Cu-free 1,3-dipolar cycloaddition click reactions to form isoxazole linkers in chelating ligands for fac -[MI(CO)3]+ centers (M = Re, 99mTc)

Article abstract of DOI:10.1021/ic402825t

Isoxazole ring formation was examined as a potential Cu-free alternative click reaction to Cu^I-catalyzed alkyne/azide cycloaddition. The isoxazole reaction was explored at macroscopic and radiotracer concentrations with the fac-[M^I(CO)₃]⁺ (M = Re, ^99mTc) core for use as a noncoordinating linker strategy between covalently linked molecules. Two click assembly methods (click, then chelate and chelate, then click) were examined to determine the feasibility of isoxazole ring formation with either alkyne-functionalized tridentate chelates or their respective fac-[M^I(CO)₃]⁺ complexes with a model nitrile oxide generator. Macroscale experiments, alkyne-functionalized chelates, or Re complexes indicate facile formation of the isoxazole ring. ^99mTc experiments demonstrate efficient radiolabeling with click, then chelate; however, the chelate, then click approach led to faster product formation, but lower yields compared to the Re analogues.

Full text of DOI:10.1021/ic402825t

Communication
pubs.acs.org/IC
Cu-Free 1,3-Dipolar Cycloaddition Click Reactions To Form Isoxazole
Linkers in Chelating Ligands for fac-[MI(CO)3]+ Centers (M = Re, 99mTc)
Shalina C. Bottorff,† Benjamin B. Kasten,† Jelena Stojakovic,‡ Adam L. Moore,† Leonard R. MacGillivray,‡
and Paul D. Benny*,†
†Department of Chemistry, Washington State University, P.O. Box 644630, Pullman, Washington 99164, United States
‡Department of Chemistry, University of Iowa, Iowa City, Iowa 52242-1294, United States
S
* Supporting Information
building strategy (click to chelate) to incorporate 1,2,3-triazole as
ABSTRACT: Isoxazole ring formation was examined as a
potential Cu-free alternative click reaction to CuI-catalyzed
alkyne/azide cycloaddition. The isoxazole reaction was
explored at macroscopic and radiotracer concentrations
with the fac-[MI(CO)3]+ (M = Re, 99mTc) core for use as a
noncoordinating linker strategy between covalently linked
molecules. Two click assembly methods (click, then chelate
and chelate, then click) were examined to determine the
feasibility of isoxazole ring formation with either alkyne-
functionalized tridentate chelates or their respective fac-
[MI(CO)3]+ complexes with a model nitrile oxide
generator. Macroscale experiments, alkyne-functionalized
chelates, or Re complexes indicate facile formation of the
isoxazole ring. 99mTc experiments demonstrate efficient
radiolabeling with click, then chelate; however, the chelate,
then click approach led to faster product formation, but
lower yields compared to the Re analogues.
part of a chelate system in peptides/small molecules for targeted
in vivo delivery when subsequently complexed with fac-
[MI(CO)3]+.8,12−18 While our group has demonstrated the
potency of the CuAAC reaction to couple fac-[MI(CO)3]+
complexes under mild conditions (15 min, 37 °C) using 1,2,3-
triazole as a linker between the metal and targeting vector,
uncoordinated 1,2,3-triazole can impact the coordination mode
and stability of the complex.12,17
Since their structural determination by Claisen and Stock,19
isoxazoles have been widely used in therapeutics (i.e., anticancer,
immune suppressor, cardiovascular) as agonists and antagonists
because of their hydrolytic stability, π-stacking, and hydrogen-
bonding capacities.20 Similar in molecular shape, volume, and
electron distribution, isoxazoles present a potential alternative
for 1,2,3-triazoles. Analogous to CuAAC, isoxazoles are prepared
from an alkyne and nitrile oxide in good-to-excellent yields
without the presence of Cu.21−23 Nitrile oxide is generated from
a reactive oxime or from in situ activation of an oxime with
chloramine-T or N-chlorosuccinimide.24,25
he CuI-catalyzed azide/alkyne cycloaddition (CuAAC)
In this work, the isoxazole 1,3-dipolar cycloaddition was
explored as a linker-based click reaction with fac-[MI(CO)3]+.
The isoxazole orientation was designed to exclude N-
coordination of the isoxazole ring to the metal center, unlike
N-coordinated bidentate bis(isoxazole) chelates with fac-
[ReI(CO)3]+.22 Two strategies, click, then chelate and chelate,
then click, were utilized to investigate the versatility of the
cycloaddition reaction between phenylchlorooxime and a
terminal alkyne tethered to a tridentate chelate. In the click,
then chelate method, the isoxazole click reaction was conducted
prior to chelation with fac-[MI(CO)3]+, while alkyne-tethered
fac-[MI(CO)3]+ complexes were utilized in the chelate, then click
method.
T
reaction to yield 1,2,3-triazole has become analogous with
the phrase “click chemistry” despite limitations (i.e., toxicity,
transchelation) from residual Cu that require additional
purification steps for biological applications.1 To circumvent
the use of a CuI catalyst, Cu-free reactions have been developed
with sterically strained cyclooctynes to perform an analogous
cycloaddition reaction.2−4 However, these reactive alkynes
consist of bulky, lipophilic, and high-molecular-weight molecules
that may adversely impact the pharmacokinetic behavior,
particularly with small molecules and peptide drugs. Alternative
approaches to CuAAC are being explored with other reactive
group pairs that maintain specificity, eliminate a metal catalyst,
and avoid bulky functional groups incorporated into the
assembled or click product.5−7
The general preparation of the ligands is depicted in Scheme 1.
Several of the precursor compounds (1−4) were synthesized as
previously reported.12,17,25 Formation of the isoxazole linkers (5
and 6) was carried out at room temperature with 2 equiv of 4 in
the presence of a weak base in moderate-to-good yields (45 and
In recent years, the CuAAC reaction has gained particular
prominence as a rapid assembly technique for radiopharmaceut-
ical preparation through the pairing of fast reaction times with
the short half-lives of diagnostic and therapeutic radionuclides to
incorporate these probes into biological targeting molecules.8−11
In particular, the CuAAC reaction has been extensively utilized
with group VII congeners, diagnostic 99mTc (t1/2 = 6.0 h; γ = 140
keV), and radiotherapeutic 186/188Re (β−, 1.071 and 2.118 MeV).
Several CuAAC strategies have been developed for the
organometallic fac-[MI(OH2)3(CO)3]+ (M = Re, 99mTc)
precursor. Schibli and Mindt pioneered an innovative chelate
1
50%, respectively). H NMR confirmed cycloaddition by the
disappearance of the alkyne triplet and the appearance of the
isoxazole proton (6.63 and 6.79 ppm, respectively) and
additional aromatic resonances that correlated with the 13C
Received: November 11, 2013
© XXXX American Chemical Society
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Scheme 1. Synthetic Strategy for Generating Isoxazole
Ligands and fac-[MI(CO)3]+ Complexes
Figure 1. X-ray structure of 9 with H atoms omitted for clarity.
Ellipsoids are drawn at 30% probability. Selected bond lengths (Å):
Re1−O4 2.125(4), Re1−N4 2.172(4), Re1−N2 2.233(4), Re1−C2
1.909(5), Re1−C3 1.915(5), Re1−C1 1.914(6). Selected bond angles
(deg): O4−Re1−N1 78.4(1), O4−Re1−N2 78.6(1), O4−Re1−C2
174.7(2), O4−Re1−C3 96.8(2), O4−Re1−C1 94.7(2), N1−Re1−N2
77.1(1), N1−Re1−C2 97.7(2), N1−Re1−C3 173.4(2), N1−Re1−C1
96.4(2), N2−Re1−C2 97.1(2), N2−Re1−C3 97.5(2), N2−Re1−C1
171.4(2), C2−Re1−C3 86.7(2), C2−Re1−C1 89.3(2), C2−Re1−C1
88.6(2).
chelate (Table S1 in the Supporting Information, SI).12 The
structure clearly confirms noncoordination of the isoxazole linker
with the Re center. IR analysis gave the expected CO peaks for
fac-[ReI(CO)3]+ complexes12,17 and showed a shift in the IR of
the isoxazole ring peak (from 1620 cm−1 in 5 to 1611 cm−1 in 8
and from 1620 cm−1 in 7 to 1609 cm−1 in 9). MS analyses of 8
and 9 were consistent with the anticipated structures and Re
splitting pattern at m/z 627.2 and 594.2, respectively.
a
Methyl ester deprotection of 2 and 6 with LiOH/methanol (1:3) was
conducted prior to complexation with fac-[MI(OH2)3(CO)3]+.
NMR data. Deprotection of 6 was carried out through base
hydrolysis to produce 7 in reasonable yields (49%) after
isolation. The synthesis of 7 was confirmed by 1H NMR analysis,
demonstrating loss of the methyl ester signal of 6 at 3.73 ppm and
loss of CH3 in mass spectrometry (MS) analysis. 7 could also be
directly prepared from 3 in significantly lower yields (<10%).
The possible flexidentate nature of the ligands was examined
using two synthetic approaches (click, then chelate and chelate,
then click) at macroscopic concentrations to inspect speciation of
the fac-[ReI(CO)3]+ products based on assembly of the ligand
(Scheme 1). The click, then chelate strategy was centered on
formation of the full ligands prior to complexation with the metal
centers. Reaction of the ligands (5 and 7) with fac-
[ReI(OH2)3(CO)3]+ yielded one product each (8 and 9)
regardless of the pH conditions. Complexation of the ligand
was carried out at room temperature (5) and 40 °C (7) for 12 h,
producing 8 and 9 in excellent-to-moderate yields (75 and 59%,
respectively). 1H NMR analysis of 8 and 9 exhibited a downfield
shift in all resonances relative to the free ligand. In 8, the adjacent
pyridine CH2’s exhibit a symmetric AB quartet splitting,
indicating that both pyridines are coordinated to the metal
center.17 The CH2 adjacent to the isoxazole ring was observed as
a singlet at 5.14 ppm, indicating that isoxazole is not involved in
coordination with the metal. In 9, similar shifts and splitting
patterns were also observed upon metal complexation. However,
the asymmetric nature of the chelate impacted the splitting
patterns of the CH2’s adjacent to the tertiary amine. The three
different CH2’s exhibited AB quartet splitting patterns, indicating
a unique magnetic environment for each proton as expected
because of the asymmetric nature of the chelate.12 The ambiguity
of the NMR splitting patterns does not clearly elucidate the
coordination environment of the metal center; consequently, X-
ray crystallography was used to determine the structure. Single
crystals of 9 were grown by slow evaporation of a dichloro-
methane/methanol (2:1) mixture for X-ray diffraction analysis
(Figure 1).26 Re bond distances (Å) and angles (deg) are
analogous to those of previously reported complexes with this
The chelate, then click approach was examined to determine if
the metal center would have any steric or electrochemical
interactions that would perturbate isoxazole formation. An
increased reactivity in CuAAC reactions performed with 10 and
11 was proposed because of the presence of fac-[MI(CO)3]+;
similar effects due to the metal center may also impact the
isoxazole reaction.12,17 In both cycloaddition reactions with 4 in
the present study, the corresponding Re complexes (8 and 9)
were formed in moderate-to-good yields after isolation.
Analytical characterization data of 8 and 9 prepared by the
chelate, then click approach from 10 and 11 corresponded to the
data obtained via the click, then chelate method from 5 and 7,
showing that both strategies successfully produced the final Re
complexes. The two synthetic approaches (click, then chelate and
chelate, then click) were also examined at radiotracer levels. In the
click, then chelate approach, assessment of the speciation and
complexation of 5 and 7 with fac-[99mTcI(OH2)3(CO)3]+ was
achieved by varying the ligand concentrations (10−3−10−5 M) at
constant temperature and reaction time (70 °C, 30 min). The
chromatograms of 8A and 9A exhibited a single peak that
correlated with the tR of the corresponding Re analogues (Figure
2). Compounds 5 and 7 displayed labeling efficiencies similar to
those of previously reported systems.12,17 Evaluation of the
stability of the metal complexes and isoxazole linker were
examined using cysteine (1 mM) and histidine (1 mM) in a
phosphate buffer (10 mM, pH 7.4). The challenge assays showed
>99% stability of 8A and 9A through 24 h, with both amino acids
indicating minimal transchelation or coordination rearrange-
ment correlating with Re stability studies (Table S2 in the SI).
Evaluation of the chelate, then click method gave unanticipated
results with 99mTc compared to the Re analogues. The reaction of
10A with 4 rapidly produced 8A with tR similar to that of 8 in
∼60% yield after 2.5 min at room temperature. In efforts to
B
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Inorganic Chemistry
Communication
versity for financial assistance. We greatly appreciate Dr. Mary
Dyszlewski at Covidien for providing the IsoLink kits.
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S
* Supporting Information
Experimental details. This material is available free of charge via
the Internet at http://pubs.acs.org.
(26) Crystal data for 9, C21H16N3O6Re,CH4O, Mr = 624.61 g mol−1,
triclinic, space group P1, a = 8.1342(8) Å, b = 12.5836(13) Å, c =
̅
12.8618(13) Å, α = 117.169(5)°, β = 101.227(5)°, γ = 97.439(5)°, V =
1112.28 Å3, T = 293(2) K, Z = 2, μ(Mo Kα) = 5.512 mm−1, 9749
reflections measured, 5274 independent reflections (Rint = 0.029). The
final R(F) values were 4562 [I > 2σ(I)]. The final Rw(F2) values were
0.066 [I > 2σ(I)]. The final R(F) values were 0.044 (all data). The final
Rw(F2) values were 0.071 (all data). The goodness of fit on F2 was 1.051.
CCDC deposition number: 962881.
AUTHOR INFORMATION
Corresponding Author
*E-mail: bennyp@wsu.edu.
Notes
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The authors declare no competing financial interest.
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Taylor, J. P. Org. Biomol. Chem. 2008, 6, 787−796.
ACKNOWLEDGMENTS
■
The authors thank the Office of Science, U.S. Department of
Energy, Radiochemistry and Radiochemistry Instrumentation
Program (Grant DE-FG02-08ER64672), NIH/NIGMS (Institu-
tional Award T32-GM008336), and Washington State Uni-
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dx.doi.org/10.1021/ic402825t | Inorg. Chem. XXXX, XXX, XXX−XXX