Synthesis and evaluation of radioiodinated acyloxymethyl ketones as activity-based probes for cathepsin B

Article abstract of DOI:10.1021/jm501357r

Dipeptidyl (acyloxy)methyl ketones (AOMKs) were functionalized with different iodine-containing prosthetic groups to generate a library of candidate cathepsin B probes. Compound 23a, (S)-20-[(S)-2-{[(benzyloxy)carbonyl]amino}-3-phenylpropanamido]-1-(4-iod

Full text of DOI:10.1021/jm501357r

Article
pubs.acs.org/jmc
Synthesis and Evaluation of Radioiodinated Acyloxymethyl Ketones
as Activity-Based Probes for Cathepsin B
Patricia E. Edem,† Shannon Czorny,† and John F. Valliant*,†,‡
†Department of Chemistry & Chemical Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada
‡Centre for Probe Development and Commercialization, 1280 Main Street West, Hamilton, Ontario L8S 4K1, Canada
S
* Supporting Information
ABSTRACT: Dipeptidyl (acyloxy)methyl ketones (AOMKs)
were functionalized with different iodine-containing prosthetic
groups to generate a library of candidate cathepsin B probes.
Compound 23a, (S)-20-[(S)-2-{[(benzyloxy)carbonyl]-
amino}-3-phenylpropanamido]-1-(4-iodophenyl)-1,14,21-tri-
oxo-5,8,11-trioxa-2,15-diazadocosan-22-yl 2,4,6-trimethylben-
zoate, was identified as a potential lead through in vitro
screening, having a Ki = 181 9 nM and demonstrating the ability to effectively label active cathepsin B in vitro. Its less potent
analogue 11a, (S)-3-[(S)-2-{[(benzyloxy)carbonyl]amino}-3-phenylpropanamido]-7-[6-(4-iodobenzamido)hexanamido]-2-oxo-
heptyl 2,4,6-trimethylbenzoate, was also tested as a comparison. Biodistribution studies of the iodine-125-labeled compounds in
MDA-MB-231 mouse xenografts exhibited tumor uptake of 0.58% 0.06% injected dose per gram (ID/g) for [125I]11a and
1.12% 0.08% ID/g for [125I]23a at 30 min. The tumor-to-blood ratios reached 1.2 for [125I]23a and 1.6 for [125I]11a after 23 h.
The more hydrophilic [125I]23a showed an improved clearance profile with a superior tumor-to-muscle ratio of 7.0 compared to
3.4 for [125I]11a at 23 h. Iodinated AOMK ligands are suitable in vitro probes for cathepsin B and hold promise as a platform to
develop molecular imaging probes.
the enzyme by reacting with the active-site cysteine, resulting in
a thioether linkage between the ABP and cathepsin B.1,27,30,31
Phe-Lys-AOMKs have been fluorescently labeled at the lysine
ε-amine and have shown specific tumor uptake in vivo.14,15
Radiolabeled variants have also been developed where 64Cu and
the macrocyclic ligand 1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetraacetic acid (DOTA) was also incorporated at the P1
site.14,32 This construct exhibited a tumor uptake of 0.35%
0.13% injected dose per gram (ID/g) at 30 min postinjection
(pi) and the activity remained in the tumor at 24 h pi (0.27%
0.05% ID/g). High liver and kidney uptake was also observed,
which is likely associated with loss of copper in vivo.33
The present study describes the preparation and in vivo
evaluation of novel dipeptidyl AOMK cathepsin B inhibitors
bearing different iodine-containing prosthetic groups linked to
the lysine ε-amine of a Phe-Lys-AOMK derivative. One of the
advantages of working with iodine is that a single construct can
be used to prepare agents for single photon emission
tomography (SPECT), positron emission tomography (PET),
or radiotherapy, simply by varying the isotope used (e.g., 123I,
124I, or 131I). This is exemplified by the use of aryl iodides to
synthesize diagnostic and therapeutic radiopharmaceuticals for
melanoma, glioma, and prostate cancer.34−38 Radioiodinated
AOMKs have been used as ABPs to label diverse families of
cysteine proteases in vitro but not cathepsin B, and they
employed prosthetic groups that are not stable in vivo.29 To
INTRODUCTION
■
Cathepsin B is a ubiquitously expressed lysosomal cysteine
protease that is involved in normal physiological processes such
as protein turnover, wound healing, and cell differentiation and
growth.1 The enzyme is overexpressed in a variety of cancers
including breast carcinoma, prostate carcinoma, glioblastoma,
and melanoma,2−5 with higher levels of active cathepsin B
residing in tumors having high metastatic potential.6−8 In
normal cells cathepsin B is localized in the lysosomes, yet in
tumors it distributes throughout the cytoplasm and cell
periphery and is secreted into the extracellular matrix as
well.9−11 These features make cathepsin B an attractive target
for developing molecular imaging probes for detecting cancer
and assessing metastatic potential.
A number of near-infrared fluorescent (NIRF) probes for
cathepsin B have been developed.12−15 Substrate-based probes
such as Cat B 750 FAST and Cat B 680 FAST are commercially
available and have been used in a number of cancer
models.13,16−19 Most recently, substrate-based probes incorpo-
rating a self-immolative spacer linked to a fluorophore have
been developed and used for in vitro and in vivo imaging of
cathepsin B.12,20,21 Irreversible inhibitors have also been
employed to develop activity-based probes (ABPs) for
cathepsin B imaging.14,15,22−25
ABPs for cathepsin B have been prepared from a number of
different constructs including dipeptidyl (acyloxy)methyl
ketones (AOMKs). The AOMK derivatives are attractive
because of their specificity and high affinity for cysteine
proteases in vitro and in vivo.26−29 These inhibitors inactivate
Received: August 20, 2014
© XXXX American Chemical Society
A
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Figure 1. Structures of AOMK cathepsin B inhibitors. (a) Structures of parent AOMKs (6a/b) and initial iodinated AOMKs: iodobenzamides (7a/
b) and iodophenylureas (8a/b). (b) Structures of AOMKs with alkyl spacer: iodobenzamides (11a/b), iodophenylureas (15a/b), and iodotriazole
(21). (c) Structures of AOMKs with PEG spacer: iodobenzamides (23a/b) and iodotriazole (29) and the known optical probe GB123 (30).
evaluate the potential utility of iodinated AOMKs as molecular
imaging probes, a library of candidates derived from robust
iodine synthons were prepared and screened as inhibitors of
cathepsin B. Two compounds were subsequently radiolabeled
and tested in vitro and in vivo.
coupled to 3- or 4-iodobenzoic acid by use of IBCF in the
presence of N-methylmorpholine (NMM) (Scheme 1). The
Scheme 1. Synthesis of 7a/b
RESULTS AND DISCUSSION
■
Synthesis of Parent Inhibitors 6a and 6b. The parent
AOMK inhibitors 6a and 6b (Figure 1a) were prepared from
the protected dipeptide Cbz-Phe-Lys(ε-Boc)-OH (3). Follow-
ing modified published procedures, isobutyl chloroformate
(IBCF) (Scheme S1, route A) or benzotriazole (Scheme S1,
route B; see Supporting Information) was used to couple the
protected phenylalanine and lysine amino acids, giving 3.39−41
The dipeptide 3 was activated with IBCF and treated with
ethereal diazomethane to generate the diazomethyl ketone in
situ. Treatment with HBr gave the bromomethyl ketone 4,
which was then converted to 5a or 5b, following the method of
Blum et al.14 The amount of potassium fluoride used in the
final step was reduced to help minimize epimerization of the
lysine residue, which was evident by both HPLC and 1H NMR
in most compounds.27,4042 The tert-butoxycarbonyl (Boc)
group was subsequently removed by use of trifluoroacetic
acid (TFA), giving 6a or 6b as TFA salts.
desired products 7a and 7b were isolated following simple
extraction in yields of 29% and 45%, respectively. To generate
the ureas, 6a was combined with 3- or 4- iodophenyl isocyanate
in the presence of triethylamine, giving 8a and 8b in 9% and
14% yields, respectively, following silica gel chromatography
(Scheme 2).
Scheme 2. Synthesis of 8a/b
Synthesis of Initial Iodinated AOMKs. The first series of
iodinated inhibitors prepared involved linking aryl iodides to
the free amine of 6a, giving iodobenzamides (7a/b) and
iodophenylureas (8a/b) (Figure 1a). Compound 6a was
B
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It has been reported that inhibitors containing bulky amino
acids at the P1 position exhibit reduced affinity for cathepsin
B.43 To minimize possible steric interactions, a six-carbon
spacer was also used to incorporate the aryl iodides (Figure
1b). 6-Aminohexanoic acid was combined with the active esters
9a and 9b, giving the aryl iodide spacers 10a and 10b in 82%
and 87% yields, respectively (Scheme 3). These were linked to
Scheme 5. Synthesis of 21
Scheme 3. Synthesis of 11a/b
various amounts of enzyme (Figure S1A, Supporting
Information). The rate of hydrolysis (ν0) was linear when the
enzyme concentration [E] was between 5 and 15 nM (R2 >
0.99) (Figure S1B, Supporting Information). The reaction rate
(ν0) was also monitored when varying the substrate
concentration in order to determine the Michaelis−Menten
constant (Km) (Figure S2, Supporting Information). A value of
390 20 μM was found, which is lower than the previously
reported value of 900 60 μM.46 The variance is likely due to
differences in the amount of dithiothreitol (DTT) used to
activate the enzyme and the incubation time for this activation,
as well as the enzyme source, which can influence the observed
Km for this substrate.46,47
Inhibition constants (Ki) were determined by monitoring the
substrate hydrolysis over time in the presence of 0−1 μM
inhibitor (Figure S3A, Supporting Information). The observed
rate constant (kobs) was determined and plotted against the
inhibitor concentration (Figure S3B, Supporting Information).
By use of nonlinear regression, Ki values were determined
where possible. In cases where full inhibition was not observed,
only the second-order rate constant (ki/Ki) was determined
(Table 1).
6a by use of benzotriazol-1-yloxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP) in the presence of triethylamine
to generate compounds 11a and 11b in 45% and 30% yields,
respectively. For the urea derivatives, it was necessary to use the
protected methyl 6-aminohexanoate 12 and combine it with 3-
or 4-iodophenyl isocyanate (Scheme 4). Following depro-
tection, 14a and 14b were coupled with 6a by use of IBCF in
the presence of NMM to give 15a and 15b in 35% and 30%
yields, respectively.
Scheme 4. Synthesis of 15a/b
The known inhibitor 6a was evaluated as reference point to
compare all iodinated derivatives. The measured Ki value was
Table 1. Inhibitory Activities of Iodinated AOMK
a
Derivatives
compd
Ki (nM)
80 10
52
ki/Ki (s−1·M−1
)
We have shown that iodinated triazoles are more polar than
iodobenzene-based derivatives, have minimal nonspecific bind-
ing in vivo, and can clear readily via renal excretion.44 To
prepare a triazole-derived synthon to derivatize 6a, ethynyl
tributyltin was used in a 1,3-dipolar cycloaddition with 17,
giving the triazole 18 (Scheme 5). 1H and 13C NMR
spectroscopy confirmed that a single isomer was obtained,
which is consistent with the literature.45 Iododestannylation
was achieved by treating 18 with molecular iodine to give 19 in
69% yield.45 Following hydrolysis, 20 was coupled to 6a by use
of PyBOP in the presence of triethylamine to generate 21 in
33% yield.
Inhibition of Cathepsin B by Iodinated AOMKs. The
binding affinity of the iodinated AOMKs was determined by a
kinetic assay similar to that reported by Krantz et al.,27
employing Cbz-Arg-Arg-p-nitroanilide (Z-RR-pNA) as the
substrate and human liver cathepsin B (Calbiochem). Optimal
enzyme concentration range and linearity was determined by
monitoring substrate hydrolysis over time in the presence of
6a
40 000 3000
40 000 6000
810 80
6b
6
7a
2000 1000
b
7b
nd
630 10
8a
nd
nd
nd
nd
0.013 0.002
8b
0.0035 0.0002
310 20
11a
11b
15a
15b
21
280 20
c
ni
ni
ni
ni
1000 50
4430 70
21 100 300
12 700 600
9900 200
19 000 2000
23a
23b
29
181
9
370 25
350 40
120 20
30
a
Inhibition constants were determined at 37 °C and pH 6.0 with
human liver cathepsin B. Full inhibition was not observed; therefore,
the value was not determined. No inhibition observed.
b
c
C
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80 10 nM, which differed from the reported value (Ki = 170
50 nM), again likely due to difference in enzyme sources.27
The simple aryl iodide derivatives (7a/b and 8a/b) showed
poor affinity for the protease, where a Ki value could be
determined only for 7a (Ki = 2 1 μM) and the ureas (8a/b)
exhibited no detectable inhibition. Incorporation of the alkyl
spacer did not show an improvement for the iodophenylureas
(15a/b) or the iodobenzamides (11a/b). These compounds
formed heterogeneous solutions when added to the assay
buffer, indicating poor solubility, which in turn made it difficult
to obtain accurate measures of the binding constants. The
iodotriazole derivative 21 had improved solubility and showed
improved binding affinity (Ki = 1 0.05 μM) but was over 10
times less potent than the parent compound and was therefore
regarded as a poor candidate.
Synthesis of Iodinated AOMKs Containing a Hydro-
philic Linker. A more hydrophilic linker was used in an
attempt to improve the solubility of the inhibitors and enhance
their binding affinities. This was achieved by replacing the six-
carbon spacer with a three-unit poly(ethylene glycol) (PEG)
linker (Figure 1c). By use of similar methods, 9a and 9b were
coupled to the tri(ethylene glycol) bifunctional linker to give
the iodobenzamide-PEG derivatives 22a and 22b (Scheme 6).
Compound 6a was coupled to the new linkers by use of PyBOP
in the presence of triethylamine, giving 23a and 23b in 22%
and 37% yields, respectively.
linker 28 was then coupled to 6a by use of PyBOP in the
presence of triethylamine, giving 29 in 37% yield.
Inhibition of Cathepsin B by Iodinated-PEG-AOMKs.
Inhibitors containing the PEG spacer (23a, 23b, and 29)
showed improved binding affinities over their alkyl counterparts
(11a, 11b, and 21) with nanomolar Ki values (Table 1). The p-
iodobenzamide 23a exhibited the best affinity with a Ki of 181
9 nM (Table 1). As an additional comparison, a known Cy5-
labeled AOMK, GB123 (30, Figure 1c), was synthesized and
tested. Reports of GB123 have shown good visualization of
cathepsin B-positive tumors through optical imaging.14,48,49
A
Ki value of 120 20 nM was determined for 30, suggesting that
the Ki value for 23a was sufficient to proceed to radiolabeling
and biodistribution studies.
Synthesis of Radioiodinated AOMKs. To compare the
effect of the hydrophilic spacer in vitro and in vivo, 11a was
selected alongside 23a for radiolabeling and further testing. We
have previously demonstrated that fluorous tin benzamides and
phenylureas can be used to radioiodinate small molecules, and
the products can be isolated without HPLC via a simple
fluorous solid-phase extraction (FSPE).50,51 To convert the
selected cathepsin B inhibitors to their radioactive analogues,
the iodobenzamide precursors 10a and 22a were converted to
the corresponding arylstannanes by use of fluorous distannane
and a Pd-catalyzed cross-coupling reaction (Schemes S3 and
S4, Supporting Information).52 Addition of the fluorous tin
moiety was performed on the prosthetic group as opposed to
the iodinated inhibitors to avoid degradation of the peptide
backbone due to the harsh reaction conditions. The fluorous tin
compounds 31 and 33 were coupled to 6a by use of PyBOP in
the presence of triethylamine, giving the precursors for
radiolabeling 32 and 34 in 14% and 12% yields, respectively.
Radiolabeling was performed by treating 32 and 34 with
Scheme 6. Synthesis of 23a/b
[
125I]NaI in the presence of iodogen (Scheme 8). Following
Scheme 8. Synthesis of [125I]11a and [125I]23a
To prepare the bifunctional triazole linker, triethylene glycol
was combined with tert-butyl acrylate, generating the protected
carboxylic acid 24 (Scheme 7). Compound 24 was converted to
the mesylate and combined with sodium azide to generate 25.
Ethynyl tributyltin was used in a 1,3-dipolar cycloaddition with
25, producing the triazole 26 as a single isomer. Treatment of
26 with iodine gave the iodinated triazole 27, which was
deprotected to generate 28. The resultant iodotriazole PEG
FSPE purification, isolated radiochemical yields were 32−36%
(n = 3) for [125I]11a and 26−35% (n = 3) for [125I]23a. The
modest radiochemical yields can be attributed to nonspecific
binding of inhibitors to the reaction vessel and to the FSPE
cartridge. The identities of the radiolabeled compounds were
confirmed by coinjection with nonradioactive standards and
correlation of retention times of peaks in the radiochromato-
grams and UV−visible chromatograms (Figures 2 and 3).
Because of the long reaction times needed to couple the
arylstannane spacers to 6a, precursors 32 and 34 underwent
some degree of epimerization. Consequently [125I]11a and
Scheme 7. Synthesis of 29
[
125I]23a also contained the associated epimers (∼7% and 20%,
respectively; Figures 2 and 3), similar to what has been
observed for other AOMK derivatives.28
Activity-Based Labeling of Cathepsin B. The affinity of
the radiolabeled inhibitors for cathepsin B was evaluated by
sodium dodecyl sulfate−polyacrylamide gel electrophoresis
(SDS−PAGE). Equivalent amounts of cathepsin B were
D
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gram (ID/g) in various tissues, organs, and fluids was
determined (Table 2).
After 30 min pi, bladder/urine (90%
20% ID/g), gall
bladder (170%
30% ID/g), and liver (16% 1% ID/g)
showed the highest uptake for [125I]11a, which was reduced
after 23 h pi (0.9% 0.2%, 0.4% 0.1%, and 1.6% 0.2% ID/
g respectively). Areas with the highest uptake at 23 h pi were
thyroid/trachea (17%
9% ID/g), small intestine (1.6%
0.2% ID/g), and liver (1.6% 0.2% ID/g). The accumulation
in thyroid/trachea is indicative of deiodination, as the sample
did not contain any free iodide at the time of administration.55
The initial tumor uptake was 0.58% 0.06% ID/g at 30 min pi
and decreased over time. Despite this, the tumor/blood ratios
increased from 0.5, reaching a maximum of 1.6 after 23 h pi
(Figure 5a). Similarly the tumor/muscle ratios increased from
2.3, reaching a peak value of 4 at 5 h pi.
Figure 2. HPLC chromatograms of [125I]11a coinjected with the
reference standard 11a. Radiochromatogram (upper trace) and UV−
visible (λ = 254 nm) chromatogram (lower trace) are shown.
In the case of the more potent and hydrophilic derivative
[
125I]23a, bladder/urine (400% 200% ID/g) and gall bladder
(42% 7% ID/g) showed the highest uptake after 30 min pi.
The liver uptake was lower in comparison to [125I]11a (3.4%
0.3% vs 16% 1% ID/g, 30 min pi), and the radioactivity was
cleared from the majority of the tissues after 23 h pi. Again,
accumulation in the thyroid/trachea was observed, indicating in
vivo deiodination. The observed tumor uptake was higher for
Figure 3. Radiochromatogram (upper trace) and UV−visible (λ = 254
nm) HPLC chromatogram (lower trace) of [125I]23a coinjected with
the reference standard 23a.
[
125I]23a (1.12% 0.08% ID/g) than that of [125I]11a at 30
pretreated with saturating amounts of the known cathepsin B-
specific inhibitor L-3-trans-(propylcarbamyl)oxirane-2-carbonyl-
L-isoleucyl-L-proline (CA-074)53 or with the vehicle (dimethyl
sulfoxide, DMSO). The samples were then treated with [125I]
11a or [125I]23 and subjected to autoradiography. Despite
different binding affinities, cathepsin B labeling was observed
with both [125I]11a and [125I]23 (Figure 4). The activity in
min, and the tumor/blood ratios showed an increase from 0.5
to 1.2 after 23 h pi (Figure 5b). In addition the tumor/muscle
ratios increased from 2.1 to 7.3 for 23a, reaching a maximum at
23 h.
Compound [125I]23a showed lower accumulation in non-
target tissues in comparison with the previously reported 64Cu
AOMK deriviative.32 When tested in MDA-MB-435 xenografts,
it exhibited <0.5% ID/g tumor uptake, a tumor-to-blood ratio
of 0.61, and a tumor-to-muscle ratio of 3.03 at 2 h pi.
Compound [125I]23a exhibited similar tumor uptake at 5 h pi
(0.27% ID/g) in the MDA-MB-231 xenografts, which have
comparable levels of cathepsin B activity as the MDA-MB-435
model,14 while the tumor-to-blood (0.98) and tumor-to-muscle
(5.31) ratios were higher for [125I]23a.
CONCLUSION
■
This work explored the utility of the AOMK construct as a
targeting vector to develop radioiodinated ABPs for cathepsin
B. Attachment of bulky aromatic groups to the lysine side chain
with and without an aliphatic spacer had a detrimental impact
on cathepsin B binding when compared to the parent ligand.
However, inhibitors containing a PEG spacer showed high
affinity for the enzyme. Despite different binding affinities and
lipophilicities, [125I]11a and [125I]23a were able to label
cathepsin B in vitro, which was blocked in the presence of a
known cathepsin B inhibitor. Evaluation of [125I]23a in a
human MDA-MB-231 breast cancer model showed improved
tumor-to-nontumor ratios in comparison to other P1 radio-
labeled AOMKs but modest total tumor uptake.
Figure 4. Phosphor images of an SDS−polyacrylamide gel showing
the specific labeling of active cathepsin B. Solutions of cathepsin B
were pretreated with CA-074 (+) or DMSO (−), followed by (a)
[
125I]11a or (b) [125I]23a.
each band was blocked quantitatively by the addition of CA-
074, indicating site-specific labeling of cathepsin B. It has be
established that Phe-Lys-AOMKs can inactivate other cysteine
cathepsins such as cathepsin L and cathepsin S.14,22,23,32 It is
therefore expected that the iodine derivatives described here
would show similar inhibition and selectivity.
Biodistributions of Activity-Based Probes in Tumor-
Bearing Mice. The labeled probes, [125I]11a and [125I]23a,
were administered intravenously into CD1 nu/nu mice bearing
the MDA-MB-231 xenograft. MDA-MB-231 cells are known to
express cathepsin B, and the xenografts have been used to
evaluate other AOMK-based ABPs.14,54 After 0.5, 5, and 23 h
pi, animals were sacrificed and the percent injected dose per
EXPERIMENTAL SECTION
■
Materials and Instrumentation. All chemicals, unless otherwise
stated, were purchased from Sigma−Aldrich, Novabiochem, or
Bachem and used without further purification. Cy5 succinimidyl
ester was obtained from GE Healthcare. Tris(1H,1H,2H,2H-
perfluorooctyl)tin hydride was obtained from Fluorous Technologies
Inc. Diazomethane was prepared by use of the Diazald glassware set
with system 45 connections (Aldrich) following the procedures
E
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a
Table 2. Biodistribution of [125I]11a and [125I]23a in MDA-MB-231 Tumor-Bearing Mice
b
c
[
125I]11a (% ID/g)
[
125I]23a (% ID/g)
organ
30 min
5 h
23 h
30 min
5 h
23 h
blood
heart
lungs
liver
1.14 0.09
0.68 0.06
2.2 0.2
0.43 0.05
0.19 0.02
0.84 0.09
4.5 0.4
0.030 0.003
0.030 0.004
0.21 0.02
1.6 0.2
2.3 0.2
0.9 0.2
3.5 0.5
3.4 0.3
0.28 0.03
0.11 0.01
0.59 0.08
0.71 0.07
0.04 0.00
0.02 0.00
1
1
16
1
0.29 0.04
0.17 0.09
0.13 0.01
0.4 0.1
gall bladder
spleen
170 30
60 20
0.4 0.1
42
7
5
1
8
2
1.3 0.2
0.6 0.1
1.5 0.2
0.37 0.04
0.55 0.06
2.2 0.2
kidneys
2.9 0.3
3.8 0.7
0.80 0.05
4.2 0.6
0.20 0.02
0.15 0.06
1.6 0.2
6
1
3
stomach
11
0.09 0.01
0.07 0.02
0.12 0.02
0.05 0.01
20 10
small intestine
large intestine
tumor
12
1
10
21
1
2
5.9 0.2
0.7 0.1
0.7 0.2
0.30 0.02
0.58 0.06
3.2 0.8
0.63 0.07
0.040 0.002
4.9 0.1
0.5 0.1
19
1.12 0.08
0.27 0.07
thyroid/trachea
adipose
5
17
9
11
3
21
9
0.22 0.05
0.52 0.07
0.25 0.03
0.09 0.01
90 20
0.100 0.004
0.15 0.01
0.11 0.02
0.04 0.01
0.030 0.005
0.030 0.004
0.010 0.001
0.003 0.001
1.1 0.9
0.55 0.07
0.5 0.2
0.03 0.01
0.09 0.01
0.05 0.00
0.01 0.00
0.01 0.00
0.01 0.00
0.01 0.00
0.00 0.00
0.7 0.2
bone
skeletal muscle
brain
0.19 0.02
400 200
bladder and urine
38
9
0.9 0.2
31
3
a
b
c
Values are the mean % ID/g standard error from the mean. n = 5. n = 3.
spectrometry (HRMS), the chemical purity of all compounds screened
for cathepsin B inhibition was >95%.
Microwave reactions were performed by use of a Biotage Initiator
microwave synthesizer. Automated flash chromatography was
performed on a Biotage SP1 flash purification system. Analytical
thin-layer chromatography (TLC) was performed on silica gel plates
with fluorescent indicator UV254 (Macherey-Nagel) and visualized by
1
use of UV light and ninhydrin or vanillin in EtOH. H, 13C, and two-
dimensional NMR spectra were recorded on either a Bruker AV500 or
AV600 spectrometer. 1H NMR signals are reported in parts per
million (ppm) measured relative to the residual proton signal of the
deuterated solvent. Coupling constants (J) are reported in Hertz (Hz).
13C signals are reported in ppm relative to the carbon signal from the
solvent. HRMS was performed on a Waters/Micromass Q-Tof Ultima
Global spectrometer. Analytical high-performance liquid chromatog-
raphy (HPLC) was performed by use of an Agilent/Varian Pro Star
model 330 photodiode array (PDA) detector, model 230 solvent
delivery system, and Phenomenex Gemini (L × i.d. = 100 × 4.6 mm)
column (5 μm C18). Semipreparative HPLC was performed by use of
an Agilent/Varian Pro Star model 325 PDA detector, model 24 solvent
delivery system, and Phenomenex Gemini (L × i.d. = 250 × 10 mm)
column (5 μm C18). The elution conditions were as follows: (Method
A) Solvent A = CH3CN with 0.1% trifluoroacetic acid (TFA), solvent
B = H2O with 0.1% TFA. Gradient: 30% A to 100% A, 0−12 min;
100% A, 12−24 min; 100% A to 90% A, 24−28 min; 90% A to 30% A,
28−30 min. (Method B) Solvent A = CH3CN, solvent B = H2O.
Gradient: 30% A to 100% A, 0−12 min; 100% A, 12−24 min; 100% A
to 90% A, 24−28 min; 90% A to 30% A, 28−30 min. (Method C)
Solvent A = CH3CN, solvent B = H2O. Gradient: 10% A, 0−2 min;
10% A to 90% A, 2−20 min; 90% A, 20−22 min; 90% A to 10% A,
22−23 min; 10% A, 23−25 min. The flow rate was set at 1 mL/min
for analytical methods and 4 mL/min for semipreparative methods;
monitoring occurred at 254 nm. Autoradiography was measured on a
GE Storm 840 phosphor imager, and absorbance readings were
performed on a Bio-Rad EL 808 plate reader. Radioactivity
measurements for biodistribution and log P studies were made on a
Wizard 1470 automated γ counter (PerkinElmer, Woodbridge, ON,
Canada).
Figure 5. Average tumor/blood and tumor/muscle ratios for (a) [125I]
11a (n = 5) and (b) [125I]23a (n = 3).
provided by the supplier. As this is a toxic and explosive substance, the
appropriate precautions should be exercised. 1,3,5,7-Tetramethyl-6-
(2,4-dimethoxyphenyl)-2,4,8-trioxa-6-phosphaadamantane was pro-
vided by the Capretta research group (McMaster University).
[
125I]NaI (specific activity 629 GBq/mg) was provided by the
McMaster nuclear reactor. As this is a radioactive isotope, appropriate
facilities, licenses, and procedures should be in place prior to use.
For screening studies, human liver cathepsin B and Cbz-Arg-Arg-
pNA were purchased from Calbiochem and Enzo Life Sciences,
respectively. Reagents used in the assay buffer were from Sigma−
Aldrich. Inhibitors were dissolved in biological-grade DMSO and
diluted in the assay buffer. Black, clear-bottom 96-well plates were
obtained from BD Biosciences. L-3-trans-(propylcarbamyl)oxirane-2-
carbonyl-L-isoleucyl-L-proline (CA-074) was obtained from EMD
Biosciences, precast gels were from Bio-Rad, and the gel drying kit was
from Promega. 1,3,4,6-Tetrachloro-3a,6a-diphenylglycoluril (iodogen)
was obtained from Thermo Scientific. According to HPLC analysis,
supported by 1H and 13C NMR spectroscopy and high-resolution mass
Synthetic Procedures for New Compounds. (S)-3-[(S)-2-
{[(Benzyloxy)carbonyl]amino}-3-phenylpropanamido]-7-(3-iodo-
benzamido)-2-oxoheptyl 2,4,6-Trimethylbenzoate (7a). To a
solution of 4-iodobenzoic acid (72 mg, 290 μmol) in N,N-
dimethylformamide (DMF) (12 mL) at −16 °C, NMM (32 μL, 290
μmol) and IBCF (38 μL, 290 μmol) were added and left to stir until
F
dx.doi.org/10.1021/jm501357r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
dissolved. To this, 6a (204 mg, 290 μmol) was added, and the solution
was left to stir overnight while warming to room temperature. The
reaction mixture was diluted to 25 mL with water and extracted with
EtOAc (3 × 50 mL), and the combined organic layers were extracted
with water (5 × 20 mL), saturated Na2CO3 (3 × 20 mL), water (20
mL), 5% (w/v) citric acid (3 × 20 mL), water (20 mL), and brine (3 ×
20 mL) and then dried over MgSO4. The solvent was removed by
rotary evaporation and the product was isolated as a white powder.
Yield 68 mg, 29%; mp 163−165 °C; TLC (2:1 EtOAc/Hex) Rf = 0.53;
HPLC (method B) tR = 18.1 min. 1H NMR [600.13 MHz,
(CD3 )2 SO] 8.51−8.48 (m, 2H, NHCHCONH and
CH2CH2CH2NH), 7.79 (d, J = 8.2 Hz, 2H, 2,6-C6H2H2I), 7.63−
7.60 (m, 1H, NHCHCOCH2), 7.61 (d, J = 8.5 Hz, 2H, 3,5-C6H2H2I),
7.33−7.18 (m, 10H, Ph), 6.91 (s, 2H, Me3C6H2CO), 4.95 (s, 2H,
PhCH2OCO), 4.91 (d, J = 17.4 Hz, 1H, COCHHO), 4.82 (d, J = 17.2
nHz, 1H, COCHHO), 4.38−4.31 (m, 2H, HNCHCO Phe and Lys),
3.25−3.22 (m, 2H, CH2CH2CH2NH), 3.01 (dd, J = 13.4, 5.0 Hz, 1H,
CHCHHPh), 2.80 (dd, J = 13.1, 10.4 Hz, 1H, CHCHHPh), 2.26 [s,
6H, 2,6-(CH3)3C6H2CO], 2.25 [s, 3H, 4-(CH3)3C6H2CO], 1.84−1.82
(m, 1H, CHCHHCH2CH2), 1.62−1.56 (m, 1H, CHCHHCH2CH2),
1.55−1.47 (m, 2H, CH2CH2CH2NH), 1.40−1.30 (m, 2H,
CH2CH2CH2NH). 13C NMR [150.92 MHz, (CD3)2SO] δ 202.7,
172.0, 168.2, 165.4, 155.8, 139.1, 137.8, 137.0, 136.9, 134.9, 134.1,
129.9, 129.2, 129.1, 128.3, 128.2, 128.1, 127.7, 127.5, 126.3, 98.5, 66.6,
65.3, 56.0, 55.9, 54.9, 37.3, 29.2, 28.6, 22.4, 20.7, 19.3. HRMS calcd for
C41H45N3O7I [M + H]+, 818.2302; found, 818.2280.
8.3 Hz, 1H, CONHCHCO), 7.51−7.48 (m, 2H, CONHC6H2H2I),
7.33−7.19 (m, 12H, ArH), 6.91 (s, 2H, Me3C6H2CO), 6.20 (t, J = 5.4
Hz, 1H, CH2CH2CH2NH), 4.96 (s, 2H, PhCH2OCO), 4.91 (d, J =
17.2 Hz, 1H, COCH2O), 4.81 (d, J = 17.2 Hz, 1H, COCHHO), 4.38−
4.31 (m, 2H, HNCHCO Phe and Lys), 3.07−3.01 (m, 3H,
CH2CH2CH2NH, CHCHHPh), 2.81 (dd, J = 13.7, 9.8 Hz, 1H,
CHCHHPh), 2.27 [s, 6H, 2,6-(CH3)3C6H2CO], 2.25 [s, 3H, 4-
(CH3)3C6H2CO], 1.85−1.78 (m, 1H, CHCHHCH2CH2), 1.62−1.54
(m, 1H, CHCHHCH2CH2), 1.46−1.39 (m, 2H, CH2CH2CH2NH),
1.39−1.33 (m, 1H, CHHCH2CH2NH), 1.32−1.25 (m, 1H,
CHHCH2CH2NH). 13C NMR [150.92 MHz; (CD3)2SO] δ 202.7,
172.0, 168.2, 155.9, 154.9, 140.5, 139.1, 137.8, 137.1, 134.9, 129.9,
129.2, 128.3, 128.3, 128.2, 128.1, 127.7, 127.5, 126.3, 119.9, 83.3, 66.6,
65.3, 56.1, 55.9, 38.9, 37.2, 29.3, 29.2, 22.4, 20.7, 19.3. HRMS calcd for
C41H46N4O7I [M + H]+, 833.2411; found, 833.2451.
(S)-3-[(S)-2-{[(Benzyloxy)carbonyl]amino}-3-phenylpropanami-
do]-7-[3-(4-iodophenyl)ureido]-2-oxoheptyl 2,4,6-Trimethylben-
zoate (8b). Synthesized in a similar manner as 8a with 3-iodophenyl
isocyanate in place of 4-iodophenyl isocyanate. Yield 20 mg, 14%; mp
166−169 °C; TLC (2:1 EtOAc/Hex) Rf = 0.85; HPLC (method B) tR
= 18.0 min. 1H NMR [600.13 MHz, (CD3)2SO] δ 8.60 (s, 1H,
CONHC6H4I), 8.53 (d, J = 7.4 Hz, 1H, CONHCHCO), 7.96 (dd, J =
1.8, 1.8 Hz, 1H, 5-NHC6H2H2I), 7.65 (d, J = 8.2 Hz, 1H,
CONHCHCO), 7.34−7.20 (m, 12H, ArH), 6.99 (m, 1H, 2-
NHC6H2H2I), 6.92 (s, 2H, Me3C6H2CO), 6.25 (t, J = 4.6 Hz, 1H,
CH2CH2CH2NH), 4.96 (s, 2H, PhCH2OCO), 4.92 (d, J = 17.2 Hz,
1H, COCHHO), 4.81 (d, J = 17.2 Hz, 1H, COCHHO), 4.39−4.32
(m, 2H, HNCHCO Phe and Lys), 3.08−3.02 (m, 3H,
CH2CH2CH2NH and CHCH2HPh), 2.82 (dd, J = 13.6, 9.8 Hz, 1H,
CHCHHPh), 2.27 [s, 6H, 2,6-(CH3)3C6H2CO], 2.25 [s, 3H, 4-
(CH3)3C6H2CO], 1.85−1.80 (m, 1H, CHCHHCH2CH2), 1.63−1.55
(m, 1H, CHCHHCH2CH2), 1.47−1.40 (m, 2H, CH2CH2CH2NH),
1.39−1.33 (m, 1H, CHHCH2CH2NH), 1.32−1.27 (m, 1H,
CHHCH2CH2NH). 13C NMR [150.92 MHz, (CD3)2SO] δ 202.7,
172.0, 168.2, 155.9, 154.9, 142.1, 139.1, 137.8, 134.9, 130.6, 129.3,
129.3, 129.2, 128.3, 128.2, 128.1, 127.7, 127.7, 127.5, 126.3, 125.6,
116.8, 94.7, 66.6, 65.3, 56.1, 55.9, 38.9, 37.2, 29.3, 29.2, 22.4, 20.7,
19.4. HRMS calcd for C41H46N4O7I [M + H]+, 833.2411; found,
833.2416.
(S)-3-[(S)-2-{[(Benzyloxy)carbonyl]amino}-3-phenylpropanami-
do]-7-(3-iodobenzamido)-2-oxoheptyl 2,4,6-Trimethylbenzoate
(7b). To a solution of 3-iodobenzoic acid (72 mg, 290 μmol) in
DMF (12 mL) at −24 °C, NMM (33 μL, 300 μmol) and IBCF (39
μL, 300 μmol) were added and left to stir until dissolved. To this, 6a
(180 mg, 260 μmol) was added and the solution was left to stir
overnight while warming to room temperature. The reaction mixture
was diluted to 25 mL with water and extracted with EtOAc (3 × 20
mL), and the combined organic layers were extracted with water (5 ×
10 mL), saturated Na2CO3 (3 × 10 mL), water (20 mL), 5% (w/v)
citric acid (3 × 10 mL), water (20 mL), and brine (3 × 10 mL) and
then dried over MgSO4. Yield 95 mg, 45%; mp 135−140 °C; TLC
t
1
(2:1 EtOAc/Hex) Rf = 0.83; HPLC (method B) R = 18.0 min. H
NMR [600.23 MHz, (CD3)2SO] δ 8.52 (m, 2H, NHCHCONH and
CH2CH2CH2NH), 8.18 (s, 1H, 2-C6HH3I), 7.86−7.83 (m, 2H, 4,6-
C6H2H2I), 7.63 (d, J = 8.2, 1H, NHCHCOCH2), 7.33−7.18 (m, 10H
Ph), 6.91 (s, 2H, Me3C6H2CO), 4.95 (s, 2H, PhCH2OCO), 4.91 (d, J
= 17.2 Hz, 1H, COCHHO), 4.81 (d, J = 17.2 Hz, 1H, COCHHO),
4.39−4.32 (m, 2H, HNCHCO Phe and Lys), 3.24 (m, 2H,
CH2CH2CH2NH), 3.02 (dd, J = 13.7, 5.3 Hz, 1H, CHCHHPh),
2.81 (dd, J = 13.5, 10.0 Hz, 1H, CHCHHPh), 2.26 (s, 6H, 2,6-
(CH3)3C6H2CO), 2.25 (s, 3H, 4-(CH3)3C6H2CO), 1.87−1.78 (m, 1H,
CHCHHCH2CH2), 1.64−1.56 (m, 1H, CHCHHCH2CH2), 1.56−
1.47 (m, 2H, CH2 CH2 CH2 NH), 1.41−1.29 (m, 2H,
CH2CH2CH2NH); 13C NMR (150.92 MHz, (CD3)2SO) δ 202.6,
172.0, 168.2, 164.5, 155.8, 139.5, 139.1, 137.8, 136.9, 136.6, 135.6,
134.9, 130.4, 129.9, 129.2, 128.2, 128.2, 128.1, 127.7, 127.5, 126.6,
126.3, 94.6, 66.6, 65.2, 56.0, 55.9, 40.0, 37.2, 29.1, 28.6, 22.5, 20.7,
19.3. HRMS calcd for C41H45N3O7I [M + H]+, 818.2302; found,
818.2291.
(S)-3-[(S)-2-{[(Benzyloxy)carbonyl]amino}-3-phenylpropanami-
do]-7-[6-(4-iodobenzamido)hexanamido]-2-oxoheptyl 2,4,6-Trime-
thylbenzoate (11a). To a solution of 10a (49 mg, 137 μmol) in
anhydrous DMF (2 mL), benzotriazol-1-yloxytripyrrolidinophospho-
nium hexafluorophosphate (PyBOP) (71 mg, 140 μmol) was added
and dissolved at 0 °C under a stream of Ar. Et3N (38 μL, 270 μmol)
was then added and allowed to stir for 5 min. Afterward 6a (96 mg,
140 μmol) and Et3N (19 μL, 140 μmol) were added and the solution
was stirred overnight at room temperature. The reaction mixture was
diluted to 25 mL with water and extracted with CH2Cl2 (3 × 15 mL),
and the organic layers were combined and further extracted with water
(5 × 15 mL) and brine (30 mL) and then dried over Na2SO4. The
solvent was removed by rotary evaporation, leaving an oily residue,
which was then purified by flash chromatography (1−10% MeOH/
CH2Cl2). The product was isolated as a white powder following
lyophilization. Yield 57 mg, 45%; mp 190−196 °C; TLC (5% MeOH/
t
1
CH2Cl2) Rf = 0.44; HPLC (method B) R = 17.3 min. H NMR
[600.13 MHz, (CD3)2SO] δ 8.48 (m, 2H, NHCHCONH and
CH2NHCOAr), 7.83 (d, J = 8.4 Hz, 2H, COC6H2H2I), 7.71 (t, J = 5.7
Hz, 1H, CH2NHCOCH2), 7.63 (d, J = 8.4 Hz, 1H, NHCHCOCH2),
7.61 (d, J = 8.4 Hz, 2H, COC6H2H2I), 7.34−7.19 (m, 10H, Ph), 6.91
(s, 2H, Me3C6H2CO), 4.97 (s, 2H, PhCH2OCO), 4.91 (d, J = 17.2 Hz,
1H, COCHHO), 4.81 (d, J = 17.2 Hz, 1H, COCHHO), 4.36−4.32
(m, 2H, HNCHCO Phe and Lys), 3.23−3.19 (m, 2H, CH2NHCOAr),
3.04−2.97 (m, 3H, CHCHHPh and CH2NHCOCH2), 2.84−2.79 (m,
1H, CHCHHPh), 2.27 [s, 6H, 2,6-(CH3)3C6H2CO], 2.25 [s, 3H, 4-
(CH3)3C6H2CO], 2.04 (t, J = 7.5 Hz, 2H, CH2NHCOCH2), 1.82−
1.76 (m, 1H, CHCHHCH2CH2), 1.60−1.54 (m, 1H,
CHCHHCH2CH2), 1.49 (m, J = 7.4 Hz, 5H, CHCH2CHHCH2 and
NHCOCH2CH2), 1.40−1.30 (m, 3H, CHCH2CHHCH2 and
CH2CH2NHCO Lys), 1.28−1.22 (m, 2H, COCH2CH2CH2). 13C
(S)-3-[(S)-2-{[(Benzyloxy)carbonyl]amino}-3-phenylpropanami-
do]-7-[3-(3-iodophenyl)ureido]-2-oxoheptyl 2,4,6-Trimethylben-
zoate (8a). To a solution of 4-iodophenyl isocyanate (42 mg, 170
μmol) in anhydrous CH2Cl2 (5 mL) were added Et3N (24 μL, 170
μmol) and 6a (119 mg, 170 μmol), and the solution was left to stir for
3 h under a stream of Ar. The solvent was removed by rotary
evaporation and the residue was dissolved in DMF (1 mL). This
solution was then added dropwise to rapidly stirring cold water
(approximately 100 mL), forming a white powder that was collected
by centrifugation. The product was isolated as a white powder
following flash chromatography (2:1 EtOAc/Hex). Yield 13 mg, 9%;
mp 149−159 °C; TLC (2:1 EtOAc/Hex) Rf = 0.72; HPLC (method
t
1
B) R = 17.3 min. H NMR [600.13 MHz, (CD3)2SO] δ 8.55 (s, 1H,
CONHC6H4I), 8.51 (d, J = 7.5 Hz, 1H CONHCHCO), 7.63 (d, J =
G
dx.doi.org/10.1021/jm501357r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
NMR [150.92 MHz, (CD3)2SO] δ 202.6, 172.0, 171.8, 168.2, 165.3,
155.8, 139.1, 137.8, 137.1, 136.9, 134.9, 134.1, 129.9, 129.2, 129.1,
128.3, 128.2, 128.1, 127.7, 127.5, 126.3, 98.5, 66.6, 65.3, 56.0, 55.9,
38.1, 38.1, 37.2, 35.4, 29.1, 28.8, 28.8, 26.2, 25.1, 22.4, 20.7, 19.3.
HRMS calcd for C47H56N4O8I [M + H]+, 931.3143; found, 931.3104.
(S)-3-[(S)-2-{[(Benzyloxy)carbonyl]amino}-3-phenylpropanami-
do]-7-[6-(3-iodobenzamido)hexanamido]-2-oxoheptyl 2,4,6-Trime-
thylbenzoate (11b). Synthesized in a similar manner as 11a with 10a
in place of 10b. Yield 38 mg, 30%; mp 159−162 °C; TLC (5%
MeOH/CH2Cl2) Rf = 0.46; HPLC (method B) R = 17.4 min. H
NMR [600.13 MHz, (CD3)2SO] δ 8.54−8.51 (m, 2H, NHCHCONH
and CH2NHCOAr), 8.17 (s, 1H, NHCOC6HH3I), 7.87 (d, J = 7.8 Hz,
1H, NHCOC6HH3I), 7.83 (d, J = 7.9 Hz, 1H, NHCOC6HH3I), 7.74
(t, J = 5.5 Hz, 1H, NHCHCOCH2), 7.67 (d, J = 8.3 Hz, 1H,
CH2NHCOCH2), 7.33−7.18 (m, 11H, Ph and NHCOC6HH3I), 6.91
(s, 2H, Me3C6H2CO), 4.96 (s, 2H, PhCH2OCO), 4.90 (d, J = 17.3 Hz,
1H, COCHHO), 4.80 (d, J = 17.2 Hz, 1H, COCHHO), 4.35−4.31
(m, 2H, HNCHCO Phe and Lys), 3.22−3.19 (m, 2H, CH2NHCOAr),
3.04−2.99 (m, 3H, CHCHHPh and CH2NHCOCH2), 2.80 (dd, J =
13.6, 9.8 Hz, 1H, CHCHHPh), 2.27 [s, 6H, 2,6-(CH3)3C6H2CO],
2.24 [s, 3H, 4-(CH3)3C6H2CO], 2.04 (t, J = 7.4 Hz, 2H,
CH2NHCOCH2), 1.82−1.75 (m, 1H, CHCHHCH2CH2), 1.59−1.52
(m, 1H, CHCHHCH2CH2), 1.52−1.46 (m, 5H, CHCH2CHHCH2
and NHCOCH2CH2), 1.39−1.30 (m, 3H, CHCH2CHHCH2 and
CH2CH2NHCO Lys), 1.28−1.22 (m, 2H, COCH2CH2CH2). 13C
NMR [125.77 MHz, (CD3)2SO] δ 202.8, 172.15, 172.02, 168.4, 164.7,
156.0, 139.7, 139.3, 138.0, 137.1, 136.8, 135.7, 135.1, 130.6, 130.1,
129.4, 128.41, 128.39, 128.25, 127.86, 127.68, 126.8, 126.5, 94.8, 66.8,
65.5, 56.24, 56.09, 38.28, 38.25, 37.4, 35.5, 29.3, 28.9, 26.3, 25.2, 22.6,
20.8, 19.5, 19.1. HRMS calcd for C47H56N4O8I [M + H]+, 931.3143;
found, 931.3161.
°C under a stream of Ar. After 1 min, 6a (106 mg, 150 μmol) and
NMM (13 μL, 125 μmol) were added and the solution was left to stir
overnight while warming to room temperature. The reaction mixture
was made up to 25 mL with water and extracted with EtOAc (4 × 25
mL). The combined extracts were extracted with saturated Na2CO3 (3
× 25 mL), water (25 mL), 5% (w/v) citric acid (3 × 25 mL), water
(25 mL), and brine (3 × 25 mL) and then dried over MgSO4. The
solvent was removed by rotary evaporation, leaving an oily residue,
which was purified by flash chromatography (12−100% EtOAc/Hex).
The product was isolated as a white powder following lyophilization.
Yield 0.047 g, 30%; TLC (1:1 EtOAc/Hex) Rf = 0.60; HPLC (method
t
1
t
1
B) R = 17.0 min. H NMR [600 MHz, (CD3)2SO] δ 8.54 (s, 1H,
NHCONHAr), 8.49 (d, J = 7.3 Hz, 1H, NHCHCONH), 7.96 (s, 1H,
CONHC6HH3I), 7.72−7.70 (m, 1H, CH2NHCOCH2), 7.64−7.62
(m, 1H, NHCHCOCH2), 7.33−7.19 (m, 12H, Ph and
NHCOC6H2H2I), 7.01−6.98 (m, 1H, NHCOC6HH3I), 6.92 (s, 2H,
Me3C6H2CO), 6.20−6.17 (m, 1H, NHCONHAr), 4.97 (s, 2H,
PhCH2OCO), 4.91 (d, J = 17.2 Hz, 1H, COCHHO), 4.81 (d, J = 17.3
Hz, 1H, COCHHO), 4.36−4.31 (m, 2H, HNCHCO Phe and Lys),
3.07−3.01 (m, 6H, CH2NHCONH, CHCHHPh, CH2NHCOCH2,
and CHCHHCH2CH2), 2.83−2.79 (m, 1H, CHCHHPh), 2.28 [s, 6H,
2,6-(CH3)3C6H2CO], 2.25 [s, 3H, 4-(CH3)3C6H2CO], 2.21 (t, J = 7.4
Hz, 2H, CH2NHCOCH2), 2.06−2.03 (m, 1H, CHCHHCH2CH2),
1.52−1.49 (m, 1H, CHCHHCH2CH2), 1.43−1.37 (m, 6H,
CHCH2CH2CH2 and CH2CH2NHCO), 1.31−1.25 (m, 2H,
COCH2CH2CH2). 13C NMR [151 MHz, (CD3)2SO] δ 174.8, 174.8,
142.5, 139.5, 138.2, 135.3, 133.5, 131.0, 129.7, 129.7, 128.7, 128.6,
128.5, 128.5, 128.1, 127.9, 126.0, 117.2, 95.1, 67.0, 65.7, 65.7, 56.3,
38.5, 37.7, 35.8, 34.1, 29.9, 29.8, 26.5, 26.3, 25.5, 24.7, 22.8, 20.1, 19.8.
HRMS calcd for C47H57N5O8I [M + H]+, 946.3252; found, 946.3223.
Methyl 6-[4-(Tributylstannyl)-1H-1,2,3-triazol-1-yl]hexanoate
(18). Tributyl(ethynyl)stannane (680 mg, 2.2 mmol) and 16 (310
mg, 1.8 mmol) were combined in toluene (2 mL) and heated to reflux
at 120 °C for 14 h. The solvent was evaporated and the product was
isolated as a colorless oil following flash chromatography (8−66%
EtOAc/hexanes). Yield 680 mg, 78%; TLC (1:2 EtOAc/Hex) Rf =
0.55. 1H NMR (600.13 MHz, CDCl3) δ 7.41 (s, 1H, triazole-H), 4.36
(t, J = 7.2 Hz, 2H, NCH2CH2CH2), 3.63 (s, 3H, CH2CH2COOCH3),
2.28 (t, J = 7.4 Hz, 2H, CH2CH2CH2COOMe), 1.93−1.88 (m, 2H,
NCH2CH2CH2CH2), 1.67−1.60 (m, 2H, NCH2CH2CH2CH2), 1.59−
1.47 [m, 6H, Sn(CH2CH2CH2)3], 1.37−1.27 [m, 8H, NCH2CH2CH2
and Sn(CH2CH2CH2)3], 1.15−1.06 [m, 6H, Sn(CH2CH2CH2)3], 0.86
[t, J = 7.3 Hz, 9H, (CH2CH2CH2CH3)3]. 13C NMR (150.92 MHz,
CDCl3) δ 173.8, 144.3, 129.7, 51.5, 49.3, 33.7, 30.2, 29.0, 27.2, 26.0,
24.3, 13.7, 9.9. HRMS calcd for C21H42N3O2Sn [M + H]+, 488.2303;
found, 488.2280.
(S)-3-[(S)-2-(Benzyloxycarbonyl)-3-phenylpropanamido]-7-{6-[3-
(4-iodophenyl)ureido]hexanamido}-2-oxoheptyl 2,4,6-Trimethyl-
benzoate (15a). To a solution of 14a (56 mg, 150 μmol) in
anhydrous DMF (5 mL) were added NMM (17 μL, 150 μmol) and
IBCF (21 μL, 150 μmol), and the mixture was stirred at −10 °C under
a stream of Ar. After 1 min, 6a (95 mg, 140 μmol) and NMM (17 μL,
150 μmol) were added and the solution was left to stir overnight while
warming to room temperature. The reaction mixture was diluted to 25
mL with water and extracted with EtOAc (3 × 25 mL). The combined
extracts were extracted with saturated Na2CO3 (3 × 25 mL), water (25
mL), 5% (w/v) citric acid (3 × 25 mL), water (25 mL), and brine (3 ×
25 mL) and then dried over MgSO4. The solvent was removed by
rotary evaporation, leaving an oily residue, which was then purified by
flash chromatography (1:1 EtOAc/Hex). The product was isolated as a
white powder following lyophilization. Yield 45 mg, 35%; TLC (1:1
t
1
Methyl 6-(4-Iodo-1H-1,2,3-triazol-1-yl)hexanoate (19). To a
solution of 18 (490 g, 1.0 mmol) in tetrahydrofuran (THF, 8 mL)
was added I2 (280 g, 1.1 mmol), and the mixture was left to stir for 2 h.
The solution was diluted to 25 mL with EtOAc, extracted with
saturated Na2S2O3 (3 × 20 mL) and brine (20 mL), and dried over
MgSO4. The solvent was removed by rotary evaporation, and the
product was isolated as a white solid following flash chromatography
(8−66% EtOAc/hexanes). Yield 220 mg, 69%; mp 49−51 °C; TLC
EtOAc/Hex) Rf = 0.60; HPLC (method B) R = 16.8 min. H NMR
[600 MHz, (CD3)2SO] δ 8.54−8.47 (m, 2H, NHCONHAr and
NHCHCONH), 7.73−7.70 (m, 1H, CH2NHCOCH2), 7.65−7.61 (m,
1H, NHCHCOCH2), 7.50 (d, J = 8.7 Hz, 1H NHCOC6H2H2I), 7.34−
7.18 (m, 12H, Ph and NHCOC6H2H2I), 6.91 (s, 2H, Me3C6H2CO),
6.20−6.16 (m, 1H, NHCONHAr), 4.96 (s, 2H, PhCH2OCO), 4.90
(d, J = 16.7 Hz, 1H, COCHHO), 4.80 (d, J = 17.4 Hz, 1H,
COCHHO), 4.35−4.30 (m, 2H, HNCHCO Phe and Lys), 3.04−3.00
(m, 3H, CHCHHPh and CH2NHCOCH2), 2.99−2.93 (m, 1H,),
2.83−2.80 (m, 1H, CHCHHPh), 2.27 [s, 6H, 2,6-(CH3)3C6H2CO],
2.25 [s, 3H, 4-(CH3)3C6H2CO], 2.04−2.02 (m, 2H, CH2NHCOCH2),
1.79−1.78 (m, 1H, CHCHHCH2CH2), 1.58−1.54 (m, 1H,
CHCHHCH2CH2), 1.49−1.47 (m, 2H, NHCOCH2CH2), 1.40−1.33
(m, 6H, CHCH2CH2CH2 and CH2CH2NHCO), 1.25−1.21 (m, 2H,
COCH2CH2CH2). 13C NMR [150.92 MHz, (CD3)2SO] δ 156.8,
154.9, 142.2, 137.1, 135.4, 134.9, 130.6, 129.3, 129.3, 128.3, 128.2,
128.1, 125.6, 119.8, 116.8, 109.1, 94.7, 65.3, 40.1, 33.99, 33.79, 29.5,
26.0, 24.3, 18.9. HRMS calcd for C47H57N5O8I [M + H]+, 946.3252;
found, 946.3266.
1
(1:2 EtOAc/Hex) Rf = 0.23. H NMR (600.13 MHz, CDCl3) δ 7.64
(s, 1H, triazole-H), 4.30 (t, J = 7.1 Hz, 2H, NCH2CH2CH2), 3.55 (s,
3H, CH2 CH2 COOCH3 ), 2.21 (t,
J = 7.4 Hz, 2H,
CH2CH2CH2COOMe), 1.85−1.80 (m, 2H, NCH2CH2CH2CH2),
1.58−1.53 (m, 2H, NCH2CH2CH2CH2), 1.28−1.22 (m, 2H,
NCH2CH2CH2). 13C NMR (150.92 MHz, CDCl3) δ 173.6, 129.1,
86.9, 51.5, 50.4, 33.6, 29.8, 25.8, 24.1. HRMS calcd for C9H15N3O2I
[M + H]+, 324.0209; found, 324.0218.
6-(4-Iodo-1H-1,2,3-triazol-1-yl)hexanoic acid (20). To 19 (190
mg, 600 μmol) dissolved in CH3CN (30 mL) was added 1 N NaOH
(6 mL), and the solution was left to stir overnight. After the solvent
was evaporated, the residue was dissolved in water (25 mL), and 1 N
HCl (6 mL) was added. The product was then extracted with CH2Cl2
(3 × 20 mL), and the combined organic layers were extracted with
brine (20 mL) and dried over Na2SO4. The product was obtained as a
white wax following rotary evaporation. Yield 150 g, 80%; mp 73−77
(S)-3-[(S)-2-(Benzyloxycarbonyl)-3-phenylpropanamido]-7-{6-[3-
(3-iodophenyl)ureido]hexanamido}-2-oxoheptyl 2,4,6-Trimethyl-
benzoate (15b). To a solution of 14b (47 mg, 125 μmol) in
anhydrous DMF (5 mL) were added NMM (25 μL, 250 μmol) and
IBCF (16 μL, 125 μmol), and the mixture was allowed to stir at −10
H
dx.doi.org/10.1021/jm501357r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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1
°C; TLC (5% MeOH/CH2Cl2) Rf = 0.52. H NMR (600.13 MHz,
CDCl3) δ 7.62 (s, 1H, triazole-H), 4.40 (t, J = 7.1 Hz, 2H,
NCH2CH2CH2), 2.38 (t, J = 7.3 Hz, 2H, CH2CH2CH2COOH), 1.97−
1.92 (m, 2H, NCH2CH2CH2CH2), 1.72−1.67 (m, 2H,
NCH2CH2CH2CH2), 1.42−1.37 (m, 2H, NCH2CH2CH2). 13C
NMR (150.92 MHz, CDCl3) δ 178.5, 128.8, 86.9, 50.3, 33.4, 29.8,
25.7, 23.8. HRMS calcd for C8H13N3O2I [M + H]+, 310.0052; found,
310.0045.
NHCH2CH2O), 3.67−3.60 [m, 12H, (CH2OCH2)3], 2.57 (t, J = 6.1
Hz, 2H, CH2CH2COOH). 13C NMR (151 MHz, CDCl3) δ 175.5,
166.9, 140.8, 136.97, 136.82, 130.7, 127.0, 94.7, 71.08, 70.92, 70.80,
70.75, 70.4, 67.0, 40.6, 35.5. HRMS calcd for C16H23NO6 [M + H]+,
452.0570; found, 452.0566.
(S)-20-[(S)-2-{[(Benzyloxy)carbonyl]amino}-3-phenylpropanami-
do]-1-(4-iodophenyl)-1,14,21-trioxo-5,8,11-trioxa-2,15-diazadoco-
san-22-yl 2,4,6-Trimethylbenzoate (23a). To a solution of 22a (36
mg, 80 μmol) in anhydrous DMF (5 mL), PyBOP (42 mg, 80 μmol)
was added and dissolved at 0 °C under a stream of Ar. Et3N (33 μL,
240 μmol) was added and the solution was left to stir for 5 min.
Afterward, 6a (56 mg, 80 μmol) was added and the reaction mixture
was stirred while warming to room temperature overnight. The
solution was made up to 25 mL with water and extracted with CH2Cl2
(3 × 10 mL), and the organic layers were combined and then extracted
with water (5 × 15 mL) and brine (5 × 10 mL). The solvent was
evaporated and the product was obtained as a white solid following
flash chromatography (2−20% MeOH/CH2Cl2) as a white wax. Yield
18 mg, 22%; TLC (5% MeOH/CH2Cl2) Rf = 0.22; HPLC (method B)
(S)-3-[(S)-2-{[(Benzyloxy)carbonyl]amino}-3-phenylpropanami-
do]-7-[6-(4-iodo-1H-1,2,3-triazol-1-yl)hexanamido]-2-oxoheptyl
2,4,6-Trimethylbenzoate (21). To a solution of 20 (42 mg 140 μmol)
in anhydrous DMF (10 mL), PyBOP (71 mg, 140 μmol) was added
and allowed to dissolve while the reaction mixture was kept at 0 °C
under a stream of Ar. Et3N (38 μL, 270 μmol) was added and the
solution was left to stir for 5 min. Afterward, 6a and Et3N (19 μL 140
μmol) were added and the reaction mixture was allowed to warm to
room temperature overnight with stirring. The solution was made up
to 25 mL with water and extracted with CH2Cl2 (5 × 15 mL), and the
combined organic layers extracted with water (5 × 15 mL) and brine
(3 × 25 mL). The solvent was evaporated and the product was
obtained following flash chromatography (1−10% MeOH/CH2Cl2) as
a white solid. Yield 40 mg, 33%; mp 102−109 °C; TLC (5% MeOH/
CH2Cl2) Rf = 0.15. 1H NMR [600.13 MHz, (CD3)2SO] δ 8.48 (d, J =
7.5 Hz, 1H, NHCHCONH), 8.33 (s, 1H, triazole-H), 7.71 (t, J = 5.5
Hz, 1H, CH2NHCOCH2), 7.62 (d, J = 8.3 Hz, 1H, NHCHCOCH2),
7.33−7.18 (m, 10H, Ph) 6.91 (s, 2H, Me3C6H2CO), 4.96 (s, 2H,
PhCH2OCO), 4.91 (d, J = 17.3 Hz, 1H, COCHHO), 4.80 (d, J = 17.3
Hz, 1H, COCHHO), 4.36−4.31 (m, 4H, CH2N3C2HI and HNCHCO
Phe and Lys), 3.04−2.99 (m, 4H CH2NHCOCH2 and CHCHHPh),
2.81 (dd, J = 13.8, 9.8 Hz, 1H, CHCHHPh), 2.26 [s, 6H 2,6-
(CH3)3C6H2CO], 2.25 [s, 3H, 4-(CH3)3C6H2CO], 2.02 (t, J = 7.4 Hz,
2H, CH2NHCOCH2), 1.79−1.72 (m, 3H, CHCHHCH2CH2 and
CH2CH2N3C2HI), 1.58−1.55 (m, 1H, CHCHHCH2CH2), 1.51−1.46
(m, 2H, NHCOCH2CH2), 1.40−1.23 (m, 4H CHCH2CH2CH2),
1.18−1.12 (m, 2H, COCH2CH2CH2). 13C NMR [150.91 MHz,
(CD3)2SO] δ 202.6, 172.0, 171.7, 168.2, 155.9, 139.1, 137.8, 136.9,
136.1, 134.9, 129.9, 129.2, 128.26, 128.22, 128.08, 127.69, 127.50,
126.3, 88.3. HRMS calcd for C42H52N6O7I [M + H]+, 879.2943;
found, 879.2915.
1-(4-Iodophenyl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic
Acid (22a). 3-{2-[2-(2-Aminoethoxy)ethoxy]ethoxy}propanoic acid
(332 mg, 1.5 mmol) and 9a (173 mg, 0.5 mmol) were added to DMF
(2 mL) in a 2−5 mL microwave vial. The solution was heated in a
microwave at 120 °C for 8 min. Afterward, water (5 mL) was added
and the mixture was extracted with CH2Cl2 (3 × 10 mL); the organic
layers were combined and then extracted with water (5 × 10 mL) and
brine (3 × 10 mL) and dried over Na2SO4. The product was isolated
as a white solid following flash chromatography [1−10% MeOH/
CH2Cl2 (with 0.1% AcOH)]. Yield 150 mg, 67%; mp 56−64 °C; TLC
[5% MeOH/CH2Cl2 (with AcOH)] Rf = 0.25. 1H NMR (600.13
MHz, CDCl3) δ 7.77 (d, J = 8.3 Hz, 2H, NHCOC6H2H2I), 7.56 (d, J =
8.4 Hz, 2H, NHCOC6H2H2I), 7.04 (br s, NHCH2CH2O), 3.74 (t, J =
6.0 Hz, 2H, NHCH2CH2O), 3.69−3.63 [m, 12H, (CH2OCH2)3], 2.59
(t, J = 5.7 Hz, 2H, CH2CH2COOH). 13C NMR (150.92 MHz, CDCl3)
δ 167.3, 137.6, 133.7, 129.0, 98.5, 70.36, 70.26, 70.21, 70.08, 69.9, 66.6,
58.1, 40.0. HRMS calcd for C16H23NO6 [M + H]+, 452.0570; found,
452.0555.
1
tR = 16.7 min. H NMR [600.13 MHz, (CD3)2SO] δ 8.58 (t, J = 5.6
Hz, 1H, CH2NHCOAr), 8.50 (d, J = 7.4 Hz, 1H, NHCHCONH),
7.85 (d, J = 8.5 Hz, 2H, NHCOC6H2H2I), 7.79 (t, J = 5.6 Hz, 1H,
CH2NHCOCH2), 7.65−7.62 (m, 3H, NHCOC6H2H2I and
NHCHCOCH2), 7.35−7.20 (m, 10H, Ph), 6.92 (s, 2H,
Me3C6H2CO), 4.98 (s, 2H, PhCH2OCO), 4.92 (d, J = 17.2 Hz, 1H,
COCHHO), 4.81 (d, J = 17.2 Hz, 1H, COCHHO), 4.37−4.32 (m,
2H, HNCHCO Phe and Lys), 3.57 (t, J = 6.5 Hz, 2H, COCH2CH2O),
3.53−3.39 (m, 12H, CH2CH2OCH2CH2OCH2CH2), 3.05−3.01 (m,
3H, CHCHHPh and CH2NHCOCH2), 2.82 (dd, J = 13.6, 9.7 Hz, 1H,
CHCHHPh), 2.30 (m, 2H, COCH2CH2O), 2.28 [s, 6H, 2,6-
(CH3)3C6H2CO], 2.26 [s, 3H, 4-(CH3)3C6H2CO], 1.83−1.77 (m,
1H, CHCHHCH2CH2), 1.61−1.53 (m, 1H, CHCHHCH2CH2),
1.41−1.24 (m, 4H, CHCH2CH2CH2). 13C NMR (150.92 MHz;
(CD3)2SO): δ 202.6, 172.0, 169.8, 168.2, 165.6, 155.8, 139.1, 137.8,
137.1, 136.9, 134.9, 133.8, 129.9, 129.2, 129.1, 128.3, 128.2, 128.1,
127.7, 127.5, 126.3, 98.8, 69.7, 69.6, 69.6, 69.5, 68.8, 66.8, 66.6, 65.3,
56.0, 55.9, 40.1, 38.2, 37.2, 36.1, 29.1, 28.7, 22.3, 20.7, 19.3. HRMS
calcd for C50H62N4O11I [M + H]+, 1021.3460; found, 1021.3456.
(S)-20-[(S)-2-{[(Benzyloxy)carbonyl]amino}-3-phenylpropanami-
do]-1-(3-iodophenyl)-1,14,21-trioxo-5,8,11-trioxa-2,15-diazadoco-
san-22-yl 2,4,6-Trimethylbenzoate (23b). To a solution of 22b (26
mg 57 μmol) in anhydrous DMF (5 mL), PyBOP (30 mg, 57 μmol)
was added and dissolved at 0 °C under a stream of Ar. Et3N (8 μL, 57
μmol) was added and the solution was left to stir for 5 min. Afterward,
6a (40 mg, 57 μmol) and Et3N (16 μL, 114 μmol) were added, and
the reaction mixture was stirred and allowed to warm to room
temperature overnight. The solution was made up to 25 mL with water
and extracted with CH2Cl2 (3 × 10 mL); the organic layers were
combined and then extracted with water (5 × 15 mL) and brine (5 ×
10 mL). The solvent was evaporated and the product was obtained as
a white solid following flash chromatography (5% MeOH/CH2Cl2).
Yield 0.216 g, 37%; mp 79−86 °C; TLC (10% MeOH/CH2Cl2) Rf =
t
0.51; HPLC (method B) R = 16.7 min. 1H NMR [600.13 MHz,
(CD3)2SO] δ 8.61 (t, J = 5.5 Hz, 1H, CH2NHCOAr), 8.50 (d, J = 7.4
Hz, 1H, NHCHCONH), 8.19 (m, 1H, NHCOC6HH3I), 7.89−7.87
(m, 1H, NHCOC6HH3I), 7.86−7.85 (m, 1H, NHCOC6HH3I), 7.79
(t, J = 5.7 Hz, 1H, CH2NHCOCH2), 7.64 (d, J = 8.2 Hz, 1H,
NHCHCOCH2), 7.34−7.18 (m, 11H, Ph and NHCOC6HH3I), 6.92
(s, 2H, Me3C6H2CO), 4.97 (d, J = 5.2 Hz, 2H, PhCH2OCO), 4.91 (d,
J = 17.2 Hz, 1H, COCHHO), 4.80 (d, J = 17.2 Hz, 1H, COCHHO),
4.36−4.33 (m, 2H, HNCHCO Phe and Lys), 3.57 (t, J = 6.5 Hz, 2H,
COCH2CH2O), 3.53−3.38 (m, 12H, CH2CH2OCH2CH2OCH2CH2),
3.05−3.01 (m, 3H, CHCHHPh and CH2NHCOCH2), 2.82 (dd, J =
13.6, 9.7 Hz, 1H, CHCHHPh), 2.29 (d, J = 6.5 Hz, 1H,
COCH2CHHO), 2.27 [s, 6H, 2,6-(CH3)3C6H2CO], 2.25 [s, 3H, 4-
(CH3)3C6H2CO], 1.82−1.78 (m, 1H, CHCHHCH2CH2), 1.60−1.53
(m, 1H, CHCHHCH2CH2), 1.40−1.24 (m, 4H, CHCH2CH2CH2).
13C NMR [150.92 MHz, (CD3)2SO] δ 202.6, 172.0, 172.0, 169.8,
168.2, 155.8, 139.6, 139.1, 137.8, 136.9, 136.4, 135.6, 134.9, 130.5,
129.9, 129.2, 128.3, 128.2, 128.1, 127.7, 127.5, 126.6, 126.3, 94.6, 69.7,
1-(3-Iodophenyl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic
acid (22b). 3-{2-[2-(2-Aminoethoxy)ethoxy]ethoxy}propanoic acid
(120 mg 540 μmol) and 9b (620 mg 180 μmol) were added to DMF
(1.5 mL) in a 0.5−2 mL microwave vial. The solution was heated in a
microwave at 120 °C for 8 min. Afterward water (5 mL) was added
and the mixture was extracted with CH2Cl2 (3 × 10 mL); the organic
layers were combined and then extracted with water (5 × 10 mL) and
brine (10 mL) and dried over Na2SO4. The solvent was removed by
rotary evaporation and the product was isolated as a white wax. Yield
34 mg, 42%; TLC (5% MeOH/CH2Cl2) Rf = 0.30. 1H NMR (600.13
MHz, CDCl3) δ 8.16 (dd, J = 1.5, 1.5 Hz, 1H, NHCOC6HH3I), 7.79−
7.77 (m, 2H, NHCOC6H2H2I), 7.24 (br s, NHCH2CH2O), 7.14 (dd, J
= 7.8, 7.8 Hz, 1H, NHCOC6HH3I), 3.71 (t, J = 6.1 Hz, 2H,
I
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69.7, 69.6, 69.5, 68.7, 66.8, 66.6, 65.3, 56.0, 55.9, 40.0, 38.2, 37.2, 36.1,
29.1, 28.7, 22.4, 20.7, 19.3. HRMS calcd for C50H62N4O11I [M + H]+,
1021.3460; found, 1021.3484.
CH2CH2OCH2CH2OCH2CH2), 3.03−2.99 (m, 3H, CHCHHPh and
CH2NHCOCH2), 2.82−2.81 (m, 1H, CHCHHPh), 2.27 [s, 6H, 2,6-
(CH3)3C6H2CO], 2.25 [s, 3H, 4-(CH3)3C6H2CO], 1.63−1.54 (m, 2H,
CHCH2CH2CH2), 1.40−1.32 (m, 4H, CHCH2CH2CH2). 13C NMR
[150.92 MHz, (CD3)2SO] δ 202.8, 172.1, 169.9, 168.4, 156.0, 139.3,
137.9, 137.1, 135.1, 130.6, 130.1, 128.41, 128.37, 128.23, 128.14,
127.84, 127.66, 126.5, 88.5, 69.85, 69.80, 69.77, 69.71, 69.68, 69.65,
69.62, 67.0, 65.4, 56.20, 56.05, 38.3, 37.4, 36.3, 30.9, 29.2, 28.9, 20.8,
19.5. HRMS calcd for C45H58N6O10I [M + H]+, 969.3259; found,
969.3243.
tert-Butyl 3-(2-{2-[2-(4-Tributylstannyl-1H-1,2,3-triazol-1-yl)-
ethoxy]ethoxy}ethoxy)propanoate (26). Tributyl(ethynyl)stannane
(0.432 g, 1.37 mmol) and 25 (0.308 g, 1.80 mmol) were combined in
toluene (3 mL) and heated to reflux at 120 °C for overnight. The
solvent was evaporated and the product was isolated as a colorless oil
following flash chromatography (12−100% EtOAc/hexanes). Yield
0.468 g, 66%; TLC (1:1 EtOAc/Hex) Rf = 0.33. 1H NMR (600 MHz,
CDCl3): δ 7.60 (s, 1H, triazole-H), 4.57 (t, J = 5.3 Hz, 2H,
NCH2CH2O), 3.87 (t, J = 5.3 Hz, 2H, NCH2CH2O), 3.70 (t, J = 6.6
Hz, 2H, CH2CH2COOtBu), 3.59−3.58 [m, 8H, (OCH2CH2)2], 2.49
(t, J = 6.7 Hz, 2H, CH2CH2COOtBu), 1.58−1.52 [m, 6H,
Sn(CH2CH2CH2)3], 1.44 [s, 9H, (CH3)3COOC], 1.32 [m, 6H,
Sn(CH2CH2CH2)3], 1.12−1.09 [m, 6H, Sn(CH2CH2CH2)3], 0.88 [t, J
= 7.3 Hz, 9H, (CH2CH2CH2CH3)3]. 13C NMR (150.92 MHz, CDCl3)
δ 171.2, 144.5, 131.3, 80.9, 70.96, 70.89, 70.73, 70.1, 67.3, 49.9, 36.6,
29.4, 28.4, 27.6, 10.2. HRMS calcd for C27H53N3O5Sn [M + H]+,
620.3091; found, 620.3077.
6-{4-[Tris(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)stannyl]-
benzamido}hexanoic Acid (31). 1,3,5,7-Tetramethyl-6-(2,4-dime-
thoxyphenyl)-2,4,8-trioxa-6-phosphaadamantane (8.0 mg, 27 μmol)
and Pd(OAc)2 (3.0 mg, 13 μmol), each dissolved in degassed THF (1
mL), were added to a 2−5 mL microwave vial purged with Ar and
mixed together for 15 min. Tris(1H,1H,2H,2H-perfluorooctyl)tin
hydride (850 mg, 730 μmol) dissolved in THF (1 mL) was added, and
the solution was stirred for 15 min. A solution of 10a (120 mg, 332
μmol) dissolved in THF (1 mL) was then added, and the reaction
mixture was heated by microwave irradiation at 85 °C for 30 min. The
mixture was filtered through silica/KF (3:1) and then further purified
by column chromatography [1−5% MeOH/CH2Cl2 (with 1%
AcOH)]. The desired fractions were then diluted with water and
the product, a white wax, was obtained following rotary evaporation.
tert-Butyl 3-(2-{2-[2-(4-Iodo-1H-1,2,3-triazol-1-yl)ethoxy]ethoxy}-
ethoxy)propanoate (27). To a solution of 26 (0.303 g, 0.49 mmol) in
THF (10 mL), iodine (0.137 g, 0.54 mmol) was added and left to stir
for 2 h. The solution volume was made up to 25 mL with EtOAc and
extracted with saturated Na2S2O3 (3 × 20 mL) and brine (20 mL) and
dried over MgSO4. The solvent was removed by rotary evaporation,
and the product was isolated as a clear colorless oil following flash
chromatography (12−100% EtOAc/hexanes). Yield 0.187 mg, 84%;
1
Yield 117 mg, 35%; TLC (5% MeOH/CH2Cl2) Rf = 0.57. H NMR
(600.13 MHz, CDCl3) 1H NMR (600 MHz; CDCl3) δ 7.77 (d, J = 8.1
Hz, 2H ArH), 7.51−7.42 (m, 2H, ArH), 6.25 (t, J = 5.8 Hz, 1H,
NHCH2CH2), 3.48−3.45 (m, J = 6.6 Hz, 2H, NHCH2CH2), 2.37 (t, J
= 7.3 Hz, 2H, CH2CH2COOH), 2.62−2.35 [m, 6H, Sn(CH2CH2)3],
1.71−1.67 (m, 2H, CH2CH2COOH), 1.66−1.62 (m, 2H,
NHCH2CH2), 1.48−1.42 (m, 2H, CH2CH2CH2COOH), 1.38−1.33
[m, 6H, Sn(CH2CH2)3]. 13C NMR (150.92 MHz; CDCl3) δ 167.6,
141.5, 136.4, 135.8, 127.1, 40.1, 33.9, 29.5, 27.8, 26.6, 24.5, −1.0.
HRMS calcd for C37H29NO3F39Sn [M + H]+, 1396.0554; found,
1396.0468.
1
TLC (1:1 EtOAc/Hex) Rf = 0.18. H NMR (600.13 MHz, CDCl3) δ
7.90 (s, 1H, triazole-H), 4.57 (t, J = 4.9 Hz, 2H, NCH2CH2O), 3.85 (t,
J = 4.9 Hz, 2H, NCH2CH2O), 3.72 (t, J = 6.5 Hz, 2H,
CH2CH2COOtBu), 3.62−3.60 [m, 8H, (OCH2CH2)3)] 2.50 (t, J =
6.5 Hz, 2H, CH2CH2COOtBu), 1.44 [s, 9H, (CH3)3COOC]. 13C
NMR (150.92 MHz, CDCl3) δ 171.2, 130.6, 87.2, 80.9, 70.91, 70.80,
70.79, 69.6, 67.3, 51.0, 36.6, 28.5. HRMS calcd for C15H27N3O5I [M +
H]+, 456.0995; found, 456.0981.
(S)-3-[(S)-2-{[(Benzyloxy)carbonyl]amino}-3-phenylpropanami-
do]-2-oxo-7-[6-({4-[tris(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-
stannyl]phenyl}amino)hexanamido]heptyl 2,4,6-Trimethylben-
zoate (32). To a solution of 31 (42 mg, 30 μmol) in anhydrous
DMF (5 mL) was added PyBOP (16 mg, 30 μmol), and the solids
were allowed to dissolve at 0 °C under a stream of Ar. Et3N (4.0 μL,
30 μmol) was added and the solution was left to stir for 5 min.
Afterward, 6a (21 mg, 30 μmol) and Et3N (8 μL, 60 μmol) were
added and the solution was stirred and allowed to warm to room
temperature overnight. Water (20 mL) was added and the solution
was extracted with CH2Cl2 (3 × 10 mL); the combined organic layers
were extracted with water (5 × 10 mL) and brine (3 × 10 mL) and
then dried over Na2SO4. The solvent was evaporated and the product
was obtained as a white wax following semipreparative HPLC (method
B). Yield 0.008 g, 14%; TLC (5% MeOH/CH2Cl2) Rf = 0.36; HPLC
(method B) tR = 25.1 min. 1H NMR [500.13 MHz, (CD3)2SO] δ 8.48
(d, J = 7.3 Hz, 1H NHCHCONH), 8.40 (t, J = 4.8 Hz, 1H,
CH2NHCOAr), 7.80 (d, J = 7.8 Hz, 2H, COC6H2H2Sn), 7.70 (t, J =
5.4 Hz, 1H, CH2NHCOCH2), 7.63−7.59 (m, 3H, NHCHCOCH2 and
COC6H2H2Sn), 7.33−7.18 (m, 10H, Ph), 6.90 (s, 2H, Me3C6H2CO),
4.96 (d, J = 4.4 Hz, 2H, PhCH2OCO), 4.90 (d, J = 17.5 Hz, 1H,
COCHHO), 4.80 (d, J = 17.0 Hz, 1H, COCHHO), 4.35−4.32 (m,
2H, HNCHCO Phe and Lys), 3.24−3.20 (m, 2H, CH2NHCOAr),
3.04−2.99 (m, 3H, CHCHHPh and CH2NHCOCH2), 2.83−2.79 (m,
1H, CHCHHPh), 2.47−2.36 [m, 6H, Sn(CH2CH2)2], 2.26 [s, 6H,
2,6-(CH3)3C6H2CO], 2.24 [s, 3H, 4-(CH3)3C6H2CO], 2.04 (t, J = 7.3
Hz, 2H, CH2NHCOCH2), 1.82−1.75 (m, 1H, CHCHHCH2CH2),
1.57−1.46 (m, 4H, CHCHHCHHCH2 and NHCOCH2CH2), 1.39−
1.21 [m, 11H, CHCH2CHHCH2, CH2CH2NHCO, and Sn-
(CH2CH2)3]. HRMS calcd for C71H68N4O8Sn [M + H]+,
1965.3455; found, 1965.2988.
3-(2-{2-[2-(4-Iodo-1H-1,2,3-triazol-1-yl)ethoxy]ethoxy}ethoxy)-
propanoic Acid (28). To a solution of 27 (0.150 g, 0.33 mmol) in
CH2Cl2 (10 mL) was added TFA (5 mL) dropwise at 0 °C, and the
reaction mixture was left to stir for 3 h. The solvent was coevaporated
with Et2O, giving the product as a clear colorless oil. Yield 0.161 g,
1
>99%; TLC (5% MeOH/CH2Cl2) Rf = 0.23. H NMR (600 MHz,
CDCl3) δ 11.02 (br s, CH2COOH), 7.94 (s, 1H, triazole-H), 4.60 (t, J
= 4.9 Hz, 2H, NCH2CH2O), 3.87 (t, J = 4.9 Hz, 2H, NCH2CH2O),
3.80 (t, J = 6.1 Hz, 2H, CH2CH2COOH), 3.68−3.67 (m, 2H, CH2),
3.64−3.60 (m, 6H, CH2), 2.67 (t, J = 6.1 Hz, 2H, CH2CH2COOH).
13C NMR (150.92 MHz, CDCl3) δ 176.4, 131.1, 86.0, 70.5, 70.4, 70.4,
70.3, 69.1, 66.4, 50.9, 34.8. HRMS calcd for C11H19N3O5I [M + H]+,
400.0370; found, 400.0368.
(S)-18-[(S)-2-{[(Benzyloxy)carbonyl]amino}-3-phenylpropanami-
do]-1-(4-iodo-1H-1,2,3-triazol-1-yl)-12,19-dioxo-3,6,9-trioxa-13-
azaicosan-20-yl 2,4,6-Trimethylbenzoate (29). To a solution of 28
(0.037 g, 0.091 mmol) in anhydrous DMF (10 mL), PyBOP (0.047 g,
0.091 mmol) was added and dissolved at 0 °C under a stream of Ar.
Et3N (0.020 mL, 0.182 mmol) was added and the solution was left to
stir for 5 min. Afterward, 6a (0.040 g, 0.057 mmol) was added and the
reaction mixture was stirred and allowed to warm to room temperature
overnight. The solution was made up to 25 mL with water and
extracted with CH2Cl2 (3 × 20 mL), and the combined organic layers
were extracted with water (5 × 10 mL) and brine (3 × 20 mL). The
solvent was evaporated and the product was obtained as a white solid
following flash chromatography (1−20% MeOH/CH2Cl2). Yield 0.22
g, 37%; mp 91−95 °C; TLC (10% MeOH/CH2Cl2) Rf = 0.67; HPLC
(method B) tR = 17.1 min. 1H NMR [600.13 MHz, (CD3)2SO] δ 8.50
(d, J = 7.5 Hz, 1H, NHCHCONH), 7.79 (t, J = 5.6 Hz, 1H,
CH2NHCOCH2), 7.64 (d, J = 8.2 Hz, 1H, NHCHCOCH2), 7.34−
7.18 (m, 10H, Ph), 6.91 (s, 2H, Me3C6H2CO), 4.97 (s, 2H,
PhCH2OCO), 4.91 (d, J = 17.2 Hz, 1H, COCHHO), 4.80 (d, J =
17.2 Hz, 1H, COCHHO), 4.36−4.32 (m, 2H, HNCHCO Phe and
Lys), 3.63−3.56 (m, 4H, COCH2CH2O), 3.47 (m, 12H,
1-Oxo-1-{4-[tris(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-
stannyl]phenyl}-5,8,11-trioxa-2-azatetradecan-14-oic acid (33).
1,3,5,7-Tetramethyl-6-(2,4-dimethoxyphenyl)-2,4,8-trioxa-6-phosphaa-
damantane (4 mg, 15 μmol) and Pd(OAc)2 (2 mg, 7 μmol), each
J
dx.doi.org/10.1021/jm501357r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
dissolved in degassed THF (1 mL), were added to a 2−5 mL
microwave vial purged with Ar and mixed together for 15 min.
Tris(1H,1H,2H,2H-perfluorooctyl)tin hydride (470 mg, 400 μmol)
dissolved in THF (1 mL) was added, and the solution was stirred for
15 min. A solution of 22a (82 mg, 182 μmol) dissolved in THF (1
mL) was then added, and the solution was heated by microwave
irradiation at 80 °C for 20 min. The mixture was filtered through
Celite and the product was isolated as a white wax following reverse-
phase flash chromatography (30−100% CH3CN/H2O; 100% THF).
Determination of Inhibition Constants. Cathepsin B (200 nM)
was preincubated for 30 min at 37 °C in a solution of 5 mM DTT and
0.01% (v/v) Tween-20. The substrate, Cbz-Arg-Arg-pNA (25 or 40
μL), and the AOMKs (50 μL) were added to the wells of a 96-well
microplate containing the assay buffer. The reaction was initiated by
addition of cathepsin B, resulting in substrate concentrations of 500 or
800 μM, inhibitor concentrations from 25 nM to 1 μM, and an
enzyme concentration of 5 nM. Formation of the pNA product was
monitored for 60 min at 405 nm at 37 °C. Measurements were
obtained in triplicate. Absorbance versus time measurements were
analyzed by nonlinear regression to determine the pseudo-first-order
1
Yield 0.095 g, 35%; TLC (5% MeOH/CH2Cl2) Rf = 0.58. H NMR
(600.13 MHz, CDCl3) δ 7.79 (d, J = 8.1 Hz, 2H, ArH), 7.46 (d, J = 8.1
Hz, 2H, ArH), 6.91 (t, J = 0.5 Hz, 1H, NHCH2CH2O), 3.76−3.56 [m,
14H NHCH2CH2O and (CH2OCH2)3], 2.55 (t, J = 6.4 Hz, 2H,
CH2CH2COOH), 2.35−2.29 [m, 6H, Sn(CH2CH2)3], 1.32 [t, J = 8.2
Hz, 6H, Sn(CH2CH2)3]. HRMS calcd for C49H35NO6F39Sn [M + H]+,
1486.0873; found, 1486.0826.
obst
rate constant (kobs) as Abs = Ae−k + B. The second-order rate
constant (ki/Ki), the apparent inactivation rate (ki), and the inhibition
constant (Ki) (where possible) were determined from kobs = ki[I]/(Ki
+ [I]) for hyperbolic relationships or kobs = (ki/Ki)[I] for linear
relationships. Equations were solved by use of GraphPad Prism
software.
(S)-20-[(S)-2-{[(Benzyloxy)carbonyl]amino}-3-phenylpropanami-
do]-1,14,21-trioxo-1-{4-[tris(3,3,4,4,5,5,6,6,7,7,8,8,8-
tridecafluorooctyl)stannyl]phenyl}-5,8,11-trioxa-2,15-diazadoco-
san-22-yl 2,4,6-Trimethylbenzoate (34). To a solution of 33 (38 mg,
26 μmol) in anhydrous DMF (5 mL) was added PyBOP (140 mg, 26
μmol), and the solids were allowed to dissolve at 0 °C under a stream
of Ar. Et3N (7 μL, 52 μmol) was added and the solution was left to stir
for 5 min. Afterward, 6a (21 mg, 30 μmol) and Et3N (4 μL, 26 μmol)
were added and the solution was stirred while warming to room
temperature overnight. Water (20 mL) was added and the solution
was extracted with CH2Cl2 (3 × 10 mL); the combined organic layers
were extracted with water (5 × 10 mL) and brine (3 × 10 mL) and
then dried over Na2SO4. The solvent was evaporated and the product
was obtained as a white wax following semipreparative HPLC (method
B). Yield 6 mg, 12%; TLC (5% MeOH/CH2Cl2) Rf = 0.09; HPLC
(method B) tR = 25 min. 1H NMR [600.13 MHz, (CD3)2SO] δ 8.51−
8.47 (m, 2H, CH2NHCOAr and NHCHCONH), 7.81 (d, J = 8.1 Hz,
2H, NHCOC6H2H2Sn), 7.77 (t, J = 5.6 Hz, 1H, CH2NHCOCH2),
7.62 (d, J = 8.2 Hz, 1H, NHCHCOCH2), 7.60−7.55 (m, 2H,
NHCOC6H2H2Sn), 7.32−7.18 (m, 10H, Ph), 6.89 (s, 2H,
Me3C6H2CO), 4.96 (s, 2H, PhCH2OCO), 4.90 (d, J = 17.2 Hz, 1H,
COCHHO), 4.79 (d, J = 17.3 Hz, 1H, COCHHO), 4.35−4.31 (m,
2H, HNCHCO Phe and Lys), 3.55 (t, J = 6.5 Hz, 2H, COCH2CH2O),
3.51−3.39 (m, 12H, CH2CH2OCH2CH2OCH2CH2), 3.04−3.00 (m,
3H, CHCHHPh and CH2NHCOCH2), 2.81 (dd, J = 13.6, 9.5 Hz, 1H,
CHCHHPh), 2.46−2.37 [m, 6H, Sn(CH2CH2)3], 2.28 (d, J = 6.5 Hz,
2H, COCH2CH2O), 2.24 [s, 6H, 2,6-(CH3)3C6H2CO], 2.23 [s, 3H, 4-
(CH3)3C6H2CO], 1.80−1.77 (m, 1H, CHCHHCH2CH2), 1.59−1.52
(m, 1H, CHCHHCH2CH2), 1.38−1.22 [m, 10H, CHCH2CH2CH2
and Sn(CH2CH2)3]. HRMS calcd for C74H77F39N5O11Sn [M +
NH4]+, 2072.4038; found, 2072.3989.
Screening Studies. Optimization of Enzyme Concentration.
Cathepsin B (500 nM) was preincubated for 30 min at room
temperature in a solution of 5 mM dithiothreitol (DTT) and 0.01%
(v/v) Tween-20. Cathepsin B was added to the wells of a 96-well
microplate containing the assay buffer [25 mM K2PO4, 1 mM
ethylenediaminetetraacetic acid (EDTA), 250 mM NaCl, and 3% (v/
v) DMSO, pH = 6.0], and Cbz-Arg-Arg-pNA (50 μL) was added,
resulting in enzyme concentrations from 1.25 to 25 nM and substrate
concentration of 1 mM. Formation of the p-nitroanilide (pNA)
product was monitored for 73 min at 405 nm at 37 °C. Measurements
were obtained in triplicate. Initial rates (ν0) were measured from the
slope obtained during the first 10 min.
Determination of Km. Cathepsin B (200 nM) was preincubated for
30 min at 37 °C in a solution of 5 mM DTT and 0.01% (v/v) Tween-
20. Cathepsin B (5 μL) was added to the wells of a 96-well microplate
containing the assay buffer. Cbz-Arg-Arg-pNA (50 μL) was then
added, resulting in an enzyme concentration of 5 nM and substrate
concentrations from 125 μM to 3 mM. Formation of the pNA product
was monitored for 15 min at 405 nm at 37 °C. Measurements were
obtained in triplicate. Initial rates (ν0) were measured from the slope
Radiochemical Methods. 125I-(S)-3-[(S)-2-{[(Benzyloxy)-
carbonyl]amino}-3-phenylpropanamido]-7-[6-(4-iodobenzamido)-
hexanamido]-2-oxoheptyl 2,4,6-Trimethylbenzoate ([125I]11a). To a
tube containing iodogen (5 μg, 0.01 μmol) in EtOH (25 μL) was
added 32 (100 μg, 0.05 μmol) in EtOH (100 μL). AcOH (5 μL) and
[
125I]NaI (18.5 MBq) in 0.1% NaOH (10 μL) were added, and after
10 min the reaction was quenched with 0.1 M Na2S2O5 (25 μL). The
reaction mixture was diluted with water (1.5 mL) and loaded onto a
FSPE cartridge (1 g). The FSPE cartridge was preactivated by washing
with dimethylformamide (1 mL) and water (6 mL). Unreacted
[
125I]NaI was eluted with water (10 mL) and the pure product was
eluted with 80% EtOH (10 mL), while the excess precursor remained
on the cartridge. Radiochemical yield (RCY) = 32−36% (n = 3);
t
specific activity >23.6 GBq/μmmol; HPLC R = 14.8 min; log P (pH
7.4) = 1.05 0.01.
125I-(S)-20-[(S)-2-{[(Benzyloxy)carbonyl]amino}-3-phenylpropa-
namido]-1-(4-iodophenyl)-1,14,21-trioxo-5,8,11-trioxa-2,15-diaza-
docosan-22-yl 2,4,6-Trimethylbenzoate ([125I]23a). To a tube
containing iodogen (2.5 μg, 0.005 μmol) in EtOH (25 μL) was
added 34 (100 μg, 0.05 μmol) in EtOH (100 μL). AcOH (5 μL) and
[
125I]NaI (18.5 MBq) in 0.1% NaOH (10 μL) were added, and after
10 min the reaction was quenched with 0.1 M Na2S2O5 (25 μL). The
reaction mixture was diluted with water (1.5 mL) and loaded onto a
FSPE cartridge (1 g). The FSPE cartridge was preactivated by washing
with dimethylformamide (1 mL) and water (6 mL). Unreacted
[
125I]NaI was eluted with water (10 mL) and the pure product was
eluted with 50% EtOH (10 mL), while the excess precursor remained
on the cartridge. RCY = 26−35% (n = 3); specific activity >23.1 GBq/
t
μmmol; HPLC R = 14.1 min; log P (pH 7.4) = 0.95 0.01.
Determination of Log P (pH 7.4). A solution of [125I]23 or [125I]11
(444 kBq) in 10% EtOH in phosphate-buffered saline (PBS, 300 μL)
was added to a 1:1 mixture of n-octanol/PBS (pH 7.4) (2 mL); the
mixture was vortexed for 20 min and centrifuged for 30 min at 6000
rpm. Aliquots (60 μL) were removed from each layer in triplicate and
added to preweighed tubes. The tubes were reweighed and the amount
of radioactivity was measured on a γ counter. The partition coefficient,
P, was calculated as P = activity concentration in n-octanol/activity
concentration in aqueous buffer.
Labeling of Cathepsin B with ABPs. Human liver cathepsin B (1
μg) was incubated in 1× binding buffer (5 mM Tris, pH = 5.5, 5 mM
MgCl2, and 2 mM DTT) containing RIPA buffer (100 mM Tris, pH =
8, 50 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, and
0.1% SDS) in the presence of either of CA-074 or DMSO for 1 h on
ice. [125I]11a or [125I]23a (approximately 1 × 106 counts per minute,
cpm) was added to each sample to give a total volume of 30 μL, and
the mixtures were incubated for 2 h at 0 °C. The assay was stopped by
the addition of 6× sample buffer (9% SDS, 60% glycerol, 375 mM
Tris, pH 6.8, 0.015% bromophenol blue, and 12% β-mercaptoethanol)
to a final concentration of 1× and incubated at room temperature for
30 min. Following incubation, aliquots (30 μL) of the mixtures were
loaded onto a 10% Mini-Protean TGX precast gel and analyzed by
SDS−PAGE. Gels were incubated overnight in gel-shrinking solution
(65% methanol, 0.5% glycerol) while shaking at 4 °C. Gels were dried
obtained during the first 10 min. Km was determined from ν0
=
Vmax[S]/(Km + [S]) and solved by use of GraphPad Prism software.
K
dx.doi.org/10.1021/jm501357r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
for 2 h at room temperature by use of a commercial gel-drying kit and
then exposed to a phosphor imaging screen for 72 h.
triazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate;
SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel
electrophoresis; SPECT, single photon emission computed
tomography; TFA, trifluoroacetic acid
Animal Model. MDA-MB-231 cells were purchased from the
American Type Culture Collection (ATCC) and cultured in
accordance with supplier guidelines. Animal studies were approved
by the Animal Research Ethics Board at McMaster University in
accordance with Canadian Council on Animal Care (CCAC)
guidelines. Female CD1 nu/nu mice (5−6 weeks old) were obtained
from Charles River Laboratories (Senneville, QC) and were
maintained under specific-pathogen-free (SPF) conditions with 12 h
light/dark cycles and given food and water ad libitum. To create the
tumor xenograft model, CD1 nu/nu female mice were injected with
2.0 × 106 MDA-MB-231 cells in 100 μL of PBS/Matrigel (1:1; BD
Biosciences, Mississauga, ON, and Invitrogen, Burlington, ON,
respectively) subcutaneously into the right flank. Tumors were
allowed to grow for approximately 2−3 weeks prior to biodistribution
studies.
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Biodistribution Studies. [125I]11a. CD1 nu/nu mice (n = 15) were
administered approximately 259 kBq of [125I]11a (3.7 GBq/mL) in
10% EtOH in sterile PBS via tail vein injection. After 30 min, 5 h, and
23 h, groups of mice (n = 5) were anesthetized with 3% isoflurane and
euthanized via exsanguination, and the indicated organs and tissues
were collected by dissection and weighed. Activities were measured on
a γ counter and reported as percent injected dose per gram (ID/g) of
each organ, tissue, or fluid.
[
125I]23a. These studies were performed in a similar manner as for
[
125I]11a, except CD1 nu/nu mice (n = 9) were administered
approximately 74 kBq of [125I]23a (3.7 GBq/mL).
ASSOCIATED CONTENT
* Supporting Information
■
S
Four schemes depicting synthesis of 6a/6b, 30, 32, and 34;
three figures showing optimization of enzyme concentration
and determination of Km, Ki, and ki/Ki; additional text with
1
synthetic details; and 151 aqdditional figures with H NMR,
13C NMR, and HRMS spectroscopic data for 1−34. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Telephone 905-525-9140, ext 20182; fax 905-522-2509; e-
mail valliant@mcmaster.ca.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Professor Fred Capretta (McMaster University) for
providing samples of the phosphaadamantane ligand. Financial
support for this research was provided by NSERC (Canada),
Ontario Research Fund, the Canadian Institutes of Health
Research, and the Ontario Institute for Cancer Research
through funding provided by the Province of Ontario.
ABBREVIATIONS USED
■
AOMK, acyloxymethyl ketone; ABP, activity-based probe; Boc,
tert-butoxycarbonyl; Cbz, carboxybenzyl; DMSO, dimethyl
sulfoxide; DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tet-
raacetic acid; DTT, dithiothreitol; FSPE, fluorous solid-phase
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