Fluorine and rhenium substituted ghrelin analogues as potential imaging probes for the growth hormone secretagogue receptor

Article abstract of DOI:10.1021/jm8014519

In our effort to create imaging probes targeting the growth hormone secretagogue receptor (GHSR), we now report on the design and synthesis of fluorine and rhenium containing ghrelin analogues through modification of the n-octanoyl Ser-3 side chain. Fluor

Full text of DOI:10.1021/jm8014519

2196
J. Med. Chem. 2009, 52, 2196–2203
Articles
Fluorine and Rhenium Substituted Ghrelin Analogues as Potential Imaging Probes for the
Growth Hormone Secretagogue Receptor
Dina Rosita,† Matthew A. DeWit,† and Leonard G. Luyt*,†,‡
Department of Chemistry, UniVersity of Western Ontario, 1151 Richmond Street, London, Ontario N6A 5B7, Canada, and Department of
Oncology, London Regional Cancer Program, UniVersity of Western Ontario, 790 Commissioners Road East,
London, Ontario N6A 4L6, Canada
ReceiVed NoVember 17, 2008
In our effort to create imaging probes targeting the growth hormone secretagogue receptor (GHSR), we
now report on the design and synthesis of fluorine and rhenium containing ghrelin analogues through
modification of the n-octanoyl Ser-3 side chain. Fluorine analogues were designed whereby the fluorine
atom is situated at the terminus of an aliphatic chain using diaminopropionic acid (Dpr) as residue-3. Truncated
ghrelin(1-5) and ghrelin(1-14) fluorine-bearing analogues were prepared, the best of which had a 28 nM
IC50 for GHSR. Ghrelin(1-14) analogues were also prepared containing rhenium, as a surrogate metal for
technetium-99m, with a cyclopentadienylrhenium tricarbonyl being situated at the terminus of the residue-3
side chain, yielding compounds the best of which had a 35 nM IC50. This represents a rare case of incorporating
rhenium into a peptide structure where the metal complex is required for biological activity. These fluorine
and rhenium derivatives demonstrate the ability to modify the Ser-3 side chain of ghrelin in order to create
imaging probes for the GHSR.
Introduction
The role of radiolabeled peptides in clinical diagnosis and
therapy, especially in oncology, has been increasing in the past
few decades. Peptides as targeting agents have several advan-
tages over other types of targeting agents such as small
molecules and antibodies.1-3 The use of peptides as tumor
Figure 1. Structure of human ghrelin.
imaging probes is based on targeting by the peptide ligand to
specific cell surface receptors that are highly expressed in tumor
imaging probes and marketed for clinical purposes, and their
cells but not in the surrounding normal cells. The binding of
use in different types of tumors is limited.1 Here we report on
the peptide ligand to its receptor is often followed by internal-
a preliminary investigation of the potential use of radiolabeled
ization, leading to a high tumor-to-background ratio, which is
ghrelin analogues as cancer imaging probes targeting the growth
further augmented by rapid tissue penetration and fast blood
clearance. Peptides can be synthesized using an automated
hormone secretagogue receptor (GHSRa).
GHSR, a member of the G protein-coupled receptor (GPCR)
synthesizer from readily available starting materials which leads
to simple and inexpensive large scale production.1,4 However,
family, was first identified in 1996 as a seven-transmembrane
domain 366 amino acid protein responsible for the regulation
despite the large number of peptides and their receptors that
of growth hormone (GH) secretion.5 This receptor is mainly
are currently under investigation, only a few are approved as
expressed in the hypothalamus, pituitary cells, and a number
of peripheral tissues.5,6 Expression of the GHSR has been
* To whom correspondence should be addressed. Phone: +1-519-685-
8600, extension 53302. Fax: +1-519-685-8646. E-mail: lluyt@uwo.ca.
reported in various types of tumors, including breast carcinomas,
prostate cancer cell lines, ovarian tumors, testicular tumors,
pancreatic endocrine tumors, and intestinal carcinoids.7-12 The
presence of high affinity and specific binding sites in the
neoplastic cells, but absence in the corresponding normal tissues,
has been demonstrated in at least three different human breast
carcinoma cell lines and pancreatic endocrine tumors.7,11 This
suggests that GHSR may be a suitable target for tumor imaging.
According to our knowledge, the potential use of GHSR as a
target in cancer imaging has not previously been reported.
Ghrelin, a 28 amino acid peptide hormone with a unique
lipophilic n-octanoyl post-translational modification in its
serine-3 residue (Figure 1), was discovered in 1999 by Kojima
et al. and determined to be the endogenous ligand for the
GHSR.13 This hormone plays an important role in the stimula-
†
Department of Chemistry, University of Western Ontario.
London Regional Cancer Program, University of Western Ontario.
‡
a Abbreviations: Boc, tert-butoxycarbonyl; Cp, cyclopentadienyl; DIPEA,
N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; Dpr, diamino-
propionic acid; EI, electron ionization; ESI, electrospray ionization; Fmoc,
9-fluorenylmethoxycarbonyl; GH, growth hormone; GHSR, growth hormone
secretagogue receptor; GPCR, G protein-coupled receptor; HBTU, 3-[bis-
(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluorophos-
phate; HRMS, high resolution mass spectrometry; IC50, half-maximal
inhibitory concentration; MBHA, 4-methylbenzhydrylamine; Ms, mesyl;
Mtt, methyltrityl; PET, positron emission tomography; Pyr, pyridine; RP
HPLC, reverse phase high performance liquid chromatography; SPPS, solid
phase peptide synthesis; SPECT, single photon emission computed tomog-
raphy; TBAF, tetra-n-butylammonium fluoride; TFA, trifluoroacetic acid;
THF, tetrahydrofuran; TIS, triisopropylsilane; Trt, trityl; Ts, tosyl. The
abbreviations for the common amino acids are in accordance with the
recommendations of the IUPAC-IUB Joint Commission on Biochemical
Nomenclature.
10.1021/jm8014519 CCC: $40.75
2009 American Chemical Society
Published on Web 03/26/2009

Fluorine and Rhenium Ghrelin Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2197
Table 1. Various Modifications to Human Ghrelin and Their Binding
Affinities, As Reported by Bednarek et al15
compd
peptide sequence
R
IC50 (nM)a
0.25
1
human ghrelin
ghrelin(1-28)
ghrelin(1-28)
ghrelin(1-28)
ghrelin(1-28)
ghrelin(1-28)
ghrelin(1-28)
ghrelin(1-28)
ghrelin(1-28)
ghrelin(1-14)
-CH2O-CO-(CH2)6CH3
2
3
4
5
6
7
8
9
-CH2OH
>10000
>2000
0.87
0.12
0.08
>2000
0.42
9.6
-CH2O-CO-CH3
-CH2O-CO-(CH2)14CH3
-CH2O-CO-CH2-adamantyl
-CH2O-CO-(CH2)7Br
-CH2O-CO-(CH2)6NH2
-CH2NH-CO-(CH2)6CH3
-CH2O-CO-(CH2)6CH3
Figure 2. Design of ghrelin analogues for radioimaging: (A) fluorine-
bearing ghrelin analogues for PET imaging; (B) rhenium or technetium-
bearing ghrelin analogues for SPECT imaging.
In our design, the radionuclide 18F will be incorporated
directly into the end of the Ser-3 side chain (Figure 2A). The
rationale behind this was that the similar bromine modification
6 is known to be well tolerated.15 For the γ-emitting radionuclide
99mTc, we decided that a cyclopentadienyltechnetium tricarbonyl
(Cp99mTc(CO)3) complex would be a good choice for this
integrated design, since not only is it small, nonchiral, and
10
11
ghrelin(1-5)-NH2 -CH2O-CO-(CH2)6CH3
ghrelin(1-4)-NH2 -CH2O-CO-(CH2)6CH3
55
889
a IC50 values determined by [35S]MK-0677 competitive binding assay.15
tion of GH secretion through binding with GHSR along with
other functions including appetite regulation.14 Ghrelin binds
to the GHSR with high affinity and specificity, resulting in a
complex formation of ghrelin-GHSR followed by internaliza-
tion of the complex.14 We intend to utilize the binding and
internalization of the ghrelin-GHSR complex by developing a
class of ghrelin based imaging agents targeting GHSR that are
suitable for cancer imaging.
chemically and metabolically stable but it is also lipophilic,18
a
key requirement for the ghrelin side chain. For this reason, the
Cp99mTc(CO)3 complex was designed to be incorporated into
the end of the side chain (Figure 2B). For chemical and
biological analysis purposes, nonradioactive 19F was used in the
place of 18F and 185/187Re was used in the place of 99mTc.
Technetium does not have a stable isotope but is known to have
similar chemical and biological properties to rhenium.19 For
binding optimization, the ghrelin analogues were designed with
several different lengths of side chain in order to determine the
optimal length.
The in vivo suitability of the peptides, including stability,
tissue penetration, and fast clearance, is an important aspect of
an imaging probe. In order to keep the size compact for better
tissue penetration, as well as to reduce complication in synthesis,
the minimum number of amino acid residues required for
binding was used in the probe design. Previous structure-activity
relationship studies have shown that the first four or five residues
from the N-terminus of ghrelin are necessary for binding,
although the binding affinity is greater when the chain is
extended to include additional residues of the endogenous
ghrelin, as shown in the cases of 9, 10, and 11.15-17 For that
purpose, two different truncation lengths were chosen in this
study: 5-mer and 14-mer peptides from the N-terminus (Figure
2). In order to increase the stability toward exopeptidases in
vivo, the carboxylic acid of the C-terminus is replaced by an
amide (Figure 2). In human ghrelin, the hydrophobic side chain
is attached to the Ser-3 via an ester bond; however, this ester
bond can undergo rapid hydrolysis.15,16 According to a previous
study, replacing the ester with an amide as in 8 has no
detrimental effect on the binding capability;15 therefore, in our
design this ester bond was replaced with an amide bond. The
non-natural amino acid diaminopropionic acid (Dpr), structurally
similar to serine with an amine in the side chain instead of
alcohol, was used in the place of Ser-3 to allow amide formation.
Design of Ghrelin Analogues as Imaging Probes
The relationship between ghrelin (1) structure and its binding
activity to the GHSR has been previously studied by several
groups, and the n-octanoyl side chain at Ser-3 was found to be
crucial for binding.15-17 These studies demonstrated that des-
acyl ghrelin 2 (Table 1), ghrelin without the n-octanoyl side
chain, does not bind to the GHSR. Various modifications to
ghrelin’s n-octanoyl group were studied, and it was concluded
that this side chain could be replaced by other groups as long
as its lipophilicity and approximate size are maintained as in 4
and 5.15 When a short side chain was used as in 3, poor binding
was observed. In addition, a neutral halogen modification such
as a bromo in 6 is well tolerated, while a charged group
modification such as an amine in 7 leads to poor binding.15
There are two radionuclides that we are interested in
incorporating as part of ghrelin based imaging agents: fluo-
rine-18 (18F) for application in positron emission tomography
(PET) and technetium-99m (99mTc) for application in single
photon emission computed tomography (SPECT). Since modi-
fication to the Ser-3 n-octanoyl is permitted as long as the
requisite hydrophobic interaction to GHSR is maintained, we
decided to integrate the radionuclide directly in the side chain
of Ser-3. Careful design is needed to minimize negative
interference caused by the addition of the radionuclides to this
binding sensitive region of the molecule. The approach to
integrate a radionuclide in a site that is crucial for the binding
is not common for radiolabeled peptides, as the more common
approach is to place the radionuclide far from the active binding
site, in a pendant manner, thus reducing interference from the
radionuclide complex with receptor binding. However, by
placement of the radionuclide directly in the active binding site,
the molecular weight of the agent will be minimized and further
development into a peptidomimetic agent may be realized.
Results and Discussion
Fluorine Bearing Ghrelin Analogues. The first class of
molecules that we developed were fluorine bearing ghrelin
analogues for a preliminary study to investigate their potential

2198 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Rosita et al.
Scheme 2. Synthesis of Fluorine Bearing Ghrelin(1-5)a
a Reagents and conditions: (a) 2% TFA, 5% TIS, CH2Cl2, room temp;
(b) 16a, HBTU, DIPEA, DMF, room temp; (c) 2% TFA, 5% TIS, CH2Cl2,
room temp; (d) TsCl, pyridine, CH2Cl2, room temp; (e) TBAF, THF, room
temp; (f) 88% TFA, 5% H2O, 5% phenol, 2% TIS, room temp.
Scheme 3. Synthesis of Fluorine Bearing Ghrelin(1-14)a
Figure 3. Truncated ghrelin analogues prior to modification.
Scheme 1. Synthesis of Side Chain Precursorsa
a Reagents and conditions: (a) 2% TFA, 5% TIS, CH2Cl2, room temp;
(b) 16a-c, HBTU, DIPEA, DMF, room temp; (c) 2% TFA, 5% TIS,
CH2Cl2, room temp; (d) MsCl, NEt3, CH2Cl2, room temp; (e) TBAF, THF,
room temp; (f) 88% TFA, 5% H2O, 5% phenol, 2% TIS, room temp.
material for 9C, were then tritylated to obtain weak acid labile
protection for the alcohol group, which was necessary during
the side chain coupling to the peptide. Purification of the
tritylated products 15a, 15b, and 15c was problematic, and
attempts to perform chromatography were made. The existence
of excess trityl chloride and pyridinium salt made it difficult to
dissolve the crude material, and in our early attempts methanol
was added to help the solvation. Unfortunately methanol reacted
with unused trityl chloride, caused acid formation, and resulted
in trityl (Trt) deprotection from the product that reformed the
starting materials. Instead, the byproducts were removed by
filtration with organic solvents at 0 °C to obtain 15a and 15b
or by flash chromatography without methanol to obtain 15c.
Hydrolysis of the methyl ester of 15a, 15b, and 15c was
carried out under basic conditions, followed by an acidic
workup, during which special attention was given to maintain
the pH above 4 to prevent trityl deprotection. After recrystal-
lization for solid product or flash chromatography for oil
products, 6-trityloxyhexanoic acid 16a, 9-trityloxynonanoic acid
16b, and 12-trityloxydodecanoic acid 16c were isolated, provid-
ing aliphatic side chains that were ready to be coupled to 12
and 13.
The on-resin reaction series started with a selective depro-
tection of the acid labile amine-Mtt of Dpr-3, which was done
with a series of treatments with a dilute trifluoroacetic acid (2%
TFA) solution in CH2Cl2. After the free amine was obtained,
the carboxylic acid group of the side chain precursor was
coupled to this amine via O-benzotriazole (OBt) ester formation,
following standard coupling procedures in peptide synthesis.20
For the 5-mer 12, only 16a was coupled to the peptide (Scheme
2), while for the 14-mer 13, all precursors previously made,
16a, 16b, and 16c, were used (Scheme 3). Reaction completion
was followed by the Kaiser test21 and in some cases micro-
cleavage to allow HPLC analysis and MS analysis to confirm
the product mass.
a Reagents and conditions: (a) H2SO4, MeOH, room temp; (b) TrtCl,
pyridine, room temp; (c) NaOHaq, THF, room temp.
use as PET imaging agents for cancer. The peptides were
assembled following standard 9-fluorenylmethoxycarbonyl (Fmoc)
solid phase peptide synthesis (SPPS) methods20 on a polystyrene-
based insoluble support, Rink amide 4-methylbenzhydrylamine
(MBHA) resin, which provided an amide C-terminus upon
cleavage. The Ser-3 of human ghrelin was replaced with Dpr,
protected by the weak acid labile methyltrityl (Mtt), to provide
an amide linkage in this position upon further modification. The
fully protected resin-bound ghrelin(1-5) 12 and ghrelin(1-14)
13 prior to modification are shown in Figure 3.
At this point, the Dpr-3 of the peptide was ready for
functionalization. Fluorine bearing analogues were designed with
the fluorine attached to the end of the aliphatic side chain with
variable length. Nonradioactive analogues using 19F in the place
of 18F were used to study their chemical and biological
properties, and side chain precursors were prepared in solution
phase prior to attachment to the peptide, with fluorination of
the side chain being conducted on-resin. Prior to attachment,
side chain precursors had three important features: an aliphatic
chain with length adjusted for binding affinity optimization, a
carboxylic acid on one end of the chain as the attachment site
to the peptide, and a protected alcohol group on the other end
as the fluorination site.
Three different side chain lengths were prepared: 6, 9, and
12 carbon length, indicated as 6C, 9C, and 12C, respectively.
The 6C and 12C precursors were prepared from their cyclic
lactones, with the first synthetic step being the opening of the
lactone rings in methanol using an acid catalyst (Scheme 1).
Methanol was chosen as media to provide methyl protection to
the carboxylate group, as that was necessary during the next
reaction step. The formed alcohols 14a and 14c, as well as
commercially available methyl 9-hydroxynonanoate, the starting
After the side chain was attached to the peptide, the protected
alcohol was transformed to a fluoro group via several steps.
The first step was the selective deprotection of the alcohol-Trt
with dilute acid (2% TFA) solution in CH2Cl2. The obtained

Fluorine and Rhenium Ghrelin Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2199
Table 2. Characterization and Binding Affinity Data of Fluorine
Bearing Ghrelin Analogues
of the fluorine bearing ghrelin(1-14) derivatives was evaluated
on the basis of the binding data of three peptides 18a, 18b, and
18c that had different side chain lengths: 6C, 9C, and 12C,
respectively. All of these derivatives were found to have
satisfactory nanomolar binding affinities that make them possible
candidates for use as imaging agents. These analogues’ IC50
values were found to decrease as the side chain length increases,
indicating that the longer aliphatic chain is preferred for binding.
This finding was in accordance with the previous study by
Matsumoto et al. that to a certain length, binding affinity of
ghrelin analogues improved as the side chain got longer.16 On
the basis of our findings in this preliminary study, we conclude
that fluorine bearing 14-mer ghrelin analogues have potential
to be developed as PET cancer imaging agents, with the 12C
side chain analogue 18c as the best candidate.
m/zb
compd purity (%)a
calcd
obsd
IC50 (nM)c
17
97
98
99
99
[M + Na]+
[M + 2H]2+
[M + H]+
646.3
857.4
1755.9 1756.0
899.5 899.5
646.3
857.5
>2000
147
18a
18b
18c
39.6
27.9
[M + 2H]2+
a Percent purity determined by RP HPLC with detection at 220 nm. b The
m/z calculated and observed values are based on the prominent observed
signals as determined by MS (ESI). c IC50 values (nM) determined by
competitive radioligand binding assay to the GHSR (mean of two experi-
ments) derived from human recombinant CHO-K1 cells according to
published procedures.23,24
alcohol was activated prior to fluorination. For the ghrelin(1-5)-
6C-OH, activation was done via tosylation in basic conditions.
HPLC monitoring and MS analysis of the microcleaved product
showed conversion of the starting material to the desired product
ghrelin(1-5)-OTs (55%) and various byproducts.
Rhenium Bearing Ghrelin Analogues. The second class of
molecules designed were rhenium bearing ghrelin analogues for
a preliminary study to investigate the suitability of 99mTc-
radiolabeled ghrelin analogues as SPECT imaging agents for
the GHSR. Re(I) and Tc(I) tricarbonyl complexes have been
reported to have great stability in vivo and have been used in
a variety of peptide and non-peptide imaging agents.25 Among
these systems, cyclopentadienyl tricarbonyl organometallic
(CpM(CO)3) species appear to be the best candidate for our
imaging purpose because of the neutral, lipophilic, and stable
properties of this complex. The ability to use the cyclopenta-
dienylmetal complex in an integrated fashion has been dem-
onstrated by Katzenellenbogen and co-workers through their
investigation of rhenium containing estrogens, where the CpRe-
(CO)3 unit is directly associated with the estrogen receptor
binding site.26 The neutral and lipophilic properties of the metal
complex are important in our design because we plan to integrate
this organometallic system as part of the residue-3 side chain
that participates in the binding to the GHSR.
The tosylation was also attempted on the ghrelin(1-14)-6C-
OH; however, after several attempts HPLC and MS analyses
clearly showed that tosylation did not work for the 14-mer
peptide. This was likely caused by aggregation of the material
on-resin, which could lead to incomplete solvation, matrix
shrinkage, and poor reagent penetration. Since the nature of the
peptide and the side chain could not be changed at this point,
more reactive reagents were sought. Mesylation was chosen for
the reason that mesyl chloride (MsCl) forms a highly reactive
intermediate sulfene (SO2CH2), in which the sulfur is highly
electrophilic and will react with any alcohol, even with tertiary
alcohols that react very slowly with TsCl.22 HPLC and MS
analyses of the microcleaved products showed that mesylation
was successfully applied to the 14-mer peptides with the 6C,
9C, and 12C side chains.
Fluorination for both ghrelin(1-5) and ghrelin(1-14) deriva-
tives was performed using tetra-n-butylamonium fluoride (TBAF)
in tetrahydrofuran (THF), monitored by HPLC and MS analyses
of microcleaved products. Even though a competing reaction
of elimination by F- as a base during the treatment with TBAF
was anticipated, this was not observed. The fluorination of the
14-mer analogues went more slowly than for the 5-mer
analogue, likely because of some degree of aggregation that
occurred in the 14-mer peptides as previously discussed.
Fluorination was the last step that needed to be done on-resin,
and at this point the peptides were ready to be cleaved off the
resin and fully deprotected. The desired fluorine bearing
ghrelin(1-5)-6C-F 17, ghrelin(1-14)-6C-F 18a, ghrelin(1-14)-
9C-F 18b, and ghrelin(1-14)-12C-F 18c were isolated with
yields of 9%, 7%, 2%, and 12%, respectively (Table 2).
The binding affinities of these fluorine bearing ghrelin(1-5)
and ghrelin(1-14) derivatives to the GHSR were evaluated
according to the half-maximal inhibitory concentration (IC50)
values (Table 2) as determined by a radioligand binding
assay.23,24 The effect of the sequence length of the peptide was
determined from the comparison of the 5-mer 17 and the 14-
mer 18a that had an identical side chain in their Dpr-3 residue.
The IC50 value of 17 was determined to be greater than 2 µM,
based on only a 20% inhibition at that concentration. This result
showed that a 5-mer peptide with the fluorine modification lacks
adequate affinity to the GHSR and therefore is not a suitable
design for an imaging probe. In contrast, the IC50 of 18a was
found to be satisfactory with a value of 147 nM, showing its
potential to be used as an imaging tracer. On the basis of these
results, all optimizations throughout the study were conducted
on 14-mer ghrelin analogues. Side chain length optimization
Metal complex incorporation into a site that is crucial for
ligand-receptor interaction is rare for peptide based technetium
radiopharmaceuticals. Typically, a metal complex is located far
from the active binding site in a pendant fashion, in order to
minimize the interference it might cause to receptor binding.
Many such examples are reported in the literature for Tc/Re,
including 99mTc-EDDA/HYNIC octreotide, Cp-TR-octreotide,
and [99mTc(CO)3-NR-histidinyl acetate]bombesin(7-14).27-29 In
contrast to this pendant (also referred to as conjugate) design,
only a few examples are reported of an integrated design
whereby the metal complex is part of the receptor binding region
of the ligand. Bigott-Hennkens et al. reported on a Re(V) oxo
inorganic complex that was successfully coordinated directly
to the octreotide peptide backbone, and this metal coordination,
replacing the function of a disulfide bridge, cyclized the
peptide.30 Although not located within the receptor binding
pocket, the metal complex is a critical element for binding
because of the resultant formation of a cyclic constraint to the
peptide. Prior work reported on a similar cyclic strategy for
rhenium and technetium R-melanocyte stimulating hormone
analogues.31 Thus, the success of our design would contribute
to the rare cases of metal ligand incorporation into a region
crucial for receptor binding of a peptide based pharmaceutical.
The side chains for the rhenium bearing ghrelin were prepared
using solution-phase chemistry prior to attachment to the
peptide. For optimization, two different lengths were prepared:
four and six carbons long, counted from the amide carbon to
the carbon in which Cp ring is attached and will be addressed
as 4C and 6C. Both precursors were prepared from their cyclic
anhydride, succinic anhydride, and adipic anhydride for 4C and

2200 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Scheme 4. Synthesis of Rhenium Side Chain Precursorsa
Rosita et al.
Table 3. Characterization and Binding Affinity Data of Rhenium
Bearing Ghrelin Analogues
m/zb
calcd
[M + 2H]2+ 1008.4 1008.4
[M + H]+
2043.8 2043.8
compd purity (%)a
obsd
IC50 (nM)c
20a
20b
98
99
35
174
a Percent purity determined by RP HPLC with detection at 220 nm.
b Both 185Re and 187Re peaks were observed; however, values reported are
based on the more abundant 187Re. The m/z calculated and observed values
are based on the prominent observed signals as determined by MS (ESI or
MALDI-TOF). c IC50 values (nM) determined by competitive radioligand
binding assay to the GHSR (mean of two experiments) derived from human
recombinant CHO-K1 cells according to published procedures.23,24
a Reagents and conditions: (a) AlCl3, CH2Cl2, room temp for 19a or reflux
for 19b.
Scheme 5. Synthesis of Rhenium Bearing Ghrelin(1-14)a
adds to the bulkiness of the overall side chain, and by
comparison of their binding affinity, it was found that the shorter
20a has the optimal binding compared to the longer 20b. This
finding is in contrast with the general trend of fluorine bearing
ghrelin(1-14) analogues 18a, 18b, and 18c, for which the longer
side chain leads to better binding. A possible cause is that the
CpRe(CO)3 has already contributed to a certain volume, and
the extra length of the side chain of 20b causes the overall size
to be larger than what is required for optimal binding. Indeed,
the previous study done by Matsumoto et al. showed that the
binding affinity of ghrelin analogues improved as the side chain
was increased, until at some point it started to decrease.16
Here we demonstrated that the incorporation of a metal
complex in the crucial binding region of a peptide ligand was
a success, with the key component being the neutral and
lipophilic CpRe(CO)3 mimicking the lipophilic property of the
original ghrelin side chain. On the basis of these findings, we
conclude that technetium-99m bearing 14-mer ghrelins have
potential to be developed as SPECT cancer imaging agents, with
the ghrelin-rhenium surrogate 20a as our best candidate.
a Reagents and conditions: (a) 2% TFA, 5% TIS, CH2Cl2, room temp;
(b) 19a,b, HBTU, DIPEA, DMF, room temp; (c) 88% TFA, 5% H2O, 5%
phenol, 2% TIS, room temp.
6C, respectively, and rhenium tricarbonyl cyclopentadienyl
(CpRe(CO)3) through Friedel-Crafts acylation using aluminum
chloride (AlCl3) (Scheme 4). The reaction for the 4C precursor
went slowly and did not go to completion because of the low
reactivity of the Cp ring when it is coordinated to Re(I). The
starting material CpRe(CO)3 was recovered during the extrac-
tion, and the product 4-(cyclopentadienylrhenium tricarbonyl)-
4-oxobutanoic acid 19a was isolated upon chromatography.
In the synthesis of 6C precursor, adipic anhydride was
prepared from adipic acid through dehydration according to a
literature procedure.32 Various attempts to optimize the
Friedel-Craft step were performed, including the use of
different solvents (chloroform and dichloroethane), increased
reaction temperature, portionwise addition of reagents, different
Lewis acids, and increased reagent equivalencies. The best result
was achieved when 2 equiv of adipic anhydride and 4 equiv of
AlCl3 were used, CH2Cl2 was used as the solvent, and the
mixture was refluxed. This caused all starting material to be
consumed within 2 days, and upon purification, 6-(cyclopen-
tadienylrhenium tricarbonyl)-6-oxohexanoic acid 19b and the
dimer 1,6-di(CpRe(CO)3)-1,6-dioxohexane as a byproduct were
isolated.
The side chain precursors 19a and 19b, already containing
rhenium, were ready to be incorporated into the peptide at this
point. Since the 14-mer binding data for fluorine bearing
analogues were found to be superior to its 5-mer equivalent,
rhenium bearing ghrelin analogues were made only with the
14-mer sequence. Fully protected on-resin 13 was prepared, and
the Mtt group was removed from the amine of Dpr-3 using 2%
TFA solution (Scheme 5). The side chains 19a and 19b were
then coupled to this amine followed by cleaving from resin and
complete deprotection. The rhenium bearing ghrelin(1-14)-4C-
CpRe(CO)3 20a and ghrelin(1-14)-6C-CpRe(CO)3 20b were
obtained with purified yields of 24% and 36%, respectively
(Table 3).
Conclusions
The aim of this preliminary study was to investigate the
appropriateness of fluorine and rhenium functionalized ghrelin
analogues as ligands for the growth hormone secretagogue
receptor (GHSR). Several 5- and 14-mer ghrelin analogues were
designed and synthesized with fluorine, or rhenium as CpRe-
(CO)3 complex, that was attached to the end of the aliphatic
side chain of residue-3, a region that is crucial for the binding
activity. From the GHSR binding evaluation, it is revealed that
14-mer ghrelin analogues have high binding affinity, while a
5-mer analogue showed poor binding affinity. The fluorine
derivative 18c, containing a 12 carbon aliphatic chain at
position-3 of the 14-mer ghrelin, has the best receptor affinity
and is our lead candidate for PET imaging. The rhenium
derivative 20a has excellent receptor affinity and is, to our
knowledge, the first example of incorporating rhenium into a
peptide structure where the metal complex itself is critical for
receptor binding. This sets the stage for future investigation by
PET and SPECT of the ability to use the analogous 18F and
99mTc ghrelin derivatives for the noninvasive imaging of GHSR
expression.
Experimental Section
Materials and Equipment. Reagents and solvents were pur-
chased from Sigma-Aldrich, Fluka, NovaBiochem, Peptides Inter-
national, Strem Chemicals, Toronto Research Chemicals, Chem-
Impex, Fisher Scientific, or VWR and were used without further
purification unless noted. Dry CH2Cl2 was prepared by distillation
from CaH under argon. Oven-dried or flame-dried apparatus and
argon flow were used in all water sensitive reactions. Analytical
HPLC was performed using a Grace Vydac protein/peptide RP-
The evaluation of 20a and 20b showed that both molecules
have good binding affinity to the GHSR, marked by their
nanomolar IC50 values: 35 and 174 nM, respectively (Table 3).
The aliphatic chain between the Dpr-3 and the rhenium complex

Fluorine and Rhenium Ghrelin Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2201
C18 column 4.6 mm × 250 mm, 5 µm, and preparative HPLC
was performed using a Grace Vydac protein/peptide RP-C18
column 22.0 mm × 250 mm, 10 µm. The absorbance was detected
at wavelengths of 220 and 254 nm. A gradient system was used:
H2O + 0.1% of TFA (solvent A) and CH3CN + 0.1% of TFA
(solvent B). Flash column chromatography was performed using
Merck silica gel 60 (230-400 mesh). Analytical TLC was carried
reduced pressure to yield 874 mg (88%) of 14c as a white solid.
1H NMR (400 MHz, CDCl3, δH ppm): 3.65 (3H, s, CO2CH3), 3.63
(2H, t, JH-H ) 6.6 Hz, HO-CH2), 2.29 (2H, t, JH-H ) 7.6 Hz,
CH2CO2), 1.50-1.66 (4H, m, 2CH2), 1.22-1.38 (14H, m, 7CH2).
HRMS (EI): m/z calcd 231.1955 ([M + H]+, C13H27O3), found
231.1954 [M + H]+.
3
3
Methyl 6-Trityloxyhexanoate (15a). Trityl chloride (18.8 g, 67.4
mmol) was added to an ice cold (0 °C) stirring solution of 14a
(9.9 g, 67.4 mmol) in 80 mL of pyridine. The reaction mixture
was warmed to room temperature and stirred under argon for 2
days, during which time a white byproduct formed. The solvent
was removed under reduced pressure, and the resulting material
was redissolved in ice cold THF. The insoluble byproduct was
removed by filtration, and the filtrate was dried under reduced
1
out on EMD silica gel 60 F254 plates. H and 13C NMR data were
obtained using a Varian Mercury 400, and the chemical shifts were
referenced to solvent signals (CDCl3, 1H 7.25 ppm, 13C 77.23 ppm)
relative to TMS. Mass spectra were obtained using Finnigan MAT
8200 (HRMS-EI), Micromass LCT (MS-ESI), and Micromass
MALDI-LR (MALDI-TOF) mass spectrometers. For compounds
containing rhenium, both 185Re and 187Re peaks are observed, and
the more abundant 187Re mass is reported in this section. Melting
points were determined in open capillary tubes on Mel-Temp
apparatus without correction.
Peptide Assembly. Fully protected resin-bound peptides were
synthesized according to the general procedures in Fmoc solid phase
peptide synthesis20 either manually or automatically using an APEX
396 peptide synthesizer. Fmoc protected rink amide MBHA resin
(loading of 0.27 or 0.47 mequiv/g) was used as the solid support.
N-Fmoc amino acids, with strong acid labile protecting groups for
side chain functional groups, were used in general, and N-Boc amino
acid was used for the N terminus. Fmoc-diaminopropanoic acid
(Dpr), with the ꢀ-amine protected with methyltrityl (Mtt), was used
for residue-3. Fmoc removal was achieved with treatments of 20%
piperidine in N,N-dimethylformamide (DMF) for 10 and 20 min
and successive washes using DMF and CH2Cl2 after each treatment.
For each amino acid coupling, resin was treated once or twice with
3 equiv of Fmoc or Boc amino acids, 3 equiv of 3-[bis(dimethy-
lamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluorophosphate
(HBTU), and 6 equiv of N,N-diisopropylethylamine (DIPEA) in 2
mL of DMF for 30 min to 4 h. Successive washes with DMF,
CH2Cl2, and THF were done following the coupling. Using this
general procedure, 12 and 13 were prepared.
Peptide Deprotection and Resin Cleavage. Selective depro-
tections of amine-Mtt and alcohol-Trt were achieved by shaking
the resin with 2% TFA and 5% triisopropylsilane (TIS) in CH2Cl2
for 2 min, followed by successive washes with CH2Cl2. This
treatment was repeated five times. During the solid phase reaction
steps, the presence or absence of a free amine group was monitored
by the Kaiser test.21 When necessary, cleaving a small sample of
resin beads (microcleave) was performed to obtain a small quantity
of representative peptide, for which HPLC and MS analyses were
conducted. After all modifications were done, the peptide was
deprotected and cleaved from the resin by TFA containing the
scavengers water (5% v/v), phenol (5% m/v), TIS (2% v/v) for
2-4 h. Resin was filtered and rinsed with a small amount of TFA.
Peptide was precipitated from the TFA solution using tert-butyl
methyl ether (TBME) and collected after centrifugation and
decantating. Peptide was then rinsed using TBME and collected
again. The resulting solid was redissolved in water with additional
CH3CN when necessary, frozen, and lyophilized to obtain crude
peptide as a fine powder. Purification of the peptide was conducted
through preparative HPLC runs, and the purity of the isolated
material was determined by analytical HPLC.
1
pressure to obtain 22.6 g (86%) of an orange oil, 15a. H NMR
(400 MHz, CDCl3, δH ppm): 7.43 (6H, m, p-Ar), 7.29 (6H, m,
m-Ar), 7.22 (3H, m, o-Ar), 3.65 (3H, s, CO2CH3), 3.05 (2H, t, 3JH-H
3
) 6.6 Hz, HO-CH2), 2.29 (2H, t, JH-H ) 7.5 Hz, CH2CO2),
1.54-1.71 (4H, m, 2CH2), 1.33-1.45 (m, 2H, CH2). HRMS (EI):
m/z calcd 388.2038 (C26H28O3), found 388.2039 [M]+.
Methyl 9-Trityloxynonanoate (15b). The synthesis procedure
of 15a was followed, with 1.37 g (4.9 mmol) of trityl chloride,
0.46 g (2.5 mmol) of methyl 9-hydroxy-nonanoate, and 10 mL of
pyridine used in the reaction. An insoluble byproduct was removed
by filtration in CH2Cl2 and 10% ethyl acetate in hexanes. Upon
solvent removal, 1.02 g (96%) of 15b was obtained as a pale-yellow
1
oil. H NMR (400 MHz, CDCl3, δH ppm): 7.44 (6H, m, p-Ar),
7.28 (6H, m, m-Ar), 7.22 (3H, m, o-Ar), 3.65 (3H, s, CO2CH3),
3
3
3.03 (2H, t, JH-H ) 6.6 Hz, HO-CH2), 2.29 (2H, t, JH-H ) 7.5
Hz, CH2CO2), 1.55-1.65 (4H, m, 2CH2), 1.18-1.40 (8H, m, 4CH2).
HRMS (EI): m/z calcd 430.2508 (C29H34O3), found 430.2514 [M]+.
Methyl 12-Trityloxydodecanoate (15c). The synthesis proce-
dure of 15a was followed. Trityl chloride (1.41 g, 5.1 mmol), 14c
(0.58 g, 2.5 mmol), and pyridine (10 mL) were used, and the
reaction was prolonged to 3 days. The filtration step was omitted;
instead, the crude material was purified by flash column chroma-
tography (10% EtOAc in hexanes) yielding 477 mg (40%) of a
1
pale-yellow oil, 15c. H NMR (400 MHz, CDCl3, δH ppm): 7.44
(6H, m, p-Ar), 7.28 (6H, m, m-Ar), 7.21 (3H, m, o-Ar), 3.66 (3H,
3
s, CO2CH3), 3.03 (2H, t, JH-H ) 6.6 Hz, HO-CH2), 2.29 (2H, t,
3JH-H ) 7.5 Hz, CH2CO2), 1.55-1.65 (4H, m, 2CH2), 1.10-1.39
(14H, m, 7CH2). HRMS (EI): m/z calcd 472.2977 (C32H40O3), found
472.2968 [M]+.
6-Trityloxyhexanoic Acid (16a). An aqueous solution of 5 M
NaOH (17.5 mL, 87.5 mmol) was added to a stirring solution of
15a (20.1 g, 51.6 mmol) in 130 mL of THF and 52.5 mL of water
at room temperature. After the mixture was stirred for 2 days, 3.1
mL of 5 M NaOH (15.5 mmol) was added, and stirring was
continued for another 2 days. The THF was removed under reduced
pressure. Then the aqueous residue was acidified with 1 M HCl to
pH 5 and extracted with diethyl ether. The combined organic layers
were washed with brine and dried over MgSO4. The diethyl ether
was removed under reduced pressure, and the resulting crude
powder was recrystallized in hexanes to yield 15.1 g (78%) of white
1
powder, 16a. Mp 114-116 °C. H NMR (400 MHz, CDCl3, δH
ppm): 7.43 (6H, m, p-Ar), 7.28 (6H, m, m-Ar), 7.22 (3H, m, o-Ar),
3
3
3.05 (2H, t, JH-H ) 6.4 Hz, HO-CH2), 2.33 (2H, t, JH-H ) 7.5
Hz, CH2CO2), 1.56-1.68 (4H, m, 2CH2), 1.36-1.47 (2H, m, CH2).
13C NMR (100 MHz, CDCl3, δH ppm): 180.26, 144.36, 128.62,
127.66, 126.79, 86.30, 63.24, 33.98, 29.63, 25.74, 24.48. HRMS
(EI): m/z calcd 374.1882 (C25H26O3), found 374.1883 [M]+.
9-Trityloxynonanoic Acid (16b). Aqueous 0.5 M NaOH (13.2
mL, 6.6 mmol) was added to a stirring solution of 15b (1.41 g, 3.3
mol) in 25 mL of THF. The reaction mixture was stirred for 3
days at room temperature, and then the solvent was removed by
rotary evaporation. The purification was carried out by flash column
chromatography (gradient 10% EtOAc in hexanes to 100% EtOAc)
Methyl 6-Hydroxyhexanoate (14a). This compound was pre-
pared from ε-caprolactone (10.01 g, 87.7 mmol) according to a
literature procedure.33 The colorless oil 14a was obtained with a
1
yield of 77%. H NMR (400 MHz, CDCl3, δH ppm): 3.63 (3H, s,
CO2CH3), 3.61 (2H, t, 3JH-H ) 6.5 Hz, HO-CH2), 2.30 (2H, t, 3JH-H
) 7.4 Hz, CH2CO2), 1.50-1.68 (4H, m, 2CH2), 1.31-1.44 (2H,
m, CH2).
Methyl 12-Hydroxydodecanoate (14c). Concentrated H2SO4
(0.2 mL) was added to a solution of oxacyclotridecan-2-one (850
mg, 4.3 mmol) in 20 mL of methanol and stirred for 1 day.
Methanol was removed under reduced pressure, and the aqueous
residue was extracted with diethyl ether 3 times. The combined
organic layers were washed with saturated NaHCO3, saturated NaCl
and then dried over MgSO4. The diethyl ether was removed under
1
to obtain 1.28 g (93%) of a pale-yellow oil, 16b. H NMR (400
MHz, CDCl3, δH ppm): 7.43 (6H, m, p-Ar), 7.28 (6H, m, m-Ar),
3
7.21 (3H, m, o-Ar), 3.03 (2H, t, JH-H ) 6.6 Hz, HO-CH2), 2.33
3
(2H, t, JH-H ) 7.5 Hz, CH2CO2), 1.56-1.66 (4H, m, 2CH2),

2202 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Rosita et al.
1.20-1.40(8H, m, 4CH2). 13C NMR (100 MHz, CDCl3, δH ppm):
180.26, 144.46, 128.63, 127.64, 126.74, 86.22, 63.55, 34.04, 29.95,
29.24, 29.13, 28.96, 26.15, 24.60. HRMS (EI): m/z calcd 416.2351
(C28H32O3), found 416.2360 [M]+.
anhydrous CH2Cl2 under argon flow. CpRe(CO)3 (1.68 g, 5.0 mmol)
was added, and mixture was refluxed for 1 day. Ice cold 5 M HClaq
was added to the reaction mixture, and the organic layer was
removed. The aqueous layer was extracted by CH2Cl2 three times,
the combined organic layers were dried over MgSO4, and the
solvent was removed under reduced pressure. After addition of
EtOAc, the insoluble byproducts were removed by filtration and
purification was performed by column chromatography (20%
EtOAc in hexanes, 50%, EtOAc in hexanes, then 1% HOAc in
EtOAc) to obtain 1.30 g (56%) of yellow solid, 19b. 1H NMR (400
MHz, CDCl3, δH ppm): 5.96-6.00 (2H, m, Cp), 5.34-5.41 (2H,
12-Trityloxydodecanoic Acid (16c). The synthesis and purifica-
tion procedures of 16b were followed. Aqueous 0.5 M NaOH (4.6
mL, 2.3 mmol), 15c (548 mg, 1.2 mmol), and THF (9.3 mL) were
used, and the reaction duration was shortened to 2 days. After
purification, 463 mg (87%) of 16c was obtained as a colorless oil.
1H NMR (400 MHz, CDCl3, δH ppm): 7.43 (6H, m, p-Ar), 7.28
3
(6H, m, m-Ar), 7.21 (3H, m, o-Ar), 3.02 (2H, t, JH-H ) 6.7 Hz,
HO-CH2), 2.34 (2H, t, 3JH-H ) 7.5 Hz, CH2CO2), 1.55-1.67 (4H,
m, 2CH2), 1.19-1.39 (14H, m, 7CH2). 13C NMR (100 MHz, CDCl3,
δH ppm): 180.02, 144.53, 128.69, 127.64, 126.75, 86.25, 63.66,
34.02, 30.04, 29.52, 29.50, 29.48, 29.39, 29.22, 29.05, 26.25, 24.66.
HRMS (EI): m/z calcd 458.2821 (C31H38O3), found 458.2814 [M]+.
Ghrelin(1-5)-6C-F (17). Selective Mtt amine deprotection was
performed to residue-3 of 12 according to the general procedure,
and the side chain precursor 16a was coupled following the general
procedure for coupling. Deprotection of the trityl containing side
chain was conducted according to the general procedure to obtain
free alcohol. This alcohol was then activated by an overnight
treatment with tosyl chloride (47.66 mg, 0.25 mmol) in 2 mL of
50% CH2Cl2/50% pyridine mixture under argon to provide tosylated
alcohol. Following this reaction, the resin was washed successively
with CH2Cl2 and THF. Fluorination was then conducted by shaking
the resin in 4 equiv of 0.1 M anhydrous TBAF in THF under argon
for 2.5 h, twice. Successive THF and CH2Cl2 washes were
performed after each fluorination step. Following this, final cleavage
and deprotection were conducted according to the general procedure
to obtain the crude peptide. Purification was performed by HPLC
(gradient 20-40% solvent B in A) to obtain 17 as a white powder
with an overall yield of 9% (3.4 mg). MS (ESI): m/z calcd 623.3,
found 646.3 [M + Na]+.
m, Cp), 2.59-2.66 (2H, t, JH-H ) 7.0 Hz, CH2CO), 2.36-2.43
3
3
(2H, t, JH-H ) 6.9 Hz, CH2CO), 1.63-1.79 (4H, m, 2CH2). 13C
NMR (100 MHz, CDCl3, δH ppm): 194.66 (CO), 191.72 (CO),
178.90 (CO2H), 95.86 (Cp-CO), 87.85 (Cp), 85.22 (Cp),
38.28(CH2CO), 33.58 (CH2CO), 23.99 (CH2), 23.52 (CH2). HRMS
(EI): m/z calcd 485.0139 ([M + Na]+, C14H13O6185ReNa); found
485.0144 [M + Na]+.
General Procedure for Rhenium Bearing Ghrelin(1-14)
Analogues (20a and 20b). Selective amine-Mtt deprotection was
conducted for residue-3 of 13 according to the general procedure,
and then the side chain precursors 19a and 19b were coupled using
the general coupling procedure. The peptides were cleaved off the
resin and deprotected according to the general procedure to obtain
crude peptides 20a and 20b.
Ghrelin(1-14)-4C-CpRe(CO)3 (20a). Purification was per-
formed by HPLC (gradient 25-50% of solvent B in A) to obtain
white powder 20a with an overall yield of 24% (55.6 mg). MS
(ESI): m/z calcd 2014.8, found 1008.4 [M + 2H]2+
.
Ghrelin(1-14)-6C-CpRe(CO)3 (20b). Purification was per-
formed by HPLC (gradient 20-40% of solvent B in A) to obtain
white powder 20b with an overall yield of 36% (43.3 mg). MS
(MALDI-TOF): m/z calcd 2042.8, found 2043.8 [M + H]+.
Radioligand Binding Assay. Determination of IC50 values of
ghrelin analogues 17, 18a-c, and 20a,b for GHSR was conducted
by radioligand binding assays according to published literature
procedures.23,24 Assays were performed using human recombinant
CHO-K1 cells as receptor source and 125I-ghrelin (human) as
radioligand. Reference standards using ghrelin (human) were run
to ensure the validity of the results. IC50 values were determined
by a nonlinear, least-squares regression analysis using MathIQ (ID
Business Solutions Ltd., U.K.). Exact IC50 value of ghrelin(1-5)-
6C-F 17 was not determined, as at a concentration of 2000 nM
only 20% inhibition was achieved; thus, these data are reported as
>2000 nM. For ghrelin(1-14) analogues 18a-c and 20a,b, IC50
values were determined semiquantitatively according to the %
inhibition at 10-5, 10-6, 10-7, and 10-8 M concentration (all data
duplicated). IC50 values are reported in Tables 2 and 3.
General Procedure for Fluorine Bearing Ghrelin(1-14)
Analogues (18a-c). These peptides were made from on-resin
peptide 13 following a procedure similar to the synthesis of 17.
Side chains 16a, 16b, and 16c were coupled to the peptide to obtain
18a, 18b, and 18c, respectively, after alcohol deprotection, activa-
tion, and fluorination steps. Mesylation was performed instead of
tosylation as follows: resin was shaken with 5 equiv of MsCl and
15 equiv of NEt3 in anhydrous CH2Cl2 under argon for 4 h, and
then after washing the process was repeated once more overnight.
Fluorination was conducted in a similar manner using up to 9 equiv
of 0.1 M anhydrous TBAF/THF, and the process was repeated up
to 6 times, as necessary for maximal fluorine incorporation.
Ghrelin(1-14)-6C-F (18a). Purification was performed by
HPLC (gradient 12-30% solvent B in A) to obtain 18a as a white
powder with an overall yield of 7% (7.4 mg). MS (ESI): m/z calcd
1712.9, found 857.5 [M + 2H]2+, 572.0 [M + 3H]3+ (100%).
Ghrelin(1-14)-9C-F (18b). Purification was performed by
HPLC (gradient 25-50% solvent B in A) to obtain 18b as a white
powder with an overall yield of 2% (2.2 mg). MS (ESI): m/z calcd
1754.9, found 1756.0 [M + H]+, 889.5 [M + Na + H]2+ (100%).
Ghrelin(1-14)-12C-F (18c). Purification was performed by
HPLC (gradient 25-40% solvent B in A) to obtain 18c as a white
powder with an overall yield of 12% (12.1 mg). MS (ESI): m/z
calcd 1797.0, found 899.5 [M + 2H]2+ (100%).
Acknowledgment. This work was supported by the London
Regional Cancer Program and the Natural Sciences and
Engineering Research Council of Canada (NSERC). Dina Rosita
gratefully acknowledges scholarship support from the Transla-
tional Breast Cancer Studentship from the London Regional
Cancer Program.
Supporting Information Available: 1H NMR spectra for
compounds 16a-c and 19a,b; MS spectra and HPLC chromato-
grams for compounds 17, 18a-c, and 20a,b. This material is
available free of charge via the Internet at http://pubs.acs.org.
4-(Cyclopentadienylrhenium Tricarbonyl)-4-oxobutanoic
Acid (19a). This compound was prepared from CpRe(CO)3 (1.00
g, 3.0 mmol) according to a literature procedure.34 The yellow solid
1
19a was obtained with a yield of 37% (0.49 g). H NMR (400
References
MHz, CDCl3, δH ppm): 6.00-6.04 (2H, m, Cp), 5.38-5.42 (2H,
m, Cp), 2.87-2.93 (2H, m, CH2), 2.72-2.78 (2H, m, CH2). 13C
NMR (100 MHz, CDCl3, δH ppm): 192.97 (CO), 191.62 (CO),
177.12 (CO2H), 95.15 (Cp-CO), 87.94 (Cp), 85.22 (Cp), 33.15
(CH2), 27.44 (CH2). HRMS (EI): m/z calcd 437.0029 ([M + H]+,
C12H10O6187Re), found 437.0022 [M + H]+.
6-(Cyclopentadienylrhenium Tricarbonyl)-6-oxohexanoic
Acid (19b). Anhydrous AlCl3 (2.67 g, 20.0 mmol) and succinic
anhydride (1.28 g, 10.0 mmol) were dissolved in 100 mL of
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