Cyclopentadienyl-based amino acids (Cp-aa) as phenylalanine analogues for tumor targeting: Syntheses and biological properties of [(Cp-aa)M(CO) 3](M = Mn, Re, 99mTc)

Article abstract of DOI:10.1021/om300695k

Due to an enhanced demand for amino acids, the l-type amino acid transporter 1 (LAT1) is overexpressed in many tumor cell lines. LAT1 represents therefore an attractive target for cancer therapy and diagnosis. On the basis of our reported aqueous synthesis of [(Cp-R)^99mTc(CO)₃]-type complexes,(1-5) we describe the preparation of unnatural amino acid analogues [(Cp-CH₂CH(NH₂)COOH)Mn(CO)₃] and [(Cp-CH(NH₂)COOH)M(CO)₃] (M = Mn, Re, ^99mTc). Starting from fully protected HC₅H₅-aa (aa = amino acid), [(Cp-aa)^99mTc(CO)₃] complexes are accessible in quantitative yields and in a one-step synthesis from [^99mTcO ₄]^-. The rhenium and manganese analogues were prepared and structurally characterized to confirm the authenticity of the ^99mTc complex. The inhibition constant of natural phenylalanine (phe) for LAT1 is in the range 70 ± 10 μM. The K_i value of [(Cp-CH(NH ₂)COOH)Mn(CO)₃] (1a) is 53 ± 11 μM, whereas K_i for the true phe analogue [(Cp-CH ₂CH(NH₂)COOH)Mn(CO)₃] (2) was surprisingly high at 277 ± 37 μM. Complex 1a caused efflux when exposed to cells, underlining its active transport by LAT1 into the cell. ^99mTc analogues of small biological lead structures such as amino acids are generally not recognized anymore by their targets, in particular by trans-membrane transporters. The bioorganometallic analogues presented here are, however, actively transported and corroborate the importance of organometallic complexes as mimics of organic lead structures in life sciences.

Full text of DOI:10.1021/om300695k

Article
pubs.acs.org/Organometallics
Cyclopentadienyl-Based Amino Acids (Cp-aa) As Phenylalanine
Analogues for Tumor Targeting: Syntheses and Biological Properties
of [(Cp-aa)M(CO)3](M = Mn, Re, 99mTc)
Samer Sulieman,† Daniel Can,† John Mertens,‡ Harmel W. Peindy N’Dongo,† Yu Liu,† Paul Schmutz,†
Matthias Bauwens,§ Bernhard Spingler,† and Roger Alberto*,†
†Institute of Inorganic Chemistry, University of Zurich, Winterthurerstraße 190, CH-8057 Zurich, Switzerland
̈
̈
‡Radiopharmaceutical Chemistry, Vrije Universiteit Brussel, Laarbeeklaan103, 1090 Brussel, Belgium
§Nuclear Medicine, NUTRIM, Maastricht University, P. Debeyelaan 25, 6229 HX Maastricht, The Netherlands
S
* Supporting Information
ABSTRACT: Due to an enhanced demand for amino acids,
the L-type amino acid transporter 1 (LAT1) is overexpressed
in many tumor cell lines. LAT1 represents therefore an
attractive target for cancer therapy and diagnosis. On the basis
of our reported aqueous synthesis of [(Cp-R)99mTc(CO)3]-
type complexes,1−5 we describe the preparation of unnatural
amino acid analogues [(Cp-CH2CH(NH2)COOH)Mn(CO)3]
and [(Cp-CH(NH2)COOH)M(CO)3] (M = Mn, Re, 99mTc).
Starting from fully protected HC5H5-aa (aa = amino acid),
[(Cp-aa)99mTc(CO)3] complexes are accessible in quantitative
yields and in a one-step synthesis from [99mTcO4]−. The rhenium and manganese analogues were prepared and structurally
characterized to confirm the authenticity of the 99mTc complex. The inhibition constant of natural phenylalanine (phe) for LAT1
is in the range 70 10 μM. The Ki value of [(Cp-CH(NH2)COOH)Mn(CO)3] (1a) is 53 11 μM, whereas Ki for the “true”
phe analogue [(Cp-CH2CH(NH2)COOH)Mn(CO)3] (2) was surprisingly high at 277 37 μM. Complex 1a caused efflux
when exposed to cells, underlining its active transport by LAT1 into the cell. 99mTc analogues of small biological lead structures
such as amino acids are generally not recognized anymore by their targets, in particular by trans-membrane transporters. The
bioorganometallic analogues presented here are, however, actively transported and corroborate the importance of organometallic
complexes as mimics of organic lead structures in life sciences.
pioneered by, for example, Jaouen and co-workers, is an
additional important feature with regard to structure recog-
nition by receptors.2,5,23−26
INTRODUCTION
■
Among the different modalities for diagnostic molecular
imaging, single photon emission computed tomography
(SPECT) with 99mTc is, quantitatively, still the most important
one and represents roughly 85% of clinically administered
radiopharmaceuticals. For targeted imaging, larger biomolecules
can conveniently be labeled with relatively bulky 99mTc
complexes without substantial loss of affinities.6−11 The labeling
of small biomolecules with 99mTc complexes represents a
distinct challenge since the label often governs the structure,
the biological pathways, and the affinities of the vector. Central
nervous system receptor ligands,12,13 carbohydrates (glu-
cose),14−19 amino acids,20 or pharmaceutically active lead
structures are examples of such small molecules.21,22 Since
complex size plays a crucial role in affecting bioactivity of small
molecules, the compact cyclopentadienyl (Cp) ligand, one of
the smallest tridentate entities, is particularly attractive. Our
recent “one-pot” syntheses of [(Cp-R)99mTc(CO)3]-type
complexes enables the application of Cp in aqueous systems
as a complementation to the generally used Werner-type
ligands. Besides being small, the mimicking of phenyl rings by
piano-stool-type [CpM(CO)3] in organic lead structures, as
We are particularly interested in labeling the α-amino acid
function with 99mTc complexes for targeting the L-type amino
acid transporter 1 (LAT1). LAT1 was shown to be overex-
pressed in many tumor cell lines, such as PA-1 tetratocarcinoma
cells, T24 bladder carcinoma cells, RERF-LC-MA lung small-
cell carcinoma cells, and HeLa uterine cervical carcinoma
cells.27 It plays an essential role in the cellular supply of
important amino acids, needed for cell growth and tissue
development. LAT1 is the transporter for, for example, tyrosine
and phenylalanine, mainly labeled with 18F or 123/125I but rarely
with 99mTc.28−31 Previously, we could show that L-lysine
derivatized with 2,3-diaminopropionic acid (DAP) as ligand at
its ε-position and labeled with the fac-{99mTc(CO)3}+ core was
still recognized by LAT1, albeit at a low Ki = 300 μM, and
actively internalized into cells.20 Despite being the first small
molecule labeled with 99mTc and actively transported through a
Received: July 23, 2012
Published: September 24, 2012
© 2012 American Chemical Society
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Scheme 1. Unnatural Amino Acid Analogues Recognized and Transported by LAT1, DAP Derivative of L-Lysine (left),20 and
Ferrocene Mimic of Phenylalanine (right)37
Scheme 2. Synthesis of Cyclopentadiene-Based Unnatural Amino Acid Precursors Cp-CH(NHR)COOCH3 (R = Bz, Boc) (4a,b)
Scheme 3. Syntheses of the Metal-Based Amino Acids 1a and 1b and the Corresponding −NH2-Protected Analogues 8a and 8b
membrane, Ki values could not be improved by structural
alterations. In order to achieve a better structural match
between the natural substrate phenylalanine and the 99mTc-
labeled mimic, we adopted the phenyl-cyclopentadienyl analogy
and replaced the phenyl ring by piano-stool-type [(Cp-
R)M(CO)3] complexes. A variety of such analogues, e.g.,
[Fc-CH2-CH(NH2)COOH] and others, have been prepared,
but their biological activity has not been investigated.32−37 We
report here the synthesis of organometallic amino acid
analogues [(Cp-CH(NH2)COOH)M(CO)3] (1a, M = Mn,
1b, M = Re, 1c, M = 99mTc) and [(Cp-CH2CH(NH2)COOH)-
Mn(CO)3] (2) and their interactions with LAT1 in comparison
to Fc-ala and the DAP derivative of L-lysine. The synthesis of
the fully protected organic Cp precursor is described together
with labeling studies, yielding [(Cp-CH(NH2)COOH)99mTc-
(CO)3] (1c) in quantitative yields and under moderate
conditions. Since Ki with LAT1 of 1c is slightly lower than
that of phenylalanine, complex 1c represents a true, 99mTc-
labeled amino acid analogue. The basic structures of organo-
metallic amino acid analogues are depicted in Scheme 1.
lowed a previously published procedure and was adapted for
the corresponding Boc-protected molecule 4b.36Accordingly,
fully protected and brominated glycine 3a and 3b were reacted
with TlCp in THF at −78 °C, which gave 4a and 4b in about
1
75% yield (Scheme 2). The H NMR of 4a/b showed an
isomeric mixture of the 2′- or the 3′-substituted products in a
ratio of about 1:1 (Supporting Information, SI). The olefinic
Cp signals appear as multiplets from 6.6 to 6.3 ppm. Notably,
whereas 4a underwent Diels−Alder dimerization to 5 in hexane
within 24 h, such a reaction was not observed for 4b, likely due
to steric constraints as imposed by the bulky Boc protecting
groups. It was not possible to fully deprotect 4a or 4b in order
to access the respective free amino acid form. Cyclopentadiene
is sensitive to the deprotection conditions and underwent cross
reactions such as amination of the double bonds or polymer-
ization. For this reason, labeling studies were performed with
the fully protected amino acid precursors (vide infra).
For structural characterization of the microscopic amounts of
99mTc complexes (t1/2 = 6 h), comparison of HPLC retention
times of the fully characterized Re or Mn homologues with
99mTc complexes is an accepted methodology.38−42 Accord-
ingly, [(Cp-CH(NH2)COOH)Re(CO)3] (1b), [(Cp-CH-
(NHBz)COOH)Re(CO)3] (8a), and [(Cp-CH(NHBoc)-
COOH)Re(CO)3] (8b) were synthesized and characterized
by ESI-MS, 1H NMR, IR, and X-ray crystallography for 1a, 1b,
RESULTS AND DISCUSSION
■
{
The labeling of cyclopentadienyl derivatives with the fac-
99mTc(CO)3}+ core requires either a monomeric precursor or
its Diels−Alder dimer. The preparation of the benzoyl-
protected compound C5H5-CH(NHBz)COOCH3 (4a) fol-
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and 7a (Scheme 3 and SI). The Mn complex 1a was also
synthesized for studying the competitive binding and uptake
with LAT1.
1b is given in Figure 1. The Re center is η5-coordinated to the
C5H4-CH(NH2)(COOH) ligand, and the three CO groups
complete the distorted octahedral coordination geometry. The
structure of the manganese complex 1a is isomorphous to 1b.
All three compounds crystallized in a nonracemic space group.
Complexes 1a and 1b are the models for their 99mTc-labeled
homologues. They serve for the determination of Ki values with
LAT1 and for HPLC confirmation of the 99mTc structure of
complex 1c.
The reaction of 1b with benzoyl chloride or di-tert-butyl-
dicarbonate (Boc2O) gave [(Cp-CH(NHBz)(COOH)Re-
(CO)3] (8a) and [(Cp-CH(NHBoc)(COOH)Re(CO)3]
(8b) in 41% and 95% yield, respectively. Since we expected
that labeling of 4a or 4b under alkaline conditions would result
in ester hydrolysis but not in cleavage of the respective NH2
protecting group, the carboxylate groups in 8a and 8b were not
esterified. Compounds 8a and 8b represent therefore models of
99mTc intermediates during the preparation of 1c. For later
comparison of Ki values, the unnatural amino acids 2 and Fc-
CH2-CH(NH2)COOH were prepared according to literature
methods (Fc = ferrocene).33,44,46
[(Cp-CHO)M(CO)3] 6a,b were synthesized from [CpM-
(CO)3]43 and compounds 7a,b from a modified Strecker
synthesis44,45 as a mixture of enantiomers in 56% yield. The 1H
NMR spectrum of 7b in CDCl3 showed the aromatic Cp
signals as multiplets at 5.69, 5.67, 5.38, and 5.31 ppm,
respectively, instead of the often found pseudotriplets due to
the adjacent stereocenter at the α-position. The enantiotopic α-
proton CH(CN)(NH2) was observed as a singlet at 4.79 ppm
(SI).44,45 Acidic hydrolysis of 7b led to the unnatural amino
acid analogue [(Cp-CH(NH2)(COOH)Re(CO)3] (1b) in 70%
1
yield (Scheme 3). The H NMR spectrum in DMSO showed
the Cp signals as multiplets at 6.02, 5.89, 5.67, and at 5.62 ppm,
respectively. A singlet at 4.88 ppm indicated the α-proton
CH(NH2)(COOH) and a broad singlet the NH2 at 8.73 ppm.
Complexes 1a, 1b, and 7a were crystallized and their structures
elucidated by X-ray analysis (Figure 1 and SI). An ORTEP of
The preparation of Cp complexes in water is not an obvious
method. For complexes comprising 99mTc however, direct
synthesis in water is crucial in light of routine application. As
reported, Diels−Alder dimers of the Cp framework are versatile
starting materials for preparing [(Cp-R)99mTc(CO)3] in
water,3−5,25 although starting directly from the corresponding
monomer would be more convenient. Indeed, some cyclo-
pentadiene derivatives such as 4b dimerize very slowly, while
others, such as 4a, dimerize in relatively short time at rt.
For the formation of [(Cp-CH(NHBz)COOH)99mTc-
(CO)3] (9a), [(Cp-CH(NHBoc)COOH)99mTc(CO)3] (9b),
and [(Cp-CH(NH2)COOH)99mTc(CO)3] (1c), two different
methods were employed (Scheme 4). In the “one-pot” method,
[
99mTcO4]− is mixed with all the components as in the Isolink
Kit and the cyclopentadiene derivatives 4a or 4b. At 90−95 °C
the 99mTc complexes were received in 70−98% yield. In the
“two-step” method, [99mTc(OH2)3(CO)3]+ is prepared in >98%
yield after 30 min at 95 °C and was then mixed with the
corresponding cyclopentadiene derivative. Both methods
exhibited the same labeling behavior and product distribution.
It is remarkable that these organometallic piano-stool
complexes are received under fully aqueous conditions and at
boiling water temperature without relevant side products. It
seems that, for example, product 1c is a strong thermodynamic
Figure 1. ORTEP presentation of [(Cp-CH(NH2)COOH)Re(CO)3]
xHCl (contains one HCl) 1b. Proton of COOH, chloride anion and
water molecule omitted for clarity. Important bond lengths (Å) and
angles (deg) are as follows: Re(1)−C(21) = 1.925(9); Re(1)−C(22)
= 1.929(9); Re(1)−C(23) = 1.918(11); Re(1)−C(1) = 2.282(8);
Re(1)−C(5) = 2.303(8); C(23)−Re(1)−C(21) = 90.5(4); C(23)−
Re(1)−C(22) = 89.8(4); C(21)−Re(1)−C(22) = 89.9(4).
Scheme 4. Two-Step and One-Pot Syntheses of Piano-Stool Complexes with 99mTc Yielding the Ester Hydrolyzed but −NH2-
Protected Acid Precursors 9a and 9b; Complete Deprotection under Mild Acidic Conditions to 1c
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sink, representing the only (stable) product under the synthetic
conditions.
same products (Figure 3). Thus, 1b and 1c are homologues
despite the differences in retention times.
The 99mTc complex 9a was obtained after 30 min in 91%
yield by the “one-pot” method with 4a. The reaction of 4b with
[
99mTc(OH2)3(CO)3]+ at 90 °C for 30 min gave 9b in 80%
yield, and prolonged heating (60 min) increased the yield to
94% for both reactions. Compound 4b reacted directly with
[
99mTcO4]− to give 9b in 77% yield. About 7% Boc
deprotection from 9b occurred during the reaction, which
directly led to 1c (Scheme 4). Prolonged heating (180 min)
increased the yield of 1c (50% after 3 h and >95% after 4 h,
Figure 2). Ester groups were rapidly and completely hydrolyzed
Figure 3. Conversion rates of 9b → 1c (99mTc) and 8b → 1b
(rhenium).
We investigated the biological behavior by quantitatively
assessing the competitive inhibition constant Ki to and
transport by LAT1 as described in the Experimental Section
and SI. Lineweaver−Burk plots were used to study the type of
inhibition of the amino acid analogue from their inhibition
potential of the [3H]-L-phe/L-phe couple. The apparent Km and
corresponding Ki values were calculated by means of Eadie−
Hofstee plots. In addition, as LAT1 is an amino acid antiport
system, cell uptakes of the organometallic amino acids 1a, 2,
and Fc-ala (enantiomeric mixtures) were measured by
monitoring the efflux of [3H]-L-phe, preloaded into the cells
before incubation with substrates. Compound 1a, [(Cp-
CH(NH2)COOH)Mn(CO)3], displayed a Ki = 53 11 μM,
thus, a higher affinity than the natural substrate phe (73 10
μM) itself.20 Unexpectedly, the “true” phe analogue 2, [(Cp-
CH2CH(NH2)COOH)Mn(CO)3], has a Ki value of 277 37
μM, about 4 times higher than phe (Figure 4). The ferrocene-
Figure 2. (A) HPLC γ-trace of the reaction products received after
one-pot reaction (180 min, 90 °C) of [99mTcO4]− with 4b; peak at
16.9 min represents 1c, peak at 22.2 min 9b; (B) co-injection of Re
(bottom-up, 8b) and 99mTc homologue (top-down, 9b); (C) co-
injection of Re (bottom-up, 1b) with 99mTc (top-down, 1c).
in all cases during the labeling procedure. We noted that the
reaction with the Diels−Alder product 5 did not give any
product at all, in sharp contrast to reactions with all other
Thiele’s acid derivatives reported so far.3−5 The relatively bulky
benzoyl protecting group is probably responsible for this
unreactivity.
The authenticity of the 99mTc complexes was confirmed by
comparing the HPLC retention times with their rhenium
homologues. Figure 2A shows the trace as received after the
one-pot reaction of [99mTcO4]− with 4b. The peak at about 22
min represents 9b with the hydrolyzed ester function. The peak
at 16.9 min corresponds to fully deprotected 1c. Both peaks
were separated by HPLC and co-injected with the respective
rhenium homologues (Figure 2B and C). Whereas 8b and 9b
exhibited the same retention times (difference due to detector
separation), 1b and 1c differed by slightly more than one
minute. Occasionally, ReI and 99mTcI homologues are not fully
identical in structure or, for example, lipophilicity, exhibiting
thus different retention times and rendering authenticity
questionable. To corroborate the identity of peaks 1b and 1c
(Figure 2C), we compared the rate of acidic Boc deprotection
in homologues 8b (Re) and 9b (99mTc). If the conversion of 9b
→ 1c corresponds to a reaction not involving Boc deprotection,
thus leading to a compound different from 1b, kinetics should
be different as compared to conversion of 8b → 1b. Therefore,
9b was isolated by HPLC, the pH was adjusted to 2−3, and the
solution was heated to 90 °C for 4 h. The same procedure was
applied to the Re homologue 8b. The rates of conversion for
Re and 99mTc are identical, implying the same Boc deprotection
reaction taking place and, consequently, the formation of the
Figure 4. Lineweaver−Burk plot: competitive binding assays of 1a and
2 to LAT1 (Ki values). The common intercept on the y-axis indicates a
competitive interaction between L-phe and the inhibitors 1a and 2,
meaning they bind to the same binding pocket on the LAT1 transport
sytem.
based amino acid Fc-ala on the other hand showed again a high
Ki value of 60 μM, well comparable with phenylalanine. Since
1a and Fc-CH2-CH(NH2)COOH stimulate efflux of [3H]-L-
phe, comparable to the DAP derivative of L-lysine,20 they are
actively transported into the cells. Quantitatively, the highest
efflux is caused by Fc-ala (25 7%), followed by 1a or the DAP
derivative (≈14
4%). 2 was not actively transported into
cells. We noticed that 2 reduced the efflux stimulating capacity
of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH).
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then allowed to reach room temperature. Solvents were removed
under reduced pressure, and the residue was purified by silica gel
column chromatography (CH2Cl2) to give an isomeric mixture of 4b
as a colorless oil (72%).
This can only point to an antagonistic effect, where 2 binds to
the outward binding sites of the amino acid transport system,
but fails to be actually transported across the membrane. This
in vitro behavior clearly indicates compounds 1a and Fc-ala to
be substrates for LAT1 and not just inhibitors of the protein,
whereas 2 can be regarded as a competitive inhibitor.
1H NMR (500 MHz, d-CDCl3) δ/ppm: 6.52−6.30 (m, 6H, Cp),
5.30 (d, JHH = 16.50 Hz, 2H, NH), 5.20 (d, JHH = 16.50 Hz, 2H, CH),
3.75 (s, 6H, CH3), 3.00 (s, 4H, Cp), 1.48 (s, 18H, Boc). IR (KBr):
1616 cm−1 (−COOMe). Calcd for Mr (C13H19NO4): 253.13. ESI-MS
[m/z] (CH3OH, pos. mode): 276.2 [M + Na]+. Anal. Calcd for
C13H19NO4: C, 61.64; H, 7.56; N, 5.53. Found: C, 61.27; H, 7.31; N,
5.25.
CONCLUSION
■
Small, natural biomolecules such as amino acids generally do
not tolerate structural changes without substantial or, mostly,
complete loss of their biological functions. Replacing a phenyl
ring in such small molecules by cyclopentadienyl complexes
may reduce the structural impact to the lead structure (phenyl-
Cp analogy). This has been shown by the syntheses and the
biological behavior of the organometallic amino acid analogues
[(Cp-CH(NH2)COOH)M(CO)3] (M = Mn, Re, 99mTc). This
compound is recognized by LAT1 with a Ki comparable to the
natural substrate phenylalanine. Since the homologue with
99mTc can be prepared in a one-pot reaction, the corresponding
complex may well serve as a molecular imaging agent for
visualizing rapidly growing cells such as cancer. Corresponding
in vivo investigations are ongoing. We also extend the
previously reported methods for the preparation of cyclo-
pentadienyl complexes of technetium to derivatives that do not
need to be Diels−Alder dimerized but for which the
monomeric precursors can yield piano-stool complexes in
water directly. With this study, we also introduce a new
example for the use of identical compounds for combined
therapy and imaging in a theranostics sense.4,38,39 Cold
rhenium-based compound 1b has potential for therapy
(inhibitor), and the homologous complex with 99mTc 1c can
serve as imaging agents for single photon emission computed
tomography.
[(Cp-CH(NH2)CN)Re(CO)3] (7b). Following a modified Strecker
synthesis,44,45 NaCN (16.33 mg, 0.33 mmol), NH4Cl (8.92 mg, 0.17
mmol), and MgSO4 (20.06 mg, 0.17 mmol) were added to a saturated
solution of NH3 in MeOH (5 mL) and stirred at 0 °C. 6b was added,
and the reaction mixture was stirred for 1 h at room temperature and
for 3 h at 30 °C. The solvent was removed in vacuo, and the residue
was taken into diethyl ether. Filtration and evaporation of the solvent
afforded an orange solid, which was purified by silica gel column
chromatography (diethyl ether/hexane, 4:1) to give 7b (56%).
1H NMR (500 MHz, d-CDCl3) δ/ppm: 5.69−5.65 (m, 2H, Cp),
5.38−5.30 (m, 2H, Cp), 4.79 (s, 1H, CH). IR (KBr): 2022, 1918 cm−1
(Re-CO). Calcd for Mr (C10H10N2O3Re): 393.02. ESI-MS [m/z]
(CH3OH, pos. mode): 415.9 [M + Na]+. ESI-MS [m/z] (CH3OH,
neg. mode): 391.9 [M − H]−. Anal. Calcd for C10H10N2O3Re: C,
30.61; H, 2.57; N, 7.14. Found: C, 30.13; H, 2.27; N, 6.98.
[(Cp-CH(NH3Cl)COOH)Re(CO)3] (1b). 7b (200 mg, 0.45 mmol)
was hydrolyzed by refluxing in HCl (5 mL, 32%). The reaction was
followed by HPLC. After 3 h, the mixture was filtered and extracted
with methyl tert-butyl ether. The aqueous layer was then dried under
reduced pressure, and 1b was obtained (70%).
1H NMR (400 MHz, d6-DMSO) δ/ppm: 8.73 (s, 2H, NH2), 6.02
(s, 1H, Cp), 5.88 (s, 1H, Cp), 5.67 (s, 1H, Cp), 5.62 (s, 1H, Cp), 4.88
(s, 1H, CH). 13C NMR (125 MHz, d6-DMSO) δ/ppm: 168.48 (CO),
101.91 (COOH), 99.66 (Cp1), 93.43 (Cp2), 87.35 (Cp3), 87.00
(Cp4), 83.11 (Cp5), 81.02 (Cp-CH). IR (KBr): 2021, 1941, 1908
cm−1 (Re-CO), 1636 cm−1 (−COOH). Calcd for Mr (C10H8NO5Re):
408.38. ESI-MS [m/z] (CH3OH, pos. mode): 392.9 [M − NH2 +
H]+, 431.9 [M + Na]+. ESI-MS [m/z] (CH3OH, neg. mode): 407.9
[M − H]−, 364.0 [M + COOH]−, 443.8 [M + Cl]−. Anal. Calcd for
C10H8NO5Re: C, 27.00; H, 2.04; N, 3.15. Found: C, 26.94; H, 2.01; N,
3.09.
[(Cp-CH(NHBz)COOH)Re(CO)3] (8a). A solution of 1b (50 mg,
0.112 μmol) in NaOH (2.0 M, 25 mL) was cooled to 0 °C with an ice
bath. Benzoyl chloride (15 μL, 124 μmol) was added, and the reaction
mixture was allowed to warm to room temperature. After 2 h, the pH
was adjusted to 5−6 by adding HCl (1.0 M), and the reaction mixture
was extracted with ethyl acetate (3 × 25 mL). The combined organic
phases were dried over magnesium sulfate and evaporated in vacuo.
The solid residue was purified by silica gel column chromatography
(EtOAc/hexane, 2:1) to afford 8a (41%).
EXPERIMENTAL SECTION
■
General Procedures. Reactions were carried out in dried
glassware under a N2 atmosphere. Solvents were dried using standard
techniques and stored over molecular sieves. All chemicals were
obtained from commercial sources and used without further
purification. Compounds 4a, 5,36 methyl 2-bromo-2-((tert-
butoxycarbonyl)amino)acetate,47 and rhenium complex 7b43 were
prepared according to literature procedures. Manganese complexes
(1a, 6a, 7a) were prepared in the same way as the corresponding
1
rhenium complexes. H and 13C NMR spectra were recorded on a
Bruker Advance 400 or 500 MHz spectrometer. Mass spectra were
measured on a Bruker Esquire HCT (ESI) instrument. Elemental
analyses were performed on a Leco CHNS-932 elemental analyzer. IR
spectra were recorded as KBr pellets on a Perkin-Elmer BX II IR
spectrometer.
1H NMR (400 MHz, d4-MeOH) δ/ppm: 7.88−7.85 (m, 2H, Ar),
7.57−7.52 (m, 1H, Ar), 7.49−7.44 (m, 2H, Ar), 5.93−5.84 (m, 2H,
Cp), 5.54 (s, 1H, CH), 5.45−5.39 (m, 2H, Cp). IR (KBr): 2020, 1921
cm−1 (Re-CO), 1696, 1642 cm−1 (−COOH). Anal. Calcd for
C17H12NO6Re: C, 39.84; H, 2.36; N, 2.73. Found: C, 39.63; H,
2.27; N, 2.56.
[(Cp-CH(NHBoc)COOH)Re(CO)3] (8b). NaHCO3 (56.7 mg, 674.4
μmol) and di-tert-butyl dicarbonate (58.9 mg, 269.8 μmol) were added
consecutively to a solution of 1b (100 mg, 224.8 μmol) in a 1:1
mixture of THF/H2O (10 mL) at 0 °C. After 45 min, the solution was
allowed to reach room temperature and was stirred for 18 h. The
solution was then extracted two times with methyl tert-butyl ether. The
aqueous layer was acidified to pH = 4−5 with 0.1 M HCl at 0 °C and
then extracted two times with CH2Cl2. The combined CH2Cl2 phases
were dried over MgSO4 and evaporated in vacuo to give 8b (95%).
1H NMR (400 MHz, d4-MeOH) δ/ppm: 5.73−5.70 (m, 2H, H),
5.35−5.32 (m, 2H, H), 3.35 (s, 1H, H), 1.46 (s, 9H, H). IR (KBr):
2016, 1926, 1893 cm−1 (Re-CO), 1636, 1614 cm−1 (−COOH). Anal.
RP-HPLC was performed on a Merck Hitachi LaChrom L7200
tunable UV detector and a radiodetector, separated by a Teflon tube,
which causes about a 0.4−0.7 min delay compared to UV/vis
detection. UV/vis detection was performed at 250 nm. The detection
of radioactive 99mTc complexes was performed with a Berthold LB 507
radiodetector equipped with a NaI(Tl) scintillation detector.
Analytical separations were performed on a Nucleosil C-18 column
(100 Å, 5 μm, 250 × 4 mm). Analytical columns were eluted with a
flow rate of 0.5 mL min−1 using 0.1% TFA in H2O (solvent A) and
methanol (solvent B) as eluents with a variable gradient (0−3 min,
100% A; 3−3.1 min, 0 to 25% B; 3.1−9 min, 25% B; 9−9.1 min, 25%
B to 34% B; 9.1−20 min, 34% B to 100% B; 20−25 min, 100% B; 25−
25.1 min, 100% B to 100% A; 25.1−30 min, 100% A).
[Cp-CH(NHBoc)COOCH3] (4b). According to the literature
procedure,36 4b was synthesized as follows: CpTl (435.8 mg, 1.62
mmol) was added to the THF (10 mL) solution of 3b (412 mg, 1.54
mmol) at −78 °C. The mixture was stirred in the dark for 5 h and was
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Article
Calcd for C15H16NO7Re: C, 36.50; H, 4.21; N, 2.66. Found: C, 36.37;
H, 4.15; N, 2.46.
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AUTHOR INFORMATION
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Corresponding Author
*E-mail: ariel@aci.uzh.ch.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank University of Zurich and the Atomic Energy
Commission of Syria (AECS) (grant no. 513/2010) for
̈
financial support.
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