Multifunctional Cyclopentadienes as a Scaffold for Combinatorial Bioorganometallics in [(η5-C5H2R1R2R3)M(CO)3] (M=Re, 99mTc) Piano-Stool Complexes

Article abstract of DOI:10.1002/chem.201801271

Multifunctional cyclopentadiene (Cp) ligands and their rhenium and ^99mTc complexes were prepared by a versatile synthetic route. The properties of these Cp ligands can be tuned on demand, either during their synthesis (variation of R₁) or through post-synthetic functionalization with two equal or different vectors (V₁ and V₂). Variation of these groups enables a combinatorial approach in the synthesis of bioorganometallic complexes. This is demonstrated by the preparation of Cp ligands containing both electron-donating and electron-withdrawing groups at the R₁ position and their subsequent homo- or heterofunctionalization with biovector models (benzylamine and phenylalanine) under standard amide bond-formation conditions. All ligands can be coordinated to the fac-[Re(CO)₃]⁺ and fac-[^99mTc(CO)₃]⁺ cores to give tetrafunctional complexes in straightforward and functional-group-tolerant procedures. The ^99mTc complexes were prepared in one step, in 30 min, and under aqueous conditions from generator-eluted [^99mTcO₄]^?.

Full text of DOI:10.1002/chem.201801271

A Journal of
Accepted Article
Title: Multifunctional Cyclopentadiene as a Scaffold for Combinatorial
Bioorganometallics in [(η5-C5H2R1R2R3)M(CO)3] (M = Re,
99mTc) Piano-stool Type Complexes
Authors: Angelo Frei, Bernhard Spingler, and Roger Alberto
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Multifunctional Cyclopentadiene as a Scaffold for Combinatorial
Bioorganometallics in [(η5-C5H2R1R2R3)M(CO)3] (M = Re, 99mTc)
Piano-stool Type Complexes
Angelo Frei*,[a] Bernhard Spingler[a] and Roger Alberto[a]
Abstract: Multifunctional cyclopentadiene ligands and their rhenium
and 99mTc complexes were prepared along a versatile synthetic route.
The properties of these Cp-ligands can be tuned on demand, either
during their synthesis (variation of R1) or through post-synthetic
functionalization with two equal or different vectors (V1 and V2).
Variation of these groups enables a combinatorial approach in the
synthesis of bioorganometallic complexes. This is demonstrated by
the preparation of Cp-ligands containing both electron-donating and
withdrawing groups at the R1 position and their subsequent homo- or
hetero-functionalization with biovector models (benzylamine and
phenylalanine) under standard amide bond formation conditions. All
ligands can be coordinated to the fac-[Re(CO)3]+ and fac-
still core in Single Photon Emission Tomography (SPECT)[6] due to its
half-life (t1/2 = 6.02 h), decay characteristics and, of growing
importance, its price.[7] Since 2001 only a limited number of new 99mTc
imaging agents have been introduced into clinical routine.[8] This
mirrors the challenges encountered when aiming at introducing a well-
defined transition metal complex into a targeting molecule in
quantitative yields and in one-step as required for routine clinical
application.[9] The preparation of fac-[99mTc(OH2)3(CO)3]+ was a step in
this direction and a wide variety of complexes based on the fac-
[
99mTc(CO)3]+ core have been explored ever since.[5a, 10] Recently, a
PSMA targeting biovector coupled to the tricarbonyl core made it into
phase III clinical trials.[11] The general concept of targeted radiotracers
is the conjugation of a receptor-binding molecule with a ligand for
coordination to the respective metal centre or complex fragment, also
referred to as the bifunctional ligand or pendent approach.
[
99mTc(CO)3]+ core, leading to tetra-functional complexes via straight-
forward and functional-group tolerant procedures. Importantly, the
99mTc complexes were prepared in one-step, in 30 min and under
aqueous conditions from generator eluted [99mTcO4]-.
Introduction
The combination of organometallic complexes with biologically active,
organic molecules or fragments has become an intensely researched
field in medicinal inorganic chemistry. These bioorganometallic
complexes play a distinct role and benefit from a series of properties
different from coordination compounds.[1] They may form integrated
substructures in pharmaceutically active lead compounds such as in
the tamoxifen/ferrocifen couple,[2] exert cytotoxicity through metal-
mediated interactions with proteins or nucleic acids as in the case of
the [(η6-arene)Ru]2+ fragment[3] or form de novo parts as e.g. in
bioorganometallic kinase inhibitors.[4] Ferrocene (Fc) represents one
of the main building blocks in this field and the cyclopentadienyl (Cp)
ligand plays an important role in all Fc-based bioorganometallic
concepts. The majority of Cp-based bioorganometallic complexes
comprises one single additional functionality, a pendent targeting
moiety, a cytotoxic or otherwise biologically active agent. Cp-ligands
are not only found in ferrocene but also in Re/99mTc piano-stool type
complexes. We recently reported two examples in which such
Re/99mTc complexes were conjugated to carbonic anhydrase inhibitors
or cytotoxic doxorubicin.[5] Since biological activities of group 7 piano-
stool complexes are rather overall structure- than metal-based, a
combination of rhenium and 99mTc complexes is a complementary
strategy in theranostics; the rhenium complex for therapy and its 99mTc
homologue for imaging. This combination is interesting since 99mTc is
Figure 1. Overview of the different poly-modalities accessible with the new
multifunctional cyclopentadienyl ligands.
Current pharmaceutical research aims at combining multiple
modalities such as targeting and cytotoxicity, in one single molecule.
This approach has not yet found the consideration it deserves in
bioorganometallic chemistry, probably since the modalities are ideally
conjugated to one single ligand. With respect to imaging, the concept
is further affected by the fact that a multi-modality 99mTc complex must
be synthesized in one single step from [99mTcO4]- and in water.
Cp-ligands represent a scaffold to which multiple functionalities can
be bound according to elaborate organic synthetic strategies. In
addition, Cp is a small and strongly binding ligand with a low molecular
weight. After coordination to the fac-[M(CO)3]+ core, the complexes
are highly inert and of zero charge. Our group reported a one-pot
synthesis of singly functionalized {(η5-C5H5)99mTc} complexes in water
under moderate heating.[12] By derivatizing di-cyclopentadiene
“Thiele’s acid” through amid bond formation, chemically stable and
functionalized Cp-precursors and complexes of the type [(η5-
C5H4R)99mTc(CO)3] became accessible.[5a, 13]
[a]
Prof. R. Alberto, Prof. B. Spingler, A. Frei
Department of Chemistry
University of Zurich
Winterthurerstr. 190, CH-8057 Zurich, Switzerland
E-mail: angelo.frei@chem.uzh.ch
Supporting information for this article is given via a link at the end of
the document.
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The bifunctional ligand approach, with or without Cp, does
not immediately comprise the option of combining multiple,
different or equal, targeting vectors. Multimeric molecular
imaging agents would offer many new opportunities such as
targeting of different sites, enhancing binding affinities
and/or addition of a cytotoxic payload. Only a few multimeric
molecular 99mTc imaging agents have been reported thus
far, mainly by Liu et al. on integrin αvβ3 targeting multimeric
cyclic-RGD peptide oligomers.[14] For Cp, all approaches
described above are synthetically incompatible with multi-
functionalization as required for multimeric targeting agents.
We reported recently the synthesis of water soluble
Re/99mTc complexes with a Cp-ligand for coordination to the
Scheme 1. General synthesis of multifunctional Cp-ligands (top) and the synthetic strategy
pursued in this study (bottom): A) variation of R1 to modify the chemical properties of the Cp.
B) variation of the conjugated vectors (V1, V2) to modify biological properties of the Cp.
{
99mTc(CO)3}+ core and two carboxylato groups in 1,2-
position for potential conjugation to two biomolecules.[15] In
thereby constant with the option of conjugating a vector V2 to this
position.
this study, we extend the flexibility of the multi-functional Cp-approach
to a C5H3R1R2R3-type scaffold, which can be mono- (Figure 1A) or
homo (Figure 1B) and hetero (Figure 1B) bi-functionalized with
respect to targeting molecules or other biovectors. The choice of
biovectors or molecular functionalities attached to the central Cp-core
is highly flexible which allows for a systematic exploration of new
imaging compounds. The ligands as such do not undergo Diels-Alder
dimerization and are water and air-stable at room temperature and up
to 220 °C. Mono-, homo- and hetero-functionalization of the ligand is
shown with model vectors on both the free ligand and the “cold”
rhenium complexes. The corresponding 99mTc complexes were
The (CH2)2-spacer in R2 was introduced in this building block design
to reduce interference of the terminal ester group with the reactivity of
the α-ketone. Furthermore, this spacer suppresses the undesired
rearrangement of the Cp-double bond in the final ligand, as previously
observed in our group in mono-functional Cp’s with one CH2 group
between Cp and the ester group. The ester group on the
phosphonium building block (R4, green in Scheme 1) was also kept
constant and will enable the conjugation of a 2nd bioactive moiety V1
to the Cp-scaffold after cyclization. In its essence, the approach
towards multi-functionalized Cp derivatives focuses on the building
block A in Scheme 1, prepared from ethyl 5-bromo-4-oxopentanoate
and phosphonium salts with a methyl (1a), a phenyl (4a) and a
methoxy (6a) group as R1, red in Scheme 1 to show that both electron
donating and withdrawing groups can equally be bound to the Cp-ring.
Introduction of V1 and V2 will lead to the trifunctional Cp derivatives as
shown with B in Scheme 1. Since metal complexes with Cp-ligands of
type A have already been reported for iron[17], ruthenium[18], cobalt[19]
and rhodium[17a, 20], we suggest that Cp derivatives of type B can
equally be applied to other metal centres.
prepared in
a one-pot reaction from [
99mTcO4]- and in high
radiochemical yields. Altogether, we present a ligand platform that
allows for the synthesis of tetra-functional Re/99mTc complexes but
also for other elements. Combinatorial variation of all pendent groups
will lead to a wide variety of structurally different but functionally
identical matched-pair complexes.
Results and Discussion
Design of Cp-Ligands. The syntheses of the multi-functional
cyclopentadiene ligands in this study as shown in Figure 1 are based
on a procedure by Hatanaka et al.[16]. A variety of triphenyl-
phosphonium salts with different substituents R1
react with α-bromo-ketones under mild conditions to
form triply substituted Cp’s in good yields. The most
general concept towards such multi-functionalized
Cp's is shown in Scheme 1 (top). The carboxylate
group directly attached to the Cp-ring (R4, green in
Scheme 1) and the terminal carboxylate in R2 (blue)
allow for later conjugation of two equal or different
biological functions V1 and V2. This synthetic
approach thus enables a versatile alteration of three
functionalities; R1 for guiding the chemical properties
and the two carboxylate groups for conjugation of
bioactive functionalities (Scheme 1, A and B). The
original literature also highlights the possibility to
vary R2 and to introduce further functionalities at R3,
while modifications of R4 and R5 are also
conceivable.
Scheme 2. Synthetic route to the Cp-ligands 1a, 4a, 6a. Reaction conditions: a) Br2, MeOH, 0 °C,
24 h, 23%. b) cat. H2SO4, EtOH, reflux, overnight, 87%. c) NBS, cat. AIBN, MeOAc, MW; 110 °C,
15 min (1.2, 83%); 110 °C 12 min (4.2, 97%); 85 °C, 30 s (6.2, 73%). d) PPh3, dry toluene, 25 °C,
24 h (1.3, 84%; 4.3, 65%; 6.3, 54%). e) 0.1, DCM/sat. NaHCO3, 25 °C, 48 h (1a, 66%; 4a, 27%; 6a,
23%). f) Trimethoxymethane, H2SO4, 25 °C, 25 h, 82%.
We did not alter all these positions on the central
cyclopentadiene scaffold but choose ethyl 5-bromo-
4-oxopentanoate (0.1 Scheme 2) as the standard α-
bromo-ketone for the bi-functionalization, keeping R2
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Synthesis and Characterization. The ligands 1a, 4a and 6a
represent the cores of the later trifunctional Cp-systems. These
precursors were synthesized following the reported procedure with
minor changes to improve yields and isolations (see Supplementary
Bis-hetero functionalization of the Cp-ligand 1a: To assess the full
potential of this approach and to open a scope towards two different
functions V1 and V2, i.e. bis-hetero functionalized Cp-ligands and
complexes, the individual carboxylato groups must be conjugated
Information for details).[16] Notably, the radical brominations of 1.1, 4.1, stepwise to biovectors. We exemplify this strategy with the Cp ligand
and 6.1 (Scheme 2) to yield 1.2, 4.2 and 6.2 were carried out under
microwave conditions in less than 15 min (30 s at 85 °C for 6.1). The
harmful solvent CCl4 could be replaced by methylacetate without loss
of performance. Subsequent reactions with triphenyl-phosphine and
cyclizations with 5-bromo-4-oxopentanoate lead straight to the Cp-
ligands 1a, 4a and 6a in moderate to good yields and in up to gram
quantities after purification by silica column chromatography.
1a, which comprises the methyl group at the R1 position. The first
mono-functionalization was achieved by hydrolysis of 1a with slightly
less than one equivalent of NaOH, yielding exclusively 8a (Scheme
4). One could expect a statistical distribution of hydrolysis at both
ester functions but the one at the R2 position is obviously more
susceptible, leaving R4 untouched. Subsequent amide bond formation
under the previously mentioned conditions gave 9a in good yields. By
further and carefully tuning the reaction conditions, 9a could
selectively be hydrolyzed to 10a. Reaction times longer than 5
min and pressures over 5 bar usually resulted in decomposition
of the compound. For subsequent bis-hetero-functionalization,
we chose O-tbutyl-L-phenylalanine to demonstrate that our
approach is substrate tolerant with the option of extension to
e.g. peptides. Along a standard peptide bond formation
strategy (see ESI), 10a was converted into 11a, containing two
different functionalities on the cyclopentadiene ring.
Bis-hetero functionalization of the rhenium complex 2b:
Unlike the Cp-ligand 1a itself, selective mono-hydrolysis of 1b
(Scheme 3) was not possible. Addition of 1 eq. of NaOH and
subsequent heating gave
a statistical mixture of ester-
hydrolyzed products. Thus, to accomplish the rhenium complex
8b, a direct reaction between the mono-acid ligand 8a and
Re2CO10 in DMF under microwave irradiation was performed.
The obtained crude mixture was directly reacted under
standard peptide coupling conditions to give 9b after
purification by preparative HPLC (Figure 4). Complex 9b was
Scheme 3. Synthetic route towards bis-homo functionalized Re and 99mTc complexes.
Reaction conditions: a) Re2CO10, o-xylene, MW, 220 °C, 15 min (1b, 76%; 4b, 83%; 6b,
44%). b) NaOH, MeOH, MW, 120 °C, 15 min (2b, 68%; 5b, 92%; 7b, 48%).
c) benzylamine, HOBt, EDC, DMF, 25 °C, 24 h, (3a, 38%; 3b, 16%). d) [TcO4]-, sodium
boranocarbonate, sodium tartrate, sodium tetraborate, H2O, MW, 120 °C, 30 min.
The rhenium complexes 1b, 4b and 6b with all three Cp-derivatives
were obtained by refluxing the respective ligand 1a, 4a and 6a with
Re2CO10 for multiple days in o-xylene (Scheme 3). Under microwave
conditions, the reaction solutions were heated to 220 °C and the
reaction time reduced to 15 min (as described in the supplementary
information) without loss of yields. For 1b, UPLC analysis indicated
complete consumption of starting material and sufficiently pure
complex could be obtained without further purification. Complexes 4b
and 6b were purified by column chromatography on silica. Ester
hydrolysis to 2b, 5b and 7b was achieved under basic conditions by
either refluxing overnight under basic conditions or by microwave
heating at 120 °C for 15 min. To demonstrate the possibility of bis-
homo functionalization, ligand 2a and complex 2b were reacted with
two equivalents of a model biovector, benzylamine, yielding the
respective bis-amide compounds 3a and 3b after purification by
preparative HPLC. Single crystals suitable for X-ray analysis were
obtained for 2b and 3b (Figure 2).
Scheme 4. Synthetic route towards hetero-functionalized Re and 99mTc
complexes. Reaction conditions: a) NaOH, MeOH, MW, 120 °C, 15 min.
b) benzylamine, HOBt, EDC, DMF, 25 °C, 48 h, 69%. c) i) NaOH, MeOH,
MW, 120 °C, 15 min. ii) O-tertbutyl-L-Phenylalanine, HOBt, EDC, DMF,
25 °C, 24 h, (11a, 26%; 11b, 46%). d) [TcO4]-, sodium boranocarbonate,
sodium tartrate, sodium tetraborate, H2O, MW, 140 °C, 30 min. e)
Re2CO10, DMF, MW, 220 °C, 15 min. f) TFA, DCM, MW, 80 °C, 15 min,
80%.
Figure 2. Crystal structures of 2b and 3b. Displacement ellipsoid
representation. Ellipsoids are drawn at 50% probability. Important bond lengths
then easily further hydrolyzed and functionalized with O-tbutyl-L-
(Å): 2b Re-centroid of Cp: 1.950(2), 3b Re-centroid of Cp: 1.945(3).
phenylalanine, yielding 11b after preparative HPLC in 26% yield. The
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tert-butyl protecting group of 11b could be
cleaved by treatment with TFA in DCM at
80 °C, yielding 12b as a model for the later
99mTc complexes.
The chiral centre in the attached amino
acid together with the chiral planarity of the
asymmetric Cp-ligand leads to the
formation of two diastereomers (see Figure
SI88). This was confirmed by the
observation of two sets of NMR signals in
1
both H and 13C NMR spectra of 11b and
12b (Figures SI83-84). All compounds
were fully characterized by NMR, HPLC
and HR ESI-MS. These synthetic results
Figure 4. UV/vis- and γ-traces of the co-injection of the rhenium compounds 3b and 12b with the 99mTc
compounds 3c and 12c respectively. Time differences are due to detector separation.
clearly
imply that more complex
biomolecules can be conjugated to both,
the free ligand and to the rhenium
microwave heating at 120 °C for 30 min (see experimental part).
complexes themselves. For rhenium, ligand 8a and complex 8b are
key since they will allow the consecutive introduction of two different
functionalities on the ligand (8a) for later labelling with 99mTc (vide
infra) or on the rhenium complex (8b) for biological evaluation and
comparison with the 99mTc complex.
Having shown that radiolabelling of these ligands with fac-
99mTc(OH2)3(CO)3]+ was straight-forward, we attempted the
[
direct synthesis of the 99mTc complexes (2c
, 5c and 7c) directly
from [TcO4]- in a “one-pot” approach. By mixing all components
in a single vial and adding directly the [99mTcO4]- generator eluate,
complexes 2c, 5c and 7c formed quantitatively by an improved
Radiolabelling with 99mTc: It is the common notion that Cp-
procedure under microwave conditions in 30 min at 120 °C
(Figure 3). We emphasize that these are conditions compatible
with routine applications in clinics.
systems, capable of binding in aqueous solution to the
[
99mTc(CO)3]+ fragment, have to comprise electron withdrawing
groups directly on the cyclopentadiene scaffold. This will
sufficiently acidify the ring to have at least small amounts of the
ligand deprotonated and therefore prone to coordination.
As typical 99mTc concentrations are too low for standard chemical
characterizations of the formed complexes by e.g. NMR, the accepted
procedure for their characterizations is HPLC co-injection with the
“cold” rhenium homologue and comparison of retention times (γ-trace
for 99mTc, UV-trace for Re). Figure 3 shows the HPLC traces of the
respective 99mTc complexes as obtained after direct preparations in
quantitative yields with all three ligands from [99mTcO4]-. Further
purification was not required. Due to the basic conditions, the ester
groups hydrolysed during the labelling process, wherefore co-
injections were performed with the di-acid rhenium homologues (2b,
5b and 7b). The retention times match, corroborating the identity of
the two homologous complexes with Re and 99mTc.
Accordingly, most of the approaches towards [(
5-C5H4-
R)99mTc(CO)3] type complexes comprise a carbonyl function
directly on the Cp ring.[5a, 10c, 13a, 21] First, we radiolabelled the
underivatized ligands 1a, 4a and 6a which follow this concept of
acidification and should therefore readily react with the
[
[
99mTc(OH2)3(CO)3]+ precursor. In a two-step approach, fac-
99mTc(OH2)3(CO)3]+ was prepared and then reacted with ligands
1a
,
4a and 6a at millimolar concentrations. Along this path, the
5c and 7c (Scheme 3) could all be obtained
99mTc complexes 2c
,
in high radiochemical yields under alkaline conditions and
The
Cp
compounds
3a
11a
two
(homo-
(hetero-
model
difunctionalized)
difunctionalized)
and
bear
functionalities V1 and V2 at positions R2 and R4
in the basic cyclopentadiene structure,
conjugated
cyclopentadiene ring. These ligands represent
the basic structure for combinatorial
via
an
amide
to
the
a
approach since essentially any two amine-
bearing biovectors can be added. The labeling
of such more complex systems in general and
3a and 11a in particular is an exemplary step
in the preparation of multi-functional molecular
imaging agents. Accordingly, by following the
preparations as described above, ligands 3a
and 11a could be labelled along the one-pot
procedure directly from [99mTcO4]-, giving the
corresponding 99mTc complexes 3c and 12c in
Figure 3. General reaction scheme for the “one-pot” synthesis of 99mTc compounds (top). UV- and y-traces
of the co-injection of the rhenium compounds 2b, 5b and 7b with the 99mTc compounds 2c, 5c and 6c
respectively (bottom). Differences in retention times are due to detector separation.
good yields (Figure 4).
Slightly higher
temperatures and reaction times were
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required. As a convenient "side effect", the tert-butyl protective group
was concertedly cleaved during the reaction in case of 11a. Taken
together, these results demonstrate the possibility to synthesize 99mTc
piano-stool type molecular imaging agents with structurally complex
and multifunctional Cp ligands in one step directly from [99mTcO4]- in a
straight-forward and efficient way. The option of altering V1 and V2
(phenyl-alanine and benzyl-amine in our case) opens a path towards
further combinations of biologically active moieties.
temperatures. High-resolution mass spectrometry (HR-MS) was
performed on a Thermo DFS double-focusing system (ThermoFisher
Scientific, Germany).
Preparative HPLC was performed on a Varian ProStar 320 system,
using a Dr. Maisch Reprosil C18 100-7 (40 x 250 mm) column (flow
rate: 40.0 ml/min) or a LiChroCART RP-18e (10 x 250 mm) column.
HPLC solvents were 0.1% trifluoroacetic acid (solvent A) and
acetonitrile (solvent B), HPLC grade. Microwave reactions were
performed using a Biotage Initiator+ Robot Eight or Anton Paar (AP)
Monowave 200 instrument. Analytical HPLC was performed on a
Merck Hitachi L7000 system, equipped with a L-7400 UV-detector and
an in-line radio-detector Berthold FlowStar LB513, using an analytical
Macherey-Nagel Nucleosil C18 5 μm (4.6 × 250 mm) column. HPLC
solvents were 0.1% trifluoroacetic acid (solvent A) and acetonitrile,
HPLC grade (solvent B). Analytical UPLC was performed on a VWR
Hitachi Chrommaster Ultra, using an Acquity UPLC BEH C18 1.7μm
(2.1 x 50 mm) column. UPLC solvents were trifluoroacetic acid (0.1%
in millipore water) (solvent A) and acetonitrile UPLC grade (solvent
B). HPLC and UPLC gradients are given in the Supporting
Information.
Conclusions
We present versatile synthetic routes towards multiply substituted
cyclopentadiene, which are the basis for the subsequent preparation
of multifunctional ligands. Both, the physico-chemical (group R1) as
well as the biological (V1 and V2) properties can be tuned individually
in the same ligand. We have furthermore shown that these
cyclopentadiene scaffolds serve as
a
ligand platform in
bioorganometallic 99mTc chemistry that allows the introduction of
multiple modalities in a straightforward way. The ligands as such can
be prepared separately with functionalities V1 and V2 according to the
target requirements. The "innocent" group R1 on the Cp scaffold
influences the physico-chemical properties such as lipo/hydrophilicity
while leaving the targeting properties untouched. Three
representative ligands (1a, 3a, 5a) with different R1 groups have been
synthesized, fully characterized and coordinated to the fac-[Re(CO)3]
moiety. As a further proof-of-concept, 1a was both bis-homo - and bis-
hetero functionalized with two simple biovector-models. Established
amide bond formation chemistry for the vector conjugation, ensures
its applicability to a wide variety of modalities. The combination of
these ligands with the theranostic pair Re and 99mTc leads to tetra-
functional complexes. Their synthesis is both straightforward, highly
tolerant to different functionalities and, most importantly, the 99mTc
homologues are prepared in one step directly from generator eluted
Synthesis of Cp-ligands and Re-complexes.
General Procedure for the synthesis of complexes 1b, 4b and 6b
A solution of the complex (1 equiv.) and Re2(CO)10 (0.52 equiv) in o-
Xylene (2 ml) in a Anton Paar microwave vial (10 ml) equipped with a
stirring bar was heated to 220 °C for 15 min (Careful, toxic CO gas is
released during the reaction, take appropriate precautions). The
solvent was evaporated in vacuo, yielding pure complex after column
chromatography on silica gel (no further purification was necessary
for complex 1b).
General procedure for the synthesis of complexes 2b, 5b and 7b
A solution of the complex (1 equiv) in MeOH (1 ml) and 1 M NaOH
(0.5 ml) in a Anton Paar microwave vial (10 ml) was heated by
microwave to 120 °C for 15 min. The crude was diluted with 1 M HCl
(20 ml) and washed with DCM twice (2 x 20 ml). The combined
organic phase was washed once with water (20 ml) dried over MgSO4
and the solvent removed in vacuo yielding pure complex.
[
99mTcO4]-. We envision that this ligand platform will enable a
combinatorial approach towards targeted and multimodal
bioorganometallic complexes.
2a. 1a (31 mg, 0.13 mmol) was dissolved in MeOH (1 ml) in a Anton
Paar microwave vial (10 ml) equipped with a stirring bar. 1 M NaOH
was added (0.4 ml) and the solution was heated by microwave to 120
°C for 15 min. UPLC measurement showed complete consumption of
the starting material. The reaction solution was diluted with H2O (5 ml)
and neutralized to pH = 3 by dropwise addition of 1 M HCl. 2a was
obtained by evaporating the solvent in vacuo and was used without
further purification. UPLC (gradient U1): RT = 1.91 min.
Experimental Section
Materials. All chemicals were of reagent-grade quality or higher and
were obtained from commercial suppliers. Solvents were used as
received or dried over molecular sieves. Sodium boranocarbonate
was a gift from Mallinckrodt Medical B.V. (The Netherlands).
Na[99mTcO4] in 0.9% saline was eluted from a 99Mo/99mTc UTK FM
generator purchased from Mallinckrodt Medical B.V.
3a. 2a (48 mg, 0.21 mmol) was dissolved in DMF (2 ml). Benzylamine
(0.53 ml, 0.49 mmol) and HOBt (66 mg, 0.49 mmol) were added under
stirring. After 5 min, EDC (93 mg, 0.49 mmol) and DIPEA (0.170 ml,
0.98 mmol) were added and the solution was stirred at room
temperature for 48 h. The solvent was removed in vacuo and the
crude was purified by preparative HPLC (Method A) and 3a was
obtained as a yellowish powder (30 mg, 0.08 mm, 38 %). UPLC
(gradient U1): RT = 2.65 min. 1H NMR (400 MHz, CDCl3): δ (ppm)
2.25 (m, 3H), 2.49 (m, 2H), 2.72 – 2.75 (m, 2H), 3.25 – 3.26 (m, 2H),
4.34 (s, 2H), 4.46 (s, 2H), 6.11 (s, 1H), 7.19 – 7.30 (m, 10H). 13C NMR
(125 MHz, CDCl3): δ (ppm) 15.39, 27.54, 36.51, 43.78, 44.05, 44.69,
127.99, 128.18, 128.40, 128.48, 129.45, 129.52, 130.64, 133.87,
Instrumentation and methods. 1H and 13C-NMR spectra were
recorded in deuterated solvents on a Bruker DRX 400 (1H: 400 MHz,
13C: 100.6 MHz) or DRX 500 (1H: 500 MHz 13C: 125.8 MHz) MHz
spectrometer at 300 K. The chemical shifts, 훿, are reported in ppm
(parts per million) relative to residual solvent peaks. IR spectra were
obtained with a PerkinElmer Spectrum Two spectrometer. UPLC-ESI-
MS was performed on a Waters Acquity UPLC System coupled to a
Bruker HCTTM, using an Acquity UPLC BEH C18 1.7μm (2.1 x 50 mm)
column. UPLC solvents were formic acid (0.1% in millipore water)
(solvent A) and acetonitrile UPLC grade (solvent B). The temperature
was regulated with a Peltier thermostatic system to the specified
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10.1002/chem.201801271
Chemistry - A European Journal
FULL PAPER
139.95, 140.68, 152.27, 152.95, 168.03, 174.92. HR-ESI mass
spectrum (MeOH): found 375.20669; calcd. for [C24H27O2N2]
375.20670.
removed in vacuo and the crude was purified by preparative HPLC
(Method A) and 11a was obtained as a yellow oil (15 mg, 0.03 mmol,
26 % from 9a). UPLC (gradient U1): RT = 3.19 min. H NMR (500
1
MHz, CDCl3) δ (ppm) 1.41 (s, 9H), 2.22 (t, J3 = 2.2 Hz, 3H), 2.43 (t, J3
= 7.6 Hz, 3H), 2.75 (t, J3 = 7.6 Hz, 2H), 3.12 – 3.16 (m, 4H), 4.44 (d,
J3 = 5.7 Hz, 2H), 4.86 (m, 1H), 5.80 (m, 1H), 5.98 (d, J3 = 7.5 Hz, 1H),
6.04 (s, 1H), 7.15 – 7.33 (m, 10H). 13C NMR (126 MHz, CDCl3): δ
(ppm): 15.37, 26.50, 28.13, 35.98, 38.35, 43.83, 53.40, 76.90, 77.16,
77.41, 82.48, 127.05, 127.73, 127.94, 128.46, 128.60, 128.89, 129.57,
129.74, 133.12, 136.54, 138.26, 150.68, 151.24, 164.70, 171.23,
171.79. HR-ESI mass spectrum (MeOH): found 489.27518; calcd. for
[C30H35N2O4+H+] 489.27478.
3b. 2b (47 mg, 0.1 mmol) was dissolved in DMF (5 ml). Benzylamine
(0.023 ml, 0.21 mmol) and HOBt (29 mg, 0.21 mmol) were added
under stirring. After 5 min, EDC (41 mg, 0.21 mmol) and DIPEA (0.073
ml, 0.42 mmol) were added and the solution was stirred for 24 h. The
solvent was removed in vacuo and the crude was purified by
preparative HPLC (Method A) and 3a was obtained as a pale yellow
powder (10 mg, 0.016 mmol, 16 %). Single crystals were obtained by
slow evaporation of a MeOH solution. UPLC (gradient U1): RT = 3.20
min. 1H NMR (400 MHz, CDCl3): δ (ppm) 2.45 (t, 3J = 7.2 Hz, 2H), 2.48
(s, 3H), 2.64 – 2.78 (m, 2H), 4.32 – 4.51 (m, 4H), 5.40 (d, J3 = 2.0 Hz,
1H), 5.97 (d, J3 = 2.0 Hz, 1H) 7.21 – 7.30 (m, 10H) 8.40 – 8.44 (m,
2H). 13C NMR (125 MHz, CDCl3): δ (ppm) 14.17, 25.30, 38.73, 43.98,
44.12, 49.00, 84.82, 86.78, 90.93, 108.57, 111.42, 128.14, 128.26,
128.38, 128.56, 129.50, 129.57, 139.85, 139.99, 166.28, 173.85,
195.29. IR bands (Golden Gate, cm-1): 2017, 1906. HR-ESI mass
spectrum (MeOH): found 645.13941; calcd. for [C27H26O5N2Re+H+]
645.13937.
8b. 8a (80 mg, 0.34 mmol) was dissolved in DMF (4 ml) in an Anton
Paar microwave vial (10 ml). Re2(CO)10 (110 mg, 0.17 mmol) was
added and the mixture was heated by microwave to 220 °C for 15 min
(Careful, toxic CO gas is released during the reaction, take
appropriate precautions). UPLC showed complete consumption of the
starting material. The crude solution was dried in vacuo and used
without further purification in the next step. UPLC (gradient UMS1):
RT = 2.88 min.
8a. 1a (38 mg, 0.16 mmol) was dissolved in MeOH (1 ml) in a Anton
Paar microwave vial (10 ml) equipped with a stirring bar. 1 M NaOH
was added (0.150 ml, 0.150 mmol) and the solution was heated to
120 °C for 15 min. UPLC measurement showed complete
consumption of the starting material. The reaction solution was diluted
with H2O (5 ml) and neutralized to pH = 3 by dropwise addition of 1 M
HCl. 8a was obtained by evaporating the solvent in vacuo and was
used without further purification. UPLC (gradient U1): RT = 2.49 min.
9b. 8b (154 mg, 0.31 mmol) was dissolved in DMF (5 ml).
Benzylamine (0.037 ml, 0.34 mmol) and HOBt (46 mg, 0.34 mmol)
were added under stirring. After 5 min, EDCI (66 mg, 0.34 mmol) and
DIPEA (0.120 ml, 0.69 mmol) were added and the solution was stirred
for 24 h. The solvent was removed in vacuo and the crude was purified
by preparative HPLC (Method A) and 9b was obtained as an orange
oil (45 mg, 0.08 mmol, 26%). UPLC (gradient U1): RT = 3.23 min. 1H
NMR (400 MHz, CDCl3): δ (ppm): 1.31 (t, J3 = 7.1 Hz, 3H), 2.40 (m,
J = 3.3 Hz, 2H), 2.48 (s, 3H) 2.68 – 2.88 (m, 2H), 4.17 – 4.34 (m, 2H),
4.45 (d, J= 5.7, 2 H), 5.20 (d, J = 2.1 Hz, 1H), 5.77 (d, J = 2.2 Hz, 1H),
5.85 (t, 1 H), 7.25 – 7.36 (m, 5H). 13C NMR (125 MHz, CDCl3): δ (ppm):
13.87, 14.28, 23.75, 38.07, 44.10, 61.13, 84.61, 85.73, 86.06, 107.58,
110.76, 127.95, 128.04, 128.99, 137.78, 164.92, 171.19, 193.56. IR
bands (Golden Gate, cm-1): 2021, 1918. HR-ESI mass spectrum
(MeOH): found 584.10780; calcd. for [C22H23O6NRe+H+] 584.10774.
9a. 8a (59 mg, 0.26 mmol) was dissolved in DMF (4 ml). Benzylamine
(0.031 ml, 0.29 mmol) and HOBt (39 mg, 0.29 mmol) were added
under stirring. After 5 min, EDC (55 mg, 0.29 mmol) and DIPEA (0.100
ml, 0.57 mmol) were added and the solution was stirred for 48 h. The
solvent was removed in vacuo and the crude was purified by
preparative HPLC (Method A) and 9a was obtained as a yellow oil (57
1
mg, 0.18 mmol, 69 %). UPLC (gradient U1): RT = 2.76 min. H NMR
(400 MHz, CDCl3): δ (ppm): 1.29 (t, J3 = 7.1 Hz, 3H), 2.27 (t, J3 = 2.3
Hz, 3H), 2.48 (t, J3 = 7.5 Hz, 2H), 2.76 (t, J3 = 7.5 Hz, 2H), 3.21 (m,
2H), 4.18 (q, J3 = 7.1 Hz, 2H), 4.42 (d, J = 5.7 Hz, 2H), 6.08 (s, 1H),
6.13 (t, J = 5.3 Hz, 1H), 7.20 – 7.33 (m, 5H). 13C NMR (126 MHz,
CDCl3): δ (ppm): 14.57, 15.59, 26.73, 35.89, 43.93, 44.42, 59.63,
127.20, 127.79, 127.89, 128.88, 133.07, 137.86, 153.11, 155.85.
165.26, 172.61. HR-ESI mass spectrum (MeOH): found 314.17497;
calcd. for [C19H23NO3+H+] 314.17507.
10b. A solution of 9b (43 mg, 0.074 mmol) in MeOH (2 ml) and 1 M
NaOH (0.15 ml) in a Anton Paar microwave vial (10 ml) was heated
by microwave to 110 °C for 15 min. The crude was diluted with 1 M
HCl (10 ml) and washed with DCM twice (2 x 10 ml). The combined
organic phase was washed once with water (10 ml) dried over MgSO4
and the solvent removed in vacuo yielding 10b as an orange solid (32
mg, 0.058 mmol, 78 %). The product was used in the next step without
further purification. UPLC (gradient U1): RT = 2.81 min. 1H NMR (400
MHz, CD3OD): δ (ppm): 2.45 (s, 3H), 2.45 (m, 2H), 2.61 – 2.68 (m,
1H), 2.75 – 2.82 (m, 1H), 4.37 (m, 2H), 5.42 (d, J3 = 2.1 Hz, 1H), 5.89
(d, J3 = 2.2 Hz, 1H), 7.21 – 7.32 (m, 5H). 13C NMR (101 MHz, CD3OD):
δ (ppm): 12.63, 23.71, 37.17, 42.77, 84.60, 86.65, 107.46, 110.82,
126.87, 127.26, 128.18, 138.49, 166.58, 172.44, 193.60. IR bands
(Golden Gate, cm-1): 2021, 1917. HR-ESI mass spectrum (MeOH):
found 554.06196; calcd. for [C20H18O6NRe-H+] 554.06189.
10a. 9a (50 mg, 0.16 mmol) was dissolved in MeOH (2 ml) in a Anton
Paar microwave vial (10 ml) equipped with a stirring bar. 1 M NaOH
(1 ml) was added and the solution was heated to 120 °C for 5 min.
UPLC measurement showed complete consumption of the starting
material. The reaction solution was diluted with H2O (5 ml) and
neutralized to pH = 3 by dropwise addition of 1 M HCl. 10a was
obtained by evaporating the solvent in vacuo and was used without
further purification. UPLC (gradient U1): RT = 2.33 min.
11b. 10b (181 mg, 0.326 mmol) was dissolved in DMF (2 ml). O-
tertbutyl-L-Phenylalanine (93 mg, 0.36 mmol) and HOBt (49 mg, 0.36
mmol) were added under stirring. After 5 min, EDCI (69 mg, 0.36
mmol) and DIPEA (0.125 ml, 0.72 mmol) were added and the solution
was stirred for 24 h. The solvent was removed in vacuo and the crude
was purified by preparative HPLC (Method A) and 11b was obtained
11a. The crude of 10a obtained from 9a (36 mg, 0.115 mmol) was
dissolved in DMF (3 ml). O-tertbutyl-L-Phenylalanine (33 mg, 0.13
mmol) and HOBt (17 mg, 0.13 mmol) were added under stirring. After
5 min, EDC (24 mg, 0.13 mmol) and DIPEA (0.044 ml, 0.25 mmol)
were added and the solution was stirred for 24 h. The solvent was
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10.1002/chem.201801271
Chemistry - A European Journal
FULL PAPER
as a pale red solid and directly used for the next step (112.8 mg,
0.149 mmol, 46%). UPLC (gradient U1): RT = 3.68 min.
H2O (2 ml). Pure 12c could be eluted by washing the cartridge with
MeOH (2 ml).
12b. 11b (15 mg, 0.02 mmol) was dissolved in DCM (2 ml) in an Anton
Paar microwave vial (10 ml). TFA (0.5 ml) was added and the solution
was heated to 80 °C by microwave for 15 min. The solvent was
evaporated and the crude was purified by preparative HPLC (Method
A) and 12b was obtained as an orange oil (15 mg, .0.16 mmol, 80 %).
X-ray Crystallography. Crystallographic data were collected at
183(2) K with Mo Kα radiation (λ = 0.7107 Å) that was graphite-
monochromated on an Oxford Diffraction CCD Xcalibur system with a
Ruby detector. Suitable crystals were covered with oil (Infineum
V8512, formerly known as Paratone N), placed on a nylon loop that is
mounted in a CrystalCap Magnetic™ (Hampton Research) and
immediately transferred to the diffractometer. The program suite
CrysAlisPro was used for data collection, multi-scan absorption
correction and data reduction.[22] The structures were solved with
direct methods using SIR97[23] and were refined by full-matrix least-
squares methods on F2 with SHELXL-2014.[24] (CCDC numbers
1828105-1828106)
1
UPLC (gradient U1): RT = 3.08 min, 3.13 min. H NMR (500 MHz,
CD3OD): δ (ppm): 2.31 (s, 3 H), 2.37 (s, 3H), 2.42 – 2.45 (m, 4H), 2.61
– 2.67 (m, 2H), 2.72 – 2.79 (m, 2H), 3.04 – 3.09 (m, 2H), 3.25 – 3.29
(m, 2H), 4.34 – 4.35 (m, 4H), 4.71 – 4.75 (m, 2H), 5.35 (d, J3 = 2.0 Hz,
1H), 5.38 (d, J3 = 2.0 Hz, 1H), 5.95 (d, J3 = 2.0 Hz, 1H ), 5.96 (d, J3 =
2.0 Hz, 1H), 7.16 – 7.32 (m, 20H). 13C NMR (101 MHz, CD3OD): 13.98,
25.21, 25.25, 37.90, 38.07, 38.62, 38.78, 44.17, 44.19, 55.16, 55.23,
85.50, 85.65, 86.61, 87.26, 90.94, 91.47, 107.56, 108.37, 110.41,
110.74, 127.81, 128.27, 128.36, 128.65, 128.67, 129.53, 129.59,
130.15. 130.25, 138.46, 138.49, 139.84, 165.84, 166.12, 173.85,
173.88, 174.37, 174.56, 195.12. IR bands (Golden Gate, cm-1): 2021,
1921. HR-ESI mass spectrum (MeOH): found 725.12675; calcd. for
[C29H27N2O7Re+Na+] 725.12680.
AUTHOR INFORMATION
Corresponding Author
*E-mail: angelo.frei@chem.uzh.ch
99mTc experiments. Caution! 99mTc is a γ-emitter (E= 140 keV, t1/2
=
Author Contributions
6.02 h) which should only be handled in a licensed and appropriately
shielded facility. Sodium boranocarbonate releases CO gas, which is
highly toxic, it is recommended to be handled only in ventilated hoods.
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
[
99mTc(OH2)3(CO)3]+
Acknowledgements
A Biotage microwave vial (2 – 5 ml) was charged with sodium
boranocarbonate (4 mg, 38.5 μmol), sodium tartrate dihydrate (7 mg,
30.4 μmol) and sodium tetraborate decahydrate (7 mg, 18.5 μmol).
The vial was sealed and flushed with N2 for 5 min before adding
Financial assistance from the University of Zurich. We thank
Giuseppe Meola for help with the crystal structures. We thank the
Swiss National Science Foundation (SNF Project IZLSZ2_149029/1
within the SSAJRP program) and the South African National
Research Foundation for financial support.
[
99mTcO4]- eluate (1-2 ml) from a commercial generator. The solution
was heated by microwave to 110 °C for 7 min. In order to normalize
the overpressure, evolving gases were released with a 1 ml
disposable syringe. Excess sodium boranocarbonate was quenched
via dropwise addition of 1 M HCl to pH = 2 and the solution was
subsequently basified by addition of 1 M NaOH to pH = 8.
Keywords: Technetium • Rhenium • Cyclopentadiene •
Bioorganometallic • Theranostics
General procedure for the labelling of Cp-Ligands 1a, 4a, 6a, 3a
and 12a.
Generally, 0.5 ml of a 5 mM stock solution of the respective ligand in
MeOH was added to a Biotage microwave vial (2 – 5 ml). The vial was
sealed, and the solvent was removed by passing a stream of N2
through the vial via two syringe needles for 30 min.
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120 °C for 30 min (1a, 4a, 6a) or 140 °C for 30 min (3a) or 2 x 30 min
(12a). Complexes 2c, 5c, 7c and 3c were obtained in radiochemical
puritiy > 95%. For 12c, some [99mTcO4]- and [99mTc(OH2)3(CO)3]+ was
observed (< 20 %, c.f. Figure SI89). 12c was purified by loading the
reaction mixture onto a Chromafix C18 cartridge and washing it with
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10.1002/chem.201801271
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Title
Multifunctional cyclopentadiene ligands with tuneable properties and their rhenium
and 99mTc complexes were prepared following a versatile synthetic route.
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