Cyclic-RGD penta-peptides cRGDyK derivatized with cyclopentadienyl complexes of technetium and rhenium as radiopharmaceutical probes

Article abstract of DOI:10.1002/jlcr.3515

The present study reports the syntheses of half-sandwich complexes of the type [M(η⁵-C₅H₄CONH-R)(CO)₃] (M═Re,^99mTc;R═cyclic RGD peptide (cRGDyK) for potential imaging of α_vβ₃ int

Full text of DOI:10.1002/jlcr.3515

Cyclic-RGD penta-peptides cRGDyK derivatized with cyclopentadienyl complexes of
technetium and rhenium as radiopharmaceutical probes
Qaisar Nadeem,a* Yunjun Shen,b Muhammad Farooq Warsi,a Gulfam Nasar,c Muhammad Abdul Qadir,d and Roger Albertoe*
a)
Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur
63100, Pakistan. Email: qaisar.nadeem@iub.edu.pk
b) Department of Chemistry, Zhejiang University, Zheda Road 38, 310027 Hangzhou, P. R. China.
c)
Department of Chemistry, Balochistan University of Information Technology, Engineering and Management
Sciences Quetta Pakistan.
d)
e)
Institute of Chemistry, University of the Punjab, Quaid-e-Azam Campus 54590, Lahore, Pakistan.
Department of Chemistry, University of Zurich, Winterthurerstr. 190, 8057Zurich, Switzerland. E-mail:
ariel@aci.uzh.ch
Abstract
The present study reports the syntheses of half-sandwich complexes of the type [M(η5-
C5H4CONH-R)(CO)3] (M=Re,99mTc;R=cyclic RGD peptide (cRGDyK) for potential imaging
of αvβ3 integrin expression. The 99mTc complex was prepared directly from the reaction of
[
99mTc(OH2)3(CO)3]+ with cRGDyK, doubly conjugated to Thiele’s acid [(C5H5COOH)2] in
water. This approach extends the viability of metal-mediated retro Diels-Alder reactions for
the preparation of small molecules such as linear tripeptides to a more complex cyclic peptide
carrying a [(η5-C5H4)99mTc(CO)3] tag. The Diels-Alder product [(C5H5CONH-cRGDyK)2]
was prepared from Thiele’s acid via double peptide coupling. The Re-complex [Re(η5-
C5H4CONH-cRGDyK)(CO)3] was obtained by attaching [Re(η5-C5H4COOH)(CO)3] directly
to the N-terminus of cRGDyK. The identity of the 99mTc-complex is confirmed by
chromatographic comparison with the corresponding rhenium complex, fully characterized
by spectroscopic techniques.
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/jlcr.3515
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Introduction
In SPECT or PET imaging, peptides labeled with radioisotopes received much attention due
to their important roles in imaging cellular functions. 111In-DTPA-octreotide (OctreoScan;
Mallinckrodt Inc.) is a well-known example in routine oncology for diagnosis and staging of
somatostatin tumours.1 Peptides sequences which have high affinities and specificities for
receptors, have been regarded as active at nanomolar level.2 To date, numerous peptides have
been identified and employed for disease detection.3-5 For example, somatostatin (SST)
peptide, vasoactive intestinal peptide (VIP), Arg-Gly-Asp (RGD) peptide, bombesin/gastrin-
releasing peptide (BBN/GRP), cholecystokinin (CCK)/gastrin peptide, α-melanocyte-
stimulating hormone (α-MSH), neutrotensin (NT), T140, exendin-4, Neuropeptide Y (NPY),
substance P and tumour molecular targeted peptide 1 (TMTP1) are studied for imaging
tumour receptors and some are amongst the subjects of current (pre) clinical evaluation.6-8
The retention of peptides in tumours is induced by their high binding affinities to specific
integrins and/or cell surface transmembrane glycoproteins,9,10 which act as targets for cancer
treatment11,12, thrombosis13 and other diseases.14,15
The integrins like heterodimers αvβ3, αvβ5, αvβ6, α6β4, α4β1 and α5β1 are related to a variety of
tumors.16,17 The αvβ3 for instance is expressed in comparatively low concentration on mature
endothelial cells and epithelial cells as well as in breast, lung, prostate and other
carcinomas.18,19 Therefore, the αvβ3 has been recognized as target in the beginning of fast
rising tumors.20,21 The affinity and selectivity of the cyclic penta-peptide (cRGDfV) for αvβ3
has been recognized to be greater than that of linear RGD peptide.22-24 The valine in position
5 can be replaced by lysine to afford (cRGDfK), with comparable affinity for αvβ3. The RGD
peptide can carry radioisotopes to integrins on tumors. Various radiolabeled cyclic RGD
peptides have been reported.21,25 Haubner et al.26 was the first to initiate 125I-radiolabling and
to study biodistributions of the RGD peptides, (cRGDfV) and (cRGDyV). Another example
is [18F]Galacto-RGD and [18F]AH111585, which is presently in clinical investigation.27,28
The choice of bifunctional chelators (BFC) is essential for labeling with metallic
radionuclides. Chelators such as DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid) and DTPA (diethylenetriaminepentaacetic acid) have been coupled to RGD through Lys
for coordinating a variety of radioisotopes.29,30 Despite the progress of labeling with
radionuclides belonging to the “3+” family, more than 80 % of nuclear medicine imaging
investigations are still done with 99mTc.68,69 In this regard, 99mTc-NC100692, 99mTc-labeled
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cyclic RGD peptide monomer, currently under preclinical evaluation, has excellent αvβ3
affinity.31-33 The [99mTc(OH2)3(CO)3]+ represents a versatile precursor for the labeling of
biomolecules in an aqueous conditions34 with which the cyclic RGD compound cyclo-[Arg-
Gly-Asp-D-Tyr-Lys(PZ)] has been labeled. This conjugate is in vitro and in vivo stable and
retains its biological activity and high selectivity for αvβ3.35,36
Alterations in the 99mTc chelate results in significant uptake differences in normal
organs (kidney, lungs spleen etc) but should have little impact on tumor uptake.37,38 A small
ligand is therefore desirable for small peptides.39,40 Thus, the cyclopentadienyl (Cp) ligand is
an obvious choice due to its small size and low molecular weight. Notably, it mimics phenyl
rings in pharmaceuticals or biomolecules such as amino acids and tripeptides.41-46 In
extending ongoing research towards the labeling of biomolecules with [M(5-C5H4-R)(CO)3]
(R=targeting group, M=99mTc), the syntheses of a cyclic RGD peptide, radiolabeled by
metal mediated retro Diels-Alder reaction was carried out in aqueous solution.46,47 In here,
the labeling study shows that the concept can be extended from small molecules to more
complex, cyclic peptides as well.
EXPERIMENTAL
General materials and methods
Fmoc-amino acids with side chain protecting groups, Fmoc-Gly-OH, Fmoc-Arg(pbf)-OH,
Fmoc-Lys(Z)-OH, Fmoc-D-Tyr(tBu)-OH, Fmoc-Asp(tBu)-OH, 2-chlorotrityl chloride resin
were obtained from Iris Biotech GmbH, HOBt from Acros organics, HBTU from
Fluorochem, sodium cyclopentadienide (Cp), HATU and DMA from Sigma Aldrich. Thiele’s
acid and [Re(5-C5H4COOH)(CO)3] were prepared according to literature procedure.20,23
1
ESI-MS, Bruker Esquire HCT (ESI) instrument, H NMR and 13C NMR spectra were
recorded on a Bruker Advance 500 MHz or 400 MHz spectrometers. RP-HPLC was
performed on Merck Hitachi LaChrom L7200 with tunable UV detector, radiodetector,
separated by a Teflon tube causing about a 0.4-0.7 min delay compared to UV/vis detection.
Analytical separations were performed on a Nucleosil C-18 column (100 Å, 5 µm, 250×4
mm) which was eluted with a flow rate of 0.5 ml/min, using 0.1 % TFA in H2O (solvent A)
and methanol (solvent B) as eluents with variable gradients (0-3 min, 100 % A; 3-3.1 min, 0
to 25 % B; 3.1-9min, 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). Preparative
HPLC was performed on a Varian Pro Star system by using a Macherey-Nagel VP 250/30
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Nucleosil 100-7 C18 column with flow rates of 30 mL min-1. The solvents were 0.1%
trifluoroacetic acid (solvent A) and methanol (solvent B). The microwave oven was a power
controlled Biotage Initiator EXP microwave system instrument. Labeling was performed in a
septum closed vial at constant temperature. The compound 1 was prepared according to
literature with some modification.48
Synthetic procedures
Synthesis of cyclo-[Arg(Pbf)-Gly-Asp(OtBu)-D-Tyr(OtBu)-Lys-H] (cRGDyK, 1)
The preparation of compound 1is given in SI1and scheme 1.
Synthesis of [(C5H5CONH-cRGDyK)2] (2)
Compound 1 (1eq.), Thiele’s acid [(C5H5COOH)2] (0.5 eq.), HOBt (2 eq.) and HBTU (2 eq.)
was dissolved in 10 ml of DMF and DIEA (5 eq.) was added (Scheme 1). The reaction
mixture was stirred for 22 h. This resulted in covalent attachment of the N-terminal amino
groups of the peptides to the two carboxylic groups of Thiele’s acid. DMF was removed in
vacuo to obtain the crude dimer with protecting groups (as attached to 1). The protecting
groups were removed under N2 after treatment with 20 ml of 95 % TFA, 2.5 % H2O and 2.5
% TIPS for 2 h. The reaction solutions were concentrated and precipitated with ice-cold
MTBE. After centrifugation, purification by preparative RP-HPLC and lyophilization, the
dimer 2 was obtained as white solid in 50 % yield. RP-HPLC: retention time (tR) = 17.3 min;
MS-ESI (m/z): [M + H]+ calcd. for C66H91N18O18, 1423.7; found, 1423.1.
Synthesis of [Re(η5-C5H4CONH-cRGDyK)(CO)3] (3)
[Re(5-C5H4COOH)(CO)3] (2 eq.), compound 1 (1eq.), HOBt (2 eq.) and HBTU (2 eq.) were
dissolved in 10 ml DMF and 5 eq. DIEA were added. The reaction solution was stirred for 22
h, resulting in covalent attachment of the N-terminus of the cyclic-peptides to the carboxylic
group of [Re5-C5H4COOH)(CO)3]. Removal of protecting groups and purification as
described for 2, compound 3 was obtained as white solid in 52 % yield. RP-HPLC: retention
1
time (tR) = 18.6 min; IR (cm-1, KBr): 2027, 1934, 1648, 1545, 1384, 1205, 1141; H NMR
(500 MHz, d4-MeOD) δ ppm: 7.01 (d, J = 8.5 Hz, 2H, ArH), 6.70 (d, J = 8.5 Hz, 2H, ArH),
6.18 (t, J = 2.3 Hz, 2H, Cp), 5.59 (t, J = 2.3 Hz, 2H, Cp), 4.78-4.75 (m, 1H, CH), 4.71-4.44
(m, 1H, CH), 4.30-4.25 (m, 2H, CH), 3.92-3.89 (m, 1H, CHaHb), 3.33 (m, 1H, CHaHb), 3.30-
3.15 (m, 4H, CH2), 2.89-2.57 (m, 4H, CH2), 1.90-1.37 (m, 8H, CH2), 1.08-1.02 (m, 2H, CH2);
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13C NMR (125 MHz, d4-MeOD) δ ppm: 194.4, 175.2, 174.4, 173.9, 173.7, 172.8, 172.3,
165.1, 158.7, 157.5, 131.6, 128.6, 116.6, 96.1, 88.0, 87.8, 86.8, 86.7, 57.3, 56.6, 53.8, 51.2,
45.0, 42.1, 40.3, 37.9, 36.2, 32.0, 29.9, 29.6, 26.5, 24.5; MS-ESI (m/z): [M + H]+ calcd. for
C36H45N9O12Re, 982.3; found, 981.9.
Labeling with [99mTc(OH2)3(CO)3]+
[
99mTc(η5-C5H4CONH-cRGDyK)(CO)3] (4). A vial was charged with 2 mg of compound 2,
4mg Na[H3BCOOH], 7mg Na2[C4H4O6].2H2O and 7mg Na2B4O7·10H2O under N2.
0
Na[99mTcO4] (1 ml, saline) was injected and the vial was heated to 110 C for 50-60 minutes
under microwave. The product was identified by coinjection with the Re-complex 3 using
HPLC coupled with gamma detector. This method was also performed for thermal heating at
85-90 0C for 30-300 minutes.
RESULTS AND DISCUSSIONS
Synthesis of Diels-Alder conjugate 2
A previous study showed that small biomolecules can be conjugated to Thiele’s acid to yield
compounds of the form [(C5H5CONH-R)2].46,47,49-51 Following this procedure, different linear
tri-peptide sequences such as Leu-Gly-Gly were coupled to (C5H5COOH)2. Subsequent
reactions with [99mTc(OH2)3(CO)3]+ allowed the convenient labeling of these tri-peptides with
piano-stool complexes to yield [99mTc(5-C5H4CONH-R)(CO)3]. In here, this labeling
scheme to cyclic peptides is extended. As depicted in scheme 1 and SI 1, the basic linear
pentapeptide was obtained by stepwise elongation on 2-chlorotrityl chloride resin with
standard SPPS techniques. After cleavage from the resin, cyclization of the linear peptide in
solution gave fully protected (cRGDyK) [cyclo-(Arg(Pbf)-Gly-Asp(OtBu)-D-Tyr(OtBu)-Lys-
Z)]. Removal of protecting group Z on Lys by H2/Pd and purification by preparative HPLC
gave 1. Two compounds 1 were then conjugated at the -amino groups of Lys to both
carboxylic acid groups on Thiele’s acid by HBTU/HOBt (scheme 1). Complete removal of all
protecting groups with 20 % piperidine in DMF and purification gave the final product 2 in
50 % yield as nicely be identified by HPLC. Compound 1 elutes with 21.8 min retention time
and 2 17.3 min respectively. MS analyses confirmed the products with main peaks at 984.3 of
([1+H]+) and 1423.1 of ([2+H]+) (see SI 2 to SI 5).
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Synthesis of the Rhenium complex 3
The Rhenium complexes [Re(5-C5H4COOH)(CO)3] can be obtained along different
pathways.46,52,53 Originally, [M(5-C5H4COOCH3)(CO)3] (M=Re, Tc) was synthesized from
[MO4]- by the so-called double ligand transfer reaction in the presence of CrCl3 and a
ferrocene (Fc) derivative which provides the Cp-system. Additional [Cr(CO)6] acts as the
CO source and the reducing agent. This procedure gave [M(C5H4COOCH3)(CO)3] which was
hydrolysed and conjugated finally to a peptide.52 Alternatively, reaction of NH4ReO4 with
[Re2(CO)10] and Thiele’s acid gave [M(5-C5H4COOH)(CO)3].53 In a previous approach,
[Re(5-C5H4CONH-R)(CO)3] (R=tri-peptides) were synthesized by conjugating [Re(5-
C5H4COOH)(CO)3] to resin bound, protected tri-peptides, followed by deprotection and
cleavage from the resin.46
The rhenium compounds [Re(5-C5H4CONH-cRGDyK)(CO)3] are essential for identifying
the nature of their 99mTc homologues [99mTc(5-C5H4CONH-cRGDyK)(CO)3] by comparing
their respective HPLC retention times. The precursor, [Re(5-C5H4COOH)(CO)3] was
synthesized according to a reported procedure.53 The amino group in [Cyclo-(Arg(Pbf)-Gly-
Asp(OtBu)-D-Tyr(OtBu)-Lys)] (1) was coupled to [Re(5-C5H4COOH)(CO)3] (scheme 2) in
the same way after HPLC purification as for 2. Accordingly, 3 was obtained in 52 % yields as
white solid, fully characterized by spectroscopic techniques and the purity confirmed by RP-
HPLC (tR=18.6 min). The 1H NMR shows two triplets between 6.2 and 5.6 ppm indicative for
the Cp-ring protons. The aromatic protons appear doublets between 7.0 and 6.7 ppm. The 13C
NMR displays well resolved signals (in CD3OD) between 86-96 ppm for the carbon atoms of
Cp. The two characteristic strong vibration bands at 2027 and 1933 cm-1 confirm the fac-
[(Re(CO)3]+ fragment. Furthermore, ESI-MS in methanol assesses the correct m/z at 981.9 in
accordance to ([3+H]+) (see SI 6 to SI 10).
Synthesis of the 99mTc-complex - labeling
For routine 99mTc labeling of peptides, the post-labeling method is the most convenient
one.54,55 In this method, the peptide conjugate with a suitable chelator is separately prepared,
followed by the labeling with 99mTc. Post-labelling is favorable over pre-labeling approach
which involves the separate formation of 99mTc-chelate, followed by conjugation to the
peptide/biomolecule and purification from unlabeled biovectors. Labeling biomolecules with
the cyclopentadienyl ligand is challenging since not immediately compatible with aqueous
conditions. Originally, half-sandwich complexes of the type [99mTc(5-C5H4-R)(CO)3] were
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synthesized by Wenzel et al.56,57 by double ligand transfer reaction from [TcO4]-, ferrocene,
Mn(CO)5Br and SnCl2 as reducing agent in organic solvents at 150 0C or by
Katzenellenbogen et al.52 in one step. Reaction conditions were however not ideal due to high
temperatures and the extensive formation of manganese homologues. In another approach,
the direct reaction of Thiele’s acid derivatives with [99mTcO4]-or [99mTc(OH2)3(CO)3]+ in
aqueous medium is more versatile as it gives directly access to a multitude of
[
99mTc(C5H4CONH-R)(CO)3] compounds with a variety of biomolecules “R”.47,49,46,50-51 as
reported e.g. for the direct labeling of tri-peptides-Cp in one step from [99mTcO4]-.46 The
present work extended the same approach now to [99mTc(C5H4CONH-cRGDyK)(CO)3] (4)
by a post-labeling method. Radiolabeling of 2 was achieved in a “one pot” reaction with
[
99mTcO4]- in the presence of boranocarbonate [H3BCOOH]- without adjustment of the pH
(under alkaline condition). The metal mediated retro-Diels-Alder reaction occurred but the
radiolabeling yield of 99mTc complex 4 was only 10 % after 1 h thermal heating of 2 at 90 0C
(scheme 2). Side products started to form after prolonged heating. The labeling in a one pot
reaction was slow, leading to side products. Therefore, the thermal heating process is
inappropriate for the synthesis 4. In addition to the slow thermal process, Cp-coordination has
to compete with other binding sites such as the guanidine group in arginine (NH2C=NH) or
other functionalities in more complex peptide sequences.
Often, better yields with fewer side products are obtained by microwave heating.58,59 Under
these heating conditions, compound 4 was obtained in 49 % yield from reacting only 0.7 mg
of 2 at 110 0C for 1 h. The one pot labeling by the microwave process is therefore superior to
thermal heating. The resulting 99mTc complex 4 was easily purified by RP-HPLC to achieve
purity > 98 %. The substantial retention time difference of 4 from the peptide precursor
supports radio-purity and specific activity of the product. The identity of 99mTc complex 4
was assessed by coinjection with the rhenium homologue 3 (Figure 1). It should be noted that
the retro Diels-Alder reaction produces one non-labeled peptide per 99mTc carrying product.
The former is more difficult to separate, however, considering the extremely low amount of a
few picomol, it is obvious that purification of this material is not of a major concern for the
later imaging agent.
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Conclusion
The radiolabeling of peptides with small 99mTc complexes is an ongoing field of research. In
particular, the current need for better αvβ3-specific 99mTc radiotracers from inexpensive kit
formulation prompted us to extend the concept of metal mediated retro-Diels-Alder reaction
to more complex biomolecules such as cyclic RGD peptides. The penta-peptide cRGDyK,
doubly conjugated to Thiele’s acid, forms dimer as prepared in this study. It acts as the direct
precursors for labeling the RGD peptide and [99mTc(η5-C5H4CONH-cRGDyK)(CO)3)] was
obtained. Considering the relative complexity of this peptide, the presented labeling method
is general and can be applied to other peptides as well. Direct labeling in one step from
[
99mTcO4]- by thermal and microwave heating resulted in the desired tracer in 10 and 49 %
yields, respectively. HPLC purification gives radiochemically pure 99mTc labeled cyclic RGD
peptide. Thus, presented work revealed with potentially αvβ3 binding RGD peptide that the
concept of retro-Diels-Alder reactions can be extended to more complex biomolecules such
as cRGDyK.
Acknowledgements
This work was financially supported by Higher Education Commission, Pakistan (PIN no.
IRSIP 18 Ps 06) and the University of Zürich, Switzerland.
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Scheme 1. Synthesis of [(C5H5CONH-cRGDyK)2] (2) (a) 20 % piperidine/DMF; (b) Fmoc-AA-OH:
[Fmoc-Arg(pbf), Fmoc-Lys(Z)-OH, Fmoc-D-Tyr(tBu)-OH, Fmoc-Asp(tBu) respectively], HOBt,
HBTU, DIEA/DMF; (c) TFA/DCM; (d) cyclization: HOBt, HBTU, DIEA/DMF; (e) H2, Pd/DMA; (f)
HOBt, HBTU, DIEA/DMF; (g) TFA, TIPS, Water
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Scheme 2. Synthesis of [Re(η5-C5H4CONH-cRGDyK)(CO)3] (3) and [99mTc(η5-C5H4CONH-
cRGDyK)(CO)3] (4) (a) HOBt, HBTU, DIEA/DMF; (b) TFA, TIPS, Water; (c) Na[H3BCO2H],
Na2B4O7.10H2O and Na2tartrate. 2H2O
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0
5
10
15
20
25
30
(4)
(3)
0
5
10
15
20
25
30
Minutes
Fig. 1 HPLC traces of Re-complex (3) and the corresponding 99mTc-complex (4) as received by
coinjection of the two compounds. Time difference is due to detector separation.
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