Thrombus imaging using technetium-99m-labeled high-potency GPIIb/IIIa receptor antagonists. Chemistry and initial biological studies

Article abstract of DOI:10.1021/jm950112e

Platelet-specific compounds which are radiolabeled with γ-emitting radionuclides may be particularly useful for the noninvasive in vivo detection of thrombi. The synthesis of peptides which are potent inhibitors of platelet aggregation and which contain a chelator for the radionuclide technetium-99m are described. The target compounds were designed such that stable, oxotechnetium(V) species could be prepared where the site of metal coordination was well defined. A strategy was employed where the pharmacophore -Arg-Gly-Asp-(RGD), or RGD mimetic, was constrained in a ring which was formed by the S-alkylation of a cysteine residue with an N-terminal chloroacetyl group. Binding affinities were enhanced by the replacement of arginine with the arginine mimetics S-(3-aminopropyl)cysteine and 4- amidinophenylalanine. Further enhancements could be obtained by the synthesis of oligomers which contained two or more rings containing receptor binding regions. The increase in binding affinity seen was more than that expected from a simple stoichiometric increase of pharmacophore. The most potent compounds described had IC₅₀s of approximately 0.03 μM for the inhibition of human platelet aggregation. Two of the more potent peptides (P280 and P748) were labeled with technetium-99m and assessed in a canine thrombosis model. The ^99mTc complexes of the peptides prepared in this work hold promise as thrombus imaging agents due to their high receptor binding affinity, ease of preparation, and expected rapid pharmacokinetics.

Full text of DOI:10.1021/jm950112e

1372
J . Med. Chem. 1996, 39, 1372-1382
Th r om bu s Im a gin g Usin g Tech n etiu m -99m -La beled High -P oten cy GP IIb/IIIa
Recep tor An ta gon ists. Ch em istr y a n d In itia l Biologica l Stu d ies
Daniel A. Pearson,* J ohn Lister-J ames, William J . McBride, David M. Wilson, Lawrence J . Martel,
Edgar R. Civitello, and Richard T. Dean
Chemistry Department, Diatide, Inc., 9 Delta Drive, Londonderry, New Hampshire 03053
Received February 13, 1995X
Platelet-specific compounds which are radiolabeled with γ-emitting radionuclides may be
particularly useful for the noninvasive in vivo detection of thrombi. The synthesis of peptides
which are potent inhibitors of platelet aggregation and which contain a chelator for the
radionuclide technetium-99m are described. The target compounds were designed such that
stable, oxotechnetium(V) species could be prepared where the site of metal coordination was
well defined. A strategy was employed where the pharmacophore -Arg-Gly-Asp-(RGD), or RGD
mimetic, was constrained in a ring which was formed by the S-alkylation of a cysteine residue
with an N-terminal chloroacetyl group. Binding affinities were enhanced by the replacement
of arginine with the arginine mimetics S-(3-aminopropyl)cysteine and 4-amidinophenylalanine.
Further enhancements could be obtained by the synthesis of oligomers which contained two
or more rings containing receptor binding regions. The increase in binding affinity seen was
more than that expected from a simple stoichiometric increase of pharmacophore. The most
potent compounds described had IC50s of approximately 0.03 µM for the inhibition of human
platelet aggregation. Two of the more potent peptides (P280 and P748) were labeled with
technetium-99m and assessed in a canine thrombosis model. The 99mTc complexes of the
peptides prepared in this work hold promise as thrombus imaging agents due to their high
receptor binding affinity, ease of preparation, and expected rapid pharmacokinetics.
In tr od u ction
the diagnosis of DVT of the lower extremities above the
knee, Duplex ultrasonography is considered highly
accurate8 and has become widely used over impedance
plethysmography.9 For diagnosis of DVT below the
knee, Duplex US is also used. However, approximately
40% of studies are technically inadequate, requiring
either a confirming contrast venogram or a repeat study
after 2-3 days.5 Although the contrast venogram is
considered the most accurate method for detecting DVT,
this uncomfortable procedure has significant associated
complications and is decreasingly used.
Anticoagulant therapy is available for the treatment
of thromboembolic disorders, but poses additional risks
such as hemorrhaging and thrombocytopenia. It has
been estimated that heparin is the most common cause
of drug-related death in reasonably healthy patients10
and that heparin-associated morbidity and mortality is
quite high (30 and 2%, respectively11). Therefore, it is
undesirable to unnecessarily anticoagulate.
Two major conclusions can be drawn from this review
of the literature: (i) that a rapid, noninvasive, cost-
effective, and accurate method for the diagnosis of PE
is highly desirable and (ii) that a rapid, noninvasive,
cost-effective, and accurate method for the diagnosis of
DVT in any part of the lower extremities is also highly
desirable, both to indicate treatment and to aid in the
diagnosis of PE.
Radionuclide imaging offers considerable hope for a
successful diagnostic agent which would address all of
the above criteria. 99mTc is the radionuclide of choice
due to the fact that it has a convenient 6 h physical half-
life, is relatively inexpensive, and is available 24 h a
day as a solution of [99mTc]pertechnetate from an in-
house 99Mo/99mTc generator. In addition, 99mTc yields
greater photon flux per unit of radiation dose delivered
Deep vein thrombosis (DVT) and pulmonary embo-
lism (PE) are thromboembolic disorders which consti-
tute a major health risk. In the United States, approxi-
mately 100 000 deaths per year result from the estimated
5 million patients who experience one or more episodes
of DVT and the 500 000 estimated cases of PE.1 It is
generally agreed that untreated PE is associated with
high mortality (18-35%) which can be reduced sub-
stantially (to 2-8%) with prompt treatment.2 But,
prompt treatment requires prompt diagnosis. Unfor-
tunately, the current state-of-the-art medicine does not
provide rapid, noninvasive, cost-effective, definitive
diagnosis of all cases of PE. Pulmonary angiography
is the most sensitive and specific method for the
diagnosis of PE.2a However, it is a technically demand-
ing procedure which, despite relatively low morbidity
and mortality (1.96 and 0.2%3), is often avoided. The
most common diagnostic procedure for PE is the V/Q
scan, which is a combination of a scintigraphic lung
perfusion scan and a scintigraphic lung ventilation scan
in which a mismatch in image defects is considered to
be diagnostic of PE.4 A recent study5 showed that V/Q
scans had high sensitivity but low specificity, and an
editorial which followed this report stated that “One of
the most difficult diagnoses to make in medicine today
is that of pulmonary embolic disease”.6
It has been estimated that approximately 70% of cases
of PE arise from DVT of the lower extremities.7 There-
fore, the accurate detection of DVT is important both
to allow prompt treatment to prevent formation of an
embolus and as an adjunct to the diagnosis of PE. For
X Abstract published in Advance ACS Abstracts, February 1, 1996.
0022-2623/96/1839-1372$12.00/0 © 1996 American Chemical Society

Thrombus Imaging
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1373
Sch em e 1. Preparation of GPIIb/IIIa-Binding Analogs
to the patient than other radionuclides used clinically.
Early work in thrombus imaging focused on the radio-
labeling of fibrinogen, fibrin, or platelets.12 However,
the success of these approaches was thwarted by the
slow blood clearance of the large macromolecules or
platelets, resulting in poor target to background ratios
over the course of several hours after injection. Fre-
quently, useful images could not be obtained until 24 h
post injection. Monoclonal antibodies have been raised
against fibrin and the Fab′ fragments labeled with
99mTc.13 Recently, encouraging results have been ob-
tained from a monoclonal antibody reactive with the DD
domain of cross-linked fibrin.14 However, even Fab′
fragments have been shown to leave the blood pool too
slowly to provide a reliably rapid diagnosis.15 In addi-
tion, it is apparent that the development of radiophar-
maceuticals from monoclonal antibodies faces a number
of technical and regulatory barriers which make it
unlikely that this approach will easily lead to a com-
mercial product.
Containing 99mTc Chelator
An alternate approach which has been investigated
recently involves the radiolabeling of smaller bioactive
peptides. Since the discovery of the platelet glycoprotein
IIb/IIIa complex (GPIIb/IIIa) as the final common
pathway for all agonists in platelet adhesion,16 there
has been much effort toward the development of small
molecule GPIIb/IIIa antagonists for the treatment of
thromboembolic disorders. Most of the structure-
activity work has revolved around the arginine-gly-
cine-aspartic acid (RGD) sequence, or mimetics of this
tripeptide sequence.17 New approaches aimed at iden-
tifying a commercially viable radiotracer to image both
PE and DVT have also involved radiolabeling of peptides
containing the RGD sequence. Promising results were
seen when bitistatin, an RGD-containing 9000 Da
polypeptide derived from a snake venom, was labeled
with the γ-emitting radionuclide 123I and tested in a dog
model of PE/DVT.18 However, although the IC50 of
bitistatin for inhibition of dog platelet aggregation is
0.028 µM, the IC50 for inhibition of human platelet
aggregation is 0.24 µM,19 indicating 10 times less
potency in binding to human platelets. Furthermore,
the biological origin of this compound, and the fact that
it contains 14 pairs of cysteine residues and so may be
difficult to label with 99mTc without loss of receptor
affinity, combine to make it unclear whether this
approach will lead to a commercial product. Also, some
smaller peptides (17-32 residues) have been synthe-
sized which contained one or more RGD sequences in
addition to containing a chelator for 99mTc.20 While
blood clearance of the 99mTc-labeled peptides was rapid,
with excretion through the kidneys, thrombus imaging
was poor. We believe this may have been a consequence
affinity for this receptor on activated human platelets
is high enough that sufficient thrombus uptake for
imaging purposes could be achieved, potentially without
interference from the bloodstream background. We also
designed these peptides to incorporate chelators for
99mTc at discrete locations such that the coordination
of this radionuclide would not interfere with receptor
binding. For this purpose, we have developed peptide
sequences which are effective technetium chelators.22
The details of the synthesis of these GPIIb/IIIa receptor-
binding peptides which contain sites for the chelation
of 99mTc and their activity in vitro and in vivo are
described herein.
Ch em istr y
All compounds were prepared by the routes outlined
in Schemes 1-3. For peptide synthesis, standard solid-
phase methodology23 was used in which the N-R-amino
groups were Fmoc-protected and the side chains (except
cysteines not involved in cyclization) were protected
with TFA-labile groups. It should be noted that in
several cases a branched lysine approach was taken in
which both the R- and ꢀ-amines were Fmoc-protected.
Synchronous removal of the Fmoc groups followed by
continuation of the synthesis resulted in identical
sequences attached to lysine at both the R- and ꢀ-amines.
At the end of the synthesis, the N-terminal D-tyrosine
was capped with either a bromo- or chloroacetyl moiety.
After cleavage and deprotection, the portion of the
peptide containing the XGD sequence was cyclized by
subjecting the peptide to basic conditions, thereby
affecting the intramolecular S-alkylation between the
free cysteine and the haloacetyl.24 Cysteines not in-
volved in this cyclization were S-protected with either
the acetamidomethyl (Acm) group or the p-methoxy-
benzyl (Mob) group. Acm protection was used on those
cysteines which could later comprise part of the 99mTc-
chelator donor set in Tc-labeling experiments. In those
cases where a cysteine was to be used to further
of their low affinity for the GPIIb/IIIa receptor (IC50
g
10 µM for the inhibition of human platelet aggrega-
tion).
We have directed our research to the preparation of
high-potency GPIIb/IIIa receptor-binding peptides la-
beled with 99mTc for evaluation as thrombus-imaging
agents. In addition to its role in thrombus formation,
this receptor is an attractive target for imaging blood
clots due to the fact that its behavior on the activated
platelets found in thrombi is different than that on
quiescent platelets circulating in the blood stream.21
This allows for the design of peptides in which their

1374 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Pearson et al.
Sch em e 2. Preparation of Multimers via S-Alkylation
Sch em e 3. Preparation of Multimers via S-Alkylation
of Maleimides
of Haloacetyls
elaborate the peptide via an S-alkylation, the 4-meth-
oxybenzyl (Mob) protecting group was used. In this
orthogonal protection scheme, the peptide was first
cyclized as described above, followed by removal of the
Mob group with boron trifluoride. The resulting free
cysteine was reacted with either maleimide-containing
compounds (Scheme 2) or compounds containing chlo-
roacetyl moieties (Scheme 3). In those cases where the
chloroacetyl groups resided at the end of a peptide
sequence (precursors to compounds 15 and 17-20), the
same solid-phase protocol described above was used to
prepare these intermediates. In addition to the chlo-
roacetyl groups, these peptides contained an S-trityl-
protected cysteine which remained protected through
the subsequent S-alkylation reaction. Removal of the
trityl group with TFA at the end of the synthesis
provided the final products. In addition to X ) arginine
in the XGD sequence, we also used the arginine mimet-
ics S-(3-aminopropyl)cysteine (Apc) and p-amidinophe-
nylalanine (Amp) at the X position. The syntheses of
these intermediates, suitably protected for peptide
synthesis, are shown in Schemes 4 and 5.
Oxorhenium(+5) complexes were prepared by react-
ing peptides 15 and 20 with Bu4NReOBr425 in DMF. The
resulting compounds were purified by reversed-phase
chromatography, characterized by electrospray mass
spectroscopy, and found to be different from the uncom-
plexed peptides by coinjection on analytical HPLC. The
technetium-99m complexes of compounds 10 (P280) and
20 (P748) were prepared by reacting these peptides with
99mTc-labeled glucoheptonate in a buffered solution. The
radiochemical purity of these complexes was measured
by analytical HPLC.
Sch em e 4.a Preparation of Fmoc-Apc(Boc)-OH
a
(i) Boc2O, DIEA, CH3CN; (ii) L-cysteine, NaOMe, MeOH; (iii)
FmocOSu, dioxane/water, pH ) 10.
Biology
The inhibition of ADP-induced platelet aggregation
by compounds 1-20 was measured in human platelet-
rich citrated plasma (PRP) using 10-20 µM ADP. The
progress and the extent of the platelet aggregation
reaction was monitored by measuring transparency of
the platelet suspension in an aggregometer. The results
are summarized in Table 1.
The 99mTc complexes of compounds 10 and 20, along
with 99mTc-labeled glucoheptonate as a negative control,
were assessed in a canine thrombosis model. The
radiotracers were administered to anesthetized dogs
which contained an induced thrombus in a femoral vein,
and the animals were sacrificed after 4 h. The thrombus-
containing vessels were carefully dissected out, weighed,
and, along with known fractions of the injected doses,
counted in a γ well counter. A tissue sample of a similar

Thrombus Imaging
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1375
Sch em e 5.a Preparation of Fmoc-Amp(STrt)-OH
TrtS
F igu r e 1. Peptidyl chelating systems.
compounds could not be reductively labile due to the
reducing conditions necessary to convert Tc(VII) to Tc-
(V) in the labeling process. Cyclic RGD-containing
peptides have already been described in the literature
in which the ring is formed via a thioether linkage.26
Recognizing that this approach to constraining the
pharmacophore in a nonreducible cyclic structure fit our
needs, we synthesized novel analogs modified to incor-
porate peptide sequences to serve as technetium chela-
tors. The use of the synthetic strategy described above
insured that the chelator could be incorporated at
known positions during the synthesis. Several different
chelating systems were utilized. These chelators in-
cluded (see Figure 1) bisamide bisthiols, such as are
provided by the novel chelating sequence -Cys-Gly-Cys-
(e.g. compound 1), and triamide thiols, such as are
provided by the chelating sequence -Lys-Gly-Cys- (e.g.
compound 7). Bisamide bisthiols and triamide thiols
have been shown to form anionic oxotechnetium com-
plexes.27 A novel diamide-amine-thiol chelator system
of the type -(ꢀ-Lys)-Gly-Cys- (e.g. compound 8) was also
prepared. In this system it is postulated that the
R-amine of lysine becomes part of the coordination
complex while the ꢀ-amine is used to form an amide
bond to the next amino acid in the sequence. This
system is similar to the triamide thiols above in that
the metal chelator contains three nitrogens and one
sulfur. However, unlike the triamide thiols, one of the
nitrogens is a less readily ionizable primary amine. We
expect metal coordination by this amine to be through
its lone pair electrons, thereby forming neutral oxotech-
netium(+5) complexes.
TrtS
TrtS
a
(i) EtONa, EtOH; (ii) 4-cyanobenzyl bromide; (iii) NaOH,
EtOH; (iv) HCl, ∆; (v) porcine kidney acylase; (vi) FmocOSu,
dioxane/aqueous Na2CO3; (vii) HCl, EtOH; (viii) NH3, NH4OAc,
EtOH; (ix) TrtSCl, DIEA, DMF; (x) 50% Et2NH/DMF; (xi) 1 M
LiOH(aq)/MeOH; (xii) FmocOSu, dioxane/aqueous Na2CO3.
Ta ble 1. IC50’s of Compounds 1-20 in Platelet Aggregation
Experiments
a
a
compd
IC50 (µM)
compd
12
13
14
15
15‚ReO
16
IC50 (µM)
1
2
3
4
5
6
7
8
1.4
1.3
0.3
0.04
0.14
0.07
0.11
0.09
0.03
0.11
0.03
0.05
0.03
0.04
0.16
0.17
0.14
0.08
0.11
0.08
0.09
0.04
17
18
19
9
10 (P280)
11
20 (P748)
20‚ReO
section of vein of the contralateral (control) legs and
samples of thigh muscle were also dissected out and
counted. Percent injected dose (%ID)/g in the thrombus,
muscle, and blood obtained just prior to euthanasia were
determined and thrombus-to-blood and thrombus-to-
muscle ratios were calculated from the %ID/g values.
Tissue data are presented in Table 2.
The binding affinities of these compounds to the
GPIIb/IIIa receptor were measured as a function of their
ability to inhibit platelet aggregation. Although an
indirect measure of receptor binding, this in vitro assay
employs activated platelets and we postulated it to be
a reasonable measure of the ability or our peptides to
Resu lts a n d Discu ssion
Our goal was to synthesize peptides containing a
technetium chelator which would also have a high
binding affinity for the GPIIb/IIIa receptor. An ad-
ditional consideration was the fact that the target
Ta ble 2. 4 h Femoral Vein Data in the Canine Thrombosis Model
compound
%ID/g thrombus
%ID/g blood
thrombus/blood
thrombus/muscle
[
[
[
99mTc]glucoheptonate
99mTc]-10 (P280)
99mTc]-20 (P748)
0.0026 ( 0.0002
0.0059 ( 0.0025
0.017 ( 0.0086
0.0015 ( 0.0007
0.0012 ( 0.0003
0.0025 ( 0.0006
2.2 ( 0.8
4.4 ( 0.7
7.2 ( 4.1
4.3 (2.4
11 ( 7
41 ( 24

1376 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Pearson et al.
F igu r e 2. Labeling of P748 with 99mTc. The bottom chromatogram (a) is after 15 min incubation with [99mTc]glucoheptonate. The
top chromatogram (b) is the oxorhenium complex of P748.
localize at regions of active thrombus formation. This
assay is also reliable for very potent GPIIb/IIIa antago-
nists whereas it has been reported26 that measurement
of the binding of RGD-containing peptides to the isolated
GPIIb/IIIa receptor in an ELISA assay shows some
discrepancies in the range in which we were most
interested (IC50 < 0.5 µM in platelet aggregation experi-
ments). As seen from the results (Table 1), receptor
binding affinity was not compromised by the incorpora-
tion of technetium-binding peptide sequences. To fur-
ther validate our approach, metal complexes of com-
pounds 15 and 20 were prepared. Because only
radioactive nuclides of technetium exist, rhenium, which
is very similar to technetium in its (+5) oxo coordination
chemistry,28 was used as a surrogate for the correspond-
ing technetium complex in the in vitro assay. As seen
in Table 1, the oxorhenium complexes retained the
receptor binding affinity of the parent peptide.
single species with a radiochemical purity of >90% by
reversed-phase HPLC analysis. Compound 20 (P748)
was labeled using the same ligand exchange technology
but at room temperature. An example of an early HPLC
trace of [99mTc]P748 is shown in Figure 2. An HPLC
comparison of the oxorhenium complex of compound
P748 (lower trace) showed it to elute close to the 99mTc-
labeled species.
In a canine thrombosis model, the 99mTc complex of
P280 showed thrombus-to-blood and thrombus-to-
muscle ratios of 4.4 and 11 (see Table 2). In comparison,
[
99mTc]glucoheptonate, studied as a negative control,
gave much lower uptake and lower thrombus-to-blood
and thrombus-to-muscle ratios (2.2 and 4.3, respec-
tively). The more potent GPIIb/IIIa antagonist, P748,
when administered as the 99mTc complex, showed much
higher uptake and higher thrombus-to-blood and throm-
bus-to-muscle ratios (7.2 and 41, respectively) compared
to P280. The ratios seen above for both P280 and P748
are well within the range of that expected for a suc-
cessful imaging agent.
In order to produce higher binding antagonists, we
used S-(3-aminopropyl)cysteine (Apc) in place of argi-
nine in the pharmacophore. This substitution gave a
5-fold increase (compound 3 vs compound 1) in binding
affinity so this substitution was retained in the majority
of the compounds which we prepared. We also prepared
dimers, trimers, and tetramers of the XGD-containing
pharmacophore in an effort to boost receptor binding
by increasing the local concentration of this tripeptide
sequence. Indeed, we found that peptides containing
dimeric Apc-Gly-Asp’s had more than the expected
2-fold increase in activity in platelet aggregation experi-
ments (e.g. compound 4 vs compound 12). This was also
shown to be the case by others in a recent study on a
different family of RGD-containing molecules.29 We also
found that the substitution of the p-amidinophenylala-
nine (Amp) for arginine30 in the RGD sequence in-
creased the binding affinity by another factor of 2
relative to compounds containing Apc (e.g. compound
19 vs compound 15).
Con clu sion s
Low molecular weight peptides were prepared which
possessed both a high affinity for the GPIIb/IIIa receptor
and a chelator for the radioisotope 99mTc. In experi-
ments inhibiting the aggregation of human platelets,
our best compounds had an IC50 of 0.03 µM, which is
comparable to the most potent fibrinogen antagonists
prepared to date. Furthermore, the technetium-99m
complexes of P280 and P748 studied above were found
to have good thrombus uptake in canine in vivo ex-
periments. The 99mTc complexes of the peptides pre-
pared in this work offer promise as useful thrombus
imaging agents due to their high receptor binding
affinity, ease in preparation, and expected rapid phar-
macokinetics.
Exp er im en ta l Section
Compound 10 (P280) was labeled with 99mTc within
15 min at 100 °C by ligand exchange using [99mTc]-
glucoheptonate. The resulting 99mTc complex was a
Symbols and abbreviations generally follow the IUPAC-
IUB recommendations (Int. J . Pept. Protein Res. 1984, 24,
9-37). Amino acids used were of the L-configuration unless

Thrombus Imaging
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1377
stated otherwise. All protected amino acids used (except
N-Fmoc-S-tritylhomocysteine and N-R-Boc-N-â-Fmoc-diami-
nopropionic acid) were purchased from either Midwest Bio-
Tech, Bachem, CA, Novabiochem, or Advanced ChemTech and
used as is. Bis-maleimidomethyl ether was purchased from
Cal Biochem. Bis(1,6-maleimido)hexane was purchased from
Pierce. Other chemicals and solvents were purchased from
either J . T. Baker, Advanced ChemTech, Fluka, or Aldrich and
used as is. Aldrich SureSeal solvents were used when
anhydrous conditions were necessary. When indicated, an
automated Applied Biosystems 431A peptide synthesizer was
used. For manual peptide synthesis, the reaction vessel used
was purchased from Safe-Lab, Santee, CA. Analytical HPLCs
were performed on a Waters system using a Delta-Pak C18
column (300 Å, 5 µm, 3.9 × 150 mm) at a flow rate of 1.2 mL/
min or on a Waters system using a radial compression Nova-
Pak C18 column (4 µm, 8 × 100 cm) at a flow rate of 3.0 mL/
min. Preparative HPLCs were performed on a Waters LC
4000 system using a 4 × 32.5 cm C18 Delta-Pak preparative
HPLC column. For normal-phase chromatographic separa-
tions Merck grade 9385 silica gel was used with a mesh of
230-400. Rf values were determined with silica gel TLC
plates (Kieselgel 60 F254, 0.25 mm layer thickness, Merck). 1H-
NMR spectra were obtained on a Varian Gemini 200 spec-
trometer at 200 MHz using TMS as an internal standard.
13C-NMR spectra were obtained on a Varian Gemini 200
spectrometer at 100 MHz using TMS as an internal standard.
Fast atom bombardment mass spectra (FABMS) were obtained
by M-Scan using a VG Analytical ZAB 2-SE or by Scripps
Research Institute using a VG ZAB VSE. Electrospray mass
spectra (ESMS) were obtained at Scripps Research Institute
using an API III Pe Sciex triple-quadrupole mass spectrom-
eter.
P la telet Aggr ega tion Assa y. A 27 mL sample of blood
was drawn into a 30 mL syringe, containing 3 mL of 3.8%
sodium citrate, from the antecubital vein of a donor using a
19 G butterfly. The blood was mixed gently in the syringe
and then placed in a 50 mL polypropylene centrifuge tube and
spun at 800 rpm (ca. 100g) for 10 min in an IEC Centra-8
centrifuge. The platelet rich plasma (PRP) was drawn off
using a pipette, the residual blood was recentrifuged at 3800
rpm (ca. 200g), and the supernatant platelet poor plasma (PPP)
was drawn off and placed in a 50 mL centrifuge tube. PRP
was stored at room temperature during the course of the assay.
Ex vivo platelet aggregation was determined by recording
the increase in light transmission through a stirred suspension
of PRP maintained at 37 °C. Peptide solution (or control) (15-
40 µL) was added to 450 µL of PRP, followed by the addition
of 25 µL of 0.2 mM ADP (in phosphate buffered saline) to give
a final solution volume of approximately 0.5 mL which was
10 µM in ADP. Platelet aggregation was recorded for 3 min.
Percent inhibition was calculated as follows: 100 × [maximum
percent aggregation (control) - observed percent aggregation
(inhibitor)] divided by [maximum percent aggregation (con-
trol)].
min) as one species for compound 10. HPLC analysis of the
99mTc complex of compound 20 showed a peak which eluted at
12.7 min. This compares with a retention time of 12.0 min
for the ReO complex of 20.
Ca n in e Th r om bosis Stu d y. The model used was that
reported by Knight.31 Mongrel dogs (25-57 lb, fasted over-
night) were sedated (ketamine and acepromazine, im) and then
anesthetized with sodium pentobarbital iv. In each animal,
an 18 gauge angiocath was inserted into the distal half of the
right femoral vein and an 8 mm Dacron-entwined stainless
steel embolisation coil (Cook Co., Bloomington, IN) was placed
in the femoral vein at approximately the mid-femur. The
catheter was removed, the wound was sutured, and the
placement of the coil was documented by X-ray. The animals
were allowed to recover overnight. On the day following coil
placement, each animal was re-anesthetized, iv saline drips
were placed in each foreleg, and a urinary bladder catheter
was inserted to collect urine. 99mTc-labeled 10 (0.2-0.4 mg at
5-10 mCi, 6 dogs) or 99mTc-labeled 20 (0.05-0.4 mg at 10-40
mCi, 7 dogs) was injected as a bolus into a foreleg iv line and
flushed in with saline. As a negative control, two dogs were
studied with [99mTc]glucoheptonate (8 mCi). After 4 h each
animal was deeply anesthetized with pentobarbital. Two blood
samples were collected by cardiac puncture using a heparin-
ized syringe followed by a euthanising dose of saturated
potassium chloride solution administered by bolus iv injection.
The femoral vein containing the thrombus, a similar section
of vein of the contralateral (control) leg, and samples of thigh
muscle were carefully dissected out. The thrombus, coil, and
coil fibers were dissected free of the vessel. The thrombus,
saline-washed vessel samples, coil, and coil Dacron fibers were
separated, weighed, and, along with known fractions of the
injected doses, counted in a γ well counter in the 99mTc channel.
Fresh thrombus weight, percent injected dose (%ID)/g in the
thrombus and blood obtained just prior to euthanasia, %ID in
the coil, excised blood vessels, muscle, and blood were deter-
mined. Thrombus-to-blood and thrombus-to-muscle ratios
were calculated from %ID/g values.
cyclo-[CH2CO-D-Tyr -Apc-Gly-Asp-Cys(S-)]-Lys-Gly-Cys-
(Acm )-Gly-Cys(Acm )-NH 2 (4). Fmoc-protected Rink resin
(0.25 mmol) was placed in the reaction vessel of an automated
peptide synthesizer. The standard FastMoc protocol32 was
used, and appropriately protected amino acids were added
sequentially until the desired sequence had been synthesized.
Briefly, the N-terminal Fmoc was removed with 20% piperi-
dine/DMF, and the resin or resin-supported peptide was
washed with NMP. Coupling was affected in NMP by reacting
4 equiv (1 mmol) of amino acid derivative with 4 equiv (1
mmol) of 1:1 HBTU/HOBt solution (0.45M in DMF) and 8
equiv (2 mmol) of DIEA. After adding the activated amino
acid to the reaction vessel, the resin was agitated for an
appropriate amount of time (usually 40 min). The resin was
then washed with additional NMP and any unreacted amine
capped with excess acetic anhydride. The order of addition of
protected amino acids for the synthesis of 4 was Fmoc-Cys-
(Acm)-OH, Fmoc-Gly-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH,
Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(tBu)-OH,
Fmoc-Gly-OH, Fmoc-Apc(Boc)-OH (22), and Fmoc-D-Tyr(tBu)-
OH. After the last amino acid in the sequence had been added,
the N-terminal Fmoc protecting group was once again removed
as indicated above and the resulting free amine reacted with
excess chloroacetic anhydride, thus placing a chloroacetyl
moiety at the N-terminus. After additional washes with NMP
and then methylene chloride, the resin was transferred to a
round bottom flask and 10 mL of trifluoroacetic acid/water/
triethylsilane (92.5:5:2.5) was added. The deprotection/cleav-
age mixture was stirred at room temperature for 1.5 h and
filtered through a sintered glass funnel. The resin was washed
with TFA (2 mL) which was added to the filtrate. Cold ethyl
ether (200 mL) was added to precipitate out the chloroacetyl
intermediate. The solid was filtered and washed with ethyl
ether to yield 123 mg of crude peptide. This peptide was taken
up in 1.2 L of argon-degassed water, and 1 M ammonium
hydroxide was added until a pH of 8.0 was achieved. The
reaction mixture was stirred for 20 h and concentrated in
vacuo to a volume of about 100 mL. The solution was loaded
Ra d iola belin g of P 280 a n d P 748 w ith Tech n etiu m -
99m . Peptide (0.1 mg) was dissolved in 0.1 mL of 0.9% saline.
[
99mTc]Glucoheptonate was prepared by reconstituting a Glu-
coscan vial (E. I. Dupont de Nemours, Inc; this kit contains
stannous chloride as a reducing agent and glucoheptonate as
a transfer ligand) with 1.0 mL of [99mTc]sodium pertechnetate
(from a 99Mo/99mTc generator) containing up to 200 mCi. The
vial was allowed to stand at room temperature for 15 min. A
25 µL aliquot of [99mTc]gluceptate from this vial was then
added to the peptide solution above and the reaction allowed
to proceed at room temperature for 15-30 min (for compound
20) or at 100 °C for 15 min (for compound 10). After filtration
through a 0.2 µm filter, the purity of the 99mTc-labeled peptide
was assessed by analytical reversed-phase HPLC (10-40% B/A
over 20 min using a Waters Delta-Pak C18 column, 5 µm, 39
× 150 mm; A ) 0.1% TFA in water, B ) 0.1% TFA in 90%
acetonitrile/water). Radioactive components were detected
using an in-line radiometric detector linked to an integrating
recorder. [99mTc]Gluceptate and [99mTc]sodium pertechnetate
elute between 1 and 4 min under these conditions, whereas
the 99mTc-labeled peptides eluted later (retention time ) 11.65

1378 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Pearson et al.
onto a preparative reversed-phase HPLC column at a flow rate
of 20 mL/min. The peptide was eluted as follows at 75 mL/
min (solvent A ) 0.1% TFA in water, solvent B ) 0.1% TFA
in 90% acetonitrile water): 0-5 min, 0% B/A; 5-10 min,
0-20% B/A; 10-30 min, 20-50% B/A; 30-34 min, 50% B/A.
Fractions were collected while monitoring at 230 nm and
analyzed by analytical HPLC (Delta-Pak, 0-100% B/A over
20 min, 4 had a retention time of 9.28 min). Fractions
containing pure product were combined, and the acetonitrile
was removed in vacuo on the rotovap. Lyophilization yielded
4 (100 mg) as the trifluoroacetate salt. FABMS indicated a
molecular ion peak (MH+) at 1247 which corresponds to the
molecular formula of C48H75N15O16S4 with a mass of 1246.
Compounds 1-3 and 5-9 were produced by the same protocol
outlined above and were confirmed by FAB mass spectral
analysis unless otherwise noted: compound 1 (C46H69N17O17S3)
in 5 mL of acetonitrile and 7 mL of water. Then, 38 mL of 50
mM sodium phosphate buffer (pH ) 7.0) containing 0.5 mM
EDTA was added and the solution degassed with argon.
Bismaleimidomethyl ether (11.8 mg, 0.045 mmol) in 150 µL
of DMF was added, and the reaction mixture was stirred at
room temperature under an atmosphere of argon. When
HPLC analysis indicated that the reaction was complete
(retention time shift from 8.83 min to 12.09 min in 10-40%
B/A, 20 min) the reaction mixture was loaded onto a prepara-
tive reversed-phase HPLC column at a flow rate of 20 mL/
min. The peptide was eluted as follows at 75 mL/min (solvent
A ) 0.1% TFA in water, solvent B ) 0.1% TFA in 90%
acetonitrile water): 0-5 min, 0% B/A; 5-25 min, 10-40% B/A;
25-35 min, 50% B/A. Fractions were collected while monitor-
ing at 230 nm and analyzed by analytical HPLC (Delta-Pak,
10-40% B/A over 20 min, the product had a retention time of
12.09 min). Fractions containing pure product were combined,
and the acetonitrile was removed on the rotovap. Lyophiliza-
tion yielded 169 mg (62% yield) of product. ESMS indicated
a molecular ion peak at 3021 which corresponds to the
molecular formula of C112H162N36O43S10 with an average mass
of 3021.3.
Compounds 11-13 were produced by the same protocol
outlined above by reacting the appropriate free cysteine-
containing peptides (see Scheme 2) with the following male-
imide electrophiles. For compound 11 tris(2-maleimidoethyl)-
amine (preparation below) was used. For compound 12
bismaleimidomethyl ether was used. For compound 13 bis-
(1,6-maleimido)hexane was used. Final products were con-
firmed by FAB mass spectral analysis: compound 11
(C171H249N55O63S15) expected 4461, found 4586 (M + Na+);
compound 12 (C120H180N38O43S10) expected 3162, found 3163
(M + H+); compound 13 (C116H170N36O42S10) expected 3060,
found 3062 (M + H+).
(cyclo-[CH 2CO-D-Tyr -Ap c-Gly-Asp -Cys(S-)]-Gly-Gly-
Cys(Acm )-Gly-Cys(Acm )-Gly-Gly-Cys-[S-CH2CO-]-NH2)2-
Lys-(E-Lys)-Gly-Cys-NH2 (15). (ClCH2CO)2Lys-(ꢀ-Lys)-Gly-
Cys(Trt)-NH2 was synthesized on a 0.25 mmol scale by the
same solid-phase peptide synthesis protocol used above in the
synthesis of 4. The order of addition for the synthesis of this
intermediate was Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, N-R-Boc-
N-ꢀ-Fmoc-Lys-OH, and N-R-Fmoc-N-ꢀ-Fmoc-Lys-OH. After
the last amino acid in the sequence had been added, the
N-terminal Fmoc protecting groups were removed as indicated
above and the resulting free amines reacted with excess
chloroacetic anhydride, thus placing a chloroacetyl moiety at
the R- and ꢀ-amines of the N-terminal lysine. The resin-
supported peptide was treated with 10 mL of TFA/water (95:
5) for 1 h. The resin was filtered and washed with 5 mL of
CHCl3. The filtrates were combined, and the volatiles were
removed in vacuo on the rotovap. The crude product was
taken up in 20 mL of CHCl3, and this was also removed in
vacuo. This chloroform treatment was repeated several times
until the color of the crude peptide in solution was no longer
red. This color change indicates removal of trace amounts of
TFA and concomitant reattachment of the trityl protecting
group to the cysteine sulfhydryl. This material was used
directly in the next reaction. Therefore 13.9 mg (16.7 µmol)
of this intermediate and 47 mg (33.5 µmol) of cyclo-[CH2CO-
D-Tyr-Apc-Gly-Asp-Cys(S-)]-Gly-Gly-Cys(Acm)-Gly-Cys(Acm)-
Gly-Gly-Cys-NH2 were dissolved in 1 mL of acetonitrile and 3
mL of 0.1 M Na2CO3 buffer (pH ) 10.0) containing 0.5 mM
EDTA. The reaction mixture was stirred for 4 h under an
atmosphere of argon followed by removal of the volatiles on
the rotovap. The crude product was treated with 10 mL of
TFA/water/triethylsilane (10:0.5:0.2) for 1.5 h. Ethyl ether
(200 mL) was added to precipitate the product which was
filtered and washed with ethyl ether. Crude product was then
dissolved in 5 mL of acetonitrile/water (1:1), and the resulting
solution was diluted with 10 mL of 0.1% TFA/water. The
solution was loaded onto a preparative reversed-phase HPLC
column at a flow rate of 20 mL/min. The peptide was eluted
as follows at 75 mL/min (solvent A ) 0.1% TFA in water,
solvent B ) 0.1% TFA in 90% acetonitrile water): 0-5 min,
0% B/A; 5-25 min, 10-30% B/A; 25-35 min, 50% B/A.
Fractions were collected while monitoring at 220 nm and
expected 1229, found 1229 (M
+ 2
H+); compound
(C44H66N16O16S3) expected 1172, found 1171 (M + H+); com-
pound 3 (C46H69N15O17S4) expected 1233, found 1232 (M + H+);
compound 5 (C67H101N19O21S5) expected 1670, found 1669 (M
+ H+); compound 6 (C37H57N11O12S3) expected 945, found 945
(M + H+); compound 7 (C43H58N13O13S2) expected 1030, found
1030 (M + H+); compound 8 (C147H216N42O49S9) ESMS, ex-
pected 3644.1, found 3643.5 (M+); compound 9 (C92H144N28O29S5)
expected 2268, found 2267 (M + H+).
cyclo-[CH2CO-D-Tyr -Apc-Gly-Asp-Cys(S-)]-Gly-Gly-Cys-
(Acm )-Gly-Cys(Acm )-Gly-Gly-Cys-NH2. The title peptide
was synthesized on a 0.25 mmol scale using the same solid-
phase peptide synthesis protocol as that used above in the
preparation of compound 4. The order of addition of protected
amino acids was Fmoc-Cys(Mob)-OH, Fmoc-Gly-OH, Fmoc-
Gly-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Cys(Acm)-
OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, Fmoc-
Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Apc(Boc)-OH (24), and
Fmoc-D-Tyr(tBu)-OH. After the last amino acid in the se-
quence had been added, the N-terminal Fmoc protecting group
was once again removed as indicated above and the resulting
free amine reacted with excess chloroacetic anhydride, thus
placing a chloroacetyl moiety at the N-terminus. After ad-
ditional washes with NMP and then methylene chloride, the
resin was transferred to a round bottom flask and 10 mL of
trifluoroacetic acid/water/triethylsilane (92.5:5:2.5) was added.
The deprotection/cleavage mixture was stirred at room tem-
perature for 1.5 h and filtered through a sintered glass funnel.
The resin was washed with TFA (2 mL) which was added to
the filtrate. Cold ethyl ether (200 mL) was added to precipitate
out the chloroacetyl intermediate. The solid was filtered and
washed with ethyl ether to produce 190 mg of crude peptide.
Then 100 mg of this peptide was taken up in 1 L of argon-
degassed water, and 1 M ammonium hydroxide was added
until a pH of 8.0 was achieved. The reaction mixture was
stirred for 20 h followed by concentrating it in vacuo to a
volume of about 200 mL. Lyophilization yielded 100 mg of
crude Mob-protected material. This material was treated with
10 mL of TFA/m-cresol/BF3‚OEt2 (8:1:1) for 1 h to remove the
Mob protecting group. Cold ethyl ether (200 mL) was added
to precipitate the product, and the solid was filtered, washed
with ether, and dissolved in acetonitrile/water (1:1). The
solution was loaded onto a preparative reversed-phase HPLC
column at a flow rate of 20 mL/min. The peptide was eluted
as follows at 75 mL/min (solvent A ) 0.1% TFA in water,
solvent B ) 0.1% TFA in 90% acetonitrile water): 0-5 min,
0% B/A; 5-25 min, 10-40% B/A; 25-35 min, 50% B/A.
Fractions were collected while monitoring at 230 nm and
analyzed by analytical HPLC (Delta-Pak, 10-40% B/A over
20 min, the product had a retention time of 8.83 min).
Fractions containing pure product were combined, and the
acetonitrile was removed on the rotovap. Lyophilization
yielded 41.5 mg of product. FABMS indicated a molecular ion
peak at 1392 which corresponds to the molecular formula of
C51H77N17O19S5 with a monoisotopic mass of 1391.4.
(cyclo-[CH 2CO-D-Tyr -Ap c-Gly-Asp -Cys(S-)]-Gly-Gly-
Cys(Acm )-Gly-Cys(Acm )-Gly-Gly-Cys[S-Ma leim id o-CH2-
]-NH2)2O (10) [P 280]. A 126 mg (0.09 mmol) portion of the
cyclo-[CH2CO-D-Tyr-Apc-Gly-Asp-Cys(S-)]-Gly-Gly-Cys(Acm)-
Gly-Cys(Acm)-Gly-Gly-Cys-NH2 produced above was dissolved

Thrombus Imaging
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1379
analyzed by analytical HPLC (Delta-Pak, 10-40% B/A over
20 min, the product had a retention time of 11.41 min).
Fractions containing pure product were combined, and the
acetonitrile was removed on the rotovap. Lyophilization
yielded 32 mg of product. ESMS indicated a molecular ion
peak at 3298 which corresponds to the molecular formula of
C123H187N41O44S11 with an average mass of 3296.7.
mol) of cysteine and 100 mL of anhydrous methanol. The
atmosphere of the reaction was flushed with argon followed
by the addition of 65 mL (0.35 mol) of a 5.4 M solution of
sodium methoxide in methanol. After stirring for 10 min,
39.27 g (0.165 mol) of 21 was added. The reaction mixture
was stirred under an atmosphere of argon for 5 h. TLC
analysis (AcOH/MeOH/CH2Cl2, 5:20:75) indicated that the
reaction was complete. NaHCO3 (1.9 g) was added followed
by the dropwise addition of acetic acid until a pH of 11 was
achieved. Then 55.6 g (0.165 mol) of FmocOSu was dissolved
in 200 mL of anhydrous THF, and this solution was added to
the reaction mixture. Stirring was continued under argon for
an additional 16 h. The volatiles were then removed in vacuo,
and the resulting slurry was taken up in 500 mL of ethyl
acetate. The organics were washed with 1 M H3PO4 (250 mL),
water (250 mL), and saturated NaCl (250 mL). After drying
over MgSO4, the organics were filtered and concentrated in
vacuo on the rotovap to yield an oil. The product was
crystallized from EtOAc/hexanes to yield 63.9 g (77% yield) of
pure product with a melting point of 116 -18 °C: 1H NMR
(CD3OD) δ 1.40 (9H, s), 1.70 (2H, quint, J ) 7 Hz), 2.56 (2H,
t, J ) 7 Hz), 2.7-3.2 (4H, m), 4.15-4.4 (4H, m), 7.2-7.8 (9H,
m); 13C NMR (CD3OD) δ 29.1, 30.8, 31.1, 35.6, 40.7, 48.7, 56.4,
68.4, 80.3, 121.1, 126.6, 128.5, 129.1, 142.8, 145.5, 158.7, 176.3;
FABMS (C26H32N2O6S) expected 501.6, found 501.6 (M + H+);
Compounds 14 and 16-20 were produced by the same
protocol outlined above by reacting the appropriate free
cysteine-containing peptides (see Scheme 2) with the following
bischloroacetyl electrophiles. For compound 14 2,2′-(ethyl-
enedioxy)bis(chloroacetamidoethane) [(ClCH2CONHCH2CH2-
OCH2)2] was used. For compound 16 tris(2-chloroacetamido-
ethyl)amine [(ClCH2CONHCH2CH2)3N] was used. For com-
pounds 17-20 (ClCH2CO)2Lys-(ꢀ-Lys)-Gly-Cys(Trt)-NH2 was
used. Final products were confirmed by electrospray mass
spectral analysis: compound 14 (C112H170N36O42S10) expected
3013, found 3013 (M+); compound 16 (C165H249N55O60S15
)
expected 4444, found 4443 (M+); compound 17 (C91H137N29O32S7)
expected 2374, found 2373 (M+); compound 18
(C243H370N80O87S21) expected 6477, found 6478 (M+); compound
19 (C131H187N43O44S9) expected 3357, found 3357 (M+); com-
pound 20 (C107H151N33O32S5) expected 2572, found 2573 (M+).
(cyclo-[CH 2CO-D-Tyr -Ap c-Gly-Asp -Cys(S-)]-Gly-Gly-
Cys(Acm )-Gly-Cys(Acm )-Gly-Gly-Cys[S-CH2CO-]-NH2)2Lys-
(E-Lys)-Gly-Cys-NH2‚Oxor h en iu m Com plex (15‚ReO). Com-
pound 15 (20 mg, 6 µmol) and 5.6 mg (7.2 µmol) of Bu4NReOBr4
were taken up in 1 mL of DMF, and the reaction mixture was
stirred at room temperature under argon for 16 h. The
solution was diluted with 1 mL of water and loaded directly
onto a preparative reversed-phase HPLC column at a flow rate
of 2 mL/min. The peptide was eluted as follows at 75 mL/min
(solvent A ) 0.1% TFA in water, solvent B ) 0.1% TFA in
90% acetonitrile water): 0-5 min, 0% B/A; 5-25 min, 10-
30% B/A; 25-35 min, 50% B/A. Fractions were collected while
monitoring at 220 nm and analyzed by analytical HPLC
(Delta-Pak, 10-40% B/A over 20 min, the product had a
retention time of 11.20 min). Fractions containing pure
product were combined, and the acetonitrile was removed on
the rotovap. Lyophilization yielded 9.0 mg of product. ESMS
[R]22 ) -26.3 ( 0.7 (c ) 1.0, DMF).
D
N-Acet yl-r-ca r b oet h oxy-4-cya n o-D,L-p h en yla la n in e,
Eth yl Ester (23). A 5-L 3-neck round bottom flask was
equipped with a mechanical stirrer and a reflux condenser.
The apparatus was flame-dried under a stream of argon. After
cooling, the flask was charged with 1400 mL of absolute
ethanol. Sodium metal (46.0 g 2.00 mol) was weighed out in
hexane and cut up into cubic portions. The sodium was then
added to the ethanol portionwise over 1 h under a blanket of
argon. After all of the sodium was added, the solution was
stirred until sodium ethoxide formation was complete. The
reflux condenser was removed and the flask fitted with a
thermometer. The ethoxide solution was then cooled to 0 °C
(ice bath), and diethyl acetamidomalonate (434 g, 2.00 mol)
was added portionwise under an atmosphere of argon, keeping
the temperature between 0 and 5 °C. After all the malonate
was added, the reaction mixture was stirred an additional 45
min. The resulting white slurry was kept at 0-5 °C, and
R-bromo-p-toluidine (392 g, 2.00 mol) was added. The ice bath
was removed, and a heating mantle was fitted on. The
reaction mixture was brought to a gentle reflux and held there
for 2.5 h. At this time the progress of the reaction was checked
by TLC (50% EtOAc/hexane), and it was found to be complete.
The reaction mixture was cooled, and 1.4 L of water was added.
Stirring was momentarily discontinued and the solid broken
up with a large spatula. Stirring was initiated again and
continued until a mostly fine slurry resulted (about 1 h). The
solid was filtered, and the any chunks that were remaining
in the reaction flask were removed and crushed with a mortar
and pestle. The resulting slurry was suspended in water and
filtered with the rest of the crude product. This crude product
was then washed with water (2 × 400 mL) and left standing
overnight in the filter funnel to dry. The crude product was
divided approximately in half and each half dissolved in about
1300 mL of hot ethanol. After slow cooling overnight the
needles were filtered, and any residual solvent was removed
in vacuo at 50 °C. The product (605.7 g, 91% yield) was
produced as white needles with a melting point of 170-72 °C:
1H NMR (CDCl3) δ 1.30 (6H, t, J ) 7 Hz), 2.04 (3H, s), 3.72
(2H, s), 4.28 (4H, q, J ) 7 Hz), 6.54 (1H, s), 7.12 (2H, d,
J ) 8 Hz), 7.56 (2H, d, J ) 8 Hz); 13C NMR (CDCl3) δ 13.9,
22.9, 37.9, 62.9, 66.9, 111.4, 118.5, 130.6, 132.0, 141.1, 167.1,
169.2.
indicated a molecular ion peak at 1166.9 [(M + 3H+)3+
,
deconvoluting to 3497.7] which corresponds to a molecular
formula of C123H184N41O45S11Re with an average mass of
3495.9. The oxorhenium complex of compound 20 was pro-
duced by the same protocol outlined above. ESMS analysis
of this compound indicated a molecular ion peak (M) of 2772
which corresponds to a molecular formula of C107H150N33O33S5-
Re1with an average mass of 2773.1. By analytical HPLC
(Delta-Pak, 10-40% B/A over 20 min) the oxorhenium complex
of 20 had a retention time of 11.65 min as compared to the
free peptide which had a retention time of 10.97 min.
N-Boc-3-a m in o-1-br om op r op a n e (21). 3-Aminopropyl
bromide hydrobromide (50.0 g, 0.228 mol) was weighed out in
a 500 mL round bottom flask, and the solid was dissolved in
200 mL of anhydrous acetonitrile. The solution was cooled to
0 °C with an ice bath, and 39.7 mL (0.228 mol) of diisopropy-
lethylamine was added with stirring. This was followed by
the addition of 49.7 g (0.228 mol) of di-tert-butyl dicarbonate.
The ice bath was removed, and the reaction mixture was
stirred for 3 h. At this time TLC (10% EtOAc/hexane)
indicated that the reaction was complete. The volatiles were
removed in vacuo on the rotovap to produce an oil, and 500
mL of diethyl ether was added. The salts were filtered off and
washed with an additional 250 mL of ether. The combined
organics were washed with 1 M NaH2PO4 (250 mL), 5%
NaHCO3 (250 mL), and saturated NaCl (250 mL). The
organics were dried over MgSO4 and filtered and the volatiles
removed in vacuo on the rotovap to yield an oil. Hexanes were
added to the crude product, and the solution was left standing
at -20 °C for 20 h. The resulting solid was filtered and
washed with hexanes. Trace solvents were removed under
high vacuum to yield 41.55 g (77% yield) of product: 1H NMR
(CDCl3) δ 1.45 (9H, s), 2.06 (2H, quint, J ) 7 Hz), 3.28 (2H,
quart, J ) 7 Hz), 3.45 (2H, t, J ) 7 Hz), 4.73 (1H, br s).
N-r-F m oc-S-(N-Boc-3-a m in op r op yl)-L-cystein e (22). A
500 mL round bottom flask was charged with 19.97 g (0.165
N-Acetyl-4-cya n o-D,L-p h en yla la n in e (24). Sodium hy-
droxide (146 g, 3.64 mole) was taken up in 950 mL of absolute
ethanol in a 5-L three-neck round bottom flask equipped with
a mechanical stirrer. The mixture was stirred until all of the
hydroxide went into solution, followed by the addition of 605.4
g (1.82 mol) of 23 as a solid. The reaction mixture was then
refluxed for 18 h with stirring. After this time, stirring was
discontinued and the reaction mixture was cooled in an ice

1380 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Pearson et al.
bath. A 400 mL sample of 6 M HCl was added slowly while
the precipitate was broken up with a large spatula. After all
of the HCl was added (pH ) 3) the precipitate was sufficiently
broken up to continue mechanical stirring again. Concen-
trated HCl was added in 25 mL portions until a pH of 2 was
reached (four portions). The reaction mixture was heated and
800 mL of ethanol was distilled off. The solution was left to
cool overnight, seed crystals were added in the morning, and
the solution was cooled in an ice bath. After crystallization
was well underway the ice bath was removed and the solution
was allowed to stand at room temperature for 3 h. The
resulting white solid was filtered off and washed with 1 L of
water. Drying in vacuo at 50 °C yielded 546.4 g of a white
solid. A second crop of 50.0 g was collected from the filtrate.
The two crops were combined and divided into approximately
two equal portions. Each was dissolved in about 1200 mL of
hot ethyl acetate with stirring. Hot hexane was added (about
200 mL) and the solution set aside to cool. A first crop of 170.5
g of white needles were collected by filtration (mp 174-76 °C).
After concentration of the filtrate and cooling, a second crop
was taken of 31.6 g (mp 173-76 °C) for a total of 202.1 g of
product (48% yield): 1H NMR (acetone-d5) δ 1.91 (3H, s), 3.11
(1H, q, J ) 15, 9 Hz), 3.32 (1H, q, J ) 15, 5.5 Hz), 4.80 (1H,
m), 7.42 (1H, d, J ) 8 Hz), 7.51 (2H, d, J ) 8 Hz), 7.73 (2H, d,
J ) 8 Hz); 13C NMR (acetone-d5) δ 22.3, 38.0, 53.5, 111.1, 119.1,
131.0, 132.6, 144.1, 170.0, 172.3.
4-Cya n o-L-p h en yla la n in e (25). In a 5-L three-neck flask
equipped with a mechanical stirrer was suspended 196.6 g
(0.846 mol) of 24 in 2 L of distilled water at 40 °C(water bath).
As the solution was being deoxygenated by sparging with
argon gas the pH was adjusted with 1 M LiOH to a pH of 7.2-
7.5. As the hydroxide was added, the suspension gradually
went into solution. Next, 500 mg of porcine kidney acylase
was added and the reaction mixture was stirred for 16 h. After
this time the reaction was monitored by HPLC (Delta-Pak
column, 0-100% B/A over 20 min, 100% B 20-25 min; solvent
A ) 0.1% TFA in water, solvent B ) 0.1% TFA in 90%
acetonitrile/10% water) and found to be 50% complete (the
retention time of starting material was 5.55 min, and the
retention time of product was 4.64 min). The pH was adjusted
from 6.5 to 7.3 with 1 M LiOH, and another 200 mg of enzyme
was added. After another 24 h the progress was again checked
by HPLC. Since it appeared as if the progress had slowed
down considerably, 714 mg of cobolt chloride hexahydrate was
added to make the reaction mixture 1 mM in Co2+. The pH
was adjusted to 7.5, and another 300 mg of enzyme was added.
The reaction was allowed to proceed as before for an additional
16 h. After this time HPLC analysis indicated that the ratio
of H-L-Phe(CN)OH to Ac-Phe(CN)OH was 46:54. The pH was
adjusted to 5.0 with concentrated HCl, and the reaction
mixture was heated to 60 °C. About 6 g of activated carbon
was added, and the reaction mixture was stirred at 60 °C for
10 min. It was then filtered through a pad of Celite while
still warm, and the pH of the filtrate was adjusted to 1.5 with
concentrated HCl. The aqueous solution was split into two
1800 mL portions, and each was extracted with EtOAc (6 ×
300 mL). The aqueous fractions (HPLC analysis indicated that
there was still 2.6% N-acetyl compound) were then recombined
and applied to a column of Dowex 50 × 8 (1 kg of Dowex). The
column was washed with water until pH ) 4 was reached,
and then the desired amino acid was eluted with 1 M aqueous
ammonia. Fractions containing product were concentrated in
vacuo to yield the desired product as a white solid (56.9 g,
70.7% yield based on theoretical amount of N-Ac-L-isomer in
racemic mixture) with a melting point of 242 °C (sublimed):
1H NMR (D2O, dioxane ref ) δ 3.63) δ 3.08 (1H, q, J ) 15, 9
Hz), 3.21 (1H, q, J ) 15, 5.5 Hz), 3.89 (1H, m), 7.33 (2H, d, J
) 8 Hz), 7.65 (2H, d, J ) 8 Hz); 13C NMR (D2O, dioxane ref )
δ 67.8) δ 37.7, 56.8, 111.4, 120.9, 131.4, 134.2, 142.6, 174.4.
N-F m oc-4-cya n o-L-p h en yla la n in e (26). Sodium carbon-
ate (27.9 g, 0.263 mol) was dissolved in 260 mL of water, and
25 (25.0 g, 0.131 mol) was added with stirring. After all solids
went into solution, the reaction mixture was cooled with an
ice bath, and FmocOSu (42.1 g, 0.125 mol) in 520 mL of
anhydrous dioxane was added dropwise over 2 h. After
addition was complete the ice bath was removed and the
reaction mixture was allowed to come to room temperature
over 1.5 h. TLC analysis (BuOH/HOAc/water, 4:1:1) indicated
that the reaction was complete so the reaction mixture was
transferred to a 4 L Ehrlenmeyer equipped with mechanical
stirring and cooled once again in an ice bath. About 1 L of
ethyl acetate was added, and 1 M HCl was added until the
pH of the aqueous layer was 1.5. The ethyl acetate separated
off in a separatory funnel, and the aqueous layer was washed
with 600 mL of additional EtOAc. The combined organics were
washed with water (500 mL) and saturated NaCl (500 mL).
After drying over sodium sulfate, the organics were decanted
and the volatiles removed in vacuo on the rotovap to yield a
clear oil. This was taken up in about 300 mL of ethyl acetate
and heated to reflux with stirring. Hot hexane (about 300 mL)
was added until the solution was cloudy. Cooling yielded the
product as white crystals (49.15 g, 89% yield, mp 191-92 °C):
1H NMR (acetone-d6) δ 3.18 (1H, q, J ) 17, 9.5 Hz), 3.40 (1H,
q, J ) 17, 4.5 Hz), 4.28 (3H, m), 4.60 (1H, m), 6.84 (1H, d, J )
8.5 Hz), 7.3-7.9 (12H, m); 13C NMR (acetone-d6) δ 37.8, 47.6,
55.3, 66.8, 111.0, 119.1, 120.5, 125.7, 127.6, 128.2, 131.0, 132.6,
141.8, 144.1, 144.6, 156.5, 172.5.
N-r-F m oc-4-(eth oxyim in o)-L-p h en yla la n in e, Eth yl Es-
ter (27). 26 (49.11 g, 0.119 mol) was suspended in 1420 mL
of anhydrous ethanol in a 5-L three-necked flask equipped with
a mechanical stirrer and a reflux condenser. Anhydrous HCl
gas was bubbled in (absorption of HCl caused the reaction
mixture to reflux) until the solution was saturated. Reflux
was maintained for 6 h with a heating mantle followed by slow
cooling overnight. Two liters of absolute ether were added,
and the reaction mixture was left standing for 0.5 h. Filtration
yielded the product as a white solid (34.8 g) after drying under
high vacuum. The filtrate was concentrated in vacuo to a
volume of about 500 mL and diluted with 1200 mL of ethyl
ether. A second crop was obtained which was dried under high
vacuum to yield an additional 22.5 g of product for a total of
57.3 g (92% yield): 1H NMR (DMSO-d6) δ 1.15 (3H, t, J ) 7
Hz), 1.45 (3H, t, J ) 7 Hz), 3.12 (1H, q, J ) 17, 9.5 Hz), 3.20
(1H, q, J ) 17, 4.5 Hz), 4.0-4.4 (6H, m), 4.61 (2H, t, J ) 7
Hz), 7.25-8.10 (13H, m); 13C NMR (DMSO-d6) δ 13.8, 18.6,
36.2, 46.5, 55.2, 60.5, 65.6, 69.3, 119.9, 125.0, 126.9, 127.3,
127.5, 128.8, 132.5, 140.6, 140.7, 143.6, 155.7, 167.6, 171.5.
N-r-Fm oc-4-am idin o-L-ph en ylalan in e, Eth yl Ester (28).
27 (55.9 g, 0.107 mol) and 150 g of ammonium acetate were
suspended in 1.5 L of absolute ethanol in a three-necked flask
equipped with a mechanical stirrer. To this stirred suspension
was added 40 mL of a saturated ethanolic ammonia solution
(the pH was checked and found to be 8). The reaction mixture
was brought to reflux and held there for 30 min, at which time
the reaction was judged complete by TLC analysis (5% MeOH/
CH2Cl2). The reaction mixture was cooled in a water bath to
room temperature, and 2 L of water was added. After stirring
for 10 min, the resulting suspension was filtered and the white
solid which was collected was washed with 700 mL of water
followed by 1 L of ether. Residual solvent was removed under
high vacuum to yield 31.97 g of product as a very slightly
yellow solid. A second crop (8.75 g) was collected from the
aqueous filtrate above for a total of 40.7 g of product (74%
yield) as the acetate salt: 1H NMR (DMSO-d6) δ 1.13 (3H, t,
J ) 7 Hz), 1.78 (HOAc, 3H, s), 2.8-3.2 (2H, m), 4.0-4.4 (6H,
m), 7.25-8.0 (13H, m), 9.15 (3H, br s); 13C NMR (DMSO-d6) δ
13.9, 23.0 (HOAc), 36.1, 36.9, 55.3, 60.0, 60.6, 109.4, 119.8,
121.2, 127.2, 128.7, 129.6, 137.3, 139.3, 142.5, 143.9, 159.6,
165.7, 172.6, 174.2 (HOAc).
N-r-F m oc-4-[(Nam -tr itylsu lfen yl)a m id in o]-L-p h en yla la -
n in e, Eth yl Ester (29). 28 (14.64 g, 28.3 mmol) was
suspended in 150 mL of anhydrous DMF, and tritylsulfenyl
chloride (11.43 g, 36.8 mmol) was added as a solid with
stirring. Most of the suspension went into solution after the
addition of the sulfenyl chloride. The reaction mixture was
stirred at room temperature under argon, and diisopropyl-
ethylamine (14.7 mL, 84.6 mmol) was added dropwise via
syringe. The reaction mixture was stirred at room tempera-
ture for 1 h. The volatiles were removed in vacuo on the
rotovap, and the crude product was taken up in 400 mL of
ethyl acetate. The organics were washed briefly with 1 M
KHSO4 (100 mL), water (100 mL), saturated NaHCO3 (100

Thrombus Imaging
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1381
mL), and saturated NaCl (50 mL). The organics were quickly
passed through sodium sulfate and concentrated in vacuo to
yield a yellow foam (23.19 g). This was taken up in a minimum
amount of methylene chloride and applied to a silica gel
column which had been equilibrated with 20% EtOAc/hexane,
with the top 1 cm equilibrated with methylene chloride. The
column was eluted with 20-30% ethyl acetate/hexane. Frac-
tions containing product were pooled and the volatiles removed
in vacuo. The resulting yellow oil was taken up in chloroform,
and this was likewise removed in vacuo to rid the product of
any trace amounts of ethyl acetate. Product was produced as
a yellow foam (17.90 g, 86% yield): 1H NMR (CDCl3) δ 1.25
(3H, t, J ) 7 Hz), 3.10 (2H, m), 4.1-4.7 (6H, m), 4.93 (2H, br
s), 5.28 (1H, d, J ) 8 Hz), 6.9 -7.8 (27H, m); 13C NMR (CDCl3)
δ 14.1, 38.1, 47.3, 54.7, 61.6, 66.9, 70.1, 120.0, 125.0, 126.1,
126.8, 127.1, 127.6, 127.7, 129.7, 130.2, 134.2, 137.5, 141.4,
143.8, 144.8, 152.3, 155.4, 171.2.
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4-[(Na m -Tr itylsu lfen yl)a m id in o]-L-p h en yla la n in e, Eth -
yl Ester (30). 29 (17.90 g, 24.5 mmol) was dissolved in 200
mL of anhydrous DMF, and 40 mL of diethylamine was added.
After 45 min the solvents were removed on the rotovap. The
crude yellow oil remaining was triturated with hexanes (3 ×
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1.51 (2H, br s), 2.83 (1H, q, J ) 16, 8 Hz), 3.17 (1H, q, J ) 16,
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The reaction mixture was stirred for 30 min after addition was
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and the pH was checked and found to be 10.5. FmocOSu (6.70
g, 19.9 mmol) was added portionwise as a solid while the
reaction mixture was being stirred. The pH was checked after
addition was complete and found to be 8.5. The reaction
mixture was stirred an additional 30 min, 140 mL of 1 M HCl
was added, and the pH was checked and found to be 7. The
solvents were removed in vacuo, and the crude product was
partitioned between 400 mL of 20% 2-propanol/chloroform and
100 mL of 0.5 M KHSO4. The organics were washed with
water (2 × 200 mL), brine (100 mL) and dried briefly over
MgSO4. Filtration and concentration in vacuo yielded a yellow
foam (15.99 g) which was taken up in 50 mL of chloroform
and added dropwise to 600 mL of ether with stirring. The
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removed under high vacuum to yield the product (12.41 g, 89%
yield) as a white solid: 1H NMR (1:1 CD3OD/CDCl3) δ 3.02
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