Synthesis of leukotriene B4 antagonists labeled with In-111 or Tc-99m to image infectious and inflammatory foci

Article abstract of DOI:10.1021/jm050383h

In previous studies we demonstrated that lipophilic ^99mTc- labeled LTB₄ antagonist 1 (RP517) accumulated in infectious foci in rabbits, but hepatobiliary clearance hampered imaging of abdominal lesions. We now report the use of cyste

Full text of DOI:10.1021/jm050383h

6442
J. Med. Chem. 2005, 48, 6442-6453
Synthesis of Leukotriene B4 Antagonists Labeled with In-111 or Tc-99m to
Image Infectious and Inflammatory Foci
Matthias Broekema,§,† Jullie¨tte J. E. M. van Eerd,‡ Wim J. G. Oyen,‡ Frans H. M. Corstens,‡
Rob M. J. Liskamp,† Otto C. Boerman,‡ and Thomas D. Harris§,*
Bristol-Myers Squibb Medical Imaging, North Billerica, Massachusetts 01862, Department of Medicinal Chemistry, Utrecht
Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands, and Department of Nuclear Medicine,
University Medical Center Nijmegen, The Netherlands
Received April 22, 2005
In previous studies we demonstrated that lipophilic 99mTc-labeled LTB4 antagonist 1 (RP517)
accumulated in infectious foci in rabbits, but hepatobiliary clearance hampered imaging of
abdominal lesions. We now report the use of cysteic acid as a pharmacokinetic modifier to
improve the water solubility and renal clearance of three hydrophilic analogues of 1. Divalent
LTB4 antagonist 17 (DPC11870-11) is a DTPA conjugate for radiolabeling with In-111.
Monovalent LTB4 antagonists 15 (BMS57868-88) and divalent LTB4 antagonist 18 (BMS57868-
81) are conjugated to bifunctional chelator HYNIC for radiolabeling with 99mTc. The three
compounds labeled efficiently with 111In or 99mTc with high radiochemical purity and specific
activities. Scintigraphic images obtained in New Zealand White rabbits having acute
intramuscular E. coli infection demonstrated that all agents were able to clearly visualize the
abscess, and clearance was exclusively renal. The biodistribution of the 99mTc-labeled LTB4
antagonists was affected by the coligands used with the HYNIC chelator and by the monovalent
or divalent nature of the receptor binding moiety. The best scintigraphic images were obtained
with monovalent HYNIC conjugate 15 using tricine and isonicotinic acid as coligands with
HYNIC for coordination with 99mTc.
Introduction
far from ideal, resulting in poor image quality and
increased radiation burden to the patient.
The leukotriene B4 (LTB4) receptors are G-protein-
coupled receptors (GPCR) that are involved in recruit-
ment and activation of leukocytes during an inflamma-
tory response.1 At present, two LTB4 receptor types are
identified, BLT1 and BLT2, which have different affini-
ties for LTB4.2,3 The high affinity receptor BLT1 is
mainly expressed on neutrophils, while BLT2 is ex-
pressed more ubiquitously. LTB4 plays an essential
regulatory role during the inflammatory response.
However, its overproduction is reported to play a role
in pathological conditions such as asthma, inflammatory
bowel disease and arthritis.4,5 Consequently, a large
number of LTB4 antagonists have been developed for
clinical application as antiinflammatory therapeutics.6
The rapid detection of infectious and inflammatory foci
in patients is essential for timely and adequate thera-
peutic intervention. One of the current methods for
detection of infection and inflammation is scintigraphic
imaging. The generally used radiopharmaceuticals for
this purpose, 99mTc-labeled leukocytes and 67Ga-citrate,
have several disadvantages. The blood handling re-
quired for the preparation of radiolabeled leukocytes is
accompanied by the risk of infection from blood-borne
pathogens, and is a time-consuming preparation.7,8 The
nuclear and biodistribution properties of 67Ga-citrate are
We previously reported on the synthesis and imaging
properties of 99mTc-labeled LTB4 antagonist 1 (Figure
1).9 LTB4 antagonist 2 (RPR69698)10 was modified with
a short PEG spacer to give compound 3, and the Boc
group of 3 was replaced with the bifunctional chelator
hydrazinonicotinic acid (HYNIC) to give drug precursor
4. The hydrazine was protected as the hydrazone of
sodium 2-formylbenzenesulfonate to avoid reaction with
trace quantities of formaldehyde and other carbonyl
compounds commonly found in the research and manu-
facturing environment.11 The radiolabeling of 4 utilized
99mTc[TcO4]- in the presence of coligands tricine
and trisodium triphenylphosphine-3,3′,3′′-trisulfonate
(TPPTS).12 This radiolabeling procedure gave a rapid
and clean formation of ternary ligand complex 1. This
tracer showed rapid accumulation in the infected thigh
muscle in the rabbit E. coli model.13 However, 1 clears
almost exclusively via the hepatobiliary route, and
physiologic uptake in the intestines limits clinical
applicability of this agent. On the basis of these obser-
vations, more hydrophilic compounds were designed,
using cysteic acid (Csa) as a pharmacokinetic modifier
(PKM). Initially, compound 3 was modified with four
cysteic acids, converted to a divalent ligand using
glutamic acid, and conjugated to DTPA giving 17
(Scheme 3). The 111In complex of 17 (23 in Figure 2)
was found to rapidly visualize infectious foci in the
rabbit E. coli infected thigh muscle model.14 Uptake in
the infectious foci was reduced 88% by preinjection with
an excess of nonradioactive [In(17)]. This indicated that
the interaction of the radiolabeled agent in the infected
* To whom correspondence should be addressed: Thomas Harris,
Bristol-Myers Squibb Medical Imaging, 331 Treble Cove Rd., North
Billerica, MA 01862. E-mail: thomas.d.harris@bms.com. Phone: 978-
671-8266. Fax: 978-436-7500.
§ Bristol-Myers Squibb Medical Imaging.
† Utrecht University.
‡ University Medical Center Nijmegen.
10.1021/jm050383h CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/01/2005

Radiolabeled LTB4 Antagonists for Infection Imaging
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6443
Figure 1. Structures of lead LTB4 antagonist 2, PEG-modified LTB4 antagonists 2, protected HYNIC derivative 4, and 99mTc
ternary ligand complex 1.
6 h half-life) and the general availability of portable
99mTc generators. Herein we describe the synthesis and
initial biological evaluation of two analogues of 17. One
compound (18) is structurally almost identical to 17,
with HYNIC replacing DTPA, while the other compound
(15) is a monovalent version of 18. Both of these LTB4
antagonists were radiolabeled with 99mTc, and the in
vivo imaging characteristics were compared with those
of 23. Furthermore, using divalent HYNIC conjugate
18, we have compared the biodistribution properties of
three coligand systems and shown that the coligand
system can have a profound effect on the biodistribution
properties of these 99mTc complexes. We also describe
the complete synthesis of 17 and provide an improved
synthesis of PEGylated LTB4 antagonist 3.
Results
Synthesis. PEG-modified LTB4 antagonist 3 was
synthesized as outlined in Scheme 1. The tetrazole ring
of 2 was alkylated with ethyl 5-bromovalerate in cyclo-
hexane. Selectivity for alkylation at the N1 position was
increased by blocking the N2 position with the tri-n-
butyltin group.15 Flash chromatography gave a 36%
yield of the desired N1-alkylation product 5a, along with
22% of the unwanted N2 isomer 5b. The ester group of
5a was hydrolyzed with LiOH in aqueous THF to give
a quantitative yield of compound 6. Conjugation of 6
with N-(tert-butoxycarbonyl)-4,7,10-trioxa-1,13-tride-
canediamine using HBTU coupling reagent afforded a
Figure 2. Comparison of the structures of [99mTc(HYNIC)-
99% yield of 3.
(L1)(L2)] complexes and [111In(17)] (23).
The tetracysteic acid derivative of 3 was synthesized
tissue was receptor-specific and that the interaction was
saturable. The agent clears rapidly from nontarget
tissues, and excretion is almost exclusively renal.
Although the biodistribution and imaging properties
of 23 are very promising, a 99mTc-labeled tracer is
generally preferred for scintigraphic imaging over an
111In-labeled tracer because of the more favorable
nuclear properties of 99mTc (140 keV gamma emissions,
by the sequential coupling of four Boc-cysteic acid-mono
sodium salt residues (Boc-Csa(Na)-OH) as shown in
Scheme 1. The Boc-protecting group of 3 was removed
using a mixture of trifluoroacetic acid (TFA), dichlo-
romethane (DCM), water, and triisopropylsilane (TIS).
The amine TFA salt (7) resulting after concentration of
the reaction solution was used without purification. Boc-
Csa(Na)-OH was preactivated using HBTU, and DIEA

6444 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Broekema et al.
Scheme 1. Synthesis of Tetra-Cysteic Acid-Modified LTB4 Antagonist 13a
a Reagents: (i) bis(tri-n-butyltin)oxide, EtOH; (ii) ethyl 5-bromovalerate, cyclohexane, 36%; (iii) aq LiOH, THF, 100%; (iv) N-(tert-
butoxycarbonyl)-4,7,10-trioxa-1,13-tridecanediamine, HBTU, DIEA, 99%; (v) TFA, DCM, TIS, water; (vi) Boc-Csa(Na)-OH, HBTU, DIEA,
DMF.
Scheme 2. Synthesis of Monovalent HYNIC Conjugate 15a
a Reagents: (i) benzaldehyde, DMF; (ii) DCM wash, 66%; (iii) 13, PyBOP, DIEA, DMF, 30%.
in DMF solution, and the resulting N-hydroxybenzo-
triazole ester (8) was added to a solution of 7 in DMF.
The resulting Boc-protected cysteic acid derivative (9)
was purified my means of extraction into CHCl3 and
washing with H2O and saturated NaHCO3. The satu-
rated NaHCO3 wash removed the 1-hydroxybenzotria-
zole (HOBt) from the organic layer, and the crude
product was used without further purification. The Boc
group of compound 9 was removed as described above
giving 10. The products of the second, third, and fourth
deprotection/cysteic acid coupling cycles (11, 12, and 13,
respectively) were too hydrophilic for purification by
partitioning between aqueous and organic phases, and
therefore were purified by preparative reverse phase
HPLC on a C18 column. Products were recovered from
the aqueous eluants by lyophilization and were obtained
as flocculent colorless solids.
6-Hydrazinonicotinic acid was converted to 6-(2-
benzaldehydehydrazono)nicotinic acid (14) using an
excess of benzaldehyde as shown in Scheme 2.11,16
Unreacted benzaldehyde was removed by trituration
with DCM to give pure 14 in 66% yield. The conjugation
reaction between 13 and 14 was mediated with benzo-
triazol-1-yl-oxytripyrrolidinophosphonium hexafluoro-

Radiolabeled LTB4 Antagonists for Infection Imaging
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6445
Scheme 3. Synthesis of Divalent DTPA Conjugate 17 and Divalent HYNIC Conjugate 18a
a Reagents: (i) Boc-Glu(OTfp)-OTfp, HOAt, DIEA, DMF; (ii) TFA, DCM, 62%; (iii) DTPA dianhydride, DIEA, DMF, 96%; (iv) 6-(2-
benzaldehydehydrazono)nicotinic acid (14), PyBOP DIEA, DMF, 78%.
phosphate (PyBOP) and DIEA in DMF solution to give
a 30% purified yield of 15.
Preparation of the divalent LTB4 antagonist was
Table 1. IC50 Values (nM) of Chelator Conjugates
competing agent
compound
[3H]LTB4
[
111In(17)]
achieved by the conjugation of tetracysteic acid deriva-
tive 13 to both of the carboxylic groups of Boc-glutamic
acid, using Boc-Glu(OTfp)-OTfp as the activated form
(Scheme 3). The addition of HOAt significantly in-
creased the reaction rate, and the reaction was complete
in 6 h at ambient temperatures. The Boc group was
removed using a cocktail of TFA/DCM/water/anisole,
and the resulting amine was purified by HPLC to give
a 62% yield of divalent LTB4 antagonist 16 as a
flocculent, colorless solid. Compound 16 was used for
the preparation of divalent DTPA conjugate 17 and
divalent HYNIC conjugate 18. Reaction of 16 with
DTPA dianhydride in DMF gave a 96% yield of 17 after
HPLC purification. HYNIC conjugate 18 was isolated
in 78% yield by a PyBOP-mediated coupling of 16 and
14 in DMF.
2
4
16
17
15
18
20a
45 (n ) 4)b
8 (n ) 2)b
54 (n ) 2)b
44 (n ) 2)
30 (n ) 2)
16 (n ) 2)
230 (n ) 2)
a Reference 10. b NovaScreen, Hanover, MD.
in a competition assay with [111In(17)]. As shown in
Table 1, the receptor affinities of monovalent conjugates
4 and 15, and divalent DTPA conjugate 17, were nearly
identical to that of parent LTB4 antagonist 2. Divalent
HYNIC conjugate 18 showed a greater than 10-fold loss
of receptor affinity.
Radiolabeling. HPLC analyses, performed after
99mTc-labeling of divalent HYNIC-conjugated compound
18, indicated that with all three coligand systems the
compound labeled at a specific activity of 37 MBq/µg
Receptor Binding Assay. The IC50 values for all of
the chelator conjugates described here were obtained

6446 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Broekema et al.
Figure 4. Biodistribution data obtained 8 h pi using divalent
imaging agent [99mTc(18)] radiolabeled using tricine/TPPTS
(19), tricine2 (20), and tricine/ISO (21) coligand systems. Each
bar represents the mean values ( SD.
Figure 3. Anterior images of rabbits induced with an
intramuscular infection. Images were acquired immediately,
4, and 8 h pi of 37 MBq divalent imaging agent [99mTc(18)],
radiolabeled using tricine/TPPTS (19) (A), tricine2 (20) (B), and
tricine/ISO (21) (C) coligand systems.
(110 MBq/nmol) with a labeling efficiency >95%. Mon-
ovalent HYNIC-conjugated LTB4 antagonist 15 labeled
at a specific activity of 110 MBq/nmol with a labeling
efficiency >95%. Divalent DTPA-conjugated LTB4 an-
tagonist 17 was labeled with 111In at a specific activity
of 12 MBq/nmol with a labeling efficiency >95%.
Animal Studies. Divalent HYNIC-modified LTB4
antagonist 18 was radiolabeled with 99mTc using three
different coligand systems, tricine/TPPTS, tricine2, and
tricine/ISO, to give complexes of the general formula
Figure 5. Comparison of divalent imaging agent [99mTc(18)-
(tricine)(ISO)] (21) (A), monovalent imaging agent [99mTc(15)
(tricine)(ISO)] (22) (B), and divalent imaging agent [111In(17)]
(23) (C). All are anterior images of rabbits induced with an
intramuscular infection. Images were acquired 8 h pi of 37
MBq for (A) and (B) and 11 MBq for (C).
visualization was best after injection of [99mTc(18)-
(tricine)(ISO)] (21), because this preparation combined
high uptake in the abscess with low radioactivity
concentration in nontarget tissues, particularly in the
kidneys.
[
99mTc(18)L1L2]. The structures of these three ligand
systems are shown in Figure 2. Pharmacokinetics of
these three tracers were studied in three groups of
rabbits with E. coli infection, with images acquired 5
min, 4 and 8 h postinjection (pi). As shown in Figure 3,
immediately after injection the distribution of [99mTc-
(18)L1L2] was similar for all three coligand systems.
Uptake of the tracer was observed in the heart, lungs,
liver, and kidneys and in the bone marrow. At 4 h and
especially at 8 h after injection of the compounds, the
radioactivity had accumulated in the abscess resulting
in clear delineation of the infectious lesions. The images
acquired at 8 h pi clearly indicated that the coligand
used during the labeling procedure affected the in vivo
distribution of the LTB4 antagonist. Visualization of the
abscess was better when TPPTS or ISO was used as
coligand in combination with tricine, than when tricine
was used alone.
Ex vivo biodistribution determination of radioactivity
concentration in the dissected tissues confirmed the
observations in the imaging experiment (Figure 4).
Radioactivity concentrations in the abscess were highest
when [99mTc(18)] was labeled with the tricine/TPPTS
and tricine/ISO coligand systems. Uptake in nontarget
organs was also affected by the coligand system. The
radioactivity concentration in the spleen and kidneys
was significantly higher using tricine/TPPTS coligands,
as compared to the uptake in these organs using tricine/
ISO or tricine alone (p < 0.01). Altogether, the abscess
In the next imaging experiment, rabbits with intra-
muscular infection were injected with either the diva-
lent or monovalent 99mTc-labeled LTB4 antagonist [99mTc-
(18)(tricine)(ISO)] (21) or [99mTc(15)(tricine)(ISO)] (22),
respectively) or with the 111In-labeled divalent LTB4
antagonist [111In(17)] (23). The images acquired at 8 h
pi showed that all three tracers visualized the intra-
muscular abscess (Figure 5). When the three agents
were compared, it appeared that the distribution of both
divalent analogues was very similar. All three analogues
clearly delineated the intramuscular abscess. Nontarget
uptake of radioactivity for the three agents was similar
in bone marrow, liver, and spleen. However, the kidney
uptake of monovalent agent [99mTc(15)(tricine)(ISO)]
(22) appeared to be significantly lower.
Discussion
Leukotriene B4 (LTB4) is a potent pro-inflammatory
lipid mediator derived from arachidonic acid via the
5-lipoxygenase pathway. It is produced by neutrophils,
monocytes, macrophages, keratinocytes, lymphocytes,
and mast cells. The physiological responses to LTB4
include potent neutrophil chemotactic activity, adhesion
of PMNs to the vascular endothelium, stimulation of the
release of lysosomal enzymes and superoxide radicals
by PMNs, and an increase in vascular permeability.
Increased levels of LTB4 have been detected in patients

Radiolabeled LTB4 Antagonists for Infection Imaging
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6447
with asthma, acute respiratory distress syndrome,
chronic obstructive pulmonary disease, contact derma-
titis, cystic fibrosis, inflammatory bowel disease, gout,
myocardial ischemia, psoriasis, rheumatoid arthritis,
and cancer. Thus, it is widely believed that LTB4 is an
important mediator of acute and chronic inflammatory
events. This has made the LTB4 receptor the target of
intense research by major pharmaceutical companies for
therapeutic applications, and in recent years, a number
of highly potent and selective LTB4 antagonists have
been reported. These compounds have been shown to
be safe and efficacious in both animal models and in
humans.6,17,18 This wealth of information on LTB4
antagonists made the LTB4 receptor the most attractive
of the various approaches available for the development
of a neutrophil-binding radiolabeled infection/inflam-
mation imaging agent.
In the present study the syntheses of three structur-
ally related LTB4 antagonists are described. These
antagonists were designed for scintigraphic visualiza-
tion of infectious and inflammatory foci. In the field of
nuclear medicine, there is an ongoing search for the
ideal radiopharmaceutical to visualize infectious and
inflammatory foci. For this purpose many radiolabeled
peptides, cytokines, and antagonists have been studied.8
In our original work in this area we synthesized LTB4
antagonist 4 (Figure 1), for the visualization of infec-
tious and inflammatory lesions. This compound was
based on 2, a lipophilic BLT1 receptor-binding ligand.10
Compound 4 is a conjugate of bifunctional chelator
hydrazinonicotinic acid (HYNIC) to allow efficient ra-
diolabeling with 99mTc. The ternary ligand complex of
4, utilizing TPPTS and tricine as coligands (1 in Figure
1), accumulated rapidly in infected tissues containing
infiltrated BLT1-positive neutrophils.12,13 However, due
to the lipophilicity of the receptor binding moiety of 1,
the tracer cleared via the hepatobiliary route, and the
rapid physiologic uptake of the tracer in the liver and
the intestines interfered with visualization of inflam-
matory lesions in the abdomen.13
Various strategies have been used to enhance the
hydrophilicity of radiopharmaceutical drugs, to increase
their water solubility and/or renal clearance. These
include the introduction of polar function groups (car-
boxyl, amine, etc.)19 and PEG chains,20 and the use of
different radionuclide chelators.21 Our experience with
4 indicated that we were unlikely to achieve a signifi-
cant change in solubility or biodistribution properties
by the introduction of simple PEG tethers. Here we
present the synthesis and characterization of three
hydrophilic tracers (17, 18, and 15 in Schemes 2 and 3)
based on the same receptor binding moiety used in the
synthesis of 4. The lipophilicity of 4 was reduced by the
introduction of multiple cysteic acids as a PKM. Cysteic
acid was chosen for this role because of the high polarity
of the sulfonic acid group and because it is an unnatural
amino acid and is therefore less likely to undergo
metabolism. We have also examined the effect of two
different radionuclide chelators (DTPA for coordination
to 111In, and HYNIC for coordination to 99mTc) because
it is known from other studies that the chelator can
influence the biodistribution of the tracer.21 Finally, we
have examined the effect of different coligands with
HYNIC because previous reports have shown that the
nature of the coligand can have a marked effect on
biodistribution.22,23
In our original synthesis of 4 the tetrazole ring of 2
was alkylated with ethyl 5-bromovalerate in refluxing
ACN in the presence of DIEA as base.24 Alkylation of
5-substituted tetrazoles under these conditions always
produces the N2 isomers in greater proportion than the
N1 isomers. In our original synthesis the N1/N2 ratio
was a very unfavorable 0.14. We subsequently learned
that the product ratio can be shifted in favor of the N1
isomer by blocking the N2 position with the tri-n-
butyltin group.15 Preparation of the 2-(tri-n-butylstan-
nyl)tetrazole was accomplished by treatment of 2 with
bis(tri-n-butyltin)oxide in refluxing EtOH (Scheme 1).
The EtOH solvent was replaced with cyclohexane, ethyl
5-bromovalerate was added, and the solution was
heated to reflux. The reaction solution was then diluted
with ether and washed with aqueous KF to remove tin
byproducts. Final purification by flash chromatography
gave a 36% yield of the N1 isomer (5a) and a more
favorable N1/N2 ratio of 1.64. We discovered that
formation of the N1 isomer is favored by the use of
nonpolar solvents, and that even trace quantities of
EtOH remaining from the formation of the 2-(tri-n-
butylstannyl)tetrazole have a negative effect on the yield
of the N1 isomer. Structure assignments were based
upon the observation that the NMR chemical shift of
the N1-alkyl substituents is 0.15-0.35 ppm upfield from
that of the N2 isomer.25 With this improvement over
our original synthesis we were able to carry out the
tetrazole alkylation on a relatively large scale, producing
16.58 g of purified 5a. The remaining steps in the
synthesis of compound 3 followed our original proce-
dures, as described in the Experimental Section. The
four cysteic acid PKMs were added stepwise to 3 in a
series of deprotection/coupling cycles, as shown in
Scheme 1. Boc-Csa(Na)-OH was preactivated using
HBTU, and the resulting N-hydroxybenzotriazole ester
(8) was added to a DMF solution of the amine. After
the addition of one cysteic acid residue (i.e., 9, R ) Boc),
the product was still sufficiently lipophilic that polar
byproducts could be removed by water washes. But
subsequent cysteic acid coupling cycles gave products
that were too hydrophilic for an aqueous workup
procedure. These compounds were deprotected, and the
products (i.e., 11, 12, 13) of these reactions were purified
by reverse phase HPLC and isolated from the eluants
by lyophilization. The overall yield for these four
coupling cycles was 37%.
The biomolecules employed as the basis for develop-
ment of radiopharmaceuticals are frequently very potent
and elicit a biological response even at low doses.
Radiolabeling in the clinical setting must therefore be
carried out using very small quantities of the biomol-
ecule, and a substantial effort has been made to find
radionuclide chelators that can be easily radiolabeled
in aqueous solution at very low concentrations of the
biomolecule and with high specific activity. One of the
best chelators for 99mTc is 6-hydrazinonicotinic acid
(HYNIC), which is efficiently labeled in aqueous solu-
tions having a ligand concentration as low as 10-5 M.12
Protection of the hydrazine group of HYNIC during
conjugation to the targeting molecule is essential in
order to prevent unwanted conjugation reactions. Pro-

6448 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Broekema et al.
tection is also desirable during manufacturing of the
lyophilized kit used for preparation of the radiophar-
maceutical, because the hydrazine moiety reacts readily
with aldehydes and ketones found in the manufacturing
environment. Such materials are extracted from various
rubber and plastic materials and are also used in
common disinfectants. Formaldehyde is particularly
ubiquitous. In our initial work in this area we discovered
that hydrazones of aromatic aldehydes were particularly
useful as hydrazine protecting groups on HYNIC.11
These hydrazones are stable during synthesis and
HPLC purification and protect the hydrazine moiety
from reaction with common carbonyl impurities. The
hydrazone is spontaneously cleaved during the radio-
labeling process allowing formation of the diazenido-
technetium bond. We found that the hydrazone of
sodium 2-formylbenzesulfonate was especially useful
because the sulfonate group greatly improved water
solubility of the HYNIC conjugate (see Figure 1). With
multiple cysteic acid PKMs present on the LTB4 an-
tagonists described here, a further increase in water
solubility was unnecessary, and we used the simpler
hydrazone of benzaldehyde for protection of the hydra-
zine moiety (14 in Scheme 2). Conjugation of 14 to
tetracysteic acid derivative 13 was accomplished using
PyBOP to give a 30% purified yield of 15.
and 15) suggests this is not due solely to the HYNIC
chelator. This value does not appear to be an outlier as
the duplicate numbers are nearly identical. At this time
we have no explanation for the relatively low receptor
affinity of 18.
Divalent DTPA conjugate 17 was radiolabeled with
111In as described in a previous publication to give [111In-
(17)] (23) as shown in Figure 2.14 The 99mTc complexes
of divalent HYNIC conjugate 18 were prepared from Na-
99mTcO4, SnSO4, and one of three coligand systems by
heating at 100 °C for 30 min in phosphate-buffered
saline, pH 7.0. The three coligand systems (tricine/
TPPTS, tricine2, and tricine/ISO) gave complexes 19, 20,
and 21 as shown in Figure 2. Radiolabeling was rapid
and efficient using each of the three coligand systems.
The radiochemical purity of all complexes was verified
by the analytical methods described in the Experimental
Section. While the binary complex (20) formed from
tricine alone has been used extensively in preclinical
research, it suffers from the drawback that it exists in
solution in several isomeric forms, many of which are
interconverting at room temperature.28 It would be very
difficult to develop such a system for clinical use. In
contrast, ternary complexes formed from TPPTS and
tricine (e.g., 19) form only two non-interconverting
diastereomeric radiolabeled products when the biomol-
ecule is itself chiral.12
Many interactions in biological systems are polyvalent
in nature (e.g., DNA helix, carbohydrate interactions,
virus-cell wall interactions). However, it is only recently
that polyvalency has begun to be explored in drug
development.26 In previous studies we have shown that
divalency of a cyclic RGD peptide targeting the vit-
ronectin receptor on tumors caused enhanced receptor
affinity and enhanced retention in the target tissue in
vivo.27 We have explored the effect of divalency in these
radiolabeled LTB4 antagonists by preparing the pseudo-
dimer of 13. Tetracysteic acid derivative 13 was ef-
ficiently converted to a divalent ligand by reaction with
Boc-Glu(OTfp)-OTfp and HOAt in DMF (Scheme 3). The
HOAt greatly accelerated the reaction rate, reducing the
reaction t1/2 from 12 to 1 h. Removal of the Boc
protecting group gave a 62% yield of 16 after HPLC
purification. Conjugation of 16 to DTPA was accom-
plished by using a 10-fold excess of DTPA dianhydride
and gave a 96% purified yield of 17. Conjugation of 16
to 14 gave a 78% purified yield of divalent HYNIC
conjugate 18.
The IC50 values of the chelator conjugates reported
in this study are shown in Table 1. Lipophilic HYNIC
conjugate 4 has a receptor affinity of 45 nM, which is
only slightly weaker than the 20 nM value of parent
LTB4 antagonist 2. Divalent water-soluble amine 16
shows a slight increase in receptor affinity to 8 nM,
while the corresponding DTPA conjugate (17) shows a
slight loss of affinity to 54 nM. These data confirm our
original hypothesis that the LTB4 receptor is very
tolerant of substitutions on the tetrazole ring. Monova-
lent water-soluble HYNIC conjugate 15 has affinity of
16 nM, but surprisingly the divalent version (18) shows
a large loss of receptor affinity with an IC50 value of
230 nM. Comparison with the affinities of the other
divalent LTB4 antagonist (17) suggests this is not due
solely to the divalent nature of the molecule, and
comparison with the other two HYNIC conjugates (4
Scintigraphic images after injection of divalent 99mTc
complexes 19, 20, and 21 are shown in Figure 3. Whole
body distribution of the three agents was similar im-
mediately after injection, with significant activity in the
lungs and heart (blood pool) and in the kidneys. By 4 h
pi, uptake in the abscesses was clearly visible with all
three agents, and there was high activity in the kidneys.
Images acquired 8 h pi clearly showed that uptake in
the abscesses was greater with the tricine/TPPTS and
tricine/ISO complexes (19 and 21). It was also apparent
that kidney uptake was higher with tricine/TPPTS
complex 19. Thus, the images obtained with ternary
complex 21 were qualitatively superior to the images
obtained with the other divalent 99mTc complexes. The
biodistribution data for complexes 19, 20, and 21 at 8
h pi (Figure 4) confirmed these visual impressions.
Uptake of 19, 20, and 21 in the abscesses was 0.21, 0.08,
and 0.22%ID/g, respectively, and the kidney uptake of
19 was nearly twice that of 20 and 21 (0.81, 0.40, and
0.48%ID/g, respectively). In addition, the levels of 19
were slightly higher in the spleen and marrow, and 20
showed slightly slower clearance from the blood. Thus,
for this divalent LTB4 antagonist platform, the tricine/
ISO coligand system (21) combined the highest uptake
in the abscess with the lowest uptake in nontarget
tissues.
In a final experiment we compared divalent tricine/
ISO ternary complex 21 with monovalent tricine/ISO
ternary complex 22 and with divalent 111In-DTPA
complex 23. Scintigraphic images 8 h pi are shown in
Figure 5. The radioactivity concentration in the ab-
scesses is similar for all three agents, but the kidney
concentration appears significantly lower for monova-
lent complex 22. This combination of high uptake in the
target tissue and low uptake in nontarget tissues makes
monovalent [99mTc(15)(tricine)(ISO)] (22) the qualita-
tively superior imaging agent. Initial biodistribution

Radiolabeled LTB4 Antagonists for Infection Imaging
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6449
evaporator. Reaction monitoring and low resolution mass data
were obtained with an Agilent Model 1100 LC/MSD API-
electrospray system using either a Phenomenex Synergi Polar-
RP C18 column (4 µm, 80 Å, 4.6 × 150 mm) or a Zorbax SB-
CN column (5 µm, 4.6 × 150 mm), operated at 50 °C with 0.1%
formic acid-modified ACN/water mobile phases. Final HPLC
purity determination was made with an Agilent Model 1100
HPLC system, using either a Waters Atlantis dC18 column
(5 µm, 4.6 × 100 mm) or a Zorbax SB-CN column (5 µm, 4.6 ×
150 mm) and 0.1% TFA or 0.1 M NaOAc-modified ACN/water
as mobile phases. UV detection was carried out at 220 and
254 nm. Preparative HPLC purifications were performed on
a Varian PrepStar system using SD-1 pumps equipped with
a UV/vis detector model 320, using a Waters Atlantis dC18
column (5 µm, 30 × 100 mm) except as noted otherwise and
using the gradients and mobile phases given in the text. UV
detection was carried out at 220 nm. All chromatography was
carried out with HPLC grade organic solvents.
data for 22 confirms these visual impressions, with
abscess uptake 8 h pi of 0.36% ID/g, vs 0.22%ID/g for
21, as described above. More detailed studies of the
pharmacokinetic and imaging properties of 22 will be
described elsewhere (manuscript submitted). It is in-
teresting that there is little difference in the images
resulting from divalent 99mTc-HYNIC complex 21 and
divalent 111In-DTPA complex. Structurally, the two
chelator-radionuclide complexes are very different. It
is also interesting that the potential divalent interaction
with LTB4 receptors on the cell membrane of the
neutrophils did not enhance the accumulation of the
either 21 or 23 in the abscess. However, divalent agent
[
99mTc(18)(tricine)(ISO)] (21) did showed enhanced re-
tention in the kidneys as compared to monovalent
compound [99mTc(15)(tricine)(ISO)] (22). In this instance,
divalency did not result in enhanced uptake in the
target, but apparently the divalent compound was more
efficiently reabsorbed in the renal tubules as has been
observed with other HYNIC-conjugated tracers.29 As a
result, abscess-to-background ratios of monovalent com-
pound 22 were higher, resulting in scintigraphic images
with improved contrast. See ref 26 for a discussion of
the various mechanisms of polyvalent interactions in
biological systems.
Exact mass determination was performed on a 7.0 T Fourier
transform ion cyclotron resonance (FTICR) mass spectrometer
(IonSpec Corporation, Lake Forest, CA) equipped with an
electrospray ionization source. A mixture of sample and
internal calibrant (poly(ethylene glycol) or polypropylene
glycol) was prepared at approximately 10 µM each in ACN:
water:acetic acid (500:500:1) and infused at 1 µL/min. A
spectrum was obtained using the IonSpec OMEGA data
system by averaging five transients of 512K data points per
transient. Mass calibration was derived from internal calibrant
ions bracketing the m/z of interest for the sample. FT-NMR
spectra were obtained on a Bruker Avance 600 MHz spec-
1
trometer at 600 MHz for H and 150 MHz for 13C using the
Conclusions
indicated solvent. Residual solvent signals were used for
internal calibration. Chemical shifts are reported in parts per
million (δ), and signals are expressed as s (singlet), d (doublet),
t (triplet), q (quartet), p (pentet), m (multiplet), or br (broad).
Elemental analyses were performed at Oneida Research
Services, Inc, Whitesboro, NY; found values agree favorably
with the calculated ones.
This study describes the syntheses of three new
radiolabeled LTB4 antagonists for imaging infection and
inflammation. All three of these tracers visualized
intramuscular infection in rabbits within a few hours
after injection, and the incorporation of multiple cysteic
acid PKMs allowed these tracers to be cleared exclu-
sively via the kidneys. The structural parameters
investigated with these tracers included the use of either
99mTc-HYNIC or 111In-DTPA complexes, monovalent vs
divalent LTB4 antagonists, and the coligands used in
the 99mTc-HYNIC complexes. The coligand system had
a marked effect on biodistribution in the rabbit model,
with the ternary complex using tricine and ISO as
coligands (i.e., 21) providing the best scintigraphic
images and the best biodistribution data of the divalent
HYNIC derivatives. Monovalent ternary 99mTc-HYNIC
complex 22 gave better scintigraphic images than either
divalent 99mTc-HYNIC complex 21 or divalent 111In-
DTPA complex 23. Tracer 22 appears to be the most
promising of these new tracers, combining the highest
uptake in abscesses with lowest uptake in nontarget
tissues.
Ethyl 5-(5-(5-(4,6-Diphenyl(2-pyridyloxy))-1,1-dimeth-
ylpentyl)-1,2,3,4-tetraazolyl)pentanoate (5a). A solution
of 2 (35.0 g, 84.6 mmol) and bis(tri-n-butyltin) oxide (21.5 mL,
42.3 mmol) in absolute EtOH (1035 mL) was heated at reflux
under nitrogen for 30 min. The solution was concentrated
under vacuum, and the resulting was dissolved in cyclohexane
(3 × 200 mL) and again concentrated to remove traces of
EtOH. The oil was dissolved in cyclohexane (35 mL), treated
with ethyl 5-bromovalerate (40.0 mL, 254 mmol), and heated
at 85 °C in an oil bath under nitrogen for 28 h. The reaction
was cooled to ambient temperatures, diluted with ether (350
mL), washed sequentially with 10% KF solution (125 mL) and
saturated NaCl (400 mL), dried (MgSO4), and concentrated
to give a yellow oil. This oil was purified by silica gel flash
chromatography. The column was initially eluted with hexane/
EtOAc (75/25) until the unwanted N2 isomer was off the
column, and the mobile phase was switched to hexane/EtOAc
(60/40) to collect the desired N1 isomer. Product fractions were
concentrated and the resulting solid was recrystallized (EtOH)
to give 16.58 g (36%) of the title compound as a colorless solid,
mp 99.5-101.5 °C. 1H NMR (CDCl3): δ 8.10-8.01 (m, 2H),
7.69-7.62 (m, 2H), 7.55-7.36 (m, 7H), 6.85 (s, 1H), 4.43 (t, J
) 6.3 Hz, 2H), 4.34 (t, J ) 7.5 Hz, 2H), 4.09 (q, J ) 7.1 Hz,
2H), 2.32 (t, J ) 7.2 Hz, 2H), 2.07-1.92 (m, 2H), 1.91-1.60
(m, 8H), 1.50 (s, 6H), 1.21 (t, J ) 7.1 Hz, 3H); 13C NMR
(CDCl3): δ 172.7, 164.1, 159.8, 155.1, 152.1, 139.1, 138.7, 129.0,
128.9, 128.6, 127.0, 126.8, 111.8, 107.1, 65.30, 60.49, 48.57,
41.32, 34.84, 33.40, 29.38, 29.30, 27.17, 21.90, 21.29, 14.18;
HRMS: Calcd for C32H40N5O3 [M + H]+: 542.3131, Found:
542.3140; Anal. (C32H39N5O3) C,H,N.
Experimental Section
General Methods. Previously published procedures were
used for the synthesis of Boc-Csa(Na)-OH,30 Boc-Glu(OTfp)-
Otfp,31 6-hydrazinonicotinic acid,32 LTB4 antagonist 2,10 and
N-(tert-butoxycarbonyl)-4,7,10-trioxa-1,13-tridecanedi-
amine.33 HPLC-grade ACN was obtained from J. T. Baker.
Absolute ethanol was obtained from Quantum Chemical Corp.
All other reagents and solvents were purchased from Aldrich
Chemical Corporation, Lancaster Synthesis, Inc., or Fluka
Chemical Corporation and used as received. Merck silica gel,
grade 9385, 230-400 mesh, 60 Å was used for flash chroma-
tography. Na-99mTcO4 (Mo-generator) and 111InCl3 were ob-
tained from Tyco Mallinckrodt, Netherlands. All reactions were
carried out at ambient temperatures under a nitrogen atmo-
sphere unless noted otherwise. Organic reaction mixtures were
concentrated under reduced pressure at 40-65 °C on a rotary
Ethyl 5-(4-(5-(4,6-Diphenyl(2-pyridyloxy))-1,1-dimeth-
ylpentyl)-1,2,3,5-tetraazolyl)pentanoate (5b). Fractions
from the above flash chromatography containing the unwanted
N2 isomer were concentrated to give 10.2 g (22%) of the title
compound as a colorless solid. 1H NMR (CDCl3): 8.10-8.01
(m, 2H), 7.69-7.61 (m, 2H), 7.55-7.35 (m, 7H), 6.86 (s, 1H),

6450 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Broekema et al.
4.54 (t, J ) 7.1 Hz, 2H), 4.42 (t, J ) 6.6 Hz, 2H), 4.09 (q, J )
7.1 Hz, 2H), 2.31 (t, J ) 7.3 Hz, 2H), 2.09-1.95 (m, 2H), 1.90-
1.70 (m, 4H), 1.70-1.56 (m, 2H), 1.45-1.28 (m, 8H), 1.21 (t, J
) 7.1 Hz, 3H); 13C NMR (CDCl3): 173.50, 172.81, 164.32,
155.12, 151.97, 139.18, 138.82, 128.94, 128.84, 128.57, 127.03,
126.81, 111.66, 107.10, 65.83, 60.40, 52.32, 42.40, 34.77, 33.32,
29.59, 28.63, 27.21, 21.74, 21.31, 14.18; MS: m/e 542.4 [M +
H]; HRMS: Calcd for C32H40N5O3 [M + H]+: 542.3131,
Found: 542.3143; Anal. (C32H39N5O3) C,H,N.
5-(5-(5-(4,6-Diphenyl(2-pyridyloxy))-1,1-dimethylpen-
tyl)-1,2,3,4-tetraazolyl)pentanoic Acid (6). A solution of
ester 5a (16.2 g, 29.9 mmol) in inhibitor free, peroxide free
THF (450 mL) was treated with 3 M LiOH (120 mL), and the
resulting mixture was stirred rapidly for 19 h. The mixture
was concentrated until most of the THF was removed. The
resulting aqueous mixture was adjusted to pH 3 with 6 N HCl
and extracted with EtOAc (1 × 450, 1 × 60 mL). The combined
EtOAc extracts were washed with saturated NaCl (100 mL),
dried (MgSO4), and concentrated to give 15.3 g (100%) of the
title compound as a viscous colorless oil. 1H NMR (CDCl3): 8.02
(d, J ) 8.4 Hz, 2H), 7.65(d, J ) 8.2 Hz, 2H), 7.55-7.35 (m,
7H), 6.84 (s, 1H), 4.43-4.31 (m, 4H), 2.36 (t, J ) 7.0 Hz, 2H),
2.10-1.92 (m, 2H), 1.89-1.62 (m, 6H), 1.49 (s, 6H), 1.42-1.20
(m, 2H); HRMS: Calcd for C30H36N5O3 [M + H]+; 514.2818,
Found: 514.2819.
N-(3-(2-(2-(3-((tert-Butoxy)carbonylamino)propoxy)-
ethoxy)ethoxy)propyl)-5-(5-(5-(4,6-diphenyl(2-pyri-
dyloxy))-1,1-dimethylpentyl)(1,2,3,4-tetraazolyl))pentan-
amide (3). A solution of 6 (5.60 g, 10.9 mmol), DIEA (4.75
mL, 27.3 mmol), and HBTU (5.68 g, 15.0 mmol) in anhydrous
DMF (75 mL) was stirred for 5 min and treated with a solution
of N-(tert-butoxycarbonyl)-4,7,10-trioxa-1,13-tridecanediamine
(4.81 g, 15.0 mmol) in anhydrous DMF (35 mL). Stirring was
continued for 6 h. The DMF was removed, and the resulting
viscous oil was partitioned between EtOAc (330 mL) and water
(75 mL) at pH 3. The organic phase was washed consecutively
with dilute (pH 3) HCl (2 × 75 mL), water (75 mL), saturated
NaHCO3 (75 mL), and saturated NaCl (75 mL). The organic
phase was dried (MgSO4) and concentrated to give 8.80 g (99%)
of 3 as an amber viscous oil. 1H NMR (CDCl3): δ 8.03 (d, J )
8.4 Hz, 2H), 7.65 (d, J ) 8.4 Hz, 2H), 7.53 (d, J ) 1.2 Hz, 1H),
7.49-7.37 (m, 6H), 6.83 (d, J ) 1.2 Hz, 1H), 6.30 (bs, 1H),
4.95 (bs, 1H), 4.41 (t, J ) 6.4 Hz, 2H), 4.34 (t, J ) 7.5 Hz, 2H),
3.65-3.48 (m, 12H), 3.36-3.25 (m, 2H), 3.21-3.11 (m, 2H),
2.16 (t, J ) 7.2 Hz, 2H), 2.02-1.94 (m, 2H), 1.88-1.66 (m,
10H), 1.48 (s, 6H), 1.40 (s, 9H), 1.37-1.28 (m, 2H); 13C NMR
(CDCl3): δ 172.06, 170.25, 164.10, 159.91, 157.14, 156.10,
154.98, 152.41, 138.84, 138.58, 129.01, 128.64, 127.59, 127.07,
126.85, 111,90, 78.94, 70.45, 70.42, 70.14, 70.01, 69.93, 69.43,
65.59, 48.72, 41.28, 38.41, 37.88, 35.53, 34.84, 29.70, 29.48,
29.30, 28.90, 28.44, 27.15, 22.59, 21.33; HRMS: Calcd for
C45H66N7O7 [M + H]: 816.5024, Found: 816.5044.
General Procedure for Preactivation of Boc-Csa(Na)-
OBt (8). Boc-cysteic acid mono sodium salt in anhydrous DMF
(6 mL DMF for 1 mmol Boc-Csa(Na)-OH) was treated with 1
equiv of HBTU and 2 equiv of DIEA, and stirred for 25 min.
The resulting solution of active ester was transferred to a
solution of the reactive amine using anhydrous DMF to
complete the transfer.
LTB4-(Csa)-H (10). A solution of 3 (1.68 g, 2.1 mmol) in
50/40/5/5 DCM/TFA/TIS/H2O (40 mL) was stirred for 30 min
and concentrated yielding 7 as a yellow viscous oil. This oil
was dissolved in DMF (25 mL) and made basic with DIEA (2.90
mL, 16.6 mmol). A solution of active ester Boc-Csa(Na)-OBt
(8) was prepared in a separate flask from Boc-Csa(Na)-OH
(1.20 g, 4.1 mmol), HBTU (1.56 g, 4.1 mmol), and DIEA (1.5
mL, 8.6 mmol) in DMF (25 mL). This solution of 8 was added
to the solution of 7, and after 2 h of stirring, the reaction
mixture was concentrated, yielding a yellow oil. This oil was
redissolved in CHCl3 (180 mL), washed with water (2 × 100
mL), and saturated NaHCO3 (1 × 80 mL). The aqueous layer
was back-extracted with CHCl3 (1 × 100 mL). The combined
organic layers were dried (Na2SO4) and concentrated to give
9 as a pale yellow oily foam. This foam was dissolved in 50/
40/5/5 DCM/TFA/TIS/H2O (40 mL), stirred for 20 min, and
concentrated to give mono-cysteic acid derivative 10 as a yellow
oil. This oil was used in the subsequent reaction without
further purification. MS: m/z 867.5 [M + H]+.
LTB4-(Csa)2-H (11). A solution of 10 in DMF (25 mL) and
DIEA (2.70 mL, 15.5 mmol) was treated with a solution of Boc-
Csa(Na)-OBt (8) prepared from Boc-Csa(Na)-OH (1.04 g, 3.6
mmol), HBTU (1.36 g, 3.6 mmol), and DIEA (1.25 mL, 7.2
mmol) in DMF (20 mL). The solution was stirred for 1 h and
concentrated to give a viscous yellow oil. This oil was dissolved
in 50/40/5/5 DCM/TFA/TIS/H2O (50 mL), stirred for 20 min,
and concentrated. Crude product was purified by HPLC using
a 3%/min gradient of 36-72% ACN containing 0.1% TFA at a
flow rate of 40 mL/min. The product fraction eluting at 5.7
min was lyophilized to give dicysteic acid derivative 11 (1.03
1
g, 0.91 mmol, 44% from compound 3) as a colorless solid. H
NMR (CD3OD): δ 7.97-7.94 (m, 2H), 7.94-7.91 (m, 2H), 7.88
(d, J ) 1.2 Hz, 1H), 7.63-7.57 (m, 6H), 7.53 (d, J ) 1.2 Hz,
1H), 4.87-4.83 (m, 1H), 4.56 (t, J ) 6.3 Hz, 2H), 4.50 (t, J )
7.2 Hz, 2H), 4.30 (t, J ) 6.0 Hz, 1H), 3.61-3.56 (m, 4H), 3.55-
3.50 (m, 4H), 3.48-3.37 (m, 7H), 3.25-3.16 (m, 4H), 3.11-
3.05 (m, 1H), 2.24 (t, J ) 7.2 Hz, 2H), 1.22-1.88 (m, 6H), 1.75-
1.66 (m, 6H), 1.51 (s, 6H), 1.39-1.32 (m, 2H); 13C NMR
(CD3OD): δ 175.4, 171.8, 168.7, 163.4, 161.7, 160.8, 153.4,
137.5, 134.3, 132.7, 132.6, 130.8, 130.6, 129.4, 129.2, 116.1,
106.9, 72.10, 71.63, 71.32, 71.30, 70.06, 69.88, 53.02, 52.74,
51.98, 51.47, 50.21, 42.12, 38.15, 38.11, 36.41, 36.21, 30.63,
30.46, 30.36, 30.06, 27.67, 24.08, 22.3; HRMS: Calcd for
C46H68N9O13S2 [M + H]: 1018.4372; Found: 1018.4386.
LTB4-(Csa)3-H (12). A solution of 11 (911 mg, 0.81 mmol)
and DIEA (625 µL, 3.6 mmol) in DMF (10 mL) was treated
with solution of Boc-Csa(Na)-OBt (8) prepared from Boc-Csa-
(Na)-OH (521 mg, 1.8 mmol), HBTU (679 mg, 1.8 mmol), and
DIEA (625 µL, 3.6 mmol) in DMF (10 mL). The reaction was
stirred for 2 h and concentrated. The residue was dissolved in
50/40/5/5 DCM/TFA/TIS/H2O (20 mL), stirred for 25 min, and
concentrated. Crude product was purified by HPLC using a
3%/min gradient of 31.5-54% ACN containing 0.1% TFA at a
flow rate of 40 mL/min. The product fraction eluting at 4.8
min was lyophilized to give tricysteic acid derivative 12 (1.01
1
g, 0.78 mmol, 97%) as a colorless solid. H NMR (CD3OD): δ
8.03-7.99 (m, 2H), 7.93-7.88 (m, 3H), 7.69-7.59 (m, 7H), 4.72
(dd, J ) 3.5, 9.7 Hz, 1H), 4.65 (dd, J ) 3.6, 9.6 Hz, 1H), 4.60
(t, J ) 6.7 Hz, 2H), 4.54 (t, J ) 7.2 Hz, 2H), 4.37 (dd, J ) 3.6,
9.6 Hz, 1H), 3.63-3.58 (m, 4H), 3.58-3.53 (m, 4H), 3.53-3.39
(m, 6H), 3.36-3.30 (m, 1H), 3.30-3.19 (m, 7H), 2.36 (t, J )
7.5 Hz, 2H), 2.06-1.99 (m, 2H), 1.99-1.90 (m, 4H), 1.79-1.70
(m, 6H), 1.52 (s, 6H), 1.41-1.33 (m, 2H); 13C NMR (CD3OD):
δ 176.1, 172.4, 171.7, 168.9, 162.9, 162.1, 161.7, 152.6, 136.9,
133.2, 133.0, 132.9, 130.8, 130.7, 129.8, 129.5, 116.7, 106.8,
73.30, 71.53, 71.21, 71.14, 69.91, 69.63, 53.61, 53.04, 52.30,
52.03, 51.73, 50.99, 50.16, 41.93, 38.57, 38.04, 36.18, 36.03,
30.53, 30.19, 30.09, 29.92, 27.63, 24.15, 22.23; HRMS: Calcd
for C49H73N10O17S3 [M + H]: 1169.4312; Found: 1169.4309.
LTB4-(Csa)4-H (13). A solution of 12 (705 mg, 0.55 mmol)
and DIEA (630 µL, 3.6 mmol in DMF (12 mL)) was treated
with a solution of Boc-Csa(Na)-OBt (8) prepared from Boc-Csa-
(Na)-OH (351 mg, 1.2 mmol), HBTU (457 mg, 1.2 mmol), and
DIEA (420 µL, 2.4 mmol) in DMF (7 mL). The reaction was
stirred for 40 min and concentrated. The residue was dissolved
in 50/40/5/5 DCM/TFA/TIS/H2O (20 mL), stirred for 25 min,
and concentrated. Crude product was purified by HPLC using
a 3%/min gradient of 27-54% ACN containing 0.1% TFA at a
flow rate of 40 mL/min. The product fraction eluting at 5.3
min was lyophilized to give tetracysteic acid derivative 13
(674.8 mg, 0.47 mmol, 86%) as a colorless solid. 1H NMR (CD3-
OD and CD3CN): δ 7.88-7.83 (m, 4H), 7.77 (d, J ) 1.2 Hz,
1H), 7.61-7.53 (m, 4H), 7.34 (d, J ) 1.2 Hz, 1H), 4.71 (dd, J
) 3.9, 9.3 Hz, 1H), 4.57-4.51 (m, 2H), 4.46-4.39 (m, 4H), 4.37
(dd, J ) 4.2, 9.0 Hz, 1H), 3.56-3.34 (m, 14H), 3.31-3.14 (m,
8H), 3.11 (t, J ) 6.9 Hz, 2H), 2.17 (t, J ) 7.2 Hz, 2H), 1.90 (p,
J ) 7.5 Hz, 2H), 1.86-1.78 (m, 4H), 1.69 (p, J ) 6.6 Hz, 2H),
1.66-1.57 (m, 4H), 1.44 (s, 6H), 1.27-1.20 (m, 2H); 13C NMR
(CD3OD and CD3CN): δ 175.6, 172.4, 172.0, 171.9, 168.8,

Radiolabeled LTB4 Antagonists for Infection Imaging
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6451
163.3, 161.8, 159.9, 153.2, 137.2, 134.1, 132.7, 132.6, 130.8,
130.6, 129.2, 129.1, 115.8, 106.9, 71.71, 71.11, 70.91, 70.87,
69.80, 69.53, 53.06, 52.88, 52.51, 51.86, 51.67, 51.53, 51.23,
50.95, 50.13, 41.66, 37.87, 37.76, 36.32, 35.98, 30.28, 30.02,
29.84, 29.68, 27.58, 23.83, 21.99; HRMS: Calcd for C52H78N11-
O21S4 [M + 2H]: 660.7162; Found: 660.7163.
25 mL/min, followed by a 5.7%/min gradient of 4.5-90% ACN
containing 0.1% TFA at a flow rate 25 mL/min. Product
fraction eluting a 10 min was lyophilized to give divalent
tetracysteic acid derivative 16 (134 mg, 0.047 mmol, 62%) as
a colorless solid. 1H NMR (CD3CN and D2O): δ 7.78-7.70 (m,
8H), 7.64 (d, J ) 1.2 Hz, 2H), 7.59-7.47 (m, 12H), 7.26 (d, J
) 1.2 Hz, 2H), 4.72 (dd, J ) 3.9 and 9.3 Hz, 1H), 4.65-4.55
(m, 5H), 4.55-4.49(m, 2H), 4.38 (t, J ) 7.5 Hz, 4H), 4.33 (t, J
) 6.3 Hz, 4H), 3.99 (dd, J ) 5.4 and 7.8 Hz, 1H), 3.53-3.43
(m, 16H), 3.42-3.35 (m, 9H), 3.32-3.14 (m, 15H), 3.12 (t, J )
7.2 Hz, 4H), 3.07 (t, J ) 7.2 Hz, 4H), 2.51 (t, J ) 6.9 Hz, 2H),
2.21-2.11 (m, 5H), 2.09-2.01 (m, 1H), 1.85 (p, J ) 7.6 Hz,
4H), 1.81-1.71 (m, 8H), 1.65 (p, J ) 6.8 Hz, 4H), 1.60 (p, J )
6.6 Hz, 4H), 1.55 (p, J ) 7.4 Hz, 4H), 1.40 (s, 12H), 1.21-1.12
(m, 4H); 13C NMR (CD3CN and D2O): δ 175.8, 175.2, 172.3,
172.1, 171.7, 170.2, 162.4, 161.7, 160.2, 152.1, 136.2, 132.9,
132.7, 132.6, 130.6, 130.5, 128.9, 128.8, 115.6, 106.2, 72.02,
70.58, 70.40, 70.35, 69.41, 69.14, 53.94, 52.43, 52.26, 52.17,
52.00, 51.73, 51.56, 51.45, 51.38, 51.23, 50.88, 49.86, 41.12,
37.53, 37.42, 35.99, 35.63, 32.18, 29.75, 29.39, 29.27, 29.10,
27.24, 26.82, 23.42, 21.52; HRMS: Calcd for C109H159N23O44S8-
Na2 [M + 2Na]+2: 1397.9231; Found: 1397.920.
6-(2-Benzaldehydehydrazono)nicotinic Acid (14). A
suspension of 6-hydrazinonicotinic acid (274 mg, 1.8 mmol) in
DMF (11 mL) was treated with benzaldehyde (1.1 mL, 11
mmol) and stirred for 2 days. Filtration of the reaction mixture
gave 14 as a pale yellow powder. The filtrate was diluted with
DCM (100 mL) to give an additional crop of 14. The combined
solids were washed with DCM and gave the title compound
1
(284 mg, 1.2 mmol, 66%) as a light yellow powder. H NMR
(DMSO-d6 and CD3OD): δ 8.53 (s, 1H), 8.42 (s, 1H), 8.30 (d, J
) 9.0 Hz, 1H), 7.96 (s, 2H), 7.47 (dd, J ) 1.8 and 5.4 Hz, 3H),
7.38 (d, J ) 9.0 Hz,1H); 13C NMR (DMSO-d6 and CD3OD): δ
164.5, 141.9, 133.4, 130.7, 128.8, 127.8, 117.5; HRMS: Calcd
for C13H12N3O2 [M + H]: 242.0924; found: 242.0923.
HYNIC Conjugate 15. A solution of 14 (10.2 mg, 42.3
µmol), PyBOP (19.3 mg, 37.1 µmol), and DIEA (20 µL, 115
µmol) in DMF (0.75 mL) was added to 13 (14.0 mg, 10.6 µmol)
in DMF (1.0 mL), and the reaction was stirred for 40 min.
Concentration gave crude 15, which was purified by HPLC
using isocratic conditions of 27% ACN containing 0.1 M
NaOAc, pH 7 for 2 min at a flow rate of 25 mL/min, followed
by a 1.0%/min gradient of 27-54% ACN containing 0.1 M
NaOAc, pH 7 at a flow rate of 25 mL/min. The product fraction
eluting at 18.1 min was diluted with 3 volumes of H2O and
reloaded on the same preparative HPLC column equilibrated
with 4.5% ACN containing 0.1% TFA. The product on the
column was desalted by elution with 4.5% ACN containing
0.1% TFA for 15 min at a flow rate of 25 mL/min, followed by
a 5.7%/min gradient of 4.5-90% ACN containing 0.1% TFA
at a flow rate 25 mL/min. Product fraction eluting a 10.4 min
was lyophilized to give HYNIC conjugated monovalent tetra-
cysteic acid derivative 15 (4.9 mg, 3.2 µmol, 30%) as a colorless
DTPA Conjugate 17. A solution of 16 (50.1 mg, 0.0182
mmol) and DIEA (64 µL, 0.364 mmol) in anhydrous DMF (2.0
mL) was treated dropwise with a solution of DTPA dianhydride
(65.0 mg, 0.182 mmol) and DIEA (16 µL, 0.091 mmol) in
anhydrous DMF (3.0 mL) over 10 min. The reaction was
quenched after 1.5 h by the addition of water (0.1 mL) and
concentrated under vacuum to give a colorless glassy solid.
The crude product was purified by HPLC on a Phenomenex
Jupiter C18 column (10 µ, 21.2 × 250 mm) using a 0.9%/min
gradient of 27-63% ACN containing 0.1% TFA at a flow rate
of 20 mL/min. Product fraction eluting at 25.1 min was
lyophilized to give 17 as a colorless solid (55.0 mg, 0.0176
1
mmol, 96%). H NMR (D2O:CD3CN 1:9): δ 7.58 (d, J ) 7.62
Hz, 8H), 7.52-7.36 (m, 14H), 7.04 (s, 2H), 4.74-4.69 (m, 1H),
4.67-4.62 (m, 4H), 4.62-4.56 (m, 4H), 4.54-4.50 (m, 2H),
4.41-4.33 (m, 5H), 4.22-4.08 (m, 12H), 3.64 (s, 2H), 3.53-
3.41 (m, 20H), 3.41-3.13 (m, 25H), 3.09 (t, J ) 6.75 Hz, 4H),
3.02 (t, J ) 6.75 Hz, 4H), 2.42-2.27 (m, 2H), 2.16-2.08 (m,
5H), 1.94-1.86 (m, 1H), 1.84-1.73 (m, 8H), 1.72-1.47 (m,
16H), 1.39 (s, 12H), 1.12-1.04 (m, 4H); HRMS: Calcd for
1
powder. H NMR (CD3CN and D2O): δ 8.50 (d, J ) 2.1 Hz,
1H), 8.32 (dd, J ) 2.1 and 9.0 Hz, 1H), 8.21 (s, 1H), 8.01-7.96
(m, 2H), 7.85-7.80 (m, 2H), 7.78-7.74 (m, 2H), 7.69 (d, J )
1.2 Hz, 1H), 7.53-7.41 (m, 9H), 7.71 (d, J ) 9 Hz, 1H), 7.00
(d, J ) 1.2 Hz, 1H), 4.88 (dd, J ) 4.2 and 9.3 Hz, 1H), 4.65
(dd, J ) 4.2 and 8.4 Hz, 1H), 4.58 (dd, J ) 4.2 and 8.4 Hz,
1H), 4.52 (dd, J ) 4.2 and 8.4 Hz, 1H), 4.37 (t, J ) 7.2 Hz,
2H), 4.35 (t, J ) 7.8 Hz, 2H), 3.53-3.44 (m, 8H), 3.44-3.35
(m, 5H), 3.35-3.25 (m, 3H), 3.25-3.15 (m, 4H), 3.13 (t, J )
6.6 Hz, 2H), 3.07 (t, J ) 6.7 Hz, 2H), 2.11 (t, J ) 7.6 Hz, 2H),
1.88-1.78 (m, 4H), 1.74 (p, J ) 7.2 Hz, 2H), 1.65 (p, J ) 6.6
Hz, 2H), 1.60 (p, J ) 6.7 Hz, 2H), 1.57 (p, J ) 7.6 Hz, 2H),
1.40 (s, 6H), 1.22-1.14 (m, 2H); 13C NMR (CD3CN and D2O):
δ 175.9, 172.3, 166.2, 165.1, 163.0, 162.1, 155.7, 152.4, 144.0,
139.1, 138.8, 138.4, 134.5, 132.9, 131.6, 131.5, 130.8, 130.6,
130.5, 129.6, 128.8, 128.7, 122.5, 114.1, 113.7, 107.7, 71.21,
71.17, 71.02, 70.97, 69.98, 69.74, 68.62, 53.14, 53.01, 52.75,
52.17, 52.05, 51.76, 51.70, 50.32, 41.86, 38.07, 37.87, 36.60,
36.20, 30.44, 30.17, 30.12, 29.92, 27.83, 23.99, 22.29; HRMS:
Calcd for C65H88N14O22S4 [M + 2H]2+: 772.2535; found 772.2535.
C
123H182N26O53S8 [M + 2H]2+: 1563.5056; Found: 1563.505.
HYNIC Conjugate 18. A solution of 14 (17.5 mg, 73 µmol),
PyBOP (37.8 mg, 72.7 µmol), and DIEA (20 µL, 115 µmol) in
DMF (1.0 mL) was stirred for 15 min and added to a solution
of 16 (50.0 mg, 17.4 µmol) and DIEA (30 µL, 172 µmol) in DMF
(1.0 mL). Stirring was continued for 20 h, and the reaction
mixture was concentrated. Crude product was purified by
HPLC using isocratic conditions of 40.5% ACN containing 0.1
M NaOAc, pH 7 for 2 min at a flow rate of 25 mL/min, followed
by a 0.39%/min gradient of 40.5-49.5% ACN containing 0.1
M NaOAc, pH 7 at a flow rate of 25 mL/min. The product
fraction eluting at 11 min was diluted with 3 volumes of H2O
and reloaded on the same preparative HPLC column equili-
brated with 4.5% ACN containing 0.1% TFA. The product on
the column was desalted by elution with 4.5% ACN containing
0.1% TFA for 15 min at a flow rate of 25 mL/min, followed by
a 5.7%/min gradient of 4.5-90% ACN containing 0.1% TFA
at a flow rate 25 mL/min. Product fraction eluting a 10.6 min
was lyophilized to give HYNIC-conjugated divalent tetracysteic
acid derivative 18 (40.2 mg, 13.5 µmol, 78%) as a colorless
solid. 1H NMR (D2O:CD3CN 1:1): δ 8.58-8.55 (m, 1H), 8.12-
8.07 (m, 1H), 8.05-8.00 (m, 4H), 7.98 (s, 1H), 7.73-7.65 (m,
6H), 7.62 (d, J ) 1.2 Hz, 2H), 7.50-7.38 (m, 12H), 7.35 (t, J )
7.2 Hz, 2H), 7.32-7.24 (m, 2H), 6.83 (d, J ) 1.2 Hz, 2H), 4.75-
4.68 (m, 3H), 4.66 (dd, J ) 4.6 and 8.3 Hz, 1H), 4.61 (dd, J )
4.6 and 8.1 Hz, 2H), 4.55-4.50 (m, 2H), 4.45 (dd, J ) 4.8 and
8.3 Hz, 1H), 4.33 (t, J ) 6.9 Hz, 8H), 3.53-3.42 (m, 16H), 3.42-
3.10 (m, 28H), 3.07 (t, J ) 6.9 Hz, 4H), 2.42 (t, J ) 7.6 Hz,
2H), 2.22-2.16 (m, 1H), 2.13-2.07 (m, 4H), 2.07-2.00 (m, 1H),
1.86-1.75 (m, 8H), 1.70 (t, J ) 7.2 Hz, 4H), 1.66 (t, J ) 6.6
Hz, 4H), 1.60 (t, J ) 6.6 Hz, 4H), 1.54 (t, J ) 7.6 Hz, 4H), 1.38
(s, 12H), 1.21-1.12 (m, 4H); 13C NMR (D2O:CD3CN 1:1): δ
(LTB4-(Csa)4)2-Glu-H (16). A solution of 13 (264 mg, 0.18
mmol), Boc-Glu(OTfp)-OTfp (41.2 mg, 0.076 mmol), HOAt (22.7
mg, 0.17 mmol), and DIEA (130 µL, 0.75 mmol) in DMF (2
mL) was stirred for 6 h and concentrated to give a viscous
yellow oil. This oil was dissolved in DCM (6.5 mL) and treated
with a solution of anisole/H2O/TFA (0.75 mL/0.75 mL/7 mL)
and stirred for 30 min. The reaction solution was concentrated
to give crude title compound as a viscous yellow oil. Crude
product was purified by HPLC using isocratic conditions of
36% ACN containing 0.1 M NaOAc, pH 7 for 2 min at a flow
rate of 25 mL/min, followed by a 0.78%/min gradient of 36-
54% ACN containing 0.1 M NaOAc, pH 7 at a flow rate of 25
mL/min. The product fraction eluting at 15 min was diluted
with 3 volumes of H2O, and reloaded on the same preparative
HPLC column equilibrated with 4.5% ACN containing 0.1%
TFA. The product on the column was desalted by elution with
4.5% ACN containing 0.1% TFA for 15 min at a flow rate of

6452 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Broekema et al.
181.22, 175.76, 175.33, 174.19, 172.47, 172.18, 172.00, 171.88,
171.78, 168.97, 165.27, 161.58, 159.24, 156.23, 153.30, 143.78,
139.73, 138.96, 135.84, 130.40, 130.30, 130.19, 129.80, 128.06,
127.78, 121.42, 118.75, 116.81, 112.70, 107.69, 107.46, 70.64,
70.73, 70.47, 70.41, 69.45, 69.25, 66.61, 54.99, 52.40, 52.39,
52.22, 52.05, 52.00, 51.90, 51.68, 51.62, 49.76, 41.39, 37.57,
37.34, 36.08, 35.66, 32.96, 29.93, 29.81, 29.62, 29.41, 27.30,
24.45, 23.45, 21.84; HRMS: Calcd for C122H170N26O45S8 [M +
2H]+2: 1487.4784; Found: 1487.482.
were immobilized in a mold and placed prone on a gamma
camera (Orbiter, Siemens, Hoffman Estates, IL) using a low-
energy parallel hole collimator in case of the 99mTc-labeled
compounds, whereas a medium-energy collimator was used
during acquisition of rabbits injected with [111In(17)]. Images
(300 000 cts/image) were obtained at several time points after
injection starting immediately after injection until 8 h postin-
jection (pi). Images were stored digitally in a 256 × 256 matrix.
All images were windowed identically, allowing a fair com-
parison among the various experiments. At 8 h pi all rabbits
were euthanized. A blood sample was taken by cardiac
puncture. Tissues were dissected and weighed. The activity
in tissues was measured in a shielded well-type gamma
counter together with the injection standards and was ex-
pressed as the percentage of the injected dose per gram (%ID/
g).
Receptor Binding Assay. In vitro binding studies were
performed on purified human granulocytes, purified as de-
scribed previously.34 Briefly, increasing amounts of 4, 15, 17,
or 18, (0-20 µM) were added to 1 × 108 cells in the presence
of 10 000 cpm [111In(17)] (0.5 nM) in 0.25 M Tris/HCl, pH 7.2.
The cell suspensions were incubated for 1 h at 37 °C, the cells
were washed twice (5 min, 5000g), supernatant was discarded,
and the radioactivity in the pellet (total bound activity) was
measured in a shielded well-type gamma counter (Wizard,
Pharmacia-LKB, Uppsala, Sweden). The specifically bound
fraction versus the LTB4 antagonist concentration was plotted.
The IC50 (50% inhibitory concentration) was determined as
being the concentration of the LTB4 antagonist that caused
50% inhibition of the maximum binding of [111In(17)].
General Procedure for 99mTc-Radiolabeling of 18. A
solution of 18 (10 µg), SnSO4 (25 µL of a 1 mg/mL solution in
0.1 N HCl), coligand(s), and 110 MBq Na-99mTcO4 in 350 µL of
PBS, pH 7.0 was heated for 30 min at 100 °C. Radiochemical
purity was checked by instant thin-layer chromatography
(ITLC) on silica gel strips (Gelman Sciences, Inc., Ann Arbor,
MI) in 0.1 M Na-citrate buffer, pH 6.0. Strips were analyzed
in a well-type gamma counter (Wizard, Pharmacia-LKB,
Uppsala, Sweden). In addition, RP-HPLC was performed on
a C18 column (Zorbax Rx-C18 4.6 mm × 25 cm) on an Agilent
1100 HPLC system equipped with an in-line radio detector
(Canberra Packard, Brussels, Belgium), using 5%/min gradient
of 0-100% ACN containing 10 mM ammonium acetate pH 7.2
at a flow rate of 1.0 mL/min. The radiolabeling efficiency was
>95% in all cases.
Statistical Analysis. All mean values are presented as
mean ( standard deviation. Statistical analysis was performed
using one-way ANOVA. Results were corrected for multiple
comparisons with the Bonferroni Multiple Comparisons Test.
The level of significance was set at 0.05.
Acknowledgment. The authors thank Mr. Gerry
Grutters and Mr. Hennie Eijkholt (University of
Nijmegen, Central Animal Laboratory) for technical
assistance, and John Pietryka (BMS Medical Imaging)
for analytical support.
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