[(Cp-R)M(CO)3] (M= Re or 99mTc) conjugates for theranostic receptor targeting

Article abstract of DOI:10.2533/chimia.2013.267

Cyclopentadienyl complexes of ^99mTc became accessible via a retro Diels-Alder synthetic approach of dimerized cyclopentadiene derivatives. So far, this approach was limited to derivatives comprising a carboxylic acid group, directly conjugated to the Cp-ring, leading to complexes [(C ₅H₅COOH)^99mTc(CO)₃] and [(C ₅H₅CONHR) ^99mTc(CO)₃], respectively. The introduction of an-NCO group via Curtius rearrangement and subsequent in situ reactions with alcohols or amines gave [(C₅H₅NHCO-OR) ₂] and [(C₅H₅NHCO-NHR)₂]. To increase the spacer lengths between the Cp-ring and the functional groups, methylene and ethylene spacers were introduced to yield C₅H ₅-CH₂COOH and C₅H₅-C ₂H₄COOH respectively. The latter Cp-derivatives reacted with [^99mTcO₄)]- and in the presence of CO releasing/reducing agents to the corresponding [(C₅H ₅-spacer-COOH)^99mTc(CO)₃] complexes. The carboxylato groups can be derivatized with targeting functions, leading to structurally altered receptor binding complexes, with ^99mTc for imaging and with rhenium for therapy. The nature of the ^99mTc complexes was assessed by HPLC comparison with the corresponding rhenium compounds. Schweizerische Chemische Gesellschaft.

Full text of DOI:10.2533/chimia.2013.267

Laureates: awards and Honors, sCs FaLL Meeting 2012
CHIMIA 2013, 67, Nr. 4 267
doi:10.2533/chimia.2013.267
Chimia 67 (2013) 267–270 © Schweizerische Chemische Gesellschaft
[(Cp-R)M(CO)3] (M= Re or 99mTc)
Conjugates for Theranostic Receptor
Targeting
Daniel Can§, Paul Schmutz, Samer Sulieman, Bernhard Spingler, and Roger Alberto*
§SCS-DSM Award for best poster presentation
Abstract: Cyclopentadienyl complexes of 99mTc became accessible via a retro Diels-Alder synthetic approach of
dimerized cyclopentadiene derivatives. So far, this approach was limited to derivatives comprising a carboxylic
acid group, directly conjugated to the Cp-ring, leading to complexes [(C5H5COOH)99mTc(CO)3] and [(C5H5CONH-
R)99mTc(CO)3], respectively. The introduction of an –NCO group via Curtius rearrangement and subsequent in
situ reactions with alcohols or amines gave [(C5H5NHCO-OR)2] and [(C5H5NHCO-NHR)2]. To increase the spacer
lengths between the Cp-ring and the functional groups, methylene and ethylene spacers were introduced to yield
C5H5-CH2COOH and C5H5-C2H4COOH respectively. The latter Cp-derivatives reacted with [99mTcO4 )]- and in the
presence of CO releasing/reducing agents to the corresponding [(C5H5-spacer-COOH)99mTc(CO)3] complexes.
The carboxylato groups can be derivatized with targeting functions, leading to structurally altered receptor
binding complexes, with 99mTc for imaging and with rhenium for therapy. The nature of the 99mTc complexes was
assessed by HPLC comparison with the corresponding rhenium compounds.
Keywords: Bioorganometallic chemistry · Carbonic anhydrase inhibitors · Radiopharmaceuticals ·
Technetium · Theranostic
very stable under physiological condi- tivity for a specific target is a key criterion
tions and can synthetically be treated like for the quality of an inhibitor.[14] Meggers
Introduction
aromatic organic molecules. Focussing on and coworkers showed recently that high
Organometallic complexes with bioac-
tive ligands are of interest for noninvasive
imaging of biological events and therapeu-
tic treatment of diseases. While several d-
element cations are studied and used for
the synthesis of 99mTc-labelled radiophar-
selectivity not only depends on intermo-
maceuticals and the corresponding cold lecular interactions but also on a versatile
rhenium compounds, a variety of organic and directed three-dimensional arrange-
and inorganic cyclopentadienyl derivatives ment of different functionalities. As exem-
therapy, 99mTc is the most prominent in nu-
has been synthesized and their complexes plified by organometallic protein kinase
biologically investigated.
inhibitors,[15–17] chemically inert organo-
clear medical diagnostics. It would be de-
sirable in a theranostic sense (therapy and
diagnostics), to have identical homologous
compoundsforcombinedtherapyanddiag-
nosis.[1–3] Rhenium and technetium belong
to the same traid; therefore it is possible
to use respective congener compounds for
matched therapy and imaging.[4–6] While
Medicinal inorganic chemistry has pro- metallic complexes offer the opportunity
gressed tremendously since the successful to occupy biologically relevant chemical
introduction of Cisplatin in the late 1960s. space as compared to similar inhibitors
During the past 50 years, a multitude of with a purely organic scaffold. Therefore,
metal-containing anticancer, anti-inflam- bioorganometallic complexes exhibit high
matory, antomicrobial, antimalarial and potential as chemical probes.[15,18–21]
antibacterial compounds, enzyme inhibi-
tors and MRI-contrast agents have been ing metal complexes into small molecules
exploited and investigated.[8,9] The vast
without affecting their biological activ-
The introduction of sterically demand-
Re-based compounds can be used for ther-
99m
apy, the Tc homologs can serve as im-
majority of clinically applied pharmaceu- ity is challenging. One possible strategy
aging agents for Single Photon Emission
[1,2,7]
ticals are of organic nature. Many of the was pioneered by Jaouen and coworkers.
Computed Tomography (SPECT).
organometallic or inorganic drug candi- In the late 1970s, Hanzlik et al. replaced
dates are believed to act in the same way a phenyl ring in phenylalanine by ferro-
as Cisplatin whereas metal complexes cene and observed that this organometallic
for structural recognition of receptors are Cp- sandwich-complex was accepted as a
rarely found. Only recently, organometal- substrate in the binding pocket of phenyl-
lic compounds came into the scope of such alanine decarboxylase.[22] Based on this
[(CP-R)M(CO)3]-type compounds are
applications. The structural complexity of
observation, Jaouen developed a variety of
metal-containing compounds that are sta- highly potent estrogen receptor inhibitors
ble in biological systems was recognized by replacing a phenyl ring in Tamoxifen
to have an underestimated and unexplored with [(Cp-R)Re(CO) ]. The resulting mol-
*Correspondence: Prof. Dr. R. Alberto
University of Zürich
Institute of Inorganic Chemistry
Winterthurerstrasse 190
CH-8057 Zürich
potential for medicinal applications.[9–13]
ecules retained a hig3h binding affinity for
Since nature has developed an enor- the estrogen receptor.[12,23–28]
mous realm of functionally and structur-
Combining the observations made by
Tel.: +41 44 635 46 31
ally similar receptors and enzymes, selec- Meggers and by Jaouen, we studied the
E-mail: ariel@aci.uzh.ch

268 CHIMIA 2013, 67, Nr. 4
Laureates: awards and Honors, sCs FaLL Meeting 2012
Scheme 1. Synthetic
conditions: i) diphe-
nylphosphoryl azide
(DPPA), NEt3, toluene
80%; ii) heated to 80
°C, toluene; iii) ad-
dition of the nucleo-
philes a/b, toluene.
biological activity and the selectivity of
[(Cp-R)Re(CO)3]-complexes with differ-
ent targeting vectors. In order to follow the
theranostic concept, we prepared the cor-
responding 99mTc-complexes in water. For
future projects, we investigated different
Cp-building blocks comprising different
functional groups bound to the Cp-ring.
Results and Discussion
Fig. 1. Time-resolved
The derivatization of cyclopentadiene
IR-measurement
(HCp) or its complexes has a long history
of the Curtius rear-
in organic and organometallic chemistry.
Many different substituents have been de-
scribed in literature.[29–35] However, reports
for coupling biologically active targeting
vectors are comparably scarce. Being part
of the spacer between the chelator and the
biovector, the functional group attached to
Cp is of great importance for a bifunctional
chelator (BFC).
rangement of 1 to 2
(complete after 20.52
min) and the following
nucleophilic addition
of MeOH (after 38.51
min) to form 3a.
We reported the unexpected forma-
tion of [(CpCOOH)99mTc(CO)3]
from
‘Thiele’s acid’, the Diels-Alder dimer of
HCp-COOH,[36–38] and subsequently a
general aqueous synthetic route to [(Cp-
R)99mTc(CO)3]-type complexes was devel-
oped by derivatization of the acid function
via amide coupling to biotargeting vectors.
This procedure, where the carbonyl group
is assumed to play the important role of
a metal-anchoring group, allows a fully
aqueous preparation of a variety of bioac-
tive compounds.[6,37,39]
Scheme 2. Synthetic
conditions: i) bro-
moacetic acid, THF,
30–50%; ii) methyl-
3-brompropionate,
THF, 30–50%; iii)
LiOH, THF/MeOH/
H2O 69–74%.
Following this approach, we recently
reported organometallic carbonic anhy-
drase inhibitors (CAI) with superior selec-
tivity for the pharmaceutically relevant iso-
forms IX and XII.[6] One of the described
examples consisted of a [(CpNHCO-R)
Re(CO)3] building block derivatized with
an arylsulfonamide targeting vector and
showed the most pronounced selectivity
profile. The corresponding
99mTc-complex
however, has not yet been described, as
the required compound HCp-NH2 is still
unknown.
Besides CAIs, many pharmacological
active species and natural substances are
aniline-based and contain the amino group
in the form of amides or as internal aryl-
Scheme 3. Synthetic
conditions: i)
Na2B4O7*10H2O
(Borax), H2O, 95 °C,
20%.
sulfonamides. The replacement of such
motives by the organometallic congener
[(CpNHCO-R)M(CO) ] (M = Re or 99mTc)
would therefore incre3ase application op-
portunities in therapy and in diagnosis.
isocyanate reacted in situ with an alcohol
or an amine, to form 3a or 3b respectively.
With MeOH, the reaction was followed in
a time-resolved IR-experiment, evidencing
the successful transformation (Fig. 1).
HCpNHCO-R, the carbonyl group would
Coupling of a hypothetical HCp-NH be in the β-position, still close enough to
to carboxylic acids would yield a carbony2l act as an anchoring group.
group close to the HCp-ring. As shown
Aiming at the synthesis of HCp-NH ,
with ketones[36] and carboxylic acids,[37] compounds 3a and 3b were synthesize2d
Another motive in bioactive phar-
the carbonyl group in α-position to the by Curtius rearrangement (Scheme 1). The
maceuticals is Ar-(CH ) -CONHR.
Organometallic analogues of2tnhis moiety
Tc(OH2)3(CO) ] and initializing the of Thiele’s acid with diphenylphosphoryl
HCp is an anchoring group, binding to acid-azide 1 was obtained after treatment
99m
+
[
retro Diels-Alder3reaction and formation azide in toluene. Heating to 80 °C induced
could represent biomimetics for new phar-
maceuticals or imaging agents.
5
of η -coordinated Cp–-complexes. With the Curtius rearrangement. The resulting

Laureates: awards and Honors, sCs FaLL Meeting 2012
CHIMIA 2013, 67, Nr. 4 269
Scheme 4. Synthetic
conditions: i) triethyl
phosphonoacetate,
MeOH, 80%; ii) H2/
Pd/C, EtOAc (10a).
iii) LiOH, H2O/MeOH/
THF 98% (10b).
of [Re(OH2)3(CO)3]+ under basic aqueous complexes. Since 99mTc complexes are
conditions.[41]
present in nanomol quantities only, com-
Complex 8 was analyzed by IR (KBr), parison of retention times (radio- (99mTc)
NMR (CDCl3), ESI-MS (CH3OH) and and UV/vis detection (Re) are the only
elemental analysis. Crystals were grown methods to assess the nature of 99mTc com-
from CH Cl2/hexane and the structure was pounds. Formation rates of the 99mTc prod-
elucidate2d (Fig. 2).
ucts are different for ligands 5 (hydrolyzed
Fig. 2. ORTEP representation of 8. Relevant
bond lengths (Å): Re(1)–C(1) 1.912(2),
Re(1)–C(2) 1.917(2), Re(1)–C(3) 1.913(2),
Re(1)–C(7) 2.299(2), Re(1)–C(6) 2.3002(19),
Re(1)–C(5) 2.301(2), Re(1)–C(4) 2.3053(19),
Re(1)–C(8) 2.308(2).
Carboxylic acid complex 10b was not 4) and 7 (hydrolyzed 6). With 5, formation
accessible along the same route by us- was quantitative after 30 min (Scheme
ing ligand 7. Instead, 10b was obtained 5) whereas the reaction with 7 gave only
by Horner-Wardsworth-Emmons ole- 7% product after the same time and 62%
fination[42] of [(CpCOH)Re(CO) ].[43] after 5 h. Higher rates were achieved
Treatment with triethyl phosphonoac3etate
when the labeling with 7 was performed
in MeOH gave olefin 9 in 80% yield. in a microwave reactor; at 130 °C and
As aforementioned, HCp-compounds Hydrogenation of the double bond with 30 min 65% product 12, 16% [99mTcO ]–
of the form C5H5-COOH can be labelled H2 on Pd, followed by ester hydrolysis of and 19% [99mTc(OH2)3(CO)3]+
were o4b-
as monomers or as dimers with 99mTc.[36–38]
10a with LiOH gave the desired complex tained (Scheme 6).
In the ligands 5 and 7 with an extended 10b (Scheme 4). Crystals were grown from
In order to prepare organometallic CAI
spacer, this anchoring group is retained. CH2Cl2/hexane and the expected structure with Tc and based on 5 and 7,[6]
The carbonyl group is now in β- or in
was confirmed (Fig. 3). pounds 13 and 14 were prepared from 8
γ-position relative to the HCp-ring, likely
Compounds 5 and 7 were subjected to and 10b with rhenium. Compounds 13
still close enough to serve as an anchoring labeling studies with 99mTc. Ligands were
and 14 extend our list of CAI, containing
group. Like Thiele’s acid, 5 and 7 can be
present as monomers and in mM concen- a methylene spacer and an ethylene spacer
derivatized with bioactive functions.
trations. The reactions were studied ‘all in respectively between the carbonyl group
Compounds 4 and 6 have both been one’. The ligand and an Isolink Kit were
and the Cp-ring. These methylene groups
described before.[30,40] With bromoacetic sealed in a vial and [99mTcO4]– was added.
alter the distance between receptor binding
acid methylester or bromopropionic acid Alternatively, a two-step procedure was motive and pendent complex and will al-
methylester respectively, 4 and 6 were syn- employed; [99mTc(OH2)3(CO) ]+ was syn-
low a more profound insight into SARs for
thesized and basic hydrolysis gave 5 and 7 thesized separately and added 3to the ligand. this important class of bioorganometallic
(Scheme 2).
Both procedures resulted in the clean for- CA targeting agents (Scheme 7).
The Re-complex 8 was synthesized
mation of the 99mTc-complexes 11 and 12
directly in water. Similar to 99mTc-labeling
respectively (Scheme 5 and 6). No relevant
99m
com-
of HCp-compounds, ligand 5 was reacted amounts of side products were observed in Conclusion
with [ReBr3(CO) ]2– in water with Borax either case. The products were analyzed
(Na B4O7*10H2O3) at 95 °C, representing by coinjection with the corresponding Re
Derivatives of HCp comprising a CP-
NHCO-R spacer were prepared in their
one2 of the rare examples of an aqueous
synthesis of a complex [(CpR)Re(CO)3]
(Scheme 3). Yields were low (20%) due
dimeric form from Thiele’s acid by use of
the Curtius rearrangement. The reaction
to a competing cluster formation reaction
progress was investigated by time-resolved
IR-measurement. Addition of nucleophiles
like alcohols or amines to isocyanate 2,
generated in situ the compounds 3a and 3b.
Compounds 5 and 7 contain a carboxyl-
ic acid function in β- or in γ-position, with
respect to the HCp-ring. Labelling experi-
ments with [99mTc(OH )3(CO)3]+
proved
that these positions are 2still close enough
to allow anchoring of the metal center by
the carboxylic acid portion. Therefore,
5
bringing the metal in close proximity, η -
coordination of the HCp-portion is sim-
plified. However, conversion rates of the
99mTc starting compounds were very differ-
ent for the ligand 5 compared to 7, which is
in accordance with the increasing distance
of the anchoring group to the HCp-moiety.
Fig. 3. ORTEP representation of 10b. Relevant
bond lengths (Å) and angles (°): Re(1)–C(2)
Scheme 5. Bottom-up trace: γ-trace of the
one-pot labeling of 5 with [99mTcO4 ]– and Isolink
Kit. Top-down trace: UV-trace of Re-compound
8. Quantitative and radiochemically pure con-
version of [99mTcO4 ]– to complex 11.
1.910(3), Re(1)–C(3) 1.919(3), Re(1)–C(1)
1.919(3), Re(1)–C(6) 2.287(3), Re(1)–C(5)
2.291(3), Re(1)–C(7) 2.304(3), Re(1)–C(8)
2.311(3), Re(1)–C(4) 2.324(2), C(2)-Re(1)-C(1)
90.10(12), C(3)-Re(1)-C(1) 90.61(12).
Compound 8 was synthesized in a similar
fashion to the 99mTc-compound 11 using
ligand 5 with [Re(Br)3(CO)3]2– in water,

270 CHIMIA 2013, 67, Nr. 4
Laureates: awards and Honors, sCs FaLL Meeting 2012
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Scheme 6. Bottom-
up trace: γ-trace of
the one-pot labeling
of 7 with [99mTcO4 ]–
and Isolink Kit. Top-
down trace: UV-trace
of Re-compound 10b.
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