Bis(thiosemicarbazones) as bifunctional chelators for the room temperature 64-copper labeling of peptides

Article abstract of DOI:10.1039/b925128f

A range of new carboxylate functionalised bis(thiosemicarbazone) ligands and their Cu(ii) complexes have been prepared, fully characterised and radiolabeled in high yield with both ⁶⁴Cu and ^99mTc. Conjugation to a bombesin derivative was achieved using standard solid phase synthetic methodologies and the ⁶⁴Cu-labeled conjugate was shown to have good tumour uptake in mice with xenografted PC-3 tumours.

Full text of DOI:10.1039/b925128f

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Bis(thiosemicarbazones) as bifunctional chelators for the room temperature
64-copper labeling of peptides†
Rebekka Hueting,a,c Martin Christlieb,*c Jonathan R. Dilworth,*a Elisa Garc´ıa Garayoa,b
Ve´ronique Gouverneur,a Michael W. Jones,a Veronique Maes,d Roger Schibli,b Xin Suna and Dirk A. Tourwe´d
Received 2nd December 2009, Accepted 28th January 2010
First published as an Advance Article on the web 3rd March 2010
DOI: 10.1039/b925128f
A range of new carboxylate functionalised bis(thiosemicarbazone) ligands and their Cu(II) complexes
have been prepared, fully characterised and radiolabeled in high yield with both 64Cu and 99mTc.
Conjugation to a bombesin derivative was achieved using standard solid phase synthetic methodologies
and the 64Cu-labeled conjugate was shown to have good tumour uptake in mice with xenografted PC-3
tumours.
We have previously reported the synthesis of the bis(thiosemi-
carbazone)ligand H2ATSR/A which can be functionalised via the
Introduction
exocyclic pendant amine substituent (Fig. 1).5 However, solid
phase peptide synthesis mostly proceeds in a C-terminus to
N-terminus fashion, and some peptides and proteins require con-
jugation via their N-terminus or amine residues in order to preserve
the biological function of the C-terminus.9 We have also earlier de-
scribed the synthesis of a bifunctional 64Cu bis(thiosemicarbazone)
with a pendant carboxylic acid functionality for conjugation to
biologically active molecules. This particular ligand system was
found to suffer from purification and solubility problems.10
Target specific delivery of radionuclides for imaging of biological
function has been of interest for several decades. The choice of
radioisotope and bifunctional chelator are crucial for successful
imaging and therapy, chelators should be easily attached to
the peptide or protein and radiolabeling conditions must be
+
biocompatible. 64Cu (t1 = 12.7 h, Eav = 278 keV) is both a b
2
and a b- emitter allowing it to be used for both imaging and
radiotherapy.1,2 There are many reports of the use of 64Cu-labeled
macrocyclic chelators such as DOTA and TETA for the labeling of
biomolecules.1-3 However, nearly all require elevated temperatures
over prolonged periods for efficient labeling. A chelating system
with fast labeling kinetics at room temperature, at near neutral pH
and giving high yields would provide advantages in the labeling of
sensitive biomolecules unable to withstand elevated temperatures
and extreme pH ranges.
N2S2-type bis(thiosemicarbazone) ligands form stable neutral
and planar complexes with Cu(II) ions. This type of complex has
been investigated extensively for the imaging of hypoxia in the
form of copper(II)-diacetyl-bis(N-4-methylthiosemicarbazone),
Cu[ATSM]. Radiolabeling occurs instantly at room tempera-
ture and near physiological pH.4,5 This enables facile labeling
post conjugation and avoids multi-step radiosynthesis. Bis-
(thiosemicarbazones) offer advantages for practical kit for-
mulation when a copper isotope is the desired radionuclide and
this would facilitate successful translation into routine clinical
diagnosis and therapy.6-8
Fig. 1 Schematic diagram of a functionalised bis(thiosemicarbazone)
conjugated to a biologically active molecule (BAM) for receptor targeted
imaging.
In this paper, we report the synthesis of an entirely new series
of bis(thiosemicarbazone) based copper(II) chelators bearing pen-
dant COOH groups for the radiolabeling of peptides and proteins.
We have opted for derivatisation of the ATSM core via the carboxyl
functionality as this is compatible with traditional solid phase
peptide synthesis methods. A number of chelating systems bearing
aliphatic and aromatic spacers based on the H2ATSR/A proligand
have been synthesised, characterised and radiolabeled. Their
suitability as bifunctional chelators and practical convenience have
been assessed by subjecting them to standard solid phase peptide
synthesis as well as solution phase coupling conditions commonly
used for protein bioconjugation.
We chose a modified bombesin (BBS) sequence as model
system as this provides substantially enhanced pharmacokinetics
compared to the native form of the protein. Bombesin binds with
high affinity to the gastrin releasing peptide receptors that are
overexpressed by a variety of tumour cells such as prostate, breast,
ovarian and small cell lung cancer.11 Compatibility of the new
aDepartment of Chemistry, Chemistry Research Laboratory, University of
Oxford, 12 Mansfield Road, Oxford, OX1 3TA, United Kingdom. E-mail:
jon.dilworth@chem.ox.ac.uk; Fax: +44 (0)1865 272690; Tel: +44 (0)1865
272689
bPaul Scherrer Institute, Centre for Radiopharmaceutical Science, CH-5232,
Villigen, Switzerland
cGray Institute for Radiation Oncology and Biology, University of Oxford,
Old Road Campus Research Bldg., Old Road Campus, Oxford, OX3 7DQ,
United Kingdom. E-mail: martin.christlieb@rob.ox.ac.uk
dDepartment of Organic Chemistry, Vrije Universiteit Brussels, 1050,
Brussels, Belgium
† Electronic supplementary information (ESI) available: Fig. S1–S3. See
DOI: 10.1039/b925128f
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ATSM derivatives with solution phase coupling conditions was
demonstrated by reacting GlyGlyGly under aqueous conditions
with the sulfo-NHS ester of the imine and amide bonded ligands.
Radiolabeling experiments with the thiosemicarbazone derived
ligands and 99mTc were also successful. Initial evaluations of
stability and serum binding show that the Tc species are stable
in solution, show low protein binding and suggest that it may be
possible to expand the scope of this ligand system to encompass
SPECT as well as PET imaging. This provides a rare example of a
ligand system that can be used for both technetium and copper in
a radiopharmaceutical context.
m/z of 342. The extremely low solubility prevented further
characterisation.
Previous derivatives of 2 or 3 have been mainly based on
aromatic imine conjugates, prepared by heating 2 under reflux
in MeOH or EtOH with an aldehyde or ketone followed by
Cu-transmetallation.12,13 This prompted us to investigate phenyl-
carboxylate imine derivatives which we anticipated would have
improved solubility characteristics compared to the previously
reported carboxylate derivatives.10 Unfortunately, reaction of zinc
complex 3 with 2- or 4-carboxybenzaldehyde proved problematic,
giving multiple products based on 1H NMR and HPLC analysis.
Trial reactions with the corresponding methyl ester analogues, in
contrast to the free acids, were found to proceed cleanly. Thus,
the problems encountered may be due to the unprotected acid
group binding to the free apical (fifth) coordination site on Zn
either during reaction, isolation or analysis. We proceeded with
the metal-free proligand 1 which was successfully reacted with 2-
and 4-carboxybenzaldehyde at room temperature (Scheme 1). We
were in fact able to show that the aromatic imine derivatives 4a/b
and 11a (vide supra) mentioned within this paper, were formed
in MeOH with the free proligands at room temperature or 40 ◦C
overnight in yields of 70-85%. This completely avoided ligand self-
cyclisation which was reported previously when carrying out such
reactions at higher temperatures.5 In contrast to other derivatives
previously reported, complexation and transmetallation of 4a/b,
5a/b and 8 proceeded most efficiently with ZnCl2 and CuCl2 rather
than the more commonly used metal acetates. Although the Zn
complexes do not serve as synthetic precursors in this instance,
we were still interested in their synthesis as we recently reported a
solid supported method for purifying the 64Cu-labeled complexes
from their Zn precursors using controlled axial coordination.8
It was found throughout these experiments that derivatives of
1 proved easier to purify than those of 2 due to their slightly
improved solubility in DMSO as well as protic solvents such as
EtOH and MeOH at elevated temperatures.
Results and discussion
Ligand synthesis - imine conjugates
The H2ATSR/A proligands 1 and 2 and the corresponding Zn
complex 3 (Fig. 2) were synthesised as previously reported.5
Fig. 2 Amine functionalised proligands and Zn(II) complex.
We were interested in routes to bis(thiosemicarbazone) ligands
with reactive pendant carboxylic acid groups. We hoped that a
simple ring-opening of succinic anhydride by ZnATSM/A 3 would
provide the desired aliphatic carboxyl derivative 10a (Fig. 3).
However, we isolated a mixture of unidentified products over a
range of temperatures from ambient to reflux in various solvent
systems (THF, MeOH, DMF). Similarly, reaction of proligands
of type 1 or 2 with succinic anhydride at room temperature
resulted in an unidentifiable range of products. Reaction at higher
temperatures resulted in a sparingly soluble precipitate. 1H NMR
and ESI-MS analysis of this compound suggested formation of a
ring closed product 10b (Fig. 3), indicated by one singlet resonance
for both methylene protons at 2.8 ppm and a corresponding
Ligand synthesis - amide conjugates
As the aromatic imine derivatives could be susceptible to hy-
drolysis in the presence of water, we also investigated the
synthesis of amide linked derivatives for improved stability.
1,4-benzenedicarboxylate motifs were used to avoid the possi-
bility of cyclisation and the OtBu-monoprotected diacid was
reacted with 1 or 2 in the presence of a range of coupling
agents. Attempts to react 1 or 2 with the N-hydroxysuccinimide
(NHS) activated esters of the monoprotected aromatic dicar-
boxylates resulted in incomplete reactions or multiple products.
Coupling with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)
(EDCI) was also unsuccessful. Eventually we found that effi-
cient coupling to 1 could be achieved with O-benzotriazole-
N,N,N¢,N¢-tetramethyl-uronium hexafluorophosphate (BOP) and
N,N-diisopropylethylamine. Compound 7 was synthesised cleanly
in a yield of 96% using 4-(tert-butoxy-carbonyl) benzoic acid which
was found to be more suitable than the mono-Me or Et esters which
were partially hydrolysed during the coupling procedure and led
to a mixture of products. Deprotection of 7 in neat TFA over 2.5 h
proceeded efficiently to give 8. It is noteworthy that exposure to
TFA was found not to affect the bis(thiosemicarbazone) ligand
system for periods of up to 4 h.
Fig. 3 Products of the reaction of H2ATSM/A with succinic anhydride.
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Scheme 1 Synthesis of imine and amide bonded functionalised bis(thiosemicarbazones) from the H2ATSE/A proligand. (i) 1.2 eq aldehyde, MeOH,
room temperature, 16 h (ii) 1.1 eq Zn(OAc)2·2H2O or Cu(OAc)2·H2O, MeOH, rt, 30 min (iii) 1.1 eq BOP, 1.1 eq diisopropylethylamine, DMF, rt, 4 h (iv)
trifluoroacetic acid (TFA), rt. 2.5 h (v) Cu(OAc)2·H2O (for radiolabeling conditions for 9a and 9b vide supra).
Amino acid conjugation and extended linker synthesis
for 7 afforded the extended linker 11c (Fig. 4). In the case of
4a, the extended linker was obtained by condensation of 1 with
the appropriate aldehyde methyl 2-(4-formylbenzamido)acetate.14
Copper complexation to give 12a and 12c was quantitative for all
the ATSE proligands with extended linkers and occurred rapidly
at room temperature in MeOH.
To assess the aqueous phase coupling behaviour of our ligands
with peptides and proteins we wanted to couple 4a, 4b and 8
(Scheme 1) to glycine and triglycine as model amino acid and
peptide sequence mimic respectively. The activated NHS esters of
4a/b and 8 were readily formed using EDCI in DMF. However
these intermediates did not react readily with glycine or triglycine
sequences and were recovered unchanged. This may be due to
the general lack of reactivity of aromatic carboxylates compared
to their aliphatic counterparts compounded by a stabilising
conjugation with the ATSE ligand system. Interestingly, coupling
of the pendant aromatic COOH group of 8 to glycine(tert-
butylester) was acomplished as previously with BOP and N,N-
diisopropylethylamine to give the corresponding conjugate 11b in
98% yield (Fig. 4). Deprotection conditions analogous to those
Our extended linkers 11a and 11c were successfully converted
to their water soluble sulfo-NHS ester analogues using EDCI and
sulfo-NHS in DMSO. Coupling was achieved within 15 min as
confirmed by ESI-MS, and the sulfo-NHS esters of ligands 11a and
11c were reacted with triglycine in DMSO or in buffered aqueous
conditions. Reactions were monitored by ESI-MS and showed that
after one hour the desired conjugate could already be detected
and after 12 h only product and residual 11a (when conducted
in H2O) were detected but no starting active ester as in the
case of the non-extended linkers (see Supplementary Information
for structures). These preliminary experiments demonstrated that
our extended linker derivatives are in principle suitable for
conjugation to pendant amino groups on larger biomolecules such
as proteins under aqueous conditions using a frequently employed
EDC/sulfo-NHS approach.15
Bifunctional derivatisation–conjugation with glutamic acid
We were also interested in preparing a bifunctional chelator
that, in addition to being capable of conjugation to a targeting
biomolecule, could potentially accommodate another orthogonal
biomarker.
In order to further enhance the solubility we wanted to provide
ligands bearing aliphatic spacers. Thus, we chose to couple our
proligands 1 and 2 to glutamic acid with orthogonally-protected
NH2 and CO2H groups. Attempts to couple 1 or 2 via a NHS-
activated glutamic acid derivative led to incomplete reactions
or non-purifiable products. The synthesis of 14 (Scheme 2) was
realised by coupling of 1 with FmocGlu(OtBu)OH in an analogous
fashion to the conditions used for ligand 7. Selective deprotection
Fig. 4 Extended linkers.
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Scheme 2 Synthesis, selective deprotection and bioconjugation of aliphatic linked bis(thiosemicarbazone) bifunctional chelators. 14 and 16 were prepared
under the same conditions as for 7 using BOP. (i) 20% piperidine, DMF, 45 min then H2O, then Et2O. (ii) TFA, 3 h, rt then Et2O. (iii) Cu(OAc)2·H2O,
MeOH, rt, 30 min. (iv) Biotin, BOP, DIPEA, DMF, 4 h.
to give the free amine or free acid was easily achieved with
standard Fmoc/tBu deprotection methods using 20% piperidine
in DMF to give 15 and neat TFA over 3 h to give 17 followed
by Cu-metallation to give 18. As proof-of-principle, 15 was
conjugated to the commonly used biomarker biotin. Subsequent
Cu-complexation to form 19 proceeded with a 96% yield.
The successful, selective deprotection, conjugation and facile
complexation with copper suggest that 15 and 17 may be suitable
for simultaneous attachment of further pretargeting groups as
well as offering the possibility of incorporating a radiotracer at
any point within a peptide sequence.
for other radionuclides. Kethoxal-bis(thiosemicarbazone) (KTS)
is reported to have been radiolabeled with 99mTcO4 to form an
-
uncharged 99mTc(IV) complex, 99mTc-KTS, which it was suggested
to be stable in aqueous media.16 The labeling of ATSM and
simple alkylated analogues with 99mTc has also been reported.17
Interestingly the ATSM complex was shown to be hypoxic selective
in isolated ischaemic heart tissue.17 However, in neither paper was
the structure of the Tc complex established. Ligands 8 and 11c were
radiolabeled according to a modified procedure by complexation
with 99mTc using SnCl2 as a reducing agent in sodium carbonate
◦
buffer at 95 C.18 Pertechnetate and other hydrophilic impurities
were removed using a Sep-pakTM C18 cartridge19 to give the
products 9b and 13c in non-optimised radiochemical yields of 30–
40% (Table 1). The Sep-pakTM method potentially removes some
of the target 99mTc-complexes and hence the actual radiolabeling
efficiency might be higher.19 After purification, the radiochemical
purities of both complexes were >95% as determined by radio-
TLC using MeOH:10% NH4Ac (aq) (1 : 1) as solvent.16 The
Tc-colloid remains at the starting point, while the radiotraces of
both complexes moved with the solvent front (see Supplementary
Information†).
Radiolabeling
64Cu. 8, 11a, and 11c were radiolabeled by direct reaction of
[64Cu](OAc)2 with the corresponding pro-ligand as reported for
previous systems.5 All complexes were labeled with radiochemical
yields >99% and high radiochemical purities (radio-TLC, >95%).
As anticipated for bis(thiosemicarbazone) systems, all labeling
occurred at room temperature virtually instantaneously. Radio-
HPLC analysis within a few minutes of labeling indicated that
no peaks resulting from unbound residual 64Cu(OAc)2 were
present.
This suggests that both derivatives labeled successfully, but
further optimisation is needed to increase the yield. Experiments
are currently underway to determine the structures of the Tc
complexes using the long-lived 99Tc isotope and the biological
99mTc. We were also interested to see whether our
bis(thiosemicarbazone) ligand systems could also be employed
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Table 1 Radiolabeling yields and a comparison of serum binding and log
P values for 99mTc and 64Cu labeled carboxylate derivatives
Table 2 Chemical characterisation of bis(thiosemicarbazone) bombesin
conjugates
Compound
RCY
Serum Binding (120 min)
Log P
Compd
m/z found(calcd) % yield
purity HPLC tR/min
CuATSM
99%
>99%
60%
>99%
50%
20%
20%
26%
19%
32%
1.5
1.48
0.28
0.88
-0.41
ATSM-BBS-1 1638.6 (1637.8)
ATSM-BBS-2 1694.7 (1694.8)
53.4%
19.4%
94%
95%
14.1 min
14.0 min
9a
9b
12c
13c
chosen was the minimum active sequence BBS(7–14) modified to
achieve enhanced pharmacokinetics. It bears a b hAspbAlabAla
3
spacer between peptide and chelator as well as Leu13 and Met14
replacement by Cha and Nle respectively.24,25 A higher tumour-
to-background ratio and increased stability were also reported.
The chosen sequence has been previously labeled with 99mTc using
an (NaHis)Ac chelator for the radiometal thus a platform for
comparison of future in vitro and in vivo evaluations would be
available.26,27 The protected peptide was synthesised manually on a
Rink Amide Resin using the Fmoc-strategy previously described.25
Again, coupling of chelator 8 with standard DIC coupling
conditions posed difficulties but coupling to the resin-bound
BBS sequence using BOP proved successful (Scheme 3). Chelator
11c coupled easily using standard DIC coupling conditions as
anticipated due to the introduction of the Gly residue. Both
conjugates of 8 and 11c were then cleaved from the resin by
treatment with TFA for 3.5 h and lyophilised to afford 20
(ATSM-BBS-1) and 21 (ATSM-BBS-2) respectively. Preparative
HPLC and lyophilisation of the conjugates resulted in chemical
purities that compared well with previously synthesised BBS-
chelate systems (Table 2).28
behaviour of the 99mTc labeled bis(thiosemicarbazone) bifunctional
chelators in vitro and in vivo.
Serum binding studies of 64Cu and 99mTc labeled derivatives
The serum stability and protein binding of our new derivatives
were determined for comparison with the parent compound
Cu[ATSM]. The 64Cu and 99mTc-labeled analogues 9a, 9b, 12c and
13c and were incubated in mouse serum for 120 min and samples
were taken at different time points. After 5 min, approximately 20%
of the activity was bound to the proteins for the 64Cu analogues.
This is comparable to what has been observed for CuATSM and
other bis(thiosemicarbazones) previously which have been tested
in vivo.4,12
The 99mTc-labeled analogues had a higher degree of binding
to serum proteins at around 30% but the total activity bound
remained constant over time, suggesting little or no decomposition
over the time course by analogy with the Cu-labeled ATSM
derivatives. The slightly lower log P values of the 99mTc-labeled
complexes are consistent with the more hydrophilic nature of a
proposed Tc oxo-core and the Tc complex may be charged
EPR studies
Before proceeding to radiolabel our ATSM-BBS conjugates, we
wished to see whether metal binding at ambient temperature
would still result in specific Cu complexation at the chelator site
rather than unspecific binding to the peptide sequence. We titrated
both 8 and ATSM-BBS-1 with 1 eq of Cu(OAc)2·H2O at room
temperature.
SPPS synthesis of chelator-BBS conjugates
The ability to survive standard solid phase peptide synthesis
conditions is important if the chelator is to be incorporated as
a tag in a peptide sequence. The stable amide-bonded chelators 8
and 11c were deemed most suitable for SPPS.
BBS sequences have been extensively labeled with various
radionuclides such as 18F, 64Cu, 67Ga, 99mTc and 177Lu to provide
tumour specific tracers for receptor imaging.20-23 The sequence
The EPR spectra of both 9 and CuATSM-BBS-1 (Fig. 5) were
indicative of a single copper(II) species being formed, with no
evidence for other binding modes observed. The copper is d9
Scheme 3 Solid Phase Synthesis of the bisthiosemicarbazone-BBS conjugates: Reagents and Conditions: (i) 1 ml 25% 4-methylpiperidine in DMF, 2
¥10 min, rt then ATSM-BBS-1: 2 eq BOP, DIPEA, DMF, rt, 8 h; ATSM-BBS-2: 2 eq DIC, HOBt, 3 h, rt (ii) thioanisole (TA)/ethanedithiol (EDT) 7 : 3
in TFA, 3.5 h.
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Receptor binding and internalisation studies
The binding affinity of the ligand-peptide conjugate is an im-
portant prerequisite parameter for high in vivo tumour uptake.
We performed binding studies with the unlabeled ATSM-BBS-1
and ATSM-BBS-2 on human prostate adenocarcinoma PC-3 cells
which express a high level of GRPR. The affinity of both analogues
was similar (see Fig. 6). The IC50 values for 20 (ATSM-BBS-1) and
21 (ATSM-BBS-2) were 2.9 nM and 3.8 nM, respectively, which
is comparable to that of natural bombesin (1.9 nM). Furthermore
these values compared well to the analogous (NaHis)chelator
containing sequence previously used for 99mTc (5.1 nM).26,27 The
bis(thiosemicarbazone) chelating system thus does not seem to
have any adverse effect on the receptor affinity of the peptide.
Fig. 5 X-band EPR spectra of 9a (red) and Cu-labeled 20 (blue) in
anhydrous DMF/ethylene glycol 4 : 1 at 60 K.
square planar and the EPR spectra at X-band bear close similarity
to other Cu(II)ATSM derivatives previously studied.29-34 The gꢀ
values of 2.1299 were less than 2.3, which indicates covalent
character for the metal–ligand bonds.34 This was found to be
the case for Cu(II)ATSM and was supported by multiple DFT
calculations.32 NPA (natural population analysis) depictions of
the spin densities of Cu(II)ATSM indicated that a high degree of
covalency was present in the coordination sphere.32 As expected,
the gz and ACuz are the largest components to the hyperfines as
the unpaired electron is held in the dx2-y2 orbital and the largest
component is in the gz direction due to the cancelling out symmetry
of the orbital in the xy plane. The superhyperfine couplings
were not well resolved, as the molecules were not completely
symmetrical, making the coordinating nitrogens inequivalent.
This is in contrast to another derivative Cu(atsm/apyrene),33 in
which the two nitrogens were reported to be equivalent. However,
since the reported spectrum was measured at room temperature,
this is likely to be a result of molecular tumbling averaging out the
anisotropy of the g-tensors.
Fig. 6 Displacement curves of ATSM-BBS-1 and ATSM-BBS-2 in PC-3
cells.
Receptor internalisation was somewhat slower for the
64/67CuATSM-BBS conjugates than for the comparable 99mTc
containing systems. The non-specific binding was 40% for 64/67Cu-
ATSM-BBS-1 and 20-30% for Cu-ATSM-BBS-2 and is higher
than for BBS labeled with 99mTc(NaHis).
Overall, CuATSM-BBS-2 showed much better internalisation
than CuATSM-BBS-1 (see Supporting Information). Pleasingly,
the internalisation of CuATSM-BBS-2 at 40% is comparable to the
99mTc radiolabeled analogues which gave values of 30-45% (15%
if there are two negative charges involved in the spacer between
peptide and chelator).
EPR analysis thus confirmed site specific labeling for our
isolated CuATSM-BBS-1 conjugate and its Cu-labeled congener.
64/67Cu-radiolabeling of ATSM-BBS conjugates
Labeling was carried out in ammonium acetate buffer at pH 5.5
and as anticipated proceeded cleanly at room temperature. Both
derivatives labeled in high radiochemical yield, the yield for
64/67CuATSM-BBS-2‡ however was slightly higher than that for
64/67CuATSM-BBS-1. The radiochemical yields for 64/67CuATSM-
BBS-1 and 64/67CuATSM-BBS-2 were 91% and 85% respectively.
Labelings done at 75 ◦C resulted in yields > 90-95%. HPLC
analysis after 24 h showed that the complexes were stable in
Preliminary In vivo biodistribution
Preliminary biodistribution data was obtained using
64/67CuATSM-BBS-2 in nude mice with PC-3 tumour xenografts.
Two groups of 3 mice were injected intravenously with 100 KBq
of the 64/67Cu complex and sacrificed 1 and 24 h post injection
(p.i.). Fig. 7 shows the biodistribution of the complex at the two
time intervals as % injected dose/gm (%ID/gm) averaged over
3 mice.
solution. Log P values were 0.42
0.04 and 0.39
0.01 for
64/67CuATSM-BBS-1 and 64/67CuATSM-BBS-2 respectively.
At 1 h p.i. there is good uptake in the tumour with a
tumour/muscle ratio of 9.6 : 1. Significantly this value was reduced
by about 52% in a third group of animals pretreated with
natural bombesin at a dose of 0.1 mg per mouse. This reduction
‡ The radiocopper used for the protein labeling and in vitro and in vivo
experiments contained small amounts of 67Cu. Pure 64Cu was used for all
other labeling work.
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behaviour of the conjugates by further modifications of the ATSE
ligand system.
Experimental
General experimental techniques
Chemicals were obtained from Acros, Aldrich, Alfa Aesar, Apollo
Scientific or Bachem unless otherwise stated. All solvents used
were HPLC grade. TLC, where applicable, was performed on pre-
coated aluminium-backed plates (Merck Kieselgel) and spots were
made visible by quenching of UV fluorescence (l = 254 nm)
and/or by staining with potassium permanganate. Flash chro-
matography was performed according to the method by Still,
using silica gel (0.040-0.063 mm; Merck) and air pressure. NMR
spectra were recorded on a Varian Mercury VX300 or a Bruker
AVANCE AVC500 at frequencies of 300 MHz and 500 MHz
Fig. 7 Biodistribution of 64/67Cu-ATSM-BBS-2 in nude mice with PC-3
tumour xenografts (100 KBq/mouse i.v.).
1
for H and 75.5 MHz and 126 MHz for 13C respectively, using
is comparable with values obtained in blocking studies with
bombesin conjugates using other radioisotopes. Lung uptake was
high at early p.i. times, probably due to the low solubility of
CuATSM-BBS-2 in aqueous biological media but it was rapidly
cleared. Liver uptake is high, which is in accordance with the high
lipophilicity, and the possibility that some copper is sequestered
from the ATSM ligand by the liver. The relatively high uptake in
both pancreas and gastrointestinal tract is consistent with the high
expression of bombesin receptors in these tissues. Both these values
were substantially reduced in the blocking experiment, confirming
the specificity of the in vivo uptake. The nature of the radiometal
chelator, the linking group and the structural variant of bombesin
used all have a impact on the biodistribution of the labelled BBS
peptide.
However the tumour uptake here appears higher than with any
other copper chelators that have been used with the same bombesin
structural motif.
Further experiments are in progress with conjugates designed
to be more hydrophilic to assess the impact on both cell internal-
isation and biodistribution. The results of this work and a more
detailed comparison of the data with other radioisotopes will be
published at a later stage.
residual solvent peaks as internal reference.35 1H-1H NOESY,
1
1H-13C HSQC and H-13C HMBC. Nominal mass spectra (m/z)
were recorded on a Fisons Platform II, under the conditions of
positive or negative electrospray ionization (ESI-MS). High reso-
lution mass spectra (HRMS) were recorded under ESI conditions
on a BrukerMicroTOF (resolution = 5000 FWHM). The lock
masses used for calibration were tetraoctylammonium bromide
and sodium dodecyl sulfate in positive and negative ion mode
respectively. Elemental analysis of solid compounds for C,H,N
was obtained by the Mr Stephen Boyer, London Metropolitan
University. Where elementals are not reported, these were metal
compounds showing consistently low C and N values, indicative of
the presence of H2O. Single peak HPLC traces and high resolution
mass spectrometry were obtained for these compounds.
Analytical HPLC spectra were recorded on a Gilson instrument
with a C-18 column and UV/vis detection at 254 nm. Solvent
conditions were as follows. Method I (M I) solvent A = H2O +
0.1% TFA, solvent B = CH3CN+ 0.1% TFA, flow rate =
1 ml min-1, gradient (min,% of B): 0, 5; 15, 95; 20, 95; 25, 5;
30, 5. Method II (M II) solvent A = H2O, solvent B = CH3CN,
flow rate = 1 ml min-1, gradient (min,% of B): 0, 5; 15, 95; 20, 95;
25, 5; 30, 5. Analytical HPLC conditions for peptide conjugates
are detailed below.
Conclusion
Synthesis of precursors
We have synthesised and characterised
a new series of
bis(thiosemicarbazone) bifunctional chelators, incorporating both
rigid aromatic and aliphatic linkers to carboxylate groups. We have
successfully conjugated them using both solid and solution phase
techniques. Preliminary biological data for the new bombesin
bioconjugates described above demonstrate their suitability for
use in biological systems. Room temperature radiolabeling and
full EPR characterisation demonstrate that rapid labeling and
site specificity are maintained when conjugated to a biologically
active molecule. Initial in vitro and in vivo evaluations of these first
generation bis(thiosemicarbazone) functionalised chelators show
that they are in principle suitable for protein targeted PET imaging.
Moreover the 99mTc-labeling experiments show that these new
carboxylate derivatised ATSM derivatives are also promising for
the SPECT labeling of proteins. These new derivatives thus offer a
viable and flexible route for labeling of sensitive biomolecules for
targeted imaging. Experiments are ongoing to optimise the in vivo
Proligands H2ATSM/A and H2ATSE/A (1 and 2) and the Zn
complex 3 were synthesised according to previously reported
procedures.5
Synthesis of linkers
General procedure A: Coupling reactions of H2ATSE/A with car-
boxylic acid linkers. H2ATSE/A was suspended in the minimum
anount of DMF. The carboxylic acid (1.1 eq), diisopropylethy-
lamine (1.1 eq) and benzotriazole-1-yl-oxy-tris-(dimethylamino)-
phosphonium hexafluorophosphate (BOP) (1.1 eq) were added
and the mixture was stirred at room temperature for 4 h until a
clear solution was formed. H2O was then added until formation of
a white precipitate. The suspension was sonicated, the precipitate
collected by filtration and washed with copious amounts of H2O,
EtOH and, if solubility allowed, Et2O.
3626 | Dalton Trans., 2010, 39, 3620–3632
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General procedure B: Copper complexation. The proligand
and CuCl2 (1.2 eq) or Cu(OAc)2·H2O (1.2 eq) were stirred in a
minimum amount of MeOH for 30 min at rt. The solvent was
removed in vacuo and H2O was added. The solid formed was
collected by filtration and washed with H2O and, where solubility
allowed, with a few drops of ice-cold Et2O before being dried
in vacuo.
(5a). 4a (70 mg, 0.172 mmol) was suspended in MeOH (5 ml)
at rt. ZnCl2 (28 mg, 0.206 mmol) was dissolved in the minimum
amount of H2O and added dropwise. The solution turned yellow
immediately and was stirred for 3 h at rt. The solvent was then
removed in vacuo and H2O added. The resulting precipitate was
filtered off, washed with cold Et2O and dried in vacuo to afford
1
the desired product (68 mg, 85%) as an orange solid. H NMR
(300 MHz, DMSO-d6): d 13.05 (1H, s, COOH), 8.49 (1H, t,
J = 5.7 Hz, NHEt), 8.18-8.16 (1H, m, ArCH=N), 8.01-7.98
(2H, m, ArH), 7.87-7.76 (d, J = 8.2 Hz, ArH) 3.66-3.57 (2H,
Diacetyl-2-(4-N-ethyl-3-thiosemicarbazone)-3-[4-N (amino)-(4-
carboxyphenylmethylidene)-3-thiosemicarbazone] (4a). 1 (500 mg,
1.82 mmol) was suspended in MeOH (20 ml). 4-formylbenzoic
acid (354 mg, 3.33 mmol) was added and the suspension stirred
overnight at rt. The residue was filtered and dissolved in DMF.
MeOH was added to induce precipitation of 4a (655 mg, 82%) as
a pale yellow solid.
=
=
m, CH2CH3), 2.29 (3H, s, CH3C N), 2.23 (3H, s, CH3C N) and
1.15 (3H, t, J = 6.93 Hz, CH2CH3);13C NMR (126 MHz, DMSO-
d6): d 177.5 (C-S), other (C-S) not observed, 167.0 and 168.9
=
=
two isomers (CO2H), (HC NNH(C S)NHN=C) not observed,
=
=
147.8 (EtNH(C S)NHN=C), 142.2 (HC NNH), 137.9 (ArC)
1H NMR (300 MHz, DMSO-d6): d 13.02 (1H, s, COOH),
131.8 (ArC), 129.8 (ArCH), 127.3 (ArCH), 38.6 (CH2CH3), 14.4
=
=
12.31 (1H, s, HC NNH(C S)NHN=), 10.74 (1H, s,
-
-
=
(CH2CH3), 11.6 and 11.5 (2 ¥ CH3C N); HRMS (ESI ): (M-H)
=
=
=
HC NNH(C S)NHN=), 10.22 (1H, s, EtNH(C S)NHN=),
8.48 (1H, t, J = 5.7 Hz, NHEt), 8.24 (1H, s, ArCH=N), 8.00 (2H,
d, J = 7.9 Hz, ArCH), 7.86 (2H, d, J = 7.9 Hz, ArCH), 3.62 (2H,
calcd for C16H18N7O2S2Zn- 468.0260; found 468.0267; HPLC (M
I) Rt 11.75 min
=
=
m, CH2CH3), 2.31 (3H, s, CH3C N), 2.25 (3H, s, CH3C N)
Diacetyl-2-(4-N-ethyl-3-thiosemicarbazonato)-3-[4-N-(amino)-
(2-carboxyphenylmethylidene)-3-thiosemicarbazonato]zinc(II) (5b).
4b (70 mg, 0.172 mmol) was suspended in MeOH (5 ml) at rt. ZnCl2
(27.7 mg, 0.206 mmol) was dissolved in the minimum amount of
H2O and added dropwise. The solution turned yellow immediately
and was stirred for 3 h at rt. The solvent was then removed in vacuo
and H2O added. The resulting precipitate was filtered off, washed
with cold Et2O and dried in vacuo to afford the desired product 8a
(73 mg, 91%) as an orange solid.
1.15 (3H, t, J = 7.3 Hz, CH2CH3); 13C NMR (75 MHz,
=
DMSO-d6): d 177.5 (EtNHC=S), 174.9 (HC NNH(C=S),
=
=
166.8 (CO2H), 153.4 (HC NNH(C S)NHN=C), 147.9
=
(EtNH(C S)NHN=C), 142.1 (HC=NNH), 137.9 (ArC),
131.7 (ArC), 129.7 (ArCH), 127.2 (ArCH), 38.6 (CH2CH3),
=
=
14.3 (CH2CH3), 11.6 (CH3C NNH(C S)NHEt), 11.5
(CH3C NNH(C S)NHNH); HRMS (ESI-): (M-H)- calcd
for C16H20N7O2S2 (M-H)- 406.1125; found 406.1137; HPLC (M
I) Rt 11.85 min; Elemental Analysis Found: C, 47.2; H, 5.1; N,
24.0. C16H21N7O2S2 requires: C, 47.2; H, 5.2; N, 24.1%
=
=
1H NMR (500 MHz, DMSO-d6): d 13.12 (1H, s, COOH),
8.86 (1H, s, ArCH=N), 8.47 (1H, t, J = 5.7 Hz, NHEt), 8.07
(1H, d, J = 7.7 Hz, ArCH-3 or -6), 7.88 (1H, d, J = 7.7 Hz,
3-ArCH or 6-ArCH), 7.63 (1H, t, J = 7.3 Hz, ArCH-4 or H-
5), 7.52 (1H, t, J = 7.3 Hz, ArCH-4 or H-5), 3.66-3.59 (2H,
Diacetyl-2-(4-N-ethyl-3-thiosemicarbazone)-3-[4-N-(amino)-
(2-carboxyphenylmethylidene)-3-thiosemicarbazone] (4b). was
prepared according to the procedure described for 4a using
1 (0.250 g, 0.91 mmol) and 2-formylbenzoic acid (177 mg,
1.18 mmol) in MeOH (20 ml) to afford 4b (305 mg, 82%) as a pale
yellow solid.
=
=
m, CH2CH3), 2.29 (3H, s, CH3C N), 2.23 (3H, s, CH3C N)
and 1.15 (3H, t, J = 6.9 Hz, CH2CH3); 13C NMR (75 MHz,
=
DMSO-d6): d (EtNHC=S) not observed, 177.4 (HC NNH(C=S),
=
=
168.1 (CO2H), (HC NNH(C S)NHN=C) not observed, 147.8
1H NMR (500 MHz, DMSO-d6): d 12.85 (1H, s, COOH),
=
=
(EtNH(C S)NHN=C), 142.5 (HC NNH), 131.9 (ArC), 131.7
=
=
12.31 (1H, s, HC NNH(C S)NHN=), 10.66 (1H, s,
(ArC), 131.0 (ArCH), 130.1 (ArCH), 129.8 (ArCH), 127.0
=
=
=
HC NNH(C S)NHN=), 10.20 (1H, s, EtNH(C S)NHN=),
8.88 (1H, s, ArCH=N), 8.46 (1H, t, J = 5.7 Hz, NHEt), 8.06
(1H, d, J = 7.62 Hz, ArCH-4 or ArCH-6), 7.86 (1H, d, J =
7.61 Hz, ArCH-3 or ArCH-6), 7.62 (1H, dt, J = 7.6 Hz, J =
8.5 Hz, ArCH-4 or ArCH-5), 7.52 (1H, dt, J = 7.6 Hz, J =
8.5 Hz, ArCH-4 or ArCH-5), 3.66-3.57 (2H, m, CH2CH3), 2.27
=
(ArCH), 38.5 (CH2CH3), 14.3 (CH2CH3), 11.5 (CH3C N), 11.5
-
-
-
=
(CH3C N); HRMS (ESI ): (M-H) calcd for C16H18N7O2S2Zn
468.0260; found 468.0259; HPLC (M II) Rt 11.75 min, Elemental
Analysis Found C, 40.9; H, 4.1; N, 20.8. C16H19N7O2S2Zn requires
C, 40.8; H, 4.1; N, 20.8%
=
=
(3H, s, CH3C N), 2.23 (3H, s, CH3C N) and 1.13 (3H, t, J =
Diacetyl-2-(4-N-ethyl-3-thiosemicarbazonato)-3-[4-N-(amino)-
(4-carboxyphenylmethylidene)-3-thiosemicarbazonato]copper(II)
(6a). 6a was prepared following General Procedure B using 4a
(70 mg, 0.171 mmol) and CuCl2 (28 mg, 0.177 mmol) in MeOH
(5 ml) to yield the desired product as a brown solid (73 mg, 91%).
HRMS (ESI-): (M-H)- calcd for C16H18N7O2S2Cu- 467.0265;
found 467.0253; HPLC (M I) Rt 12.25 min
7.0 Hz, CH2CH3); 13C NMR (75 MHz, DMSO-d6): d 179.8
=
(EtNHC=S), 177.9 (HC NNH(C=S), 168.7 (CO2H), 149.8
=
=
=
(HC NNH(C S)NHN=C), 147.9 (EtNH(C S)NHN=C),
142.6 (HC=NNH), 136.7 (ArC), 133.4 (ArC), 129.4 (ArCH),
129.3 (ArCH), 127.8 (ArCH), 124.4 (ArCH) 38.6 (CH2CH3),
=
=
14.3 (CH2CH3), 11.9 (CH3C NNH(C S)NHEt), 11.6
(CH3C NNH(C S)NHNH); HRMS (ESI-): (M-H)- calcd
for C16H20N7O2S2 (M-H)- 406.1125, found 406.1123; HPLC (M I)
Rt 12.05 min, Elemental Analysis Found C, 47.1; H, 5.1; N, 24.0.
C16H21N7O2S2 requires C, 47.2; H, 5.2; N 24.1%
=
=
Diacetyl-2-(4-N-ethyl-3-thiosemicarbazonato)-3-[4-N-(amino)-
(2-carboxyphenylmethylidene)-3-thiosemicarbazonato]copper(II)
(6b). 6b was prepared following General Procedure B using 4b
(60 mg, 0.147 mmol) and CuCl2 (23.7 mg, 0.177 mmol) in MeOH
(5 ml) to afford the desired product as a dark brown solid (66 mg,
96%).
Diacetyl-2-(4-N-ethyl-3-thiosemicarbazonato)-3-[4-N-(amino)-
(4-carboxyphenylmethylidene)-3-thiosemicarbazonato]zinc(II ))
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Dalton Trans., 2010, 39, 3620–3632 | 3627
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HRMS (ESI-): (M-H)- calcd for C16H18N7O2S2Cu- 467.0265;
found 467.0255; HPLC (M I) Rt 12.05 min,; Elemental Analysis
Found C, 40.9; H, 4.0; N, 20.9. C16H19CuN7O2S2 requires C, 41.0;
H, 4.1; N, 20.9%
(M-H)- calcd for C16H18CuN7O3S2 483.0214; found 483.0213;
HPLC (M I) Rt 10.30 min, Elemental Analysis Found C, 39.6;
H, 4.0; N, 20.1. C16H19CuN7O3S2 requires C, 39.6; H, 4.0; N, 20.2
-
Diacetyl-2-(4-N-ethyl-3-thiosemicarbazone)-3-[4-N-(amino)-
methyl 2-(4-formylbenzamido)acetate)-3 thiosemicarbazone] (11a).
1b (230 mg, 0.62 mmol) and methyl 2-(4-formylbenzamido) acetate
(230 mg, 1.11 mmol) were stirred in MeOH (15 ml) overnight. The
suspension was then heated at 50 ◦C for 2 h. The solvent was
removed in vacuo and the solid was washed with cold MeOH
(10 ml) and H2O (10 ml) and dried to afford 3c (195 mg, 68%).
Methyl 2-(4-formylbenzamido) acetate was synthesised following
a previously reported procedure.14
Diacetyl-2-(4-N-ethyl-3-thiosemicarbazone)-3-[4-N-(amino)-(4-
tertbutoxycarboxybenzamide)-3-thiosemicarbazone] (7). 7 was
synthesized according to General Procedure A, using H2ATSE/A
(300 mg, 1.1 mmol), 4-tert-butylbenzoic acid (266 mg, 1.2 mmol),
BOP (530 mg, 1.2 mmol) and diisopropylethylamine (209 ml,
1.2 mmol). The product was isolated as an off-white solid (503 mg,
96%).
1
=
H NMR (500 MHz, DMSO-d6): 10.78 (1H, s, NH(C O)Ar),
10.71 (1H, s, NHNH(C S)NHN=), 10.19 (1H, s,
1H NMR (500 MHz, DMSO-d6): d 12.64 (brs, COOH), 12.3
=
=
=
=
=
NHNH(C S)NHN=), 10.17 (1H, s, EtNHC SNHN=),
8.45 (1H, t, J = 5.8 Hz, NHCH2CH3), 8.02 (2H, d, J = 8.5 Hz,
ArCH), 8.01 (2H, d, J = 8.5 Hz, ArCH), 3.66-3.59 (2H, m, J =
(NHN CH), 10.75 and 10.23 (2 ¥ 1H, s, C SNH-N=), 8.94
(1H, t, J = 5.7 Hz, NHCH2CO2H), 8.49 (1H, t, J = 5.8 Hz,
=
NHEt), 8.26 (1H, brs, NHN CH), 7.95 (2H, d, J = 8.2 Hz,
=
=
6.9 Hz, CH2CH3), 2.27 (3H, s, CH3C N), 2.25 (3H, s, CH3C N),
ArCH), 7.85 (2H, d, J = 7.9 Hz, ArCH), 3.96 (2H, d, J =
1.57 (9H, s, CO2 Bu), 1.15 (3H, t, J = 7.3 Hz, CH2CH3); 13C
5.7 Hz, CH2CO2H), 3.66-3.57 (2H, m, CH2CH3), 2.31 (3H, s,
t
=
=
NMR (75 MHz, DMSO-d6): d 179.8 (NHNHC=S), 177.4
CH3C N), 2.25 (3H, s, CH3C N), 1.15 (3H, t, J = 7.2 Hz,
CH2CH3); 13C NMR (126 MHz, DMSO-d6): d 177.5 (EtNHC=S),
175.1 (C=S), 171.3 (CO2H), 165.9 (CONHCH2), 153.3
t
(EtNHC=S), 166.7 (NHNHC=O), 164.9 (CO2 Bu), 149.8
=
=
(EtNH(C S)NHN=C), 147.9 (NHNH(C S)NHN=C), 136.7
=
=
=
(ArCCO2H), 133.9 (ArC(C O)NH), 129.3 (ArCH), 127.8
(NHNH(C S)NHN=C), 147.8 (EtNH(C S)NHN=C), 142.1
(HC=NNH), 136.8 (ArC), 134.8 (ArC), 127.8 (ArCH), 127.1
(ArCH), 41.3 (CH2CO2H), 38.6 (CH2CH3), 14.4 (CH2CH3), 11.6,
(ArCH), 81.3 (C(CH3)3), 38.6 (CH2CH3), 27.8 (C(CH3)3),
=
=
14.4 (CH2CH3), 11.9 (CH3C NNH(C S)NHEt), 11.7
-
+
=
=
=
=
=
=
(CH3C NNH(C S)NHNH); HRMS (ESI ) (M+Na) calcd
for- C20H29N7NaO3S2 502.1666; found 502.1667; HPLC (MII) Rt
13.18 min Elemental Analysis Found C, 50.0; H, 6.0; N, 20.4.
C20H29N7O3S2 requires C, 50.1; H, 6.1; N 20.4%
11.1 (CH3C NNH(C S)NHEt), (CH3C NNH(C S)NHNH);
HRMS (ESI-): (M+Na)+ calcd for C18H24NaN8O3S2 487.1305;
+
found 487.1306; Elemental Analysis Found C, 46.6; H, 5.2; N,
24.1. C18H24N8O3S2 requires C, 46.5; H, 5.2; N 24.1%
Diacetyl-2-(4-N-ethyl-3-thiosemicarbazone)-3-[4-N-(amino)-
Diacetyl-2-(4-N-ethyl-3-thiosemicarbazone)-3-[4-N-(amino)-
4(N-(2-tertbutylacetate)benzamide))-3-thiosemicarbazone (11b).
11b was synthesised according to General Procedure A using
8 (250 mg, 0.59 mmol), Glycine tert-butyl ester hydrochloride
(110 mg, 0.65 mmol), diisopropylethylamine (168 mg, 1.30 mmol)
and BOP (288 mg, 0.65 mmol) in DMF (5 ml). The product was
isolated as an off-white solid (283 mg, 98%).
(4-carboxybenzamide)-3-thiosemicarbazone] (8).
7 (500 mg,
1.2 mmol) was suspended in TFA (2 ml) and stirred for 2.5 h
at rt. TFA was then removed in vacuo and the resulting residue
was sonicated in Et2O, filtered and washed with more Et2O. The
resulting solid was suspended in warm EtOH and sonicated,
filtered and dried to afford 8 (300 mg, 65%) as an off-white solid.
1H NMR (500 MHz, DMSO-d6):
d
13.18 (1H, br s,
1H NMR (500 MHz, DMSO-d6):
d 10.78 (2H, br s,
=
=
=
=
=
COOH), 10.78 (1H, br s, NH(C O)Ar), 10.70 (1H, s,
(C S)NHNH-C O) and (C S)NHNH–C O), 10.17 (2H, brs s,
t
=
=
=
NHNH(C S)NHN=), 10.20 (1H, s, NHNH(C S)NHN=),
2 ¥ (C S)-NH–N), 9.00 (1H, t, J = 5.8 Hz, NHCH2CO2 Bu),
=
10.16 (1H, s, EtNHC SNHN=), 8.45 (1H, t, J = 5.8 Hz,
8.45 (1H, t, J = 5.8 Hz, NHEt), 8.01 (2H, d, J = 8.2 Hz,
ArCH), 7.98 (2H, d, J = 8.5 Hz, ArCH), 3.92 (2H, d, J =
NHEt), 8.07 (2H, d, J = 8.5 Hz, ArCH), 8.01 (2H, d, J =
t
=
8.5 Hz, ArCH), 3.61 (2H, m, CH2CH3), 2.27 (3H, s, CH3C N),
5.8 Hz, CH2CO2 Bu), 3.66-3.57 (2H, m, CH2CH3), 2.27 (3H, s,
t
=
=
=
2.25 (3H, s, CH3C N), 1.15 (3H, t, J = 7.3 Hz, CH2CH3);
CH3C N), 2.25 (3H, s, CH3C N), 1.43 (9H, s, CO2 Bu),
13C NMR (126 MHz, DMSO-d6): d 179.8 (NHNHC=S),
177.4 (EtNHC=S), 166.7 (NHNHC=O), 164.9 (CO2H),
1.15 (3H, t, J = 6.6 Hz, CH2CH3); 13C NMR (125.8 MHz,
DMSO-d6):
d
179.7 (NHNHC=S), 177.4 (EtNHC=S),
t
=
=
149.8 (EtNH(C S)NHN=C), 147.9 (NHNH(C S)NHN=C),
168.7 (CO2 Bu), 165.8 (CONHCH2), 164.9 (CONHNH),
=
=
=
136.7 (ArCCO2H), 133.4 (ArC(C O)NH), 129.3 (ArCH),
149.7 (EtNH(C S)NHN=C), 147.9 (NHNH(C S)NHN=C),
127.8 (ArCH), 38.6 (CH2CH3), 14.3 (CH2CH3), 11.9
136.5 (ArC), 133.4 (ArC), 127.6 (ArCH), 127.3 (ArCH),
t
=
=
=
=
(CH3C NNH(C S)NHEt), 11.6 (CH3C NNH(C S)NHNH);
80.72 (C(CH3), 41.9 (CH2CO2 Bu), 38.6 (CH2CH3), 27.7
HRMS (ESI-): (M-H)- calcd for C16H20N7O3S2 422.1075; found
(C(CH3), 14.3 (CH2CH3), 11.9 (CH3C NNH(C S)NHEt), 11.6
-
=
=
-
-
=
=
422.1076; HPLC (M I) Rt 10.35 min; Elemental Analysis Found
C, 45.3; H, 5.0; N, 23.2. C16H21N7O3S2 requires C, 45.4; H 5.0; N,
23.2%
(CH3C NNH(C S)NHNH); HRMS (ESI ): (M-H) calcd for
-
C22H31N8O4S2 535.1915; found 535.1921; HPLC (M I) Rt
11.8 min Elemental Analysis Found C, 49.4; H, 6.1; N, 20.9.
C22H32N8O4S2 requires C, 49.2; H, 6.0; N 20.9%
Diacetyl-2-(4-N-ethyl-3-thiosemicarbazonato)-3-[4-N-(amino)-
(4-carboxybenzamide)-3 thiosemicarbazonato]copper(II) (9a). 9a
was prepared following General Procedure B, using 8 (38 mg,
0.09 mmol) and CuCl2·H2O (0.1 mmol, 1.1 eq) to afford the
desired product as a red-brown solid (37 mg, 85%). HRMS (ESI+):
Diacetyl-2-(4-N-ethyl-3-thiosemicarbazone)-3-[4-N-(amino)-
4(N-(2-acetic acid)benzamide)-3-thiosemicarbazone (11c). 11b
(280 mg, 0.052 mmol) was suspended in TFA (3 ml) and stirred at
room temperature for 2.25 h. TFA was then removed in vacuo and
3628 | Dalton Trans., 2010, 39, 3620–3632
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the resulting residue was sonicated in Et2O, filtered and washed
with more Et2O and dried to afford the desired product (178 mg,
71%) as a pale yellow solid.
(CH2Fmoc), 52.4 (Ca), 46.6 (C-9), 31.4 (Cg ), 31.2 (NHCH3), 27.7
=
=
(C(CH3)), 27.5 (Cb), 11.8 (CH3(C N)NH(C S)NHNH), 11.6
+
+
=
=
(CH3(C N)NH(C S)NHMe); HRMS (ESI ): (M+Na) calcd
1H NMR (500 MHz, DMSO-d6): d 12.59 (brs, COOH), 10.74-
for C31H40N8NaO5S2 691.2455; found 691.2437; HPLC (MII) Rt
+
=
=
=
=
10.70 (2H, br s, (C S)NHNH-C O) and (C S)NHNH–C O),
14.55 min; Elemental Analysis Found C, 55.6; H, 5.9; N, 16.6.
C31H40N8O5S2 requires C, 55.7; H, 5.9; N, 16.6%
=
10.17 (2H, brs s, 2 ¥ (C S)-NH–N), 8.99 (1H, t, J = 5.8 Hz,
NHCH2CO2H), 8.45 (1H, t, J = 5.8 Hz, NHEt), 8.01 (2H, d,
J = 8.2 Hz, ArCH), 7.98 (2H, d, J = 8.5 Hz, ArCH), 3.96
(2H, d, J = 5.8 Hz, CH2CO2H), 3.65-3.57 (2H, m, CH2CH3),
H2ATSM/A-a-NH2-L-Glu(OtBu)
(15). 14
(100
mg,
0.150 mmol) was stirred in DMF (600 ml) and piperidine
(120 ml) for 45 min. The solution was concentrated and H2O
was added until the formation of a white precipitate. The white
precipitate was filtered off, washed with a few more drops of H2O.
The aqueous phase was concentrated and Et2O was added until
formation of a precipitate. Filtration and drying under vacuum
afforded 15 as an off-white solid (40 mg, 60%).
=
=
2.27 (3H, s, CH3C N), 2.25 (3H, s, CH3C N), 1.15 (3H, t,
J = 6.6 Hz, CH2CH3); 13C NMR (125.8 MHz, DMSO-d6): d
179.8 (NHNHC=S), 177.4 (EtNHC=S), 171.17 (CO2H), 165.8
=
(CONHCH2), 164.9 (CONHNH), 149.7 (EtNH(C S)NHN=C),
=
147.9 (NHNH(C S)NHN=C), 136.5 (ArC), 135.4 (ArC),
127.6 (ArCH), 127.3 (ArCH), 41.2 (NHCH2CO2H) 38.6
1H NMR (300 MHz, DMSO-d6): d 10.20 (4H, superimposed
=
=
(CH2CH3), 14.4 (CH2CH3), 11.9 (CH3C NNH(C S)NHEt),
=
=
broad singlets, MeNH(C S)NHN=, NHNH(C S)NHN=,
-
-
=
=
11.6 (CH3C NNH(C S)NHNH); HRMS (ESI ): (M-H) calcd
=
=
NHNH(C S)NHN=, NHNH(C S)NHN=), 8.40 (d, 1H, J =
4.7 Hz, CH3NH), 3.33 (obscured by H2O, CHa) 3.02 (3H,
d, J = 4.7 Hz, CH3NH), 2.37 (2H, t, J = 6.6 Hz, CHg ),
-
for C18H23N8O4S2 479.1289; found 479.1278; HPLC (M I) Rt
9.48 min Elemental Analysis Found C, 44.9; H, 5.1; N, 23.4.
C18H24N8O4S2 requires C, 50.0; H, 5.0; N, 23.3%
=
=
2.23 (3H, s, CH3C N), 2.21 (3H, s, CH3C N), 1.94-1.87
(1H, m, CHb), 1.75-1.69 (1H, m, CHb), 1.14 (9H, s, C(CH3));
Diacetyl-2-(4-N-ethyl-3-thiosemicarbazonato)-3-[4-N-(amino)-
Methyl 2-(4-formylbenzamido)acetate)-3 thiosemicarbazonato]-
copper(II) (12a). 12a was prepared following General Procedure
B, using 11a (30 mg, 0.06 mmol) and CuCl2 (12 mg, 0.07 mmol)
to afford the desired product as a black solid (25 mg, 76%).
13C NMR (75 MHz, DMSO-d6):
d
179.2 (NHNHC=S),
178.4 (EtNHC=S), 171.7 (CO2tBu), 170.1 (NHNHCO),
=
=
149.5 (NHNH(C S)NH(C=N)), 147.8 MeNH(C S)NH(C=N),
79.5 (CCH3), 52.5 (Ca), 31.4 (Cg ), 31.2 (NHCH3), 27.7
HRMS (ESI+): (M-H)- calcd for C18H19CuN7O3S2 522.0323;
-
=
=
(C(CH3)), 27.5 (Cb), 11.8 (CH3(C N)NH(C S)NHNH), 11.6
+
+
=
=
(CH3(C N)NH(C S)NHMe); HRMS (ESI ): (M+H) calcd
found 522.0317; HPLC (M I) Rt 10.30 min
+
for C16H31N8O3S2 447.1955; found 447.1964; HPLC (M II) Rt
Diacetyl-2-(4-N-ethyl-3-thiosemicarbazonato)-3-[4-N-(amino)-
4(N-(2-acetic acid)benzamide)-3-thiosemicarbazonato]copper(II)
(12c). 12c was prepared following General Procedure B, using
11c (60 mg, 0.125 mmol) and Cu(OAc)2·H2O (33 mg, 1.3 eq) to
afford the desired product as a black solid (65 mg, 96%). HRMS
12.40 min Elemental Analysis Found C, 42.9; H, 6.8; N, 25.0.
C16H30N8O3S2 requires C, 43.0; H, 6.8; N, 25.1%
H2ATSM/A-a-NH-Biotin-L-Glu(OtBu) (16). 16 was synthe-
sised according to General Procedure A using 15 (40 mg,
0.089 mmol), biotin (23 mg, 0.09 mmol), diisopropylethylamine
(16 ml, 0.09 mmol) and BOP (42 mg, 0.09 mmol) in DMF (5 ml).
The product was isolated as a beige solid (38.2 mg, 63%).
(ESI-): (M-H)- calcd for C18H21CuN8O4S2 540.0418; found
-
540.0429; HPLC (M I) Rt 9.07 min
H2ATSM/A-a-Fmoc-L-Glu(OtBu) (14). 14 was prepared ac-
cording to General Procedure A using 2 (543 mg, 2.08 mmol),
FmocL-GluOtBu (885 mg, 2.08 mmol), diisopropylethylamine
(362 ml, 2.08 mmol) and BOP (920 mg, 2.08 mmol) in DMF (7 ml).
The precipitate was then stirred in hot EtOH (5-10 ml) before being
filtered off and washed with cold Et2O (10 ml) to afford 975 mg
(70%) of 14 as a white solid.
1H NMR (300 MHz, DMSO-d6):
d
10.58 (1H, s,
=
=
NHNH(C S)NHN=), 10.23 (1H, s, MeNH(C S)NHN=),
=
10.17 (1H, s, NHNH(C S)NHN=), 10.00 (1H, s,
=
NHNH(C S)NHN=), 8.41 (d, 1H, J = 4.5 Hz, CH3NH),
7.89 (2H, d, J = 7.5 Hz, H-4, H-5), 7.75 (2H, m, H-1, H-8),
7.99 (1H, d, J = 8.5 Hz, NHbiotin), 6.41 and 6.35 (2 ¥
=
1H, s, CHNHC O), 4.45 (1H, dd, J = 13.8 Hz, J = 8.5 Hz,
1H NMR (300 MHz, DMSO-d6):
d
10.60 (1H, s,
NHNH(C O)CH), 4.30-4.13 (2H, m, 2¥CHNH(C O), 3.09
(1H, m, CH2CHSCH2), 3.02 (3H, d, J = 4.6 Hz, CH3NH),
2.81 (1H, dd, J = 12.6 Hz, J = 5.2 Hz, 1¥SCH2CH),
=
=
=
=
NHNH(C S)NHN=), 10.25 (1H, s, MeNH(C S)NHN=),
=
10.20 (1H, s, NHNH(C S)NHN=), 10.02 (1H, s,
=
NHNH(C S)NHN=), 8.40 (d, 1H, J = 4.5 Hz, CH3NH),
2.54 (1H, m, obscured by DMSO, 1¥SCH2CH),2.36-2.31 (2H,
t
=
=
7.90 (2H, d, J = 7.5 Hz, H-4, H-5), 7.75 (2H, m, H-1, H-8),
7.62 (1H, d, J = 8.88 Hz, NHFmoc), 7.42 (2H, t, J = 7.5 Hz,
H-3, H-6), 7.33 (2H, dt, J = 7.5 Hz, J = 1.4 Hz, H-2, H-7),
4.27-4.17 (4H, m, CHa, CH2Fmoc, H-9), 3.02 (3H, d, J =
4.5 Hz, CH3NH), 2.37 (2H, t, J = 6.6 Hz, CHg ), 2.23 (3H, s,
CH2CH2CO2 Bu), 2.23 (3H, s, CH3C N), 2.21 (3H, s, CH3C N),
2.15-2.13 (2H, m, (C O)CH2(CH2)3), 2.04-1.24 (8H,m,
=
t
=
(C O)CH2(CH2)3 and CH2CH2CO2 Bu), 1.40 (9H,s, CO2tBu);
13C NMR (75 MHz, DMSO-d6): d 179.21 (NHNHC=S), 178.47
(EtNHC=S), 172.03 (CO), 171.7 (CO2tBu), 170.11 (NHNHCO),
=
=
= =
162.66 (NH(C O)NH), 149.78 (NHNH(C S)NH(C=N)),
CH3C N), 2.21 (3H, s, CH3C N), 2.08 (1H, m, CHb), 1.85
(1H, m, CHb), 1.14 (9H, s, C(CH3)); 13C NMR (75 MHz,
DMSO-d6): d 179.2 (NHNHC=S), 178.4 (EtNHC=S), 171.7
=
147.92 MeNH(C S)NH(C=N), 79.57 (CO2C(CH3)3), 61.01
=
=
(CHCHNH(C O)), 59.18 (SCH2CHNH(C O)), 55.41 (CHS),
t
t
=
(CO2 Bu), 170.1 (NHNHCO), 155.8 ((C=O)Fmoc), 149.7
50.02 (CHCH2CH2CO2 Bu), 34.8 (NH(C O)CH2(CH2)3),
=
=
(NHNH(C S)NH(C=N)), 147.8 MeNH(C S)NH(C=N),
143.8 (ArC), 143.7 (ArC), 140.6 (ArC), 127.6 ( C-3, C-6), 127.0 (
C-2, C-7), 125.3 ( C-1, C-8), 120.1 ( C-4, C-5), 79.5 (CCH3), 65.7
28.14 (CH2), 28.01 (CH2), 27.78 (CO2C(CH3)3, 25.24
=
=
(CH2),
11.84
(CH3(C N)NH(C S)NHNH),
11.65
(CH3(C N)NH(C S)NHMe); HRMS (ESI+): (M+Na)+
=
=
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The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 3620–3632 | 3629
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+
calcd for C26H44N10NaO5S3 695.2550; found 695.2554; HPLC Rt
in duplicate (2 ¥ 90 min) with 4 eq Fmoc-amino acid, 4 eq DIC and
4 eq HOBt in DMF. A stock solution of HOBt in DMF was used
to dissolve the amino acid ([AA] = 0.5 mM). After each coupling
(M II) 11.26 min, Elemental Analysis Found C, 46.4; H, 6.5; N,
20.9. C26H44N10O5S3 requires C, 46.4; H, 6.6; N, 20.8%
i
the resin was washed with 3 ¥ DMF, 3 ¥ PrOH and 3 ¥ DMF. The
H2ATSM/A-a-Fmoc-L-Glu(OH)
(17). 14
(400
mg,
completion of each coupling was checked with the NF31 color
0.60 mmol) was stirred in TFA (3 ml) at room temperature
for 3 h. The solvent was then evaporated in vacuo. Et2O was added
and the suspension sonicated before evaporating to dryness. The
residue was stirred in warm EtOH (5 mL) and left to cool before
being filtered, washed with Et2O and dried in vacuo to afford 17
(216 mg, 59%) as a white solid.
test. Chelators 8 (2 eq) and 11c (2 eq) were coupled in a single
3
coupling to b hAsp using 2 eq of BOP, 2 eq diisopropylethylamine
(8 h) or 2 eq DIC and 2 eq HOBt (3 h) respectively. At the end of
the synthesis the resin was washed several times with CH2Cl2 and
Et2O and dried in vacuo before cleavage.
For cleavage from the resin,
a mixture of thioanisole
1H NMR (300 MHz, DMSO-d6):
d
10.61 (1H, s,
(TA)/ethanedithiol (EDT) 7 : 3 was added as scavenger to the
TFA. The ligand- peptide-resin (0.2 mmol) was shaken in 900 ml
TFA and 100 ml TA/EDT in a syringe fitted with a glass frit
for 3.5 h. The filtrate was added dropwise to ice-cooled Et2O
(10 ml) and the mixture was left in an ice-bath for 15 min before
centrifugation for 5 min. The Et2O was decanted and the solid
resuspended in Et2O, left to settle in the ice-bath and centrifuged
again. This operation was repeated twice more to eliminate all
remaining TFA and scavengers. The resulting precipitate was
dried, dissolved in CH3CN–H2O with 5% DMSO and lyophilized.
Analytical HPLC for BBS-conjugates was performed on a
Waters BreezeTM HPLC system with UV detection (215 nm) using
an RP-column (Supelco Discovery Bio Wide Pore, C 18, 5 mm,
250 mm ¥ 4.6 mm). Gradient 1: 3% B to 100%B in 30 min, flow
rate 1 ml min-1, the gradient was finished by washing with 100%
B. (solvent B = 0.1% TFA in CH3CN).
=
=
NHNH(C S)NHN=), 10.25 (1H, s, MeNH(C S)NHN=),
=
10.20 (1H, s, NHNH(C S)NHN=), 10.03 (1H, s,
=
NHNH(C S)NHN=), 8.41 (d, 1H, J = 4.6 Hz, CH3NH),
7.89 (2H, d, J = 7.5 Hz, H-4, H-5), 7.75 (2H, m, H-1, H-8), 7.65
(1H, d, J = 8.2 Hz, NHFmoc), 7.42 (2H, t, J = 7.5 Hz, H-3,
H-6), 7.33 (2H, t, J = 7.0 Hz, H-2, H-7), 4.27-4.17 (4H, m, CHa,
CH2Fmoc, H-9), 3.03 (3H, d, J = 4.6 Hz, CH3NH), 2.42 (2H, t,
=
=
J = 6.6 Hz, CHg ), 2.23 (3H, s, CH3C N), 2.21 (3H, s, CH3C N),
2.10 (1H, m, CHb), 1.89 (1H, m, CHb); 13C NMR (75 MHz,
DMSO-d6): d 179.27 (NHNHC=S), 178.50 (EtNHC=S), 174.06
(CO2H), 170.36 (NHNHCO), 155.90 ((C=O)Fmoc), 149.78
=
=
(NHNH(C S)NH(C=N)), 147.92 MeNH(C S)NH(C=N),
143.92 (ArC), 143.79 (ArC), 140.73 (ArC), 127.70 ( C-3, C-6),
127.12 ( C-2, C-7), 125.41 ( C-1, C-8), 120.15 ( C-4, C-5),
65.81 (CH2Fmoc), 52.57 (Ca), 46.67 (C-9), 31.28 (Cg ), 30.39
=
=
(NHCH3), 27.54 (Cb), 11.83 (CH3(C N)NH(C S)NHNH),
Analysis was performed by coupling the mass spectrometer to
a Waters system equipped with a UV detector (Waters, 215 nm), a
manual injector and Waters 600E pump. The runs were performed
on an RP-column (Supelco Discovery Bio Wide Pore, C 18, 5 mm,
250 mm ¥ 4.6 mm) at a flow rate of 1 ml min-1. The outlet of the
HPLC column passed through a splitter with a split ratio of 1/10.
Preparative HPLC was carried out on a Gilson System fitted
with a reverse phase C18 Column, Supelco Discovery Bio Wide
Pore, 10 m, 250 mm ¥ 21.2 mm, flow rate 20 ml min-1.
+
-
=
=
11.64 (CH3(C N)NH(C S)NHMe); HRMS (ESI ): (M-H)
+
calcd for C27H33N8O5S2 613.2010; found 613.1989; HPLC (M I)
Rt 12.45 min, Elemental Analysis Found C, 52.9; H, 5.2; N, 18.2
C27H32N8O5S2 requires C, 52.9; H, 5.3; N, 18.3%
CuATSM/A-a-L-FmocGlu(OH)
(18). Cu(OAc)2·H2O
(12 mg, 0.059 mmol) was dissolved in H2O (1 ml) and added
dropwise to a stirring suspension of 15 (30 mg, 0.048 mmol) in
MeOH (5 ml). The suspension turned brown immediately and
was left to stir for 1 h. The solvent was then removed in vacuo and
H2O was added, the residue was filtered, washed with cold Et2O
and dried in vacuo to afford 18 (25 mg, 77%) as a dark brown
solid. HPLC (M I) Rt 12.40 min, HRMS (ESI+): (M-H)- calcd
Electron paramagnetic resonance
CW EPR experiments were performed on an X-band Bruker
BioSpin GmbH EMX spectrometer equipped with a high sensitiv-
ity Bruker probehead and a low-temperature Oxford Instruments
CF935 helium-flow cryostat. The magnetic field was calibrated at
room temperature with an external 2,2-diphenyl-1-picrylhydrazyl
standard (/g/= 2.0036). X-band CW experiments were conducted
at 60 K, with 0.20 mW microwave power, and 2.0 G modulation
amplitude at 100 kHz.
-
for C27H29CuN8O5S2 672.1004; found 672.0983
CuATSM/A-a-N-Biotin-L-Glu(OtBu) (19). 19 was prepared
following General Procedure B, using 16 (38 mg, 0.056 mmol) and
Cu(OAc)2·H2O (14 mg, 1.2 eq) to afford the desired product as
a black solid (40 mg, 96%). HRMS (ESI+): (M+Na)+ calcd for
C26H42CuN10NaO5S3+ 756.1690; found 756.1691; HPLC (M II) Rt
11.84 min
Synthesis of the BBS-conjugates
Radiolabeling and octanol/water partition coefficients of
ATSM-BBS conjugates
The synthesis of the Bombesin analogues was performed manually
as previously described24,25 by solid-phase peptide synthesis on
a Fmoc Rink Amide resin (Novabiochem). The synthesis was
carried out in a 2.5 mL plastic syringe fitted with a PE frit
(MultiSynTech GmbH, Germany) with a maximum of 50 mg
Fmoc-Rink Amide resin per syringe. The Fmoc group was
removed by treatment with 25% 4-methylpiperidine in DMF (2 ¥
20 ml of 1 mM stock solution ATSM-BBS-1 or 2 in DMSO were
transferred into 280 ml of 0.25 M ammonium acetate at pH 5.5.
2.5 ml 64/67Cu (equivalent to 0.045mg Cu) was added and the
solution was shaken for 30 min at room temperature. The labeling
was purified on a Sep-PakTM C18 or Chromafixꢀ C18ec. by washing
R
with 2 ml H2O and eluting with 2 ¥ 1 ml EtOH–H2O 9 : 1.
The octanol/water partition coefficients were determined at
pH 7.4 by adding 5 ml of radiolabeled 20 and 21 in PBS to a vial
i
10 min), the resin was washed with 3 ¥ DMF, 3 ¥ PrOH and 3 ¥
DMF. Unless mentioned otherwise, all couplings were carried out
3630 | Dalton Trans., 2010, 39, 3620–3632
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containing 1.2 mL of 1 : 1 octanol:PBS. Vortex mixing for 1 min
was followed by centrifugation at 10 000 rpm for 5 min. 40 ml from
each separated layer were sampled into a pre-weighed vial and
measured in a g-counter. Counts per unit weight of sample were
calculated and log D values were obtained using the formula log
D = log (counts in 1 g of octanol/counts in 1 g of water).
the Swiss Academy of Natural Sciences. Female CD-1 nu/nu mice
6-8 week-old (Charles River Laboratories, Sulzfeld, Germany)
were subcutaneously injected with 8 ¥ 106 PC-3 cells. Three weeks
after tumour implantation, the mice received i.v. 100 kBq 64/67Cu-
ATSM-BBS-2. At 1 and 24 h postinjection (p.i.), the animals were
killed by cervical dislocation. Blood and different organs were
collected, weighed, and the radioactivity measured in a g -counter.
Results are presented as percentage of injected dose per gram of
tissue (%ID/g). To determine the specificity of the in vivo uptake,
one group of mice received a co-injection of 100 mg of unlabeled
BBS(1–14) and the radiolabeled analogue and were sacrificed at
1 h p.i.
Cell culture
The human prostate adenocarcinoma cell line PC-3 was purchased
from the European Collection of Cell Culture (ECACC, Salis-
bury, UK). Cells were maintained in DMEM GLUTAMAX-I
(Invitrogen) supplemented with 1-10% FCS, 100 IU/ml penicillin
G sodium, 100 mg ml-1 streptomycin sulfate and 0.25mg ml-1
◦
Acknowledgements
amphotericin B (Bioconcept). Cells were cultured at 37 C in an
atmosphere containing 5% CO2. The cells were subcultured weekly
after detaching them with trypsin/EDTA 0.25% (Invitrogen).
The authors thank Mr Alain Blanc and Miss Alice Bowen for
technical assistance. We also thank Prof Antony Gee for helpful
discussion and Dr Robert King for assistance with the cell work.
We are also indebted to Dr Simon Bayly for his expert supervision
of radiolabeling of the ligands. RH thanks the Gray Lab Cancer
Research Trust (Prof Peter Wardman) and GlaxoSmithKline for
a studentship. MC thanks Cancer Research UK for a Project
Grant C5255/A8591. Biological Investigations were funded by
The Swiss Cancer League (project number KLS-02040-02-2007).
The collaboration within this project is part of COST action
BM0607.
Binding assays
The binding assays were carried out as previously reported.36
All experiments were carried out twice in triplicate. PC-3 cells
were placed in 48-well plates (250,000 cells/well). A protease
inhibitor containing binding buffer was used (50 mM HEPES,
125 mM NaCl, 7.5 mM KCl, 5.5 mM MgCl2, 1 mM EGTA,
5 g l-1 BSA, 2 mg l-1 chymostatin, 100 mg l-1 soybean trypsin
inhibitor and 50 mg l-1 bacitracin). Cells were incubated at
◦
37 C with 15,000-25,000 cpm of [125I-Tyr4]BBS per well and
increasing concentrations (0-3000nM) of the non-labeled ATSM-
BBS analogues. After 1 h incubation the cells were rinsed twice
with cold PBS. Cells with bound activity were solubilised by adding
2 ¥ 400 ml of 1 M NaOH at 37 ◦C. The final suspension was
measured in a g-counter. IC50 values were calculated by nonlinear
regression analysis using GraphPad Prism.
References
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The internalisation assays were carried out as previously
reported.36 PC-3 cells at confluence were placed in 6-well plates
at ~106 cells/well. Cells were incubated with ~1 kBq of the 64/67Cu-
labeled analogues in culture medium at a total volume of 1 ml/well
for 5, 15, 30, 60 and 120 min at 37 ◦C to allow binding and
internalisation. Non-specific internalisation was determined in the
presence of 1 mM non-labeled BBS(7–14). After each incubation
time, cells were washed three times with PBS to remove any
unbound peptide. Surface-bound activity was removed by two
steps of 5 min acid wash (50 mM glycine-HCl, 100 mM NaCl,
pH2.8, 600 ml per well) at room temperature. Cells containing
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given as a percentage of the total activity added per milligram of
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All animal experiments were conducted in compliance with the
Swiss animal protection laws and guidelines for scientific animal
trials established by the Swiss Academy of Medical Sciences and
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The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 3620–3632 | 3631
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