Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
PHARMACEUTICALS FOR THE IMAGING OF ANGIOGENIC DISORDERS
FOR USE IN COMBINATION THERAPY
FIELD OF THE INVENTION
The present invention provides novel
pharmaceuticals useful for the diagnosis and treatment
of cancer, methods of imaging tumors in a patient, and
methods of treating cancer in a patient. The invention
is also directed to novel pharmaceutical compositions
and combination therapy comprising a compound of the
invention or a pharmaceutically acceptable salt thereof,
and at least one agent selected from the group
consisting of an anti-cancer agent, a photosensitizer
agent and a radiosensitizer agent. The present invention
also provides novel pharmaceuticals useful for
monitoring therapeutic angiogenesis treatment and
destruction of new angiogenic vasculature. The
pharmaceuticals are comprised of a targeting moiety that
binds to a receptor that is upregulated during
angiogenesis, an optional linking group, and a
therapeutically effective radioisotope or diagnostically
effective imageable moiety. The therapeutically
effective radioisotope emits a particle or electron
sufficient to be cytotoxic. The imageable moiety is a
gamma ray or positron emitting radioisotope, a magnetic
resonance imaging contrast agent, an X-ray contrast
agent, or an ultrasound contrast agent.
BACKGROUND OF THE INVENTION
Cancer is a major public health concern in the
United States and around the world. It is estimated
that over 1 million new cases of invasive cancer will be
diagnosed in the United Stakes in 1998. The most
prevalent forms of the disease are solid tumors of the
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lung, breast, prostate, colon and rectum. Cancer is
typically diagnosed by a combination of in vitro tests
and imaging procedures. The imaging procedures include
X-ray computed tomography, magnetic resonance imaging,
ultrasound imaging and radionuclide scintigraphy.
Frequently, a contrast agent is administered to the
patient to enhance the image obtained by X-ray CT, MRI
and ultrasound, and the administration of a
radiopharmaceutical that localizes in tumors is required
for radionuclide scintigraphy.
Treatment of cancer typically involves the use of
external beam radiation therapy and chemotherapy, either
alone or in combination, depending on the type and
extent of the disease. A number of anti-cancer agents
are available, but generally they all suffer from a lack
of specificity for tumors versus normal tissues,
resulting in considerable side-effects. The
effectiveness of these treatment modalities is also
limited, as evidenced by the high mortality rates for a
number of cancer types, especially the more prevalent
solid tumor diseases. More effective and specific
treatment means continue to be needed.
Despite the variety of imaging procedures available
for the diagnosis of cancer, there remains a need for
improved methods. In particular, methods that can
better differentiate between cancer and other pathologic
conditions or benign physiologic abnormalities are
needed. One means of achieving this desired improvement
would be to administer to the patient a
metallopharmaceutical that localizes specifically in the
tumor by binding to a receptor expressed only in tumors
or expressed to a significantly greater extent in tumors
than in other tissue. The location of the
metallopharmaceutical could then be detected externally
either by its imageable emission in the case of certain
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radiopharmaceuticals or by its effect on the relaxation
rate of water in the immediate vicinity in the case of
magnetic resonance imaging contrast agents.
This tumor specific metallopharmaceutical approach
can also be used for the treatment of cancer when the
metallopharmaceutical is comprised of a particle
emitting radioisotope. The radioactive decay of the
isotope at the site of the tumor results in sufficient
ionizing radiation to be toxic to the tumor cells. The
20 specificity of this approach for tumors minimizes the
amount of normal tissue that is exposed to the cytotoxic
agent and thus may provide more effective treatment with
fewer side-effects.
Previous efforts to achieve these desired
l5 improvements in cancer imaging and treatment have
centered on the use of radionuclide labeled monoclonal
antibodies, antibody fragments and other proteins or
polypeptides (i.e., molecular weight over 10,000 D) that
bind to tumor cell surface receptors. The specificity
20 of these radiopharmaceuticals is frequently very high,
but they suffer from several disadvantages. First,
because of their high molecular weight, they are
generally cleared from the blood stream very slowly,
resulting in a prolonged blood background in the images.
25 Also, due to their molecular weight they do not
extravasate readily at the site of the tumor and then
only slowly diffuse through the extravascular space to
the tumor cell surface. This results in a very limited
amount of the radiopharmaceutical reaching the receptors
30 and thus very low signal intensity in imaging and
insufficient cytotoxic effect for treatment.
Alternative approaches to cancer imaging and
therapy have involved the use of small molecules, such
as peptides, that bind to tumor cell surface receptors.
35 An 111=n labeled somatostatin receptor binding peptide,
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lllln-DTPA-D-Phe1-octeotide, is in clinical use in many
countries for imaging tumors that express the
somatostatin receptor (Baker, et al., Life Sci., 1991,
49, 1583-91 and Krenning, et al., Eur. J. Nucl. Med.,
1993, 20, 716-31). Higher doses of this
radiopharmaceutical have been investigated for potential
treatment of these types of cancer (Krenning, et al.,
Digestion, 1996, 57, 57-61). Several groups are
investigating the use of Tc-99m labeled analogs of
111In-DTPA-D-Phe1-octeotide for imaging and Re-186
labeled analogs for therapy (Flanagan, et al., U.S.
5,556,939, Lyle, et al., U.S. 5,382,654, and Albert et
al.,U.S. 5,650,134).
Angiogenesis is the process by which new blood
vessels are formed from pre-existing capillaries or post
capillary venules; it is an important component of a
variety of physiological processes including ovulation,
embryonic development, wound repair, and collateral
vascular generation in the myocardium. It is also
central to a number of pathological conditions such as
tumor growth and metastasis, diabetic retinopathy, and
macular degeneration. The process begins with the
activation of existing vascular endothelial cells in
response to a variety of cytokines and growth factors.
Tumor released cytokines or angiogenic factors stimulate
vascular endothelial cells by interacting with specific
cell surface receptors for the factors. The activated
endothelial cells secrete enzymes that degrade the
basement membrane of the vessels. The endothelial cells
then proliferate and invade into the tumor tissue. The
endothelial cells differentiate to form lumens, making
new vessel offshoots of pre-existing vessels. The new
blood vessels then provide nutrients to the tumor
permitting further growth and a route for metastasis.
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Under normal conditions, endothelial cell
proliferation is a very slow process, but it increases
for a short period of time during embryogenesis,
ovulation and wound healing. This temporary increase in
5 cell turnover is governed by a combination of a number
of growth stimulatory factors and growth suppressing
factors. In pathological angiogenesis, this normal
balance is disrupted resulting in continued increased
endothelial cell proliferation. Some of the pro-
angiogenic factors that have been identified include
basic fibroblast growth factor (bFGF), angiogenin, TGF-
alpha, TGF-beta, and vascular endothelium growth factor
(VEGF), while interferon-alpha, interferon-beta and
thrombospondin are examples of angiogenesis suppressors.
The proliferation and migration of endothelial
cells in the extracellular matrix is mediated by
interaction with a variety of cell adhesion molecules
(Folkman, J., Nature Medicine, 2995, 1, 27-31).
Integrins are a diverse family of heterodimeric cell
surface receptors by which endothelial cells attach to
the extracellular matrix, each other and other cells.
The integrin oc,~(33 is a receptor for a wide variety of
extracellular matrix proteins with an exposed tripeptide
Arg-Gly-Asp moiety and mediates cellular adhesion to its
ligands: vitronectin, fibronectin, and fibrinogen,
among others. The integrin ocV(33 is minimally expressed
on normal blood vessels, but, is significantly
upregulated on vascular cells within a variety of human
tumors . The role of the ocv(33 receptors is to mediate
the interaction of the endothelial cells and the
extracellular matrix and facilitate the migration of the
cells in the direction of the angiogenic signal, the
tumor cell population. Angiogenesis induced by bFGF or
TNF-alpha depend on the agency of the integrin Oc,~,(33,
while angiogenesis induced by VEGF depends on the
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integrin oc~,(35 (Cheresh et. al., Science, 1995, 270,
1500-2 ) . Induction of expression of the integrins 0c1(31
and OG2~1 on the endothelial cell surface is another
important mechanism by which VEGF promotes angiogenesis
(Senger, et. al., Proc. Natl. Acad, Sci USA, 1997, 94,
13612-7).
Angiogenic factors interact with endothelial cell
surface receptors such as the receptor tyrosine kinases
EGFR, FGFR, PDGFR, Flk-1/KDR, Flt-1, Tek, Tie,
neuropilin-1, endoglin, endosialin, and Axl. The
receptors Flk-1/KDR, neuropilin-1, and Flt-1 recognize
VEGF and these interactions play key roles in VEGF-
induced angiogenesis. The Tie subfamily of receptor
tyrosine kinases are also expressed prominently during
blood vessel formation.
Because of the importance of angiogenesis to tumor
growth and metastasis, a number of anti.-cancer
approaches are being developed to interfere with or
prevent this process. One of these approaches, involves
the use of anti-angiogenic proteins such as angiostatin
and endostatin. Angiostatin is a 38 kDa fragment of
plasminogen that has been shown in animal models to be a
potent inhibitor of endothelial cell proliferation.
(0'Reilly et. al., Cell, 1994, 79, 315-328) Endostatin
is a 20 kDa C-terminal fragment of collagen XVIII that
has also been shown to be a potent inhibitor. (0'Reilly
et. al., Cell, 1997, 88, 277-285)
Systemic therapy with endostatin has been shown to
result in strong anti-tumor activity in animal models.
However, human clinical trials of these two anti-cancer
agents of biological origin have been hampered by lack
of availability.
Another approach to anti-angiogenic therapy is to
use targeting moieties that interact with endothelial
cell surface receptors expressed in the angiogenic
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vasculature to which are attached anti-cancer agents.
Burrows and Thorpe (Pros. Nat. Acad. Sci, USA, 1993, 90,
8996-9000) described the use of an antibody-immunotoxin
conjugate to eradicate tumors in a mouse model by
destroying the tumor vasculature. The antibody was
raised against an endothelial cell class II antigen of
the major histocompatibility complex and was then
conjugated with the cytotoxic agent, deglycosylated
ricin A chain. The same group (Clin. Can. Res., 1995,
1, 1623-1634) investigated the use of antibodies raised
against the endothelial cell surface receptor, endoglin,
conjugated to deglycosylated ricin A chain. Both of
these conjugates exhibited potent anti-tumor activity in
mouse models. However, both still suffer drawbacks to
routine human use. As with most antibodies or other
large, foreign proteins, there is considerable risk of
immunologic toxicity which could limit or preclude
administration to humans. Also, while the vasculature
targeting may improve the local concentration of the
attached anti-cancer agents, the agents still must be
cleaved from the antibody carrier and be transported or
diffuse into the cells to be cytotoxic.
Thus, it is desirable to provide a combination of
angiogenesis-targeted therapeutic radiopharmaceuticals
and an anti-cancer agents or a radiosensitizer agent, or
a pharmaceutically acceptable salt thereof, which target
the luminal side of the neovasculature of tumors, to
provide a surprising, and enhanced degree of tumor
suppression relative to each treatment modality alone
without significant additive toxicity.
Photodynamic therapy has also been used in the
treatment of cancer. Photodynamic therapy involves the
administration of a photosensitive agent and subsequent
irradiation with light to excite the photosensitizer,
thus producing a cytotoxic effect. Spears, U.S. Pat. No.
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4,512,762, and U.S. Pat. No. 4,566,636, Kelly, et al.
United States Patent 6,235,767.
In photodynamic therapy, the photosensitizers used
are capable of localizing in malignant cells, either by
natural tendency or because they have been intentionally
targeted to a specific type of tissue, or both. When
irradiated, they may be capable of fluorescing and,
thus, may also be useful in diagnostic methods related
to detecting target tissue. However, even more
importantly, the photosensitizer has the capacity, when
irradiated with light at a wavelength which the compound
absorbs, of causing a cytotoxic effect against whatever
cells or other tissue in which the photosensitizer has
localized.
In one form of this therapy, a photosensitizer
agent having a characteristic light absorption waveband
is first administered to the patient, typically either
orally or by injection. Abnormal tissue in the body is
known to selectively absorb certain photosensitizer
agents to a much greater extent than normal tissue. More
effective selectivity can be achieved using a
photoreactive agent that is bound to an antibody, which
links with antigens on targeted cells. The cancerous or
abnormal tissue that has absorbed or linked with the
photosensitizer dye is then destroyed by administering
light of an appropriate wavelength or waveband
corresponding to the absorption wavelength or waveband
of the photosensitizer agent.
Photosensitizing agents such as Photofrin, a
haematoporphyrin derivative, are known. ( Dougherty, T.
J. (1987) Photosensitizers: therapy and detection of
malignant tumours. Photochem. Photobiol., 45, 879-889,
and Boyle R. W. and D. David (1996) Structure and
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biodistribution relationships of photodynamic
sensitizers. Photochem. Photobiol. 64, 469-485) Also,
Rodgers,et al., United States Patent No. 6,225,333,
discloses treating cancers with a variety of
photosensitizing agents for example naphthalocyanine
photosensitizing agents; tetrapyrrole-based
photosensitizers; including porphyins; chlorins;,
phthalocyanines; napthalocyanines; coumarins and
psoralens. Furthermore, Mazur, et al., United States
Patent No. 6,229,048, discloses a method for treatment
of solid tumors by photodynamic therapy comprising
administering a photosensitizer selected from the group
consisting~of: 1,3,4,6-tetrahydroxyhelianthrone;
1,3,4,6-tetramethoxyhelianthrone; 10,13-dimethyl-
1,3,4,6-tetrahydroxyhelianthrone; 10,13-
di(methoxycarbonyl)-1,3,4,6-tetramethoxyhelianthrone;
1,6-di-N-butylamino-3,4-dimethoxy-helianthrone; 1,6-di-
N-butylamino-3,4-dimethoxy-10,13-dimethyl-helianthrone;
1,6-di-(N-hydroxyethylamino)-3,4-dimethoxy-helianthrone;
2,5-dibromo-1,3,4,6-tetrahydroxyhelianthrone; and 2,5-
dibromo-10,13-dimethyl-1,3,4,6-tetrahydroxyhelianthrone.
In another method, "green porphyrins" have been
used in photodynamic therapy with light having a
wavelength range around 670-780 nm. See for example,
Levy et al., U.S. Pat. No. 5,399,583 Levy et al., U.S.
Pat. No. 4,920,143, Levy et al., U.S. Pat. No.
5,095,030; and Levy et al., and U.S. Pat. No. 5,171,749.
In most photodynamic therapy protocols, a method
must be found for the irradiating light to reach the
targeted tissue where the photosensitizer has been
localized. For example, a light-emitting balloon
catheter may be used or alternatively, a form of "liquid
light" may be injected into the vascular tree such that
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the "liquid light", perfuses the vasculature at the
target site. Spears, U.S. Pat. No. 4,512,762.
Alternatively
The targeted tissues are visually located by
5 imaging the treatment site through a fiber optic system
so that light from a laser source can be accurately
directed through the optical fiber to destroy the
abnormal tissue. Even when the internal treatment site
is accessible through natural body orifices, an
10 endoscope is usually required to visualize the targeted
tissue and accurately direct the light therapy
administered to the treatment site. Chen, United States
Patent No. 6,210,425 discloses an apparatus and a method
to identify an internal treatment site within a
patient's body for administration of light therapy and
treatment of the site.
Thus, it is also desirable to provide a combination
of a photosensitizer agent (as part of photodynamic
therapy), an angiogenesis-targeted therapeutic
radiopharmaceutical and an anti-cancer agent or a
radiosensitizer agent, or a pharmaceutically acceptable
salt thereof, which target the luminal side of the
neovasculature of tumors, to provide a surprising, and
enhanced degree of tumor suppression relative to each
treatment modality alone without significant additive
toxicity.
There is also a growing interest in therapeutic
angiogenesis to improve blood flow in regions of the
body that have become ischemic or poorly perfused..
Several investigators are using growth factors
administered locally to cause new vasculature to form
either in the limbs or the heart. The growth factors
VEGF and bFGF are the most common for this application.
Recent publications include: Takeshita, S., et. al., J.
Clin. Invest., 1994, 93, 662-670; and Schaper, W. and
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Schaper, J., Collateral Circulation: Heart, Brain,
Kidney, Limbs, Kluwer Academic Publishers, Boston, 1993.
The main applications that are under investigation in a
number of laboratories are for improving cardiac blood
flow and in improving peripheral vessal blood flow in
the limbs. For example, Henry, T. et. al. (J. Amer.
College Cardiology, 1998, 31, 65A) describe the use of
recombinant human VEGF in patients for improving
myocardial perfusion by therapeutic angiogenesis.
Patients received infusions of rhVEGF and were monitored
by nuclear perfusion imaging 30 and 60 days post
treatment to determine improvement in myocardial
perfusion. About 50% of patients showed improvement by
nuclear perfusion imaging whereas 5/7 showed new
collatoralization by angiography.
Thus, it is desirable to discover a method of
monitoring improved cardiac blood flow which is targeted
to new collatoral vessels themselves and not, as in
nuclear perfusion imaging, a regional consequence of new
eollatoral vessels.
SUMMARY OF THE INVENTION
It is one object of the present invention to
provide anti-angiogenic pharmaceuticals, comprised of a
targeting moiety that binds to a receptor that is
expressed in tumor neovasculature, an optional linking
group, and a radioactive metal ion that emits ionizing
radiation such as beta particles, alpha particles and
Auger or Coster-Kronig electrons. The receptor binding
compounds target the radioisotope to the tumor
neovasculature. The beta or alpha-particle emitting
radioisotope emits a cytotoxic amount of ionizing
radiation which results in cell death. The penetrating
ability of radiation obviates the requirement that the
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cytotoxic agent diffuse or be transported into the cell
to be cytotoxic.
It is another object of the present invention to
provide pharmaceuticals to treat rheumatoid arthritis.
These pharmaceuticals comprise a targeting moiety that
binds to a receptor that is upregulated during
angiogenesis, an optional linking group, and a
radioisotope that emits cytotoxic radiation (i.e., beta
particles, alpha particles and Auger or Coster-Kronig
electrons). In rheumatoid arthritis, the ingrowth of a
highly vascularized pannus is caused by the excessive
production of angiogenic factors by the infiltrating
macrophages, immune cells, or inflammatory cells..
Therefore, the radiopharmaceuticals of the present
invention that emit cytotoxic radiation could be used to
destroy the new angiogenic vasculature that results and
thus treat the disease.
It is another object of the present invention to
provide kits and therapeutic radiopharmacutical
compositions for use in combination therapy comprising a
radiopharmacutical of the invention and at least one
agents selected from the group consisting of an anti-
cancer agent and a radiosensitizer agent.
It is another object of the present invention to
provide kits and therapeutic radiopharmacutical
compositions for use in combination therapy comprising a
radiopharmacutical of the invention and a
photosensitising agent.
It is another object of the present invention to
provide a method of treating cancer comprising
administering to a patient in need of such treatment a
therapeutic radiopharmaceutical composition of the
invention in combination with photodynamic therapy..
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It is another object of the present invention to
provide tumor imaging agents, comprised of targeting
moiety that binds to a receptor that is upregulated
during angiogenesis, an optional linking group, and an
imageable moiety, such as a gamma ray or positron
emitting radioisotope, a magnetic resonance imaging
contrast agent, an X-ray contrast agent, or an
ultrasound contrast agent.
It is another object of the present invention to
provide imaging agents for monitoring the progress and
results of therapeutic angiogenesis treatment. These
agents comprise of targeting moiety that binds to a
receptor that is upregulated during angiogenesis, an
optional linking group, and an imageable moiety.
Imaging agents of the present invention could be
administered intravenously periodically after the
administration of growth factors and imaging would be
performed using standard techniques of the affected
areas, heart or limbs, to monitor the progress and
results of the therapeutic angiogenesis treatment (i.e.,
image the formation of new blood vessels).
It is another object of the present invention to
provide compounds useful for preparing the
pharmaceuticals of the present invention. These
compounds are comprised of a peptide or peptidomimetic
targeting moiety that binds to a receptor that is
upregulated during angiogenesis, Q, an optional linking
group, Ln, and a metal chelator or bonding moiety, Ch.
The compounds may have one or more protecting groups
attached to the metal chelator or bonding moiety. The
protecting groups provide improved stability to the
reagents for long-term storage and are removed either
immediately prior to or concurrent with the synthesis of
the radiopharmaceuticals. Alternatively, the compounds
of the present invention are comprised of a peptide or
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peptidomimetic targeting moiety that binds to a receptor
that is upregulated during angiogenesis, Q, an optional
linking group, Ln, and a surfactant, Sf.
The pharmaceuticals of the present invention may be
used for diagnostic and/or therapeutic purposes.
Diagnostic radiopharmaceuticals of the present invention
are pharmaceuticals comprised of a diagnostically useful
radionuclide (i.e., a radioactive metal ion that has
imageable gamma ray or positron emissions). Therapeutic
radiopharmaceuticals of the present invention are
pharmaceuticals comprised of a therapeutically useful
radionuclide, a radioactive metal ion that emits
ionizing radiation such as beta particles, alpha
particles and Auger or Coster-Kronig electrons.
The pharmaceuticals comprising a gamma ray or
positron emitting radioactive metal ion are useful for
imaging tumors by gamma scintigraphy or positron
emission tomography. The pharmaceuticals comprising a
gamma ray or positron emitting radioactive metal ion are
also useful for imaging therapeutic angiogenesis by
gamma scintigraphy or positron emission tomography. The
pharmaceuticals comprising a particle emitting
radioactive metal ion are useful for treating cancer by
delivering a cytotoxic dose of radiation to the tumors.
The pharmaceuticals comprising a particle emitting
radioactive metal ion are also useful for treating
rheumatoid arthritis by destroying the formation of
angiogenic vasculature. The pharmaceuticals comprising
a paramagnetic metal ion are useful as magnetic
resonance imaging contrast agents. The pharmaceuticals
comprising one or more X-ray absorbing or "heavy" atoms
of atomic number 20 or greater are useful as X-ray
contrast agents. The pharmaceuticals comprising a
microbubble of a biocompatible gas, a liquid carrier,
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and a surfactant microsphere, are useful as ultrasound
contrast agents.
DETAILED DESCRTPTION OF THE INVENTION
5 [1~ Thus, in a first embodiment, the present
invention provides a kit for treating cancer, comprising
a compound of the formula (I) and at least one agent
selected from the group consisting of an anti-cancer
agent and a radiosensitizer agent, or a pharmaceutically
10 acceptable salt thereof, and a pharmaceutically
acceptable carrier, wherein the compound of the formula
(I) is:
(Q)d-Ln-Ch ~r (Q)d-Ln-(Ch)d'
(I)
15 wherein, Q is a peptide independently selected from the
group:
K M K R
~1 2 3
R R , R R , L M , and
R3 R5
K is an L-amino acid independently selected at each
occurrence from the group: arginine, citrulline,
N-methylarginine, lysine, homolysine,
2-aminoethylcysteine, 8-N-2-imidazolinylornithine,
8-N-benzylcarbamoylornithine, and
~3-2-benzimidazolylacetyl-1,2-diaminopropionic acid;
K' is a D-amino acid independently selected at each
occurrence from the group: arginine, citrulline,
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N-methylarginine, lysine, homolysine,
2-aminoethylcysteine, 8-N-2-imidazolinylornithine,
8-N-benzylcarbamoylornithine, and
(3-2-benzimidazolylacetyl-1,2-diaminopropionic acid;
L is independently selected at each occurrence from the
group: glycine, L-alanine, and D-alanine;
M is L-aspartic acid;
M' is D-aspartic acid;
R1 is an amino acid substituted with 0-1 bonds to Ln,
independently selected at each occurrence from the
group: glycine, L-valine, D-valine, alanine,
leucine, isoleucine, norleucine, 2-aminobutyric
acid, 2-aminohexanoic acid, tyrosine,
phenylalanine, thienylalanine, phenylglycine,
cyclohexylalanine, homophenylalanine,
1-naphthylalanine,. lysine, serine, ornithine,
1,2-diaminobutyric acid, 1,2-diaminopropionic acid,
cysteine, penicillamine, and methionine;
R2 is an amino acid, substituted with 0-1 bonds to Ln,
independently selected at each occurrence from the
group: glycine, valine, alanine, leucine,
isoleucine, norleucine, 2-aminobutyric acid,
2-aminohexanoic acid, tyrosine, L-phenylalanine, D-
phenylalanine, thienylalanine, phenylglycine,
biphenylglycine, cyclohexylalanine,
homophenylalanine, L-1-naphthylalanine,
D-1-naphthylalanine, lysine, serine, ornithine,
1,2-diaminobutyric acid, 1,2-diaminopropionic acid,
cysteine, penicillamine, methionine, and
2-aminothiazole-4-acetic acid;
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R3 is an amino acid, substituted with 0-1 bonds to Ln,
independently selected at each occurrence from the
group: glycine, D-valine, D-alanine, D-leucine,
D-isoleucine, D-norleucine, D-2-aminobutyric acid,
D-2-aminohexanoic acid, D-tyrosine,
D-phenylalanine, D-thienylalanine, D-phenylglycine,
D-cyclohexylalanine, D-homophenylalanine,
D-1-naphthylalanine, D-lysine, D-serine,
D-ornithine, D-1,2-diaminobutyric acid,
D-1,2-diaminopropionic acid, D-cysteine,
D-penicillamine, and D-methionine;
R4 is an amino acid, substituted with 0-1 bonds to Ln,
independently selected at each occurrence from the
group: glycine, D-valine, D-alanine, D-leucine,
D-isoleucine, D-norleucine, D-2-aminobutyric acid,
D-2-aminohexanoic acid, D-tyrosine,
D-phenylalanine, D-thienylalanine, D-phenylglycine,
D-cyclohexylalanine, D-homophenylalanine,
D-1-naphthylalanine, D-lysine, D-serine,
D-ornithine, D-1,2-diaminobutyric acid,
D-1,2-diaminopropionic acid, D-cysteine,
D-penicillamine, D-methionine, and
2-aminothiazole-4-acetic acid;
R5 is an amino acid, substituted with 0-1 bonds to Ln,
independently selected at each occurrence from the
group: glycine, L-valine, L-alanine, L-leucine,
L-isoleucine, L-norleucine, L-2-aminobutyric acid,
L-2-aminohexanoic acid, L-tyrosine,
L-phenylalanine, L-thienylalanine, L-phenylglycine,
L-cyclohexylalanine, L-homophenylalanine,
L-1-naphthylalanine, L-lysine, L-serine,
L-ornithine, L-1,2-diaminobutyric acid,
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L-1,2-diaminopropionic acid, L-cysteine,
L-penicillamine, L-methionine, and
2-aminothiazole-4-acetic acid;
provided that one of R1, R~, R3, R4, and R5 in each Q is
substituted with a bond to Ln, further provided
that when R~ is 2-aminothiazole-4-acetic acid, K is
N-methylarginine, further provided that when R4 is
2-aminothiazole-4-acetic acid, K and K' are
N-methylarginine, and still further provided that
when R5 is 2-aminothiazole-4-acetic acid, K' is
N-methylarginine;
d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
Ln is a linking group having the formula:
(CR6R7)g-(W)h_(CR6aR7a)g,_(Z)k-(W)h'-(CR8R9)g~~-(W)h~~-
(CR8aR9a) g.. ,
provided that g+h+g'+k+h'+g"+h"+g"' is other than 0;
W is independently selected at each occurrence from the
group: O, S, NH, NHC(=0), C(=0)NH, C(=0), C(=0)0,
OC(=O), NHC(=S)NH, NHC(=O)NH, 502, (OCH~CH~)S,
(CH2CH~0)s~, (OCH~CH~CH2)5~~, (CH~CH2CH~0)t, and
(aa) t' ;
as is independently at each occurrence an amino acid;
Z is selected from the group: aryl substituted with 0-3
R1~~ C3-10 cYcloalkyl substituted with 0-3 Rlo, and
a 5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and 0 and substituted with 0-3 R1~;
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R6, R6a, R7, R7a, R8, R8a, R9 and R9a are independently
selected at each occurrence from the group: H, =0,
COON, S03H, P03H, CZ-C5 alkyl substituted with 0-3
R~-~, aryl substituted with 0-3 R1~, benzyl
substituted with 0-3 R1~, and C1-C5 alkoxy
substituted with 0-3 R1~, NHC(=0)R11, C(=0)NHR11,
NHC ( =0 ) NHR11, NHR11, R11, and a bond to Ch;
R1~ is independently selected at each occurrence from
the group: a bond to Ch, COOR11, OH, NHR11, S03H,
P03H, aryl substituted with 0-3 R11, C1_5 alkyl
substituted with 0-1 R1~, C~_5 alkoxy substituted
with 0-1 R1~, and a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently
selected from N, S, and 0 and substituted with 0-3
R~1;
R11 is independently selected at each occurrence from
the group: H, aryl substituted with 0-1 R12, a
5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and 0 and substituted with 0-1 R1~, C3_1o cycloalkyl
substituted with 0-1 R1~, polyalkylene glycol
substituted with 0-1 R1~, carbohydrate substituted
with 0-1 R12, cyclodextrin substituted with 0-1 R1~,
amino acid substituted with 0-1 R1~,
polycarboxyalkyl substituted with 0-1 R12,
polyazaalkyl substituted with 0-1 R1~, peptide
substituted with 0-1 R12, wherein the peptide is
comprised of 2-10 amino acids, and a bond to Ch;
R12 is a bond to Ch;
k is selected from 0, 1, and 2;
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h is selected from 0, 1, and 2;
h' is selected from 0, 1, 2, 3, 4, and 5;
h" is selected from 0, 1, 2, 3, 4, and 5;
g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
5 g' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
g" is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
g"' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10 10;
s is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
s' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
s" is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
15 10;
t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
t' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
20 Ch is a metal bonding unit having a formula selected
from the group:
~E A2
A , A ,
p5
~'E
E~ AY-\As
s
~A~E- 4~~ c-.E A~ E ~E
~E E
A
A3 A5 As A
and
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A~ , A3 , A3 , A4 , A5 , A6 , A7 , and A8 are independently
selected at each occurrence from the group N, NR~-3,
NR13R14, S, SH, S(Pg), 0, OH, PR13, pR13g.14~
P(0)R15R16, and a bond to Ln;
E is a bond, CH, or a spacer group independently
selected at each occurrence from the group: C1-C10
alkyl substituted with 0-3 R1~, aryl substituted
with 0-3 R1~, C3_~o cycloalkyl substituted with 0-3
R1~, heterocyclo-C1_1o alkyl substituted with 0-3
R1~, wherein the heterocyclo group is a 5-10
membered heterocyclic ring system containing 1-4
heteroatoms independently selected from N, S, and
0, C6_1o aryl-C1_1o alkyl substituted with 0-3 R1~,
C1-1o alkyl-C6-so aryl- substituted with 0-3 R1~,
and a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected
from N, S, and O and substituted with 0-3 R17;
R13, and R1~ are each independently selected from the
group: a bond to Ln, hydrogen, C1-C10 alkyl
substituted with 0-3 R1~, aryl substituted with 0-3
R1~, C~_1o cycloalkyl substituted with 0-3 R1~,
heterocyclo-C1-1o alkyl substituted with 0-3 R1~,
wherein the heterocyclo group is a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and 0, C6-1o
aryl-C~_1o alkyl substituted with 0-3 R1~, C1_1o
alkyl-C6-1o aryl- substituted with 0-3 R1~, a 5-10
membered heterocyclic ring system containing 1-4
heteroatoms independently selected from N, S, and O
and substituted with 0-3 R17, and an electron,
provided that when one of R13 or R14 is an
electron, then the other is also an electron;
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alternatively, R13 and R14 combine to form =C(R2o)(R~1);
R15 and R16 are each independently selected from the
group: a bond to Ln, -OH, C1-C10 alkyl substituted
with 0-3 R1~, C1-C10 alkyl substituted with 0-3
R1~, aryl substituted with 0-3 R1~, C3-1o cYcloalkyl
substituted with 0-3 R1~, heterocyclo-C1-so alkyl
substituted with 0-3 R1~, wherein the heterocyclo
group is a 5-10 membered heterocyclic ring system
containing 2-4 heteroatoms independently selected
from N, S, and 0, C6-so aryl-C1-1o alkyl substituted
with 0-3 R1~, C1-1o alkyl-C6_1o aryl- substituted
with 0-3 R1~, and a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-3
R~-7 ;
R1~ is independently selected at each occurrence from
the group: a bond to Ln, =0, F, Cl, Br, I, -CF3,
-CN, -CO~R18, -C(=O)R18, -C(=0)N(R18)2, -CHO,
-CH20R18, -OC(=0)R18, -OC(=0)ORlBa, -OR18,
_OC(=O)N(R18)2~ _NR19C(=0)R18, -NR19C(=0)ORl8a~
_NR19C(=O)N(R18)2~ -NR19S02N(R18)~~ _NR19SO~R18a~
_S03H~ _Sp~Rl8a~ _SR18~ _S(=0)Rl8a~ -SO~N(R18)~~
-N(R18)2, -NHC(=S)NHR18, =NOR18, NO~, -C(=0)NHOR18,
-C(=0)NHNR18R18a, -OCH~CO,~H,
2-(1-morpholino)ethoxy, C1-C5 alkyl, C~-Cg alkenyl,
C3-C6 cycloalkyl, C3-C6 cycloalkylmethyl, C~-C6
alkoxyalkyl, aryl substituted with 0-2 R18, and a
5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and 0;
R28, RlBa, and R19 are independently selected at each
occurrence from the group: a bond to Ln, H, C1-C6
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alkyl, phenyl, benzyl, C1-C6 alkoxy, halide, nitro,
cyano, and trifluoromethyl;
Pg is a thiol protecting group;
R2o and R21 are independently selected from the group:
H, C1-C10 alkyl, -CN, -CO~R25, -C(=0)R25,
-C(=0)N(R~5)~, C~-C1o 1-alkene substituted with 0-3
R23, C~-C1o 1-alkyne substituted with 0-3 R23, aryl
substituted with 0-3 R23, unsaturated 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and O and
substituted with 0-3 R~3, and unsaturated C3_1o
carbocycle substituted with 0-3 R23;
alternatively, R2o and R21, taken together with the
divalent carbon radical to which they are attached
form:
22
23
n
R22 and R23 are independently selected from the group:
H, R~4, C1-C10 alkyl substituted with 0-3 R~4,
C2-C10 alkenyl substituted with 0-3 R24, C~-C10
alkynyl substituted with 0-3 R~4, aryl substituted
with 0-3 R24, a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently
selected from N, S, and 0 and substituted with 0-3
R24, and C3-1o carbocycle substituted with 0-3 R24;
alternatively, R22, R23 taken together form a fused
aromatic or a 5-10 membered heterocyclic ring
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system containing 1-4 heteroatoms independently
selected from N, S, and 0;
a and b indicate the positions of optional double bonds
and n is 0 or 1;
R24 is independently selected at each occurrence from
the group: =0, F, Cl, Br, I, -CF3, -CN, -C02R25,
-C(=0)R25, -C(=0)N(R25)2, -N(R25)3+, -CH20R25,
-OC(=0)R25, -OC(=0)OR25a~ _OR25~ -pC(=0)N(R25)2,
-NR26C (=0) R25, -NR26C (=0) OR25a~ _NR26C (=O) N(R25) 2,
_NR26S02N(R25)2~ _NR26~02R25a~ _S03H, -S02R25a~
_SR25~ _S(=O)R25a~ _g02N(R25)2. -N(R25)2. =NOR25,
-C(=0)NHOR25, -OCH2C02H, and
2-(1-morpholino)ethoxy; and,
R25, R25a, and R26 are each independently selected at
each occurrence from the group: hydrogen and C1-C6
alkyl;
and a pharmaceutically acceptable salt thereof.
[2] In another embodiment, the present invention
provides a kit according to Embodiment 1 wherein:
L is glycine;
R1 is an amino acid, optionally substituted with a bond
to Ln, independently selected at each occurrence
from the group: L-valine, D-valine, alanine,
leucine, isoleucine, norleucine, 2-aminobutyric
acid, tyrosine, phenylalanine, phenylglycine,
cyclohexylalanine, homophenylalanine, lysine,
ornithine, 1,2-diaminobutyric acid, and
1,2-diaminopropionic acid;
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R2 is an amino acid, optionally substituted with a bond
to Ln, independently selected at each occurrence
from the group: valine, alanine, leucine,
isoleucine, norleucine, 2-aminobutyric acid,
5 tyrosine, L-phenylalanine, D-phenylalanine,
thienylalanine, phenylglycine, biphenylglycine,
cyclohexylalanine, homophenylalanine,
L-1-naphthylalanine, D-1-naphthylalanine, lysine,
ornithine, 1,2-diaminobutyric acid,
10 1,2-diaminopropionic acid, and
2-aminothiazole-4-acetic acid;
R3 is an amino acid, optionally substituted with a bond
to Ln, independently selected at each occurrence
15 from the group: D-valine, D-alanine, D-leucine,
D-isoleucine, D-norleucine, D-2-aminobutyric acid,
D-tyrosine, D-phenylalanine, D-phenylglycine,
D-cyclohexylalanine, D-homophenylalanine, D-lysine,
D-serine, D-ornithine, D-1,2-diaminobutyric acid,
20 and D-1,2-diaminopropionic acid;
R4 is an amino acid, optionally substituted with a bond
to Ln, independently selected at each occurrence
from the group: D-valine, D-alanine, D-leucine,
25 D-isoleucine, D-norleucine, D-2-aminobutyric acid,
D-tyrosine, D-phenylalanine, D-thienylalanine,
D-phenylglycine, D-cyclohexylalanine,
D-homophenylalanine, D-1-naphthylalanine, D-lysine,
D-ornithine, D-1,2-diaminobutyric acid.,
D-1,2-diaminopropionic acid, and
2-aminothiazole-4-acetic acid;
R5 is an amino acid, optionally substituted with a bond
to Ln, independently selected at each occurrence
from the group: L-valine, L-alanine, L-leucine,
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L-isoleucine, L-norleucine, L-2-aminobutyric acid,
L-tyrosine, L-phenylalanine, L-thienylalanine,
L-phenylglycine, L-cyclohexylalanine,
L-homophenylalanine, L-1-naphthylalanine, L-lysine,
L-ornithine, L-1,2-diaminobutyric acid,
L-1,2-diaminopropionic acid, and
2-aminothiazole-4-acetic acid;
d is selected from 1, 2, and 3;
W is independently selected at each occurrence from the
group: 0, NH, NHC(=0), C(=0)NH, C(=0), C(=0)O,
OC ( =0 ) , NHC ( =S ) NH , NHC ( =0 ) NH , 502 , ( OCH2 CH2 ) S ,
( CH2CH20 ) S ~ , ( OCH2CH2CH2 ) S ~~ , and ( CH2CH2CH20 ) t,
Z is selected from the group: aryl substituted with 0-1
R~o~ C3-1o cycloalkyl substituted with 0-1 Rlo, and
a 5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and O and substituted with 0-1 Rlo;
R6, R6a, R7, R7a, R8, R8a, R9, and R9a are independently
selected at each occurrence from the group: H, =0,
COOH, S03H, C1-C5 alkyl substituted with 0-1 Rlo,
aryl substituted with 0-1 Rlo, benzyl substituted
with 0-1 Rlo, and C1-C5 alkoxy substituted with 0-1
R~-o , NHC ( =0 ) R11, C ( =0 ) NHR11, NHC ( =0 ) NHR11, NHR11,
R11, and a bond to Ch;
R1o is independently selected at each occurrence from
the group: COOR11, OH, NHR11, S03H, aryl
substituted with 0-1 R11, a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and 0 and
substituted with 0-1 R11, C1-C5 alkyl substituted
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with 0-1 R12, C1-C5 alkoxy substituted with 0-1 R12,
and a bond to Ch;
R11 is independently selected at each occurrence from
the group: H, aryl substituted with 0-1 R1~, a
5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and O and substituted with 0-1 R12, polyalkylene
glycol substituted with 0-1 R12, carbohydrate
substituted with 0-1 R12, cyclodextrin substituted
with 0-1 R12, amino acid substituted with 0-1 R12,
and a bond to Ch;
k is 0 or 1;
h is 0 or 1;
h' is 0 or 1;
s is selected from 0, 1, 2, 3, 4, and 5;
s' is selected from 0, 1, 2, 3, 4, and 5;
s" is selected from 0, 1, 2, 3, 4, and 5;
t is selected from 0, 1, 2, 3, 4, and 5;
A~-, A~ , A3 , A4 , A5 , A6 , A7 , and A8 are , independent 1y
selected at each occurrence from the group: NR13,
NR13R14, S, SH, S(Pg), OH, and a bond to Ln;
E is a bond, CH, or a spacer group independently
selected at each occurrence from the group: C1-C10
alkyl substituted with 0-3 R1~, aryl substituted
with 0-3 R1~, C3-so cYcloalkyl substituted with 0-3
R1~, and a 5-10 membered heterocyclic ring system
containing l-4 heteroatoms independently selected
from N, S, and 0 and substituted with 0-3 R17;
R13, and R14 are each independently selected from the
group: a bond to Ln, hydrogen, C1-C10 alkyl
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28
substituted with 0-3 R1~, aryl substituted with 0-3
R1~, a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected
from N, S, and 0 and substituted with 0-3 R1~, and
an electron, provided that when one of R13 or R14
is an electron, then the other is also an electron;
alternatively, R13 and R14 combine to form =C (R~~) (R~1) ;
R1~ is independently selected at each occurrence from
the group: a bond to Ln, =O, F, Cl, Br, I, -CF3,
-CN, -C02R18, -C(=0)R18, -C(=O)N(R18)2, -CH~OR18,
_OC(=O)R18~ _OC(=O)ORl8a~ _OR18~ -OC(=O)N(R18)2~
-NR19C(=0)R18, -NR19C(=0)ORl8a~ _NR19C(=O)N(R18)~~
-NR19S02N(R18)2, -NR19S02R18a~ _S03H~ _g02R18a~
_S(=0)Rl8a~ _S02N(R18)2, -N(R18)2, -NHC(=S)NHR18,
=NOR18, -C(=0)NHNR18R18a, -OCH2C02H, and '
2-(1-morpholino)ethoxy;
R18, RlBa, and R19 are independently selected at each
occurrence from the group: a bond to Ln, H, and
C1-C6 alkyl;
R~0 and R21 are independently selected from the group:
H, C1-C5 alkyl, -C02R~5, C2-C5 1-alkene substituted
with 0-3 R~3, C2-C5 1-alkyne substituted with 0-3
R~3, aryl substituted with 0-3 R~3, and unsaturated
5--10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and 0 and substituted with 0-3 R23;
alternatively, R~0 and R21, taken together with the
divalent carbon radical to which they are attached
form:
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R22 R22
a b.
R23 R23
n
R22 and R23 are independently selected from the group:
H, and R24;
alternatively, R22, R23 taken together form a fused
aromatic or a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently
selected from N, S, and 0;
R24 is independently selected at each occurrence from
the group: -C02R25, -C(=O)N(R25)2, -CH20R25,
-OC(=0)R25, -OR25, -S03H, -N(R25)2, and -OCH2C02H;
and ,
R25 is independently selected at each occurrence from
the group: H and C1-C3 alkyl.
[3] In another embodiment, the present invention
provides a kit according to Embodiment 1 wherein:
Q is a peptide selected from the group:
3
K/L'M K/R' 4
R
~1 2
R R and ~ M ;
R1 is L-valine, D-valine, D-lysine optionally
substituted on the E amino group with a bond to Ln
or L-lysine optionally substituted on the ~ amino
group with a bond to Ln;
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R~ is L-phenylalanine, D-phenylalanine,
D-1-naphthylalanine, 2-aminothiazole-4-acetic acid,
L-lysine optionally substituted on the ~ amino
group with a bond to Ln or tyrosine, the tyrosine
5 optionally substituted on the hydroxy group with a
bond to Ln;
R3 is D-valine, D-phenylalanine, or L-lysine optionally
substituted on the ~ amino group with a bond to Ln;
R4 is D-phenylalanine, D-tyrosine substituted on the
hydroxy group with a. bond to Ln, or L-lysine
optionally substituted on the ~ amino group with a
bond to Ln;
provided that one of R1 and R~ in each Q is substituted
with a bond to Ln, and further provided that when
R~ is 2-aminothiazole-4-acetic acid, K is
N-methylarginine;
d is 1 or 2;
W is independently selected at each occurrence from the
group: NHC (=0) , C (=0) NH, C (=0) , (CH2CH~0) S~ , and
(CH~CH2CH~0)t;
R6, R6a, R7, R7a, R8, R8a, R9, and R9a are independently
selected at each occurrence from the group: H,
NHC ( =O ) R11, and a bond to Ch;
k is 0;
h" is selected from 0, 1, 2, and 3;
g is selected from 0, 1, 2, 3, 4, and 5;
g' is selected from 0, 1, 2, 3, 4, and 5;
g" is selected from 0, 1, 2, 3, 4, and 5;
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31
g"' is selected from 0, 1, 2, 3, 4, and 5;
s' is 1 or 2;
t is 1 or 2 ;
~E~AZE Aa,~~~c-E A~
AE E5 E~A8
Ch is A ,
AZ is selected from the group: OH, and a bond to Ln;
A2, A4, and A6 are each N;
A3, A5, and A8 are each OH;
A~ is a bond to Ln or NH-bond to Ln;
E is a C~ alkyl substituted with 0-1 R1~;
R1~ is =0;
t~E A2
alternatively, Ch is A ;
A~- is NH2 or N=C (R2~) (R~1) ;
E is a bond;
A~ is NHR13;
R~3 is a heterocycle substituted with R17, the
heterocycle being selected from pyridine and
pyrimidine;
R17 is selected from a bond to Ln, C (=0)NHR18, and
C (=0) R18;
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R18 is a bond to Ln;
R24 is selected from the group: -C02R25, -OR25, -S03H,
and -N ( R2 5 ) ~ ;
R25 is independently selected at each occurrence from
the group: hydrogen and methyl;
p5
fE'
~1~
E~'~' AY-\A6
A \ ~A4
E \E A3S
~E
alternatively, C~ is A7
A1, A2, A3, and A4 are each N;
A5, A6, and A8 are each OH;
A7 is a bond to Ln;
E is a C2 alkyl substituted with 0-1 R1~; and,
R1~ is =0.
[4] In another embodiment, the present invention
provides a kit according to Embodiment 1 wherein a
compound of the formula (I) is selected from the
group consisting of:
(a) cyclo{Arg-Gly-Asp-D-Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfoniC acid]-
3-aminopropyl)-Val};
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33
(b) cyclo{Arg-Gly-Asp-D-Tyr((N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
18-amino-14-aza-4,7,10-oxy-15-oxo-octadecoyl)-3-
aminopropyl)-Val};
(c) [2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-Glu(cyclo{D-Tyr(3-
aminopropyl)-Val-Arg-Gly-Asp})-cyclo{D-Tyr(3-
aminopropyl)-Val-Arg-Gly-Asp};
(d) cyclo(Arg-Gly-Asp-D-Tyr-Lys([2-[[[5-[carbonyl]-2
pyridinyl]hydrazono]methyl]-benzenesulfonic
acid] ) } ;
(e) cyclo{Arg-Gly-Asp-D-Phe-Lys([2-[[[5-[carbonyl]-2
pyridinyl]hydrazono]methyl]-benzenesulfonic
acid])};
(f) [2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-Glu(cyclo{Lys-Arg-Gly-Asp-D-
Phe})-cyclo{Lys-Arg-Gly-Asp-D-Phe};
(g) [2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]
benzenesulfonic acid]-Phe-Glu(cyclo{Lys-Arg-Gly
Asp-D-Phe})-cyclo{Lys-Arg-Gly-Asp-D-Phe};
(h) cyclo{Arg-Gly-Asp-D-Nal-Lys([2-[[[5-[carbonyl]-2
pyridinyl]hydrazono]methyl]-benzenesulfonic
acid])};
(i) [2-[[[5-[carbonyl]-2-pyridinyl]-hydrazono]methyl]-
benzenesulfonic acid]-Glu(cyclo{Lys-Arg-Gly-Asp-D-
Nal})-cyclo{Lys-Arg-Gly-Asp-D-Nal};
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34
(j) cyclo{Arg-Gly-Asp-Lys([2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid])-
D-Val} ;
(k) [2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-Glu(cyclo{Lys-D-Val-Arg-Gly-
Asp})-cyclo{Lys-D-Val-Arg-Gly-Asp};
(1) {cyclo(Arg-D-Val-D-Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
3-aminopropyl)-D-Asp-Gly};
(m) cyclo{D-Lys([2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid])-
D-Phe-D-Asp-Gly-Arg};
(n) [2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-Glu(cyclo{D-Lys-D-Phe-D-Asp-
Gly-Arg})-cyclo{D-Lys-D-Phe-D-Asp-Gly-Arg};
(o) cyclo{D-Phe-D-Lys([2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid])-
D-Asp-Gly-Arg};
(p) cyclo{N-Me-Arg-Gly-Asp-ATA-D-Lys([2-[[[5-[carbonyl]-
2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid] ) } ;
(q) cyclo{Cit-Gly-Asp-D-Phe-Lys([2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic
acid] ) } ;
(r) 2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-1-
cyclododecyl)acetyl-Glu(cyclo{Lys-Arg-Gly-Asp-D-
Phe})-cyclo{Lys-Arg-Gly-Asp-D-Phe};
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(s) cyclo{Arg-Gly-Asp-D-Phe-Lys(DTPA)};
(t) cyclo{Arg-Gly-Asp-D-Phe-Lys}2(DTPA);
5
(u) Cyclo{Arg-Gly-Asp-D-Tyr(N-DTPA-3-aminopropyl)-Val};
(v) cyclo{Orn(d-N-2-Imidazolinyl)-Gly-Asp-D-Tyr(N-[2-
[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
10 benzenesulfonic acid]-3-aminopropyl)-Val};
(w) cyclo{Lys-Gly-Asp-D-Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
3-aminopropyl)-Val};
(x) cyclo{Cys(2-aminoethyl)-Gly-Asp-D-Tyr(N-[2-[[[5-
[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-Val};
(y) cyclo{HomoLys-Gly-Asp-D-Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
3-aminopropyl)-Val};
(z) cyclo{Orn(d-N-Benzylcarbamoyl)-Gly-Asp-D-Tyr(N-[2-
[([5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-Val};
(aa) cyclo{Dap(b-(2-benzimidazolylacetyl))-Gly-Asp-D-
Tyr (N- [2- [ [ [5- [carbonyl] -2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
3-aminopropyl)-Val};
(bb) cyclo{Orn(d-N-2-Imidazolinyl)-Gly-Asp-D-Phe-Lys(N-
[2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid])};
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36
(cc) cyclo{Orn(d-N-Benzylcarbamoyl)-Gly-Asp-D-Phe-Lys(N-
[2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid])};
(dd) cyclo{Lys-D-Val-D-Tyr(N-[2-[[[5-[carbonyl]-2-,-t,
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
3-aminopropyl)-D-Asp-Gly};
(ee) cyclo{0rn(d-N-Benzylcarbamoyl)-D-Val-D-Tyr(N-[2-
[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-D-Asp-Gly};
and ,
(ff) cyclo{0rn(d-N-2-Imidazolinyl)-D-Val-D-Tyr(N-[2-
[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-D-Asp-Gly};
or a pharmaceutically acceptable salt form thereof.
[5] In another embodiment, the present invention
provides a kit according to Embodiment 1, wherein the
kit further comprises one or more ancillary ligands and
a reducing agent.
[6] In another embodiment, the present invention
provides a kit according to Embodiment 5, wherein the
ancillary ligands are tricine and TPPTS.
[7] In another embodiment, the present invention
provides a kit according to Embodiment 5, wherein the
reducing agent is tin(II).
[8] In another embodiment, the present invention
provides a kit according to Embodiment 1, wherein the
anti-cancer agent is selected from the group consisting
of mitomycin, tretinoin, ribomustin, gemcitabine,
vincristine, etoposide, cladribine, mitobronitol,
methotrexate, doxorubicin, carboquone, pentostatin,
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37
nitracrine, zinostatin, cetrorelix, letrozole,
raltitrexed, daunorubicin, fadrozole, fotemustine,
thymalfasin, sobuzoxane, nedaplatin, cytarabine,
bicalutamide, vinorelbine, vesnarinone,
aminoglutethimide, amsacrine, proglumide, elliptinium
acetate, ketanserin, do~ifluridine, etretinate,
isotretinoin, streptozocin, nimustine, vindesine,
flutamide, drogenil, butocin, carmofur, razoxane,
sizofilan, carboplatin, mitolactol, tegafur, ifosfamide,
prednimustine, picibanil, levamisole, teniposide,
improsulfan, enocitabine, lisuride, oxymetholone,
tamoxifen, progesterone, mepitiostane, epitiostanol,
formestane, interferon-alpha, interferon-2 alpha,
interferon-beta, interferon-gamma, colony stimulating
factor-1, colony stimulating factor-2, denileukin
diftitox, interleukin-2, and leutinizing hormone
releasing factor.
[9] In another embodiment, the present invention
provides a kit according to Embodiment 1, wherein the
anti-cancer agent is selected from the group consisting
of mitomycin, tretinoin, ribomustin, gemcitabine,
vincristine, etoposide, cladribine, mitobronitol,
methotrexate, doxorubicin, carboquone, pentostatin,
nitracrine, zinostatin, cetrorelix, letrozole,
raltitrexed, daunorubicin, fadrozole, fotemustine,
thymalfasin, sobuzoxane, nedaplatin, cytarabine,
bicalutamide, vinorelbine, vesnarinone,
aminoglutethimide, amsacrine, proglumide, elliptinium
acetate, ketanserin, doxifluridine, etretinate,
isotretinoin, streptozocin, nimustine, vindesine,
flutamide, drogenil, butocin, carmofur, razoxane,
sizofilan, carboplatin, mitolactol, tegafur, ifosfamide,
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38
prednimustine, picibanil, levamisole, teniposide,
improsulfan, enocitabine, and lisuride.
[10] In another embodiment, the present invention
provides a kit according to Embodiment 1 wherein the
anti-cancer agent is selected from the group consisting
of oxymetholone, tamoxifen, progesterone, mepitiostane,
epitiostanol, and formestane.
[11] In another embodiment, the present invention
provides a kit according to Embodiment 2 wherein the
anti-cancer agent is selected from the group consisting
of interferon-alpha, interferon-2 alpha, interferon-
beta, interferon-gamma, colony stimulating factor-1,
colony stimulating factor-2, denileukin diftitox,
interleukin-2, and leutinizing hormone releasing factor.
[12] In another embodiment, the present invention
provides a kit according to Embodiment 1, wherein
radiosensitizer agent is selected from the group
consiting of 2-(3-vitro-1,2,4-triazol-1-yl)-N-(2-
methoxyethyl)acetamide, N-(3-vitro-4-quinolinyl)-4-
morpholinecarboxamidine, 3-amino-1,2,4-benzotriazine-
1,4-dioxide, N-(2-hydroxyethyl)-2-nitroimidazole-1-
acetamide, 1-(2-nitroimidazol-1-yl)-3-(1-piperidinyl)-
2-propanol, and 1-(2-vitro-1-imidazolyl)-3-(1-
aziridino)-2-propanol.
[13] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
comprising at least one agent selected from the group
consisting of an anti-cancer agent and a radiosensitizer
agent, or a pharmaceutically acceptable salt thereof,
and a radiopharmaceutical comprising:
a) a radioisotope;
b) a chelator capable of chelating the radioisotope; and
c) a targeting moiety;
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39
wherein the targeting moiety is bound to the chelator
through 0-1 linking groups, and the targeting moiety is
a peptide or peptidomimetic that binds to a receptor
that is upregulated during angiogenesis.
[14] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 13, wherein the targeting moiety
is a cyclic pentapeptide and the receptor is oc~,(33.
[15] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 14, wherein the
radiopharmaceutical comprises:
a) a radioisotope selected from the group 33p, 125I~
186Re~ 188Re~ 153Sm~ 166go~ 177Lu~ 149pm~ 90y~ 212Bi~
103pd~ 109pd~ 159~d~ 140La~ 198Au~ 199Au~ 169yb~
175yb~ 165Dy~ 166Dy~ 67Cu~ 105Rh~ 111Ag~ and 192Ir~
and
b) a compound of the formula (I):
(Q)d'Ln-Ch ~r (Q)d'Ln'(Ch)d'
(I)
wherein, Q is a peptide independently selected from the
group:
~R3~ 4
K ~ M K R
~1 2 3
R R , R R , L M , and
L
~~ ~M~
3 5
R R
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K is an L-amino acid independently selected at each
occurrence from~the group: arginine, citrulline,
N-methylarginine, lysine, homolysine,
2-aminoethylcysteine, 8-N-2-imidazolinylornithine,
5 8-N-benzylcarbamoylornithine, and
(3-2-benzimidazolylacetyl-1,2-diaminopropionic acid;
K' is a D-amino acid independently selected at each
occurrence from the group: arginine, citrulline,
10 N-methylarginine, lysine, homolysine,
2-aminoethylcysteine, ~-N-2-imidazolinylornithine,
~-N-benzylcarbamoylornithine, and
(3-2-benzimidazolylacetyl-1,2-diaminopropionic acid;
15 L is independently selected at each occurrence from the
group: glycine, L-alanine, and D-alanine;
M is L-aspartic acid;
20 M' is D-aspartic acid;
R1 is an amino acid substituted with 0-1 bonds to Ln,
independently selected at each occurrence from the
group: glycine, L-valine, D-valine, alanine,
25 leucine, isoleucine, norleucine, 2-aminobutyric
acid, 2-aminohexanoic acid, tyrosine,
phenylalanine, thienylalanine, phenylglycine,
cyclohexylalanine, homophenylalanine,
1-naphthylalanine, lysine, serine, ornithine,
30 1,2-diaminobutyric acid, 1,2-diaminopropionic acid,
cysteine, penicillamine, and methionine;
R~ is an amino acid, substituted with 0-1 bonds to Ln,
independently selected at each occurrence from the
35 group: glycine, valine, alanine, leucine,
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41
isoleucine, norleucine, 2-aminobutyric acid,
2-aminohexanoic acid, tyrosine, L-phenylalanine, D-
phenylalanine, thienylalanine, phenylglycine,
biphenylglycine, cyclohexylalanine,
homophenylalanine, L-1-naphthylalanine,
D-1-naphthylalanine, lysine, serine, ornithine,
1,2-diaminobutyric acid, 1,2-diaminopropionic acid,
cysteine, penicillamine, methionine, and
2-aminothiazole-4-acetic acid;
R3 is an amino acid, substituted with 0-1 bonds to Ln,
independently selected at each occurrence from the
group: glycine, D-valine, D-alanine, D-leucine,
D-isoleucine, D-norleucine, D-2-aminobutyric acid,
D-2-aminohexanoic acid, D-tyrosine,
D-phenylalanine, D-thienylalanine, D-phenylglycine,
D-cyclohexylalanine, D-homophenylalanine,
D-1-naphthylalanine, D-lysine, D-serine,
D-ornithine, D-1,2-diaminobutyric acid,
D-1,2-diaminopropionic acid, D-cysteine,
D-penicillamine, and D-methionine;
R4 is an amino acid, substituted with 0-1 bonds to Ln,
independently selected at each occurrence from the
group: glycine, D-valine, D-alanine, D-leucine,
D-isoleucine, D-norleucine, D-2-aminobutyric acid,
D-2-aminohexanoic acid, D-tyrosine,
D-phenylalanine, D-thienylalanine, D-phenylglycine,
D-cyclohexylalanine, D-homophenylalanine,
D-1-naphthylalanine, D-lysine, D-serine,
D-ornithine, D-1,2-diaminobutyric acid,
D-1,2-diaminopropionic acid, D-cysteine,
D-penicillamine, D-methionine, and
2-aminothiazole-4-acetic acid;
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42
R5 is an amino acid, substituted with 0-1 bonds to Ln,
independently selected at each occurrence from the
group: glycine, L-valine, L-alanine, L-leucine,
L-isoleucine, L-norleucine, L-2-aminobutyric acid,
L-2-aminohexanoic acid, L-tyrosine,
L-phenylalanine, L-thienylalanine, L-phenylglycine,
L-cyclohexylalanine, L-homophenylalanine,
L-1-naphthylalanine, L-lysine, L-serine,
L-ornithine, L-1,2-diaminobutyric acid,
L-1,2-diaminopropionic acid, L-cysteine,
L-penicillamine, L-methionine, and
2-aminothiazole-4-acetic acid;
provided that one of R1, R2, R3, R4, and R5 in each Q is
substituted with a bond to Ln, further provided
that when R~ is 2-aminothiazole-4-acetic acid, K is
N-methylarginine, further provided that when R4 is
2-aminothiazole-4-acetic acid, K and K' are
N-methylarginine, and still further provided that
when R5 is 2-aminothiazole-4-acetic acid, K' is
N-methylarginine;
d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
Ln is a linking group having the formula:
(CR6R~)g-(W)h-(CR6aR7a)g,_(Z)k-(W)h'-(CR8R9)g..-(W)h"-(CR8aR9a
) g..
provided that g+h+g'+k+h'+g"+h"+g"' is other than 0;
W is independently selected at each occurrence from the
group: 0, S, NH, NHC (=0) , C (=0)NH, C (=0) , C (=0) 0,
OC (=0) , NHC (=S)NH, NHC (=O)NH, 502, (OCH2CH2) S,
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43
(CH~CH20)S~, (OCH2CH2CH~)5~~, (CH~CH~CH~O)t, and
(aa)t~;
as is independently at each occurrence an amino acid;
Z is selected from the group: aryl substituted with 0-3
Rlo~ C3-so cYcloalkyl substituted with 0-3 Rlo, and
a 5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and 0 and substituted with 0-3 Rlo;
R6, R6a, R7, R7a, R8, R8a, R9 and R9a are independently
selected at each occurrence from the group: H, =0,
COOH, S03H, P03H, C1-C5 alkyl substituted with 0-3
Rlo, aryl substituted with 0-3 Rlo, benzyl
substituted with 0-3 Rlo, and C1-C5 alkoxy
substituted with 0-3 Rlo, NHC(=0)R11, C(=0)NHR11,
NHC ( =0 ) NHR11, NHR11, R11, and a bond to Ch ;
R1o is independently selected at each occurrence from
the group: a bond to Ch, COOR11, OH, NHR11, S03H,
P03H, aryl substituted with 0-3 R11, C1-5 alkyl
substituted with 0-1 R12, C1-5 alkoxy substituted
with 0-1 R1~, and a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently
selected from N, S, and 0 and substituted with 0-3
R11;
R11 is independently selected at each occurrence from
the group: H, aryl substituted with 0-1 R1~, a
5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and 0 and substituted with 0-1 R12, C3-1o cYcloalkyl
substituted with 0-1 R1~, polyalkylene glycol
substituted with 0-1 R12, carbohydrate substituted
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44
with 0-1 R1~, Cyclodextrin substituted with 0-1 R1~,
amino acid substituted with 0-1 R1~,
polycarboxyalkyl substituted with 0-1 R12,
polyazaalkyl substituted with 0-1 R1~, peptide
substituted with 0-1 R12, wherein the peptide is
comprised of 2-10 amino acids, and a bond to Ch;
R12 is a bond to Ch;
k is selected from 0, 1, and2;
h is selected from 0, 1, and2;
h' is selected from 0, 1, 2, 3, 4, and 5;
h" is selected from 0, 1, 2, 3, 4, and 5;
g is selected from 0, 1, 2, 3, 4, 5, 6, 8, 9, and
7, 10;
g' is selected from 0, 1, 2, 3, 4, 5, 6, 8, 9, and
7,
10;
g" is selected.from 0, 1, 2, 3, 4, 5, 6, 8, 9, and
7,
10;
g" ' s selected , , , , 5, 6, , ,
i from 0 1, 3 4 7 8 9,
2 and
10;
s is selected from 0, 1, 2, 3, 4, 5, 6, 8, 9, and
7, 10;
s' is selected from 0, 1, 2, 3, 4, 5, 6, 8, 9, and
7,
10;
s" is selected from 0, 1, 2, 3, 4, 5, 6, 8, 9, and
7,
10;
t is selected from 0, 1, 2, 3, 4, 5, 6, 8, 9, and
7, 10;
t' is selected from 0, 1, 2, 3, 4, 5, 6, 8, 9, and
7,
10;
Ch is a metal bonding unit having a formula selected
from the group:
~E A2 ~~A~~A~~A4
A A
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p5
fE\
A,1
E~~2'~ s
A
~A~-E Aa,-~ c-E A~ E 'E
~E E E ~ E
A i ~ ~As
A3 A5 , and A~ ;
A1, A~ , A3 , A4 , A5 , A6 , A7 , and A8 are independent 1y
selected at each occurrence from the group N, NR13,
5 NR13R14, S, SH, S(Pg), O, OH, PR13, pR13R14~
P ( 0 ) R15R16 , and a bond to Ln;
E is a bond, CH, or a spacer group independently
selected at each occurrence from the group: C1-C10
10 alkyl substituted with. 0-3 R1~, aryl substituted
with 0-3 R1~, C3-1o cYcloalkyl substituted with 0-3
R1~, heterocyclo-C1_1o alkyl substituted with 0-3
R1~, wherein the heterocyclo group is a 5-10
membered heterocyclic ring system containing 1-4
15 heteroatoms independently selected from N, S, and
0, C6_1o aryl-C1-1o alkyl substituted with 0-3 R1~,
C1-1o alkyl-C6-1o aryl- substituted with 0-3 R1~,
and a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected
20 from N, S, and 0 and substituted with 0-3 R17;
R13, and R14 are each independently selected from the
group: a bond to Ln, hydrogen, C1-C10 alkyl
substituted with 0-3 R1~, aryl substituted with 0-3
25 R1~, C1-1o cycloalkyl substituted with 0-3 R1~,
heterocyclo-C1_1o alkyl substituted with 0-3 R1~,
wherein the heterocyclo group is a 5-10 membered
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46
heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and 0, C6-10
aryl-C1-so alkyl substituted with 0-3 R1~, C~_1o
alkyl-C6_1o aryl- substituted with 0-3 R1~, a 5-10
membered heterocyclic ring system containing 1-4
heteroatoms independently selected from N, S, and 0
and substituted with 0-3 R17, and an electron,
provided that when one of R13 or R14 is an
electron, then the other is also an electron;
alternatively, R13 and R14 combine to form =C (R~o) (R21) ;
R15 and R16 are each independently selected from the
group: a bond to Ln, -OH, C1-C10 alkyl substituted
with 0-3 R1~, C1-C1p alkyl substituted with 0-3
R1~, aryl substituted with 0-3 R1~, C3-so cYcloalkyl
substituted with 0-3 R1~, heterocyclo-C~_1o alkyl
substituted with 0-3 R1~, wherein the heterocyclo
group is a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected
from N, S, and O, C6_1o aryl-C1_1o alkyl substituted
with 0-3 R1~ , C1-1o alkyl-C6_10 aryl- substituted
with 0-3 R1~, and a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-3
R17:
R1~ is independently selected at each occurrence from
the group: a bond to Ln, =0, F, C1, Br, I, -CF3,
-CN, -CO~R18, -C(=O)R18, -C(=0)N(R18)~, -CHO,
-CH~OR18, -OC(=O)R18, -OC(=0)ORlBa, -OR18,
-OC(=0)N(R18)2. -NR19C(=O)R18, -NR19C(=0)ORl8a~
-NR19C(=0)N(R18)2, -NR19S02N(R18)~, -NR19S02R18a~
_S03H~ _SO~Rl8a~ -SR18~ _S(-0)Rl8a~ _SO~N(R18)2.
-N(R18)~, -NHC(=S)NHR18, =NOR18, NO~, -C(=0)NHOR18,
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47
-C(=0)NHNRIgRIga, -OCH2C02H,
2-(1-morpholino)ethoxy, C1-C5 alkyl, C2-C4 alkenyl,
C3-C6 cycloalkyl, C3-C6 cycloalkylmethyl, C2-C6
alkoxyalkyl, aryl substituted with 0-2 R18, and a
5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and 0;
Rlg, Rl8a, and R19 are independently selected at each
occurrence from the group: a bond to Ln, H, C1-C6
alkyl, phenyl, benzyl, C1-C6 alkoxy, halide, nitro,
cyano, and trifluoromethyl;
Pg is a thiol protecting group;
R2o and R21 are independently selected from the group:
H, C1-C10 alkyl, -CN, -C02R25, -C(=0)R25,
-C(=0)N(R25)2, C2-C1o 1-alkene substituted with 0-3
R23, C~-C1o 1-alkyne substituted with 0-3 R23, aryl
substituted with 0-3 R23, unsaturated 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and 0 and
substituted with 0-3 R23, and unsaturated C3-1o
carbocycle substituted with 0-3 R~3;
alternatively, R2o and R21, taken together with the
divalent carbon radical to which they are attached
form:
R22 R22
t
.a b.
R23 R23
n
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48
R22 and R23 are independently selected from the group:
H, R24, C1-C10 alkyl substituted with 0-3 R24,.
C2-C10 alkenyl substituted with 0-3 R24, C2-C10
alkynyl substituted with 0-3 R24, aryl substituted
with 0-3 R24, a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-3
R24, and C3_1o carbocycle substituted with 0-3 R24;
alternatively, R22, R23 taken together form a fused
aromatic or a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently
selected from N, S, and 0;
a and b indicate the positions of optional double bonds
and n is 0 or 1;
R24 is independently selected at each occurrence from
the group: =0, F, Cl, Br, I, -CF3, -CN, -C02R25,
-C(=p)R25~ _C(=O)N(R25)2~ _N(R25)3+~ -CH20R25,
-OC(=0)R25, -OC(=0)OR25a~ _Og.25~ -OC(=0)N(R25)2,
-NR26C (=0) R25, -NR26C (=0) OR25a, -NR26C (=0) N (R25) 2.
-NR26S02N(R25)2, -NR26S02R25a~ _S03H~ _r~02R25a~
-SR25~ _S(=p)R25a~ _S02N(g.25)2, -N(R25)2~ =NOR25,
-C(=O)NHOR25, -OCH2C02H, and
2-(1-morpholino)ethoxy; and,
R25~ R25a~ and R26 are each independently selected at
each occurrence from the group: hydrogen and C1-C6
alkyl;
and a pharmaceutically acceptable salt thereof.
[16] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical
composition according to Embodiment 15, wherein:
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L is glycine;
R1 is an amino acid, optionally substituted with a bond
to Ln, independently selected at each occurrence
from the group: L-valine, D-valine, alanine,
leucine, isoleucine, norleucine, 2-aminobutyric
acid, tyrosine, phenylalanine, phenylglycine,
cyclohexylalanine, homophenylalanine, lysine,
ornithine, 1,2-diaminobutyric acid, and
1,2-diaminopropionic acid;
R~ is an amino acid, optionally substituted with a bond
to Ln, independently selected at each occurrence
from the group: valine, alanine, leucine,
isoleucine, norleucine, 2-aminobutyric acid,
tyrosine, L-phenylalanine, D-phenylalanine,
thienylalanine, phenylglycine, biphenylglycine,
cyclohexylalanine, homophenylalanine,
L-1-naphthylalanine, D-1-naphthylalanine, lysine,
ornithine, 1,2-diaminobutyric acid,
1,2-diaminopropionic acid, and
2-aminothiazole-4-acetic acid;
R3 is an amino acid, optionally substituted with a bond
to Ln, independently selected at each occurrence
from the group: D-valine, D-alanine, D-leucine,
D-isoleucine, D-norleucine, D-2-aminobutyric acid,
D-tyrosine, D-phenylalanine, D-phenylglycine,
D-cyclohexylalanine, D-homophenylalanine, D-lysine,
D-serine, D-ornithine, D-1,2-diaminobutyric acid,
and D-1,2-diaminopropionic acid;
R4 is an amino acid, optionally substituted with a bond
to Ln, independently selected at each occurrence
from the group: D-valine, D-alanine, D-leucine,
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D-isoleucine, D-norleucine, D-2-aminobutyric acid,
D-tyrosine, D-phenylalanine, D-thienylalanine,
D-phenylglycine, D-cyclohexylalanine,
D-homophenylalanine, D-1-naphthylalanine, D-lysine,
5 D-ornithine, D-1,2-diaminobutyric acid,
D-1,2-diaminopropionic acid, and
2-aminothiazole-4-acetic acid;
R5 is an amino acid, optionally substituted with a bond
10 to Ln, independently selected at each occurrence
from the group: L-valine, L-alanine, L-leucine,
L-isoleucine, L-norleucine, L-2-aminobutyric acid,
L-tyrosine, L-phenylalanine, L-thienylalanine,
L-phenylglycine, L-cyclohexylalanine,
15 L-homophenylalanine, L-1-naphthylalanine, L-lysine,
L-ornithine, L-1,2-diaminobutyric acid,
L-1,2-diaminopropionic acid, and
2-aminothiazole-4-acetic acid;
20 d is selected from 1, 2, and 3;
W is independently selected at each occurrence from the
group: 0, NH, NHC(=0), C(=0)NH, C(=0), C(=0)O,
OC(=0), NHC(=S)NH, NHC(=0)NH, 502, (OCH~CH~)S,
25 (CH~CH~O)S~, (OCH~CH~CH~)5~~, and (CH~CH2CH20)t,
Z is selected from the group: aryl substituted with 0-1
Rlo~ C3-ZO cYcloalkyl substituted with 0-1 Rlo, and
a 5-10 membered heterocyclic ring system containing
30 1-4 heteroatoms independently selected from N, S,
and 0 and substituted with 0-1 R10;
R6, R6a, R7, R7a, R8, R8a, R9, and R9a are independently
selected at each occurrence from the group: H, =0,
35 COOH, S03H, C1-C5 alkyl substituted with 0-1 Rlo,
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aryl substituted with 0-1 R1~, benzyl substituted
with 0-1 R1~, and C1-C5 alkoxy substituted with 0-1
R1~, NHC (=0) R11, C (=0) NHR11, NHC (=0) NHR11, NHR11,
R11, and a bond to Ch;
R1~ is independently selected at each occurrence from
the group: COOR11, OH, NHR11, S03H, aryl
substituted with 0-1 R11, a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and 0 and
substituted with 0-1 R11, C1-C5 alkyl substituted
with 0-1 R1~, C1-C5 alkoxy substituted with 0-1 R1~,
and a bond to Ch;
R11 is independently selected at each occurrence from
the group: H, aryl substituted with 0-1 R12, a
5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and 0 and substituted with 0-1 R12, polyalkylene
glycol substituted with 0-1 R1~, carbohydrate
substituted with 0-1 R12, cyclodextrin substituted
with 0-1 R1~, amino acid substituted with 0-1 R1~,
and a bond to Ch;
k is 0 or 1;
h is 0 or 1;
h' is 0 or 1;
s is selected from 0, 1, 2, 3, 4, and 5;
s' is selected from 0, 1, 2, 3, 4, and 5;
s" is selected from 0, 1, 2, 3, 4, and 5;
t is selected from 0, 1, 2, 3, 4, and 5;
A1, A2, A3, A4, A5, A6, A7, and A8 are independently
selected at each occurrence from the group: NR13,
NR13R14, S, SH, S(Pg), OH, and a bond to Ln;
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E is a bond, CH, or a spacer group independently
selected at each occurrence from the group: C1-C10
alkyl substituted with 0-3 R1~, aryl substituted
with 0-3 R1~, C3-1o cycloalkyl substituted with 0-3
R1~, and a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected
from N, S, and 0 and substituted with 0-3 R17;
R13, and R14 are each independently selected from the
group: a bond to Ln, hydrogen, C1-C10 alkyl
substituted with 0-3 R1~, aryl substituted with 0-3
R1~, a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected
from N, S, and 0 and substituted with 0-3 R1~, and
an electron, provided that when one of R13 or R14
is an electron, then the other is also an electron;
alternatively, R13 and R14 combine to form =C(R2o)(R21);
R1~ is independently selected at each occurrence from
the group: a bond to Ln, =0, F, Cl, Br, I, -CF3,
-CN, -C03R18, -C(=0)R18, -C(=0)N(R18)2, -CH20R18,
-OC(=0)R18, -OC(=0)ORlBa, -OR18, -OC(=0)N(R18)2,
-NR19C(=0)R18, -NR19C(=0)ORl8a~ _NR19C(=0)N(R18)2,
-NR19S02N(R18)2, -NR19SO~R18a~ _S03H~ _SO~Rl8a~
_S(=0)Rl8a~ -SO~N(R18)2~ _N(R18)~~ -NHC(=S)NHR18,
=NOR18, -C(=O)NHNR18R18a, -OCH2C02H, and
2-(1-morpholino)ethoxy;
R18, RlBa, and R19 are independently selected at each
occurrence from the group: a bond to Ln, H, and
C1-C6 alkyl;
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R2~ and R~1 are independently selected from the group:
H, C1-C5 alkyl, -C02R~5, C~-C5 1-alkene substituted
with 0-3 R23, C~-C5 1-alkyne substituted with 0-3
R23, aryl substituted with 0-3 R~3, and unsaturated
5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and 0 and substituted with 0-3 R~3;
alternatively, R20 and R~~-, taken together with the
divalent carbon radical to which they are attached
form:
n
R~2 and R23 are independently selected from the group:
H, and R24;
alternatively, R22, R23 taken together form a fused
aromatic or a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently
selected from N, S, and 0;
R~4 is independently selected at each occurrence from
the group: -C02R~5, -C(=0)N(R25)~, -CH20R25,
-OC(=O)R25, -ORBS, -S03H, -N(R~5)~, and -OCH2C02H;
2 5 and ,
R~5 is independently selected at each occurrence from
the group: H and C1-C3 alkyl.
[17~. In another embodiment, the present
invention provides a therapeutic radiopharmaceutical
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composition according to Embodiment 15, wherein the
radioisotope is 153Sm.
[18] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 17, wherein the
radiopharmaceutical is selected from the group:
cyclo(Arg-Gly-Asp-D-Phe-Lys(DTPA-153gm));
cyclo(Arg-Gly-Asp-D-Phe-Lys)2(DTPA-153Sm); and,
cyclo(Arg-Gly-Asp-D-Tyr(N-DTPA(153gm)-3-aminopropyl)-
Val ) .
[19] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition,
according to Embodiment 15, wherein the radioisotope is
177Lu.
[20] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 19, wherein the
radiopharmaceutical is selected from the group:
cyclo(Arg-Gly-Asp-D-Phe-Lys(DTPA-177Lu));
(DOTA-177Lu)-Glu(cyclo{Lys-Arg-Gly-Asp-D-Phe})-
cyclo{Lys-Arg-Gly-Asp-D-Phe};
cyclo(Arg-Gly-Asp-D-Phe-Lys)2(DTPA-177Lu); and,
cyclo(Arg-Gly-Asp-D-Tyr(N-DTPA(177Lu)-3-aminopropyl)-
Val ) .
[21] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 15, wherein the radioisotope is
90y.
[22] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 21, wherein the
radiopharmaceutical is:
(DOTA-90Y)-Glu(cyclo{Lys-Arg-Gly-Asp-D-Phe})-C~1'C10{Lys-
Arg-Gly-Asp-D-Phe}.
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[23] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 15, wherein the anti-cancer
agent is selected from the group consisting of
5 mitomycin, tretinoin, ribomustin, gemcitabine,
vincristine, etoposide, cladribine, mitobronitol,
methotrexate, doxorubicin, carboquone, pentostatin,
nitracrine, zinostatin, cetrorelix, letrozole,
raltitrexed, daunorubicin, fadrozole, fotemustine,
10 thymalfasin, sobuzoxane, nedaplatin, cytarabine,
bicalutamide, vinorelbine, vesnarinone,
aminoglutethimide, amsacrine, proglumide, elliptinium
acetate, ketanserin, doxifluridine, etretinate,
isotretinoin, streptozocin, nimustine, vindesine,
15 flutamide, drogenil, butocin, carmofur, razoxane,
sizofilan, carboplatin, mitolactol, tegafur, ifosfamide,
prednimustine, picibanil, levamisole, teniposide,
improsulfan, enocitabine, lisuride, oxymetholone,
tamoxifen, progesterone, mepitiostane, epitiostanol,
20 formestane, interferon-alpha; interferon-2 alpha,
interferon-beta, interferon-gamma, colony stimulating
factor-1, colony stimulating factor-2, denileukin
diftitox, interleukin-2, and leutinizing hormone
releasing factor.
25 [24] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 15, wherein radiosensitizer
agent is selected from the group consiting of 2-(3-
nitro-1,2,4-triazol-1-yl)-N-(2-methoxyethyl)acetamide,
30 N-(3-nitro-4-quinolinyl)-4-morpholinecarboxamidine, 3-
amino-1,2,4-benzotriazine-1,4-dioxide, N-(2-
hydroxyethyl)-2-nitroimidazole-1-acetamide, 1-(2-
nitroimidazol-1-yl)-3-(1-piperidinyl)-2-propanol, and 1-
(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol.
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[25] In another embodiment, the present invention
provides a method of treating cancer in a patient
comprising: administering to a patient in need thereof a
therapeutic radiopharmaceutical comprising:
a) a radioisotope;
b) a chelator capable of chelating the radioisotope l;
and
c) a targeting moiety;
wherein the targeting moiety is bound to the chelator
through a linking group, and the targeting moiety is a
peptide or peptidomimetic that binds to a receptor that
is upregulated during angiogenesis, and the radioisotope
is a radioisotope selected from the group: 33p~ 125I~
186Re~ 188Re~ 153Sm~ 166go~ 177Lu~ 149pms 90y~ 212gi,
103pd~ 109pd~ 159Gd~ 140La~ 198Au~ 199Au~ 169yb~ 175yb~
165DY~ 166DY~ 67Cu~ 105Rh~ 111Ag~ and 192Ir or a
pharmaceutically acceptable salt thereof; and
at least one agent selected from the group consisting of
an anti-cancer agent and a radiosensitizer agent, or a
pharmaceutically acceptable salt thereof.
[26] In another embodiment, the present invention
provides a method according to Embodiment 25, wherein
the targeting moiety is a cyclic pentapeptide and the
receptor is ocv(33 or ocv(35.
[27] In another embodiment, the present invention
provides a method according to Embodiment 25, wherein
the therapeutic radiopharmaceutical comprises:
a) a radioisotope selected from the group: 33p~ 125I~
186Re~ 188Re~ 153Sm~ 166Ho~ 177Lu~ 149pm~ 90y~ 212Bi~
103pd~ 109pd~ 159Gd~ 140La~ 198Au~ 199Au~ 169yb~
175yb~ 165DY~ 166DY~ 67Cu, 105Rh~ 111Ag~ and 192Ir;
and
b) a compound of the formula (I):
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(Q)d-Ln-Ch ~r (Q)d-Ln-(Ch)d~
(I)
wherein, Q is a peptide independently selected from the
group:
,~L~ /R~ 4
K ~ M K R
1 2 ~ 3
R R , R R , L M' , and
K~~L~M
R3 R5
K is an L-amino acid independently selected at each
occurrence from the group: arginine, citrulline,
N-methylarginine, lysine, homolysine,
2-aminoethylcysteine, 8-N-2-imidazolinylornithine,
~-N-benzylcarbamoylornithine, and
l5 (3-2-benzimidazolylacetyl-1,2-diaminopropionic acid;
K' is a D-amino acid independently selected at each
occurrence from the group: arginine, citrulline,
N-methylarginine, lysine, homolysine,
2-aminoethylcysteine, 8-N-2-imidazolinylornithine,
8-N-benzylcarbamoylornithine, and
(3-2-benzimidazolylacetyl-1,2-diaminopropionic acid;
L is independently selected at each occurrence from the
group: glycine, L-alanine, and D-alanine;
M is L-aspartic acid;
M' is D-aspartic acid;
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R1 is an amino acid substituted with 0-1 bonds to Ln,
independently selected at each.occurrence from the
group: glycine, L-valine, D-valine, alanine,
leucine, isoleucine, norleucine, 2-aminobutyric
acid, 2-aminohexanoic acid, tyrosine,
phenylalanine, thienylalanine, phenylglycine,
cyclohexylalanine, homophenylalanine,
1-naphthylalanine, lysine, serine, ornithine,
1,2-diaminobutyric acid, 1,2-diaminopropionic acid,
cysteine, penicillamine, and methionine;
R~ is an amino acid, substituted with 0-1 bonds to Ln,
independently selected at each occurrence from the
group: glycine, valine, alanine, leucine,
isoleucine, norleucine, 2-aminobutyric acid,
2-aminohexanoic acid, tyrosine, L-phenylalanine, D-
phenylalanine, thienylalanine, phenylglycine,
biphenylglycine, cyclohexylalanine,
homophenylalanine, L-1-naphthylalanine,
D-1-naphthylalanine, lysine, serine, ornithine,
1,2-diaminobutyric acid, 1,2-diaminopropionic acid,
cysteine, penicillamine, methionine, and
2-aminothiazole-4-acetic acid;
R3 is an amino acid, substituted with 0-1 bonds to Ln,
independently selected at each occurrence from the
group: glycine, D-valine, D-alanine, D-leucine,
D-isoleucine, D-norleucine, D-2-aminobutyric acid,
D-2-aminohexanoic acid, D-tyrosine,
D-phenylalanine, D-thienylalanine, D-phenylglycine,
D-cyclohexylalanine, D-homophenylalanine,
D-1-naphthylalanine, D-lysine, D-serine,
D-ornithine, D-1,2-diaminobutyric acid,
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D-1,2-diaminopropionic acid, D-cysteine,
D-penicillamine, and D-methionine;
R4 is an amino acid, substituted with 0-1 bonds to Ln,
independently selected at each occurrence from the
group: glycine, D-valine, D-alanine, D-leucine,
D-isoleucine, D-norleucine, D-2-aminobutyric acid,
D-2-aminohexanoic acid, D-tyrosine,
D-phenylalanine, D-thienylalanine, D-phenylglycine,
D-cyclohexylalanine, D-homophenylalanine,
D-1-naphthylalanine, D-lysine, D-serine,
D-ornithine, D-1,2-diaminobutyric acid,
D-1,2-diaminopropionic acid, D-cysteine,
D-penicillamine, D-methionine, and
2-aminothiazole-4-acetic acid;
R5 is an amino acid, substituted with 0-1 bonds to Ln,
independently selected at each occurrence from the
group: glycine, L-valine, L-alanine, L-leucine,
L-isoleucine, L-norleucine, L-2-aminobutyric acid,
L-2-aminohexanoic acid, L-tyrosine,
L-phenylalanine, L-thienylalanine, L-phenylglycine,
L-cyclohexylalanine, L-homophenylalanine,
L-1-naphthylalanine, L-lysine, L-serine,
L-ornithine, L-1,2-diaminobutyric acid,
L-1,2-diaminopropionic acid, L-cysteine,
L-penicillamine, L-methionine, anal
2-aminothiazole-4-acetic acid;
provided that one of R1, R2, R3, R4, and R5 in each Q is
substituted with a bond to Ln, further provided
that when R~ is 2-aminothiazole-4-acetic acid, K is
N-methylarginine, further provided that when R4 is
2-aminothiazole-4-acetic acid, K and K' are
N-methylarginine, and still further provided that
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when R5 is 2-aminothiazole-4-acetic acid, K' is
N-methylarginine;
d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
5
Ln is a linking group having the formula:
(CR6R7)g-(W)h_(CR6aR7a)g,-(z)k_(W)h,_(CR8R9)g~~-(W)h"-(CR8aR9a
g..
provided that g+h+g'+k+h'+g"+h"+g"' is other than 0;
W is independently selected at each occurrence from the
group: 0, S, NH, NHC(=0), C(=0)NH, C(=0), C(=0)0,
OC (=0) , NHC (=S)NH, NHC (=O)NH, 50~, (OCH~CH2) s,
(CH~CH~O)s~, (OCH~CH~CH~)s~~, (CH2CH~CH~O)t, and
(aa)t~;
as is independently at each occurrence an amino acid;
Z is selected from the group: aryl substituted with 0-3
R1~~ C3-1o cycloalkyl substituted with 0-3 Rlo, and
a 5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and O and substituted with 0-3 Rlo;
R6, R6a, R7, R7a, R8, Rga, R9 and R9a are independently
selected at each occurrence from the group: H, =0,
COOH, S03H, P03H, C1-C5 alkyl substituted with 0-3
Rlo, aryl substituted with 0-3 Rlo, benzyl
substituted with 0-3 Rlo, and C1-C5 alkoxy
substituted with 0-3 Rlo, NHC(=0)R11, C(=0)NHR11,
NHC ( =0 ) NHR11, NHR11, R11, and a bond to Ch;
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Rso is independently selected at each occurrence from
the group: a bond to Ch, COOR11, OH, NHR11, S03H,
P03H, aryl substituted with 0-3 R11, C1_5 alkyl
substituted with 0-1 R1~, C~_5 alkoxy substituted
with 0-1 R1~, and a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently
selected from N, S, and 0 and substituted with 0-3
Rll;
R11 is independently selected at each occurrence from
the group: H, aryl substituted with 0-1 R1~, a
5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and 0 and substituted with 0-1 R1~, C3-1o cycloalkyl
substituted with 0-1 R12, polyalkylene glycol
substituted with 0-1 R12, carbohydrate substituted
with 0-1 R1~, cyclodextrin substituted with 0-1 R~-2,
amino acid substituted with 0-1 R1~,
polycarboxyalkyl substituted with 0-1 R1~,
polyazaalkyl substituted with 0-1 R1~, peptide
substituted with 0-1 R12, wherein the peptide is
comprised of 2-10 amino acids, and a bond to Ch;
R1~ is a bond to Ch;
k is selected from 0, 1, and 2;
h is selected from 0, 1, and 2;
h' is selected from 0, 1, 2, 3, 4, and 5;
h" is selected from 0, 1, 2, 3, 4, and 5;
g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
g' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
g" is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
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g"' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
s is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
s' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
s" is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
t' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
Ch is a metal bonding unit having a formula selected.
from the group:
~E A2 ~~A~~A~~A4
A , A ,
p5
~'E
Ar-'A6
A8
E~A~E ~~-E-~~-E A~
A~ E I5 A8 ~ E
A A , and A
A1, A2 , A3 , A4 , A5 , A6 , A7 , and A8 are independently
selected at each occurrence from the group: N,
NR13, NR13R14, S, SH, S (Pg) , 0, OH, PR13, pR13R14~
P ( O ) R15R16 , and a bond to Ln;
E is a bond, CH, or a spacer group independently
selected at each occurrence from the group: C1-C10
alkyl substituted with 0-3 R1~, aryl substituted
with 0-3 R1~, C3_10 cycloalkyl substituted with 0-3
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R1~, heterocyclo-C~-1o alkyl substituted with 0-3
R1~, wherein the heterocyclo group is a 5-10
membered heterocyclic ring system containing 1-4
heteroatoms independently selected from N, S, and
0, C6_1o aryl-C1-1o alkyl substituted with 0-3 R1~,
C1-1o alkyl-C6-1o aryl- substituted with 0-3 R1~,
and a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected
from N, S, and 0 and substituted with 0-3 R~-7;
R13, and R14 are each independently selected from the
group: a bond to Ln, hydrogen, C1-C1p alkyl
substituted with 0-3 R1~, aryl substituted with 0-3
R1~. CZ-1o cYcloalkyl substituted with 0-3 R1~,
heterocyclo-C1-1o alkyl substituted with 0-3 R1~,
wherein the heterocyclo group is a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and 0, C6_1o
aryl-C1-1p alkyl substituted with 0-3 R1~ , C1-1o
alkyl-C6-1o aryl- substituted with 0-3 R1~, a 5-10
membered heterocyclic ring system containing 1-4
heteroatoms independently selected from N, S, and 0
and substituted with 0-3 R17, and an electron,
provided that when one of R13 or R14 is an
electron, then the other is also an electron;
alternatively, R13 and R14 combine to form =C(R2p)(R~1);
R15 and R16 are each independently selected from the
group: a bond to Ln, -OH, C1-C1o alkyl substituted
with 0-3 R1~, C1-C1o alkyl substituted with 0-3
R1~, aryl substituted with 0-3 R1~, C3_1o cycloalkyl
substituted with 0-3 R1~, heterocyclo-C1-so alkyl
substituted with 0-3 R1~, wherein the heterocyclo
group is a 5-10 membered heterocyclic ring system
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containing 1-4 heteroatoms independently selected
from N, S, and 0, C6_1o aryl-C1_1o alkyl substituted
with 0-3 R1~, C1_1o alkyl-C6_1o aryl- substituted
with 0-3 R1~, and a 5-10 membered heterocyclic ring
system containing 2-4 heteroatoms independently
selected from N, S, and 0 and substituted with 0-3
R17;
R1~ is independently selected at each occurrence from
the group: a bond to Ln, =0, F, Cl, Br, I, -CF3,.
-CN, -CO~R18, -C(=0)R18, -C(=0)N(R18)2, -CHO,
-CH20R18, -OC(=0)R18~ _OC(=0)pRl8a~ -OR18
-OC(=O)N(R18)~~ -NR19C(=0)R18~ -NR19C(=0)ORl8a~
-NR19C(=0)N(R18)2, -NR19SO~N(R18)~, -NR19S02R18a~
-S03H, -SO~Rl8a~ -SR18~ _S(=0)Rl8a~ _S02N(R18)2~
-N(R18)2, -NHC(=S)NHR18, =NOR18, N02, -C(=0)NHOR18,
-C(=0)NHNR18R18a, -OCH2CO~H,
2-(1-morpholino)ethoxy, C1-C5 alkyl, C2-C4 alkenyl,
C3-C6 cycloalkyl, C3-C6 cycloalkylmethyl, C~-C6
alkoxyalkyl, aryl substituted with 0-2 R18, and a
5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and 0 ;
R18, RlBa, and R19 are independently selected at each
occurrence from the group: a bond to Ln, H, C1-C6
alkyl, phenyl, benzyl, C1-C6 alkoxy, halide, nitro,
cyano, and trifluoromethyl;
Pg is a thiol protecting group;
R2o and R~1 are independently selected from the group:
H, C1-C1o alkyl, -CN, -COBRAS, -C (=0) R25,
-C(=0)N(R~5)~, C2-C1o 1-alkene substituted with 0-3
R~3, C~-C1o 1-alkyne substituted with 0-3 R~3, aryl
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substituted with 0-3 R23, unsaturated 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and 0 and
substituted with 0-3 R23, and unsaturated C3_1o
5 carbocycle substituted with 0-3 R23;
alternatively, R2o and R21, taken together with the
divalent carbon radical to which they are attached
form:
n
R22 and R23 are independently selected from the group:
H, R24, C1-C10 alkyl substituted with 0-3 R24,
C2-C10 alkenyl substituted with 0-3 R24, C2-C10
alkynyl substituted with 0-3 R24, aryl substituted
with 0-3 R24, a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently
selected from N, S, and 0 and substituted with 0-3
R24, and C3_2o carbocycle substituted with 0-3 R24;
alternatively, R22, R23 taken together form a fused
aromatic or a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently
selected from N, S, and 0;
a and b indicate the positions of optional double bonds
and n is 0 or 1;
R24 is independently~selected at each occurrence from
the group: =0, F, C1, Br, I, -CF3, -CN, -C02R25,
-C(=O)R25, -C(=0)N(R25)2, -N(R25)3+~ -CH20R25,
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_OC(=O)R25~ -OC(=O)OR25a~ _OR25~ -OC(=0)N(R25)2,
-NR26C (=0) R25 ~ -NR26C (=O) OR25a~ _NR26C (=p)N (R25) 2
_NR26S02N(R25)2~ _NR26S02R25a~ _S03H~ -S02R25a~
-SR25~ _S(=O)R25a~ _g02N(R25)2~ -N(R25)2, =NOR25,
-C(=0)NHOR25, -OCH2C02H, and
2-(1-morpholino)ethoxy; and,
R25, R25a, and R26 are each independently selected at
each occurrence from the group: hydrogen and C1-C6
alkyl;
and a pharmaceutically acceptable salt thereof.
[28] In another embodiment, the present invention
provides a method according to, Embodiment 27,
wherein:
L is glycine;
R~- is an amino acid, optionally substituted with a bond
to Ln, independently selected at each occurrence
from the group: L-valine, D-valine, alanine,
leucine, isoleucine, norleucine, 2-aminobutyric
acid, tyrosine, phenylalanine, phenylglycine,
cyclohexylalanine, homophenylalanine, lysine,
ornithine, 1,2-diaminobutyric acid, and
1,2-diaminopropionic acid;
R2 is an amino acid, optionally substituted with a bond
to Ln, independently selected at each occurrence
from the group: valine, alanine, leucine,
isoleucine, norleucine, 2-aminobutyric acid,
tyrosine, L-phenylalanine, D-phenylalanine,
thienylalanine, phenylglycine, biphenylglycine,
cyclohexylalanine, homophenylalanine,
L-1-naphthylalanine, D-1-naphthylalanine, lysine,
ornithine, 1,2-diaminobutyric acid,
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1,2-diaminopropionic acid, and
2-aminothiazole-4-acetic acid;
R3 is an amino acid, optionally substituted with a bond
to Ln, independently selected at each occurrence
from the group: D-valine, D-alanine, D-leucine,
D-isoleucine, D-norleucine, D-2-aminobutyric acid,
D-tyrosine, D-phenylalanine, D-phenylglycine,
D-cyclohexylalanine, D-homophenylalanine, D-lysine,
D-serine, D-ornithine, D-1,2-diaminobutyric acid,
and D-1,2-diaminopropionic acid;
R4 is an amino acid, optionally substituted with a bond
to Ln, independently selected at each occurrence
from the group: D-valine, D-alanine, D-leucine,
D-isoleucine, D-norleucine, D-2-aminobutyric acid,
D-tyrosine, D-phenylalanine, D-thienylalanine,
D-phenylglycine, D-cyclohexylalanine,
D-homophenylalanine, D-1-naphthylalanine, D-lysine,
D-ornithine, D-1,2-diaminobutyric acid,
D-1,2-diaminopropionic acid, and
2-aminothiazole-4-acetic acid;
R5 is an amino acid, optionally substituted with a bond
to Ln, independently selected at each occurrence
from the group: L-valine, L-alanine, L-leucine,
L-isoleucine, L-norleucine, L-2-aminobutyric acid,
L-tyrosine, L-phenylalanine, L-thienylalanine,
L-phenylglycine, L-cyclohexylalanine,
L-homophenylalanine, L-1-naphthylalanine, L-lysine,
L-ornithine, L-1,2-diaminobutyric acid,
L-1,2-diaminopropionic acid, and
2-aminothiazole-4-acetic acid;
d is selected from 1, 2, and 3;
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W is independently selected at each occurrence from the
group: 0, NH, NHC(=0), C(=0)NH, C(=0), C(=0)0,
OC (=O) , NHC (=S)NH, NHC (=0)NH, 502, (OCH~CH~) S,
(CH~CH~O)S~, (OCH~CH~CH~)5~~, and (CH~CH~CH~O)t,
Z is selected from the group: aryl substituted with 0-1
Rlo. C3-1o cYcloalkyl substituted with 0-1 Rlo, and
a 5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and 0 and substituted with 0-1 Rlo;
R6, R6a, R7, R7a, R8, R8a, R9, and R9a are independently
selected at each occurrence from the group: H, =0,
COON, S03H, C1-C5 alkyl substituted with 0-1 Rlo,
aryl substituted with 0-1 Rio, benzyl substituted
with 0-1 Rlo, and C1-C5 alkoxy substituted with 0-1
R10 , NHC ( =0 ) R11, C ( =0 ) NHR11, NHC ( =0 ) NHR11, NHR11,
R11, and a bond to Ch;
Rio is independently selected at each occurrence from
the group: COOR11, OH, NHR11, S03H, aryl
substituted with 0-1 R11, a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and O and
substituted with 0-1 R11, C~-C5 alkyl substituted
with 0-1 R1~, C1-C5 alkoxy substituted with 0-1 R1~,
and a bond to Ch;
R11 is independently selected at each occurrence from
the group: H, aryl substituted with 0-1 R12, a
5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and 0 and substituted with 0-1 R1~, polyalkylene
glycol substituted with 0-1 R1~, carbohydrate
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substituted with 0-1 R12, cyclodextrin substituted
with 0-1 R1~, amino acid substituted with 0-1 R1~,
and a bond to Ch;
k is 0 or 1;
h is 0 or 1;
h' is 0 or 1;
s is selected from 0, 1, 2, 3, 4, and 5;
s' is selected from 0, 1, 2, 3, 4, and 5;
s" is selected from 0, 1, 2, 3, 4, and 5.~~-
t is selected from 0, 1, 2, 3, 4, and 5;
A1, A~, A3, A4, A5, A6, A7, and A8 are independently
selected at each occurrence from the group: NR13,
NR13R14, S, SH, S(Pg), OH, and a bond to Ln;
E is a bond, CH, or a spacer group independently
selected at each occurrence from the group: C1-C10
alkyl substituted with 0-3 R1~, aryl substituted
with 0-3 R1~, C3_1p cycloalkyl substituted with 0-3
R1~, and a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected
from N, S, and 0 and substituted with 0-3 R17;
R13, and R14 are each independently selected from the
group: a bond to Ln, hydrogen, C1-C1p alkyl
substituted with 0-3 R1~, aryl substituted with 0-3
R1~, a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected
from N, S, and 0 and substituted with 0-3 R1~, and
an electron, provided that when one of R13 or R14
is an electron, then the other is also an electron;
alternatively, R13 and R14 combine to form =C (R~0) (R21) ;
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R1~ is independently selected at each occurrence from
the group: a bond to Ln, =0, F, Cl, Br, I, -CF3,
-CN, -C02R18, -C(=0)R18, -C(=0)N(R18)~, _CH~OR18,
_OC(=0)R18~ -OC(=O)ORlBa~ _OR18~ _OC(=O)N(R18)2~
5 -NR19C(=0)R18, -NR19C(=0)ORl8a~ _NR19C(=O)N(R18)2~
_NR19S02N(R18)~~ _NR19SO~R18a~ _S03g~ _SO~Rl8a~
_S(=O)Rl8a~ _S02N(R18)~~ _N(R18)2~ -NHC(=S)NHR18,
=NOR18, -C(=0)NHNR18R18a, -OCH~C02H, and
2-(1-morpholino)ethoxy;
R18, RlBa, and R19 are independently selected at each
occurrence from the group: a bond to Ln, H, and
C1-C6 alkyl;
R2~ and R~1 are independently selected from the group:
H, C1-C5 alkyl, -C02R25, C2-C5 1-alkene substituted
with 0-3 R~3, C2-C5 1-alkyne substituted with 0-3
R23, aryl substituted with 0-3 R23, and unsaturated
5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S,
and 0 and substituted with 0-3 R23;
alternatively, R~~ and R~1, taken together with the
divalent carbon radical to which they are attached
form:
n
R~2 and R23 are independently selected from the group:
H , and R~ 4 ;
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alternatively, R~~, R~3 taken together form a fused
aromatic or a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently
selected from N, S, and 0;
R24 is independently selected at each occurrence from
the group: -C02R~5, -C(=0)N(R~5)2, -CH~OR~S,
-OC(=0)R25, -OR~5, -S03H, -N(R~5)2, and -OCH2C02H;
and ,
R~5 is independently selected at each occurrence from
the group: H and C1-C3 alkyl.
[29] In another embodiment, the present invention
provides a method according to Embodiment 27,
wherein:
Q is a peptide selected from the group:
~R~ 4
K ~ K R
~1 2
R R and ~ M ;
R1 is L-valine, D-valine, D-lysine optionally
substituted on the E amino group with a bond to Ln
or L-lysine optionally substituted on the ~ amino
group with a bond to Ln;
R2 is L-phenylalanine, D-phenylalanine,
D-1-naphthylalanine, 2-aminothiazole-4-acetic acid,
L-lysine optionally substituted on the E amino
group with a bond to Ln or tyrosine, the tyrosine
optionally substituted on the hydroxy group with a
bond to Ln;
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R3 is D-valine, D-phenylalanine, or L-lysine optionally
substituted on the ~ amino group with a bond to Ln;
R4 is D-phenylalanine, D-tyrosine substituted on the
hydroxy group with a bond to Ln, or L-lysine
optionally substituted on the ~ amino group with a
bond to Ln;
provided that one of R1 and R2 in each g is substituted
with a bond to Ln, and further provided that when
R2 is.2-aminothiazole-4-acetic acid, K is
N-methylarginine;
d is 1 or 2;
W is independently selected at each occurrence from the
group: NHC(=0), C(=0)NH, C(=0), (CH2CH20)S~, and
(CH2CH2CH20)t;
R6, R6a, R~, R7a, R8, R8a, R9, and R9a are independently
selected at each occurrence from the group: H,
NHC(=O)R11, and a bond to Ch;
k is 0;
h" is selected from 0, 1, 2, and 3;
g is selected from 0, 1, 2, 3, 4, and 5;
g' is selected from 0, 1, 2, 3, 4, and 5;
g" is selected from 0, 1, 2, 3, 4, and 5;
g"' is selected from 0, 1, 2, 3, 4, and 5;
s' is 1 or 2;
t is 1 or 2;
~ E~A~E Aa~ E-~c-E A~
AE E5 E~A8
Ch is A ,
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A1 is selected from the group: OH, and a bond to Ln;
A2, A4, and A6 are each N;
A3 , A5 , and A8 are each OH;
A7 is a bond to Ln or NH-bond to Ln;
E is a C2 alkyl substituted with 0-1 R1~;
R1~ is =O;
~E A2
alternatively, Ch is A ;
A1 is NHS or N=C (R~~) (R~1) ;
E is a bond;
2 0 A~ i s NHR13 ;
R13 is a heterocycle substituted with R17, the
heterocycle being selected from pyridine and
pyrimidine;
R17 is selected from a bond to Ln, C(=O)NHR18, and
C (=O) R18;
R18 is a bond to Ln;
R~4 is selected from the group: -C02R~5, -ORBS, -S03H,
and -N ( R2 5 ) 2 ;
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R25 is independently selected at each occurrence from
the group: hydrogen and methyl;
p5
~'E
E~~ Y-\ s
A
s
A
E 'E A3/
~E
alternatively, Ch is A~ ;
A1, A~ , A3 , and A4 are each N;
A5, A6, and A8 are each OH;
A7 is a laond to Ln;
E is a C2 alkyl substituted with 0-1 R17; and,
R17 is =O.
[30] In another embodiment, the present invention
provides a method according to Embodiment 27 wherein the
compound of formula (I) is selected from the group
consisting of:
(a) cyclo{Arg-Gly-Asp-D-Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
3-aminopropyl)-Val};
(b) cyclo{Arg-Gly-Asp-D-Tyr((N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
18-amino-14-aza-4,7,10-oxy-15-oxo-octadecoyl)-3-
aminopropyl)-Val};
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(c) [2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-Glu(cyclo{D-Tyr(3-
aminopropyl)-Val-Arg-Gly-Asp})-cyclo{D-Tyr(3-
aminopropyl)-Val-Arg-Gly-Asp};
5
(d) cyclo(Arg-Gly-Asp-D-Tyr-Lys([2-[[[5-[carbonyl]-2
pyridinyl]hydrazono]methyl]-benzenesulfonic
acid] ) } ;
10 (e) cyclo{Arg-Gly-Asp-D-Phe-Lys([2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic
acid] ) } ;
(f) [2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
15 benzenesulfonic acid]-Glu(cyclo{Lys-Arg-Gly-Asp-D-
Phe})-cyc.lo{Lys-Arg-Gly-Asp-D-Phe};
(g) [2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]
benzenesulfonic acid]-Phe-Glu(cyclo{Lys-Arg-Gly
20 Asp-D-Phe})-CyClo{Lys-Arg-Gly-Asp-D-Phe};
(h) cyclo{Arg-Gly-Asp-D-Nal-Lys([2-[[[5-[carbonyl]-2
pyridinyl]hydrazono]methyl]-benzenesulfonic
acid] ) } ;
(i) [2-[[[5-[carbonyl]-2-pyridinyl]-hydrazono]methyl]-
benzenesulfonic acid]-Glu(cyclo{Lys-Arg-Gly-Asp-D-
Nal})-cyclo{Lys-Arg-Gly-Asp-D-Nal};
(j) cyclo{Arg-Gly-Asp-Lys([2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid])-
D-Val} ;
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(k) [2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-Glu(cyclo{Lys-D-Val-Arg-Gly-
Asp})-cyclo{Lys-D-Val-Arg-Gly-Asp};
(1) {cyclo(Arg-D-Val-D-Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
3-aminopropyl)-D-Asp-Gly};
(m) cyclo{D-Lys([2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid])-
D-Phe-D-Asp-Gly-Arg};
(n) [2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-Glu(cyclo{D-Lys-D-Phe-D-Asp-
Gly-Arg})-CyClO{D-Lys-D-Phe-D-Asp-Gly-Arg};
(o) cyclo{D-Phe-D-Lys([2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfoniC acid])-
D-Asp-Gly-Arg};
(p) cyclo{N-Me-Arg-Gly-Asp-ATA-D-Lys([2-[[[5-[carbonyl]-
2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid] ) } ;
(q) cyclo{Cit-Gly-Asp-D-Phe-Lys([2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic
acid])};
(r) 2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-1-
cyclododecyl)acetyl-Glu(cyclo{Lys-Arg-Gly-Asp-D-
Phe})-cyclo{Lys-Arg-Gly-Asp-D-Phe};
(s) cyclo{Arg-Gly-Asp-D-Phe-Lys(DTPA)};
(t) cyclo{Arg-Gly-Asp-D-Phe-Lys}~(DTPA);
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(u) Cyclo{Arg-Gly-Asp-D-Tyr(N-DTPA-3-aminopropyl)-Val};
(v) cyclo{Orn(d-N-2-Imidazolinyl)-Gly-Asp-D-Tyr(N-[2-
[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-Val};
(w) cyclo{Lys-Gly-Asp-D-Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
3-aminopropyl)-Val};
(x) cyclo{Cys(2-aminoethyl)-Gly-Asp-D-Tyr(N-[2-[[[5-
[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-Val};
(y) cyclo{HomoLys-Gly-Asp-D-Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
3-aminopropyl)-Val};
(z) cyclo{Orn(d-N-Benzylcarbamoyl)-Gly-Asp-D-Tyr(N-[2-
[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-Val};
(aa) cyclo{Dap(b-(2-benzimidazolylacetyl))-Gly-Asp-D-
Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
3-aminopropyl)-Val};
(bb) cyclo{Orn(d-N-2-Imidazolinyl)-Gly-Asp-D-Phe-Lys(N-
[2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid])};
(cc) cyclo{Orn(d-N-Benzylcarbamoyl)-Gly-Asp-D-Phe-Lys(N-
[2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid])};
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(dd) cyclo{Lys-D-Val-D-Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
3-aminopropyl)-D-Asp-Gly};
(ee) cyclo{Orn(d-N-Benzylcarbamoyl)-D-Val-D-Tyr(N-(2-
[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-D-Asp-Gly};
and,
(ff) cyclo{Orn(d-N-2-Imidazolinyl)-D-Val-D-Tyr(N-[2-
[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-D-Asp-Gly};
or a pharmaceutically acceptable salt form thereof.
(31] In another embodiment, the present invention
provides a method according to Embodiment 27,
wherein the radioisotope is 153Sm.
(32] In another embodiment, the present invention
provides a method according to Embodiment 31,
wherein the radiopharmaceutical is selected from
the group:
cyclo(Arg-Gly-Asp-D-Phe-Lys(DTPA-153Sm));
cyclo(Arg-Gly-Asp-D-Phe-Lys)2(DTPA-153Sm); and,
cyclo(Arg-Gly-Asp-D-Tyr(N-DTPA(153Sm)-3-aminopropyl)-
Val).
[33] In another embodiment, the present invention
provides a method according to Embodiment 27,
wherein the radioisotope is 177Lu.
[34] In another embodiment, the present invention
provides a method according to Embodiment 27,
wherein the radiopharmaceutical is selected from
the group:
cyclo(Arg-Gly-Asp-D-Phe-Lys(DTPA-177Lu));
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(DOTA-l~~Lu)-Glu(cyclo{Lys-Arg-Gly-Asp-D-Phe})-
cyclo{Lys-Arg-Gly-Asp-D-Phe};
cyclo(Arg-Gly-Asp-D-Phe-Lys)2(DTPA-177Lu); and,
cyclo(Arg-Gly-Asp-D-Tyr(N-DTPA(177Lu)-3-aminopropyl)-
Val ) .
[35] In another embodiment, the present invention
provides a method according to Embodiment 27,
wherein the radioisotope is Soy.
[36] A method according to Embodiment 35, wherein
the radiopharmaceutical is:
(DOTA-9~Y)-Glu(cyclo{Lys-Arg-Gly-Asp-D-Phe})-cyclo{Lys-
Arg-Gly-Asp-D-Phe}.
[37] In another embodiment, the present invention
provides a method according to Embodiment 25 wherein
administering the therapeutic radiopharmaceutical and
agent is concurrent.
[38] In another embodiment, the present invention
provides a method according to Embodiment 25 wherein
administering the therapeutic radiopharmaceutical and
agent is sequential.
[39] In another embodiment, the present invention
provides a method according to Embodiment 25 wherein the
cancer is selected from the group consisting of
carcinomas of the lung, breast, ovary, stomach,
pancreas, larynx, esophagus, testes, liver, parotid,
biliary tract, colon, rectum, cervix, uterus,
endometrium, kidney, bladder, prostate, thyroid, squamous
cell carcinomas, adenocarcinomas, small cell carcinomas,
melanomas, gliomas, and neuroblastomas.
[40] In another embodiment, the present invention
provides a method according to Embodiment 25 wherein the
anti-cancer agent is selected from the group consisting
of mitomycin, tretinoin, ribomustin, gemcitabine,
vincristine, etoposide, cladribine, mitobronitol,
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methotrexate, doxorubicin, carboquone, pentostatin,
nitracrine, zinostatin, cetrorelix, letrozole,
raltitrexed, daunorubicin, fadrozole, fotemustine,
thymalfasin, sobuzoxane, nedaplatin, cytarabine,
5 bicalutamide, vinorelbine, vesnarinone,
aminoglutethimide, amsacrine, proglumide, elliptinium
acetate, ketanserin, doxifluridine, etretinate,
isotretinoin, streptozocin, nimustine, vindesine,
flutamide, drogenil, butocin, carmofur, razoxane,
10 sizofilan, carboplatin, mitolactol, tegafur, ifosfamide,
prednimustine, picibanil, levamisole, teniposide,
improsulfan, enocitabine, lisuride, oxymetholone,
tamoxifen, progesterone, mepitiostane, epitiostanol,
formestane, interferon-alpha, interferon-2 alpha,
15 interferon-beta, interferon-gamma, colony stimulating
factor-1, colony stimulating factor-2, denileukin
diftitox, interleukin-2, and leutinizing hormone
releasing factor.
[41] In another embodiment, the present invention
20 provides a method according to Embodiment 25 wherein the
radiosensitizer agent is selected from the group
consisting of 2-(3-vitro-1,2,4-triazol-1-yl)-N-(2-
methoxyethyl)acetamide, N-(3-vitro-4-quinolinyl)-4-
morpholinecarboxamidine, 3-amino-1,2,4-benzotriazine-
25 1, 4-dioxide, N-(2-hydroxyethyl)-2-nitroimidazole-1-
acetamide, 1-(2-nitroimidazol-1-yl)-3-(1-piperidinyl)-
2-propanol, and 1-(2-vitro-1-imidazolyl)-3-(1-
aziridino)-2-propanol.
[42] In another embodiment, the present invention
30 provides a method according to Embodiment 25 wherein the
anti-cancer agent is an anti-cancer agent agent.
[43] In another embodiment, the present invention
provides a method of treating cancer according to
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Embodiment 25, wherein the administration is by
injection or infusion.
[44] In another embodiment, the present invention
provides the method of Embodiment 25, further comprising
treating the cancer by brachytherapy, external beam
radiation, laser therapy or surgical removal.
[45] In another embodiment, the present invention
provides a kit comprising packaging material, and a
therapeutic radiopharmaceutical composition of
Embodiment 13, contained within said packaging material,
wherein the packaging material comprises a label or
package insert which indicates that said therapeutic
radiopharmaceutical composition can be used for treating
cancer.
[46] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
of Embodiment 13, further comprising a photosensitizing
agent.
[47] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 46, wherein the photosensitizing
agent is selected from the group consisting of
photofrin; naphthalocyanine photosensitizing agents;
tetrapyrrole-based photosensitizers; porphyins;
chlorins;, phthalocyanines; napthalocyanines; coumarins,
psoralens, 1,3,4,6-tetramethoxyhelianthrone; 10,13-
dimethyl-1,3,4,6-tetrahydroxyhelianthrone; 10,13-
di(methoxycarbonyl)-1,3,4,6-tetramethoxyhelianthrone;
1,6-di-N-butylamino-3,4-dimethoxy-helianthrone; 1,6-di-
N-butylamino-3,4-dimethoxy-10,13-dimethyl-helianthrone;
1,6-di-(N-hydroxyethylamino)-3,4-dimethoxy-helianthrone;
2,5-dibromo-1,3,4,6-tetrahydroxyhelianthrone; and 2,5-
dibromo-10,13-dimethyl-1,3,4,6-tetrahydroxyhelianthrone.
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[48] In another embodiment, the present invention
provides a kit according to Embodiment 45, further
comprising a photosensitizing agent.
[49] In another embodiment, the present invention
provides a kit according to Embodiment 48, wherein the
photosensitizing agent is selected from the group
consisting of photofrin; naphthalocyanine
photosensitizing agents; tetrapyrrole-based
photosensitizers; porphyins; chlorins;, phthalocyanines;
napthalocyanines; coumarins, psoralens, 1,3,4,6-
tetramethoxyhelianthrone; 10,23-dimethyl-1,3,4,6-
tetrahydroxyhelianthrone; 10,13-di(methoxycarbonyl)-
1,3,4,6-tetramethoxyhelianthrone; 1,6-di-N-butylamino-
3,4-dimethoxy-helianthrone; 1,6-di-N-butylamino-3,4-
dimethoxy-10,13-dimethyl-helianthrone; 1,6-di-(N-
hydroxyethylamino)-3,4-dimethoxy-helianthrone; 2,5-
dibromo-1,3,4,6-tetrahydroxyhelianthrone; and 2,5-
dibromo-10,13-dimethyl-1,3,4,6-tetrahydroxyhelianthrone.
[50] In another embodiment, the present invention
provides a method of treating cancer according to
Embodiment 25, further comprising treating the patient
with photodynamic therapy.
[51] In another embodiment, the present invention
provides a method of treating cancer according to
Embodiment 50, wherein the photodynamic therapy
comprises:
a) administering a therapeutic radiopharmaceutical of
the present invention and a photosensitive agent
(photoreactive agent) to a patient, said photosensitive
agent having a characteristic light absorption waveband
and being preferentially absorbed by abnormal tissue;
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b)providing an imaging device that is integral with a
plurality of light sources and produces a signal used
for imaging abnormal tissue at the internal treatment
site, said light sources emitting light in a waveband
corresponding to the characteristic light absorption
waveband of the photosensitive agent, said waveband
including wavelengths sufficiently long to penetrate
through a dermal layer of the patient to the internal
treatment site;
(c) determir~.ing a location of the abnormal tissue at the
internal targeted site within the body of the patient
with the imaging device, by viewing an image of the
abnormal tissue at the targeted site developed in
response to the signal produced by the imaging device;
and
(d) energizing the light sources to administer light
therapy to the internal targeted site at the location
determined with the imaging device.
[52] In another embodiment, the present invention
provides a method of treating cancer according to
Embodiment 47, wherein the photosensitive agent
(photoreactive agent) is specifically targeted at the
targeted tissue by including a binding agent that
selectively links the photosensitive agent to the
targeted tissue.
[53] In another embodiment, the present invention
provides a method of treating cancer according to
Embodiment 51, wherein the photosensitizing agent is
selected from the group consisting of photofrin;
naphthalocyanine photosensitizing agents; tetrapyrrole-
based photosensitizers; porphyins; chlorins;
phthalocyanines; napthalocyanines; coumarins, psoralens,
1,3,4,6-tetramethoxyhelianthrone; 10,13-dimethyl-
1,3,4,6-tetrahydroxyhelianthrone; 10,13-
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di(methoxycarbonyl)-1,3,4,6-tetramethoxyhelianthrone;
1,6-di-N-butylamino-3,4-dimethoxy-helianthrone; 1,6-di-
N-butylamino-3,4-dimethoxy-10,13-dimethyl-helianthrone;
1,6-di-(N-hydroxyethylamino)-3,4-dimethoxy-helianthrone;
2,5-dibromo-1,3,4,6-tetrahydroxyhelianthrone; and 2,5-
dibromo-10,13-dimethyl-1,3,4,6-tetrahydroxyhelianthrone.
Another embodiment of the present invention is
diagnostic kits for the preparation of
radiopharmaceuticals useful as imaging agents for cancer
or imaging agents for imaging formation of new blood
vessels. Diagnostic kits of the present invention
comprise one or more vials containing the sterile,
non-pyrogenic, formulation comprised of a predetermined
amount of a reagent of the present invention, and
optionally other components such as one or two ancillary
ligands, reducing agents, transfer ligands, buffers,
lyophilization aids, stabilization aids, solubilization
aids and bacteriostats. The inclusion of one or more
optional components in the formulation will frequently
improve the ease of synthesis of the radiopharmaceutical
by the practicing end user, the ease of manufacturing
the kit, the shelf-life of the kit, or the stability and
shelf-life of the radiopharmaceutical. The inclusion of
one or two ancillary ligands is required for diagnostic
kits comprising reagent comprising a hydrazine or
hydrazone bonding moiety. The one or more vials that
contain all or part of the formulation can independently
be in the form of a sterile solution or a lyophilized
solid.
Another aspect of the present invention
contemplates the combination of anti-cancer agents and
angiogenesis-targeted therapeutic radiopharmaceuticals
of the invention, which target the luminal side of the
neovasculature of tumors, to provide a surprising, and
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enhanced degree of tumor suppression relative to each
treatment modality alone without significant additive
toxicity.
Another aspect of the present invention
5 contemplates the compounds of the present invention
(i.e. a compound comprising: a targeting moiety and a
chelator, wherein the targeting moiety is bound to the
chelator, is a peptide or peptidomimetic, and binds to a
receptor that is upregulated during angiogenesis and the
10 compound has 0-1 linking groups between the targeting
moiety and chelator) which is administered in
combination therapy, with one or more anti-cancer
agent(s)selected from the group consisting of mitomycin,
tretinoin, ribomustin, gemcitabine, vincristine,
15 etoposide, cladribine, mitobronitol, methotrexate,
doxorubicin, carboquone, pentostatin, nitracrine,
zinostatin, cetrorelix, letrozole, raltitrexed,
daunorubicin, fadrozole, fotemustine, thymalfasin,
sobuzoxane, nedaplatin, cytarabine, bicalutamide,
20 vinorelbine, vesnarinone, aminoglutethimide, amsacrine,
proglumide, elliptinium acetate, ketanserin,
doxifluridine, etretinate, isotretinoin, streptozocin,
nimustine, vindesine, flutamide, drogenil, butocin,
carmofur, razoxane, sizofilan, carboplatin, mitolactol,
25 tegafur, ifosfamide, prednimustine, picibariil,
levamisole, teniposide, improsulfan, enocitabine,
lisuride, oxymetholone, tamoxifen, progesterone,
mepitiostane, epitiostanol, formestane, interferon-
alpha, interferon-2 alpha, interferon-beta, interferon-
30 gamma, colony stimulating factor-1, colony stimulating
factor-2, denileukin diftitox, interleukin-2, and
leutinizing hormone releasing factor.
This combination therapy may further, optionally,
include a radiosensitizer agent, or a pharmaceutically
35 acceptable salt thereof, to enhance the radiotherapeutic
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effect together with the anti-cancer agent, said
radiosensitizer agent being selected from the group
consisting of 2-(3-nitro-1,2,4-triazol-1-yl)-N-(2-
methoxyethyl)acetamide, N-(3-nitro-4-quinolinyl)-4-
morpholinecarboxamidine, 3-amino-1,2,4-benzotriazine-
1,4-dioxide, N-(2-hydroxyethyl)-2-nitroimidazole-1-
acetamide, 1-(2-nitroimidazol-1-yl)-3-(1-piperidinyl)-
2-propanol, and 1-(2-nitro-1-imidazolyl)-3-(1-
aziridino)-2-propanol. A thorough discussion of
radiosensitizer agents is provided in the following:
Rowinsky-EK, Oncology-Huntingt., 1999 Oct; 13(10 Suppl
5): 61-70; Chen-AY et al., Oncology-Huntingt. 1999 Oct;
13(10 Suppl 5): 39-46; Choy-H, Oncology-Huntingt. 1999
Oct; 13(10 Suppl 5): 23-38; and Herscher-LL et al,
Oncology-Huntingt. 1999 Oct; 13(10 Suppl 5): 11-22,
which are incorporated herein by reference.
It is a further aspect of the invention to provide
kits having a plurality of active ingredients (with or
without carrier) which, together, may be effectively
utilized for carrying out the novel combination
therapies of the invention.
It is another aspect of the invention to provide a
novel pharmaceutical composition which is effective, in
and of itself, for utilization in a beneficial
combination therapy because it includes compounds of the
present invention, and an anti-cancer agent or a
radiosensitizer agent, which may be utilized in
accordance with the invention.
In another aspect, the present invention provides a
method for treating cancer in a patient in need of such
treatment, said method including the steps of
administering a therapeutically effective amount of a
compound of the present invention and administering a
therapeutically effective amount of at least one agent
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selected from the group consisting of an anti-cancer
agent and a radiosensitizer agent.
Methods for carrying out photodynamic therapy, and
photosensitizers which can be used, are well known in
the art. For example, they are described in the
following patents which are herein incorporated in their
entirety:U.S. Patent No.s 6,248,741, 6,248,734,
6,248,727, 6,248,117, 6,245,811, 6,238,426, 6,238,392,
6,233,481, 6,229,048, 6,232,613, 6,225,333, 6,223,071,
6,219,577, 6,219,575, 6,217,869, 6,217,848, 6,216,540,
6,212,425, 6,211,626, 6,208,886, 6,207,464, 6,207,107,
6,198,532, 6,194,415, and 6,186,628.
It is appreciated that certain features of the
invention, which are, for clarity, described in the
context of separate emlaodiments, may also be provided in
combination in a single embodiment. Conversely, various
features of the invention which are for brevity,
described in the context of a single embodiment, may
also be provided separately or in any subcombination.
DEFINITIONS
The compounds herein described may have asymmetric
centers. Unless otherwise indicated, all chiral,
diastereomeric and racemic forms are included in. the
present invention. Many geometric isomers of olefins,
C=N double bonds, and the like can also be present in
the compounds described herein, and all such stable
isomers are contemplated in the present invention. It
will be appreciated that compounds of the present
invention contain asymmetrically substituted carbon
atoms, and may be isolated in optically active or
racemic forms. It is well known in the art how to
prepare optically active forms, such as by resolution of
racemic forms or by synthesis from optically active
starting materials. Two distinct isomers (cis and
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traps) of the peptide bond are known to occur; both can
also be present in the compounds described herein, and
all such stable isomers are contemplated in the present
invention. The D and L-isomers of a particular amino
acid are designated herein using the conventional
3-letter abbreviation of the amino acid, as indicated by
the following examples: D-Leu, or L-Leu.
When any variable occurs more than one time in any
substituent or in any formula, its definition on each
occurrence is independent of its definition at every
other occurrence. Thus, for example, if a group is
shown to be substituted with 0-2 RS~, then said group
may optionally be substituted with up to two RS~, and R5~
at each occurrence is selected independently from the
defined list of possible RS~. Also, by way of example,
for the group -N(R53)~, each of the two R53 substituents
on N is independently selected from the defined list of
possible R53. Combinations of substituents and/or
variables are permissible only if such combinations
result in stable compounds. When. a bond to a
substituent is shown to cross the bond connecting two
atoms in a ring, then such substituent may be bonded to
any atom on the ring.
By "reagent" is meant a compound of this invention
capable of direct transformation into a
metallopharmaceutical of this invention. Reagents may
be utilized directly for the preparation of the
metallopharmaceuticals of this invention or may be a
component in a kit of this invention.
The term "binding agent" means a
metallopharmaceutical of this invention having affinity
for and capable of binding to the vitronectin receptor.
The binding agents of this invention preferably have
Ki<1000nM.
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Metallopharmaceutical as used herein is intended to
refer to a pharmaceutically acceptable compound
containing a metal, wherein the compound is useful for
imaging, magnetic resonance imaging, contrast imaging,
or x-ray imaging. The metal is the cause of the
imageable signal in diagnostic applications and the
source of the cytotoxic radiation in radiotherapeutic
applications. Radiopharmaceuticals are
metallopharmaceuticals in which the metal is a
radioisotope.
By "stable compound" or "stable structure" is meant
herein a compound-that is sufficiently robust to survive
isolation to a useful degree of purity from a reaction
mixture, and formulation into an efficacious
pharmaceutical agent.
The term "substituted", as used herein, means that
one or more hydrogens on the designated atom or group is
replaced with a selection from the indicated group,
provided that the designated atom's or group's normal
valency is not exceeded, and that the substitution
results in a stable compound. When a substituent is
keto (i.e., =0), then 2 hydrogens on the atom are
replaced.
The term "bond", as used herein, means either a
single or double bond.
The term "salt", as used herein, is used as defined
in the CRC Handbook of Chemistry and Physics, 65th
Edition, CRC Press, Boca Raton, Fla, 1984, as any
substance which yields ions, other than hydrogen or
hydroxyl ions. As used herein, "pharmaceutically
acceptable salts" refer to derivatives of the disclosed
compounds modified by making acid or base salts.
Examples of pharmaceutically acceptable salts include,
but are not limited to, mineral or organic acid salts of
basic residues such as amines; alkali or organic salts
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of acidic residues such as carboxylic acids; and the
like.
The phrase "pharmaceutically acceptable" is
employed herein to refer to those compounds, materials,
5 compositions, and/or dosage forms which are, within the
scope of sound medical judgment, suitable for use in
contact with the tissues of human beings and animals
without excessive toxicity, irritation, allergic
response, or other problem or complication, commensurate
10 with a reasonable benefit/risk ratio.
The phrase "pharmaceutically acceptable prodrugs"
as used herein means those prodrugs of the compounds
useful according to the present invention which are,
within the scope of sound medical judgment, suitable for
15 use in contact with the tissues of humans and lower
animals with undue toxicity, irritation, allergic
response, and the like, commensurate with a reasonable
benefit/risk ratio, and effective for their intended
use, as well as the zwitterionic forms, where possible,
20 of the compounds of the invention. The term "prodrug"
means compounds that are rapidly transformed in vivo to
yield the parent compound of the above formula, for
example by hydrolysis in blood. Functional groups which
may be rapidly transformed, by metabolic cleavage, in
25 vivo form a class of groups reactive with the carboxyl
group of the compounds of this invention. They include,
but are not limited to such groups as alkanoyl (such as
acetyl, propionyl, butyryl, and the like), unsubstituted
and substituted aroyl (such as benzoyl and substituted
30 benzoyl), alkoxycarbonyl (such as ethoxycarbonyl),
trialkylsilyl (such as trimethyl- and triethysilyl),
monoesters formed with dicarboxylic acids (such as
succinyl), and the like. Because of the ease with which
the metabolically cleavable groups of the compounds
35 useful according to this invention are cleaved in vivo,
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the compounds bearing such groups act as pro-drugs. The
compounds bearing the metabolically cleavable groups
have the advantage that they may exhibit improved
bioavailability as a result of enhanced solubility
andlor rate of absorption conferred upon the parent
compound by virtue of the presence of the metabolically
cleavable group. A thorough discussion of prodrugs is
provided in the following: Design of Prodrugs, H.
Bundgaard, ed., Elsevier, 1985; Methods in Enzymology,
K. Widder et al, Ed., Academic Press, 42, p.309-396,
1985; A Textbook of Drug Design and Development,
Krogsgaard-Larsen and H. Bundgaard, ed., Chapter 5;
"Design and Applications of Prodrugs" p.113-191, 1991;
Advanced Drug Delivery Reviews, H. Bundgard, 8, p.1-38,
1992; Journal of Pharmaceutical Sciences, 77, p. 285,
1988; Chem. Pharm. Bull., N. Nakeya et al, 32, p. 692,
1984; Pro-drugs as Novel Delivery Systems, T. Higuchi
and V. Stella, Vol. 14 of the A.C.S. Symposium Series,
and Bioreversible Carriers in Drug Design, Edward B.
Roche, ed., American Pharmaceutical Association and
Pergamon Press, 1987, which are incorporated herein by
reference.
As used herein, "pharmaceutically acceptable salts"
refer to derivatives of the disclosed compounds wherein
the parent compound is modified by making acid or base
salts thereof. Examples of pharmaceutically acceptable
salts include, but are not limited to, mineral or
organic acid salts of basic residues such as amines;
alkali or organic salts of acidic residues such as
carboxylic acids; and the like. The pharmaceutically
acceptable salts include the conventional non-toxic
salts or the quaternary ammonium salts of the parent
compound formed, for example, from non-toxic inorganic
or organic acids. For example, such conventional
non-toxic salts include those derived from inorganic
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acids such as hydrochloric, hydrobromic, sulfuric,
sulfamic, phosphoric, nitric and the like; and the salts
prepared from organic acids such as acetic, propionic,
succinic, glycolic, stearic, lactic, tartaric, citric,
ascorbic, pamoic, malefic, hydroxymaleic, phenylacetic,
glutamic, benzoic, salicylic, sulfanilic,
2-acetoxybenzoic, fumaric, toluenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, isethionic,
and the like.
The pharmaceutically acceptable salts of the
present invention can be synthesized from the parent
compound which contains a basic or acidic moiety by
conventional chemical methods. Generally, such salts
can be prepared by reacting the free acid or base forms
of these compounds with a stoichiometric amount of the
appropriate base or acid in water or in an organic
solvent, or in a mixture of the two; generally,
nonaqueous media like ether, ethyl acetate, ethanol,
isopropanol, or acetonitrile are preferred. Lists of
suitable salts are found in Remington's Pharmaceutical
Sciences, 17th ed., Mack Publishing Company, Easton, PA,
1985, p. 1418, the disclosure of which is hereby
incorporated by reference.
As used herein, "alkyl" is intended to include both
branched and straight-chain saturated aliphatic
hydrocarbon groups having the specified number of carbon
atoms. C1-1o alkyl, is intended to include C~, C2, C3,
Cg, C5, C6, C7, Cg, Cg, and C1p alkyl groups. Examples
of alkyl include, but are not limited to, methyl, ethyl,
n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl,
and s-pentyl. "Haloalkyl" is intended to include both
branched and straight-chain saturated aliphatic
hydrocarbon groups having the specified number of carbon
atoms, substituted with 1 or more halogen (for example
-CVFW where v = 1 to 3 and w = 1 to (2v+1)). Examples
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of haloalkyl include, but are not limited to,
trifluoromethyl, trichloromethyl, pentafluoroethyl, and
pentachloroethyl. "Alkoxy" represents an alkyl group as
defined above with the indicated number of carbon atoms
attached through an oxygen bridge. CZ_1o alkoxy, is
intended to include C1, C~, C3, C4, C5, C6, C7, Cg, Cg,
and C1o alkoxy groups. Examples of alkoxy include, but
are not limited to, methoxy, ethoxy, n-propoxy,
i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, and
s-pentoxy. "Cycloalkyl" is intended to include
saturated ring groups, such as cyclopropyl, cyclobutyl,
or cyclopentyl. C3_~ cycloalkyl, is intended to include
C3, Cg, C5, C6, and C7 cycloalkyl groups. Alkenyl" is
intended to include hydrocarbon chains of either a
straight or branched configuration and one or more
unsaturated carbon-carbon bonds which may occur in any
stable point along the chain, such as ethenyl and
propenyl. C2_1o alkenyl, is intended to include C~, C3,
C4, C5, C6, C7, Cg, Cg, and C1o alkenyl groups.
"Alkynyl" is intended to include hydrocarbon chains of
either a straight or branched configuration and one or
more triple carbon-carbon bonds which may occur in any
stable point.along the chain, such as ethynyl and
propynyl. C2_1o alkynyl, is intended to include C~, C3,
C4, C5, C6, C7, Cg, Cg, and C1o alkynyl groups.
As used herein, "carbocycle" or "carbocyclic
residue" is intended to mean any stable 3, 4, 5, 6, or
7-membered monocyclic or bicyclic or 7, 8, 9, 10, 11,
12, or 13-membered bicyclic or tricyclic, any of which
may be saturated, partially unsaturated, or aromatic.
Examples of such carbocycles include, but are not
limited to, cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cycloheptyl, adamantyl, cyclooctyl,
[3.3.0]bicyclooctane, [4.3.0]bicyclononane,
[4.4.0]bicyclodecane, [2.2.2]bicyclooctane, fluorenyl,
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phenyl, naphthyl, indanyl, adamantyl, and
tetrahydronaphthyl.
As used herein, the term "alkaryl" means an aryl
group bearing an alkyl group of 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10 carbon atoms; the term "aralkyl" means an alkyl
group of 1, 2, 3, 4, 5, 6, '7, 8, 9, or 10 carbon atoms
bearing an aryl group; the term "arylalkaryl" means an
aryl group bearing an alkyl group of 1-10 carbon atoms
bearing an aryl group; and the term "heterocycloalkyl"
means an alkyl group of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
carbon atoms bearing a heterocycle.
As used herein, the term "heterocycle" or
"heterocyclic system'° is intended to mean a stable 5, 6,
or 7-membered monocyclic or bicyclic or 7, 8, 9, or 10-
membered bicyclic heterocyclic ring which is saturated,
partially unsaturated or unsaturated (aromatic), and
which consists of carbon atoms and 1, 2, 3, or 4
heteroatoms independently selected from the group
consisting of N, NH, 0 and S and including any bicyclic
group in which any of the above-defined heterocyclic
rings is fused to a benzene ring. The nitrogen and
sulfur heteroatoms may optionally be oxidized. The
heterocyclic ring may be attached to its pendant group
at any heteroatom or carbon atom which results in a
stable structure. The heterocyclic rings described
herein may be substituted on carbon or on a nitrogen
atom if the resulting compound is stable. A nitrogen in
the heterocycle may optionally be quaternized. It is
preferred that when the total number of S and 0 atoms in
the heterocycle exceeds 1, then these heteroatoms are
not adjacent to one another. It is preferred that the
total number of S and 0 atoms in the heterocycle is not
more than 1. As used herein, the term "aromatic
heterocyclic system" or "heteroaryl" is intended to mean
a stable 5, 6, or 7-membered monocyclic or bicyclic or
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7, 8, 9, or 10-membered bicyclic heterocyclic aromatic
ring which consists of carbon atoms and 1, 2, 3, or 4
heterotams independently selected from the group
consisting of N, NH, 0 and S. Tt is to be noted that
5 total number of S and 0 atoms in the aromatic
heterocycle is not more than 1.
Examples of heterocycles include, but are not
limited to, acridinyl, azocinyl, benzimidazolyl,
benzofuranyl, benzothiofuranyl, benzothiophenyl,
10 benzoxazolyl, benzthiazolyl, benztriazolyl,
benztetrazolyl, benzisoxazolyl, benzisothiazolyl,
benzimidazolinyl, carbazolyl, 4aH-carbazolyl,
carbolinyl, chromanyl, chromenyl, cinnolinyl,
decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,
15 dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl,
imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl,
indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl,
isobenzofuranyl, isochromanyl, isoindazolyl,
isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl,
20 isoxazolyl, methylenedioxyphenyl, morpholinyl,
naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,
1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,
1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl,
pyrimidinyl, phenanthridinyl, phenanthrolinyl,
25 phenazinyl, phenothiazinyl, phenoxathiinyl,
phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl,
piperidonyl, 4-piperidonyl, piperonyl, pteridinyl,
purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,
pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole,
30 pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,
pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,
quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,
quinuclidinyl, tetrahydrofuranyl,
tetrahydroisoquinolinyl, tetrahydroquinolinyl,
35 tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,
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1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-
thiadiazolyl, thianthrenyl, thiazolyl, thienyl,
thienothiazolyl, thienooxazolyl, thienoimidazolyl,
thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl,
1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl.
Preferred heterocycles include, but are not limited to,
pyridinyl, furanyl, thienyl, pyrrolyl, pyrazolyl,
pyrrolidinyl, imidazolyl, indolyl, benzimidazolyl, 1H-
indazolyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl,
oxindolyl, benzoxazolinyl, and isatinoyl. Also included
are fused ring and spiro compounds containing, for
example, the above heterocycles.
A "polyalkylene glycol" is a polyethylene glycol,
polypropylene glycol or polybutylene glycol having a
molecular weight of less than about 5000, terminating in
either a hydroxy or alkyl ether moiety.
A "carbohydrate" is a polyhydroxy aldehyde, ketone,
alcohol or acid, or derivatives thereof, including
polymers thereof having polymeric linkages of the acetal
type.
A "cyclodextrin" is a cyclic oligosaccharide.
Examples of cyclodextrins include, but are not limited
to, Cc-cyclodextrin, hydroxyethyl-oc-cyclodextrin,
hydroxypropyl-oc-cyclodextrin, (3-cyclodextrin,
hydroxypropyl-(3-cyclodextrin,
carboxymethyl-~i-cyclodextrin,
dihydroxypropyl-(3-cyclodextrin,
hydroxyethyl-~3-cyclodextrin, 2,6
di-0-methyl-(3-cyclodextrin, sulfated-(3-cyclodextrin,
y-cyclodextrin, hydroxypropyl-y-cyclodextrin,
dihydroxypropyl-'y-cyclodextrin,
hydroxyethyl-y-cyclodextrin, and sulfated
y-cyclodextrin.
As used herein, the term "polycarboxyalkyl" means
an alkyl group having between two and about 100 carbon
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atoms and a plurality of carboxyl substituents; and the
term "polyazaalkyl" means a linear or branched alkyl
group having between two and about 100 carbon atoms,
interrupted by or substituted with a plurality of amine
groups.
A "reducing agent" is a compound that reacts with a
radionuclide, which is typically obtained as a
relatively unreactive, high oxidation state compound, to
lower its oxidation state by transferring electrons) to
the radionuclide, thereby making it more reactive.
Reducing agents useful in the preparation of
radiopharmaceuticals and in diagnostic kits useful for
the preparation of said radiopharmaceuticals include but
are not limited to stannous chloride, stannous fluoride,
formamidine sulfiniv acid, ascorbic acid, cysteine,
phosphines, and cuprous or ferrous salts. Other
reducing agents are described in Brodack et. al., PCT
Application 94/22496, which is incorporated herein by
reference.
A "transfer ligand" is a ligand that forms an
intermediate complex with a metal ion that is stable
enough to prevent unwanted side-reactions but labile
enough to be converted to a metallopharmaceutical. The
formation of the intermediate complex is kinetically
favored while the formation of the metallopharmaceutical
is thermodynamically favored. Transfer ligands useful
in the preparation of metallopharmaceuticals and in
diagnostic kits useful for the preparation of diagnostic
radiopharmaceuticals include but are not limited to
gluconate, glucoheptonate, mannitol, glucarate,
N,N,N',N'-ethylenediaminetetraacetic acid, pyrophosphate
and methylenediphosphonate. In general, transfer
ligands are comprised of oxygen or nitrogen donor atoms.
The term "donor atom" refers to the atom directly
attached to a metal by a chemical bond.
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"Ancillary" or "co-ligands" are ligands that are
incorporated into a radiopharmaceutical during its
synthesis. They serve to complete the coordination
sphere of the radionuclide together with the chelator or
radionuclide bonding unit of the reagent. For
radiopharmaceuticals comprised of a binary ligand
system, the radionuclide coordination sphere is composed
of one or more chelators or bonding units from one or
more reagents and one or more ancillary or co-ligands,
provided that there are a total of two types of ligands,
chelators or bonding units. For example, a
radiopharmaceutical comprised of one chelator or bonding
unit from one reagent and two of the same ancillary or
co-ligands and a radiopharmaceutical comprised of two
chelators or bonding units from one or two reagents and
one ancillary or co-ligand are both considered to be
comprised of binary ligand systems. For
radiopharmaceuticals comprised of a ternary ligand
system, the radionuclide coordination sphere is composed
of one or more chelators or bonding units from one or
more reagents and one or more of two different types of
ancillary or co-ligands, provided that there are a total
of three types of ligands, chelators or bonding units.
For example, a radiopharmaceutical comprised of one
chelator or bonding unit from one reagent and two
different ancillary or co-ligands is considered to be
comprised of a ternary ligand system.
Ancillary or co-ligands useful in the preparation
of radiopharmaceuticals and in diagnostic kits useful
for the preparation of said radiopharmaceuticals are
comprised of one or more oxygen, nitrogen, carbon,
sulfur, phosphorus, arsenic, selenium, and tellurium
donor atoms. A ligand can be a transfer ligand in the
synthesis of a radiopharmaceutical and also serve as an
ancillary or co-ligand in another radiopharmaceutical.
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Whether a ligand is termed a transfer or ancillary or
co-ligand depends on whether the ligand remains in the
radionuclide coordination sphere in the
radiopharmaceutical, which is determined by the
coordination chemistry of the radionuclide and the
chelator or bonding unit of the reagent or reagents.
A "chelator" or "bonding unit" is the moiety or
group on a reagent that binds to a metal ion through the
formation of chemical bonds with one or more donor
atoms.
The term "binding site" means the site in vivo or
in vitro that binds a biologically active molecule.
A "diagnostic kit" or "kit" comprises a collection
of components, termed the formulation, in one or more
vials which are used by the practicing end user in a
clinical or pharmacy setting to synthesize diagnostic
radiopharmaceuticals. The kit provides all the
requisite components to synthesize and use the
diagno tic radiopharmaceutical except those that are
commonly available to the practicing end user, such as
water or saline for injection, a solution of the
radionuclide, equipment for heating the kit during the
synthesis of the radiopharmaceutical, if required,
equipment necessary for administering the
radiopharmaceutical to the patient such as syringes and
shielding, and imaging equipment.
Therapeutic radiopharmaceuticals, X-ray contrast
agent pharmaceuticals, ultrasound contrast agent
pharmaceuticals and metallopharmaceuticals for magnetic
resonance imaging contrast are provided to the end user
in their final form in a formulation contained typically
in one vial, as either a lyophilized solid or an aqueous
solution. The end user reconstitutes the lyophilized
with water or saline and withdraws the patient dose or
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just withdraws the dose from the aqueous solution
formulation as provided.
A "lyophilization aid" is a component that has
favorable physical properties for lyophilization, such
as the glass transition temperature, and is added to the
formulation to improve the physical properties of the
combination of all the components of the formulation for
lyophilization.
A "stabilization aid" is a component that is added
to the metallopharmaceutical or to the diagnostic kit
either to stabilize the metallopharmaceutical or to
prolong the shelf-life of the kit before it must be
used. Stabilization aids can be antioxidants, reducing
agents or radical scavengers and can provide improved
stability by reacting preferentially with species that
degrade other components or the metallopharmaceutical.
A "solubilization aid" is a component that improves
the solubility of one or more other components in the
medium required for the formulation.
A "bacteriostat" is a component that inhibits the
growth of bacteria in a formulation either during its
storage before use of after a diagnostic kit is used to
synthesize a radiopharmaceutical.
The term "amino acid" as used herein means an
organic compound containing both a basic amino group and
an acidic carboxyl group. Included within this term are
natural amino acids (e.g., L-amino acids), modified and
unusual amino acids (e.g., D-amino acids), as well as
amino acids which are known to occur biologically in
free or combined form but usually do not occur in
proteins. Included within this term are modified and
unusual amino acids,such as those disclosed in, for
example, Roberts and Vellaccio (2983) The Peptides, 5:
342-429, the teaching of which is hereby incorporated by
reference. Natural protein occurring amino acids
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include, but are not limited to, alanine, arginine,
asparagine, aspartic acid, cysteine, glutamic acid,
glutamine, glycine, histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, serine, threonine,
tyrosine, tyrosine, tryptophan, proline, and valine.
Natural non-protein amino acids include, but are not
limited to arginosuccinic acid, citrulline, cysteine
sulfiniv acid, 3,4-dihydroxyphenylalanine, homocysteine,
homoserine, ornithine, 3-monoiodotyrosine,
3,5-diiodotryosine, 3,5,5'=triiodothyronine, and
3,3',5,5'-tetraiodothyronine. Modified or unusual amino
acids which can be used to practice the invention
include, but are not limited to, D-amino acids,
hydroxylysine, 4-hydroxyproline, an N-Cbz-protected
amino acid, 2,4-diaminobutyric acid, homoarginine,
norleucine, N-methylaminobutyric acid, naphthylalanine,
phenylglycine, f~-phenylproline, tert-leucine,
4-aminocyclohexylalanine, N-methyl-norleucine,
3,4-dehydroproline, N,N-dimethylaminoglycine,
N-methylaminoglycine, 4-aminopiperidine-4-carboxylic
acid, 6-aminocaproic acid,
trans-4-(aminomethyl)-cyclohexanecarboxylic acid, 2-,
3-, and 4-(aminomethyl)-benzoic acid,
1-aminocyclopentanecarboxylic acid,
1-aminocyclopropanecarboxylic acid, and
2-benzyl-5-aminopentanoic acid.
The term "peptide" as used herein means a linear
compound that consists of two or more amino acids (as
defined herein) that are linked by means of a peptide
bond. A "peptide" as used in the presently claimed
invention is intended to refer to a moiety with a
molecular weight of less than 10,000 Daltons, preferable
less than 5,000 Daltons, and more preferably less than
2,500 Daltons. The term "peptide" also includes
compounds containing both peptide and non-peptide
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components, such as pseudopeptide or peptidomimetic
residues or other non-amino acid components. Such a
compound containing both peptide and non-peptide
components may also be referred to as a "peptide
analog".
A "pseudopeptide" or "peptidomimetic" is a compound
which mimics the structure of an amino acid residue or a
peptide, for example, by using linking groups other than
amide linkages between the peptide mimetic and an amino
acid residue (pseudopeptide bonds) and/or by using
non-amino acid substituents and/or a modified amino acid
residue. A "pseudopeptide residue" means that portion
of an pseudopeptide or peptidomimetic that is present in
a peptide.
The term "peptide bond" means a covalent amide
linkage formed by loss of a molecule of water between
the carboxyl group of one amino acid and the amino group
of a second amino acid.
The term "pseudopeptide bonds" includes peptide
bond isosteres which may be used in place of or as
substitutes for the normal amide linkage. These
substitute or amide "equivalent" linkages are formed
from combinations of atoms not normally found in
peptides or proteins which mimic the spatial
requirements of the amide bond and which should
stabilize the molecule to enzymatic degradation.
The following abbreviations are used herein:
Acm acetamidomethyl
b-Ala, beta-Ala
or bAla 3-aminopropionic acid
ATA 2-aminothiazole-5-acetic acid or 2-
aminothiazole-5-acetyl group
Boc t-butyloxycarbonyl
CBZ, Cbz or Z Carbobenzyloxy
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Cit citrulline
Dap 2,3-diaminopropionic acid
DCC dicyclohexylcarbodiimide
DIEA diisopropylethylamine
DMAP 4-dimethylaminopyridine
EOE ethoxyethyl
HBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium
hexafluorophosphate
hynic boc-hydrazinonicotinyl group or 2-
[[[5- [carbonyl]-2-
pyridinyl]hydrazono] methyl]-
benzenesulfonic acid,
NMeArg or MeArg a-N-methyl arginine
NMeAsp a-N-methyl aspartic acid
NMM N-methylmorpholine
OcHex 0-cyclohexyl
OBzl 0-benzyl
oSu 0-succinimidyl
TBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium tetrafluoroborate
THF tetrahydrofuranyl
THP tetrahydropyranyl
Tos tosyl
Tr trityl
The following conventional three-letter amino acid
abbreviations are used herein; the conventional
one-letter amino acid abbreviations are NOT used herein:
Ala - alanine
Arg - arginine
Asn - asparagine
Asp - aspartic acid
Cys - cysteine
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Gln - glutamine
Glu - glutamic acid
Gly - glycine
His - histidine
Ile - isoleucine
Leu - leucine
Lys - lysine
Met - methionine
Nle - norleucine
Orn - ornithine
Phe - phenylalanine
Phg - phenylglycine
Pro - proline
Sar - sarcosine
Ser - serine
Thr - threonine
Trp - tryptophan
Tyr - tyrosine
Val - valine
The pharmaceuticals of the present invention are
comprised of a targeting moiety for a receptor that is
expressed or upregulated in angiogenic tumor
vasculature. For targeting the VEGF receptors, Flk-
1/K.DR, Flt-1, and neuropilin-1, the targeting moieties
are comprised of peptides or peptidomimetics that bind
with high affinity to the receptors. For example,
peptides comprised of a 23 amino acid portion of the C-
terminal domain of VEGF have been synthesized which
competitively inhibit binding of VEGF to VEGFR (Soker,
et. al., J. Biol. Chem., 1997, 272, 31582-8). Linear
peptides of 11 to 23 amino acid residues that bind to
the basic FGF receptor (bFGFR) are described by Cosic
et. al., Mol. and Cell. Biochem., 1994, 130, 1-9. A
preferred linear peptide antagonist of the bFGFR is the
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16 amino acid peptide, Met-Trp-Tyr-Arg-Pro-Asp-Leu-Asp-
Glu-Arg-Lys-Gln-Gln-Lys-Arg-Glu. Gho et. al. (Cancer
Research, 1997, 57, 3733-40) describe the identification
of small peptides that bind with high affinity to the
angiogenin receptor on the surface of endothelial cells.
A preferred peptide is Ala-Gln-Leu-Ala-Gly-Glu-Cys-Arg-
Glu-Asn-Val-Cys-Met-Gly-Ile-Glu-Gly-Arg, in which the
two Cys residues form an intramolecular disulfide bond.
Yayon et. al. (Proc. Natl. Acad. Sci, USA, 1993, 90,
10643-7) describe other linear peptide antagonists of
FGFR, identified from a random phage-displayed peptide
library. Two linear octapeptides, Ala-Pro-Ser-Gly-His-
Tyr-Lys-Gly and Lys-Arg-Thr-Gly-Gln-Tyr-Lys- Leu are
preferred for inhibiting binding of bFGF to it receptor.
Targeting moieties for integrins expressed in tumor
vasculature include peptides and peptidomimetics that
bind to oc~,~i3, oc~,~i5, a5~31, a4~1. a1~31. and a2aa
Pierschbacher and Rouslahti (J. Biol. Chem., 1987, 2&2,
17294-8) describe peptides that bind selectively to oc5(31
and oc,~,(33. U.S. 5, 536, 814 describe peptides that bind
with high affinity to the integrin a5~il. Burgess and Lim
(J. Med. Chem., 1996, 39, 4520-6) disclose the synthesis
three peptides that bind with high affinity to Oc~,(33:
cyclo[Arg-Gly-Asp-Arg-Gly-Asp], cyclo[Arg-Gly-Asp-Arg
Gly-D-Asp] and the linear peptide Arg-Gly-Asp-Arg-Gly
Asp. U.S. 5,770,565 and U.S. 5,766,591 disclose
peptides that bind with high affinity to 0(33. U.S.
5,767,071 and U.S. 5,780,426, disclose cyclic peptides
that have an exocyclic Arg amino acid that have high
affinity for oc,~,(33. Srivatsa et. al. , (Cardiovascular
Res., 1997, 36, 408-28) describe the cyclic peptide
antagonist for o~(33, cyclo[Ala-Arg-Gly-Asp-Mamb]. Tran
et. al., (Bioorg. Med. Chem. Lett., 1997, 7, 997-1002)
disclose the cyclic peptide cyclo[Arg-Gly-Asp-Val-Gly-
Ser-BTD-Ser-Gly-Val-Ala] that binds with high affinity
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to oc.~(33. Arap et. al. (Science, 1998, 279, 377-80)
describe cyclic peptides that bind to or,~,(33 and oc~,(35, Cys-
Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys, and cyclo[Cys-Asn-Gly-
Asp-Cys]. Corbett et. al. (Biorg. Med. Chem. Lett.,
1997, 7, 1371-6) describe a series of or,~,(33 selective
peptidomimetics. And Haubner et. al., (Angew. Chem. Int.
Ed. Engl., 1997, 36, 1374-89) disclose peptides and
peptidomimetic oc~,(33 antagonists obtained from peptide
libraries.
The targeting moieties of the present invention,
preferably, have a binding affinity for the integrin
oc,~,(33 of less than 1000nM. More preferably, the
targeting moieties of the present invention, preferably,
have a binding affinity for the integrin or,~,(33 of less
than 100nM. Even more preferably, the targeting
moieties of the present invention, preferably, have a
binding affinity for the integrin or~,~(33 of less than
lOnM.
The ultrasound contrast agents of the present
invention comprise a plurality of angiogenic tumor
vasculature targeting moieties attached to or
incorporated into a microbubble of a biocompatible gas,
a liquid carrier, and a surfactant microsphere, further
comprising an optional linking moiety, Ln, between the
targeting moieties and the microbubble. In this
context, the term liquid carrier means aqueous solution
and the term surfactant means any amphiphilic material
which produces a reduction in interfacial tension in a
solution. A list of suitable surfactants for forming
surfactant microspheres is disclosed in EP0727225A2,
herein incorporated by reference. The term surfactant
microsphere includes nanospheres, liposomes, vesicles
and the like. The biocompatible gas can be air, or a
fluorocarbon, such as a C3-C5 perfluoroalkane, for
example, perflouropropane, perflourobutane, or
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perflouroperitaxTv~,~ which provides the difference in
echogenicity and thus the contrast in ultrasound
imaging. The gas is encapsulated or contained in the
microsphere to which is attached the biodirecting group,
optionally via a linking group. The attachment can be
covalent, ionic or by van der Waals forces. Specific
examples of such contrast agents include lipid
encapsulated perfluorocarbons with a plurality of tumor
neovasculature receptor binding peptides or'
peptidomimetics.
Sf as used herein is a surfactant which is either a
lipid or a compound of the formula A1-E-A~, defined
above. The surfactant is intended to form a vesicle
(e. g., a microsphere) capable of containing an echogenic
gas. The ultrasound contrast agent compositions of the
present invention are intended to be capable upon
agitation (e.g., shaking, stirring, etc...) of
encapsulating an echogenic gas in a vescicle in such a
way as to allow for the resultant product to be useful
as an ultrasound contrast agent.
"Vesicle" refers to a spherical entity which is
characterized by the presence of an internal void.
Preferred vesicles are formulated from lipids, including
the various lipids described herein. In any given
vesicle, the lipids may be in the form of a monolayer or
bilayer, and the mono- or bilayer lipids may be used to
form one of more mono- or bilayers. In the case of more
than one mono- or bilayer, the mono- or bilayers are
generally concentric. The lipid vesicles des.c.ribed
herein include such entities commonly Deferred to as
liposomes, micelles, bubbles, microbubbles, microspheres
and the like. Thus, the lipids may be used to form a
unilamellar vesicle (comprised of one monolayer or
bilayer), an oligolamellar vesicle (comprised of about
two or about three monolayers or bilayers) or a
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multilamellar vesicle (comprised of more than about
three monolayers or bilayers). The internal void of the
vesicles may be filled with a liquid, including, for
example, an aqueous liquid, a gas, a gaseous precursor,
and/or a solid or solute material, including, for
example, a bioactive agent, as desired.
"Vesicular composition" refers to a composition .
which is formulate from lipids and which comprises
vesicles.
"Vesicle formulation" refers to a composition which
comprises vesicles and a bioactive agent.
Microsphere,~as used herein, is preferably a sphere
of less than or equal to 10 microns. Liposome, as used
herein,~may include a single lipid layer (a lipid
monolayer), two lipid layers (a lipid bilayer) or more
than two lipid layers (a lipid multilayer). "Lipsomes"
refers to a generally spherical cluster or aggregate of
amphipathic compounds, including lipid compounds,
typically in the form of one or more concentric layers,
for example, bilayers. They may also be referred to
herein as lipid vesicles.
The term "bubbles", as used herein, refers to
vesicles which are generally characterized by the
presence of one or more membranes or walls surrounding
an internal void that is filled with a gas or precursor
thereto. Exemplary bubbles include, for example,
liposomes, micelles and the like.
"Lipid" refers to a synthetic or naturally
occurring amphipathic compound which comprises a
hydrophilic component and a hydrophobic component.
Lipids include, for example, fatty acids, neutral fats;
phosphatides, glycolipids, aliphatic alchols and waxes,
terpenes and steroids.
"Lipid composition" refers to a composition which
comprises a lipid compound. Exemplary lipid
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compositions include suspensions, emulsions and
vesicular compositions.
"Lipid formulation" refers to a composition which
comprises a lipid compound and a bioactive agent.
Examples of classes of suitable lipids and specific
suitable lipids include: phosphatidylcholines, such as
dioleoylphosphatidylcholine,
dimyristoylphosphatidylcholine,
dipalmitoylphosphatidylcholine (DPPC), and
distearoylphosphatidylcholine;
phosphatidylethanolamines, such as
dipalmitoylphosphatidylethanolamine (DPPE),
dioleoylphosphatidylethanolamine and N-succinyl-
dioleoylphosphatidylethanolamine; phosphatidylserines;
phosphatidylglycerols; sphingolipids; glycolipids, such
as ganglioside. GM1; glucolipids; sulfatides;
glycosphingolipids; phosphatidic acids, such as
dipalmatoylphosphatidic acid (DPPA); palmitic fatty
acids; stearic fatty acids; arachidonic fatty acids;
lauric fatty acids; myristic fatty acids; lauroleic
fatty acids; physeteric fatty acids; myristoleic fatty
acids; palmitoleic fatty acids; petroselinic fatty
acids; oleic fatty acids; isolauric fatty acids;
isomyristic fatty acids; isopalmitic fatty acids;
isostearic fatty acids; cholesterol and cholesterol
derivatives, such as cholesterol hemisuccinate,
cholesterol sulfate, and cholesteryl-(4'-
trimethylammonio)-butanoate; polyoxyethylene fatty acid
esters; polyoxyethylene fatty acid alcohols;
polyoxyethylene fatty acid alcohol ethers;
polyoxyethylated sorbitan fatty acid esters; glycerol
polyethylene glycol oxystearate; glycerol polyethylene
glycol ricinoleate; ethoxylated soybean sterols;
ethoxylated castor oil; polyoxyethylene-polyoxypropylene
fatty acid polymers; polyoxyethylene fatty acid
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stearates; 12-(((7'-diethylaminocoumarin-3-yl)-
carbonyl)-methylamino)-octadecanoic acid; N-[12-(((7'-
diethylamino-coumarin-3-yl)-carbonyl)-methyl-
amino)octadecanoyl]-2-amino-palmitic acid; 1,2-dioleoyl-
sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol; 1,3-
dipalmitoyl-2-succinyl-glycerol; and 1-hexadecyl-2-
palmitoyl-glycerophosphoethanolamine and
palmitoylhomocysteine; lauryltrimethylammonium bromide;
cetyltrimethylammonium bromide;
myristyltrimethylammonium bromide;
alkyldimethylbenzylammonium chlorides, such as wherein
alkyl is a C12, C14 or C16 alkyl;
benzyldimethyldodecylammonium bromide;
benzyldimethyldodecylammonium chloride,
benzyldimethylhexadecylammonium bromide;
benzyldimethylhexadecylammonium chloride;
benzyld.imethyltetradecylammonium bromide;
benzyldimethyltetradecylammonium chloride;
cetyldimethylethylammonium bromide;
cetyldimethylethylammonium chloride; cetylpyridinium
bromide; cetylpyridinium chloride; N-[1,2,3-
dioleoyloxy)-propyl]-N,N,N-trimethylammonium chloride
(DOTMA); 1,2-dioleoyloxy-3-(trimethylammonio)propane
(DOTAP); and 1,2-dioleoyl-c-(4'-trimethylammonio)-
butanoyl-sn-glycerol (DOTE).
The echogenic gas may be one gas or mixture of
gases, such as CF4, C2F6, C3Fg, cyclo-C4Fg, C4F10,
C5F12, cyclo-C5F10,cyclo-C4F~ (1-trifluoromethyl),
propane (2-trifluoromethyl)-1,1,1,3,3,3 hexafluoro, and
butane (2-trifluoromethyl)-1,1,1,3,3,3,4,4,4 nonafluoro.
Also preferred are the the corresponding unsaturated
versions of the above compounds, for example C2F4, C3F6,
the isomers of C4Fg. Also, mixtures of these gases,
especially mixtures of perfluorocarbons with other
perfluorocarbons and mixtures of perfluorocarbons with
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other inert gases, such as air, N2, 02, He, would be
useful. Examples of these can be found in Quay, U.S.
Patent No. 5,595,723, the contents of which are herein
incorporated by reference.
X-ray contrast agents of the present invention are
comprised of one or more angiogenic tumor vasculature
targeting moieties attached to one or more X-ray
absorbing or "heavy" atoms of atomic number 20 or
greater, further comprising an optional linking moiety,
Ln, between the targeting moieties and the X-ray
absorbing atoms. The frequently used heavy atom in X-
ray contrast agents is iodine. Recently, X-ray contrast
agents comprised of metal chelates (Wallace, R., U.S.
5,417,959) and polychelates comprised of a plurality of
metal ions (Love, D., U.S. 5,679,810) have been
disclosed. More recently, multinuclear cluster
complexes have been disclosed as X-ray contrast agents
(U. S. 5,804,161, PCT W091/14460, and PCT WO 92/17215).
Examples of X-ray agents include the non-radioactive or
naturally occurring analogs of the above listed
radionuclides (e.g., Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd,
La, Au, Au, Yb, Dy, Cu, Rh, Ag, and Ir).
MRI contrast agents of the present invention are
comprised of one or more angiogenic tumor vasculature
targeting moieties attached to one or more paramagnetic
metal ions, further comprising an optional linking
moiety, Ln, between the targeting moieties and the
paramagnetic metal ions. The paramagnetic metal ions
are present in the form of metal complexes or metal
oxide particles. U.S. 5,412,148, and 5,760,191, describe
examples of chelators for paramagnetic metal ions for
use in MRI contrast agents. U.S. 5,801,228, U.S.
5,567,411, and U.S. 5,281,704, describe examples of
polychelants useful for complexing more than one
paramagnetic metal ion for use in MRI contrast agents.
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U.S. 5,520,904, describes particulate compositions
comprised of paramagnetic metal ions for use as MRI
contrast agents.
Administration of a compound of the present invention in
combination with such additional therapeutic agents, may
afford an efficacy advantage over the compounds and
agents alone, and may do so while permitting the use of
lower doses of each. A lower dosage minimizes the
potential of side effects, thereby providing an
increased margin of safety. The combination of a
compound of the present invention with such additional
therapeutic agents is preferably a synergistic
combination. Synergy, as described for example lay Chou
and Talalay, Adv. Enzyme Regul. 22:27-55 (1984), occurs
when the therapeutic effect of the compound and agent
when administered in combination is greater than the
additive effect of the either the compound or agent when
administered alone. In general, a synergistic effect is
most clearly demonstrated at levels that are
(therapeutically) sub-optimal for either the compound of
the present invention, an anti-cancer agent, a
photosensitizer agent or a radiosensitizer agent alone,
but which are highly efficacious in combination. Synergy
can be in terms of improved tumor response without
substantial increases in toxicity over individual
treatments alone, or some other beneficial effect of the
combination compared with the individual components.
The compounds of the present invention, and an
anti-cancer agent or a radiosensitizer agent, utilized
in combination therapy may be administered
simultaneously, in either separate or combined
formulations, or at different times e.g., sequentially,
such that a combined effect is achieved. The amounts and
regime of administration will be adjusted by the
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practitioner, by preferably initially lowering their
standard doses and then titrating the results obtained.
The invention also provides kits or single packages
combining two or more active ingredients useful in
treating cancer. A kit may provide (alone or in
combination with a pharmaceutically acceptable diluent
or carrier), the compound of the present invention and
additionally at least one agent selected from the group
consisting of an anti-cancer agent and a radiosensitizer
agent (alone or in combination with diluent or carrier)
and a photosensitizer agent.
The pharmaceuticals of the present invention have
the formulae, (Q)d-Ln-(Ch-X), (Q)d-Ln-(Ch-X~)d
(Q)d-Ln-(X~)d~~, and (Q)d-Ln-(X3), wherein Q represents a
peptide or peptidomimetic that binds to a receptor
expressed in angiogenic tumor vasculature, d is 1-10, Ln
represents an optional linking group, Ch represents a
metal chelator or bonding moiety, X represents a
radioisotope, X1 represents paramagnetic metal ion, X~
represents a paramagnetic metal ion or heavy atom
containing insoluble solid particle, d" is 1-100, and X3
represents a surfactant microsphere of an echogenic gas.
Preferred pharmaceuticals of the present invention are
comprised of targeting moieties, Q, that are peptides
and peptidomimetics that bind to the vitronectin
receptors oc,~,(33 and oc,~,(35. More preferred pharmaceuticals
of the present invention are comprised of targeting
moieties, Q, that are peptides and peptidomimetics that
bind to oc,~,(33. Most preferred pharmaceuticals of the
present invention are comprised of oc~,(33 targeting
moieties, Q, that are comprised of one to ten cyclic
pentapeptides or peptidomimetics, independently
attached to a therapeutic radioisotope or imageable
moiety, further comprising an optional linking moiety,
Ln, between the targeting moieties and the therapeutic
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radioisotopes or imageable moieties. The cyclic
peptides are comprised of a tripeptide sequence that
binds to the ocv(33 receptor and two amino acids either
one of which can be attached to Ln, Ch, X2, or X3. The
interaction of the tripeptide recognition sequences of
the cyclic peptide or peptidomimetic portion of the
pharmaceuticals with the ocv~i3 receptor results in
localization of the pharmaceuticals in angiogenic tumor
vasculature, which express the o~,v(33 receptor.
The pharmaceuticals of the present invention can be
synthesized by several approaches. One approach
involves the synthesis of the targeting peptide or
peptidomimetic moiety, Q, and direct attachment of one
or more moieties, Q, to one or more metal chelators or
bonding moieties, Ch, or to a paramagnetic metal ion or
heavy atom containing solid particle, or to an echogenic
gas microbubble. Another approach involves the
attachment of one or more moieties, Q, to the linking
group, Ln, which is then attached to one or more metal
chelators or bonding moieties, Ch, or to a paramagnetic
metal i.on or heavy atom containing solid particle, or to
an echogenic gas microbubble. Another approach, useful
in the synthesis of pharmaceuticals wherein d is 1,
involves the synthesis of the moiety, Q-Ln, together, by
incorporating an amino acid or amino acid mimetic
residue bearing Ln into the synthesis of the peptide or
peptidomimetic. The resulting moiety, Q-Ln, is then
attached to one or more metal chelators or bonding
moieties, Ch, or to a paramagnetic metal ion or heavy
atom containing solid particle, or to an echogenic gas
microbubble. Another approach involves the synthesis of
a peptide or peptidomimetic, Q, bearing a fragment of
the linking group, Ln, one or more of which are then
attached to the remainder of the linking group and then
to one or more metal chelators or bonding moieties, Ch,
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or to a paramagnetic metal ion or heavy atom containing
solid particle, or to an echogenic gas microbubble.
The peptides or peptidomimetics, Q, optionally
bearing a linking group, Ln, or a fragment of the
linking group, can be synthesized using standard
synthetic methods known to those skilled in the art.
Preferred methods include but are not limited to those
methods described below.
Generally, peptides and peptidomimetics are
elongated by deprotecting the alpha-amine of the
C-terminal residue and coupling the next suitably
protected amino acid through a peptide linkage using the
methods described. This deprotection and coupling
procedure is repeated until the desired sequence is
obtained. This coupling can be performed with the
constituent amino acids in a stepwise fashion, or
condensation of fragments (two to several amino acids),
or combination of both processes, or by solid phase
peptide synthesis according to the method originally
described by Merrifield, J. Am. Chem. Soc., 85,
2149-2154 (1963), the disclosure of which is hereby
incorporated by reference.
The peptides and peptidomimetics may also be
synthesized using automated synthesizing equipment. In
addition to the foregoing, procedures for peptide and
peptidomimetic synthesis are described in Stewart and
Young, "Solid Phase Peptide Synthesis", 2nd ed, Pierce
Chemical Co., Rockford, IL (1984); Gross, Meienhofer,
Udenfriend, Eds., "The Peptides: Analysis, Synthesis,
Biology, Vol. 1, 2, 3, 5, and 9, Academic Press, New
York, (1980-1987); Bodanszky, "Peptide Chemistry: A
Practical Textbook", Springer-Verlag, New York (1988);
and Bodanszky et al. "The Practice of Peptide
Synthesis" Springer-Verlag, New York (1984), the
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disclosures of which are hereby incorporated by
reference.
The coupling between two amino acid derivatives, an
amino acid and a peptide or peptidomimetic, two peptide
or peptidomimetic fragments, or the cyclization of a
peptide or peptidomimetic can be carried out using
standard coupling procedures such as the azide method,
mixed carbonic acid anhydride (isobutyl chloroformate)
method, carbodiimide (dicyclohexylcarbodiimide,
diisopropylcarbodiimide, or water-soluble carbodiimides)
method, active ester (p-nitrophenyl ester,
N-hydroxysuccinic imido ester) method, Woodward reagent
K method, carbonyldiimidazole method, phosphorus
reagents such as BOP-Cl, or oxidation-reduction method.
Some of these methods (especially the carbodiimide) can
be enhanced by the addition of 1-hydroxybenzotriazole.
These coupling reactions may be performed in either
solution (liquid phase) or solid phase.
The functional groups of the constituent amino
acids or amino. acid mimetics must be protected during
the coupling reactions to avoid undesired bonds being
formed. The protecting groups that can be used are
listed in Greene, "Protective Groups in Organic
Synthesis" John Wiley & Sons, New York (1981) and "The
Peptides: Analysis, Synthesis, Biology, Vol. 3, Academic
Press, New York (1981), the disclosure of which is
hereby incorporated by reference.
The alpha-carboxyl group of the C-terminal residue
is usually protected by an ester that can be cleaved to
give the carboxylic acid. These protecting groups
include: 1) alkyl esters such as methyl and t-butyl, 2)
aryl esters such as benzyl and substituted benzyl, or 3)
esters which can be cleaved by mild base treatment or
mild reductive means such as trichloroethyl and phenacyl
esters. In the solid phase case, the C-terminal amino
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acid is attached to an insoluble carrier (usually
polystyrene). These insoluble carriers contain a group
which will react with the carboxyl group to form a bond
which is stable to the elongation conditions but readily
cleaved later. Examples of which are: oxime resin
(DeGrado and Kaiser (1980) J. Org. Chem. 45, 1295-1300)
chloro or bromomethyl resin, hydroxymethyl resin, and
aminomethyl resin. Many of these resins are
commercially available with the desired C-terminal amino
acid already incorporated.
The alpha-amino group of each amino acid must be
protected. Any protecting group known in the art can be
used. Examples of these are: 1) acyl types such as
formyl, trifluoroacetyl, phthalyl, and
p-toluenesulfonyl; 2) aromatic carbamate types such as
benzyloxycarbonyl (Cbz) and substituted
benzyloxycarbonyls,
1-(p-biphenyl)-1-methylethoxycarbonyl, and
9-fluorenylmethyloxycarbonyl (Fmoc); 3) aliphatic
carbamate types such as tart-butyloxycarbonyl (Boc),
ethoxycarbonyl, diisopropylmethoxycarbonyl, and
allyloxycarbonyl; 4) cyclic alkyl carbamate types such
as cyclopentyloxycarbonyl and adamantyloxycarbonyl; 5)
alkyl types such as triphenylmethyl and benzyl; 6)
trialkylsilane such as trimethylsilane; and 7) thiol
containing types such as phenylthiocarbonyl and
dithiasuccinoyl. The preferred alpha-amino protecting
group is either Boc or Fmoc. Many amino acid or amino
acid mimetic derivatives suitably protected for peptide
synthesis are commercially available.
The alpha-amino protecting group is cleaved prior
to the coupling of the next amino acid. When the Boc
group is used, the methods of choice are trifluoroacetic
acid, neat or in dichloromethane, or HC1 in dioxane.
The resulting ammonium salt is then neutralized either
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prior to the coupling or in situ with basic solutions
such as aqueous buffers, or tertiary amines in
dichloromethane or dimethylformamide. When the Fmoc
group is used, the reagents of choice are piperidine or
substituted piperidines in dimethylformamide, but any
secondary amine or aqueous basic solutions can be used.
The deprotection is carried out at a temperature between
0 °C and room temperature.
Any of the amino acids or amino acid mimetics
bearing side chain functionalities must be protected
during the preparation of the peptide using any of the
above-identified groups. Those skilled in the art will
appreciate that the selection and use of appropriate
protecting groups for these side chain functionalities
will depend upon the amino acid or amino acid mimetic
and presence of other protecting groups in the peptide
or peptidomimetic. The selection of such a protecting
group is important in that it must not be removed during
the deprotection and coupling of the alpha-amino group.
For example, when Boc is chosen for the alpha-amine
protection the following protecting groups are
acceptable: p-toluenesulfonyl (tosyl) moieties and nitro
for arginine; benzyloxycarbonyl, substituted
benzyloxycarbonyls, tosyl or trifluoroacetyl for lysine;
benzyl or alkyl esters such as cyclopentyl for glutamic
and aspartic acids; benzyl ethers for serine and
threonine; benzyl ethers, substituted benzyl ethers or
2-bromobenzyloxycarbonyl for tyrosine; p-methylbenzyl,
p-methoxybenzyl, acetamidomethyl, benzyl, or
t-butylsulfonyl for cysteine; and the indole of
tryptophan can either be left unprotected or protected
with a formyl group.
When Fmoc is chosen for the alpha-amine protection
usually tert-butyl based protecting groups are
acceptable. For instance, Boc can be used for lysine,
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tart-butyl ether for serine, threonine and tyrosine, and
tart-butyl ester for glutamic and aspartic acids.
Once the elongation of the peptide or
peptidomimetic, or the elongation and cyclization of a
cyclic peptide or peptidomimetic is completed all of the
protecting groups are removed. For the liquid phase
synthesis the protecting groups are removed in whatever
manner as dictated by the choice of protecting groups.
These procedures are well known to those skilled in the
art.
When a solid phase synthesis is used to synthesize
a cyclic peptide or peptidomimetic, the peptide or
peptidomimetic should be removed from the resin without
simultaneously removing protecting groups from
functional groups that might interfere with the
cyclization process. Thus, if the peptide or
peptidomimetic is to be cyclized in solution, the
cleavage conditions need to be chosen such that a free
a-carboxylate and a free a-amino group are generated
without simultaneously removing other protecting groups.
Alternatively, the peptide or peptidomimetic may be
removed from the resin by hydrazinolysis, and then
coupled by the azide method. Another very convenient
method involves the synthesis of peptides or
peptidomimetics on an oxime resin, followed by
intramolecular nucleophilic displacement from the resin,
which generates a cyclic peptide or peptidomimetic
(Osapay, Profit, and Taylor (1990) Tetrahedron Letters
43, 6121-6124). When the oxime resin is employed, the
Boc protection scheme is generally chosen. Then, the
preferred method for removing side chain protecting
groups generally involves treatment with anhydrous HF
containing additives such as dimethyl sulfide, anisole,
thioanisole, or p-cresol at 0 °C. The cleavage of the
peptide or peptidomimetic can also be accomplished by
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other acid reagents such as trifluoromethanesulfonic
acid/trifluoroacetic acid mixtures.
Unusual amino acids used in this invention can be
synthesized by standard methods familiar to those
skilled in the art ("The Peptides: Analysis, Synthesis,
Biology, Vol. 5, pp. 342-449, Academic Press, New York
(1981)). N-Alkyl amino acids can be prepared using
procedures described in previously (Cheung et al.,
(1977) Can. J. Chem. 55, 906; Freidinger et al., (1982)
J. Org. Chem. 48, 77 (1982)), which are incorporated
herein by reference.
Additional synthetic procedures that can be used by
one of skill in the art to synthesize the peptides and
peptidomimetics targeting moieties are described in PCT
W094/22910, the contents of which are herein
incorporated by reference.
The attachment of linking groups, Ln, to the
peptides and peptidomimetics, Q; chelators or bonding
units, Ch, to the peptides and peptidomimetics, Q, or to
the linking groups, Ln; and peptides and peptidomimetics
bearing a fragment of the linking group to the remainder
of the linking group, in combination forming the moiety,
(Q)d-Ln, and then to the moiety Ch; can all be performed
by standard techniques. These include, but are not
limited to, amidation, esterification, alkylation, and
the formation of ureas or thioureas. Procedures for
performing these attachments can be found in Brinkley,
M., Bioconjugate Chemistry 1992, 3(1), which is
incorporated herein by reference.
A number of methods can be used to attach the
peptides and peptidomimetics, Q, to paramagnetic metal
ion or heavy atom containing solid particles, X2, by one
of skill in the art of the surface modification of solid
particles. In general, the targeting moiety Q or the
combination (Q)dLn is attached to a coupling group that
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react with a constituent of the surface of the solid
particle. The coupling groups can be any of a number of
silanes which react with surface hydroxyl groups on the
solid particle surface, as described in co-pending
U.S.A.N 60/092,360, and can also include
polyphosphonates, polycarboxylates, polyphosphates or
mixtures thereof which couple with the surface of the
solid particles, as described in U.S. 5,520,904.
A number of reaction schemes can be used to attach
the peptides and peptidomimetics, Q, to the surfactant
microsphere, X3. These are illustrated in following
reaction schemes where Sf represents a surfactant moiety
that forms the surfactant microsphere.
Acylation Reaction:
S f-C ( =0 ) -Y+ Q-NHS or -----------> S f-C ( =0 ) -NH-Q
Q-OH or Sf-C(=0)-0-Q
Y is a leaving group or active ester
Disulfide Coupling:
Sf-SH + Q-SH ___________> Sf_S_S_Q
Sulfonamide Coupling:
Sf-S(=0)2-Y + Q-NH2 ___________> Sf_S(=O)2_
NH-Q
Reductive Amidation:
Sf-CHO + Q-NH2___________> Sf-NH-Q
In these reaction schemes, the substituents Sf and Q can
be reversed as well.
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The linking group Ln can serve several roles.
First it provides a spacing group between the metal
chelator or bonding moiety, Ch, the paramagnetic metal
ion or heavy atom containing solid particle, X2, and the
surfactant microsphere, X3, and the one or more of the
peptides or peptidomimetics, Q, so as to minimize the
possibility that the moieties Cn-X, Ch-X~-, X2, and X3,
will interfere with the interaction of the recognition
sequences of Q with angiogenic tumor vasculature
receptors. The necessity of incorporating a linking
group in a reagent is dependent on the identity of Q,
Ch-X, Ch-X1, X2 , and X3 . I f Ch-X, Ch-X1, X2 , and X3 ,
cannot be attached to Q without substantially
diminishing its affinity for the receptors, then a
linking group is used. A linking group also provides a
means of independently attaching multiple peptides and
peptidomimetics, Q, to one group that is attached to Ch-
X, Ch-X1, X2, or X3.
The linking group also provides a means of
incorporating a pharmacokinetic modifier into the
pharmaceuticals of the present invention. The
pharmacokinetic modifier serves to direct the
biodistibution of the injected pharmaceutical other than
by the interaction of the targeting moieties, Q, with
the receptors expressed in the tumor neovasculature. A
wide variety of functional groups can serve as
pharmacokinetic modifiers, including, but not limited
to, carbohydrates, polyalkylene glycols, peptides or
other polyamino acids, and cyclodextrins. The modifiers
can be used to enhance or decrease hydrophilicity and to
enhance or decrease the rate of blood clearance. The
modifiers can also be used to direct the route of
elimination of the pharmaceuticals. Preferred
pharmacokinetic modifiers are those that result in
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moderate to fast blood clearance and enhanced renal
excretion.
The metal chelator or bonding moiety, Ch, is
selected to form stable complexes with theemetal ion
chosen for the particular application. Chelators or
bonding moieties for diagnostic radiopharmaceuticals are
selected to form stable complexes with the radioisotopes
that have imageable gamma ray or positron emissions,
such as 99mTc, 95TC~ 111In~ 62Cu~ 6oCu~ 64Cu~ 67Ga~ 68Ga~
86Y.
Chelators for technetium, copper and gallium
isotopes are selected from diaminedithiols,
monoamine-monoamidedithiols, triamide-monothiols,
monoamine-diamide-monothiols, diaminedioximes, and
hydrazines. The chelators are generally tetradentate
with donor atoms selected from nitrogen, oxygen and
sulfur. Preferred reagents are comprised of chelators
having amine nitrogen and thiol sulfur donor atoms and
hydrazine bonding units. The thiol sulfur atoms and the
hydrazines may bear a protecting group which can be
displaced either prior to using the reagent to
synthesize a radiopharmaceutical or preferably in situ
during the synthesis of the radiopharmaceutical.
Exemplary thiol protecting groups include those
listed in Greene and Wuts, "Protective Groups in Organic
Synthesis" John Wiley & Sons, New York (1991), the
disclosure of which is hereby incorporated by reference.
Any thiol protecting group known in the art can be used.
Examples of thiol protecting groups include, but are not
limited to, the following: acetamidomethyl,
benzamidomethyl, 1-ethoxyethyl, benzoyl, and
triphenylmethyl.
Exemplary protecting groups for hydrazine bonding
units are hydrazones which can be aldehyde or ketone
hydrazones having substituents selected from hydrogen,
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alkyl, aryl and heterocycle. Particularly preferred
hydrazones are described in co-pending U.S.S.N.
08/476,296 the disclosure of which is herein
incorporated by reference in its entirety.
The hydrazine bonding unit when bound to a metal
radionuclide is termed a hydrazido, or diazenido group
and serves as the point of attachment of the
radionuclide to the remainder of the
radiopharmaceutical. A diazenido group can be either
terminal (only one atom of the group is bound to the
radionuclide) or chelating. In order to have a
chelating diazenido group at least one other atom of
the group must also be bound to the radionuclide. The
atoms bound to the metal are termed donor atoms.
Chelators for 111In and 86Y are selected from cyclic
and acyclic polyaminocarboxylates such as DTPA, DOTA,
D03A, 2-benzyl-DOTA, alpha-(2-phenethyl)1,4,7,10-
tetraazazcyclododecane-1-acetic-4,7,10-
tris(methylacetic)acid, 2-benzyl-
cyclohexyldiethylenetriaminepentaacetic acid, 2-benzyl-
6-methyl-DTPA, and 6,6"-bis[N,N,N",N"-
tetra(carboxymethyl)aminomethyl)-4'-(3-amino-4-
methoxyphenyl)-2,2':6',2"-terpyridine. Procedures for
synthesizing these chelators that are not commercially
available can be found in Brechbiel, M. and Gansow, 0.,
J. Chem. Soc. Perkin Trans. 1992, 1, 1175; Brechbiel, M.
and Gansow, 0., Bioconjugate Chem. 1991, 2, 187;
Deshpande, S., et. al., J. Nucl. Med. 1990, 31, 473;
Kruper, J., U.S. Patent 5,064,956, and Toner, J., U.S.
Patent 4,859,777, the disclosures of which are hereby
incorporated by reference in their entirety.
The coordination sphere of metal ion includes all
the ligands or groups bound to the metal. For a
transition metal radionuclide to be stable it typically
has a coordination number (number of donor atoms)
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comprised of an integer greater than or equal to 4 and
less than or equal to 8; that is there are 4 to 8 atoms
bound to the metal and it is said to have a complete
coordination sphere. The requisite coordination number
for a stable radionuclide complex is determined by the
identity of the radionuclide, its oxidation state, and
the type of donor atoms. If the chelator or bonding
unit does not provide all of the atoms necessary to
stabilize the metal radionuclide by completing its
coordination sphere, the coordination sphere is
completed by donor atoms from other ligands, termed
ancillary or co-ligands, which can also be either
terminal or chelating.
A large number of ligands can serve as ancillary or
co-ligands, the choice of which is determined by a
variety of considerations such as the ease of synthesis
of the radiopharmaceutical, the chemical and physical
properties of the ancillary ligand, the rate of
formation, the yield, and the number of isomeric forms
of the resulting radiopharmaceuticals, the ability to
administer said ancillary or co-ligand to a patient
without adverse physiological consequences to said
patient, and the compatibility of the ligand in a
lyophilized kit formulation. The charge and
lipophilicity of the ancillary ligand will effect the
charge and lipophilicity of the radiopharmaceuticals.
For example, the use of 4,5-dihydroxy-1,3-benzene
disulfonate results in radiopharmaceuticals with an
additional two anionic groups because the sulfonate
groups will be anionic under physiological conditions.
The use of N-alkyl substituted 3,4-hydroxypyridinones
results in radiopharmaceuticals with varying degrees of
lipophilicity depending on the size of the alkyl
substituents.
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Preferred technetium radiopharmaceuticals of the
present invention are comprised of a hydrazido or
diazenido bonding unit and an ancillary ligand, AL1, or
a bonding unit and two types of ancillary AL1 and ALA, or
a tetradentate chelator comprised of two nitrogen and ._
two sulfur atoms. Ancillary ligands AL1 are comprised
of two or more hard donor atoms such as oxygen and amine
nitrogen (spa hybridized). The donor atoms occupy at
least two of the sites in the coordination sphere of the
radionuclide metal; the ancillary ligand AL1 serves as
one of the three ligands in the ternary ligand system.
Examples of ancillary ligands AL1 include but are not
limited to dioxygen ligands and functionalized
aminocarboxylates:~~A large number of such ligands are
available from commercial sources.
Ancillary dioxygen ligands include ligands that
coordinate to the metal ion through at least two oxygen
donor atoms. Examples include but are not limited to:
glucoheptonate, gluconate, 2-hydroxyisobutyrate,
lactate, tartrate, mannitol, glucarate, maltol, Kojic
acid, 2,2-bis(hydroxymethyl)propionic acid,
4,5-dihydroxy-1,3-benzene disulfonate, or substituted or
unsubstituted 1,2 or 3,4 hydroxypyridinones. (The names
for the ligands in these examples refer to either the
protonated or non-protonated forms of the ligands.)
Functionalized aminocarboxylates include ligands
that have a combination of amine nitrogen and oxygen
donor atoms. Examples include but are not limited to:
iminodiacetic acid, 2,3-diaminopropionic acid,
nitrilotriacetic acid, N,N'-ethylenediamine diacetic
acid, N,N,N'-ethylenediamine triacetic acid,
hydroxyethylethylenediamine triacetic acid, and
N,N'-ethylenediamine bis-hydroxyphenylglycine. (The
names for the ligands in these examples refer to either
the protonated or non-protonated forms of the ligands.)
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A series of functionalized aminocarboxylates are
disclosed by Bridger et. al. in U.S. Patent 5,350,837,
herein incorporated by reference, that result in
improved rates of formation of technetium labeled
hydrazino modified proteins. We have determined that
certain of these aminocarboxylates result in improved
yields of the radiopharmaceuticals of the present
invention. The preferred ancillary ligands AL1
functionalized aminocarboxylates that are derivatives of
glycine; the most preferred is tricine
(tris(hydroxymethyl)methylglycine).
The most preferred technetium radiopharmaceuticals
of the present invention are comprised of a hydrazido or
diazenido bonding unit and two types of ancillary
designated AL1 and AL2, or a diaminedithiol chelator.
The second type of ancillary ligands AL2 are comprised
of one or more soft donor atoms selected from the group:
phosphine phosphorus, arsine arsenic, imine nitrogen
(sp2 hybridized), sulfur (sp2 hybridized) and carbon (sp
hybridized); atoms which have p-acid character. Ligands
AL2 can be monodentate, bidentate or tridentate, the
denticity is defined by the number of donor atoms in the
ligand. One of the two donor atoms in a bidentate
ligand and one of the. three donor atoms in,a tridentate
ligand must be a soft donor atom. We have disclosed in
co-pending U.S.S.N. 08/415,908, and U.S.S.N. &0/023360
and 08/646,886, the disclosures of which are herein
incorporated by reference in their entirety, that
radiopharmaceuticals comprised of one or more ancillary
or co-ligands ALA are more stable compared to
radiopharmaceuticals that are not comprised of one or
more ancillary ligands, ALA; that is, they have a
minimal number of isomeric forms, the relative ratios of
which do not change significantly with time, and that
remain substantially intact upon dilution.
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The ligands ALA that are comprised of phosphine or
arsine donor atoms are trisubstituted phosphines,
trisubstituted arsines, tetrasubstituted diphosphines
and tetrasubstituted diarsines. The ligands AL2 that
are comprised of imine nitrogen are unsaturated or
aromatic nitrogen-containing, 5 or 6-membered
heterocycles. The ligands that are comprised of sulfur
(spy hybridized) donor atoms are thiocarbonyls,
comprised of the moiety C=S. The ligands comprised of
carbon (sp hybridized) donor atoms are isonitriles,
comprised of the moiety CNR, where R is an organic
radical. A large number of such ligands are available
from commercial sources. Isonitriles can be synthesized
as described in European Patent 0107734 and in U.S.
Patent 4,988,827, herein incorporated by reference.
Preferred ancillary ligands AL2 are trisubstituted
phosphines and unsaturated or aromatic 5 or 6 membered
heterocycles. The most preferred ancillary ligands AL2
are trisubstituted phosphines and unsaturated 5 membered
heterocycles.
The ancillary ligands ALA may be substituted with
alkyl, aryl, alkoxy, heterocycle, aralkyl, alkaryl and
arylalkaryl groups and may or may not bear functional
groups comprised of heteroatoms such as oxygen,
nitrogen, phosphorus or sulfur. Examples of such
functional groups include but are not limited to:
hydroxyl, carboxyl, carboxamide, nitro, ether, ketone,
amino, ammonium, sulfonate, sulfonamide, phosphonate,
and phosphonamide. The functional groups may be chosen
to alter the lipophilicity and water solubility of the
ligands which may affect the biological properties of
the radiopharmaceuticals, such as altering the
distribution into non-target tissues, cells or fluids,
and the mechanism and rate of elimination from the body.
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Chelators or bonding moieties for therapeutic
radiopharmaceuticals are selected to form stable
complexes with the radioisotopes that have alpha
particle, beta particle, Auger or Coster-Kronig electron
emissions, such as 186Re, lg8Re, 153Sm~ 166Ho~ 177Lu,
149pm~ 90y~ 212Bi~ 103pd~ 109pd~ 159Gd~ l4oLa~ 198Au~ 199Au,
169yb~ 175yb~ 165Dy~ 166Dy~ 67Cu~ 105Rh~ 111Ag~ and l9~Ir.
Chelators for rhenium, copper, palladium, platinum,
iridium, rhodium, silver and gold isotopes are selected
from diaminedithiols, monoamine-monoamidedithiols,
triamide-monothiols, monoamine-diamide-monothiols,
diaminedioximes, and hydrazines. Chelators for yttrium,
bismuth, and the lanthanide isotopes are selected from
cyclic and acyclic polyaminocarboxylates such as DTPA,
DOTA, D03A, 2-benzyl-DOTA, alpha-(2-phenethyl)1,4,7,10-
tetraazacyclododecane-1-acetic-4,7,10-
tris(methylacetic)acid, 2-benzyl-
cyclohexyldiethylenetriaminepentaacetic acid, 2-benzyl-
6-methyl-DTPA, and 6,6"-bis[N,N,N",N"-
tetra(carboxymethyl)aminomethyl)-4'-(3-amino-4-
methoxyphenyl)-2,2':6',2"-terpyridine.
Chelators for magnetic resonance imaging contrast
agents are selected to form stable complexes with
paramagnetic metal ions, such as Gd(III), Dy(III),
Fe(III), and Mn(II), are selected from cyclic and
acyclic polyaminocarboxylates such as DTPA, DOTA, D03A,
2-benzyl-DOTA, alpha-(2-phenethyl)1,4,7,10-
tetraazacyclododecane-1-acetic-4,7,10-
tris(methylacetic)acid, 2-benzyl-
cyclohexyldiethylenetriaminepentaacetic acid, 2-benzyl-
6-methyl-DTPA, and 6,6"-bis[N,N,N",N"-
tetra(carboxymethyl)aminomethyl)-4'-(3-amino-4-
methoxyphenyl)-2,2':6',2"-terpyridine.
The technetium and rhenium radiopharmaceuticals of
the present invention comprised of a hydrazido or
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diazenido bonding unit can be easily prepared by
admixing a salt of a radionuclide, a reagent of the
present invention, an ancillary ligand AL1, an ancillary
ligand ALA, and a reducing agent, in an aqueous solution
at temperatures from 0 to 100 °C. The technetium and
rhenium radiopharmaceuticals of the present invention
comprised of a tetradentate chelator having two nitrogen
and two sulfur atoms can be easily prepared by admixing
a salt of a radionuclide, a reagent of the present
invention, and a reducing agent, in an aqueous solution
at temperatures from 0 to 100 °C.
When the bonding unit in the reagent of the present
invention is present as a hydrazone group, then it must
first be converted to a hydrazine, which may or may not
be protonated, prior to complexation with the metal
radionuclide. The conversion of the hydrazone group to
the hydrazine can occur either prior to reaction with
the radionuclide, in which case the radionuclide and the
ancillary or co-ligand or ligands are combined not with
the reagent but with a hydrolyzed form of the reagent
bearing the chelator or bonding unit, or in the presence
of the radionuclide in which case the reagent itself is
combined with the radionuclide and the ancillary or
co-ligand or ligands. In the latter case, the pH of the
reaction mixture must be neutral or acidic.
Alternatively, the radiopharmaceuticals of the
present invention comprised of a hydrazido or diazenido
bonding unit can be prepared by first admixing a salt of
a radionuclide, an ancillary ligand AL1, and a reducing
agent in an aqueous solution at temperatures from 0 to
100 °C to form an intermediate radionuclide complex with
the ancillary ligand AL1 then adding a reagent of the
present invention and an ancillary ligand ALA and
reacting further at temperatures from 0 to 100 °C.
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Alternatively, the radiopharmaceuticals of the
present invention comprised of a hydrazido or diazenido
bonding unit can be prepared by first admixing a salt of
a radionuclide, an ancillary ligand AL1, a reagent of
the present invention, and a reducing agent in an
aqueous solution at temperatures from 0. to 100 °C to
form an intermediate radionuclide complex, and then
adding an ancillary ligand AL2 and reacting further at
temperatures from 0 to 100 °C.
The technetium and rhenium radionuclides are
preferably in the chemical form of pertechnetate or
perrhenate and a pharmaceutically acceptable cation.
The pertechnetate salt form is preferably sodium
pertechnetate such as obtained from commercial Tc-99m
generators. The amount of pertechnetate used to prepare
the radiopharmaceuticals of the present invention can
range from 0.1 mCi to 1 Ci, or more preferably from 1 to
200 mCi.
The amount of the reagent of the present invention
used to prepare the technetium and rhenium
radiopharmaceuticals of the present invention can range
from 0.01 ~g to 10 mg, or more preferably from 0.5 ~.~.g to
200 ug. The amount used will be dictated by the amounts
of the other reactants and the identity of the
radiopharmaceuticals of the present invention to be
prepared.
The amounts of the ancillary ligands AL1 used can
range from 0.1 mg to 1 g, or more preferably from 1 mg
to 100 mg. The exact amount for a particular
radiopharmaceutical is a function of identity of the
radiopharmaceuticals of the present invention to be
prepared, the procedure used and the amounts and
identities of the other reactants. Too large an amount
of AL1 will result in the formation of by-products
comprised of technetium labeled AL1 without a
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biologically active molecule or by-products comprised of
technetium labeled biologically active molecules with
the ancillary ligand AL1 but without the ancillary
ligand AL2. Too small an amount of ALA will result in
other by-products such as technetium labeled
biologically active molecules with the ancillary ligand
ALA but without the ancillary ligand AL1, or reduced
hydrolyzed technetium, or technetium colloid.
The amounts of the ancillary ligands AL2 used can
range from 0.001 mg to 1 g, or more preferably from 0.01
mg to 10 mg. The exact amount for a particular
radiopharmaceutical is a function of the identity of the
radiopharmaceuticals of the present invention to be
prepared, the procedure used and the amounts and
identities of the other reactants. Too large an amount
of AL2 will result in the formation of by-products
comprised of technetium labeled AL2 without a
biologically active molecule or by-products comprised of
technetium labeled biologically active molecules with
the ancillary ligand ALZ but without the ancillary
ligand AL1. If the reagent bears one or more
substituents that are comprised of a soft donor atom, as
defined alcove, at least a ten-fold molar excess of the
ancillary ligand AL2 to the reagent of formula 2 is
required to prevent the substituent from interfering
with the coordination of the ancillary ligand AL2 to the
metal radionuclide.
Suitable reducing agents for the synthesis of the
radiopharmaceuticals of the present invention include
stannous salts, dithionite or bisulfate salts,
borohydride salts, and formamidinesulfinic acid, wherein
the salts are of any pharmaceutically acceptable form.
The preferred reducing agent is a stannous salt. The
amount of a reducing agent used can range from 0.001 mg
to 10 mg, or more preferably from 0.005 mg to 1 mg.
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The specific structure of a radiopharmaceutical of
the present invention comprised of a hydrazido or
diazenido bonding unit will depend on the identity of
the reagent of the present invention used, the identity
of any ancillary ligand AL1, the identity of any
ancillary ligand AL2, and the identity of the
radionuclide. Radiopharmaceuticals comprised of a
hydrazido or diazenido bonding unit synthesized using
concentrations of reagents of <100 ug/mL, will be
comprised of one hydrazido or diazenido group. Those
synthesized using >1 mg/mL concentrations will be
comprised of two hydrazido or diazenido groups from two
reagent molecules. For most applications, only a
limited amount of the biologically active molecule can
be injected and not result in undesired side-effects,
such as chemical toxicity, interference with a
biological process or an altered biodistribution of the
radiopharmaceutical. Therefore, the radiopharmaceuticals
which require higher concentrations of the reagents
comprised in part of the biologically active molecule,
will have to be diluted or purified after synthesis to
avoid such side-effects.
The identities and amounts used of the ancillary
ligands AL1 and ALA will determine the values of the
variables y and z. The values of y and z can
independently be an integer from 1 to 2. In
combination, the values of y and z will result in a
technetium coordination sphere that is made up of at
least five and no more than seven donor atoms. For
monodentate ancillary ligands Av2, z can be an integer
from 1 to 2; for bidentate or tridentate ancillary
ligands ALA, z is 1. The preferred combination for
monodentate ligands is y equal to 1 or 2 and z equal to
1. The preferred combination for bidentate or
tridentate ligands is y equal to 1 and z equal to 1.
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The indium, copper, gallium, silver, palladium,
rhodium, gold, platinum, bismuth, yttrium and lanthanide
radiopharmaceuticals of the present invention can be
easily prepared by admixing a salt of a radionuclide and
a reagent of the present invention, in an aqueous
solution at temperatures from 0 to 100 °C. These
radionuclides are typically obtained as a dilute aqueous
solution in a mineral acid, such as hydrochloric, nitric
or sulfuric acid. The radionuclides are combined with
from one to about one thousand equivalents of the
reagents of the present invention dissolved in aqueous
solution. A buffer is typically used to maintain the pH
of the reaction mixture between 3 and 10.
The gadolinium, dysprosium, iron and manganese
metallopharmaceuticals of the present invention can be
easily prepared by admixing a salt of the paramagnetic
metal ion and a reagent of the present invention, in an
aqueous solution at temperatures from 0 to 100 °C.
These paramagnetic metal ions are typically obtained as
a dilute aqueous solution in a mineral acid, such as
hydrochloric, nitric or sulfuric acid. The paramagnetic
metal ions are combined with from one to about one
thousand equivalents of the reagents of the present
invention dissolved in aqueous solution, A buffer is
typically used to maintain the pH of the reaction
mixture between 3 and 10.
The total time of preparation will vary depending
on the identity of the metal ion, the identities and
amounts of the reactants and the procedure used for the
preparation. The preparations may be complete,
resulting in > 80% yield of the radiopharmaceutical, in
1 minute or may require more time. If higher purity
metallopharmaceuticals are needed or desired, the
products can be purified by any of a number of
techniques well known to those skilled in the art such
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as liquid chromatography, solid phase extraction,
solvent extraction, dialysis or ultrafiltration.
Buffers useful in the preparation of
metallopharmaceuticals and in diagnostic kits useful for
the preparation of said radiopharmaceuticals include but
are not limited to phosphate, citrate, sulfosalicylate,
and acetate. A more complete list can be found in the
United States Pharmacopeia.
Lyophilization aids useful in the preparation of
diagnostic kits useful for the preparation of
radiopharmaceuticals include but are not limited to
mannitol, lactose, sorbitol, dextran, Ficoll, and
polyvinylpyrrolidine (PVP).
Stabilization aids useful in the preparation of
metallopharmaceuticals and in diagnostic kits useful for
the preparation of radiopharmaceuticals include but are
not limited to ascorbic acid, cysteine,
monothioglycerol, sodium bisulfate, sodium
metabisulfite, gentisic acid, and inositol.
Solubilization aids useful in the preparation of
metallopharmaceuticals and in diagnostic kits useful for
the preparation of radiopharmaceuticals include but are
not limited to ethanol, glycerin, polyethylene glycol,
propylene glycol, polyoxyethylene sorbitan monooleate,
sorbitan monoloeate, polysorbates,
poly(oxyethylene)poly(oxypropylene)poly(oxyethylene)
block copolymers (Pluronics) and lecithin. Preferred
solubilizing aids are polyethylene glycol, and
Pluronics.
Bacteriostats useful in the preparation of
metallopharmaceuticals and in diagnostic kits useful for
the preparation of radiopharmaceuticals include but are
not limited to benzyl alcohol, benzalkonium chloride,
chlorbutanol, and methyl, propyl or butyl paraben.
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A component in a diagnostic kit can also serve more
than one function. A reducing agent can also serve as a
stabilization aid, a buffer can also serve as a transfer
ligand, a lyophilization aid can also serve as a
transfer, ancillary or co-ligand and so forth.
The diagnostic radiopharmaceuticals are
administered by intravenous injection, usually in saline
solution, at a dose of 1 to 100 mCi per 70 kg body
weight, or preferably at a dose of 5 to 50 mCi. Imaging
is performed using known procedures.
The therapeutic radiopharmaceuticals are
administered by intravenous injection, usually in saline
solution, at a dose of 0.1 to 100 mCi per 70 kg body
weight, or preferably at a dose of 0.5 to 5 mCi per 70
kg body weight.
The magnetic resonance imaging contrast agents of.
the present invention may be used in a similar manner as
other MRI agents as described in U.S. Patent 5,155,215;
U.S. Patent 5,087,440; Margerstadt et al., Magn. Reson.
Med., 1986, 3, 808; Runge et al., Radiology, 1988, 166,.
835; and Bousquet et al., Radiology, 1988, 166, 693.
Generally, sterile aqueous solutions of the contrast
agents are administered to a patient intravenously in
dosages ranging from 0.01 to 1.0 mmoles per kg body
weight.
For use as X-ray contrast agents, the compositions
of the present invention should generally have a heavy
atom concentration of 1 mM to 5 M, preferably 0.1 M to 2
M. Dosages, administered by intravenous injection, will
typically range from 0.5 mmol/kg to 1.5 mmol/kg,
preferably 0.8 mmol/kg to 1.2 mmol/kg. Imaging is
performed using known techniques, preferably X-ray
computed tomography.
The ultrasound contrast agents of the present
invention are administered by intravenous injection in
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an amount of 10 to 30 ~L of the echogenic gas per kg
body weight or by infusion at a rate of approximately 3
~~L/kg/min. Imaging is performed using known techniques
of sonography.
Other features of the invention will become
apparent in the course of the following descriptions of
exemplary embodiments which are given for illustration
of the invention and are not intended to be limiting
thereof.
EXAMPLES
Representative materials and methods that may be
used in preparing the compounds of the invention are
described further below.
Manual solid phase peptide synthesis was performed
in 25 mL polypropylene filtration tubes purchased from
BioRad Inc., or in 60 mL hour-glass reaction vessels
purchased from Peptides International. Oxime resin
(substitution level = 0.96 mmol/g) was prepared
according to published procedure (DeGrado and Kaiser, J.
Org. Chem. 1980, 45, 1295), or was purchased from
Novabiochem (substitution level = 0.62 mmol/g). All
chemicals and solvents (reagent grade) were used as
supplied from the vendors cited without further
purification. t-Butyloxycarbonyl (Boc) amino acids and
other starting amino acids may be obtained commercially
from Bachem Inc., Bachem Biosciences Inc. (Philadelphia,
PA), Advanced ChemTech (Louisville, KY), Peninsula
Laboratories (Belmont, CA), or Sigma (St. Louis, MO).
2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) and TBTU were purchased from
Advanced ChemTech. N-methylmorpholine (NMM), m-cresol,
D-2-aminobutyric acid (Abu), trimethylacetylchloride,
diisopropylethylamine (DIEA), 1,2,4-triazole, stannous
chloride dehydrate, and tris(3-sulfonatophenyl)phosphine
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trisodium salt (TPPTS) were purchased from Aldrich
Chemical Company. Bis(3-sulfonatophenyl)phenylphosphine
disodium salt (TPPDS) was prepared by the published
procedure (Kuntz, E., U.S. Patent 4,248,802). (3-
Sulfonatophenyl)diphenylphosphii~.e monosodium salt
(TPPMS)was purchased from TCI America, Tnc. Tricine was
obtained from Research Organics, Inc. Technetium-99m-
pertechnetate (99mTcOg-) was obtained from a DuPont
Pharma 99Mo/99mTc Technelite~ generator. In-111-chloride
(Indichlor~) was obtained from Amersham Medi-Physics,
Inc. Sm-153-chloride and Lutetium-177-chloride were
obtained from the University of Missouri Research
Reactor (MURK). Yttrium-90 chloride was obtained from
the Pacific Northwest Research Laboratories.
Dimethylformamide (DMF), ethyl acetate, chloroform
(CHC13), methanol (MeOH), pyridine and hydrochloric acid
(HCl) were obtained from Baker. Acetonitrile,
dichloromethane (DCM), acetic acid (HOAc),
trifluoroacetic acid (TFA), ethyl ether, triethylamine,
acetone, and magnesium sulfate were commercially
obtained. Absolute ethanol was obtained from Quantum
Chemical Corporation.
General Procedure for Solid Phase Peptide Synthesis
Using Boc-Chemistry on the Oxime Resin for the
Preparation of Cyclic Peptides
The appropriately protected CyCllC peptides,
described in the Examples, were prepared by manual solid
phase peptide synthesis using Boc-teabag chemistry
(Houghton, 1985) on a p-nitrobenzophenone oxime solid
support (DeGrado, 1982, Scarr and Findeis, 1990). The
5.0 cm x 5.0 cm teabags were made from 0.75 mm mesh
polypropylene filters (Spectra Filters) and filled with
0.5 g (or 1 g) of the oxime resin. The coupling and
deprotection steps were carried out in a polypropylene
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reactor using a table-top shaker for agitation.
Synthesis of the protected pentapeptide-resin
intermediate was achieved by first coupling Boc-Gly-OH
to the oxime~resin (substitution 0.69 mmol/g or 0.95
mmol/g). Attachment of Boc-Gly-OH onto the oxime resin
was achieved by using five equivalents each of the amino
acid, HBTU and diisopropylethylamine (DIPEA) in DMF.
Coupling of the. first amino acid generally occurred over
2-3 days. After thorough washing, substitution levels
were determined using the picric acid assay (Stewart and
Martin). Unreacted oxime groups on the resin were then
capped with a solution of DIPEA and trimethylacetyl
chloride in DMF. The boc-group was deprotected using
50% or 25% TFA in DCM (30 min). Coupling of the other
protected boc-amino acids were performed in a similar
manner by overnight shaking (1-2 days), and the coupling
yields for each newly added amino acid was determined
using the picric acid assay.
General Procedure for Solid Phase Peptide Synthesis
Using Fmoc-Chemistry on the HMPB-BHA Resin for the
Preparation of Cyclic Peptides
The appropriately protected linear peptide
precursors to the cyclic peptides, described in the
Examples, were also prepared by automated solid phase
peptide synthesis using Fmoc chemistry on an Advanced
ChemTech Model 90 Synthesizer and using HMPB-BHA resin
as the solid support. Synthesis of the protected
pentapeptide-resin intermediates was achieved by
coupling (for 3 h) the Fmoc-amino acids sequentially to
the commercially available (Novabiochem) Fmoc-Gly-HMPB-
BHA resin (usually 2 g, substitution 0.47 to 0.60
mmol/g) by using three to five equivalents each of the
amino acid, HBTU, HOBt and diisopropylethylamine (DIPEA)
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in DMF. The Fmoc-group was deprotected using 20%
piperidine in DMF (30 min). The peptides were cleaved
from the HMPB-BHA resin using a solution of 1% TFA/DCM
and collecting the peptide solutions in a solution of
pyridine in methanol (1:10). The linear protected v..,
peptides were isolated by removing the solvents and
reagents in vacuo and triturating the crude residue in
diethyl ether.
The syntheses of several amino acids that are not
commercially available are described in the following
procedures.
Synthesis of Tfa-amino acids
Boc-HomoLys(Tfa)-OH and Boc-Cys(2-N-Tfa-
aminoethyl)-OH are prepared via the reaction of Boc-
HomoLys-OH and Boc-Cys(2-aminoethyl)-OH, respectively,
with ethyl thioltrifluoroacetate in Aq. NaOH, and
purified by recrystallization from ethanol.
Synthesis of Boc-Orn(d-N-Benzylcarbamoyl)
To a solution of Boc-Orn (1 mmol) in DMF (30 mL) is
added benzylisocyanate (2.2 mmol), and diisopropylamine
(3 mmol). The reaction mixture is then stirred
overnight at room temperature. The volatiles are
removed in vacuo and the crude material is purified by
column chromatography to obtain the desired product.
Synthesis of Boc-Orn(d-N-1-Tos-2-Imidazolinyl)
A solution of Boc-Orn-OH (10 mmol), 1-tosyl-2-
methylthio-2-imidazoline (12 mmol, (which in turn is
prepared from the reaction of the commercially available
2-methylthio-2-imidazoline hydriodide and p-
toluenesulfonic anhydride in methylene chloride (0 ~C to
RT) in the presence of triethylamine)), and
diisopropylethylamine (12 mmol) is stirred at reflux,
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overnight. The volatiles are removed and the desired
product isolated by chromatography.
Synthesis of Dap(b-(1-Tos-2-benzimidazolylacetyl))
To a solution of 1-Tos-2-benzimidazolylacetic acid
(10 mmol, prepared using tosyl chloride and standard
reported conditions) and N-methylmorpholine (10 mmol) in
anhydrous DMF is added isobutyl chloroformate (10 mmol).
After stirring at ice bath temperature for 5-10 min.,
Boc-Orn-OH (10 mmol) and N-methylmorpholine (20 mmol) in
anhydrous DMF is added in one portion. The reaction
mixture is stirred overnight at room temperature, the
volatiles removed in vacuo, and the product is isolated
by chromatography. (Alternatively, Boc-Orn-OMe is used
and the product isolated is treated with aqueous LiOH to
obtain the acid.)
The analytical HPLC methods utilized are described
below:
HPLC Method 1
Instrument: HP1050
Column: Z7ydac C18(4.6 x 250 mm)
Detector: Diode array detector 220nm/500ref
Flow Rate: 1.0 mL/min.
Column Temp: 50 ~C
Sample Size: 15 uL
Mobile Phase: A: 0.1% TFA in water
B: 0.1% TFA in ACN/Water (9:1)
Gradient A: Time (min) %A %B
0 80 20
20 0 100
30 0 100
31 80 20
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Gradient B: Time (min) oA %B
0 98 2
l6 63.2 36.8
l8 0 100
28 0 100
30 98 2
Example 1
Synthesis of cyclo{Arg-Gly-Asp-D-Tyr(N-[2-[[[5-
[carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid]-3-aminopropyl)-Val}
~H O
TFA~H2N N N
H NH H HN OH
O
NH H O O
,, O
O ~ ~ H H
N
O / S03H
Part A: Preparation of cyclo{Arg(Tos)-Gly-Asp(OBzl)-D-
Tyr(N-Cbz-3-aminopropyl)-Val}
The N-terminus Boc- protecting group of the peptide
sequence Boc-Asp(OBzl)-D-Tyr(N-Cbz-aminopropyl)-Val-
Arg(Tos)-Gly-Oxime resin was removed using standard
deprotection (25o TFA in CH2C12). After eight washes
with DCM, the resin was treated with 10% DIEA/DCM (2 x
10 min.). The resin was subsequently washed with DCM (x
5) and dried under high vacuum. The resin (1.7474 g,
0.55 mmol/g) was then suspended in dimethylformamide (15
mL). Glacial acetic acid (55.0 uL, 0.961 mmol) was
added, and the reaction mixture was heated at 50 ~C for
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72 h. The resin was filtered, and washed with DMF (2 x
mL). The filtrate was concentrated to an oil under
high vacuum. The resulting oil was triturated with
ethyl acetate. The solid thus obtained was filtered,
5 washed with ethyl acetate, and dried under high vacuum
to give 444.4 mg of the desired product. ESMS: Calcd.
for C51H63N9012S~ 1025.43; Found, 1026.6 [M+H]+1.
Analytical HPLC, Method 1A, Rt = 14.366 min, Purity =
75%.
Part B: Preparation of cyclo{Arg-Gly-Asp-D-Tyr(3-
aminopropyl)-Val} Trifluoroacetic acid salt.
TFA~H
NH O
2N~H H
NH HN OH
O
NH HN O O
O
O ~NH2~TFA
Cyclo{Arg(Tos)-Gly-Asp(OBzl)-D-Tyr(N-Cbz-3-
aminopropyl)-Val} (0.150 g, 0.146 mmol) was dissolved in
trifluoroacetic acid (0.6 mL) and cooled to -10 ~C.
Trifluoromethanesulfonic acid (0.5 mL) was added
dropwise, maintaining the temperature at -10 ~C.
Anisole (0.1 mL) was added and the reaction mixture was
stirred at -10 ~C for 3 h. Diethyl ether was added, the
reaction mixture cooled to -35 ~C and then stirred for
min. The reaction mixture was cooled further to -50
25 ~C and stirred for 30 min. The crude product obtained
was filtered, washed with diethyl ether, dried under
high vacuum, and purified by preparative HPLC Method 1,
to give 29.7 mg (23%) of the desired product as a
lyophilized solid. ESMS: Calcd. for C2gH45NgOg,
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647.34; Found, 648.5 [M+H]+1. Analytical HPLC, Method
1B, Rt = 10.432 min, Purity = 910.
Preparative HPLC Method 1
Instrument: Rainin Rabbit; Dynamax software
Column: Vydac C-18 (21.2 mm x 25 cm)
Detector: Knauer VWM
Flow Rate: l5ml/min
Column Temp: RT
Mobile Phase: A: 0.1o TFA in H20
B: 0.1%TFA in ACN/H20 (9:1)
Gradient: Time (min) %A %B
0 98 2
16 63.2 36.8
18 0 100
28 0 100
30 98 2
Part C. Preparation of cyclo{Arg-Gly-Asp-D-Tyr(N-[2-
[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-Val}
Cyclo{Arg-Gly-Asp-D-Tyr(3-aminopropyl)-Val}
trifluoroacetic acid salt (0.020 g, 0.0228 mmol) was
dissolved in DMF (1 mL). Triethylamine (9.5 }.1L, 0.0648
mmol) was added, and after 5 min of stirring 2-[[[5-
[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid,
monosodium salt (0.0121 g, 0.0274 mmol) was added. The
reaction mixture was stirred for 7 days, and then
concentrated to an oil under high vacuum. The oil was
purified by preparative HPLC Method 1 to give 8.9 mg
(37%) of the title product as a lyophilized solid (TFA
salt). HRMS: Calcd. for C42H54N12~125 +H, 951.3783;
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Found, 951.3767. Analytical HPLC, Method 1B, Rt =
14.317 min, Purity = 95%.
Example 2
Synthesis of cyclo{Arg-Gly-Asp-D-Tyr((N-[2-[[[5-
[carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid]-18-amino-14-aza-4,7,10-oxy-15-oxo-octadecoyl)-3-
aminopropyl)-Val}
NIIH O
TFA.H2N~ H~ H~O
NH HN~OH
~O
NH HN O O
v / o~ H
~~o
H
O N
O~O
O~ O
N
H /
N
NH
H03 N
Part A: Preparation of 3-(N-(3-(2-(2-(3-((tent-butoxy)-
carbonylamino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)-
propanoic acid
N-(3-(2-(2-(3-
Aminopropoxy)ethoxy)ethoxy)propyl)(tert-butoxy)formamide
(1.5 g, 4.68 mmol) was added to DMF (15 mL). To this
solution pyridine (15 mL), succinic anhydride (0.47 g,
4.68 mmol) were added, followed by dimethylaminopyridine
(62 mL, 0.468 umol). The reaction mixture was stirred
overnight at 100 ~C. The mixture was concentrated under
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high vacuum and the residue was brought up in water,
acidified to pH 2.5 with 1N HCl, and extracted with
ethyl acetate (3x). The combined organic extracts were
dried over MgS04 and filtered. The filtrate was
concentrated in vacuo to provide 1.24 g of an oil
product (63%). The desired product was used without
further purification. 1H NMR (CDC13) 3.67-3.45 (m,
11H), 3.41-3.28 (m, 2H), 3.21-3.09 (m, 2H), 2.95-2.82
(m, 2H), 2.80-2.35 (m, 3H), 1.81-1.68 (m, 4H), 1.50-1.35
(s, 9H); ESMS: Calculated for C1gH36N20g, 420.2471
Found 419.3 [M-H]-1.
Part B: Preparation of 3-(N-(3-(2-(2-(3-((tert-butoxy)-
carbonylamino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)
propanoic acid succinimide ester
Boo-Hf~C~~ C~ OSu
H O
To a solution of 3-(N-(3-(2-(2-(3-((tent-butoxy)-
carbonylamino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)-
propanoic acid (1.12 g, 2.66 mmol), N-hydroxysuccinimide
(0.40 g, 3.46 mmol), and N,N-dimethylformamide (40 mL)
was added 1-(3-dimethylamin.opropyl)-3-ethylcarbodimide
(0.67 g, 3.46 mmol). The reaction mixture was stirred
at room temperature for 48 h. The mixture was
concentrated under high vacuum and the residue was
brought up in 0.1N HCl and extracted with ethyl acetate
(3x). The combined organic extracts were washed with
water (2x) then saturated sodium chloride, dried over
MgS04, and filtered. The filtrate was cocnentrated in
vacuo to give 1.0 g of the product as an oil (73%). The
desired product was used without further purification.
ESMS: Calculated for C23H3gN3010, 517.2635 Found 518.2
[M+H] +1 .
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Part C. Preparation of cyclo{Arg-Gly-Asp-D-Tyr(3-(3-(N-
(3-(2-(2-(3-( (tert-butoxy)-
carbonylamino)propoxy)ethoxy)-ethoxy)propyl)carbamoyl)-
propanamido)propyl)-Val}
NH O
TFA~H2N~ H~~ H~O
NH HN~OH
I1O
NH HN O O
O
N
~O
H
O N
O~O
O
~NH-BOC
Cyclo{Arg-Gly-Asp-D-Tyr(3-aminopropyl)-Val}. TFA
salt (0.040 g, 0.0457 mmol) was dissolved in DMF (2 mL).
Triethylamine (19.1 uL, 0.137 mmol) was added and after
stirring for 5 minutes 3-(N-(3-(2-(2-(3-((tert-butoxy)-
carbonylamino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)pro
panoic acid succinimide ester (0.0284 g, 0.0548 mmol)
was added. The reaction mixture was stirred under N2
for 48 h and then concentrated to an oil under high
vacuum. The oil was triturated with ethyl acetate, the
product filtered, washed with ethyl acetate, and dried
under high vacuum. The crude'~product was purified by
Preparative HPLC Method 1 to give 7.4 mg (140) of the
desired product as a lyophilized solid. ESMS: Calcd.
for C48H7gN11015, 1049.58; Found, 1050.5 [M+H]+1.
Analytical HPLC, Method 1B, Rt = 20.417 min, Purity =
100%.
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Part D. Preparation of cyclo{Arg-Gly-Asp-D-Tyr(3-(3-(N-
(3- (2- (2- (3-
(amino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)-
propanamido)propyl)-Val}
Cyclo{Arg-Gly-Asp-D-Tyr(3-(3-(N-(3-(2-(2-(3-((tert-
butoxy)-carbonylamino)propoxy)ethoxy)ethoxy)propyl)-
carbamoyl)-propanamido)propyl)-Val} (6.0 mg, 0.00515
mmol) was dissolved in methylene chloride (1 mL) and
trifluoroacetic acid (1 mL) was added. The solution
stirred for 2 h and then concentrated to an oil under
high vacuum. The oil was triturated with diethyl ether,
the product filtered, washed with diethyl ether, and
dried under high vacuum to give 6.0 mg (98%) of the
desired product. ESMS: Calcd. for C43H71N11~13~
949.52; Found, 950.6 [M+H]+1. Analytical HPLC, Method
1B, Rt = 14.821 min, Purity = 73%.
Part E. Preparation of cyclo{Arg-Gly-Asp-D-Tyr((N-[2-
[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-18-amino-14-aza-4,7,10-oxy-15-oxo-
octadecoyl)-3-aminopropyl)-Val}
Cyclo{Arg-Gly-Asp-D-Tyr(3-(3-(N-(3-(2-(2-(3-
(amino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)-
propanamido)propyl)-Val} (5.0 mg, 0.00424 mmol) was
dissolved in dimethylformamide (1 mL). Triethylamine
(1.8 uL, 0.0127 mmol) was added, and after stirring for
5 min 2-[[[5-[[(2,5-dioxo-1-pyrrolidinyl)oxy]-carbonyl]-
2-pyridinyl]hydrazono]methyl]-benzenesulfonic acid,
monosodium salt (2.2 mg, 0.00509 mmol) was added. The
reaction mixture was stirred for 24 h and then
concentrated to an oil under high vacuum. The oil was
purified by preparative HPLC Method 1 to give 2.2 mg
(38%) of the desired product as a lyophilized solid (TFA
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salt). ESMS: Calcd. for C56HgpN140175. 1252.6; Found,
1253.7 (M+H+). Analytical HPLC, Method 1B, Rt = =17.328
min, Purity = 100%.
Example 3
Synthesis of [2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
Glu(cyclo{D-Tyr(3-aminopropyl)-Val-Arg-Gly-Asp})-
cyclo{D-Tyr(3-aminopropyl)-Val-Arg-Gly-Asp}
NI'H O
TFA~H2N~ N~ N~O ~ ~ S03H O O
H NH H HN~'' - N ~ N~°~~ N NH2~TFA
I C02H NH ~ C~'~_ NH H H H
NH HN~i O ~ 2 ~ ~O
N O' - NH HN-s
i
O ~ ~ O O O O
NH
HN
~- NH
O O
Part A. Preparation of Boc-Glu(cyclo{D-Tyr(3
aminopropyl)-Val-Arg-Gly-Asp})-cyclo{D-Tyr(3
aminopropyl)-Val-Arg-Gly-Asp}
Cyclo{D-Tyr(3-aminopropyl)-Val-Arg-Gly-Asp} (0.040
g, 0.0457 mmol) was dissolved in dimethylformamide (2
mL). Triethylamine (19.1 ~L, 0.137 mmol) was added and
the reaction mixture was stirred for 5 minutes. Boc-
Glu(OSu)-OSu (0.0101 g, 0Ø229 mmol) was added and the
reaction mixture was stirred under N2 for 18 h. The
reaction mixture was then concentrated to an oil under
high vacuum. The oil was triturated with ethyl acetate.
The product was filtered, washed with ethyl acetate, and
dried under high vacuum to give 38.0 mg (55%) of the
desired product. ESMS: Calcd. for C6gH103N19020.
1505.76; Found, 1504.9 [M-H]-1. Analytical HPLC, Method
1B, Rt = 19.797 min, Purity = 73%.
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Part B. Preparation of Glu(cyclo{D-Tyr(3-aminopropyl)-
Val-Arg-Gly-Asp})-cyclo{D-Tyr(3-amiriopropyl)-Val-Arg-
Gly-Asp}. TFA salt
N''H O
TFA.H2N~ N~ N~'O O O
H NH H HN ~ ~'~~~ N NH2~TFA
O ~ C02 H
HOaC~r~. NH H H H
NH HN O O~NH H -f 0
N
o \ / ~ TFA - '
NH2 O \ ~ O
HN
~J--~ ~- N H
O O
Boc-Glu(cyclo{D-Tyr(3-aminopropyl)-Val-Arg-Gly
Asp})-cyclo{D-Tyr(3-aminopropyl)-Val-Arg-Gly-Asp} (0.035
g, 0.0232 mmol) was dissolved in methylene chloride (1
mL). Trifluoroacetic acid (1 mL) was added, and the
reaction mixture was stirred for 2 h, concentrated to an
oil under high vacuum and triturated with ether. The
product obtained was filtered, washed with diethyl
ether, and dried under high vacuum to give 30.7 mg (76%)
of the desired product. ESMS: Calcd. for C~3H95N19C18~
1405.71; Found, 1404.7 [M-H]-1. Analytical HPLC, Method
1B, Rt = 15.907 min, Purity = 77%.
Part C. Preparation of [2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
Glu(cyclo{D-Tyr(3-aminopropyl)-Val-Arg-Gly-Asp})-
cyclo{D-Tyr(3-aminopropyl)-Val-Arg-Gly-Asp}
To a solution of Glu(cyclo{D-Tyr(3-aminopropyl)-
Val-Arg-Gly-Asp})-cyclo{D-Tyr(3-aminopropyl)-Val-Arg-
Gly-Asp} (0.025 g, 0.0143 mmol) in dimethylformamide (2
mL) was added triethylamine (6.0 ~L, 0.0429 mmol) and
the reaction mixture was stirred for 5 min. 2-[[[5-
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[[(2,5-Dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid,
monosodium salt (0.0076 g, 0.0172 mmol) was added, and
the reaction mixture was stirred for 5 days, then
concentrated to an oil under high vacuum. The oil was
purified by Preparative HPLC Method 1 to give 12.0 mg
(43%) of the desired product as a lyophilized solid.
ESMS: Calcd. for C~6H104N220225~ 1708.7; Found, 1710.1
(M+H+). Analytical HPLC, Method 1B, Rt = 17.218 min,
Purity = 940.
Example 4
Synthesis of cyclo(Arg-Gly-Asp-D-Tyr-Lys([2-[[[5-
[carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid] ) }
T
H
Part A. Preparation of cyclo{Arg(Tos)-Gly-Asp(OBzl)-D-
Tyr(Bzl)-Lys(Cbz)}
The N-terminus Boc-protecting group of the peptide
sequence Boc-Asp(OBzl)-D-Tyr(Bzl)-Lys(Z)-Arg(Tos)-Gly-
oxime resin was removed using standard deprotection (250
TFA in CH2C12). After eight washes with DCM, the resin
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was treated with 10% DIEA/DCM (2 x 10 min.). The resin
was subsequently washed with DCM (x 5) and dried under
high vacuum. The resin (1.8711 g, 0.44 mmol/g) was then
suspended in DMF (15 mL). Glacial acetic acid (47.1 uL,
0.823 mmol) was added, and the reaction was heated at 60
~C for 72 h. The resin was filtered, and 'washed with
DMF (2 x 10 mL). The filtrate was concentrated to an
oil under high vacuum. The resulting oil was triturated
with ethyl acetate. The solid thus obtained was
filtered, washed with ethyl acetate, and dried under
high vacuum to give 653.7 mg of the desired product.
ESMS: Calcd. for C56H65N9012S~ 1087.45; Found,~1088.7
[M+H]+1. Analytical HPLC, Method 1A, Rt = 17.559 min,
Purity = 82%.
Part B. Preparation of cyclo{Arg-Gly-Asp-D-Tyr-Lys}
TFA~H
~H
Cyclo{Arg(Tos)-Gly-Asp(OBzl)-D-Tyr(Bzl)-Lys(Cbz)}
(0.200 g, 0.184 mmol) was dissolved in trifluoroacetic
acid (0.6 mL) and cooled to -10 ~C.
Trifluoromethanesulfonic acid (0.5 mL) was added
dropwise, maintaining the temperature at -10 ~C.
Anisole (0.1 mL) was added and the reaction mixture was
stirred at -10 ~C for 3 h. Diethyl ether was added, the
reaction was cooled to -50 ~C, and stirred for 1 h. The
crude product was filtered, washed with diethyl ether,
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and dried under high vacuum. The crude product was
purified by Preparative HPLC Method 1, to give 15.2 mg
(10%) of the desired product as a lyophilized solid.
HRMS: Calcd. for C27H41N908 +H, 620.3156; Found,
620.3145. Analytical HPLC, Method 1B, Rt = 8.179 min,
Purity = 1000.
Part C. Preparation of cyclo{Arg-Gly-Asp-D-Tyr-Lys([2-
[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid])}
Cyclo{Arg-Gly-Asp-D-Tyr-Lys} TFA salt (0.010 g,
0.0118 mmol) was dissolved in DMF (1 mL). Triethylamine
(5,0 ~.L, 0.0354 mmol) was added, and after stirring for
5 min 2-[[[5-[[(2,5-Dioxo-1-pyrrolidinyl)oxy]carbonyl]-
2-pyridinyl]hydrazono]-methyl]-benzenesulfonic acid,
monosodium salt (0.0062 g, 0.0142 mmol) was added. The
reaction mixture was stirred for 20 h and then
concentrated to an oil under high vacuum. The oil was
purified by Preparative HPLC Method 1 to give 6.2 mg
(46%) of the desired product as a lyophilized solid.
HRMS: Calcd. for C4pH50N12~125 + H, 923.3470; Found,
923.3486. Analytical HPLC, Method 1B, Rt = 11.954 min,
Purity = 1000.
Example 5
Synthesis of cyclo{Arg-Gly-Asp-D-Phe-Lys([2-[[[5
[carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid] ) }
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H
Part A. Preparation of cyclo{Arg(Tos)-Gly-Asp(OBzl)-D-
Phe-Lys(Cbz)}
The N-terminus Boc- protecting group of the peptide
sequence Boc-Asp(OBzl)-D-Phe-Lys(Z)-Arg(Tos)-Gly-Oxime
resin was removed using standard deprotection (25o TFA
in CH2C12). After eight washes with DCM, the resin was
treated with 10% DIEA/DCM (2 x 10 min.). The resin was
subsequently washed with DCM (x 5) and dried under high
vacuum. The resin (1.7053 g, 0.44 mmol/g) was then
suspended~in dimethylformamide (15 mL). Glacial acetic
acid (43.0 ~.L, 0.750 mmol) was added, and the reaction
was heated to 60 ~C for 72 h. The resin was filtered,
and washed with DMF (2 x 10 mL). The filtrate was
concentrated to an oil under high vacuum. The resulting
oil was triturated with ethyl acetate. The solid thus
obtained was filtered, washed with ethyl acetate, and
dried under high vacuum to give 510.3 mg of the desired
product. ESMS: Calcd. for C49H59N9011S~ 981.40; Found,
982.6 [M+H]+1. Analytical HPLC, Method 1A, Rt = 15.574
min, Purity = 89%.
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Part B. Preparation of cyclo{Arg-Gly-Asp-D-Phe-Lys}
TFA
Cyclo{Arg(Tos)-Gly-Asp(OBzl)-D-Phe-Lys(Cbz)} (0.200
g, 0.204 mmol) was dissolved in trifluoracetic acid (0.6
mL) and cooled to -10 ~C. Trifluoromethanesulfonic acid
(0.5 mL) was added dropwise, maintaining the temperature
at -10 ~C. Anisole (0.1 mL) was added and the reaction
was stirred at -10 ~C for 3 h. Diethyl ether was added,
the reaction was cooled to -50 ~C, and stirred for 1 h.
The crude product was filtered, washed with diethyl
ether, dried under high vacuum and purified by
Preparative HPLC Method 1, to give 121.1 mg (71%) of the
desired product as a lyophilized solid. HRMS: Calcd.
for C27H41N907 +H, 604.3207; Found, 604.3206.
Analytical HPLC, Method 1B, Rt = 11.197 min, Purity =
100%.
Part C. Preparation of cyclo{Arg-Gly-Asp-D-Phe-Lys([2-
[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid])}
Cyclo{Arg-Gly-Asp-D-Phe-Lys} TFA salt (0.040 g,
0.0481 mmol) was dissolved in DMF (2 mL). Triethylamine
(20.1 uL, 0.144 mmol) was added, and after 5 min of
stirring 2-[[[5-[[(2,5-dioxo-1-
pyrrolidinyl)oxy]carbonyl]-2-pyridinyl]hydrazono]-
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methyl]-benzenesulfonic acid, monosodium salt (0.0254
g, 0.0577 mmol) was added. The reaction mixture was
stirred for 20 h and then concentrated to an oil under
high vacuum. The oil was purified by Preparative HPLC
Method 1 to give 38.2 mg (78%) of the desired product as
a lyophilized solid. HRMS: Calcd. for C4pH5pN120115 +
H, 907.3521; Found, 907.3534. Analytical HPLC, Method
1B, Rt = 14.122 min, Purity = 910.
Example 6
Synthesis of [2-[[[5-[carbonyl]-2
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]
Glu(cyclo{Lys-Arg-Gly-Asp-D-Phe})-cyclo{Lys-Arg-Gly-Asp
D-Phe}
O N'IH NI'H O
O~ N~'~~~ N~ NH2-TFA TFA.H2N~ N~ N~O
HO~yNH H OHN"O H O O H O NHO H HN~OH
f0 O,~( N N J~'°~ N~r~~ ~ N'~~~.,~ N~ N O j[0
H H H HN O H H . H
N
HN, N
H03
Part A. Preparation of Boc-Glu(OSu)-OSu
NH-Boc
Su0 OSu
O O
To a solution of Boc-Glu-OH (8.0 g, 32.25 mmol), N-
hydroxysuccinimide (8.94 g, 77.64 mmol), and DMF (120
mL) was added 1-(3-dimethylaminopropyl)-3-
ethylcarbodimide (14.88 g, 77.64 mmol). The reaction
mixture was stirred at room temperature for 48 h. The
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mixture was concentrated under high vacuum and the
residue was brought up in 0.1 N HCl and extracted with
ethyl acetate (3x). The combined organic extracts were
washed with water, saturated sodium bicarbonate and then
saturated sodium chloride, dried over MgS04, and
filtered. The filtrate was concentrated in vacuo and
purified via reverse-phase HPLC (Vydac C18 column, 18 to
90 o acetonitrile gradient containing 0.1% TFA, Rt =
9.413 min) to afford 8.5 g (60%) of the desired product
as a white powder. 1H NMR (CDC13): 2.98-2.70 (m, 1:1H),
2.65-2.25 (m, 2H), 1.55-1.40 (s, 9H); ESMS: Calculated
for C18H23N3010. 441.1383 Found 459.2 [M+NH4]+1.
Part B. Preparation of Boc-Glu(cyclo{Lys-Arg-Gly-Asp-D-
Phe})-cyclo{Lys-Arg-Gly-Asp-D-Phe}
To a solution of cyclo(Lys-Arg-Gly-Asp-D-Phe)
(0.050 g, 0.0601 mmol) in dimethylformamide (2 mL) was
added triethylamine (25.1 ~L, 0.183 mmol). After
stirring for 5 minutes Boc-Glu(OSu)-OSu (0.0133 g,
0.0301 mmol) was added. The reaction mixture was
stirred under N2 for 20 h, then concentrated to an oil
under high vacuum and triturated with ethyl acetate.
The product thus obtained was filtered, washed with
ethyl acetate, and dried under high vacuum to give 43.7
mg (44%) of the desired product. ESMS: Calcd. for
C64H95N19018~ 1417.71; Found, 1418.8 [M+H]+1.
Analytical HPLC, Method 1B, Rt = 19.524 min, Purity =
73%.
Part C. Preparation of Glu(cyclo{Lys-Arg-Gly-Asp-D-
Phe})-cyclo{Lys-Arg-Gly-Asp-D-Phe} TFA salt.
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O NIIH NIIH O
O~ N~'~~~ N~ NH2~TFA TFA~H2N~ N~ N~O
HO~i~NH H OHN~O H O O H O NHO H HN'~JOH
(O 0,,~~( N NJ ~/~/~ N~''~~~ N~~~\~~N~ N~O ~[O
H . H H INH2.TFA H H . H
To a solution of Boc-Glu(cyclo{Lys-Arg-Gly-Asp-D-
Phe})-cyclo{Lys-Arg-Gly-Asp-D-Phe} (0.040 g, 0.0243
mmol) in methylene chloride (1 mL) was added
trifluoroacetic acid (1 mL). The reaction mixture was
stirred for 2 h, concentrated to an oil under high
vacuum and triturated with diethyl ether. The product
was filtered, washed with diethyl ether, and dried under
high vacuum to give 39.9 mg (100%) of the desired
product. ESMS: Calcd. for C59Hg7N19016. 1317.66;
Found, 1318.9 [M+H]+1. Analytical HPLC, Method 1B, Rt =
15.410 min, Purity = 73%.
Part D. Preparation of [2-[[[5-[carbonyl]-2-pyridinyl]-
hydrazono]methyl]-benzenesulfonic acid]-Glu(cyclo{Lys-
Arg-Gly-Asp-D-Phe})-cyclo{Lys-Arg-Gly-Asp-D-Phe}
To a solution of Glu(cyclo{Lys-Arg-Gly-Asp-D-Phe})-
cyclo{Lys-Arg-Gly-Asp-D-Phe} (0.030 g, 0.0183 mmol) in
dimethylformamide (3 mL) was added triethylamine (7.6
uL, 0.0549 mmol) and the reaction mixture was stirred
for 5 min. 2-[[[5-[[(2,5-Dioxo-1-
pyrrolidinyl)oxy]carbonyl]-2-pyridinyl]-
hydrazono]methyl]-benzenesulfonic acid, monosodium salt
(0.0096 g, 0.0220 mmol) was added, and the reaction
mixture was stirred for 18 h, then concentrated to an
oil under high vacuum. The oil was purified by
Preparative HPLC Method 1 to give 11.0 mg (32%) of the
desired product as a lyophilized solid. ESMS: Calcd.
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for C72H96N220205, 1620.7; Found, 1620.1 (M-H+).
Analytical HPLC, Method 1B, Rt = =16.753 min, Purity =
91%.
Example 7
Synthesis of [2-[[[5-[carbonyl]-2
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-Phe
Glu(cyclo{Lys-Arg-Gly-Asp-D-Phe})-cyclo{Lys-Arg-Gly-Asp-
D-Phe}
15
O INIH IN'H O
O~ H~~,o~ H~ NH2 H2Nx H~ H~O
HOOCH''' NH HN NH HN COOH
~O O O O
O NH HN ~.,,~ N~ N~ NH jN O
H H H J)--'a
..
O O
~NH
I / ' H03S
I/
N NH- N-
Part A. Preparation of Phe-Glu(cyclo{Lys-Arg-Gly-Asp-D-
Phe})-CyClO{Lys-Arg-Gly-Asp-D-Phe}
O 'NIH NIIH O
O~ H~~~o~ H~ NH2 H2N~ H'~ H~O
HOOC~''~ NH H O O O O NH HN'~COOH
O NH H N ..,, NH H N O
~ H H ~,
O H O O
~ vNH2
A solution of Glu(cyclo{Lys-Arg-Gly-Asp-D-Phe})-
cyclo{Lys-Arg-Gly-Asp-D-Phe} (23.4 mg, 0.014 mmol) and
triethylamine (7.8 ~L, 0.56 mmol) in DMF (2 mL) was
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stirred for 5 min. To this was added Boc-Phe-OSu (5.1
mg, 0.014 mmol) and the reaction mixture was stirred
overnight' at room temperature under nitrogen. DMF was
removed in vacuo, and the resulting residue was
dissolved in TFA (1.5 mL) and methylene chloride (1.5
mL). The solution was stirred for 2 h and concentrated
in vacuo to provide 31 mg of the desired product as the
TFA salt. ESMS: Calcd. for C6gHg6N20~17~ 1464.7;
Found, 1465.6 (M+H)+1. Analytical HPLC, Method 1B, Rt =
=15.48 min, Purity = 95%.
Part B. Preparation of [2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-Phe-
Glu(cyclo{Lys-Arg-Gly-Asp-D-Phe})-cyclo{Lys-Arg-Gly-Asp-
D-Phe}
To a solution of Phe-Glu(cyclo{Lys-Arg-Gly-Asp-D-
~Phe})-cyclo{Lys-Arg-Gly-Asp-D-Phe} (0.030 g, 0.016 mmol)
in dimethylformamide (2 mL) was added triethylamine (9
~L, 0.064 mmol) and the reaction mixture was stirred for
5 min. 2-[[[5-[[(2,5-Dioxo-1-
pyrrolidinyl)oxy]carbonyl]-2-pyridinyl]-
hydrazono]methyl]-benzenesulfonic acid, monosodium salt
(0.0099 g, 0.0220 mmol) was added, and the reaction
mixture was stirred for 18 h, then concentrated under
high vacuum. The residue was purified by preparative
RP-HPLC Method 1 to give 7 mg (22%) of the desired
product as a lyophilized solid (TFA salt). ESMS:
Calcd. for Cg1H105N23~215~ 1767.8; Found, 1768.8 (M-H+).
Analytical HPLC, Method 1B, Rt = - 17.68 min, Purity =
99%.
Example 8
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Synthesis of cyclo{Arg-Gly-Asp-D-Nal-Lys([2-[[[5
[carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid] ) }
NH O
H2N~H H~O
NH HN COOH
O O
S03H _ NH HN O
I~ H
N-HN
Part A. Preparation of cyclo{Arg(Mtr)-Gly-Asp(OtBu)-D-
Nal-Lys(Boc)}
N-Mtr O
H2N~H H~O
NH HN
~COOt-B a
O~
~nm HN O
Boc-NH~ ~ v
O
l0
The peptide Asp(OtBu)-D-Nal-Lys(Boc)-Arg(Mtr)-Gly
was obtained by automated solid phase peptide synthesis
using Fmoc chemistry. A 100 mL round bottom flask was
charged with HBTU (349 mg, 0.92 mmol) and DMF (10 mL).
The solution was stirred at 60 ~C for 5 min. To this a
solution of Asp(OtBu)-D-Nal-Lys(Boc)Arg(Mtr)-Gly (0.64
g) and Hunig's base (0.34 mL, 1.97 mmol.) in DMF (10 mL)
was added and the solution stirred at 60 ~C for 4 h
under nitrogen. The solvent was then removed in vacuo
and the residue was triturated with ethyl acetate. The
solids were filtered and washed with ethyl acetate (3 x
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mL) and dried in vacuo to give the desired product
(520 mg, 86%).
ESMS: Calcd. for C5pH71N9012S, 1021.5; Found, 1022.5
[M+H]+1. Analytical HPLC, Method 1A, Rt = 15.91 min
5 (purity 99%).
Part B. Preparation of cyclo{Arg-Gly-Asp-D-Nal-Lys} bis
TFA salt
NH O
H2N~H H~O
NH HN
~COOH
O
NH HN O
H 2N
O
A solution of cyclo{Arg(Mtr)-Gly-Asp(OtBu)-D-Nal-
Lys(Boc)} (500 mg, 0.49 mmol), TFA (7 mL),
triisopropylsilane (0.25 mL) and water (0.25 mL) was
stirred at room temperature under nitrogen for 18 h.
The solvents were removed in vacuo (over 3 h) and the
residue triturated with diethyl ether to give the
desired product as the TFA salt (426 mg, 98%). ESMS:
Calcd. for C31H43N9~7~ 653.3; Found, 654.3 [M+H]+1.
Analytical HPLC, Method 1B, Rt = 13.30 min, Purity =
97%.
Part C. Preparation of cyclo{Arg-Gly-Asp-D-Nal-Lys([2-
[((5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid])}
Cyclo{Arg-Gly-Asp-D-Nal-Lys} TFA salt (0.056 g,
0.064 mmol) was dissolved in DMF (2 mL). Triethylamine
(27 ~L, 0.19 mmol) was added, and after 5 min of
stirring 2-[[[5-[[(2,5-dioxo-1-
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pyrrolidinyl)oxy]carbonyl]-2-pyridinyl]-hydrazono]-
methyl]-benzenesulfonic acid, monosodium salt (0.039 g,
0.089 mmol) was added. The reaction mixture was stirred
overnight, under nitrogen, and then concentrated to an
oil under high vacuum. The oil was purified by
Preparative HPLC Method 1 to give 49.3 mg (720) of the
desired product as a lyophilized solid (TFA salt).
ESMS: Calcd. for C44H52N120115~ 956.4; Found, 957.5
[M+H]+1. Analytical HPLC, Method 1B, Rt = 16.19 min,
Purity = 99%.
Example 9
Synthesis of [2-[[[5-[carbonyl]-2-pyridinyl]
hydrazono]methyl]-benzenesulfonic acid]-Glu(cyclo{Lys
Arg-Gly-Asp-D-Nal})-cyclo{Lys-Arg-Gly-Asp-D-Nal}
NH NH O
~NH2 H2N~H H
NH HN
~COOH
O O O
NH HN
H NH H
O
y
N
S03H
NH-N
Part A. Preparation of Boc-Glu(cyclo{Lys-Arg-Gly-Asp-D-
Nal})-cyclo{Lys-Arg-Gly-Asp-D-Nal}
To a solution of cyclo{Lys-Arg-Gly-Asp-D-Nal}
(0.052 g, 0.059 mmol) in dimethylformamide (2 mL) was
added triethylamine (25 ~L). After stirring for 5
minutes Boc-Glu(OSu)-OSu (0.013 g, 0.029 mmol) was
added. The reaction mixture was stirred under N2 for 20
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h, then concentrated to an oil under high vacuum and
triturated with ethyl acetate. The product thus
obtained was filtered, washed with ethyl acetate, and
dried under high vacuum to give 35.2 mg of the desired
product in crude form. ESMS: Calcd. for C72H99N19O18~
1517.7; Found, 760.1 [M+2H]+2. Analytical HPLC, Method
1B, Rt = 21.07 min (65%).
Part B. Preparation of Glu(cyclo{Lys-Arg-Gly-Asp-D-
Nal})-cyclo{Lys-Arg-Gly-Asp-D-Nal}
O NH NH O
O~H~,~''~H~NH2 H2N~H H
HOOC~h'' NH HN NH HN''
~ ~O O O ~O
i'NH HN
O HN~ NH
,~H H
.O NH2 O
To a solution of the crude Boc-Glu(cyclo{Lys-Arg-
Gly-Asp-D-Nal})-CyClo{Lys-Arg-Gly-Asp-D-Nal} (35.2 mg)
in methylene chloride (1.5 mL) was added trifluoroacetic
acid (1.5 mL). The reaction mixture was stirred for 2
h, concentrated to an oil under high vacuum and
triturated with diethyl ether. The product was
filtered, washed with diethyl ether, and dried under
high vacuum to give 34.9 mg of the crude desired product
(TFA salt). ESMS: Calcd. for C67H91N1g016, 1417.69;
Found, 1418.7 [M+H]+1. Analytical HPLC, Method 1B, Rt =
19.1 min, Purity = 620.
Part C. Preparation of [2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
Glu(cyclo{Lys-Arg-Gly-Asp-D-Nal})-cyclo{Lys-Arg-Gly-Asp-
D-Nal}
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To a solution of Glu(cyclo{Lys-Arg-Gly-Asp-D-Nal})-
cyclo{Lys-Arg-Gly-Asp-D-Nal} (34.9 mg) in
dimethylformamide (2 mL) was added triethylamine (10 ~.~.L,
0.074 mmol) and the reaction mixture was stirred for 5
min. 2-[[[5-[[(2,5-Dioxo-1-pyrrolidinyl)oxy]carbonyl]-
2-pyridinyl]-hydrazono]methyl]-benzenesulfonic acid,
monosodium salt (15.2 mg, 0.0344 mmol) was added, and
the reaction mixture was stirred for 18 h, then
concentrated to an oil under high vacuum. The oil was
purified by preparative RP-HPLC Method 1 to give 3 mg of
the desired product (TFA salt). ESMS: Calcd. for
C80H100N220205. 1720.7; Found, 1722.6 (M+H)+1.
Analytical HPLC, Method 1B, Rt = =19.78 min, Purity =
92 0 .
Example 10
Synthesis of cyclo{Arg-Gly-Asp-Lys([2-[[[5-[carbonyl]-2
pyridinyl]hydrazono]methyl]-benzenesulfonic acid])-D
Val}
NH O
TFA~H2N~H H~O
NH HN OH
O
NH HN O
O O
N
H
N- N
H S03H
Part A. Preparation of cyclo{Arg(Tos)-Gly-Asp(OBzl)-
Lys(Cbz)-D-Val}
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The N-terminus Boc- protecting group of the peptide
sequence.Boc-Asp(OBzl)-Lys(Z)-D-Val-Arg(Tos)-Gly-Oxime
resin was removed using standard deprotection (25o TFA
in CH2C12). After eight washes with DCM, the resin was
treated with 10% DIEA/DCM (2 x 10 min.). The resin was
subsequently washed with DCM (x 5) and dried under high
vacuum. The resin (1.3229 g, 0.44 mmol/g) was then
suspended in dimethylformamide (10 mL). Glacial acetic
acid (33.3 ~.zL,Ø582 mmol) was added, and the reaction
was heated at 65 ~C for 72 h. The resin was filtered,
and washed with DMF (2 x 10 mL). The filtrate was
concentrated to an oil under high vacuum. The resulting
oil was triturated with ethyl acetate. The solid thus
obtained was filtered, washed with ethyl acetate, dried
under high vacuum, then purified by Preparative HPLC
Method 2 to give 93.0 mg of the desired product as a
lyophilized solid. ESMS: Calcd. for C45H59N9011S~
933.41; Found, 934.5 [M+H]+1. Analytical HPLC, Method
1A, Rt = 14.078 min, Purity = 85%.
Preparative HPLC Method 2
Instrument: Rainin Rabbit; Dynamax software
Column: Vydac C-18 (21.2 mm x 25 cm)
Detector: Knauer VWM
Flow Rate: l5ml/min
Column Temp: RT
Mobile Phase: A: 0.1% TFA in H20
B: 0.1%TFA in ACN/H20 (9:1)
Gradient: Time (min) %A %B
0 80 20
20 0 100
30 0 100
31 80 20
Part B. Preparation of cyclo{Arg-Gly-Asp-Lys-D-Val}
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TFA~H
T FA
Cyclo{Arg(Tos)-Gly-Asp(OBzl)-Lys(Cbz)-D-Val} (0.080
g, 0.0856 mmol) was dissolved in trifluoroacetic acid
(0.6 mL) and cooled to -10 ~C. Trifluoromethanesulfonic
acid (0.5 mL) was added dropwise, maintaining the
temperature at -10 ~C. Anisole (0.1 mL) was added and
the reaction mixture was stirred at -10 ~C for 3 h.
Diethyl ether was added, the reaction mixture cooled to
-50 ~C and stirred for 30 minx. The crude product
obtained was filtered, washed with ether, dried under
high vacuum and purified by Preparative HPLC Method 1,
to give 44.2 mg (66%) of the desired product as a
lyophilized solid. ESMS: Calcd. for C23H41N907~
555.31; Found, 556.3 [M+H]+1. Analytical HPLC, Method
1B, Rt = 8.959 min, Purity = 920.
Part C. Preparation of cyclo{Arg-Gly-Asp-Lys([2-[[[5-
[carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid])-D-Val}
To a solution of cyclo{Arg-Gly-Asp-Lys-D-Val}
(0.036 g, 0.0459 mmol) in dimethylformamide (3 mL) was
added triethylamine (19.2 uL, 0.0138 mmol) and stirred
for 5 min. Methyl sulfoxide was added (0.7 mL) followed
by 2-[[[5-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-
pyridinyl]-hydrazono]methyl]-benzenesulfonic acid,
monosodium salt (0.0243 g, 0.0551 mmol) and the
reaction mixture stirred for 20 h. The reaction mixture
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was concentrated to an oil under high vacuum and
purified by Preparative HPLC Method 1 to give 13.9 mg
(31%) of the desired product as a lyophilized solid.
HRMS: Calcd. for C36H5pN120115 +H, 859.3443; Found,
859.3503. Analytical HPLC, Method 1B, Rt = 13.479 min,
Purity = 92%.
Example 11
Synthesis of [2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
Glu(cyclo{Lys-D-Val-Arg-Gly-Asp})-cyclo{Lys-D-Val-Arg-
Gly-Asp}
0 0
NH O~ N~COOH HOOC~~~'~ N~O
H N~ N NH H HN NH H HN ~~ N NH
H/~~ O O O O~ ~ H
O NH HN__~~ N N~,~~ NH HN O
H~ H
NH
O \
S03H
N NH- N
Part A. Preparation of Boc-Glu(cyclo{Lys-D-Val-Arg-Gly-
Asp})-cyclo{Lys-D-Val-Arg-Gly-Asp}
To a solution of cyclo{Lys-D-Val-Arg-Gly-Asp}
(0.400 g, 0.51 mmol) in dimethylformamide (7 mL) was
added triethylamine (0.21 mL, 1.53 mmol). After
stirring for 5 minutes Boc-Glu(OSu)-OSu (115 mg, 0.26
mmol) was added. The reaction mixture was stirred under
N2 for 20 h, then concentrated to an oil. The product
thus obtained was partially purified by preparative RP-
HPLC to give 124 mg of product. ESMS: Calcd. for
C56H95N19018~ 1321.71; Found, 1322.6 [M+H]+1.
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Part B. Preparation of Glu(cyclo{Lys-D-Val-Arg-Gly-
Asp})-cyclo{Lys-D-Val-Arg-Gly-Asp}
0 0
NIfH O~ N~COOH HOOC~~'~ N~O NH
H N~ N NH H HN NH H HN n~ N~ NH
H/~~ O O O O~ ~ H
O NH HN--~~ N N~,v H HN O
H~ H
~ O NHz O
To a solution of the impure Boc-Glu(cyclo{Lys-D-
Val-Arg-Gly-Asp})-cyclo{Lys-D-Val-Arg-Gly-Asp} (0.124 g)
in methylene chloride (5 mL) was added trifluoroacetic
acid (5 mL). The reaction mixture was stirred for 2 h,
concentrated to an oil under high vacuum and triturated
with diethyl ether. The product was filtered, washed
with diethyl ether, and dried under high vacuum to give
16.2 mg of the desired product after RP-HPLC (TFA salt).
ESMS: Calcd. for C5lHg~N19016, 1221.66; Found, 1222.6
[M+H]+1. Analytical HPLC, Method 1B, Rt = 11.43 min,
Purity = 93%.
Part C. Preparation of [2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
Glu(cyclo{Lys-D-Val-Arg-Gly-Asp})-cyclo{Lys-D-Val-Arg-
Gly-Asp}
To a solution of Glu(cyclo{Lys-D-Val-Arg-Gly-Asp})-
cyclo{Lys-D-Val-Arg-Gly-Asp} (0.016 g, 0.01 mmol) in
dimethylformamide (2 mL) was added triethylamine (4.2
~L) and the reaction mixture was stirred for 5 min. 2-
[[[5-[[(2,5-Dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-
pyridinyl]-hydrazono]methyl]-benzenesulfonic acid,
monosodium salt (0.0063 g, 0.014 mmol) was added, and
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the reaction mixture was stirred for 18 h, then
concentrated to an oil under high vacuum. The residue
was purified by preparative RP-HPLC Method 1 to give the
desired product (TFA salt). ESMS: Calcd. for
C64H96N220205, 1524.7; Found, 1525.7 (M+H)+1.
Analytical HPLC, Method 1B, Rt = =13.20 min, Purity =
99%.
Example 12
Synthesis of {cyclo(Arg-D-Val-D-Tyr(N-[2-[[[5-
[carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid]-3-aminopropyl)-D-Asp-Gly}
CF3C021-'
H
O
H
S03H
N N-
~OH
Part A: Preparation of cyclo{Arg(Tos)-D-Val-D-Tyr(N-
Cbz-3-aminopropyl)-D-Asp(OBzl)-'Gly}
The N-terminus Boc-protecting group of the peptide
sequence Boc-Arg(Tos)-D-Val-D-Tyr(N-Cbz-aminopropyl)-D-
Asp(OBzl)-Gly-Oxime resin was removed using standard
deprotection (50o TFA in CH2C12). After washing with
DCM (8x), the resin was neutralized with 10% DIEA/DCM (2
x 10 min). The resin was washed with DCM (5x) and dried
under high vacuum overnight. The resin (1.08 g, 0.36
mmol/g) was then suspended in N,N-dimethylformamide (12
mL). Glacial acetic acid (67 mL, 1.16 mmol) was added
and the reaction mixture was heated to 55 °C for 72 h.
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The resin was filtered and washed with DMF (3 x 10 mL).
The filtrate was concentrated under high vacuum to give
an oil. The resulting oil was triturated with ethyl
acetate. The solid obtained was purified by reverse-
s phase HPLC (Vydac C18 column, 18 to 90o acetonitrile
gradient containing 0.1% TFA, Rt=15.243 min) to afford
101 mg of a white powdered product (30%). ESMS:
Calculated for C44H57N9012S, 935.3847 Found 936.5
[M+H]+1.
Part B: Preparation of cyclo{Arg-D-Val-D-Tyr(3-
aminopropyl)-D-Asp-Gly}
CF3C02H
H 2N
O~NH2 . CF3C02H
OOH
The protected cyclic peptide cyclo{Arg(Tos)-D-Val-
D-Tyr(N-Cbz-3-aminopropyl)-D-Asp(OBzl)-Gly} (90 mg,
0.0961 mmol) was dissolved in trifluoroacetic acid (0.95
mL) and cooled to -10 °C in a dry ice/acetone bath. To
this solution was added trifluoromethanesulfonic acid
(0.1.16 mmol), followed by anisole (190 mL). The
reaction mixture was stirred at -16 °C for 3 h. The dry
ice/acetone bath was then cooled to -35 °C and cold
ether (40 mL) was added to the solution. The mixture
was stirred for 30 min at -35 oC, then cooled to -50 °C
and stirred for another 30 min. The crude product was
filtered, redissolved in water/acetonitrile (1/1),
lyophilized, and purified by reverse-phase HPLC (Vydac
C18 Column, 1.8 to 90% acetonitrile gradient containing
0.1o TFA, Rt=13.383 min) to generate 17 mg of the title
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product (270). ESMS: Calculated for C29H45N9~8~
647.3391 Found 648.2 [M+H]+1.
Part C: Preparation of {cyclo(Arg-D-Val-D-Tyr(N-[2-
[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-D-Asp-Gly}
A solution of cyclo{Arg-D-Val-D-Tyr(3-aminopropyl)-
D-Asp-Gly} (14 mg, 0.0216 mmol) in N,N-dimethylformamide
(2 mL) was added triethylamine (15 mL, 0.108 mmol) and
stirred at room temperature for 10 min. 2-[[[5-[[(2,5-
Dioxo-1-pyrrolidinyl)oxy]carbonyl-2-pyridinyl]-
hydrazono]methyl-benzenesulfonic acid, monosodium salt
(11 mg, 0.0260 mmol) was added, and the mixture was
stirred for 18 h. The mixture was concentrated under
high vacumm and the residue was purified by reverse-
phase HPLC (Vydac C18 Column, 1.8 to 90% acetonitrile
gradient containing 0.1% TFA, Rt=16.264 min) to afford
10 mg of a white powdered product (49%). ESMS:
Calculated for C42H54N120125, 950.3705 Found 951.3
[M+H]+1.
Example 13
Synthesis of cyclo{D-Lys([2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid])-D-
Phe-D-Asp-Gly-Arg}
~I-i O ~ H I \ 03H
H2N N N~ ~ N-N
H H H HN . \ H I
O V ~ I /
N_H Hrl O
O
OH
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Part A: Preparation of cyclo{D-Lys(Cbz)-D-Phe-D-
Asp(OBzl)-Gly-Arg(Tos)}
The N-terminus Boc- protecting group of the peptide
sequence Boc-Arg(Tos)-D-Lys(Cbz)-D-Phe-D-Asp(OBzl)-Gly-
Oxime resin was removed using standard deprotection (250
TFA in CH2C12). After eight washes with DCM, the resin
was treated with 10% DIEA/DCM (2 x 10 min.). The resin
was subsequently washed with DCM (x 5) and dried under
high vacuum. The resin (1.93 g, 0.44 mmol/g) was then
suspended in dimethylformamide (15 mL). Glacial acetic
acid (77 ~L) was added, and the reaction was heated to
60 ~C for 72 h. The resin was filtered, and washed with
DMF (2 x 10 mL). The filtrate was concentrated to an
oil under high vacuum. The resulting oil was triturated
with ethyl acetate. The solid thus obtained was
filtered, washed with ethyl acetate, and dried under
high vacuum to give the desired product which was then
purified by preparative RP-HPLC (yield = 252 mg). ESMS:
Calcd. for C49H59N9011S~ 981.40; Found, 982.3 [M+H]+1.
Analytical HPLC, Method 1A, Rt = 14.577 min.
Part B: Preparation of cyclo{D-Lys-D-Phe-D-Asp-Gly-Arg}
TFA salt
~H O
H2N [~I H H .
H
O V ~ I /
N~ O
OH
Cyclo{D-Lys(Cbz)-D-Phe-D-Asp(OBzl)-Gly-Arg(Tos)}
(0.152 g, 0.155 mmol) was dissolved in trifluoracetic
acid (1.55 mL) and cooled to -16 ~C.
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Trifluoromethanesulfonic acid (1.86 mL) was added
dropwise, maintaining the temperature at -16 ~C.
Anisole (0.31 mL) was added and the reaction was stirred
at -16 ~C for 3 h. Diethyl ether was added, the reaction
was cooled to -35 ~C, and stirred for 20 min. The crude
product was filtered, washed with diethyl ether, dried
under high vacuum and purified by Preparative HPLC
Method 1 , to give 69 mg (~53%) of the desired product
as a lyophilized solid (TFA salt). ESMS: Calcd. for
C27H41N907 +H, 604.3207; Found, 604.4. Analytical HPLC,
Method 1B, Rt = 10.35 min, Purity = 93%.
Part C: Preparation of cyclo{D-Lys([2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid])-D-
Phe-D-Asp-Gly-Arg} TFA salt
Cyclo{D-Lys-D-Phe-D-Asp-Gly-Arg} TFA salt (0.056 g,
0.0673 mmol) was dissolved in DMF (2 mL). Triethylamine
(28 ~L, 0.202 mmol) was added, and after 5 min of
stirring 2-[[[5-[[(2,5-dioxo-1-
pyrrolidinyl)oxy]carbonyl]-2-pyridinyl]hydrazono]-
methyl]-benzenesulfonic acid, monosodium salt (0.029 g,
0.0673 mmol) was added. The reaction mixture was
stirred for 70 h and then concentrated to an oil under
high vacuum. The oil was purified by preparative HPLC
Method 1 to give 14 mg (78%) of the desired product as a
lyophilized solid (TFA salt). ESMS: Calcd. for
C40H50N12~115 + H, 907.3521; Found, 907.3. Analytical
HPLC, Method 1B, Rt = 14.17 min, Purity = 99%.
Example 14
Synthesis of [2-[[[5-[carbonyl]-2
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]
Glu(cyclo{D-Lys-D-Phe-D-Asp-Gly-Arg})-cyclo{D-Lys-D-Phe
D-Asp-Gly-Arg}
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N-HN N
O
S03H ~ O
OHN N O NH
H
HN O H~,aa~H~NH2
NH HN
~ O
NH O ~ Oi 'NH
'I HN
H2N~N N~O
H H HOOC~O
NH HN ~.~
O
NH HN O
O~ '=COOH
Part A. Preparation of Boc-Glu(cyclo{D-Lys-D-Phe-D-Asp-
Gly-Arg})-cyclo{D-Lys-D-Phe-D-Asp-Gly-Arg}
To a solution of cyclo(D-Lys-D-Phe-D-Asp-Gly-Arg)
(0.190 g, 0.228 mmol) in dimethylformamide (5 mL) was
added triethylamine (95 ~.L, 0.684 mmol). After stirring
for 5 minutes Boc-Glu(OSu)-OSu (0.050 g, 0.114 mmol) was
added. The reaction mixture was stirred under N2 for 20
h, then concentrated to an oil under high vacuum and
triturated with ethyl acetate. The product thus
obtained was filtered, washed with ethyl acetate, and
dried under high vacuum to give 172 mg of the desired
product in crude form. ESMS: Calcd. for C64H95N19018~
1417.71; Found, 1418.7 [M+H]+1. Analytical HPLC, Method
1B, Rt = 16.8 min.
Part B. Preparation of Glu(cyclo{D-Lys-D-Phe-D-Asp-Gly-
Arg})-cyclo{D-Lys-D-Phe-D-Asp-Gly-Arg}
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O
H 2N..
O ~'' N O NH
H II
HN NO H~,Ja~H~NH2
NH HN
~ O
NH O ~ O-' -NH
II HN
H2N~N N~ O OC~O
H NH H HN ' '' HO
O
NH HN O
O '-COOH
To a solution of the crude Boc-Glu(cyclo{D-Lys-D-
Phe-D-Asp-Gly-Arg})-cyclo{D-Lys-D-Phe-D-Asp-Gly-Arg}
(0.172 g) in methylene chloride (4.5 mL) was added
trifluoroacetic acid (4.5 mL). The reaction mixture was
stirred for 2 h, concentrated to an oil under high
vacuum and triturated with diethyl ether. The product
was filtered, washed with diethyl ether, and dried under
high vacuum to give 38 mg of the desired product after
RP-HPLC as a lyophilized solid (TFA salt). ESMS:
Calcd. for C59Hg7N1g016, 1317.66; Found, 1318.9 [M+H]+1.
Analytical HPLC, Method 1B, Rt = 13.06 min, Purity =
93%.
Part C. Preparation of [2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-
Glu(cyclo{D-Lys-D-Phe-D-Asp-Gly-Arg})-cyclo{D-Lys-D-Phe-
D-Asp-Gly-Arg}
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N-HN N
O
S03H ~ O
OHN N O NH
H II
HN NO H~,',yH~NH2
NH HN
~ O
NH O ~ Oi 'NH
II HN
H2N~N N~O HOOC~O
H NH H HN ~.~
O
NH HN O
O- =COOH
To a solution of Glu(cyclo{D-Lys-D-Phe-D-Asp-Gly
Arg})-cyclo{D-Lys-D-Phe-D-Asp-Gly-Arg} (0.025 g, 0.015
mmol) in dimethylformamide (2 mL) was added
triethylamine (6.3 ~L, 0.045 mmol) and the reaction
mixture was stirred for 5 min. 2-[[[5-[[(2,5-Dioxo-1-
pyrrolidinyl)oxy]carbonyl]-2-pyridinyl]-
hydrazono]methyl]-benzenesulfonic acid, monosodium salt
(0.0092 g, 0.0210 mmol) was added, and the reaction
mixture was stirred for 18 h, then concentrated to an
oil under high vacuum. The oil was purified by
Preparative HPLC Method 1 to give 12.5 mg of the desired
product as a lyophilized solid (TFA salt). ESMS: Calcd.
for C~2H96N22O20S, 1620.7; Found, 1622.5 (M+H)+1.
Analytical HPLC, Method 1B, Rt = =14.62 min, Purity =
96%.
Example 15
Synthesis of Cy'Clo{D-Phe-D-Lys([2-[[[5-[carbonyl]-2
pyridinyl]hydrazono]methyl]-benzenesulfonic acid])-D
Asp-Gly-Arg}
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O . / ~ \
H2N N N~O N N-N
H NH H HN .,~~N ~ / S03H
O ~ H N" O
NH , O
O
O
OH
Part A. Preparation of cyclo{D-Phe-D-Lys(Cbz)-D-
Asp(OBzl)-Gly-Arg(Tos)}
The.N-terminus Boc- protecting group of the peptide
sequence Boc-Arg(Tos)-D-Phe-D-Lys(Cbz)-D-Asp(OBzl)-Gly-
0xime resin was removed using standard deprotection (25%
TFA in CH2C12). After eight washes with DCM, the resin
was treated with 10% DIEA/DCM (2 x 10 min.). The resin
was subsequently washed with DCM (x 5) and dried under
high vacuum. The resin (1.5 g, 0.44 mmol/g) was then
suspended in dimethylformamide (12 mL). Glacial acetic
acid (61 ~~.L) was added, and the reaction was heated to
60 ~C for 72 h. The resin was filtered, and washed with
DMF (2 x 10 mL). The filtrate was concentrated to an
oil under high vacuum. The resulting oil was triturated
with ethyl acetate. The solid thus obtained was
filtered, washed with ethyl acetate, and dried under
high vacuum to give the desired product (yield = 370
mg). ESMS: Calcd. for C4gH5gNg011S, 981.40; Found,
982.4 [M+H]+1. Analytical HPLC, Method 1A, Rt = 14.32
min (purity 60%).
Part B. Preparation of cyclo{D-Phe-D-Lys-D-Asp-Gly-Arg}
bis TFA Salt
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NH O
H2N' _N N~O
H NH H HN .,~~NH2
O ~
H N"
NH , O
O
O °-
OH
The crude cyclo{D-Phe-D-Lys(Cbz)-D-Asp(OBzl)-Gly-
Arg(Tos)} (0.146 g) was dissolved in trifluoracetic acid
(1.5 mL) and cooled to -16 ~C. Trifluoromethanesulfonic
acid (1.8 mL) was added dropwise, maintaining the
temperature at -16 ~C. Anisole (0.3 mL) was added and
the reaction was stirred at -16 ~C for 3 h. Diethyl
ether was added, the reaction was cooled to -35 ~C, and
stirred for 20 min. The crude product was filtered,
washed with diethyl ether, dried under high vacuum and
purified by Preparative HPLC Method 1, to give 100 mg of
the desired product as a lyophilized solid (TFA salt).
ESMS: Calcd. for C2~H41N90~ +H, 604.3; Found, 604.3.
Analytical HPLC, Method 1B, Rt = 10.25 min, Purity =
90%.
Part C. Preparation of cyclo{D-Phe-D-Lys([2-[[[5-
[carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid])-D-Asp-Gly-Arg}
Cyclo{D-Phe-D-Lys-D-Asp-Gly-Arg} TFA salt (0.090 g,
0.108 mmol) was dissolved in DMF (2 mL). Triethylamine
(45 ~L, 0.324 mmol) was added, and after 5 min of
stirring 2-[[[5-[[(2,5-dioxo-1-
pyrrolidinyl)oxy]carbonyl]-2-pyridinyl]hydrazono]-
methyl]-benzenesulfonic acid, monosodium salt (0.048 g,
0.108 mmol) was added. The reaction mixture was stirred
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for 70 h and then concentrated to an oil under high
vacuum. The oil was purified by Preparative HPLC Method
1 to give 10 mg of the desired product as a lyophilized
solid (TFA salt). ESMS: Calcd. for C4pH5pN120115 + H,
907.4; Found, 907.3. Analytical HPLC, Method 1B, Rt =
13.47 min, Purity = 89%.
Example 16
Synthesis of cyclo{N-Me-Arg-Gly-Asp-ATA-D-Lys([2-[[[5-
[carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid])}
NH
HOAc.H2N~N
H~~~ N~O
N~ H HN OH
~HN NH ~ O
S03H
- ~ H N 0~~~--~ S
I/
Part A: Preparation of cyclo{N-Me-Arg(Tos)-Gly-
Asp(OBzl)-ATA-D-Lys(Cbz)}
The N-terminus Boc-protecting group of the peptide
sequence Boc-Asp(OBzl)-ATA-D-Lys(Z)-N-Me-Arg(Tos)-Gly-
Oxime resin was removed using standard deprotection (50%
TFA in CH2C12). After washing with DCM (8x), the resin
was treated with 10% DIEA/DCM (2 x 10 min). The resin
was washed with DCM (5x) and dried under high vacuum
overnight. The resin (1.24 g, 0.39 mmol/g) was then
suspended in DMF (12 mL). Glacial acetic acid (67 mL,
1.16 mmol) was added and the reaction mixture was heated
at 50 ~C for 72 h. The resin was filtered and washed
with DMF (3 x 10 mL). The filtrate was concentrated
under high vacuum to give an oil. The resulting oil was
triturated with ethyl acetate. The solid obtained was
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purified by reverse-phase HPLC (Vydac C18 column, 18 to
90% acetonitrile gradient containing 0.1% TFA, Rt=14.129
min) to afford 42 mg (9%) of the desired product as a
lyophilized solid. ESMS: Calculated for
C46H56N10O11S2~ 988.3571 Found 989.4 [M+H]+1.
Part B: Preparation of cyclo{N-Me-Arg-Gly-Asp-ATA-D
Lys}
'/NH
TFA .H2N~N O
H~ H~O
~~ N~ HN~OH
TFA.H2N NH ~ O O
N-
O~ S
Cyclo{N-Me-Arg(Tos)-Gly-Asp(OBzl)-ATA-D-Lys(Cbz)}
(36 mg, 0.0364 mmol) was dissolved in trifluoroacetic
acid (0.364 mL) and cooled to -10 ~C in a dry
ice/acetone bath. To this solution was added
trifluoromethanesulfonic acid (0.437 mmol), followed by
anisole (70 mL). The reaction mixture was stirred at -
10 ~C for 3 h. The dry ice/acetone bath was then cooled
to -35 ~C and cold ether (40 mL) was added to the
solution. The mixture was stirred for 30 min at -35 ~C,
then cooled further to -50 ~C and stirred for another 30
min. The crude product was filtered, redissolved in
water/acetonitrile (1/1), and lyophilized to generate 35
mg of the title product (100%). ESMS: Calculated for
C24H3gN100~S, 610.2646 Found 611.4 [M+H]+1.
Part C: Preparation of cyclo{N-Me-Arg-Gly-Asp-ATA-D-
Lys([2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid])}
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To a solution of cyclo{N-Me-Arg-Gly-Asp-ATA-D-Lys}
(31 mg, 0.051 mmol) in DMF (2 mL) was added
triethylamine (28 mL, 0.204 mmol) and the reaction
mixture stirred at room temperature for 10 min. 2-[[[5-
[[(2,5-Dioxo-1-pyrrolidinyl)-oxy]carbonyl-2-
pyridinyl]hydrazono]methyl-benzenesulfonic acid,
monosodium salt (27 mg, 0.0612 mmol) was added, the
mixture stirred for 18 h and then concentrated under
high vacumm. The residue obtained was purified by
reverse-phase HPLC (Shandon HS-BDS column, 3 to 10%
acetonitrile, Rt=13.735 min) to afford 4 mg (8.8%) of
the desired product as a lyophilized solid. ESMS:
Calculated for C37H47N1301152, 913.2959 Found 914.5
[M+H] +1 .
20
Example 17
Synthesis of cyclo{Cit-Gly-Asp-D-Phe-Lys([2-[[[5
[carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid] ) }
O O
H2N~H H~O
NH HN OH
O O
S03H NH HN O
N ~
N H O//
N-HN
Part A. Preparation of cyclo{Cit-Gly-Asp(OtBu)-D-Phe-
Lys(Boc)}
The peptide Asp(OtBu)-D-Phe-Lys(Boc)-Cit-Gly was
obtained by automated solid phase peptide synthesis
using Fmoc chemistry (see general procedure). A 100 mL
round bottom flask was charged with HBTU (271 mg, 0.71
mmol) and DMF (10 mL). The solution was stirred at 60
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~C for 5 min. To this a solution of Asp(OtBu)-D-Phe-
Lys(Boc)-Cit-Gly (0.456 g) and Hunig's base (0.27 mL,
1.53 mmol.) in DMF (10 mL) was added and the solution
stirred at 60 ~C for 4 h under nitrogen. The solvent
was then removed in vacuo and the residue was triturated
with ethyl acetate. The solids were filtered and washed
with ethyl acetate (3 x 6 mL) and dried in vacuo to give
the desired product (305 mg, 780).
ESMS: Calcd. for C36H56N8010~ 760.4; Found, 761.4
[M+H]+1. Analytical HPLC, Method 1A, Rt = 11.8 min
(purity 99%).
Part B. Preparation of cyclo{Cit-Gly-Asp(OtBu)-D-Phe-
Lys(Boc)}
O O
H2~ H H ~O
N NH HN OH
O~
''-NH HN O
H 2N ~~/~/
O
A solution of cyclo{Cit-Gly-Asp(OtBu)-D-Phe-
Lys(Boc)} (287 mg, 0.38 mmol), TFA (6 mL),
triisopropylsilane (0.25 mL) and water (0.25 mL) was
stirred at room temperature under nitrogen for 4 h. The
solvents were removed in vacuo (over 3 h) and the
residue triturated with diethyl ether, filtered and
washed with ether to give the desired product (315 mg)
(TFA salt). ESMS: Calcd. for C27H4pN80g, 604.3; Found,
605.4 [M+H]+1. Analytical HPLC, Method 1B, Rt = 9.6
min, Purity = 97%.
Part C. Preparation of cyclo{Cit-Gly-Asp-D-Phe-Lys([2-
[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid])}
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Cyclo{Cit-Gly-Asp-D-Phe-Lys} TFA salt (0.044 g) was
dissolved in DMF (2 mL). Triethylamine (22 ~.L, 0.156
mmol) was added, and after 5 min of stirring 2-[[[5-
[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-
pyridinyl]hydrazono]-methyl]-benzenesulfonic acid,
monosodium salt (0.032 g, 0.073 mmol) was added. The
reaction mixture was stirred overnight, under nitrogen,
and then concentrated under high vacuum. The residue
was purified by preparative RP-HPLC Method 1 to give 37
mg (700) of the desired product as a lyophilized solid
(TFA salt). ESMS: Calcd. for C4pH49N11~125~ 907.3;
Found, 908.4 [M+H]+1. Analytical HPLC, Method 1B, Rt =
14.15 min, Purity = 99%.
Example 18A
Synthesis of tris(t-butyl)-1,4,7,10
tetraazacyclododecane-1,4,7,10-tetraacetic acid
t-Bu-02C~N~~-C02-t-BU
HO ~N N
V C02-t-Bu
Part A. Preparation of Phenylmethyl 2-(1,4,7,10-
Tetraaza-4,7,10-tris(((tert-
butyl)oxycarbonyl)methyl)cyclododecyl)acetate
t-Bu-02C~N ~--C02-t-Bu
i
~ I o ~ N
C02-t-BU
A solution of tert-butyl (1,4,7,10-tetraaza-4,7-
bis(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)acetate
(0.922 g, 1.79 mmol), TEA (1.8 mL)~and benzyl
bromoacetate (0.86 mL, 5.37 mmol) in anhydrous DMF (24
mL) was stirred at ambient temperatures under a nitrogen
atmosphere for 24 h. The DMF was removed under vacuum
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and the resulting oil was dissolved in EtOAc (300 mL).
This solution was washed consecutively with water (2 x
50 mL) and saturated NaCl (50 mL), dried (MgS04), and
concentrated to give the title compound as an amorphous
solid (1.26 g). MS: m/e 663.5 [M+H].
Part B. Preparation of 2-(1,4,7,10-tetraaza-4,7,10-
tris(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)acetic
acid
The product from Part A, above (165 mg, 0.25 mmol)
was hydrogenolyzed over 10o Pd on carbon (50 mg) in EtOH
(15 mL) at 60 psi for 24 h. The catalyst was removed by
filtration through filter aid and washed with EtOH. The
filtrates were concentrated to give the title compound
as an amorphous solid (134 mg, 94%). MS: m/e 573.5
[M+H].
Example 18
Synthesis of 2-(1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)-1-cyclododecyl)acetyl-Glu(cyclo{Lys-
Arg-Gly-Asp-D-Phe})-cyclo{Lys-Arg-Gly-Asp-D-Phe}
O NH NH O
~H~',,,,~H~NH2 H2N~H H
HOOC~~~''~ NH HN NH HN COOH
~O O O O
O NH HN-: NH HN p
'''~~ N N
O H NH H O
O
HOOC-~ /~~
N N
HOOC-~NV ~COOH
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Part A. Preparation of 2-(1,4,7,10-tetraaza-4,7,10-
tris(t-butoxycarbonylmethyl)-1-cyclododecyl)acetyl-
Glu(cyclo{Lys-Arg-Gly-Asp-D-Phe})-cyclo{Lys-Arg-Gly-Asp-
D-Phe}
O NH NH O
~H~'',''~H~NH2 H2N~H H
HOOC~~°'' NH HN NH HN COOH
~ O O O
% 'NH ~ HN
NH O
O HN
O H NH H
O
tBu00C--~ /~~
N N
N N
tBu00C-~ ~,~/ ~COOtBu
To a solution of tris(t-butyl)-1,4,7,10-tetra
azacyclododecane-1,4,7,10-tetraacetic acid (28 mg, 0.049
mmol) and Hunig's base (14 ~.~.L) in DMF (2 mL) was added
HBTU (17 mg, 0.0456 mmol) and the mixture stirred for 5
min. To this was added a solution of Glu(cyclo{Lys-Arg-
Gly-Asp-D-Phe})-cyclo{Lys-Arg-Gly-Asp-D-Phe} (54.1 mg,
0.0326 mmol) in DMF (1 mL) and the reaction mixture
allowed to stir under nitrogen at room temperature for 4
h. The solvent was removed in vacuo and the residue
purified by preparative RP-HPLC to give the product as a
lyophilized solid (18.3 mg) (TFA salt). ESMS: Calcd.
for Cg7H137N23~23~ 1872.0; Found, 937.2 [M+2H]+2.
Analytical HPLC, Method 1B, Rt = 19.98 min, Purity =
99%.
Part B. Preparation of 2-(1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)-1-cyclododecyl)acetyl-Glu(cyclo{Lys-
Arg-Gly-Asp-D-Phe})-cyclo{Lys-Arg-Gly-Asp-D-Phe}
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A solution of 2-(1,4,7,10-tetraaza-4,7,10-tris(t-
butoxycarbonylmethyl)-1-cyclododecyl)acetyl-
Glu(cyclo{Lys-Arg-Gly-Asp-D-Phe})-cyclo{Lys-Arg-Gly-Asp-
D-Phe} (18.3 mg, 8.71 mmol) in TFA (3 mL) was stirred at
room temperature under nitrogen for 5 h. The solution
was concentrated in vacuo and the residue was purified
by preparative RP-HPLC to give 8 mg (45%) of the desired
product as the lyophilized solid (TFA salt). ESMS:
Calcd. for C75H113N23~23. 1703.8; Found, 853.0 [M+2H]+2.
Analytical HPLC, Method 1B, Rt = 13.13 min, Purity =
99%.
l5
Example 19
Synthesis of cyclo{Arg-Gly-Asp-D-Phe-Lys(DTPA)}
NH O
O O TFA~H2N~H H
O ~OH HO~ p O NH HN OH
~ ~N N_ ~
HO' v ~N~ v _H NH HN O
HO\J
O O
To a solution of cyclo{Arg-Gly-Asp-D-Phe-Lys}
(0.050 g, 0.0601 mmol) in DMF (2 mL) was added
triethylamine (41.9 ~.L, 0.301 mmol). This solution was
added dropwise over 4 h to a solution of
diethylenetriaminepentaacetic dianhydride (0.1074 g,
0.301 mmol) in DMF (2 mL) and methyl sulfoxide (2 mL).
The reaction mixture was then stirred for 16 h,
concentrated to an oil under high vacuum and purified by
Preparative HPLC Method 1 to give 29.9 mg (46%) of the
desired product as a lyophilized solid. ESMS: Calcd.
for C41H62N12~16. 978.4; Found, 977.5 (M-H+).
Analytical HPLC, Method 1B, Rt = 11.916 min. Purity =
1000.
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Example 20
Synthesis of cyclofArg-Gly-Asp-D-Phe-Lys}2(DTPA)
O NH NH O
O~ N~ ,~~ N~ NH2 H2N~ Ni~ N~O
,~~ NH H HN O H O rC02H H02C, O H O NH H HN
HOOC ~ ~,~~ ~N~ ~ N~ ~~~~~ ~COOH
O NH HN H N H NH HN O
o Ho2cJ o
i~ ~i
The oil obtained in Example 9 upon purification by
Preparative HPLC Method 1, also gave 21.5 mg (210) of
the title product as a lyophilized solid. ESMS: Calcd.
for C6gH101N21~22~ 1563.7; Found, 1562.8 (M-H+).
Analytical HPLC, Method 1B, Rt = 15.135 min, Purity =
93%.
Example 21
Synthesis of Cyclo{Arg-Gly-Asp-D-Tyr(N-DTPA-3
aminopropyl)-Val}
TFA~H
H
v
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To a solution of cyclo{Arg-Gly-Asp-D-Tyr(3-
aminopropyl)-Val} (0.050 g, 0.0571 mmol) in
dimethylformamide (2 mL) was added triethylamine (39.8
~~.L, 0.286 mmol). This solution was added dropwise over
5 h to a solution of diethylenetriamine-pentaacetic
dianhydride (0.1020 g, 0.286 mmol) in methyl sulfoxide
(2 mL). The reaction mixture was stirred for an
additional 18 h, then concentrated to an oil under high
vacuum and purified by Preparative HPLC Method 1 to give
41.9 mg (65%) of the desired product as a lyophilized
solid. ESMS: Calcd. for C43H66N12017, 1022.5; Found,
1021.4 (M-H+). Analytical HPLC, Method 1B, Rt = 15.690
min, Purity = 96%.
Example 22
Synthesis of cyclo{Orn(d-N-2-Imidazolinyl)-Gly-Asp-D
Tyr(N-[2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]
benzenesulfonic acid]-3-aminopropyl)-Val}
O O
H H NH H HN OH
O
NH HN O O
O~. ~ I O H H
~N ' N
O ,~ S03H
Part A: Preparation of cyclo{Orn(d-N-l-Tos-2-
Imidazolinyl)-Gly-Asp(OBzl)-D-Tyr(N-Cbz-3-aminopropyl)-
Val}
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The N-terminus Boc- protecting group of the peptide
sequence Boc-Asp(OBzl)-D-Tyr(N-Cbz-aminopropyl)-Val-
Orn(d-N-1-Tos-2-Imidazolinyl)-Gly-Oxime resin is removed
using standard deprotection (25% TFA in CH~C1~). After
eight washes with DCM, the resin is treated with 10%
DIEA/DCM (2 x 10 min.). The resin is subsequently
washed with DCM (x 5) and dried under high vacuum. The
resin (1.75 g, 0.55 mmol/g) is then suspended in
dimethylformamide (15 mL). Glacial acetic acid (55.0
~zL, 0.961 mmol) is added, and the reaction mixture is
heated at 50 ~C for 7~ h. The resin is filtered, and
washed with DMF (2 x 10 mL). The filtrate is
concentrated to an oil under high vacuum. The resulting
oil is triturated with ethyl acetate. The solid is
filtered, washed with ethyl acetate, and is dried under
high vacuum to obtain the desired product.
Part B: Preparation of cyclo{Orn(d-N-2-Imidazolinyl)-
Gly-Asp-D-Tyr(3-aminopropyl)-Val}. Trifluoroacetic acid
salt.
Cyclo{Orn(d-N-1-Tos-2-Imidazolinyl)-Gly-Asp(OBzl)-
D-Tyr(N-Cbz-3-aminopropyl)-Val} (0.146 mmol) is
dissolved in trifluoroacetic acid (0.6 mL) and cooled to
-10 ~C. Trifluoromethanesulfonic acid (0.5 mL) is added
dropwise, maintaining the temperature at -10 ~C.
Anisole (0.1 mL) is added and the reaction mixture is
stirred at -10 ~C for 3 h. Diethyl ether is added, the
reaction mixture cooled to -35 ~C and then stirred for
30 min. The reaction mixture is cooled further to -50
~C and stirred for 30 min. The crude product is
filtered, washed with diethyl ether, dried under high
vacuum, and is purified by preparative HPLC to obtain
the desired product.
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Part C. Preparation of cyclo{Orn(d-N-2-Imidazolinyl)-
Gly-Asp-D-Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-3-
aminopropyl)-Val}
Cyclo{Orn(d-N-2-Imidazolinyl)-Gly-Asp-D-Tyr(3-
aminopropyl)-Val} trifluoroacetic acid salt (0.0228
mmol) is dissolved in DMF (1 mL). Triethylamine (0.0648
mmol) is added, and after 5 min of stirring 2-[[[5-
[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid,
monosodium salt (0.0274 mmol) is added. The reaction
mixture is stirred for 1-2 days, and then concentrated
to an oil under high vacuum. The oil is purified by
l5 preparative HPLC to obtain the desired product.
Example 23
Synthesis of cyclo{Lys-Gly-Asp-D-Tyr(N-[2-[[[5
[carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid]-3-aminopropyl)-Val}
O
H2N N~O
NH H HN OH
O
NH HN O O
O~~ ~ ~ O H ,N H
~N N
\ ~ ~l
O S03H
Part A: Preparation of cyclo{Lys(Tfa)-Gly-Asp(OBzl)-D-
Tyr(N-Cbz-3-aminopropyl)-Val}
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The N-terminus Boc- protecting group of the peptide
sequence Boc-Asp(OBzl)-D-Tyr(N-Cbz-aminopropyl)-Val-
Lys(Tfa)-Gly-Oxime resin is removed using standard
deprotection (25% TFA in CH2C12). After eight washes
with DCM, the resin is treated with 10% DIEA/DCM (2 x 10
min.). The resin is subsequently washed with DCM (x 5)
and dried under high vacuum. The resin (1.75 g, 0.55
mmol/g) is then suspended in dimethylformamide (15 mL).
Glacial acetic acid (55.0 ~.~.L, 0.961 mmol) is added, and
the reaction mixture is heated at 50 ~C for 72 h. The
resin is filtered, and washed with DMF (2 x 10 mL). The
filtrate is concentrated to an oil under high vacuum.
The resulting oil is triturated with ethyl acetate. The
solid thus obtained is filtered, washed with ethyl
acetate, and is dried under high vacuum to obtain the
desired product.
Part B: Preparation of cyclo{Lys(Tfa)-Gly-Asp-D-Tyr(3-
aminopropyl)-Val} Trifluoroacetic acid salt.
Cyclo{Lys(Tfa)-Gly-Asp(OBzl)-D-Tyr(N-Cbz-3-
aminopropyl)-Val} (0.146 mmol) is dissolved in
trifluoroacetic acid (0.6 mL) and cooled to -10 ~C.
Trifluoromethanesulfonic acid (0.5 mL) is added
dropwise, maintaining the temperature at -10 ~C.
Anisole (0.1 mL) is added and the reaction mixture is
stirred at -10 ~C for 3 h. Diethyl ether is added, the
reaction mixture cooled to -35 ~C and then stirred for
min. The reaction mixture is cooled further to -50
30 ~C and stirred for 30 min. The crude product obtained
is filtered, washed with diethyl ether, dried under high
vacuum, and is purified by preparative HPLC to obtain
the desired product.
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Part C. Preparation of cyclo{Lys-Gly-Asp-D-Tyr(N-[2-
[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-Val}
Cyclo{Lys(Tfa)-Gly-Asp-D-Tyr(3-aminopropyl)-Val}
trifluoroacetic acid salt (0.0228 mmol) is dissolved in
DMF (1 mL). Triethylamine (0.0648 mmol) is added, and
after 5 min of stirring 2-[[[5-[[(2,5-dioxo-1-
pyrrolidinyl)oxy]carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid,
monosodium salt (0.0274 mmol) is added. The reaction
mixture is stirred for 1-2 days, and then concentrated
to an oil under high vacuum. The oil is treated with
20% piperidine in DMF, and the crude material is
purified by preparative HPLC to obtain the desired
product.
Example 24
Synthesis of cyclo{Cys(2-aminoethyl)-G1y-Asp-D-Tyr(N-[2-
[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-Val}
O
H2N~S~H~O
NH HN OH
O
NH HN O O
~s
O~~ ~ I H N H
~N
N~N
O S03H
Part A: Preparation of cyclo{Cys(2-N-Tfa-aminoethyl)-
Gly-Asp(OBzl)-D-Tyr(N-Cbz-3-aminopropyl)-Val}
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The N-terminus Boc- protecting group of the peptide
sequence Boc-Asp(OBzl)-D-Tyr(N-Cbz-aminopropyl)-Val.-
Cys(2-N-Tfa-aminoethyl)-Gly-Oxime resin is removed using
standard deprotection (25% TFA in CH2C12). After eight
washes with DCM, the resin is treated with 10%~DTEA/DCM
(2 x 10 min.). The resin is subsequently washed with
DCM (x 5) and dried under high vacuum. The resin (1.75
g, 0.55 mmol/g) is then suspended in dimethylformamide
(15 mL). Glacial acetic acid (55.0 uL, 0.961 mmol) is
added, and the reaction mixture is heated at 50 ~C for
72 h. The resin is filtered, and washed with DMF (2 x
10 mL). The filtrate is concentrated to an oil under
high vacuum. The resulting oil is triturated with ethyl
acetate. The solid thus obtained is filtered, washed
with ethyl acetate, and dried under high vacuum to
obtain the desired product.
Part B: Preparation of cyclofCys(2-N-Tfa-aminoethyl)-
Gly-Asp-D-Tyr(3-aminopropyl)-Val}. Trifluoroacetic acid
salt.
Cyclo{Cys(2-N-Tfa-aminoethyl)-Gly-Asp(OBzl)-D-
Tyr(N-Cbz-3-aminopropyl)-Val} (0.146 mmol) is dissolved
in trifluoroacetic acid (0.6 mL) and cooled to -10 ~C.
Trifluoromethanesulfonic acid (0.5 mL) is added
dropwise, maintaining the temperature at -10 ~C.
Anisole (0.1 mL) is added and the reaction mixture is
stirred at -10 ~C for 3 h. Diethyl ether is added, the
reaction mixture cooled to -35 ~C and then stirred for
30 min. The reaction mixture is cooled further to -50
~C and stirred for 30 min. The crude product obtained
is filtered, washed with diethyl ether, dried under high
vacuum, and is purified by preparative HPLC to obtain
the desired product.
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Part C. Preparation of cyclo{Cys(2-aminoethyl)-Gly-Asp-
D-Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-3-
aminopropyl)-Val}
Cyclo{Cys(2-N-Tfa-aminoethyl)-Gly-Asp-D-Tyr(3-
aminopropyl)-Val} trifluoroacetic acid salt (0.0228
mmol) is dissolved in DMF (1 mL). Triethylamine (9.5
uL, 0.0648 mmol) is added, and after 5- min of stirring
2-[[[5-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid,
monosodium salt (0.0121 g, 0.0274 mmol) is added. The
reaction mixture is stirred for 1-2 days, and then
concentrated to an oil under high vacuum. The oil is
treated with 20% piperidine in DMF, and the crude
material is purified by preparative HPLC to obtain the
desired product.
Example 25
Synthesis of cyclo{HomoLys-Gly-Asp-D-Tyr(N-[2-[[[5-
[carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid]-3-aminopropyl)-Val}
O
H 2N
O
N
~H ~N N
~N
O SO3H
Part A: Preparation of cyclo{HomoLys(Tfa)-Gly-Asp(OBzl)-
D-Tyr(N-Cbz-3-aminopropyl)-Val}
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The N-terminus Boc- protecting group of the peptide
sequence Boc-Asp(OBzl)-D-Tyr(N-Cbz-aminopropyl)-Val-
HomoLys(Tfa)-Gly-Oxime resin is removed using standard
deprotection (25% TFA in CH2C12). After eight washes
with DCM, the resin is treated with 10% DIEA/DCM (2 x 10
min.). The resin is subsequently washed with DCM (x 5)
and dried under high vacuum. The resin (1.75 g, 0.55
mmol/g) is then suspended in dimethylformamide (15 mL).
Glacial acetic acid (55.0 ~.L, 0.961 mmol) is added, and
the reaction mixture is heated at 50 ~C for 72 h. The
resin is filtered, and washed with DMF (2 x 10 mL). The
filtrate is concentrated to an oil under high vacuum.
The resulting oil is triturated with ethyl acetate. The
solid thus obtained is filtered, washed with ethyl
acetate, and dried under high vacuum to obtain the
desired product.
Part B: Preparation of cyclo{HomoLys(Tfa)-Gly-Asp-D-
Tyr(3-aminopropyl)-Val}, Trifluoroacetic acid salt.
Cyclo{HomoLys(Tfa)-Gly-Asp(OBzl)-D-Tyr(N-Cbz-3-
aminopropyl)-Val} (0.146 mmol) is dissolved in
trifluoroacetic acid (0.6 mL) and cooled to -10 ~C.
Trifluoromethanesulfonic acid (0.5 mL) is added
dropwise, maintaining the temperature at -10 ~C.
Anisole (0.1 mL) is added and the reaction mixture is
stirred at -10 ~C for 3 h. Diethyl ether is added, the
reaction mixture cooled to -35 ~C and then stirred for
30 min. The reaction mixture is cooled further to -50
~C and stirred for 30 min. The crude product obtained
is filtered, washed with diethyl ether, dried under high
vacuum, and is purified by preparative HPLC to obtain.
the desired product.
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Part C. Preparation of cyclo{HomoLys-Gly-Asp-D-Tyr(N-
[2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-Val}
Cyclo{HomoLys(Tfa)-Gly-Asp-D-Tyr(3-aminopropyl)-
Val} trifluoroacetic acid salt (0.0228 mmol) is
dissolved in DMF (1 mL). Triethylamine (9.5 ~L, 0.0648
mmol) is added, and after 5 min of stirring 2-[[[5-
[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid,
monosodium salt (0.0121 g, 0.0274 mmol) is added. The
reaction mixture is stirred for 1-2 days, and then
concentrated to an oil under high vacuum. The oil is
treated with 20% piperidine in DMF, and the crude
material is purified by preparative HPLC to obtain the
desired product.
Example 26
Synthesis of cyclo{Orn(d-N-Benzylcarbamoyl)-Gly-Asp-D
Tyr(N-[2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]
benzenesulfonic acid]-3-aminopropyl)-Val}
O O
H~H
/ NH HN OH
O
NH HN O
O H N H
~N ~ N
O S03H
Part A: Preparation of cyclo{Orn(d-N-Benzylcarbamoyl)-
Gly-Asp(OBzl)-D-Tyr(N-Cbz-3-ami.nopropyl)-Val}
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The N-terminus Boc- protecting group of the peptide
sequence Boc-Asp(OBzl)-D-Tyr(N-Cbz-aminopropyl)-Val-
Orn(d-N-Benzylcarbamoyl)-Gly-Oxime resin is removed
using standard deprotection (25% TFA in CH2C12). After
eight washes with DCM, the resin is treated with 10%
DTEA/DCM (2 x 10 min.). The resin is subsequently
washed with DCM (x 5) and dried under high vacuum. The
resin (1.75 g, 0.55 mmol/g) is then suspended in
dimethylformamide (15 mL). Glacial acetic acid (55.0
~zL, 0.961 mmol) is added, and the reaction mixture is
heated at 50 ~C for 72 h. The resin is filtered, and
washed with DMF (2 x 10 mL). The filtrate is
concentrated to an oil under high vacuum. The resulting
oil is triturated with ethyl acetate. The solid thus
obtained is filtered, washed with ethyl acetate, and
dried under high vacuum to obtain the desired product.
Part B: Preparation of cyclo{Orn(d-N-Benzylcarbamoyl)-
Gly-Asp-D-Tyr(3-aminopropyl)-Val}. Trifluoroacetic acid
salt.
Cyclo{Orn(d-N-Benzylcarbamoyl)-Gly-Asp(OBzl)-D-
Tyr(N-Cbz-3-aminopropyl)-Val} (0.146 mmol) is dissolved
in trifluoroacetic acid (0.6 mL) and cooled to -10 ~C.
Trifluoromethanesulfonic acid (0.5 mL) is added
dropwise, maintaining the temperature at -10 ~C.
Anisole (0.1 mL) is added and the reaction mixture is
stirred at -10 ~C for 3 h. Diethyl ether is added, the
reaction mixture cooled to -35 ~C and then stirred for
30 min. The reaction mixture is cooled further to -50
~C and stirred for 30 min. The crude product obtained
is filtered, washed with diethyl ether, dried under high
vacuum, and is purified by preparative HPLC to obtain
the desired product.
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Part C. Preparation of cyclo{Orn(d-N-Benzylcarbamoyl)-
Gly-Asp-D-Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-3-
aminopropyl)-Val}
Cyclo{Orn(d-N-Benzylcarbamoyl)-Gly-Asp-D-Tyr(3-
aminopropyl)-Val} trifluoroacetic acid salt (0.0228
mmol) is dissolved in DMF (1 mL). Triethylamine (9.5
~.~.L, 0.0648 mmol) is added, and after 5 min' of stirring
2-[[[5-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid,
monosodium salt (0.0121 g, 0.0274 mmol) is added. The
reaction mixture is stirred for 1-2 days, and then
concentrated to an oil under high vacuum. The oil is
purified by preparative HPLC to obtain. the desired
product.
Example 27
Synthesis of cyclo{Dap(b-(2-benzimidazolylacetyl))-Gly-
Asp-D-Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-3-
aminopropyl)-Val}
N O O
O
NH HN OH
O
NH HN O
O
H ~N H
~N N
~N
O S03H
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Part A: Preparation of cyclo{Dap(b-(1-Tos-2-
benzimidazolylacetyl))-Gly-Asp(OBzl)-D-Tyr(N-Cbz-3-
aminopropyl)-Val}
The N-terminus Boc- protecting group of the peptide
sequence Boc-Asp(OBzl)-D-Tyr(N-Cbz-aminopropyl)-Val-
Dap(b-(1-Tos-2-benzimidazolylacetyl))-Gly-Oxime resin is
removed using standard deprotection (25% TFA in CH2C12).
After eight washes with DCM, the resin is treated with
10% DIEA/DCM (2 x 10 min.). The resin is subsequently
washed with DCM (x 5) and dried under high vacuum. The
resin (1.75 g, 0.55 mmol/g) is then suspended in
dimethylformamide (15 mL). Glacial acetic acid (55.0
uL, 0.961 mmol) is added, and the reaction mixture is
heated at 50 ~C for 72 h. The resin is filtered; and
washed with DMF (2 x 10 mL). The filtrate is
concentrated to an oil under high vacuum. The resulting
oil is triturated with ethyl acetate. The solid thus
obtained is filtered, washed with ethyl acetate, and
dried under high vacuum to obtain the desired product.
Part B: Preparation of cyclo{Dap(b-(2-
benzimidazolylacetyl))-Gly-Asp-D-Tyr(3-aminopropyl)-
Val}. Trifluoroacetic acid salt.
Cyclo{Dap(b-(1-Tos-2-benzimidazolylacetyl))-Gly-
Asp(OBzl)-D-Tyr(N-Cbz-3-aminopropyl)-Val} (0.146 mmol)
is dissolved in trifluoroacetic acid (0.6 mL) and cooled
to -10 ~C. Trifluoromethanesulfonic acid (0.5 mL) is
added dropwise, maintaining the temperature at -10 ~C.
Anisole (0.1 mL) is added and the reaction mixture is
stirred at -10 ~C for 3 h. Diethyl ether is added, the
reaction mixture cooled to -35 ~C and then stirred for
30 min. The reaction mixture is cooled further to -50
~C and stirred for 30 min. The crude product obtained
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is filtered, washed with diethyl ether, dried under high
vacuum, and purified by preparative HPLC to obtain the
desired product.
Part C. Preparation of cyclo{Dap(b-(2-
benzimidazolylacetyl))-Gly-Asp-D-Tyr(N-[2-[[[5-
[carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid]-3-aminopropyl)-Val}
Cyclo{Dap(b-(2-benzimidazolylacetyl))-Gly-Asp-D-
Tyr(3-aminopropyl)-Val} trifluoroacetic acid. salt
(0.0228 mmol) is dissolved in DMF (1 mL). Triethylamine
(9.5 uL, 0.0648 mmol) is added, and after 5 min of
stirring 2-[[[5-[[(2,5-dioxo-1-
pyrrolidinyl)oxy]carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid,
monosodium salt (0.0121 g, 0.0274 mmol) is added. The
reaction mixture is stirred for 1-2 days, and then
concentrated to an oil under high vacuum. The oil is
purified by the method described below to obtain the
desired product.
Example 28
Synthesis of cyclo{Orn(d-N-2-Imidazolinyl)-Gly-Asp-D-
Phe-Lys(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid])}
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~N O
'H~H H~O
NH HN OH
O
NH HN O O
s
o --O
~'' v /
0
NH
a
"N-NH
S03H
Part A: Preparation of CyClO{Orn(d-N-1-Tos-2
Imidazolinyl)-Gly-Asp(OBzl)-D-Phe-Lys(Cbz)}
The N-terminus Boc- protecting group of the peptide
sequence Boc-Asp(OBzl)-D-Phe-Lys(Z)-Orn(d-N-2-Tos-2-
Imidazolinyl)-Gly-Oxime resin is removed using standard
deprotection (25% TFA in CH2C12). After eight washes
with DCM, the resin is treated with 10% DIEA/DCM (2 x 10
min.). The resin is subsequently washed with DCM (x 5)
and dried under high vacuum. The resin (1.75 g, 0.55
mmol/g) is then suspended in dimethylformamide (15 mL).
Glacial acetic acid (55.0 uL, 0.961 mmol) is added, and
the reaction mixture is heated at 50 ~C for 72 h. The
resin is filtered, and washed with DMF (2 x 10 mL). The
filtrate is concentrated to an oil under high vacuum.
The resulting oil is triturated with ethyl acetate. The
solid thus obtained is filtered, washed with ethyl
acetate, and dried under high vacuum to obtain the
desired product.
Part B. Preparation of cyclo{Orn(d-N-2-Imidazolinyl)-
Gly-Asp-D-Phe-Lys}
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Cyclo{Orn(d-N-1-Tos-2-Imidazolinyl)-Gly-Asp(OBzl)-
D-Phe-Lys(Cbz)} (0.204 mmol) is dissolved in
trifluoroacetic acid (0.6 mL) and cooled to -10 ~C.
Trifluoromethanesulfonic acid (0.5 mL) is added
dropwise, maintaining the temperature at -10 ~C.
Anisole (0.1 mL) is added and the reaction is stirred at
-10 ~C for 3 h. Diethyl ether is added, the reaction is
cooled to -50 ~C, and stirred for 1 h. The crude
product is filtered, washed with diethyl ether, dried
under high vacuum and purified by preparative HPLC to
obtain the desires product.
Part C. Preparation of cyclo{Orn(d-N-2-Imidazolinyl)-
Gly-Asp-D-Phe-Lys(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid])}
Cyclo{Orn(d-N-2-Imidazolinyl)-Gly-Asp-D-Phe-Lys}
TFA salt (0.0481 mmol) is dissolved in DMF (2 mL).
Triethylamine (20.1 uL, 0.144 mmol) is added, and after
5 min of stirring 2-[[[5-[[(2,5-dioxo-1-
pyrrolidinyl)oxy]carbonyl]-2-pyridinyl]hydrazono]-
methyl]-benzenesulfonic acid, monosodium salt (0.0254
g, 0.0577 mmol) is added. The reaction mixture is
stirred for 20 h and then concentrated to an oil under
high vacuum. The oil is purified by preparative HPLC to
obtain the desired product.
Example 29
Synthesis of cyclo{Orn(d-N-Benzylcarbamoyl)-Gly-Asp-D-
Phe-Lys(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid])}
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O O
I i H~H H~O
NH HN OH
O
NH HN O O
ode. v
0
NH
~N-NH
S03H
Part A: Preparation of cyclo{Orn(d-N-Benzylcarbamoyl)
Gly-Asp(OBzl)-D-Phe-Lys(Cbz)}
The N-terminus Boc- protecting group of the peptide
sequence Boc-Asp(OBzl)-D-Phe-Lys(Z)-Orn(d-N-
Benzylcarbamoyl)-Gly-Oxime resin is removed using
standard deprotection (25% TFA in CH2C12). After eight
washes with DCM, the resin is treated with 10% DIEA/DCM
(2 x 10 min.). The resin is subsequently washed with
DCM (x 5) and dried under high vacuum. The resin (1.75
g, 0.55 mmol/g) is then suspended in dimethylformamide
(15 mL). Glacial acetic acid (55.0 ~L, 0.961 mmol) is
added, and the reaction mixture is heated at 50 ~C for
72 h. The resin is filtered, and washed with DMF (2 x
10 mL). The filtrate is concentrated to an. oil under
high vacuum. The resulting oil is triturated with ethyl
acetate. The solid thus obtained is filtered, washed
with ethyl acetate, and dried under high vacuum to
obtain the desired product.
Part B. Preparation of cyclo{Orn(d-N-Benzylcarbamoyl)-
Gly-Asp-D-Phe-Lys}
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Cyclo{Orn(d-N-Benzylcarbamoyl)-Gly-Asp(OBzl)-D-Phe-
Lys(Cbz)} (0.204 mmol) is dissolved in trifluoroacetic
acid (0.6 mL) and cooled to -10 ~C.
Trifluoromethanesulfonic acid (0.5 mL) is added
dropwise, maintaining the temperature at -10 ~C.
Anisole (0.1 mL) is added and the reaction is stirred at
-10 ~C for 3 h. Diethyl ether is added, the reaction is
cooled to -50 ~C, and stirred for 1 h. The crude
product is filtered, washed with diethyl ether, dried
under high vacuum and purified by preparative HPLC to
obtain the desires product.
Part C. Preparation of cyclo{Orn(d-N-Benzylcarbamoyl)-
Gly-Asp-D-Phe-Lys(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid])}
Cyclo{Orn(d-N-Benzylcarbamoyl)-Gly-Asp-D-Phe-Lys}
TFA salt (0.0481 mmol) is dissolved in DMF (2 mL).
Triethylamine (20.1 ~.L, 0.144 mmol) is added, and after
5 min of stirring 2-[[[5-[[(2,5-dioxo-1-
pyrrolidinyl)oxy]carbonyl]-2-pyridinyl]hydrazono]-
methyl]-benzenesulfonic acid, monosodium salt (0.0254
g, 0.0577 mmol) is added. The reaction mixture is
stirred for 20 h and then concentrated to an oil under
high vacuum. The oil is purified by preparative HPLC to
obtain the desired product.
Example 30
Synthesis of cyclo{Lys-D-Val-D-Tyr(N-[2-[[[5-[carbonyl]-
2-pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-3-
aminopropyl)-D-Asp-Gly}
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H
Part A: Preparation of CyClO{Lys(Tfa)-D-Val-D-Tyr(N-
Cbz-3-aminopropyl)-D-Asp(OBzl)-Gly}
The N-terminus Boc-protecting group of the peptide
sequence Boc-Lys(Tfa)-D-Val-D-Tyr(N-Cbz-aminopropyl)-D-
Asp(OBzl)-Gly-Oxime resin is removed using standard
deprotection (50% TFA in CH2C12). After washing with
DCM (8x), the resin is neutralized with 10o DIEA/DCM (2
x 10 min). The resin is washed with DCM (5x) and dried
under high vacuum overnight. The resin (1.0 g, about
0.36 mmol/g) is then suspended in N,N-dimethylformamide
(12 mL). Glacial acetic acid (67 mL, 1.16 mmol) is
added and the reaction mixture is heated to 55 °C for 72
h. The resin is filtered and washed with DMF (3 x 10
mL). The filtrate is concentrated under high vacuum to
give an oil. The resulting oil is triturated with ethyl
acetate. The desired product is purified by reverse-
phase HPLC.
Part B: Preparation of cyclo{Lys-D-Val-D-Tyr(3-
aminopropyl)-D-Asp-Gly}, Trifuoroacetic acid salt.
The protected cyclic peptide cyclo{Lys(Tfa)-D-Val-
D-Tyr(N-Cbz-3-aminopropyl)-D-Asp(OBzl)-Gly} (0.10 mmol)
is dissolved in trifluoroacetic acid (0.95 mL) and
cooled to -10 oC in a dry ice/acetone bath. To this
solution is added trifluoromethanesulfonic acid (0.12
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mmol), followed by anisole (190 mL). The reaction
mixture is stirred at -16 °C for 3 h. The dry
ice/acetone bath is then cooled to -35 °C and cold ether
(40 mL) is added to the solution. The mixture is
stirred for 30 min at -35 °C, then cooled to -50 °C and
stirred for another 30 min. The crude product is
filtered, redissolved in water/acetonitrile (1/1),
lyophilized, and purified by reverse-phase HPLC to give
the desired product.
Part C: Preparation of cyclo{Lys-D-Val-D-Tyr(N-[2-[[[5-
[carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic
acid]-3-aminopropyl)-D-Asp-Gly}
A solution of cyclo{Lys(Tfa)-D-Val-D-Tyr(3-
aminopropyl)-D-Asp-Gly} (0.0216 mmol) in N,N-
dimethylformamide (2 mL) is added triethylamine (15 mL,
0.108 mmol) and stirred at room temperature for 10 min.
2-[[[5-[[(2,5-Dioxo-1-pyrrolidinyl)oxy]carbonyl-2-
pyridinyl]-hydrazono]methyl-benzenesulfonic acid,
monosodium salt (0.0260 mmol) is added, and the mixture
is stirred for 18 h. The mixture is concentrated under
high vacumm, the oil is treated with 20% piperidine in
DMF, and is again concntrated in vacuo. The residue is
purified by reverse-phase HPLC to give the desired
product.
Example 31
Synthesis of cyclo{Orn(d-N-Benzylcarbamoyl)-D-Val-D-
Tyr(N-[2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-D-Asp-Gly}
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0 w
w
/ H
O
O~H ~ \ So3H
~N-N-
OH H
Part A: Preparation of cyclo{Orn(d-N-Benzylcarbamoyl)-
D-Val-D-Tyr(N-Cbz-3-aminopropyl)-D-Asp(OBzl)-Gly}
The N-terminus Boc-protecting group of the peptide
sequence Boc-Orn(d-N-Benzylcarbamoyl)-D-Val-D-Tyr(N-Cbz-
aminopropyl)-D-Asp(OBzl)-Gly-Oxime resin is removed
using standard deprotection (50% TFA in CH2C12). After
washing with DCM (8x), the resin is neutralized with 10%
DIEA/DCM (2 x 10 min). The resin is washed with DCM
(5x) and dried under high vacuum overnight. The resin
(1.0 g, about 0.36 mmol/g) is then suspended in N,N-
dimethylformamide (12 mL). Glacial acetic acid (67 mL,
1.16 mmol) is added and the reaction mixture is heated
to 55 °C for 72 h. The resin is filtered and washed
with DMF (3 x 10 mL). The filtrate is concentrated
under high vacuum to give an oil. The resulting oil is
triturated with ethyl acetate. The desired product is
purified by reverse-phase HPLC.
Part B: Preparation of cyclo{Orn(d-N-Benzylcarbamoyl)-
D-Val-D-Tyr(3-aminopropyl)-D-Asp-Gly}, Trifuoroacetic
acid salt.
The protected cyclic peptide cyclo{Orn(d-N-
Benzylcarbamoyl)-D-Val-D-Tyr(N-Cbz-3-aminopropyl)-D-
Asp(OBzl)-Gly} (0.20 mmol) is dissolved in
trifluoroacetic acid (0.95 mL) and cooled to -10 °C in a
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dry ice/acetone bath. To this solution is added
trifluoromethanesulfonic acid (0.12 mmol), followed by
anisole (190 mL). The reaction mixture is stirred at -
16 °C for 3 h. The dry ice/acetone bath is then cooled
to -35 °C and cold ether (40 mL) is added to the
solution. The mixture is stirred for 30 min at -35 °C,
then cooled to -50 °C and stirred for another 30 min.
The crude product is filtered, redissolved in
water/acetonitrile (1/1), lyophilized, and purified by
reverse-phase HPLC to give the desired product.
Part C: Preparation of cyclo{Orn(d-N-Benzylcarbamoyl)-
D-Val-D-Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-3-
aminopropyl)-D-Asp-Gly}
A solution of cyclo{Orn(d-N-Benzylcarbamoyl)-D-Val-
D-Tyr(3-aminopropyl)-D-Asp-Gly} (0.0216 mmol) in N,N-
dimethylformamide (2 mL) is added triethylamine (15 mL,
0.108 mmol) and stirred at room temperature for 10 min.
2-[[[5-[[(2,5-Dioxo-1-pyrrolidinyl)oxy]carbonyl-2-
pyridinyl]-hydrazono]methyl-benzenesulfonic acid,
monosodium salt (0.0260 mmol) is added, and the mixture
is stirred for 18 h. The mixture is concentrated under
high vacumm and the residue is purified by reverse-phase
HPLC to give the desired product.
Example 32
Synthesis of cyclo{Orn(d-N-2-Imidazolinyl)-D-Val-D-
Tyr(N-[2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-3-aminopropyl)-D-Asp-Gly}
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O~
OOH
Part A: Preparation of cyclo{Orn(d-N-1-Tos-2-
Imidazolinyl)-D-Val-D-Tyr(N-Cbz-3-aminopropyl)-D-
Asp(OBzl)-Gly}
The N-terminus Boc-protecting group of the peptide
sequence Boc-Orn(d-N-1-Tos-2-Imidazolinyl)-D-Val-D-
Tyr(N-Cbz-aminopropyl)-D-Asp(OBzl)-Gly-Oxime resin is
removed using standard deprotection (50% TFA in CH2C12).
After washing with DCM (8x), the resin is neutralized
with 10% DIEA/DCM (2 x 10 min). The resin is washed
with DCM (5x) and dried under high vacuum overnight.
The resin (1.0 g, about 0.36 mmol/g) is then suspended
in N,N-dimethylformamide (12 mL). Glacial acetic acid
(67 mL, 1.16 mmol) is added and the reaction mixture is
heated to 55 °C for 72 h. The resin is filtered and
washed with DMF (3 x 10 mL). The filtrate is
concentrated under high vacuum to give an oil. The
resulting oil is triturated with ethyl acetate. The
desired product is purified by reverse-phase HPLC.
Part B: Preparation of cyclo{Orn(d-N-2-Imidazolinyl)-D
Val-D-Tyr(3-aminopropyl)-D-Asp-G1y}, Trifuoroacetic acid
salt.
The protected cyclic peptide cyclo{Orn(d-N-1-Tos-2-
Imidazolinyl)-D-Val-D-Tyr(N-Cbz-3-aminopropyl)-D-
Asp(OBzl)-Gly} (0.10 mmol) is dissolved in
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trifluoroacetic acid (0.95 mL) and cooled to -10 °C in a
dry ice/acetone bath. To this solution is added
trifluoromethanesulfonic acid (0.12 mmol), followed by
anisole (190 mL). The reaction mixture is stirred at -
16 °C for 3 h. The dry ice/acetone bath is then cooled
to -35 °C and cold ether (40 mL) is added to the
solution. The mixture is stirred for 30 min at -35 °C,
then cooled to -50 °C and stirred for another 30 min.
The crude product is filtered, redissolved in
water/acetonitrile (1/1), lyophilized, and purified by
reverse-phase HPLC to give the desired product.
Part C: Preparation of cyclo{Orn(d-N-2-Imidazolinyl)-D-
Val-D-Tyr(N-[2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-3-
aminopropyl)-D-Asp-Gly}
A solution of cyclo{Orn(d-N-2-Imidazolinyl)-D-Val-
D-Tyr(3-aminopropyl)-D-Asp-G1y} (0.0216 mmol) in N,N-
dimethylformamide (2 mL) is added triethylamine (15 mL,
0.108 mmol) and stirred at room temperature for 10 min.
2-[[[5-[[(2,5-Dioxo-1-pyrrolidinyl)oxy]carbonyl-2-
pyridinyl]-hydrazono]methyl-benzenesulfonic acid,
monosodium salt (0.0260 mmol) is added, and the mixture
is stirred for 18 h. The mixture is concentrated under
high vacumm and the residue is purified by reverse-phase
HPLC to give the desired product.
Radiopharmaceutical Examples
The following procedures (A, B) describe the
synthesis of radiopharmaceuticals of the present
invention of the formula 99mTc(VnA)(tricine)(phosphine),
in which (VnA) represents the vitronectin receptor
antagonist compound bonded to the Tc through a diazenido
(-N=N-) or hydrazido (=N-NH-) moiety. The diazenido or
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hydrazido moiety results from the reaction of the
hydrazinonicotinamido group, present either as the free
hydrazine or protected as a hydrazone, with the Tc-99m.
The other two ligands in the Tc coordination sphere are
tricine and a phosphine.
Procedure A
Synthesis of Tc-99m Vitronectin Receptor Antagonist
Complexes of the Formula 99mTc(VnA)(tricine)(phosphine)
Using Stannous Reducing Agent
10-30 ~.~.g (0.2-0.4 mL) of a reagent of the present
invention dissolved in saline or 50% aqueous ethanol, 40
mg (0.4 mL) of tr.icine in water, 1-7 mg (0.10-0.30 mL)
of phosphine dissolved in water or ethanol, 25 ug (25
~zL) SnCl2~ 2H20 dissolved in 0.1 M HCl, 0-0.25 mL
ethanol and 50-150 mCi 99mTc04- in saline were combined
in a 10 cc vial. The kit was heated in a 100°C water
bath for 10-20 minutes, then a 50 uL sample analyzed by
HPLC Method 3. If necessary, the complex was purified
by performing a 300-400 ~.L injection on the HPLC and
collecting the fraction into a shielded flask. The
collected fraction was evaporated to dryness,
redissolved in saline containing 0-5 volo Tween 80, and
then re-analyzed using HPLC Method 3.
Procedure B
Synthesis of Tc-99m Vitronectin Receptor Antagonist
Complexes of the Formula g9mTc(VnA)(tricine)(TPPTS)
Without Using Stannous Reducing Agent
To a lyophilized vial containing 4.84 mg TPPTS, 6.3
mg tricine, 40 mg mannitol and 0.25 mmol succinate
buffer, pH 4.8, was added 0.2-0.4 mL (20-40 ug) of a
reagent of the present invention dissolved in saline or
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50% aqueous ethanol, 50-100 mCi 99mTc04- in saline, and
additional saline to give a total volume of 1.3-1.5 mL.
The kit is heated in an 100°C water bath for 10-15
minutes, and a sample was then analyzed by HPLC Method
3. If necessary, the complex was purified by performing
a 300-400 uL injection on the HPLC and collecting the
fraction into a shielded flask. The collected fraction
was evaporated to dryness, redissolved in saline
containing 0-5 vol% Tween 80, and then re-analyzed using
HPLC Method 3.
Table 1. Analytical and Yield Data for
99mTc(VnA)(tricine)(Phosphine) Complexes
Complex Ex. Reagent Ex. Phosphine % Yield RT (min)
No. No.
33 1 TPPTS 88 8.2
34 2 TPPTS 96 19.5
35 3 TPPTS 91 33.7
36 4 TPPTS 92 21.8
37 5 TPPTS 65 25.1
38 6 TPPTS 91 41.7
39 7 TPPTS 89 20.4
40 8 TPPTS 93 16.4
41 9 TPPTS 90 13.4
42 10 TPPTS 93 12.9
43 12 TPPMS 94 23.5
44 12 TPPDS 93 18.1
45 12 TPPTS 93 13.6
46 13 TPPTS 93 11.2
47 14 TPPTS 79 11.0
48 15 TPPTS 94 11.2
49 16 TPPTS 81 9.2
50 17 TPPTS 97 10.4
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The following example describes the synthesis of
radiopharmaceuticals of the present invention of the
formula 99mTc(VnA)(tricine)(L) (L = Imine-Nitrogen
Containing Heterocycle), in which (VnA) represents the
vitronectin receptor antagonist compound bonded to the
Tc through a diazenido (-N=N-) or hydrazido (=N-NH-)
moiety. The other two ligands in the Tc coordination
sphere are tricine and an imine-nitrogen containing
heterocycle.
Example 51
Synthesis of Tc-99m Vitronectin Receptor Antagonist
Complex 99mTc(VnA)(tricine)(1,2,4-triazole)
30 ug of the Reagent of Example 1 (0.30 mL 50/50
EtOH/H20), 40 mg tricine (0.25 mL/H~0), 8 mg 1, 2, 4-
triazole (0.25 mL/H~O), 25 ug SnCl2 (25 ~zL/0.1 N HCl),
0.50 mL water and 0.20 mL 50~ 5 mCi 99mTcOg- were
combined in a shielded 10 cc vial and heated at 100 °C
for 10 minutes. 50 uL of the kit contents were analyzed
by HPLC using Method listed below. The product eluted
at a retention time of 8.33 min and had a radiochemical
purity of 88.1%.
Reagents of the present invention comprised of
either a DOTA (Example 18), DTPA monoamide (Examples 19
and 20) or DTPA bisamide chelator (Example 21) readily
form complexes with metal ions of elements 31, 39, 49,
and 58-71. The following examples demonstrate the
synthesis of complexes with 153Sm, 177Lu, and 9~Y, beta
particle emitting isotopes used in radiopharmaceutical
therapy, and 111In, a gamma emitting isotope used in
radiopharmaceutical imaging agents. In both types of
complexes, the metal ion is bound to the DOTA, DTPA
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monoamide or DTPA bisamide chelator moiety of the
reagents.
Examples 52 and 53
Synthesis of Y-90 and Lu-177 DOTA-Containing Vitronectin
Antagonist Complexes
To a clean sealed 10 mL vial was added 0.5 mL of
the reagent of Example 18 (200 ~g/mL in 0.25 M ammonium
acetate buffer, pH 7.0), followed by 0.05 - 0.1 mL of
gentisic acid (sodium salt, 10 mg/mL in 0.25 M ammonium
acetate buffer, pH 7.0) solution, 0.3 mL of 0.25 M
ammonium acetate buffer (pH 7.0), and 0.05 mL of
177LuC13 solution or 9~YC13 solution (100 - 200 mCi/mL)
in 0.05 N HCl. The resulting mixture was heated at 100
C for 35 min.. After cooling to room temperature, a
sample of the resulting solution was analyzed by radio-
HPLC and ITLC. The complex of Example 53 was analyzed
by mass spectroscopy (Found [M+H+] - 1877.6, Calcd.
1875.8 for C75H110N23023Lu) which confirmed identity.
Example 54
Synthesis of a 111In DOTA-Containing Vitronectin
Antagonist Complex
To a lead shielded 300 uL autosampler vial was
added 50 }1L of gentisic acid (10 mg/mL in 0.1 M ammonium
acetate buffer, pH 6.75) solution, followed by 100 ~L of
the reagent of Example 18 (200 ~g/mL in 0.2 M ammonium
acetate buffer, pH 5.0), and 50 uL of 111InC13 solution
(10 mCi/mL) in 0.04 N HCl. The pH of the reaction
mixture was about 4Ø The solution was heated at 100
C for 25 min. A sample of the resulting solution was
analyzed by radio-HPLC and ITLC.
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Table 1A: Analytical and Yield Data for Y-90, In-111,
and Lu-177 Complexes of DOTA-Conjugated Vitronectin
Receptor Antagonists.
Complex Ex. Reagent Ex. Isotope %Yield HPLC Ret. Time
No. No. (min)
52 18 Y-90 96 16.5
53 18 Lu-177 96 16.5
54 18 In-111 95 16.5
Examples 55 and 56
Synthesis of In-111 DTPA-monoamide or DTPA-bisamide
Containing Vitronectin Antagonist Complexes
0.2 mL of 111InC13 (1.7 mCi) in 0.1 N HCl, 0.2 mL of
1.0 M ammonium acetate buffer (pH 6.9) and 0.1 ml of the
reagent of the present invention dissolved in water were
combined in a l0cc glass vial and allowed to react at
room temperature for 30 min. The reaction mixture was
analyzed by HPLC Method 3.
20
Table 2. Analytical and Yield Data for 111In Complexes
Complex Ex. Reagent Ex. %Yield HPLC Ret. Time
No. No. (min)
55 19 86 11.1
56 20 96 18.8
Examples 57-59
Synthesis of Sm-153 Vitronectin Antagonist Complexes
0.25 mL of a 153SmC13 stock solution (54 mCill~mol
Sm, 40 mCi/mL) in 0.1 N HCl was combined with the
reagent of the present invention (50-fold molar excess)
dissolved in 1 N ammonium acetate buffer in a l0cc glass
vial. The reaction was allowed to proceed at room
temperature for ~30 min and was then analyzed by ITLC
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and HPLC (Method 3). If necessary, the complex was
purified by performing a 300-400 ~L injection on the
HPLC and collecting the fraction into a shielded flask.
The collected fraction was evaporated to dryness,
redissolved in saline, and then re-analyzed using HPLC
Method 3.
Table 3. Analytical and Yield Data for 153Sm Complexes
Complex Ex. Reagent Ex. %Yield HPLC Ret. Time
No. No. (min)
57 19 91 11.7
58 20 84 13.1
59 21 96 16.9
The non-radioactive (naturally occurring) samarium
analog of the Radiopharmaceutical of Example 59 was
prepared by combining 3.3. mg (2.9 ~mol) of the Reagent
of Example 21 dissolved in 2 mL of 1 M ammonium acetate
buffer, pH 7, and 0.29 mL of 0.01 M solution of SmCl3 in
0.1 N HCl. The reaction was allowed to proceed for ~ 5
h at room temperature and then the product was isolated
by HPLC Method 3. The volatiles were removed by
lyophilization. The identity of the complex was
confirmed by mass spectroscopy. (API-ESMS:Found [M+2H+ _
1172.4, Calcd. 1172.9 for C43H64N12~17Sm] A stock
solution of the complex was made in water and its
concentration determined by ICP analysis for use in
determining the binding affinity of the complex for the
vitronectin receptor oc,~(33.
The structures of representative In-111 (Example 56
), Y-90 (Example 52) and Sm-153 (Example 59)
radiopharmaceuticals of the present invention are shown
below.
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HN
,~~ ~2 H2N~N O
Ol N ; H N~O
HO~~~~, NH H HN O O NH H HN OH
[O O~~ O O
HN~~~w~ v NH HN O
N Ni
H H
O NH O
r--N / O
O
I\
~N~ O
O V ~ O
O
~~~~~ ~ NH2
H H NH H2N~~N T-
~''e NH H O NH H H O
O ~ ~
O' NN H~'~/~/~ OH
NH H
~O O
NH O
H O
2~H ~ O
NH H H ~~N/
OH O
NH H ~O
O ~ ~ ~ O O
OH2
Examples 60-62
Synthesis of Lu-177 Vitronectin .Antagonist Complexes
5 x 10'9 mol of a reagent of the present invention
was dissolved in 1.0 mL of 0.1 N acetate buffer, pH 6.8.
1 x 10-9 mol of Lu-177 (40 }.11, 3 mCi) dissolved in 0.1 N
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HCl was added and the reaction allowed to proceed at
room temperature for 30-45 min. The reaction mixtures
were analyzed by HPLC Method 3.
Table 4. Analytical and Yield Data fox 177Lu Complexes
Complex Ex. Reagent Ex. %Yield HPLC Ret. Time
No. No. (min)
60 19 98 11.0
61 20 98 15.6
62 21 98 11.7
Example 63
The gadolinium complex of the reagent of Example 21
was prepared according to the following procedure. 3-
3.5 mg of the reagent was dissolved in 2 mL 1 M ammonium
acetate buffer at pH 7.0 , and one equivalent Gd(N03)3
solution (0.02 M in water) was added to it. The
reaction mixture was allowed to stay at room temperature
for 3-5 hours and the product was isolated by HPLC
Method 4. The fraction containing the complex was
lyophilized and dissolved in 1 mL H20 resulting in a
solution approximately 2 mM in Gd as determined by ICP
analysis. The identity of the complex was confirmed by
mass spectroscopy. (API-ESMS:Found [M+2H+] - 1176.9,
Calcd. 1176.2 for C43H54N12017Gd] .
The following examples describe the synthesis of
ultrasound contrast agents of the present invention
comprised of targeting moieties for tumor neovasculature
that are or,~,(33 receptor antagonists .
Example 64
Part A. Synthesis of 1-(1,2-Dipalmitoyl-sn-glycero-3-
phosphoethanolamino)-12-(cyclo(Arg-Gly-Asp-D-Phe-Lys)-
dodecane-1,12-dione
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NH
H2N--~(
HN
O
O O NH HN
Q H O O
O~O_O O NH NH HHN
O O~ N-~ COOH
O
O
A solution of disuccinimidyl dodecane-1,12-dioate
(0.424 g, 1 mmol), 1,2-dipalmitoyl-sn-glycero-3-
phosphoethanolamine (1.489 g, 1 mmol) and cyclo(Arg-Gly-
Asp-D-Phe-Lys) TFA salt (0.831 g, l mmol) in 25 ml
chloroform is stirred for 5 min. Sodium carbonate (1
mmol) and sodium sulfate (1 mmol) are added and the
solution is stirred at room temperature under nitrogen
for 18 h. DMF is removed in vacuo and the crude product
is purified to obtain the title compound.
Part B. Preparation of Contrast Agent Composition
The Synthesis of 1-(1,2-Dipalmitoyl-sn-glycero-3-
phosphoethanolamino)-12-(cyclo(Arg-Gly-Asp-D-Phe-Lys)-
dodecane-1,12-dione is admixed with three other lipids,
1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid, 1,2-
dipalmitoyl-sn-glycero-3-phosphatidylcholine, and N-
(methoxypolyethylene glycol 5000 carbamoyl)-1,2-
dipalmitoyl-sn-glycero-3-phosphatidylethanolamine in
relative amounts of 1 wt.%:6 wt.o:54 wt.%:41 wt.%. An
aqueous solution of this lipid admixture (1 mg/mL),
sodium chloride (7 mg/mL), glycerin (0.1 mL/mL),
propylene glycol (0.1 mL/mL), at pH 6-7 is then prepared
in a 2 cc glass vial. The air in the vial is evacuated
and replaced with perfluoropropane and the vial is
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sealed. The ultrasound contrast agent composition is
completed by agitating the sealed vial in a dental
amalgamator for 30-45 see. to form a milky white
solution.
Example 65
Part A. Preparation of (c~-amino-PEG34oo-a-carbonyl) -
cyclo(Arg-Gly-Asp-D-Phe-Lys)
NH
H2N"'Q
HN
~--~~O
O NH HN
O
O HN
NH
H NCO N ~ N~COOH
z ~~H O _ O
m
~ Ph
To a solution of N-Boc-(~J-amino-PEG3400-a-
carboxylate sucinimidyl ester (1 mmol) and cyclo(Arg-
Gly-Asp-D-Phe-Lys) (1 mmol) in DMF (25 mL) is added
triethylamine (3 mmol). The reaction mixture is stirred
under nitrogen at room temperature overnight and the
solvent is removed in vacuo. The crude product is
dissolved in 50% trifluoroacetic acid/dichloromethane
and is stirred for 4 h. The volatiles are removed and
the title compound is isolated as the TFA salt via
trituration in diethyl ether.
Part B. Preparation of 1-(1,2-Dipalmitoyl-sn-glycero-3-
phosphoethanolamino)-12-((C~-amino-PEG340o-a-carbonyl)-
cyclo(Arg-Gly-Asp-D-Phe-Lys))-Dodecane-1,12-Dione
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NH
H2N
HN
O
14 O NH HN
O
O H O O NH H
O O~P-O~N NCO N N-~COOH
OH H ~~ H O~_ O
~ Ph
140
A solution of disuccinimidyl dodecanoate (1 mmol),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (1
mmol) and ((~-amino-PEG3goo-a-carbonyl)-cyclo(Arg-Gly-
Asp-D-Phe-Lys) TFA salt (1 mmol) in 25 ml chloroform is
stirred for 5 min. Sodium carbonate (1 mmol) and sodium
sulfate (1 mmol) are added and the solution is stirred
at room temperature under nitrogen for 18 h. DMF is
removed in vacuo and the crude product is purified to
obtain the title compound.
Part C. Preparation of Contrast Agent Composition
The 1-(1,2-Dipalmitoyl-sn-glycero-3-
phosphoethanolamino)-12-(((~-amino-PEG34oo-a-carbonyl)-
cyclo(Arg-Gly-Asp-D-Phe-Lys))-Dodecane-1,12-Dione is
admixed with three other lipids, 1,2-dipalmitoyl-sn-
glycero-3-phosphotidic acid, 1,2-dipalmitoyl-sn-glycero
3-phosphatidylcholine, and N-(methoxypolyethylene glycol
5000 carbamoyl)-1,2-dipalmitoyl-sn-glycero-3
phosphatidylethanolamine in relative amounts of 1 wt.%:6
wt.%:54 wt.%:41 wt.%. An aqueous solution of this lipid
admixture (1 mg/mL), sodium chloride (7 mg/mL), glycerin
(0.1 mL/mL), propylene glycol (0.1 mL/mL), at pH 6-7 is
then prepared in a 2 cc glass vial. The air in the vial
is evacuated and replaced with perfluoropropane and the
vial is sealed. The ultrasound contrast agent
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composition is completed by agitating the sealed vial in
a dental amalgamator for 30-45 sec. to form a milky
white solution.
Example 66
Part A. Preparation of Synthesis of ((~-amino-PEG34oo-a-
carbonyl)-Glu-(cyclo(Arg-Gly-Asp-D-Phe-Lys))~
NH
H2N--
HN
O
O NH HN
~O
HN
O NH O N~ N'~OOOH
O
O ~ Ph
H2N~O~O~ N O Ph
O
H HN O H-!( ''~ OOH
~i, , N ~H
HN
~ ~O
O_ _ NH HN
O
HN
H2N-~
NH
To a solution of N-Boc-CO-amino-PEG3400-a-
carboxylate sucinimidyl ester (1 mmol) and Glu-
(cyclo(Arg-Gly-Asp-D-Phe-Lys))2 (1 mmol) in DMF (25 mL)
is added triethylamine (3 mmol). The reaction mixture
is stirred under nitrogen at room temperature overnight
and the solvent is removed in vacuo. The crude product
is dissolved in 50% trifluoroacetic acid/dichloromethane
and is stirred for 4 h. The volatiles are removed and
the title compound is isolated as the TFA salt via
trituration in diethyl ether.
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Part B. Preparation of 1-(1,2-Dipalmitoyl-sn-glycero-3-
phosphoethanolamino)-12-((c~-amino-PEG34oo-a-carbonyl)-
Glu-(cyclo(Arg-Gly-Asp-D-Phe-Lys))2)-Dodecane-1,12-Dione
NH
H2N
HN
O
O NH HN
r0
HN
O NH O N~ N~ COOH
14 _- O
O H O O ~ Ph
Ph
O~O_P_O~NH~O~O~H HN O O O
OH
O H~ ~ I/ OOH
NH HN
14
~ O
_ NH HN
O
HN
H2N~
NH
A solution of disuccinimidyl dodecanoate (1 mmol), 1,2-
dipalmitoyl-sn-glycero-3-phosphoethanolamine (1 mmol)
and ((~-amino-PEG34oo-a-carbonyl)-Glu-(cyclo(Arg-Gly-Asp-
D-Phe-Lys))~ TFA salt (1 mmol) in 25 ml chloroform is
stirred for 5 min. Sodium carbonate (1 mmol) and sodium
sulfate (1 mmol) are added and the solution is stirred
at room temperature under nitrogen for 18 h. DMF is
removed in vacuo and the crude product is purified to
obtain the title compound.
Part C. Preparation of Contrast Agent Composition
The 1-(1,2-Dipalmitoyl-sn-glycero-3-
phosphoethanolamino)-12-((~-amino-PEG34oo-oc-carbonyl)-
Glu-(cyclo(Arg-Gly-Asp-D-Phe-Lys))2)-Dodecane-1,12-Dione
is admixed with three other lipids, 1,2-dipalmitoyl-sn-
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glycero-3-phosphotidic acid, 1,2-dipalmitoyl-sn-glycero-
3-phosphatidylcholine, and N-(methoxypolyethylene glycol
5000 carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-
phosphatidylethanolamine in relative amounts of 1 wt. o:6
wt.%:54 wt.%:41 wt.~. An aqueous solution of this lipid
admixture (1 mg/mL), sodium chloride (7 mg/mL), glycerin
(0.1 mL/mL), propylene glycol (0.1 mL/mL), at pH 6-7 is
then prepared in a 2 cc glass vial. The air in the vial
is evacuated and replaced with perfluoropropane and the
vial is sealed. The ultrasound contrast agent
composition is completed by agitating the sealed vial in
a dental amalgamator for 30-45 sec. to form a milky
white solution.
Analytical Methods
HPLC Method 3
Column: Zorbax C18, 25 cm x 4.6 mm or Vydac C18, 25 cm x
4.6 mm
Column Temperature: ambient
Flow: 1.0 mL/min
Solvent A: 10 mM sodium phosphate buffer pH 6
Solvent B: 100% Acetonitrile
Detector: sodium iodide (NaI) radiometric probe or beta
detector
Gradient A (Exs. 33, 51)
t (min) 0 20 30 31 40
%B 0 75 75 0 0
Gradient B (Exs. 39, 40, 43, 44, 45, 46, 48, 50)
t (min) 0 20 30 31 35 36 40
%B 0 25 25 75 75 0 0
Gradient C (Examples 34, 35, 36, 37, 38, 42):
t (min) 0 40 41 46 47 55
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%B 0 35 75 75 0 0
Gradient D (Ex. 49)
t (min) 0 20 30 31 40
%B 0 25 25 0 0
Gradient E (Exs. 55, 56):
t (min) 0 20 21 30 31 40
%B 0 20 50 50 0 0
Gradient F (Exs. 57, 58):
t (min) 0 15 16 25 26 35
oB 0 20 75 75 0 0
Gradient G (Ex. 59):
t (min) 0 20 21 30 31 40
%B 0 20 75 75 0 0
Gradient H (Exs. 60 ,61, 62):
t (min) 0 15 16 21 22 40
%B 0 20 50 50 0 0
Gradient I (Exs. 52, 53, 54)
t (min) 0 20 21 30 31 40
o Solvent B 5 20 60 60 5 5
Gradient J (Ex. 41)
t (min) 0 20 30 31 40
Solvent B 0 50 50 0 0
Gradient K (Ex. 47)
t (min) 0 20 21 30 31 40
Solvent B 10 20 60 60 10 10
HPLC Method 4
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Column: Zorbax C18, 25 cm x 4.6 mm
Flow: 1.0 mL/min
Solvent A: 10 mM ammonium acetate
Solvent B: 100% methanol
Gradient:
t (min) 0 23 26 27
%B 8 100 100 8
UV Detection
ITLC Method
Gelman ITLC-SG strips (2 cm x 7.5 cm)
Solvent System: 1:1 acetone: saline
Detection using a Bioscan System 200.
UTILITY
The pharmaceuticals of the present invention are
useful for imaging angiogenic tumor vasculature in a
patient or for treating cancer in a patient. The
radiopharmaceuticals of the present invention comprised
of a gamma emitting isotope are useful for imaging of
pathological processes involving angiogenic
neovasculature, including cancer, diabetic retinopathy,
macular degeneration, restenosis of blood vessels after
angioplasty, and wound healing. Diagnostic utilities
also include imaging of unstable coronary syndromes
(e.g., unstable coronary plaque). The
radiopharmaceuticals of the present invention comprised
of a beta, alpha or Auger electron emitting isotope are
useful for treatment of pathological processes involving
angiogenic neovasculature, by delivering a cytotoxic
dose of radiation to the locus of the angiogenic
neovasculature. The treatment of cancer is affected by
the systemic administration of the radiopharmaceuticals
resulting in a cytotoxic radiation dose to tumors.
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The compounds of the present invention comprised of
one or more paramagnetic metal ions selected from
gadolinium, dysprosium, iron, and manganese, are useful
as contrast agents for magnetic resonance imaging (MRI)
of pathological processes involving angiogenic
neovasculature.
The compounds of the present invention comprised of
one or more heavy atoms with atmic number of 20 or
greater are useful as X-ray contrast agents for X-ray
imaging of pathological processes involving angiogenic
neovasculature.
The compounds of the present invention comprised of
an echogenic gas containing surfactant microsphere are
useful as ultrasound contrast agents for sonography of
pathological processes involving angiogenic
neovasculature.
Representative compounds of the present invention
were tested in the following in vitro and in vivo assays
and models and were found to be active.
Immobilized Human Placental o~,~,(33 Receptor Assay
The assay conditions were developed and validated.
using [I-125]vitronectin. Assay validation included
Scatchard format analysis (n=3) where receptor number
(Bmax) and Kd (affinity) were determined. Assay format
is such that compounds are preliminarily screened at 10
and 100 nM final concentrations prior to IC50
determination. Three standards (vitronectin, anti-oc,~,(33
antibody, LM609, and anti-oc,~,(35, P1F6) and five reference
peptides have been evaluated for IC50 determination.
Briefly, the method involves immobilizing previously
isolated receptors in 96 well plates and incubating
overnight. The receptors were isolated from normal,
fresh, non-infectious (HIV, hepatitis B and C, syphilis,
and HTLV free) human placenta. The tissue was lysed and
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tissue debris removed via centrifugation. The lysate
was filtered. The receptors were isolated by affinity
chromatography using the immobilized oc,~,(33 antibody. The
plates are then washed 3x with wash buffer. Blocking
buffer is added and plates incubated for 120 minutes at
room temperature. During this time, compounds to be
tested and [I-125]vitronectin are premixed in a
reservoir plate. Blocking buffer is removed and
compound mixture pipetted. Competition is carried out
for 60 minutes at room temperature. Unbound material is
then removed and wells are separated and counted via
gamma scintillation.
Other Receptor Binding Assays
Whole cell assays for the determination of the
binding affinity of pharmaceuticals of the present
invention for the VEGF receptors, Flk-1/KDR and Flt-1,
are described in Ortega, et. al., Amer. J. Pathol.,
1997, 151, 1215-1224, and Dougher, et. al., Growth
Factors, 1997, 14, 257-268. An in vitro assay for
determining the affinity of pharmaceuticals of the
present invention for the bFGF receptor is described in
Yayon, et. al., Proc. Natl. Acad. Sci USA, 1993, 90,
10643-10647. Gho et. al., Cancer Research, 1997, 57,
3733-40, describe assays for angiogenin receptor binding
peptides. Senger, et. al., Proc. Natl. Acad. Sci USA,
1997, 94, 13612-13617 describe assays for antagonists of
the integrins alB1 and a2Bl. U.S. 5,536,814 describes
assays for compounds that bind to the integrin a5Bl.
Oncomouse~ Imaging
The study involves the use of the c-Neu Oncomouse~
and FVB mice simultaneously as controls. The mice are
anesthetized with sodium pentobarbital and injected with
approximately 0.5 mCi of radiopharmaceutical. Prior to
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injection, the tumor locations on each Oncomouse~ are
recorded and tumor size measured using calipers. The
animals are positioned on the camera head so as to image
the anterior or posterior of the animals. 5 Minute
dynamic images are acquired serially over 2 hours using
a 256x256 matrix and a zoom of 2x. Upon completion of
the study, the images are evaluated by circumscribing
the tumor as the target region of interest (ROI) and a
background site in the neck area below the carotid
salivary glands.
This model can also be used to assess the
effectiveness of the radiopharmaceuticals of the present
invention comprised of a beta, alpha or Auger electron
emitting isotope. The radiopharmaceuticals are
administered in appropriate amounts and the uptake in
the tumors can be quantified either non-invasively by
imaging for those isotopes with a coincident imageable
gamma emission, or by excision of the tumors and
counting the amount of radioactivity present by standard
techniques. The therapeutic effect of the
radiopharmaceuticals can be assessed by monitoring the
rate of growth of the tumors in control mice versus
those in the mice administered the radiopharmaceuticals
of the present invention.
This model can also be used to assess the compounds
of the present invention comprised of paramagnetic
metals as MRI contrast agents. After administration of
the appropriate amount of the paramagnetic compounds,
the whole animal can be placed in a commercially
available magnetic resonance imager to image the tumors.
The effectiveness of the contrast agents can be readily
seen by comparison to the images obtain from animals
that are not administered a contrast agent.
This model can also be used to assess the compounds
of the present invention comprised of heavy atoms as X-
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ray contrast agents. After administration of the
appropriate amount of the X-ray absorbing compounds, the
whole animal can be placed in a commercially available
X-ray imager to image the tumors. The effectiveness of
the contrast agents can be readily seen by comparison to
the images obtain from animals that are not administered
a contrast agent.
This model can also be used to assess the compounds
of the present invention comprised of an echogenic gas
containing surfactant microsphere as ultrasound contrast
agents. After administration of the appropriate amount
of the echogenic compounds, the tumors in the animal can
be imaging using an ultrasound probe held proximate to
the tumors. The effectiveness of the contrast agents
can be readily seen by comparison to the images obtain
from animals that are not administered a contrast agent.
Rabbit Matrigel Model
This model was adapted from a matrigel model
intended for the study of angiogenesis in mice.
Matrigel (Becton & Dickinson, USA) is a basement
membrane rich in laminin, collagen IV, entactin, HSPG
and other growth factors. When combined with growth
factors such as bFGF [500 ng/ml] or VEGF [2 ug/ml] and
injected subcutaneously into the mid-abdominal region of
the mice, it solidifies into a gel and stimulates
angiogenesis at the site of injection within 4-8 days.
In the rabbit model, New Zealand White rabbits (2.5-3.0
kg) are injected with 2.0 ml of matrigel, plus 1 ~.~.g bFGF
and 4 ~.zg VEGF. The radiopharmaceutical is then injected
7 days later and the images obtained.
This model can also be used to assess the
effectiveness of the radiopharmaceuticals of the present
invention comprised of a beta, alpha or Auger electron
emitting isotope. The radiopharmaceuticals are
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administered in appropriate amounts and the uptake at
the angiogenic sites can be quantified either non-
invasively by imaging for those isotopes with a
coincident imageable gamma emission, or by excision of
the angiogenic sites and counting the amount of
radioactivity present by standard techniques. The
therapeutic effect of the radiopharmaceuticals can be
assessed by monitoring the rate of growth of the
angiogenic sites in control rabbits versus those in the
rabbits administered the radiopharmaceuticals of the
present invention.
This model can also be used to. assess the compounds
of the present invention comprised of paramagnetic
metals as MRI contrast agents. After administration of
the appropriate amount of the paramagnetic compounds,
the whole animal can be placed in a commercially
available magnetic resonance images to image the
angiogenic sites. The effectiveness of the contrast
agents can be readily seen by comparison to the images
obtain from animals that are not administered a contrast
agent.
This model can also be used to assess the compounds
of the present invention comprised of heavy atoms as X-
ray contrast agents. After administration of the
appropriate amount of the X-ray absorbing compounds, the
whole animal can be placed in a commercially available
X-ray images to image the angiogenic sites. The
effectiveness of the contrast agents can be readily seen
by comparison to the images obtain from animals that are
not administered a contrast agent.
This model can also be used to assess the compounds
of the present invention comprised of an echogenic gas
containing surfactant microsphere as ultrasound contrast
agents. After administration of the appropriate amount
of the echogenic compounds, the angiogenic sites in the
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animal can be imaging using an ultrasound probe held
proximate to the tumors. The effectiveness of the
contrast agents can be readily seenby comparison to the
images obtain from animals that are not administered a
contrast agent.
Canine Spontaneous Tumor Model
Adult dogs with spontaneous mammary tumors were
sedated with xylazine (20 mg/kg)/atropine (1 ml/kg).
Upon sedation the animals were intubated using ketamine
(5 mg/kg)/diazepam (0.25 mg/kg) for full anethesia.
Chemical restraint was continued with ketamine (3
mg/kg)/xylazine (6 mg/kg) titrating as necessary. If
required the animals were ventilated with room air via
an endotrachael tube (12 strokes/min, 25 mllkg) during
the study. Peripheral veins were catheterized using 20G
I.V. catheters, one to serve as an infusion port for
compound while the other for exfusion of blood samples.
Heart rate and EKG were monitored using a
cardiotachometer (Biotech, Grass Quincy, MA) triggered
from a lead II electrocardiogram generated by limb
leads. Blood samples are generally taken at ~10 minutes
(control), end of infusion, (1 minute), 15 min, 30 min,
60 min, 90 min, and 120 min for whole blood cell number
and counting. Radiopharmaceutical dose was 300 ~Ci/kg
adminitered as an i.v. bolus with saline flush.
Parameters were monitored continuously on a polygraph
recorder (Model 7E Grass) at a paper speed of 10 mm/min
or 10 mm/sec.
Imaging of the laterals were for 2 hours with a
256x256 matrix, no zoom, 5 minute dynamic images. A
known source is placed in the image field (20-90 uCi) to
evaluate region of interest (ROI) uptake. Images were
also acquired 24 hours post injection to determine
retention of the compound in the tumor. The uptake is
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determined by taking the fraction of the total counts in
an inscribed area for ROI/source and multiplying the
known ~Ci. The result is ~Ci for the ROI.
This model can also be used to assess the
effectiveness of the radiopharmaceuticals of the present
invention comprised of a beta, alpha or Auger electron
emitting isotope. The radiopharmaceuticals are
administered in appropriate amounts and the uptake in
the tumors can be quantified either non-invasively by
imaging for those isotopes with a coincident imageable
gamma emission, or by excision of the tumors and
counting the amount of radioactivity present by standard
techniques. The therapeutic effect of the
radiopharmaceuticals can be assessed by monitoring the
size of the tumors over time.
This model can also be used to assess the compounds
of the present invention comprised of paramagnetic
metals as MRI contrast agents. After administration of
the appropriate amount of the paramagnetic compounds,
the whole animal can be placed in a commercially
available magnetic resonance imager to image the tumors.
The effectiveness of the contrast agents can be readily
seen by comparison to the images obtain from animals
that are not administered a contrast agent.
This model can also be used to assess the compounds
of the present invention comprised of heavy atoms as X-
ray contrast agents. After administration of the
appropriate amount of the X-ray absorbing compounds, the
whole animal can be placed in a commercially available
X-ray imager to image the tumors. The effectiveness of
the contrast agents can be readily seen by comparison to
the images obtain from animals that are not administered
a contrast agent.
This model can also be used to assess the compounds
of the present invention comprised of an echogenic gas
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containing surfactant microsphere as ultrasound contrast
agents. After administration of the appropriate amount
of the echogenic compounds, the tumors in the animal cart--
be imaging using an ultrasound probe held proximate to
the tumors. The effectiveness of the contrast agents
can be readily seen by comparison to the images obtain
from animals that are not administered a contrast agent.
Obviously, numerous modifications and variations of
the present invention are possible in light of the above
teachings. It is therefore to be understood that within
the scope of the appended claims, the invention may be
practiced otherwise that as specifically described
herein.
