.5, 28.4, 28.0, 27.5, 26.9, 22.1, 21.9, 12.7, 9.5 ppm. 119Sn NMR (CD3CN): δ ꢀ
58.8 ppm. IR (KBr): 3315, 2979, 1733 cmꢀ1; HRMS (ESI+) m/z for C49H79O10N7SnH: calcd
1046.5001, obsd 1046.5026 [M + H]+.
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Synthesis of 15. To a solution of 14 (240 mg, 0.23 mmol) in DCM (5 mL), I2 (58 mg, 0.23
mmol) was added and the mixture was stirred for 15 min at rt. The reaction mixture was
passed through a silica plug (2 g, 10 wt % KF mixed in silica), concentrated under vacuum to
dryness and the desired product was purified by silica gel column chromatography using
hexane/ethyl acetate as eluents (1:2) to provide compound 15. Colorless oil (89% yield, 180
mg, Rf = 0.38). 1H NMR (CDCl3): δ 7.85 (m, 2H, 2CH), 7.74 (m, 2H, 2CH), 5.79 (m, 1H,
NH), 5.47 (m, 3H, NH, CH2), 5.02 (m, 2H, CH2), 4.37 (m, 1H, CH), 4.30 (m, H, CH), 3.29
(m, H, CH), 3.08 (m, 1H, CH), 2.34 (m, 2H, CH2), 2.08 (m, 1H, CH), 1.89 (m, 2H, CH2),
1.75 (m, 1H, CH), 1.56 (m, 2H, CH2), 1.47 (s, 9H, CH3), 1.43 (s, 9H, CH3), 1.41 (s, 9H, CH3),
1.32 (m, 2H, CH2) ppm. 13C NMR (CDCl3): δ 174.0, 172.5, 172.3, 167.8, 165.4, 157.6, 135.9,
134.5, 131.9, 123.8, 91.6, 82.4, 81.8, 80.8, 53.6, 53.4, 53.2, 52.6, 52.0, 39.4, 32.6, 32.1, 31.9,
30.8, 28.8, 28.2, 28.0, 22.7, 22.2 ppm. IR (KBr): 3345, 2976, 2928, 1721 cmꢀ1. HRMS (ESI+)
m/z for C37H52IO10N7H: calcd 882.2894, obsd 882.2899 [M + H]+.
Synthesis of 16. Hydrazine hydrate (103 mg, 2.08 mmol) was added to a stirred solution of
phthalimide 15 (180 mg, 0.204 mmol) in THF (5 mL), and the solution was heated to reflux
for 5 h. After cooling to rt, the solvent was removed under reduced pressure. The resulting
residue was dissolved in NaOH (5 mL, 20%), and the desired product extracted with DCM (3
× 10 mL). The combined organic layers were then dried over MgSO4. The filtrate was
evaporated under reduced pressure to leave the amine 16. Colorless viscous oil (60% yield,
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92 mg, Rf = 0.23 ). H NMR (CDCl3): δ 7.64 (t, J = 6 Hz,1H, NH), 5.94 (d, J = 6 Hz, 1H,
NH), 5.61 (d, J = 6 Hz, 1H, NH), 5.25 (q, J = 18 Hz, 2H, CH2), 4.35 (m, 1H, CH), 4.27 (m,
1H, CH), 3.94 (s, 2H, CH2), 3.35 (m, 1H, CH), 3.19 (m, 1H, CH), 2.35 (m, 2H, CH2), 2.05 (m,
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5H, CH), 1.88 (m, 2H, CH2), 1.75 (m, 2H, CH2), 1.58 (m, 3H, CH), 1.47 (s, 9H, CH3), 1.45 (s,
9H, CH3), 1.43 (s, 9H, CH3), 1.35 (m, 2H) ppm. 13C NMR (CDCl3): δ 173.5, 172.5, 172.3,
165.6, 157.6, 141.0, 89.2, 82.4, 81.6, 80.6, 53.4, 53.3, 51.6, 50.6, 39.3, 35.2, 32.2, 31.7, 28.3,
28.0, 27.9, 22.4 ppm. IR (KBr) 3289, 2926, 1735, 1652 cmꢀ1. HRMS (ESI+) m/z for
C29H50IO8N7H: calcd 752.2844, obsd 752.2842 [M + H]+.
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Synthesis of 17. Compound 16 (30 mg, 3.99 mmol) was dissolved in 1:1 (v/v) TFA:DCM (3
mL) and stirred at rt for 24 h. Evaporation of volatiles gave the crude product as a TFA salt,
which was dissolved in water, lyophilized and analyzed by NMR. The free amine was
obtained by dissolving the TFA salt in MeOH (1mL) and treating it with TEA (100 µL) at rt
for 1 h. The solvent was removed under reduced pressure and the product was isolated by
semiꢀpreparative HPLC and lyophilized to provide 17. Waxy white solid (74% yield, 17 mg,
tr = 6.25 min). 1H NMR (DMSOꢀd6): δ 12.48 (bs, 3H, COOH), 8.81 (s, 1H, NH), 8.28 (bs, 3H,
NH3), 6.34 (m, 2H, NH), 5.34 (s, 2H, CH2), 4.20 (s, 2H, CH2), 4.13 (m, 2H, CH), 3.12 (m,
2H, CH2), 2.25 (m, 2H, CH2), 1.91 (m, 1H), 1.70 (m, 2H), 1.51 (m, 4H), 1.27 (m, 2H) ppm.
13C NMR (DMSOꢀd6): δ 174.5, 174.2, 173.7, 165.7, 157.4, 134.8, 95.4, 52.2, 51.7, 51.2, 31.7,
30.8, 29.8, 28.3, 28.0, 27.4, 22.6 ppm; IR (KBr): 3295, 2942, 1724, 1197 cmꢀ1. HRMS (ESIꢀ)
m/z for C17H25IO8N7: calcd 582.0809, obsd 582.0805 [M ꢀ H]ꢀ.
Synthesis of 18. 1ꢀChloroꢀ2,4ꢀdinitrobenzene (10 mg, 4.95 mmol) was added to a solution
of 16 (30 mg, 3.99 mmol) in ethanol (2 mL). The reaction mixture was heated to reflux for 8
h and concentrated under reduced pressure. The desired product was isolated by preparative
TLC using 9:1 DCM/MeOH. A yellow oil (38% yield, 14 mg, Rf = 0.62). 1H NMR (CDCl3):
δ 9.13 (s, 1H, CH), 8.93 (s, 1H, NH), 8.32 (bs, 1H, CH), 7.77 (bs, 1H, NH), 7.05 (bs, 1H,
CH), 5.84 (bs, 1H, NH), 5.47 (s, 1H, NH), 5.33 (m, 1H, CH), 5.15 (m, 1H, CH), 4.89 (m, 2H,
CH2), 4.35 (bs, 1H, CH), 4.22 (bs, 1H, CH), 3.49 (s, 2H, CH2), 3.30 (bs, 1H, CH), 3.17 (bs,
1H, CH), 2.34 (s, 2H, CH2), 2.07 (bs, 1H, CH), 2.01 (bs, 1H, CH), 1.87 (m, 1H, CH), 1.77 (m,
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2H, CH2), 1.46 (m, 29H, CH2, CH3) ppm. 13C NMR (CDCl3): δ 173.9, 172.3, 172.2, 164.9,
157.7, 147.4, 137.1, 134.9, 134.8, 131.5, 130.5, 124.0, 114.5, 90.9, 82.9, 81.9, 80.8, 53.5,
53.4, 51.8, 50.8, 39.4, 37.4, 32.4, 31.6, 29.6, 28.0, 27.9, 27.8, 27.6, 22.7 ppm. IR (KBr): 3315,
3101, 2979, 1733 cmꢀ1. HRMS (ESI+) m/z for C35H52IO12N9H: calcd 918.2858, obsd
918.2833 [M + H]+.
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Synthesis of 19. 10 mg (1.09 mmol) of 18 was dissolved in 1:1 (v/v) TFA:DCM (1 mL)
and stirred at rt for 10 h. Evaporation of the solvent gave a dark yellow waxy solid that was
dissolved in water, and lyophilized to provide 19. Yellow solid (87% yield, 7 mg, tr = 7.89).
1H NMR (DMSOꢀd6): δ 12.39 (bs, 3H, COOH), 9.12 (t, J = 4 Hz, 1H, CH), 8.87 (d, J = 4 Hz,
1H, CH), 8.37 (t, J = 12 Hz, 1H, NH), 8.25 (dd, J = 4, 6 Hz, 1H, CH), 7.18 (d, J = 6 Hz, 1H,
NH), 6.30 (dd, J = 6, 8 Hz, 2H, 2NH), 5.27 (s, 2H, CH2), 4.84 (d, J = 6, 2H, CH2), 4.08 (m,
1H, CH), 4.03 (m, 1H, CH), 3.01 (bs, 2H, CH2), 2.28 (bs, 2H, CH2), 1.91 (m, 1H, CH),1.65
(m, 2H, CH), 1.50 (m, 1H, CH), 1.38 (m, 2H, CH), 1.26 (bs, 3H, CH) ppm. 13C NMR
(DMSOꢀd6): δ 176.5, 176.2, 175.8, 160.2, 150.0, 149.5, 137.2, 131.7, 131.1, 124.7, 116.2,
83.4, 71.6, 71.4, 71.3, 70.8, 70.0, 65.1, 53.8, 53.6, 51.6, 44.1, 32.9, 31.1, 30.5, 29.0, 23.5 ppm;
IR: (neat) 3430, 3272, 2253, 1653, 1027 cmꢀ1; HRMS (ESI‒) m/z for C23H27IO12N9: calcd
748.0824, obsd 748.0788 [M ꢀ H]ꢀ.
Synthesis of 20. Hydrazine hydrate (70 mg, 1.46 mmol) was added to a stirred solution of
phthalimide 14 (148 mg, 0.143 mmol) in THF (3 mL), and the solution was heated to reflux
for 5 h. It was then cooled to rt, and the solvent removed under reduced pressure. The
resulting residue was dissolved in NaOH (3 mL, 20%), and the desired product was extracted
with DCM (3 × 5 mL). The combined organic solution was then dried over MgSO4. The
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filtrate was evaporated to produce 20. Colorless oil (54% yield, 70 mg, Rf = 0.27). H NMR
(CDCl3): δ 7.16 (t, J = 6 Hz, 1H, NH), 5.82 (d, J = 12 Hz, 1H, NH), 5.48 (d, J = 6 Hz, 1H,
NH), 5.16 (q, J = 18 Hz, 2H, CH2), 4.33 (m, 1H, CH), 4.23 (m, 1H, CH), 3.92 (s, 2H, CH2),
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3.29 (m, 1H, CH), 3.10 (m, 1H, CH), 2.31 (m, 2H, CH2), 2.04 (m, 1H, CH), 1.83 (m, 2H),
1.71 (m, 2H, CH2), 1.51 (m, 6H, CH2), 1.44 (m, 11H, CH2), 1.40 (m, 12H, CH3, CH2), 1.39
(m, 11H, CH3, CH2), 1.29 (m, 6H, CH2), 1.12 (m, 5H, CH2), 0.85 (t, J = 6 Hz, 9H, CH3) ppm.
13C NMR (CDCl3): δ 173.2, 172.4, 172.3, 167.4, 166.4, 157.7, 144.8, 144.3, 82.2, 81.5, 80.6,
53.3, 53.2, 52.4, 51.0, 39.0, 35.5, 31.8, 29.0, 28.4, 28.1, 28.0, 27.2, 22.1, 27.9, 27.5, 26.9,
22.1, 21.9, 13.7, 10.1 ppm. 119Sn NMR (CDCl3): δ ꢀ59.7 ppm. IR (KBr): 3314, 2956, 1771
cmꢀ1. HRMS (ESI+) m/z for C41H77O8N7SnH: calcd 916.4943, obsd 916.4980 [M + H]+.
Synthesis of 21. To a solution of 20 (20 mg, 2.18 mmol) in ethanol (2 mL) were added 1ꢀ
chloroꢀ2,4ꢀdinitrobenzene (8 mg, 3.96 mmol) and TEA (0.1 mL, 1.36 mmol). The reaction
mixture was heated to reflux for 12 h and concentrated under reduced pressure. Purification
of the desired product by preparative TLC using 95:5 DCM/MeOH gave compound 21.
Yellow waxy solid (52% yield, 12 mg, Rf = 0.35). 1H NMR (CDCl3): δ 9.14 (d, J = 3 Hz, 1H,
CH), 8.58 (t, J = 6 Hz, 1H, CH), 8.32 (m, 1H, CH), 7.40 (bs, 1H, NH), 7.06 (d, J = 6 Hz, 1H,
NH), 5.79 (d, J = 6 Hz, 1H, CH), 5.36 (d, J = 12 Hz, 1H, CH), 5.24 (d, J = 12 Hz, 1H, CH),
5.00 (d, J = 12 Hz, 1H, CH), 4.84 (m, 1H, CH), 4.72 (m, 1H, CH), 4.32 (m, 1H, CH), 4.26 (m,
1H, CH), 3.31 (m, 1H, CH), 3.11 (m, 1H, CH), 2.34 (m, 2H, CH2), 2.07 (m, 1H, CH), 1.87 (m,
1H, CH), 1.75 (bs, 4H, CH2), 1.42 (m, 33H, CH2, CH3), 1.27 (m, 8H, CH2), 1.11 (m, 6H,
CH2), 0.83 (t, J = 6 Hz, 9H, CH3) ppm. 13C NMR (CDCl3): δ 173.7, 172.3, 172.2, 165.6,
157.6, 147.3, 146.7, 138.8, 137.0, 131.3, 130.6, 124.1, 114.0, 82.5, 81.8, 80.7, 53.5, 53.0,
51.2, 39.2, 38.0, 32.2, 31.8, 29.0, 28.1, 28.0, 27.8, 27.7, 27.2, 22.1, 13.6, 10.0 ppm. 119Sn
NMR (CDCl3): δ ꢀ58.6 ppm. IR (KBr): 3325, 2931, 1732 cmꢀ1. HRMS (ESI+) m/z for
C47H79N9O12SnH: calcd 1082.4960, obsd 1082.4934 [M + H]+.
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For all radiolabeled compounds, purity was determined using radioHPLC in comparison to
the authentic nonꢀradioactive standard detected by UVꢀHPLC. Purity was >95% for all
compounds tested.
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Preparation of 125I-9b.25 To anhydrous copper(II) chloride (13.4 mg, 0.1 mmol) was added
anhydrous acetonitrile (4 mL), followed by anhydrous TEA (15.1 mg, 0.15 mmol), and the
mixture sonicated until a clear burgundy solution was obtained. To the alkyne 4b (1.0 µmol)
was added to the solution of the CuCl2/TEA complex in acetonitrile (40 µL). After 5 min, the
solution was added to Na125I (4 MBq, 6.0 µL) in an eppendorf tube (1.5 mL), to which the
azide 8 (1.0 µmol) in anhydrous acetonitrile (20 µL) was added. The tube was capped and the
reaction was heated at 60 °C for 90 min. The solvent was evaporated using a stream of air
and 500 µL of ACN and 2 mL of TFA were added, followed by stirring for 1 h at rt. The
reaction mixture was concentrated, diluted with acetonitrile and water (1:1 v/v, 1 mL) and the
compound purified by repeated preparative HPLC until there was no desꢀiodo side product
evident. The labeled compound was identified by coꢀinjection with the nonꢀradioactive
reference standard. 125I-9b: radiochemical yield 70% ± 12 (n = 3), and > 97% radiochemical
purity, as measured by analytical HPLC (see Figure 4).
Preparation of 125I-19. A 1 mg/mL solution of Iodogen® in CHCl3 was prepared shortly
before use and 25 µL of the solution added to an eppendorf tube. The solvent was removed
using a rotary evaporator leaving a film of Iodogen®. To this tube, 100 µL of a 1 mg/mL
solution of 21 in 5% AcOH in acetonitrile was added, followed by 92.5 MBq solution of
Na125I in sodium hydroxide (10 µL, pH 8ꢀ11). The reaction was allowed to stand for 15 min,
whereupon 0.1 M Na2S2O5 was added (20 µL). The solvent was evaporated and 500 µL of
ACN and 2 mL of TFA were added, followed by stirring for 1 h at rt. The reaction mixture
was concentrated, diluted with acetonitrile/water (1:1 v/v, 1 mL) and purified by HPLC. The
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