Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
VITRONECTIN RECEPTOR ANTAGONIST PHARMACEUTICALS FOR USE
IN COMBINATION THERAPY
FIELD OF THE INTENTION
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 y
invention 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. In addition, the invention is directed to novel
pharmaceutical compositions and combination therapy
comprising a compound of the invention or a
pharmaceutically acceptable salt thereof, and a
photosensitizing agent. The pharmaceuticals are
comprised of a targeting moiety that binds to the
vitronectin receptor that is expressed in tumor
vasculature, an optional linking group, and a
therapeutically effective radioisotope or diagnostically
effective imageable moiety. The therapeutically
effective radioisotope emits a gamma ray or alpha
particle 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
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that over 1 million new cases of invasive cancer will be
diagnosed in the United States in 1998. The most
prevalent forms of the disease are solid tumors of the
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 chemotherapeutic
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
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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
radiopharmaceuticals or by its effect on the relaxation
rate of water in the immediate vicinity in the case of
magnetic resonance imaging contrast agents. w
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
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
improvements in cancer imaging and treatment have
Centered on the use of radionuclide labeled monoclonal
antibodies, antibody fragments and other proteins or
polypeptides that bind to tumor cell surface receptors.
The specificity 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. 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 and thus very low signal intensity in imaging
and insufficient cytotoxic effect for treatment.
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Alternative approaches to cancer imaging and
therapy have involved the use of small molecules, such
as peptides, that bind to tumor cell surface receptors.
An In-111 labeled somatostatin receptor binding peptide,
In-111-DTPA-D-Phe~--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 ananl-ogs of In-
111-DTPA-D-Phe1-octeotide for imaging and Re-186 labeled
analogs for therapy (Flanagan, e~, 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 prcoess 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
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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.
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
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
proangiogenic 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 , 1995, 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 for a
wide variety of extracellular matrix proteins with an
exposed tripeptide Arg-Gly-Asp moiety and mediates
cellular adhesion to its ligand: 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
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variety of human tumors. The role of the ocV(33receptors
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 ocv(33, while angiogenesis induced
by VEGF depends on the integrin 0v(33 (Cheresh et. al.,
Science, 1955, 270, 1500-2). Induction of expression of
the integrins Ocl(31 and oc~~is on the endothelial cell
surface is another important mechanism by which VEGF
promotes angiogenesis (Senger, et. al., Proc. Natl.
Acad, Sci USA, 1997, 84, 13612-7).
Angiogenic factors interact with endothelial cell
surface receptors such as the receptor tyrosine kinases
EGFR, FGFR, PDGFR, Flk-ljKDR, Flt-1, Tek, tie,
neuropilin-1, endoglin, endosialin, and Axl. The
receptors Flk-ljKDR, 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 chemotherapeutic
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.
(O'Reilly et. al. , Cell, 1994, 79, 315-328) Endostatin
is a 20 kDa C-terminal fragment of collagen .XVIII that
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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 chemotherapeutic 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
vasculature to which are attached chemotherapeutic
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 chemotherapeutic agents, the agents still must
be cleaved from the antibody carrier and be transported
or diffuse into the cells to be cytotoxic.
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Thus, it is desirable to provide anti-angiogenic
pharmaceuticals and tumor or new vasculature imaging
agents which do not suffer from poor diffusion or
transportation, possible immunologic toxicity, limited
availability, and/or a lack of specificity.
There continues to be a need for more effective
treatment options for patients with solid tumors. This
is especially true in cases of metastatic cancer in
which current standard chemotherapy and external beam
radiation regimens only result in marginal survival
improvements.
Although improvements in cytotoxic
chemotherapeutics have been made in recent years, the
toxicity of these compounds to normal tissues has
continued to severely limit their utility in extending
survival in patients with solid tumors. Recently
developed combinations of different therapeutic
modalities, such as external beam irradiation and
chemotherapy (i.e. chemoradiation), has provided some
incremental benefit to the control of tumor progression
and quality of life. However, neither systemic
chemotherapeutics nor external beam irradiation have
acceptable therapeutic indices, and are often limited
due to unacceptable toxicity to normal tissues. The
concept of combined therapy of cancer using anti-
angiogenesis drugs in combination with chemotherapeutics
is not new. Further, the concept of combining targeted
in-vivo radiotherapy using radiolabeled antibodies and
antibody fragments with chemotherapy has been reported
(Stein R, Juweid M, Zhang C, et al., Clin. Cancer Res.,
5: 3199x-3206x, 1999. However, the combination of a
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angiogenesis-targeted therapeutic radiopharmaceutical
which is targeted to receptors, which are then
upregulated in the neovasculature of tumors, together
with anti-cancer agents has not been described before.
Therefore, there is a need for a combination of a
therapeutic radiopharmaceutical, which is targeted to
localize in the neovasculature of tumors, with an anti-
cancer agent or a radiosensitizer agent, or a
pharmaceutically acceptable salt thereof, to provide
additive or synergistic therapeutic response without
unacceptable additive toxicity in the treatment of solid
tumors.
The major advantage of combined chemotherapy and
angiogenesis-targeted therapeutic radiopharmaceuticals,
over each therapeutic modality alone, is improved tumor
response without substantial increases in toxicity over
either treatment alone. The advantage of using
neovascular-specific radiopharmaceuticals, versus a
tumor-cell targeted antibody, is that there is much
lower systemic radiation exposure to the subject being
treated.
Further, if the receptor targets for the
radiopharmaceutical compounds, used in this method of
treatment, are expressed on the luminal side of tumor
vessels, there is no requirement that these compounds
traverse the capillary bed and bind to the tumor itself.
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
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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.
4,512,762, and U.S. Pat. No. 4,566,636, Kelly, et a1.
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
1~ 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
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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. (19871 Photosensitizers: therapy and detection of
malignant tumours. Photochem. Photobiol., 45, 879-889,
and Boyle R. W. and D. David (1996) Structure and
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;
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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
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
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
endoscope is usually requi-red 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
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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.
Another application of anti-angiogenic therapy is
in treating rheumatoid arthritis (RA). In RA, 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, it is desirable to have new
pharmaceuticals to destroy the highly vascularized
pannus that results and thus treat the disease.
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
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.
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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 collatoral vessels.
The detection, imaging and diagnosis of a number of
cardiovascular diseases need to be improved, including
restenosis, atherosclerosis, myocardial reperfusion
injury, and myocardial ischemia, stunning or infarction.
It has recently been determined that in all of these
disease conditions, the integrin receptor ocv(33 plays an
important role.
For example, in the restenosis complication that
occurs in ~30-50% of patients having undergone
angioplasty or stmt placement, neointimal hyperplasia
and ultimate reocclusion is caused by aggressively
proliferating vascular smooth muscle cells that express
ccv(33. (Cardiovascular Res., 1997, 36, 408-428; DDT,
1997, 2, 187-199; Current Pharm. Design, 1997, 3, 545-
584 )
Atherosclerosis proceeds from an intial endothelial
damage that results in the recruitment and subintimal
migration of monocytes at the site of the injury. Growth
factors are released which induce medial smooth muscle
cells to proliferate and migrate to the intimal layer.
The migrating smooth muscle cells express oc~(33.
In reperfusion injury, neutrophil transmigration is
integrin dependent and the integrins moderate initial
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infiltration into the viable border zone. The induction
of oc5(31, oc4(31 and ocV(35 in infiltrating neutrophils occurs
within 3 to 5 hours after reperfusion as neutrophils
move from the border zone to the area of necrosis.
(Circulation, 1999, 100, I-275)
Acute or chronic occlusion of a coronary artery is
known to result in angiogenesis in the heart as native
collateral vessels are recruited to attempt to relieve
the ischemia. However, even a gradual occlusion. usually
results in areas of infarction as the resulting
angiogenesis is not sufficient to prevent damage.
Cardiac angiogenesis has been associated with increased
expression of the growth factors VEGF and FGF and the
upregulation of the growth factor receptors flt-1 and
flk-1/KDR. (Drugs, 1999, 5~, 391-396)
SUMMARY OF THE INVENTION
It is one object of the present invention to
provide improved anti-angiogenic pharmaceuticals,
comprised of a targeting moiety that binds to the
vitronectin receptor that is expressed in tumor
neovasculature, an optional linking group, and a
radioisotope. The vitronectin 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
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
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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
~5 administering to a patient in need of such treatment a
therapeutic radiopharmaceutical composition of the
invention in combination with photodynamic therapy.
It is another object of the present invention to
provide imaging agents, comprised of vitronectin
receptor binding compounds conjugated to 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. These imaging agents are useful for
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imaging tumor neovasculature, therapeutic angiogenesis
interventions in the heart, natural angiogenic processes
in response to acute or chronic coronary vessel
occlusion, restenosis post-angioplasty, atherosclerosis
and plaque formation, and reperfusion injury.
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 non-peptide indazole
containing targeting moiety that binds to a receptor
that is upregulated during angiogenesis or during
cardiovascular diseases, 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
peptidomimetic targeting moiety that binds to a receptor
that is upregulated during angiogenesis or during
cardiovascular diseases, Q, an optional linking group,
Ln, and a surfactant, Sf.
The pharmaceuticals of the present invention may be
used for diagnostic andlor 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
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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 and 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,
natural angiogenic processes in response to acute or
chronic coronary vessel occlusion, restenosis post-
angioplasty, atherosclerosis and plaque formation, and
reperfusion injury 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
pk~armaceuticals 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, and a surfactant
microsphere, are useful as ultrasound contrast agents:
DETAILED DESCRIPTION OF THE INVENTION
[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
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agent and a radiosensitizer agent, or a pharmaceutically
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)
wherein, Q is independently a compound of Formula (Ia)
or (Ib)
R1d Rlld
X4d
~ ~ X3d
W d-Xd Yd
~ ~2d
i
X1d
RlOd
(za)
Rlld
X4d
\ '~ X3d
Rlde N ~ Wd,-Xd Yd
~/ X2d
~Xld
RlOde
( Ib )
including stereoisomeric forms thereof, or mixtures of
stereoisomeric forms thereof, or pharmaceutically
acceptable salt or prodrug forms thereof wherein:
~1d is N, CH, C- Wd_ ~d_ yd~ or C-Ln;
19
CA 02413957 2002-12-18
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X2d is N, CH, or C- Wd- Xd- yd~
X3d is N, CRlld~ or C- Wd- Xd_ yd~
X4d is N or CRlld~
provided that when R1d is Rlde then one of X1d and X~d is
C- Wd- Xd- Yd, and when RlOd is Rlde then X3d is C- Wd-
Xd_ yd
R1d is selected from: Rlde~ C1-C6 alkyl substituted with
0-1 Rl5d or 0-1 R2ld, C3-C~ alkenyl substituted with
0-1 Rl5d or 0-1 R2ld, C3-C~ cycloalkyl substituted
with 0-1 R~-5d or 0-1 R~ld, C4-C11 cYcloalkylalkyl
substituted with 0-1 Rl5d or 0-1 R2ld, aryl
substituted with 0-1 Rl5d or 0-2 R~ld or 0-1 R2ld
and aryl(C1-C6 alkyl)- substituted with 0-1 RlSd or
0-2 Rlld or 0-1 R2ld
CA 02413957 2002-12-18
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R~-de is selected from:
N- Ad N ~~ ra
-Ud ~ ~6d ~ / Ud ~ ~6d ~ , A1 d
Bd ~ B1d
ra ,
Nw Da
6d ~ ~ d U--(NR6d)
~ t NR. ~ L ~ d
Fd~E
Ja-Kd ,
NH
~~Fa
Ua f ~sd ) ~ Ed R2dN
Dd ~
.Ud /
i
ra
NHR2d ~R2d
N ~ Dd N
a
Ua
Ud ~
wN/ y,Fd
or
a Ed
Fa.E Ni
Ud a
f
21
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Ad and Bd are independently -CHI-, -0-, -N(R~d)-, or -C(=0)-
A1d and B1d are independently -CH2- or -N(R3d)_;
Dd is -N (R~d) -, -0-, -S-, -C (=0) - or -S02-;
Ed-gd is -C (R4d) =C (R5d) - ~ _N=C (R4d) _ ~ _C (R4d) -N- ~ or
-C(R4d)2C(R5d)~-;
Jd, Kd, Ld and Md are independently selected from
-C(R4d)-, -C(R5d)- and -N-, provided that at least
one of Jd, Kd, Ld and Md is not -N-;
Rid is selected. from: H, C1-C6 alkyl, (C1-C6
alkyl)carbonyl, (C1-C6 alkoxy)carbonyl; (C1-C6
alkyl)aminocarbonyl, C3-C6 alkenyl, C3-C7
cycloalkyl, C4-C11 cycloalkylalkyl, aryl,
heteroaryl(C1-C6 alkyl)carbonyl,
heteroarylcarbonyl,
aryl(C1-C6 alkyl)-, (C1-C6 alkyl)carbonyl-,
arylcarbonyl, C1-C6 alkylsulfonyl, arylsulfonyl,
aryl(C1-C6 alkyl)sulfonyl, heteroarylsulfonyl,
heteroaryl(C1-C6 alkyl)sulfonyl, aryloxycarbonyl,
and. aryl(C1-C6 alkoxy)carbonyl, wherein said aryl
groups are substituted with 0-2 substituents
selected from the group: C1-C4 alkyl, C1-C~
alkoxy, halo, CF3, and nitro;
22
CA 02413957 2002-12-18
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R3d is selected from: H, C1-C6 alkyl, C3-C7 cycloalkyl,
C4-C11 cycloalkylalkyl, aryl, aryl(C1-C6 alkyl)-,
and heteroaryl(C1-C6 alkyl)-;
R4d and R5d are independently selected from: H, C1-C4
alkoxy, NR~dR3d, halogen, NOZr CN, CF3, C1-C6 alkyl,
C3-C6 alkenyl, C3-C~ cycloalkyl, C4-C11
cycloalkylalkyl, aryl, aryl(C1-C6 alkyl)-~ (C1-C6
alkyl)carbonyl, (C1-C6 alkoxy)carbonyl, and
arylcarbonyl, or
alternatively, when substituents on adjacent atoms, R4d
and R5d can be taken together with the carbon atoms
to which they are attached to form a 5-7 membered
carbocyclic or 5-7 membered heterocyclic aromatic
or non-aromatic ring system, said carbocyclic or
heterocyclic ring being optionally substituted with
0-2 groups selected from: C1-C4 alkyl, C1-C4
alkoxy, halo, cyano, amino, CF3, and N02;
Ud is selected from:
-(CH2)nd-,
- (CH2 ) nd (CR7d=CR8d) (CH2 ) md_
-(CH2)nd(C=C)(CH2)md-.
-(CH2)~dQ(CH2)md_~
-(CH2)nd0(CH2)md-~
-(CH2)ndN(R6d)(CH2)md-.
- ( CH2 ) ndC ( =Q ) ( CH2 ) md- f
- ( CHI ) nd ( C=0 ) N ( R6d ) ( CH2 ) md_
- ( CH2 ) ndN ( R6d ) ( C=0 ) ( CH2 ) md_ ~ and
- ( CH2 ) ndS ( 0 ) pd ( CH2 ) md- ;
23
CA 02413957 2002-12-18
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wherein one or more of the methylene groups in Ud is
optionally substituted with R7d;
Qd is selected from 1,2-cycloalkylene, 1,2-phenylene,
1.,3-phenylene, 1,4-phenylene, 2,3-pyridinylene,
3,4-pyridinylene, 2,4-pyridinylene, and 3,4-
pyridazinylene;
R6d is selected from: H, C1-C4 alkyl, and benzyl;
R7d and R8d are independently selected from: H, C1-C6
alkyl, C3-C7 cycloalkyl, Cg-C11 cycloalkylalkyl,
aryl, aryl(C1-C6 alkyl)-,
and heteroaryl(Cp-C& alkyl)-;
RlOd is selected from: H, Rlde, C1-C4 alkoxy substituted
with 0-1 R2ld, N (R6d) 2 , halogen, N02 , CN, CF3 ,
C02R17d~ C(=0)Rl7d~ CONR17dR20d~ _Sp2R17d~
-SOzNR17dR20d, C1-C6 alkyl substituted with 0-1 Rl5d
or 0-1 R2ld, C3-C6 alkenyl substituted with 0-1 RlSd
or 0-1 R~ld, C3-C7 cycloalkyl substituted with 0-1
Rl5d or 0-1 R2ld, C4-C11 cYcloalkylalkyl substituted
with 0-1 RlSd or 0-1 R2ld, aryl substituted with 0-1
Rl5d or 0-2 Rlld or 0-1 R~ld, and aryl(C1-C6 alkyl)-
substituted with 0-1 RlSd or 0-2 Rlld or 0-1 R~ld;
Rlode is selected from: H, C1-C4 alkoxy substituted with
0-1 R2ld, N ( R6d) ~ , halogen, N02 , CN, CF3 , C02R17d,
C(-0)Rl7d~ CONR17dR2od~ -aro2R17dr -Sp2NR17dR20d~ C1-C6
alkyl substituted with 0-1 RlSd or 0-1 R~ld, C3-C6
alkenyl substituted with 0-1 Rl5d or O-1 R2ld~ C3-C7
24
CA 02413957 2002-12-18
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cycloalkyl substituted with 0-1 Rl5d or O-1 R2ld
C4-C11 cycloalkylalkyl substituted with 0-1 Rl5d or
0-1 R2ld, aryl substituted with 0-1 RlSa or 0-2 R~-1d
or 0-1 R2ld, and aryl(C1-C6 alkyl)- substituted with
0-1 Rl5d or 0-2 R~-1d or 0-1 R2~d;
Rild is selected from H, halogen, CF3, CN, N02, hydroxy,
NR2dR3d, C1-C4 alkyl substituted with 0-1 R2ld, C1-C4
alkoxy substituted with 0-1 R2ld, aryl substituted
with 0-1 R2ld, aryl(C1-C6 alkyl)- substituted with
0-1 R2ld, (C1-Cg alkoxy)carbonyl substituted with 0-
1 R2ld~ (C1_C4 alkyl)carbonyl substituted with 0-1
R2ld~ C1-C4 alkylsulfonyl substituted with 0-1 R2ld,
and C1-C4 alkylaminosulfonyl substituted with 0-1
R2za;
Wd is selected from:
_(C(Rl2d)2)qdC(=0)N(Rl3d)_~ and
_C (=p) _N (Rl3d) _ (C (Rl2d) 2 ) qd_;
xd is -C (R~2d) (Rl4d.) -C (Rl2d) (RlSd) _; or
alternatively, Wd and Xd can be taken together to be
( CH2 ) qdC ( =0 ) -N' N-Rl~d
Rl2d is selected from H, halogen, C1-Cg alkyl, C2-C~
alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl,
C4-C1p cycloalkylalkyl, (C1-C4 alkyl)carbonyl, aryl,
and aryl(C1-C6 alkyl)-;
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Rl3d is selected from H, C1-C6 alkyl, C3-C7
cycloalkylmethyl, and aryl{C~-C6 alkyl)-;
Rl4d is selected from:
H, C1-C6 alkylthio(C1-C6 alkyl)-, aryl(C1-C10
alkylthioalkyl)-, aryl(C1-C1o alkoxyalkyl)-, C1-C1o
alkyl, C1-C1p alkoxyalkyl, C1-C6 hydroxyalkyl,
C2-C1o alkenyl, C~-C1o alkynyl, C3-C1o cycloalkyl,
C3-C1o cycloalkylalkyl, aryl(C1-C6 alkyl)-,
heteroaryl(C1-C6 alkyl)-, aryl, heteroaryl, CO~Rl7d,
C{=O)Rl7d, and CONR17dR2od~ provided that any of the
above alkyl, cycloalkyl, aryl or heteroaryl groups
may be unsubstituted or substituted independently
with 0-1 Rl6d or 0-2 R.lld;
Rl5d is selected from:
H, Rlod, C~-C1o alkyl, C1-C1o alkoxyalkyl,
CZ-C1o alkylaminoalkyl, C1-C~o dialkylaminoalkyl,
(C1-C1o alkyl)carbonyl, aryl(C1-C6 alkyl)carbonyl,
C1-010 alkenyl, C1-C1o alkynyl ,C3-C1o cycloalkyl,
C3-C1o cycloalkylalkyl, aryl(C1-C6 alkyl)-,
heteroaryl(C1-C6 alkyl)-, aryl, heteroaryl, C02R17d,
C (-0) Rl7d~ CONR17dR2od~ SO~Rl7d~ and S02NR17dR2odl
provided that any of the above alkyl, cycloalkyl,
aryl or heteroaryl groups may be unsubstituted or
substituted independently with 0-2 Rlld
Yd is selected from:
-CORl9d~ _S03H~ _p03H, tetra~olyl, -CONHNHS02CF3, -
CONHSO~RI7d, -CONHS02NHR17d, -NHCOCF3, -
26
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NHCONHS02R17d, -NHS02R17d, -pP03H2, -OS03H, -P03H2,
S03H, -S02NHCORI7d, -S02NHC02R17d,
N
N/ ~ N N/ ~ C F3 0
N ~ N
\ \
H , H , and HO O ;
Rl6d is selected from:
_N (R20d) _C (-O) _0_Rl7d~
_N (R20d) -C (=O) _Rl7d~
-N(R20d) _C (=O) -NH-Rl7d
-N (R2od) S02-Rl7d~ and
_N(R20d) S02-NR20dR17d~
Rl7d is selected from:
C1-C10 alkyl optionally substituted with a bond to
Ln, C3-C11 Cycloalkyl optionally substituted with a
bond to Ln, aryl(C1-Cg alkyl)- optionally
substituted with a bond to Ln, (C1-C6 alkyl)aryl
optionally substituted with a bond to Ln,
heteroaryl(C1-C6 alkyl)- optionally substituted
with a bond to Ln, (C1-C6 alkyl)heteroaryl
optionally substituted with a bond to Ln,
biaryl(C1-C6 alkyl)- optionally substituted with a
bond to Ln, heteroaryl optionally substituted with
a bond to Ln, aryl optionally substituted with a
bond to Ln, biaryl optionally substituted with a
bond to Ln, and a bond to Ln, wherein said aryl,
biaryl or heteroaryl groups are also optionally
substituted with 0-3 substituents selected from the
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CA 02413957 2002-12-18
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group consisting of: C1-C4 alkyl, C1-C4 alkoxy,
aryl, heteroaryl, halo, cyano, amino, CF3, and N02;
Rl8d is selected from:
-H,
-C (=0) -O-Rl7d
-C (=0) -Rl7d
-C ( =O ) -NH-Rl7d,
-S0~-R~7d, and
-S02-NR~odRl7d~
Rl9d is selected from: hydroxy, C1-C1o alkyloxy,
C3-C11 cycloalkyloxy, aryloxy, aryl(C1-C6 alkoxy)-,
Cg-C1o alkylcarbonyloxyalkyloxy, C3-C1o
alkoxycarbonyloxyalkyloxy,
C~-C1o alkoxycarbonylalkyloxy,
C5-C1o cycloalkylcarbonyloxyalkyloxy,
C5-C1o cycloalkoxycarbonyloxyalkyloxy,
C5-C1o cycloalkoxycarbonylalkyloxy,
C7-C11 aryloxycarbonylalkyloxy,
Cg-C12 aryloxycarbonyloxyalkyloxy,
Cg-C12 arylcarbonyloxyalkyloxy,
C5-C1p alkoxyalkylcarbonyloxyalkyloxy,
C5-C1o (5-alkyl-1,3-dioxa-cyclopenten-2-one-
yl)methyloxy, C1o-C1g (5-aryl-1,3-dioxa-
cyclopenten-2-one-yl)methyloxy, and
(Rlld) (Rl2d) N_ (C1-C1o alkoxy) -;
R2od is selected from: H, C1-C6 alkyl, C3-C7 cycloalkyl,
C4-C11 cycloalkylalkyl, aryl, aryl(C1-C6 alkyl)-,
and heteroaryl(C1-C6 alkyl)-;
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R~ld is selected from: COOH and NR6d2;
d
m is 0-4;
d
n is 0-4;
d
t is 0-4;
d
p is 0-2;
d
q is 0-2; and
d
r is 0-2;
with the following provisos:
d d d d
(1) t , n , m and q are chosen such that the number of
d
atoms connecting R1d and Y is in the range of
10-14; and
d d d
(2) n and m are chosen such that the value of n plus
d d
m is greater than one unless U is
d d d
- ( CHI ) t Q ( CH2 ) m -
or Q is a peptide selected from the group:
L R
K/ \M ~C/ R4
-1 2
R R and L M ;
R1 is L-valine, D-valine or L-lysine optionally
substituted on the E amino group with a bond to Ln;
R2 is L-phenylalanine, D-phenylalanine,
D-1-naphthylalanine, 2-aminothiazole-4-acetic acid
29
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or tyrosine, the tyrosine optionally substituted on
the hydroxy group with a bond to Ln;
R3 is D-valine;
R4 is D-tyrosine substituted on the hydroxy group with a
bond to Ln;
provided that one of R1 and RZ in each Q is substituted
with a bond to Ln, and further provided that when
R2 is 2-aminothiazole-4-acetic acid, K is
N-methylarginine;
provided that at least one Q is a compound of Formula
(Ia) or (Ib) ;
d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
d' is 1-100;
Ln is a linking group having the formula:
( (w)n-(~R6R7)g)x-(~)k' ( (CR6aR7a)g,_ (W)h, )x';
W is independently selected at each occurrence from the
group: 0, S, NH, NHC(=0), C(=0)NH, NR8C(=0),
C (=O)N R8, C (=O) , C (=0) O, OC (=O) , NHC (=S)NH,
NHC(=0)NH, 50~, S02NH, (OCH2CH2)S, (CH2CH~0)S,,
(OCH2CH2CH2)5~~, (CH~CH2CH20)t, and (aa)t~;
as is independently at each occurrence an amino acid;
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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, and R8 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 ( =O ) R11, C ( =O ) NHR1 ~ , 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, C(=O)NHR11,
NHC(=0)R11, OH, NHR11, S03H, P03H, -OP03H2, -OS03H,
aryl substituted with 0-3 R11, CZ-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
R~.1;
R11 is independently selected at each occurrence from
the group: H, -OP03H~, alkyl substituted with 0-1
R1~, 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 R12, polyalkylene glycol
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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 R~~,
polyazaalkyl substituted with 0-1 R12' peptide
substituted with -C(=0)-(CH2)5-NHR12, and peptide
substituted with 0-1 R1~, wherein the peptide is
comprised of 2-10 amino acids, C1_5 alkyl
substituted with 3,6-O-disulfo-B-D-
galactopyranosyl, bis(phosphonomethyl)glycine, and
a bond to Ch;
R1~ 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, an d ;
2
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 ;
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;
x is selected from 0, 1, 2, 3, 4, and
5;
x' is selected from 0, 1, 2, 3, 4, and ;
5
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Ch is a metal bonding unit having a formula selected
from the group:
-~E A2 1/~A2/~A~~A4
A , A ,
05
f ~'E
A1~
E~ AY-'As
s
A ~ ~Aa.
E~A2~E A't~~ ,~,-E A7 E ~E A
E E ~ E
A i ~ ~Aa
A5 , and A~ ;
A1, AZ , A3 , A4 , A5 , A6 , A7 , and A8 are independent 1y
selected at each occurrence from the group: NRl3j
NR13R14, S, SH, S(Pg), O, 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~, C3-1o 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-so alkyl substituted with 0-3 R1~,
C1_~o alkyl-C6_1o aryl- substituted with 0-3 R1~,
and a 5-10 membered heterocyclic ring system
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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-C1o alkyl
substituted with 0-3 R~-~, aryl substituted with 0-3
R~-~, C1_1o cycloalkyl substituted with 0-3 Rl~,
heterocyclo-C1_1o alkyl substituted with 0-3 R~~,
wherein the heterocyclo group is a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and 0, C6-~o
aryl-C1_1o alkyl substituted with 0-3 R1~, C1-10
alkyl-C6-so 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(R2o)(R21);
R15 and R16 are each independently selected from the
group: a bond to Ln, -OH, C1-C1o 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 R~-~,
heterocyclo-C1_so 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
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aryl-C1_1o alkyl substituted with 0-3 R1~, C1-1o
alkyl-C6_~p 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, Cl, Br, I, -CF3,
-CN, -C02R18, -C(=0)R18, -C(=0)N(R18)2, -CHO,
-CH20R18, -OC(=0)R18, -OC(=0)ORlBaj _0R18,
-OC(=O)N(R18)2~ -NR1~C(=0)R18, -NR19C(=0)ORl8a~
-NR19C(=0)N(R18)2, -NR19S02N(R18)z, -NR19SO~R18a,
_S03H~ _S02R18a~ _SR18~ _S(=O)Rl8a~ _SO~N(R18)2,
-N(R18)2, -NHC(=S)NHR18, =NOR18, N02, -C(=O)NHOR18,
-C(=0)NHNR18R18a, -OCH~CO~H,
2-(1-morpholino)ethoxy, C1-C5 alkyl, C~-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;
R18~ 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;
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R2o and R21 are independently selected from the group:
H, C1-C1o alkyl, -CN, -COBRAS, -C (=0) RCS,
-C(=0)N(R25)2, C2-C1o 1-alkene substituted with 0-3
R23, C2-C1o 1-alkyne substituted with 0-3 R23, aryl
substituted with 0-3 R~3, 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 R23;
alternatively, R2o and R21, taken together with the
divalent carbon radical to which they are attached
form:
R22 R22
.a b
R23 R23
n
R22 and R23 are independently selected from the group:
H, R24, C1-C10 alkyl substituted with 0-3 R24,
C~-C10 alkenyl substituted with 0-3 R24, C~-C10
alkynyl substituted with 0-3 R24, aryl substituted
with 0-3 R34, 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
36
CA 02413957 2002-12-18
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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, -CO~R25,
-C(=O)R25, -C(=0)N(R25)2, -N(R25)3+, -CH20R25,
-OC(=O)R25, -OC(=O)OR25a~ _OR25~ _OC(=O)N(R25)2,
-NR26C (=0) R25, -NR26C (=0) OR25a~ _NR26C (=O) N (R25) 2
_NR26S02N(R25)2~ _NR26~02R25a~ _S03H~ _SO~R25a~
_~R25~ _S(=O)R25a~ _SO~N(R25)21 _N(R25)2~ =~OR25,
-C(=O)NHOR25, -OCH2CO~H, 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.
[2] In another embodiment, the present invention
provides a kit according to Embodiment 1 wherein
Rlde is selected from:
37
CA 02413957 2002-12-18
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N-Aa N~) ra
-Ua (NR6a) ~ Ua (NR6a)--~f ~ ~Ala
Bd ) B1d
rd ~
0
N- Ma N ~. Da
Ua (ISR6a) ~ 'La -Ua (NR6a)~/ Ea
Fd ~
Ja _- Ka
NHR2 a
N '
Fa N ~ Da
Ua (NR6a)~ ~ a . or
~Da~E
' Ua
NHR2 a
N
;
Ua
Ad and Bd are independently -CH2-, -0-, -N(R2d)-, or -C(=0)-
;
A1d and B1d are independently -CH2- or -N(R3~)-;
d
D is -N(R2d)-, -O-, -S-, -C(=0)- or -S02-;
38
CA 02413957 2002-12-18
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Ed-Fd is -C (R4d) -C (R5d) _ ~ _N=C (R4d) _ ~ _C (R4d) =N-~ or -
C (R4d) 2C (R5d) 2-
d d d d
J , K , L and M are independently selected from:
C(R4d)-, -C(R5d)- and -N-, provided that at least
d d d d
one of J , K , L and M is not -N-;
R2d is selected from: H, C1-C6 alkyl, (C1-C6
alkyl)carbonyl, (C~-C6 alkoxy)carbonyl, C1-C6
alkylaminocarbonyl, C3-Cg alkenyl, C3-C7
cycloalkyl, C4-C11 cycloalkylalkyl, aryl,
heteroaryl(C1-C6 alkyl)carbonyl,
heteroarylcarbonyl, aryl(C1-C6 alkyl)-, (C1-C6
alkyl)carbonyl, arylcarbonyl, alkylsulfonyl,
arylsulfonyl, aryl(C1-Cg alkyl)sulfonyl,
heteroarylsulfonyl, heteroaryl(C1-C6
alkyl)sulfonyl, aryloxycarbonyl, and aryl(C1-C6
alkoxy)carbonyl, wherein said aryl groups are
substituted with 0-2 substituents selected from the
group consisting of C1-C4 alkyl, C1-C4 alkoxy,
halo, CF3, arid nitro;
R3d is selected from: H, C1-C6 alkyl, C3-C7 cycloalkyl,
C4-C11 cycloalkylalkyl, aryl, aryl(C1-C6 alkyl)-,
and heteroaryl(C1-C6 alkyl)-;
R4d and R5d are independently selected from: H, C1-C4
alkoxy, NR2dR3d, halogen, N02, CN, CF3, C1-C6 alkyl,
C3-C6 alkenyl, C3-C7 cycloalkyl, C4-C11
39
CA 02413957 2002-12-18
WO 01/98294 PCT/USO1/19794
cycloalkylalkyl, aryl, aryl(C1-C6 alkyl)-~ C2-C7
alkylcarbonyl, and arylcarbonyl;
alternatively, when substituents on adjacent atoms, R4d
and R5d can be taken together with the carbon atoms
to which they are attached to form a 5-? membered
carbocyclic or 5-7 membered heterocyclic aromatic
or non-aromatic ring system, said carbocyclic or
heterocyclic ring being optionally substituted with
0-2 groups selected from: C1-C4 alkyl, C1-Cg
alkoxy, halo, cyano, amino, CF3, or N02;
d
U is selected from:
d
-(CH2)n -,
- (CH2 ) nd (CR7d=CR8d) (CH2 ) md-,
d d d
-(CH2)t Q (CH2)m -,
d d
-(CH2)n O(CH2)m -,
- ( CH2 ) ndN ( R6d ) ( CH2 ) md- ,
d d
- ( CH2 ) n C ( =O ) ( CH2 ) m - , arid
d d d
-(CH2)n ~(O)p (CH2)m -.
d
wherein one or more of the methylene groups in U is
optionally substituted with R7d;
d
Q is selected from 1,2-phenylene, 1,3-phenylene, 2,3-
pyridinylene, 3,4-pyridinylene, and 2,4-
pyridinylene;
R6d is selected from: H, C1-C4 alkyl, and benzyl;
CA 02413957 2002-12-18
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R7d and Rgd are independently selected from: H, C1-C6
alkyl, C3-C7 cycloalkyl, C4-C11 cycloalkylalkyl,
aryl, aryl (C1-C6 alkyl) -, and
heteroaryl(Cp-C~ alkyl)-;
Wd is -C (=0) -N (Rl3d) _ (C (Rl2d) 2 ) qd-;
Xd 1S -C (Rl2d) (Rl4d) -C (Rl2d) ~Rl5d) _;
1~
d d
alternatively, W and X can be taken together to be
(CH2) qdC (=0) -N N-Rlsd
Rl2d is H or C1-C6 alkyl;
d
Y is selected from:
-CORl9d~ -~03H,
N
N/ ~N N/ ~ CF3
~N
v
H , H , and H~~ ~ ;
d is selected from 1, 2, 3, 4, and 5;
d' is 1-50;
41
CA 02413957 2002-12-18
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W is independently selected at each occurrence from the
group: 0, NH, NHC(=0), C(=0)NH, NR8C(=0), C(=0)N
R8, C (=O) , C (=0) 0, OC (=0) , NHC (=S)NH, NHC (=0)NH,
502, (OCH2CH2)S, (CH~CH~O)S~, (OCH2CH2CH2)5~~,
(CH~CH2CH20)t, and (aa)t~;
as is independently at each occurrence an amino acid;
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, and R8 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 Rlo,
NHC ( =0 ) R11, C ( =0 ) NHR11, NHC ( =0 ) NHR11, NHR11, R11,
and a bond to Ch;
k 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 Ag are independent 1y
selected at each occurrence from the group: NR13,
NR13R14, S, SH, S (Pg) , OH, and a bond to Ln;
42
CA 02413957 2002-12-18
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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 R1~ combine to form =C(R~~)(R~1);
R1~ is independently selected. at each occurrence from
the group: a bond to Ln, =0, F, Cl, Br, I, -CF3,
-CN, -C02R18, -C(=O)R18, -C(=O)N(R18)2. -CH20R18,
-OC(=O)R18~ _OC(=O)ORl8a~ -OR18~ _OC(=0)N(R18)2~
-NR19C(=0)R18, -NR19C(=O)ORl8a~ _NR19C(=O)N(R18)2,
_NR.19S02N(R18)2, _NR19S02R18a~ _S03g~ _S02R18a~
-S(=O)Rl8a~ _S02N(R18)2. -N(R18)2, -NHC(=S)NHR18,
=NOR18, -C(=0)NHNR18R18a, -OCH2C02H, and
2-(1-morpholino)ethoxy;
43
CA 02413957 2002-12-18
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R18, Rl8a, and R19 are independently selected at each
occurrence from the group: a bond to Ln, H, and
C1-C6 alkyl;
R2~ and R21 are independently selected from the group:
H, C1-C5 alkyl, -C02R35, C2-C5 1-alkene substituted
with 0-3 R23, 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 heteroatams independently selected from N, S,
and 0 and substituted with 0-3 R23;
alternatively, R20 arid R21, taken together with the
divalent carbon radical to which they are attached
form:
R22
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 ( =0 ) N ( R25 ) 2 , -CH20R25 ,
44
CA 02413957 2002-12-18
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-OC(=O)R~S, -OR25, -S03H, -N(R25)2, and -OCH~C02H;
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 l, wherein:
CA 02413957 2002-12-18
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Rlde is selected from:
N
-UdNR6d~ HN
N -UdNR6d~~
H , ~N ,
R4d
N
-~dNR6d~ ~ N-
UdNRsd
R5d
N N \
r~dNRsd ~ UdNR6d--
N
S , H ,
N N~ N
-~dNR6d ~ -NdNR6d~
N
S ~ ~ H ,
N- N \
-~dNR6d~~ -UdNR6d--<
N ~ S
NH2
NH2
Nw N-
-Ud ~ -~d
\ s ~ /
- ,
NH2
-Ud ~~ -UdNRsd
~NH N
NH2
N-
W /
or N ;
wherein the above heterocycles are optionally
substituted with 0-2 substituents selected from the
46
CA 02413957 2002-12-18
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group: NH2, halogen, N02, CN, CF3, C1-C4 alkoxy, C1-
C6 alkyl, and C3-C7 cycloalkyl;
d d d d d
U is -(CH2)n"~ -(CH2)t Q (CH2)m - or -C(=O)(CH2)n -1-.
wherein one of the methylene groups is optionally
substituted with R7d;
7d
R is selected from: C1-C6 alkyl, C3-C7 cycloalkyl, C4-
C11 cycloalkylalkyl, aryl, aryl(C1-Cg alkyl),
heteroaryl, and heteroaryl(C1-C6 alkyl);
Rlod is selected from: H, Rlde~ C1_C4 alkoxy substituted
with 0-1 R2ld, halogen, C02R17a, CONR17dR20d~ C1-C6
alkyl substituted with 0-1 R~-5d or 0-1 R2ld, C3-C7
cycloalkyl substituted with 0-1 Rl5d or 0-1 R2ld,
Cg-C11 cyeloalkylalkyl substituted with 0-1 Rl5d or
0-1 R2ld, and aryl(C1-C6 alkyl)- substituted with 0-
1 Rl5d or 0-2 Rlld or 0-1 R2ld;
Rlode is selected from: H, C1-C4 alkoxy substituted with
0-1 R2ld, halogen, CO~Rl7d, CONR17dR2od~ C1_C6 alkyl
substituted with 0-1 Rl5d or 0-1 R~ld, C3-C7
cycloalkyl substituted with 0-1 Rl5d or 0-1 R2ld
C4-C11 cycloalkylalkyl substituted with 0-1 RlSd Or
0-1 R~ld, and aryl(C1-C6 alkyl)- substituted with 0-
1 Rl5d or 0-2 Rlld or 0-1 R2ld;
Wd is -C (=0) -N (Rl3d) _;
47
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Xd is -CH (Rl4d) -CH (Rl5d) _
d
R13 is H or CH3;
Rl4d is selected from:
H, C1-C1p alkyl, aryl, or heteroaryl, wherein said
aryl or heteroaryl groups are optionally
substituted with 0-3 substituents selected from the
group Consisting of: C1-Cg alkyl, C1-C4 alkoxy,
aryl, halo, cyano, amino, CF3, and N02;
Rl5d is H or Rl6d
d 19d.
Y is -COR ,
Rl9d is selected from:
hydroxy, C1-C1p alkoxy,
methylcarbonyloxymethoxy-,
ethylcarbonyloxymethoxy-,
t-butylcarbonyloxymethoxy-,
cyclohexylcarbonyloxymethoxy-,
1-(methylcarbonyloxy)ethoxy-,
1-(ethylcarbonyloxy)ethoxy-,
1-(t-butylcarbonyloxy)ethoxy-,
1-(cyclohexylcarbonyloxy)ethoxy-,
i-propyloxycarbonyloxymethoxy-,
t-butyloxycarbonyloxymethoxy-,
1-(i-propyloxycarbonyloxy)ethoxy-,
1-(cyclohexyloxycarbonyloxy)ethoxy-,
1-(t-butyloxycarbonyloxy)ethoxy-,
dimethylaminoethoxy-,
diethylaminoethoxy-,
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(5-methyl-1,3-dioxacyclopenten-2-on-4-yl)methoxy-,
(5-(t-butyl)-1,3-dioxacyclopenten-2-on-4-yl)methoxy-,
(1,3-dioxa-5-phenyl-cyclopenten-2-on-4-yl)methoxy-, and
1-(2-(2-methoxypropyl)carbonyloxy)ethoxy-;
R2~d is H or CH3;
d
m is 0 or 1;
d
n is 1-4;
d
t is 0 or 1;
Ch i s
-~E~A~E Aa~E-As-E A~
AE E5 E~A8
A ,
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;
49
CA 02413957 2002-12-18
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alternatively, Ch is
p5
rE
E~~ 2'-\ s
A
s
A ~ ~Aa.
E \E
~E
A~
A1 is selected from the group: OH and a bond to Ln;
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~;
R1~ is =O;
~E A2
alternatively, Ch is A ;
A1 is NHS or N=C (R20 ) (R21 ) ;
E is a bond;
A2 i s NHR13 ;
CA 02413957 2002-12-18
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R13 is a heterocycle substituted with R17, the
heterocycle being selected from pyridine and
pyrimidine;
R1~ is selected from a bond to Ln, C(=0)NHR18 and
C ( =0 ) R18
R18 is a bond to Ln;
R~4 is selected from the group: -C02R25, -OR25, -S03H,
and -N ( R2 5 ) ~ ; and ,
R25 is independently selected at each occurrence from
the group: hydrogen and methyl.
[4] In another embodiment, the present invention
provides a kit according to Embodiment 1, wherein:
51
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Rlde is selected from:
N HN
-~dNR6d~ -~dNRsd
N
H N
R4d
N
-udNR6d~ ~ N-
UdNR6d
Rsd
N
N
-UdNRsd ~ -UdNR6d'~
N
S , H ,
N
-~dNR6d~
N
H
NH2
N NH2 N
~Ud ~ -Ud
S
, ,
N NH2
-Ud ~ -UdNR6d
NH
N
NH2
N-
!Ud
or N ;
wherein the above heterocycles are optionally
substituted with 0-2 substituents selected from the
52
CA 02413957 2002-12-18
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group: NH2, halogen, N02, CN, CF3, C1-C4 alkoxy, C1-
C6 alkyl, and C3-C7 cycloalkyl.
[5] In another embodiment, the present invention
provides a kit according to Embodiment 1, wherein
the compound of formula (I) is selected from the
group:
2- ( ( (4- (4- ( ( (3- (2- (2- (3- ( (6- ( (1-aza-2- (2-
sul f ophenyl ) vinyl ) amino ) ( 3 -
pyridyl))carbonylamino)propoxy)-
ethoxy)ethoxy)propyl)amino)sulfonyl)-
phenyl)phenyl)sulfonyl)amino)-3-((1-(3-(imidazole-
2-ylamino)propyl)(1H-indazol-5-
yl))carbonylamino)propanoic acid;
2-(2-aza-2-((5-(N-(1,3-bis(3=(2-(2-(3-(((4-(4-(((1-
carboxy-2-((1-(3-(imidazol-2-ylamino)propyl)(1H-
indazol-5-yl))carbonylamino)ethyl)amino)sulfonyl)-
phenyl)phenyl)sulfonyl)amino)propoxy)-
ethoxy)ethoxy)propyl)carbam~yl)propyl)carbamoyl)(2-
pyridyl))amino)vinyl)benzenesulfonic acid;
2-((6-((1-aza-2-(sulfophenyl)vinyl)amino)(3-
pyridyl))carbonylamino)-4-(N-(3-(2-(~-(3-(((4-(4-
(((1-carboxy-2-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonylamino)-
ethyl)amino)sulfonyl)phenyl)phenyl)sulfonyl)-
amino)propoxy)-
ethoxy)ethoxy)propyl)carbamoyl)butanoic acid;
3-((1-(3-(imidazole-2-ylamino)propyl)(1H-indazol-5-
yl))carbonylamino)-2-(((4-(4-(((3-(2-(2-(3-(2-
53
CA 02413957 2002-12-18
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(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-
cyclododecyl)-
acetylamino)propoxy)ethoxy)ethoxy)propyl)amino)sulf
onyl)phenyl)phenyl)sulfonyl)amino)propanoic acid;
2-(6-((6-((1-aza-2-(2-sulfophenyl)vinyl)-amino)(3-
pyridyl))carbonylamino)hexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonylamino)-propanoic acid;
2-((6-((1-aza-2-(2-sulfophenyl)vinyl)-amino)(3-
pyridyl))carbonylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-
yl))carbonylamino)propanoic acid;
[2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-Glu(2-(6-aminohexanoylamino)-
3-((1-(3-(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonyl-amino)propanoic acid)(2-(6-
aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid);
[2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-
benzenesulfonic acid]-Glu-bis-[Glu(2-(6-
Aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)(2-(6-aminohexanoylamino)-3-
((1-(3-(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonyl-amino)propanoic acid)];
2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-1-
cyclododecyl)acetyl-{2-(6-aminohexanoylamino)-3-
54
CA 02413957 2002-12-18
WO 01/98294 PCT/USO1/19794
((1-(3-(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))Carbonyl-amino)propanoiC acid};
2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-1-
Cyclododecyl)acetyl-Glu{2-(6-Aminohexanoylamino)-3-
((1-(3-(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonyl-amino)propanoiC acid}{2-(6-
Aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))Carbonyl-
amino)propanoiC acid};
_ _ H CO H
HO SH \ / / \ ~ N.~,~O..~O~.O~N O N N~C02H
O O
O ~v~ H
O~N C02
H H NH H
~~-N~ OQ / \ ~ / ~_N~O~.O.~O~J
N NN ~ ~ N NHOH OH
'~f
O O
O O
~N~OH
_ H CO H
H~ ~H \ / / \ S N.~.O~~ wO.~.N O ' ~ C02H
N N
H H O~O O
o~ ° Cu)COH
~'N
OH H
c
CNO ~~-COZH
O H CN NJ
~OwO.~O~N~.N~ ~, ~-CO2H
HC~3S' O
CA 02413957 2002-12-18
WO 01/98294 PCT/USO1/19794
CO N~C02H
Nor-' ~ ~ C02H
O
O
r
N ~~~ f
N>.N.J CO~H ~C02H
H H H CN N1
J~C02H
\/
H03S0
O ~ ~O
N Y OH
N ~ ~ ~ H NH
SO
Ny..N~ / 2 CO~H ~C02H
H H ~ I O O H CN N1
O.~N~O'~'O'i'O~N~"N~-' ~ J~C02H
H OH H O
O ~ ~O
N~OH
~ i H NH
N
SO
~>.,N.J z CO~H ~C02H
H H ~ I O O H CN N1
O.~N~O~.O.~O~N~.N~, ~ ~J-C02H
H H~ O
HN O
~i-Cyclodextrin
56
H03S O ~°
H03S~0~'N~O
OH H
CA 02413957 2002-12-18
WO 01/98294 PCT/USO1/19794
O
I
N
~N~N~ CO~ e-C02H
H H ~.N CN N
H _ p ~ C02H
~O~N~O n =114 ave
nH
2-(((4-(3-(N-(3-(2-(2-(3-(2-(1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)cyclododecylacetylamino)-6-
aminohexanoylamino)propoxy)ethoxy)ethoxy)propyl)-
carbamoyl)propoxy)-2,6-dimethylphenyl)-
sulfonyl)amino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonylamino)-
propionic acid salt;
O O
H H OH
N SO
N~N~ / 2 CO~H ~C02H
H H ~ I O O H CN N1
O.~JLH~O~.O.~O~H~. O ~ J~-CO H
2
O~NH
'N~P03H2
H20sPJ
HOOC1 O
N~N O
f O
HOOC~N~ H HN
HOO~H I ~ NN
HOOC.~N~COOH
~NH
~NH
NJ
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CA 02413957 2002-12-18
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2-({[4-(3-{N-[2-((2R)-3-Sulfo-2-{2-[1,4,7,10-tetraaza-
4,7,10-tris(carboxymethyl)cyclododecyl]-
acetylamino}propyl)ethyl]carbamoyl}propoxy)-2,6-
dimethylphenyl]sulfonyl}amino)(2S)-3-({1-[3-
(imidazol-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)propanoic Acid;
O O
~N~OH
HO~~ O
H H ~ I O HO~S~ O HO2C-. ,--~ ,-C02H
O~N~N~N~ H CN N1
H O H i~N~, ~N'.CO2H
O~fN'~NO N OO
O HO
s
~N NN I ~ H .N ~
~ ~ N~OH
O O
2-[({4-[4-({[2-((2R)-3-Sulfo-2-{2-[1,4,7,10-tetraaza-
4,7,10-tris(carboxymethyl)cyclododecyl]-
acetylamino}propyl)ethyl]amino}sulfonyl)phenyl]phen
yl}sulfonyl)amino](2S)-3-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)propanoic Acid;
(4S)-4-(N-{1-[N-(2-{4-[4-({[(1S)-1-carboxy-2-({1-[3-(2-
pyridylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-3-
carboxypropyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-
4,7,10-
tris(carboxymethyl)cyclododecyl]acetylamino}butanoi
c acid;
s8
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(4S)-4-(N-{1-[N-(2-{4-[4-({[(1S)-1-carboxy-2-({1-[3
(imidazol-2-ylamino)propyl](1H-indazol-5
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-3-
carboxypropyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-
4,7,10-
tris(carboxymethyl)cyclododecyl]acetylamino}butanoi
c acid;
(4S)-4-{N-[(1S)-1-(N-{1,3-bis[N-(2-{4-[4-({[(1S)-1-
carboxy-2-({1-[3-(imidazol-2-ylamino)propyl](1H-
indazol-5-yl)}carbonylamino)ethyl]amino}sulfonyl)-
3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]propy
1}carbamoyl)-3-carboxypropyl]carbamoyl}-4-(6-{2-
[1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)cyclododecyl]acetylamino}
hexanoylamino)butanoic acid;
(4S)-4-(N-{1-[N-(2-{4-[4-({[(1S)-1-carboxy-2-({1-[3-
(3,4,5,6-tetrahydropyrimidin-2-ylamino)propyl](1H-
indazol-5-yl)}carbonylamino)ethyl]amino}sulfonyl)-
3,5-dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-
3-carboxy propyl}carbamoyl)-4-{2-[1,4,7,10-
tetraaza-4,7,10-tris
(earboxymethyl)cyclododecyl]acetylamino}butanoic
acid;
(4S)-4-(N-{1-[N-(2-{4-[4-({[(1S)-1-carboxy-2-({1-methyl-
3-[3-(2-3,4,5,6-tetrahydropyridylamino)propyl] (1H-
indazol-6-yl)}carbonylamino)ethyl]amino}sulfonyl)-
3,5-dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-
3-carboxypropyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-
4,7,10-
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tris(carboxymethyl)cyclododecyl]acetylamino}butanoi
c acid;
(4S)-4-(N-{(1S)-1-[N-{2-{4-[4-{{[{1S)-1-carboxy-2-({1-
[2-(2-3,4,5,6-tetrahydropyridylamino)ethyl] {1H-
indazol-5-yl)}carbonylamino)ethyl]amino}sulfonyl)-
3,5-dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-
3-carboxy propyl}carbamoyl)-4-{2-[1,4,7,10-
tetraaza-4,7,10-tris
(carboxymethyl)cyclododecyl]acetylamino}butanoic
acid;
(2S)-2-{[(2,6-dimethyl-4-{3-[N-(2-{2-[1,4,7,10-tetraaza-
4,7,10-tris(carboxymethyl)cyclododecyl]acetyl-
amino}ethyl)carbamoyl]propoxy}phenyl)sulfonyl]amino
}-3-({2-[2-(2-3,4,5,6-
tetrahydropyridylamino)ethyl](2-hydro-1H-indazol-5-
yl)}carbonylamino)propanoic acid;
(4S)-4-{N-[(1S)-1-(N-{2-[({4-[4-({[(1S)-1-carboxy-2-({1-
[2-(2-3,4,5,6-tetrahydropyridylamino)ethyl] (1H-
indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)phenyl]
phenyl}sulfonyl)amino]ethyl}carbamoyl)-3-
carboxypropyl] carbamoyl}-4-{2-[1,4,7,10-tetraaza-
4,7,10-tris(carboxy-
methyl)cyclododecyl]acetylamino}butanoic acid;
(4S)-4-{N-[(1S)-1-(N-{2-[({4-[4-({[(1S)-1-carboxy-2-({1-
[3-(3,4,5,6-tetrahydropyrimidin-2-ylamino)
propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)
phenyl]phenyl}sulfonyl)amino]ethyl}carbamoyl)-3-
carboxy propyl]carbamoyl}-4-{2-[1,4,7,10-tetraaza-
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4,7,10-tris
(carboxymethyl)cyclododecyl]acetylamino}butanoic
acid;
(2S)-3-({3-[(imidazol-2-ylamino) methyl]-1-methyl(1H-
indazol-6-yl)}carbonylamino)-2-({[4-(4-{[(2-{2-
[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)
cyclododecyl]acetylamino}ethyl)amino]sulfonyl}pheny
1)phenyl]sulfonyl}amino)propanoic acid;
3-[(7-{3-[(6-{[(1E)-1-aza-2-(2-
sul f ophenyl ) vinyl ] amino } ( 3 -
pyridyl))carbonylamino]propoxy}-1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-yl))-
carbonylamino] (2S) -2-{ [ (2, 4, 6-
trimethylphenyl)sulfonyl]-amino}propanoic acid;
and
3-{[1-[3-(imidazol-2-ylamino)propyl]-7-(3-{2-[1,4,7,10-
tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]-
acetylamino}propoxy)(1H-indazol-5-
yl)]carbonylamino}-2-{[(2,4,6-
trimethylphenyl)sulfonyl]amino}propanoic acid;
or a pharmaceutically acceptable salt form thereof.
[6] 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.
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[7] In another embodiment, the present invention
provides a kit according to Embodiment 6, wherein
the ancillary ligands are tricine and TPPTS.
[8] In another embodiment, the present invention
provides a kit according to Embodiment 6, wherein
the reducing agent is tin(II).
[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,
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.
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[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 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,
prednimustine, picibanil, levamisole, teniposide,
improsulfan, enoeitabine, and lisuride.
[11.] 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.
[12] 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 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.
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[13] 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-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.
[14] 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;
wherein the targeting moiety is bound to the chelator
through 0-1 linking groups, and the targeting moiety is
a indazole nonpeptide that binds to a receptor that is
upregulated during angiogenesis.
[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~ 166Ho~ 177Lu~ 149pm~ 90y~ 212gi~
103pd~ 109pd~ 159Gd~ 140La~ 198Au~ 199Au~ 169yb~
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175yb~ 165Dy~ 166Dy.~ 67Cu~ 105Rh~ 111Ag~ and 192Ir;
and
b) a compound of the formula (I):
( Q ) d-Ln-Ch or ( Q ) d-Ln- ( Ch ) d'
(I)
wherein, Q is independently a compound of Formula (Ia)
or (Ib)
R1d Rlld
X4d
\ ~ X3d
W d-Xd Yd
~ X2d
i
X1d
Rlod
(Ia)
Rlld
X4d
~ X3d
Rlde I Wd-Xd Yd
~ X2d
i
X1d
R1
(Ib)
including stereoisomeriC foams thereof, or mixtures of
stereoisomeriC forms thereof, or pharmaceutically
acceptable salt or prodrug forms thereof wherein:
X1d is N, CH, C- Wd- Xd- Yd, or C-Ln;
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X2d is N, CH, or C- Wd- Xd- yd~
X3d is N, CRlld, or C- Wd- Xd- yd~
X4d is N or CRlld~
provided that when R1d is R~-de then one of X1d and X~d is
C- Wd- Xd- Yd, and when Rlod is Rlde then X3d is C- Wd-
Xd_ Yd:
R1d is selected from: Rlde, C1-C6 alkyl substituted with
0-1 RlSd or 0-1 R~ld, C3-C6 alkenyl substituted with
0-1 Rl5d or 0-1 R~ld, C3-C7 cycloalkyl substituted
with 0-1 RlSd or O-1 R~ld, C4-C11 CYcloalkylalkyl
substituted with 0-1 Rl5d or 0-1 R2ld, aryl
substituted with 0-1 Rl5d or 0-2 R~-1d or 0-1 R~~d,
and aryl(C1-C6 alkyl)- substituted with 0-1 Rl5d or
0-2 Rl~d or 0-1 R~ld:
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RZde is selected from:
-Ad N~~ ra
-Ud ~ ~6d ~ / Ud ~ ~6d ~ / A1 d
Bd ~ B1d
rd , ,
N-~ ~ WDd
Udt~6d~~
Ud ~ NR6d ~ ~ ~ Ld ~ d
Fd~E
Jd_-__Kd
NH
v~Fd
Ua f Visa ~ ~ Ed R2dN
Dd~ Ud/
i ,
rd
NHR~d ~R2d
N ~ Dd N
~/
d
Ua
Ua ~
NON/ ~ ~Fd
or
d Ed
Fd~E Ni ;
Ud
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Ad and Bd are independently -CH2-, -0-, -N(R2d)-, or -C(=0)-
A1d and B1d are independently -CH2- or -N(R3d)-;
Dd is -N(R2d)-, -0-, -S-, -C(=0)- or -S02-;
Ed_gd is _C (R4d) =C (R5d) _ ~ _N=C (R4d) _ ~ _C (R4d) -N-~ or
-C (R4d) 2C (R5d) 2-;
Jd, Kd, Ld and Md are independently selected from
-C(R4d)-, -C(R5d)- and -N-, provided that at least
one of Jd, Kd, Ld and Md is not -N-;
Rid is selected from: H, C1-C6 alkyl, (C1-C6
alkyl)carbonyl, (C1-C6 alkoxy)carbonyl; (C1-C6
alkyl)aminocarbonyl, C3-C6 alkenyl, C3-C7
cycloalkyl, C4-C11 cycloalkylalkyl, aryl,
heteroaryl(C1-C6 alkyl)carbonyl,
heteroarylcarbonyl,
aryl(C1-C6 alkyl)-, (C1-C6 alkyl)carbonyl-,
arylcarbonyl, C1-C6 alkylsulfonyl, arylsulfonyl,
aryl(C~-C6 alkyl)sulfonyl, heteroarylsulfonyl,
heteroaryl(C1-C6 alkyl)sulfonyl, aryloxycarbonyl,
and aryl(C1-C6 alkoxy)carbonyl, wherein said aryl
groups are substituted with 0-2 substituents
selected from the group: C1-Cg alkyl, C1-C4
alkoxy, halo, CF3, and nitro;
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R3d is selected from: H, C1-C6 alkyl, C3-C7 cycloalkyl,
C4-C11 cycloalkylalkyl, aryl, aryl(C1-C6 alkyl)-,
and heteroaryl(C1-C6 alkyl)-;
R4d and R5d are independently selected from: H, C1-C4
alkoxy, NR2dR3d, halogen, N02, CN, CF3, C1-C6 alkyl,
C3-C6 alkenyl, C3-C7 cycloalkyl, C4-C11
cycloalkylalkyl, aryl, aryl(C1-C6 alkyl)-~ (C1-C6
alkyl)carbonyl, (C1-C6 alkoxy)carbonyl, and
arylcarbonyl, or
alternatively, when substituents on adjacent atoms, R4d
and R5d can be taken together with the carbon atoms
to which they are attached to form a 5-7 membered
carbocyclic or 5-7 membered heterocyclic aromatic
or non-aromatic ring system, said carbocyclic or
heterocyclic ring being optionally substituted with
0-2 groups selected from: C1-C4 alkyl, C1-C4
alkoxy, halo, cyano, amino, CF3, and N02;
Ud is selected from:
-(CH2)nd-r
-{CH2)nd{CR7d=CR8d)(CH~)md-,
-(CH2)nd(C=C)(CH2)md-.
- ( CH2 ) tdS~ ( CH2 ) md- .
-{CH2)nd0(CH2)md-~
- ( CH2 ) ndN ( R6d ) ( CH2 ) md- .
- ( CH2 ) ndC ( =0 ) ( CH2 ) md- .
- ( CHI ) nd ( C=0 ) N { R6d ) ( CHI ) md_
- ( CH2 ) ndN ( R6d ) ( C=0 ) ( CH2 ) md- , and
-(CH2)nds(0)pd{CH2)md_~
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wherein one or more of the methylene groups in Ud is
optionally substituted with R7d;
Qd is selected from 1,2-cycloalkylene, 1,2-phenylene,
1,3-phenylene, 1,4-phenylene, 2,3-pyridinylene,
3,4-pyridinylene, 2,4-pyridinylene, and 3,4-
pyridazinylene;
R6d is selected from: H, C1-C4 alkyl, and benzyl;
R7d and R8d are independently selected from: H, C1-Co
alkyl, C3-C7 cycloalkyl, C4-C11 cycloalkylalkyl,
aryl, aryl(C1-C6 alkyl)-,
and heteroaryl(Cp-C& alkyl)-;
Rlod is selected from: H, Rlde~ C1-C4 alkoxy substituted
with 0-1 R2ld, N(R6d)2, halogen, N02, CN, CF3,
CO~Rl7d, C (=O) Rl7d, CONR17dR20d~ _~02R17d~
-SO~NR~-7dR20d~ C1-C6 alkyl substituted with 0-1 Rl5d
or 0-1 R2ld, C3-C6 alkenyl substituted with 0-1 RlSd
or 0-1 R~ld, C3-C7 cycloalkyl substituted with 0-1
Rl5d or 0-1 R2ld, C4-C11 cycloalkylalkyl substituted
with 0-1 Rl5d or 0-1 R2ld, aryl substituted with 0-1
Rl5d or 0-2 Rlld or 0-1 R2ld, and aryl(C1-C6 alkyl)-
substituted with 0-1 Rl5d or 0-2 Rlld or 0-1 R2ld;
Rlode is selected from: H, C1-C4 alkoxy substituted with
0-1 R2ld~ N (R6d) ~ ~ halogen, N02, CN, CF3, C02R17d,
C (=0) Rl.7d~ CONR17dR20d~ -S02R17d~ _S02NR17dR20d~ C1-C6
alkyl substituted with 0-1 Rl5a or O-1 R~ld, C3-C6
alkenyl substituted with 0-1 Rl5d or 0-1 R.2ld~ C3-C7
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cycloalkyl substituted with 0-1 Rl5d or 0-1 R2ld
C4-C11 cycloalkylalkyl substituted with 0-1 Rl5d or
O-1 R2ld, aryl substituted with 0-1 Rl5d or O-2 Rlld
or 0-1 R2ld, and aryl(C1-C6 alkyl)- substituted with
0-1 RlSd or 0-2 Rlld or 0-1 R2ld;
Rlld is selected from H, halogen, CF3, CN, N02, hydroxy,
NR2dR3d, C1-C4 alkyl substituted with 0-1 R2ld, C1-C4
alkoxy substituted with 0-1 R2ld, aryl substituted
with 0-1 R2ld, aryl(C1-C6 alkyl)- substituted with
0-1 R2ld, (C1-C4 alkoxy)carbonyl substituted with 0-
1 R2ld~ (C1-C4 alkyl)carbonyl substituted with 0-1
R2ld, C1-C4 alkylsulfonyl substituted with 0-1 R2ld,
and C1-C4 alkylaminosulfonyl substituted with 0-1
R2ldj
Wd is selected from:
_(C(Rl2d)2)~dC(=0)N(Rl3d)_~ and
_C(=O)_N(Rl3d)_(C(Rl2d)2)qd_;
Xd is -C (Rl2d) (Rl4d) _C (Rl2d) (Rl5d) _; or
alternatively, Wd and Xd can be taken together to be
( CHI ) qdC ( _~ ) -N~.. ~ N-Rl8d
Rl2d is selected from H, halogen, C1-C6 alkyl, C2-C6
alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl,
C4-C1p cycloalkylalkyl, (C1-C4 alkyl)carbonyl, aryl,
and aryl(C1-C6 alkyl)-;
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Rl3d is selected from H, C1-C6 alkyl, C3-C7
cycloalkylmethyl, and aryl(C1-Cg alkyl)-;
Rl4d is selected from:
H, C1-C6 alkylthio(C1-C6 alkyl)-, aryl(C1-C1o
alkylthioalkyl)-, aryl(C1-C1o alkoxyalkyl)-, C1-C1o
alkyl, C1-C1o alkoxyalkyl, C1-C6 hydroxyalkyl,
C2-C1p alkenyl, C2-C1o alkynyl, C3-C1p cycloalkyl,
C3-C1o cycloalkylalkyl, aryl(CZ-C6 alkyl)-,
heteroaryl(C1-C6 alkyl)-, aryl, heteroaryl, C02R17d,
C (=O) R~-7d, and CONR17dR2od, provided that any of the
above alkyl, cycloalkyl, aryl or heteroaryl groups
may be unsubstituted or substituted independently
with 0-1 Rl6d or 0-2 R1~-d
Rl5d is selected from:
H, Rl6d, C1-C1o alkyl, C1-C1o alkoxyalkyl,
C1-C1o alkylaminoalkyl, C1-C1o dialkylaminoalkyl,
(C1-C1o alkyl)carbonyl, aryl(C1-C6 alkyl)carbonyl,
C1-C1o alkenyl, C1-CZO alkynyl ,C3-C1o cycloalkyl,
C~-C1o cycloalkylalkyl, aryl(C1-C6 alkyl)-,
heteroaryl(C1-C6 alkyl)-, aryl, heteroaryl, C02R1~d,
C (=0) Rl7d~ CONR17dR2od~ S02R17d~ and S02NR17dR20d~
provided that any of the above alkyl, cycloalkyl,
aryl or heteroaryl groups may be unsubstituted or
substituted independently with 0-2 R.lld
Yd is selected from:
-CORl9d, -S03H, -P03H, tetrazolyl, -CONHNHS02CF3, -
CONHS02R17d, -CONHS02NHR17d, -NHCOCFg, -
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NHCONHS02R17d, -NHS02R17d, -OP03H2, -OS03H, -P03H2, -
S03H, -S02NHCORI7d, -S02NHC02R17d,
N
N~ ~ N N~ ~ C F3
N
H , and
Rl6d is selected from:
_N (R20d) _C (=O) -O_Rl7d~
_N(R20d) -C (;O) _Rl7d~
-N(R20d) _C (i0) _NH_Rl7d~
-N (R20d) S02-Rl7d~ and
-N(R20d) S02-NR2odR17d~
Rl7d is selected from:
C1-C10 alkyl optionally substituted with a bond to
Ln, C3-C11 cycloalkyl optionally substituted with a
bond to Ln, aryl(C1-C~ alkyl)- optionally
substituted with a bond to Ln, (C1-C6 alkyl)aryl
optionally substituted with a bond to Ln,
heteroaryl(C1-C6 alkyl)- optionally substituted
with a bond to Ln, (C1-Cg alkyl)heteroaryl
optionally substituted with a bond to Ln,
biaryl(C1-C6 alkyl)- optionally substituted with a
bond to Ln, heteroaryl optionally substituted with
a bond to Ln, aryl optionally substituted with a
bond to Ln, biaryl optionally substituted with a
bond to Ln, and a bond to Ln, wherein said aryl,
biaryl or heteroaryl groups are also optionally
substituted with 0-3 substituents selected from the
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group consisting of: C1-C4 alkyl, C1-Cg alkoxy,
aryl, heteroaryl, halo, cyano, amino, CF3, and N02;
Rl8d is selected from:
-H,
-C ( =0 ) -O-R17 d
-C ( =0 ) -R17 d
-C (=0) -NH-Rl7d,
-S02-Rl7d, and
-S02-NR~OdRl7d~
Rl9d is selected from: hydroxy, C1-C1o alkyloxy,
C3-C1~, cycloalkyloxy, aryloxy, aryl(C1-Cg alkoxy)-,
C3-C1o alkylcarbonyloxyalkyloxy, C3-C1o
alkoxycarbonyloxyalkyloxy,
C2-C1o alkoxycarbonylalkyloxy,
C5-C1o cycloalkylcarbonyloxyalkyloxy,
C5-C1o cycloalkoxycarbonyloxyalkyloxy,
C5-C1o cycloalkoxycarbonylalkyloxy,
C7-C1~ aryloxycarbonylalkyloxy,
Cg-C1~ aryloxycarbonyloxyalkyloxy,
Cg-C12 arylcarbonyloxyalkyloxy,
C5-C1o alkoxyalkylcarbonyloxyalkyloxy,
C5-C1o (5-alkyl-1,3-dioxa-cyclopenten-2-one-
yl)methyloxy, C1p-C1g (5-aryl-1,3-dioxa-
cyclopenten-2-one-yl)methyloxy, and
(Rlld) (Rl2d) N- (C1_C1p alkoxy) -.
R2od is selected from: H, C1-C6 alkyl, C3-C7 cycloalkyl,
C4-Cll cycloalkylalkyl, aryl, aryl(C1-C6 alkyl)-,
and heteroaryl(C1-C6 alkyl)-;
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R2ld is selected from: COOH and NR6d2;
d
m is 0-4;
d
n is 0-4;
d
t is 0-4;
d
p is 0-2;
d
q is 0-2; and
d
r is 0-2;
with the following provisos:
d d d d
(1) t , n , m and q are chosen such that the numlaer of
d
atoms connecting R1d and Y is in the range of
10-14; and
d d d
(2) n and m are chosen such that the value of n plus
d d
m is greater than one unless U is
d d d
- (CH2) t Q (CH2)m -~
or Q is a peptide selected from the group:
J
~~L~M K~R'R4
R R and ~ M' ;
R1 is L-valine, D-valine or L-lysine optionally
substituted on the ~ amino group with a bond to Ln;
R~ is L-phenylalanine, D-phenylalanine,
D-1-naphthylalanine, 2-aminothiazole-4-acetic acid
CA 02413957 2002-12-18
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or tyrosine, the tyrosine optionally substituted on
the hydroxy group with a bond to Ln;
R3 is D-valine;
R4 is D-tyrosine substituted on the hydroxy group with a
bond to Ln;
provided that one of R1 and R2 in each Q is substituted
with a bond to Ln, and further provided that when
R2 is 2-aminothiazole-4-acetic acid, K is
N-methylarginine;
provided that at least one Q is a compound of Formula
(Ia) or (Ib) ;
d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
d' is 1-100;
Ln is a linking group having the formula:
( (~1)h-(CR.6R7)g)x-(~)k-( (CR6aR7a)g,_ (W)h, )x':
W is independently selected at each occurrence from the
group: O, S, NH, NHC(=0), C(=0)NH, NR8C(=0),
C (=0)N Rg, C (=O) , C (=O) O, OC (=O) , NHC (=S)NH,
NHC(=O)NH, 502, S02NH, (OCH2CH2)S, (CH2CH20)s~,
(OCH2CH2CH2) 5~~, (CH2CH2CH20) t, and (aa) t-;
as is independently at each occurrence an amino acid;
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2 is selected from the group: aryl substituted with 0-3
Rlo~ C3-1o cYcloalkyl substituted with 0-3 Rlo, and
a 5-10 membered heterocyclic ring system containing
1-4 heteroatoms independe-ntly selected from N, S,
and 0 and substituted with 0-3 Rlo
R6, R6a, R~, R7a, and R8 are independently selected at
each occurrence from the group: H, =0, COON, 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 ( =O ) NHR1 ~ , NHR1 ~- , R11,
and a bond to Ch;
R1o is independently selected at each occurrence from
the group: a bond to Ch, COOR11, C(=O)NHR11~
NHC (=0) R11, OH, NHR11, S03H, P03H, -OP03H2, -OS03H,
aryl substituted with 0-3 R11, C1_5 alkyl
substituted with 0-1 R12, C1_5 alkoxy substituted
with 0-1 R12, 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, -OP03H~, alkyl substituted with 0-1
R~2, aryl substituted with 0-1 R12, a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and O and
substituted with 0-1 R12, C3-so cYcloalkyl
substituted with 0-1 R12, polyalkylene glycol
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substituted with 0-1 R12, carbohydrate substituted
with 0-1 R1~, cyclodextrin substituted with 0-1
R1~, amino acid substituted with 0-1 R1~,
polycarboxyalkyl substituted with 0-1 R1~,
polyazaalkyl substituted with 0-1 R12, peptide
substituted with -C(=0)-(CH2)5-NHR12, and peptide
substituted with 0-1 R12, wherein the peptide is
comprised of 2-10 amino acids, C1-5 alkyl
substituted with 3,6-0-disulfo-B-D-
galactopyranosyl, bis(phosphonomethyl)glycine, and
a bond to Ch;
R12 is a bond to Ch;
k is selected from 1, and2;
0,
h is selected from 1, and2;
0,
h' is selected from 1, and ;
0, 2
g is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
0,
g' is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and
0,
10 ;
s is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
0,
s' is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and
0,
10;
s" is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and
0,
10;
t is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
0,
t' is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and
0,
10;
x is selected from 1, 2, 3, 4, and
0, 5;
x' is selected from 1, 2, 3, 4, and ;
0, 5
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Ch is a metal bonding unit having a formula selected
from the group:
-~E A2
A , A ,
p5
r
AY 'As
A
~E~A~E ~4~E-~c~E A
A AE A5 A8 A
and
A1, A2 , A3 , A4 , A5 , A6 , A7 , and A8 are independent 1y
selected at each occurrence from the group: NR13,
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
alkyl substituted with 0-3 R1~, aryl substituted
with 0-3 R1~, C3_1fl 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, Cg-1o aryl-C1_1o alkyl substituted with 0-3 R1~,
C1-10 alkyl-C6_1o aryl- substituted with 0-3 R1~,
and a 5-10 membered heterocyclic ring system
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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-C1o 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-C1-1o 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 0
and substituted with 0-3 R17, and an electron,
provided that when one of R13 or R~-4 is an
electron, then the other is also an electron;
alternatively, R13 and R14 combine to form =C (R2o) (R21) ;
R15 and R16 are each independently selected from the
group: a bond to Ln, -0H, C1-C10 alkyl substituted
with 0-3 R1~, C1-C10 alkyl substituted with 0-3
R1~, aryl substituted with 0-3 R1~, C3_so
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
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aryl-C1-1o alkyl substituted with 0-3 R1~, C1_1o
alkyl-C6_~o 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, Cl, Br, I, -CF3,
-CN, -C02R18, -C(=0)R18, -C(=0)N(R18)2, -CHO,
-CH20R18, -OC(=0)R18~ _OC(=O)ORl8a~ _OR18~
-OC(=O)N(R18)2, -NR19C(=0)R18, -NR19C(=O)ORl8a~
-NR19C(=0)N(R.18)2, -NR19S02N(R18)2, -NR19SO~R18a~
_S03H~ _SO~Rl8a~ _SR18~ _S(=O)Rl8a~ -S02N(R18)~~
-N(R18)2, -NHC(=S)NHR18, =NOR18, NO~, -C(=0)NHOR18,
-C(=O)NHNR18R18a, -OCH~C02H,
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 O;
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, vitro,
cyano, and trifluoromethyl;
Pg is a thiol protecting group;
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R~o and R~1 are independently selected from the group:
H, C1-C10 alkyl, -CN, -CO~R~5, -C (=0) R25,
-C(=0)N(R25)2, C2-C1o 1-alkene substituted with 0-3
R23, C2-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 R23;
alternatively, R2o and R~1, taken together with the
divalent carbon radical to which they are attached
form:
R22 R22
.a b
R23 R23
n
R~2 and R23 are independently selected from the group:
H, R~4, C1-C10 alkyl substituted with 0-3 R~4,
C~-C1p alkenyl substituted with 0-3 R~4, C~-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
R~4, and C3_1o carbocycle substituted with 0-3 R24;
alternatively, R~~, 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 i s 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(=O)N(R25)2wN(R25)3+, -CH20R25,
-OC (=0) R25, -OC (=O) OR25a~ _OR25~ -OC (=0)N (R25) 2,
-NR26C (=0) R25, -NR26C (=0) OR25a~ _NR26C (=0) N (R25) 2
-NR26S02N(R25)2, _NR26SO~R25a~ _S03H~ _S02R25a~
_SR25~ _S(=O)R25a~ _S02N(R25)2~ _N(R25)2~ =NOR25,
-C(=0)NHOR25, -OCH~C02H, 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.
[16] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical
composition according to Embodiment 15, wherein
wherein the radioisotope is 99mTc or 95Tc, the
radiopharmaceutical further comprises a first
ancillary ligand and a second ancillary ligand
capable of stabilizing the radiopharmaceutical.
[17] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical
composition according to Embodiment 15, wherein the
radioisotope is 99mTc .
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[18] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical
composition according to Embodiment 17, wherein the
radiopharmaceutical is selected from the group:
99mTc ((((4-(4-(((3-(2-(2-(3-((6-(diazenido)(3-
pyridyl))carbonylamino)propoxy)-
ethoxy)ethoxy)propyl)amino)sulfonyl)-
phenyl)phenyl)sulfonyl)amino)-3-((1.-(3-(imidazole-
2-ylamino)propyl)(1H-indazol-5-
yl))carbonylamino)propanoic acid) (tricine)(TPPTS);
99mTc (2-(2-( (5-(N-(1,3-bis(3-(2-(2-(3-( ( (4-(4-( ( (1-
carboxy-2-((1-(3-(imidazol-2-ylamino)propyl)(1H-
indazol-5-yl))carbonylamino)ethyl)amino)sulfonyl)-
phenyl)phenyl)sulfonyl)amino)propoxy)-
ethoxy)ethoxy)propyl)carbamoyl)propyl)carbamoyl)(2-
pyridyl))2-diazenido) (tricine)(TPPTS);
g9mTC (2-((6-(diazenido)(3-pyridyl))carbonylamino)-4-tN-
(3- (2- (2- (3- ( ( (4- (4- ( ( (1-carboxy-2- ( (1- (3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))-
carbonylamino)ethyl)amino)sulfonyl)-
phenyl)phenyl)sulfonyl)amino)propoxy)-
ethoxy)ethoxy)propyl)carbamoyl)butanoic acid)
(tricine)(TPPTS);
99mTc (2-(6-((6-(diazenido)(3-
pyridyl))carbonylamino)hexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonylamino)-propanoic acid)
(tricine)(TPPTS);
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99mTc (2-{(6-{diazenido)(3-pyridyl))carbonylamino)-3-
((1-(3-(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonylamino)propanoic acid (tricine)(TPPTS);
9~mTc [2-[[[5-[carbonyl]-2-pyridinyl]diazenido]-Glu(2-
(6-aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)(2-(6-aminohexanoylamino)-3- -
{(1-(3-(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonyl-amino)propanoic acid))
(tricine)(TPPTS); and
99mTc ([2-[[[5-[carbonyl]-2-pyridinyl]diazenido]-Glu
bis-[Glu(2-(6-aminohexanoylamino)-3-((1-(3
(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonyl-amino)propanoic acid)(2-(6-
aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)]) (tricine)(TPPTS).
[19] In. another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 15, wherein the radioisotope is
111In.
[20] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 15, wherein, the
radiopharmaceutical is selected from the group:
8s
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H
H H
O O
N ~ I ~ H HOH
N~N~ O-~ O
H H \ ~ BS03H
O'~H~.N1~NH ~.O-~
O ~/'
n~ ~~~O
~~~~0
OS'-O
O O
N~OH
/ N ~ I ~ H NH
O=~ O
HRH ~ I HO S
O I~ ~ O
O.~N-w.NI~N~ H O
H O H i, N
H O
O~f N '~ N N O ~~~~~0
O HO
H H ~I s O O
~NN ~ \ H -NO
I i N~OH
O O
O O
N ~ I ~ H H ~OH / / \ S.N.~~NO N
N~N~ 002 HC~3~ C3~ ~-, ~,~
H H ~~~~0
O~-O
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O O
N~'OH
N I N ~ I ~ H S02
H~ ~ C02H
~I
O.~N~.N~NO NH ~-O-
H O H~ ~N~ I, NCO
C02H ~fLll~~O
O O
O O
N ~J I ~ H H OH
N~N~ S02
H H \ I C02H
O~NwN~NO NH ~-O-
H O H~ ~~ ~.N~O
C02H ~21I~~O
O O
O O
~N~OH
~ H NH
~J'N~N ~ SO2
H H ~ I C02H
O~N~N~NO NH
H O H~ ~f~LN~O
C02H ~fLll~~O
O O
O O
I , H HOH
~N S02
H ~I
O.~N~.N
H
and
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[21] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical
composition according to Embodiment 15, wherein the
radioisotope is 153gm.
[22] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical
composition according to Embodiment 15, wherein the
radioisotope is l7~Lu.
[23] In another embodiment, the present invention
provides a therapeutic composition according to
Embodiment 22, wherein the radiopharmaceutical is
U v .
[24] A therapeutic radiopharmaceutical composition
according to Embodiment 15, wherein the radioisotope is
90y.
[25] In another embodiment, the present invention
provides a therapeutic composition according to
Embodiment 24, wherein, the radiopharmaceutical is
selected from the group:
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O O
N ~ ~~ H N OH
O-~ O
HRH ~ ~ S03H
O H
O'~N~.N1~NH O
H O _~' ~~~
_Nl
'fin I'.f'~,~o
o~-o
0 0
N ~
N~N~ 002 HC~3~ C~-~~O
/ ~ i H HN~.SOH / / \ SN.~NO N
H H ~~I~~O
and
O ~ yO~
~ ~ N~OH
H NH
NON ~ S02
H H \ ~ CO2H
O.~N~.N~NO NH
H O H~ ~~.N10
C02H ~~1I~~O
O O
[26] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 29, wherein the targeting moiety
is a indazole and the receptor is ocv~33 or ocv~i5.
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[27] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 14, 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,
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.
[28] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 14, wherein radiosensitizer
agent is selected from the group consiting 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-
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nitroimidazol-1-Y1)-3-(1-piperidinyl)-2-propanol, and 1-
(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol.
[29] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 14, wherein the radioisotope is
selected from the group 33p, 1252, 186Re, 188Re, 153Sm,
166go, 177Lu, 149pm, 90y, 212Bi, 103pd, 109pd, 159Gd,
140La, 198Au, 199Au, 169yb, 175yb, 165DY, 166DY, 67Cu,
105Rh, 111Ag, and 192Ir, and the linking group is
present between the non-peptide targeting moiety and
chelator.
[30] 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
indazole nonpeptide that binds to a receptor that is
upregulated during angiogenesis, and the radioisotope is
a radioisotope selected from the group: 33p, 125I~
186Re, 188Re, 153gm, 166go, 177Lu, 149pm, 90y, 212$i,
103pd, 109pd, 159Gd, 140La, 198Au, 199Au, 169yb, 175yb,
165DY, 166DY, 67Cu, 105Rh, 111Ag, and 192Ir or a
pharmaceutically acceptable salt thereof; and
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at least one agent selected from the group consisting of
an anti-cancer agent and a radiosensitizer agent, or a
pharmaceutically acceptable salt thereof.
[31] In another embodiment, the present invention
provides a method according to Embodiment 30, wherein
the targeting moiety is an indazole non-peptide and the
receptor is ocv(33 or ocv(35.
[32] In another embodiment, the present invention
provides a method according to Embodiment 30, wherein
the therapeutic radiopharmaceutical comprises:
a) a radioisotope selected from the group: 33p, 125=
186Re~ 188Re, 153gm~ 166go~ 177Lu~ 149pm~ 90y~ 212gi~
103pd~ 109pd~ 159Gd~ 140La~ 198Au~ 199Au~ 169yb~
175yb~ 165DY, 166pY~ 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 independently a compound of Formula (Ia)
or (Ib)
R1d Rlld
X4d
~ X3d
W d-Xd Yd
X2d
X1d
n
(Ia)
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Rlld
X4d
~ X3d
Rlde I Wd-Xd Yd
~ X2d
X1d
Ri uae
(Ib)
including stereoisomeriC forms thereof, or mixtures of
stereoisomeric forms thereof, or pharmaceutically
acceptable salt or prodrug forms thereof wherein:
X1d is N, CH, C- Wd- Xd_ yd~ or C-Ln;
X2d is N, CH, or C- Wd- Xd_ yd~
X3d is N, CRlld~ or C- Wd- Xd_ yd~
X4d is N or CRlld~
provided that when R1d is Rlde then one of X1d and X2d is
C- Wd- Xd_ yd~ and when Rlod is Rlde then X3d is C- Wd
Xd_ yd~
R1d is selected from: Ride, C~_C6 alkyl substituted with
0-1 R~Sd or O-1 R2ld~ C3_C6 alkenyl substituted with
0-1 RlSd or 0-1 R2ld, C3-C7 CyCloalkyl substituted
with 0-1 RlSd or 0-1 R2ld~ C4_C~1 cycloalkylalkyl
substituted with 0-1 Rl5d or 0-1 R~ld, aryl
substituted with 0-1 Rl5d or 0-2 Rlld or 0-1 R2ld
and aryl(C~-C6 alkyl)- substituted with 0-1 Rl5d or
0-2 Rlld or 0-1 R2ld
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Rlde is selected from:
-Ad N~) rd
-Ud ~ ~6d ) / Ud ~ ~6d ) ~ A1 d
Bd ~ B1d
rd
_ N\ Dd
d 6d / ~ d '~ ~ ~6d )
U ( NR ) L a
Fd~E
Jd-Ka
NH
w.Fa
Ua ~ ~sd ) ~ Ed RaaN
Day a/
,U
rd
NHR.2d ~R2d
N ~ Dd N
a
Ua
s
Udr
~.N/ ~ ~Fd
°r
a ~ Ea
Fa~E ~N
Ua
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Ad and Bd are independently -CH2-, -0-, -N(R2d)-, or -C(=0)-
A1d and B1d are independently -CH2- or -N(R3d)-;
Dd is -N(R2d)-, -0-, -S-, -C(=0)- or -S02-;
Ed-gd is _C (R4d) =C (R5d) _ ~ _N=C (R4d) -~ -C (R4d) =N-~ or
-C(R4d)2C(R5d)2-;
Jd, Kd, Ld and Md are independently selected from
-C(R4d)-, -C(R5d)- and -N-, provided that at least
one of Jd, Kd, Ld and Md is not -N-;
Rid is selected from: H, C1-C6 alkyl, (C1-C6
alkyl)carbonyl, (C1-C6 alkoxy)carbonyl; (C1-C6
alkyl)aminocarbonyl, C3-C6 alkenyl, C3-C~
cycloalkyl, C4-C11 cycloalkylalkyl, aryl,
heteroaryl(C1-C6 alkyl)carbonyl,
heteroarylcarbonyl,
aryl(C1-C6 alkyl)-~ (C1-C6 alkyl)carbonyl-,
arylcarbonyl, C1-C6 alkylsulfonyl, arylsulfonyl,
aryl(C1-C6 alkyl)sulfonyl, heteroarylsulfonyl,
heteroaryl(C1-C6 alkyl)sulfonyl, aryloxycarbonyl,
and aryl(C1-Cg alkoxy)carbonyl, wherein said aryl
groups are substituted with 0-2 substituents
selected from the group: C1-C4 alkyl, C1-C4
alkoxy, halo, CF3, and vitro;
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R3d is selected from: H, C1-Cg alkyl, Cg-C7 cycloalkyl,
Cg-C11 cycloalkylalkyl, aryl, aryl(C1-C6 alkyl)-,
and heteroaryl(C1-C6 alkyl)-;
R4d and R5d are independently selected from: H, C1-C4
alkoxy, NR2dR3d, halogen, N02, CN, CF3, C1-C6 alkyl,
C3-C6 alkenyl, C3-C7 cycloalkyl, C4-C1~
cycloalkylalkyl, aryl, aryl(C1-C6 alkyl)-~ (C1-C6
alkyl)carbonyl, (CZ-C6 alkoxy)carbonyl, and
arylcarbonyl, or
alternatively, when substituents on adjacent atoms, R4d
and R5d can be taken together with the carbon atoms
to which they are attached to form a 5-7 membered
carbocyclic or 5-7 membered heterocyclic aromatic
or non-aromatic ring system, said carbocyclic or
heterocyclic ring being optionally substituted with
0-2 groups selected from: C1-C4 alkyl, C1-C4
alkoxy, halo, cyano, amino, CF3, and N02;
Ud is selected from:
-(CH2)nd-
- ( CH2 ) nd ( CR.7d=CR8d ) ( CH2 ) md-
-(CH2)nd(C=C)(CH2)md-.
- ( CH2 ) tdQ ( CH2 ) md- .
- ( CH2 ) nd0 ( CH2 ) md- .
- ( CH2 ) ndN ( R6d ) ( CH2 ) md- r
- ( CH2 ) ndC ( =0 ) ( CH2 ) md_
- ( CH2 ) nd ( C=0 ) N ( R6d ) ( CHs ) md-
- ( CH2 ) ndN ( R6d ) ( C=0 ) ( CH2 ) md_ ~ and
-(CH~)ndS(0)pd(CH2)md-J
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wherein one or more of the methylene groups in Ud is
optionally substituted with R7d;
Qd is selected from 1,2-cycloalkylene, 1,2-phenylene,
1,3-phenylene, 1,4-phenylene, 2,3-pyridinylene,
3,4-pyridinylene, 2,4-pyridinylene, and 3,4-
pyridazinylene;
R6d is selected from: H, C1-C4 alkyl, and ben~yl;
R7d and R8d are independently selected from: H, C1-C6
alkyl, C3-C7 cycloalkyl, C4-C11 cycloalkylalkyl,
aryl, aryl(C1-C6 alkyl)-,
and heteroaryl(Cp-C6 alkyl)-;
Rl~d is selected from: H, R.lde~ C1-C4 alkoxy substituted
with 0-1 R2ld, N(R6d)2, halogen, N02, CN, CF3,
C02R17d, C(=0)Rl7d, CONR17dR20d~ _S02R17d~
-S02NR17dR20d~ C1-C6 alkyl substituted with 0-1 RlSd
or O-1 R2ld~ C3-C6 alkenyl substituted with 0-1 Rl5d
or O-1 R2ld, C3-C7 cycloalkyl substituted with 0-1
Rl5d or 0-1 R2ld, C4-C11 cycloalkylalkyl substituted
with 0-1 Rl5d or 0-1 R2ld, aryl substituted with 0-1
Rl5d or 0-2 Rlld or 0-1 R2ld, and aryl(C1-C6 alkyl)-
substituted with 0-1 RlSd or 0-2 Rlld or 0-1 R2ld;
Rlode is selected from: H, C1-C4 alkoxy substituted with
0-1 R2ld, N(R6d)2, halogen, N02, CN, CF3, C02R17d,
C (=0) Rl7d~ CONR17dR20d~ -S02R17d~ _ar02NR17dR20d~ 01-C6
alkyl substituted with 0-1 Rl5d or 0-1 R2ld, C3-C6
alkenyl substituted with 0-1 Rl5d or 0-1 R2ld, C3-C7
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cycloalkyl substituted with 0-1 Rl5d or 0-1 R2ld
C4-C11 cycloalkylalkyl substituted with 0-1 Rl5d or
0-1 R2ld, aryl substituted with 0-1 Rl5d or 0-2 Rlld
or O-1 R2ld, and aryl(C1-C6 alkyl)- substituted with
0-1 RlSd or 0-2 R2ld or 0-1 R2ld;
Rlld is selected from H, halogen, CF3, CN, NO~, hydroxy,
NR2dR3ds C1_C4 alkyl substituted with 0-1 R2ld, C1-C4
alkoxy substituted with 0-1 R~ld, aryl substituted
with 0-1 R2ld, aryl(C1-C6 alkyl)- substituted with
0-1 R~ld, (C1-C4 alkoxy)carbonyl substituted with 0-
1 R2ld, (C1-C4 alkyl)carbonyl substituted with 0-1
R2ld, C1-C4 alkylsulfonyl substituted with 0-1 R2ld,
and C1-C4 alkylaminosulfonyl substituted with 0-1
R~ld;
Wd is selected from:
-(C(Rl2d)2)qdC(=0)N(Rl3d)_~ and
_C (=p) _N (Rl3d) _ (C (Rl2d) 2 ) qd-;
Xd is -C (Rl2d) (Rl4d) -C (Rl2d) (Rl5d) -; or
alternatively, Wd and Xd can be taken together to be
~- ( ~H2 ~ qdC ( -~ ~ _N N_Rlad
Rl~d is selected from H, halogen, C1-C6 alkyl, C2-C6
alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl,
C4-C1o cycloalkylalkyl, (C1-C4 alkyl)carbonyl, aryl,
and aryl(C1-C6 alkyl)-;
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Rl3d is selected from H, C1-C6 alkyl, C3-C7
cycloalkylmethyl, and aryl(C1-C~ alkyl)-;
Rl4d is selected from:
H, C1-C6 alkylthio(C1-C6 alkyl)-, aryl(C1-C1o
alkylthioalkyl)-, aryl(C1-C1p alkoxyalkyl)-, C1-C1o
alkyl, C~-C1p alkoxyalkyl, C1-C6 hydroxyalkyl,
C2-C1p alkenyl, C~-C1p alkynyl, C3-C1p cycloalkyl,
C3-C1p cycloalkylalkyl, aryl(C1-C6 alkyl)-,
heteroaryl(C1-C6 alkyl)-, aryl, heteroaryl, C02R17d,
C(=O)Rl7d, and CONR~-7dR2od, provided that any of the
above alkyl, cycloalkyl, aryl or heteroaryl groups
may be unsubstituted or substituted independently
with 0-1 Rl6d or 0-2 Rlld;
Rl5d is selected from:
H, Rl6d, C1_C1p alkyl, C1-C1p alkoxyalkyl,
C1-C1p alkylaminoalkyl, C1-C1p dialkylaminoalkyl,
(C1-C1p alkyl)carbonyl, aryl(C1-C6 alkyl)carbonyl,
C1-C1p alkenyl, C1-C1p alkynyl ,C3-C1p cycloalkyl,
C3-C1p cycloalkylalkyl, aryl(C1-C6 alkyl)-,
heteroaryl(C1-C6 alkyl)-, aryl, heteroaryl, C02R1~d,
C (=O) Rl7d, CONR17dR20d~ S02R17d~ and S02NR17dR20d~
provided that any of the above alkyl, cycloalkyl,
aryl or heteroaryl groups may be unsubstituted or
substituted independently with 0-2 R,lld;
Yd is selected from:
-CORl9d, -S03H, -P03H, tetrazolyl, -CONHNHS02CF3, -
CONHS02R17d, -CONHS02NHR17d, -NHCOCF3, -
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NHCONHS02R17d, -NHS02R17d, -0P03H2, -OS03H, -P03H2, -
S03H, -S02NHCORI7d, -S02NHC02R17d,
N
N/ ~N N' ~ CF3 0
H , H , and HO 0 ;
Rl6d is selected from:
_N (R20d) _C (=0) -0-Rl7d
_N (R20d) -C (-0) _Rl7d~
_N (R20d) _C (=0) _NH_Rl7d~
-N (R2od) S02-Rl7d~ and
-N (R2~d) S02-NR20dR17d;
Rl7d is selected from:
C1-C1o alkyl optionally substituted with a bond to
Ln, C3-C11 Cycloalkyl optionally substituted with a
bond to Ln, aryl(C1-C6 alkyl)- optionally
substituted with a bond to Ln, (C1-C6 alkyl)aryl
optionally substituted with a bond to Ln,
heteroaryl(C1-C6 alkyl)- optionally substituted
with a bond to Ln, (C1-C& alkyl)heteroaryl
optionally substituted with a bond to Ln,
biaryl(C1-Cg alkyl)- optionally substituted with a
bond to Ln, heteroaryl optionally substituted with
a bond to Ln, aryl optionally substituted with a
bond to Ln, biaryl optionally substituted with a
bond to Ln, and a bond to Ln, wherein said aryl,
biaryl or heteroaryl groups are also optionally
substituted with 0-3 substituents selected from the
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group consisting of: C1-C4 alkyl, C1-C4 alkoxy,
aryl, heteroaryl, halo, cyano, amino, CF3, and NO2;
Rl8d is selected from:
-H,
_C (=p) -p_g.l7d~
-C ( =0 ) -R17 d
-C (=O) -NH-Rl7d,
_S02-Rl7d~ and
-S02-NR20dR17d~
Rl9d is selected from: hydroxy, C1-C1p alkyloxy,
C3-C11 cycloalkyloxy, aryloxy, aryl(C1-C6 alkoxy)-,
C3-C1p alkylcarbonyloxyalkyloxy, C3-C1o
alkoxycarbonyloxyalkyloxy,
C2-C1p alkoxycarbonylalkyloxy,
C5-C10 cYcloalkylcarbonyloxyalkyloxy,
C5-C1p cycloalkoxycarbonyloxyalkyloxy,
C~-Clp cycloalkoxycarbonylalkyloxy,
C7-C11 aryloxycarbonylalkyloxy,
Cg-C12 aryloxycarbonyloxyalkyloxy,
Cg-C12 arylcarbonyloxyalkyloxy,
C5-C1p alkoxyalkylcarbonyloxyalkyloxy,
C5-C1p (5-alkyl-1,3-dioxa-cyclopenten-2-one-
yl)methyloxy, C1p-C14 (5-aryl-1,3-dioxa-
cyclopenten-2-one-yl)methyloxy, and
(Rlld) (Rl2d) N_ (C1-C1~ alkoxy) -;
R~~d is selected from: H, C1-C6 alkyl, C3-C7 cycloalkyl,
C4-C11 cycloalkylalkyl, aryl, aryl(C1-C& alkyl)-,
and heteroaryl(C1-C~ alkyl)-;
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R~ld is selected from: COON and NR6d2;
d
m is 0-4;
d
n is 0-4;
d
t is 0-4;
d
p is 0-2;
d
q is 0-2; and
d
r is 0-2;
with the following provisos:
d d d d
(1) t , n , m and q are chosen such that the number of
d
atoms connecting R1d and Y is in the range of
10-14; and
d d d
(2) n and m are chosen such that the value of n plus
d d
m is greater than one unless U is
d d d
- (CHI ) t Q (CH2 ) m -;
or Q is a peptide selected from the group:
L R'
K~ ~M KO R4
~1 2
R R and L M ;
R1 is L-valine, D-valine or L-lysine optionally
substituted on the E amino group with a bond to Ln;
R~ is L-phenylalanine, D-phenylalanine,
D-1-naphthylalanine, 2-aminothiazole-4-acetic acid
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or tyrosine, the tyrosine optionally substituted on
the hydroxy group with a bond to Ln;
R3 is D-valine;
R4 is D-tyrosine substituted on the hydroxy group with a
bond to Ln;
provided that one of Rl and R2 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;
provided that at least one Q is a compound of Formula
(Ia) or (Ib) ;
d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
d' is 1-100;
Ln is a linking group having the formula:
( (W)h-(CR6R~)g)x-(Z)k-( (CR6aR7a)g,_(W)h,)x°;
W is independently selected at each occurrence from the
group: 0, S, NH, NHC(=0), C(=0)NH, NRgC(=0),
C (=0) N R8, C (=0) , C (=0) 0, OC (=0) , NHC (=S) NH,
NHC(=O)NH, 502, S02NH, (OCH2CH2)S, (CH~CH20)s~,
(OCH2CH~CH2)S,<, (CH2CH2CH20)t, and (aa)t.;
as is independently at each occurrence an amino acid;
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is selected from the group: aryl substituted with 0-3
Rlo~ 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 0 and substituted with 0-3 Rlo
R6, R6a, R7, R7a, and R8 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 ( =O ) NHR11, NHR11, R11
and a bond to Ch;
R1o is independently selected at each occurrence from
the group: a bond to Ch, COOR11, C(=0)NHR11,
NHC (=0) R11, OH, NHR11, S03H, P03H, -OP03H2, -OS03H,
aryl substituted with 0-3 R11, C1-5 alkyl
substituted with 0-1 R12, C1-5 alkoxy substituted
with 0-1 R12, 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, -OP03H2, alkyl substituted with 0-1
R12, 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, C3-1o cychoalkyl
substituted with 0-1 R12, polyalkylene glycol
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substituted with 0-1 R1~, carbohydrate substituted
with 0-1 R1~, cyclodextrin substituted with 0-1
R12, amino acid substituted with 0-1 R12,
polycarboxyalkyl substituted with 0-1 R1~,
polyazaalkyl substituted with 0-1 R12, peptide
substituted with -C(=0)-(CH2)5-NHR12, and peptide
substituted with 0-1 R1~, wherein the peptide is
comprised of 2-10 amino acids, C1-5 alkyl
substituted with 3,6-0-disulfo-B-D-
galactopyranosyl, bis(phosphonomethyl)glycine, and
a bond to Ch;
R12 is a bond to Ch;
15k is selected from 1, and2;
0,
h is selected from 1, and2;
0,
h' is selected from 1, an d ;
0, 2
g is selected from 1, 2, 3,4, 5, 6, 7,8, 9,and
0, 10;
g' is selected from 1, 2, 3,4, 5, 6, 7,8, 9,and
0,
10 ;
s is selected from 1, 2, 3,4, 5, 6, 7,8, 9,and
0, 10;
s' is selected from 1, 2, 3,4, 5, 6, 7,8, 9,and
0,
10;
s" is selected from 1, 2, 3,4, 5, 6, 7,8, 9,and
0,
10 ;
t is selected from 1, 2, 3,4, 5, 6, 7,8, 9,and
0, 10;
t' is selected from 1, 2, 3,4, 5, 6, 7,8, 9,and
0,
10;
x is selected from 1, 2, 3,4, and5;
0,
30x' is selected from 1, 2, 3,4, an d ;
0, 5
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Ch is a metal bonding unit having a formula selected
from the group:
E A2 1/~,A~/~As/~Aa.
A , A ,
~5
~,q1
E~~2'~ s
A
s
E/A~E A~~A~E A7 E ~E A
,41° E E \ E
~ ~Aa a
A5 , and A~ ;
A1, A2 , A3 , A4 , A5 , A6 , A7 , and A8 ar a independent 1y
selected at each occurrence from the group: NR13,
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
alkyl substituted with 0-3 R1~, aryl substituted
with 0-3 R1~, C3_1o cycloalkyl substituted with 0-3
R17, 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-C6-1o aryl- substituted with 0-3 R1~,
and a 5-10 membered heterocyclic ring system
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containing 1-4 heteroatoms independently selected
from N, S, and 0 and substituted with 0-3 R17;
R~-3 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
R~-~ , 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
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_~o 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 (R2o) (R21)
R15 and R16 are each independently selected from the
group: a bond to Ln, -OH, C1-C1p 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-C1-1o 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_~o
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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 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(=O)ORlBa, -OR18,
-OC(=O)N(R18)2, -NR19C(=0)R18, -NR19C(=0)ORl8a~
-NR19C(=0)N(R18)2, -NR19S02N(R18)2, -NR19S02R18a~
-S03H~ _SO~Rl8a~ -SR18~ _S(=0)Rl8a~ _SO~N(R18)2~
-N(R18)2, -NHC(=S)NHR18, =NOR18, N02, -C(=O)NHOR18,
-C(=0)NHNR18R18a, -OCH2CO~H,
2-(1-morpholino)ethoxy, C1-C5 alkyl, C~-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 O ;
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;
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R2o and R21 are independently selected from the group:
H, C1-C10 alkyl, -CN, -CO~R25, -C(=0)R25,
-C(=0)N(R~5)2, C2-C1o 1-alkene substituted with 0-3
R23, C2-C1o 1-alkyne substituted with 0-3 R~3, 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 R23;
alternatively, R2o and R~1, taken together with the
divalent carbon radical to which they are attached
form:
n
R~2 and R~3 are independently selected from the group:
H, R24, C1-C10 alkyl substituted with 0-3 R24,
C~-C10 alkenyl substituted with 0-3 R~4, 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
R~4, and C3_1o carbocycle substituted with 0-3 R~4;
alternatively, R2~, R~3 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(=O)R25~ -O(=O)N(R25)2, _N(R25)3+~ -CH20R25,
-OC (=0) R25, -OC (=O) OR25a, -OR25, -OC (=O) N (R25) 2,
-NR26C (=0) R25, -NR26C (=0) OR25a, _NR26C (=0) N (R25 ) 2
_NR26S02N(R25)2~ _NR26S02R25a~ _S03H~ _S02R25a~
_SR25~ -S(=O)R25a~ _S02N(R25)2~ -N(R25)2~ =~'OR25,
-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.
33. In another embodiment, the present invention
provides a method according to Embodiment 30,
wherein Rlde is selected from:
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N-Act N~~ rd
-Ud (NR6d) ~ Ud (NR6d)--~~ A1d
Bd ~ B1d
rd ~ ,
0
N w, Dd
d sd j ~ a --Ud (NR6a)
U ( I~R ) L ~ Ed
Fd ~
Jd.Kd ,
NHR~ d
N~
' Fd N ~ Dd
Ud (NR6d)~ 'd =~ or
\\Dd~E
' Ua
NHR~d
N
Ua
Ad and Bd are independently -CH2-, -0-, -N(R2d)-, or -C(=0)-
;
A1d and B1d are independently -CH2- or -N(R3d)_;
Dd is -N (R2d) -, -0-, -S-, -C (=0) - or -S02-;
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Ed-Fd is -C (R4d) =C (R5d) _ ~ _N=C (R4d) _ ~ _C (R4d) =N_ ~ or -
C (R4d) 2C (R5d) 2-:
d d d d
J , K , L and M are independently selected from:
C(R4d)-, -C(R5d)- and -N-, provided that at least
d d d d
one of J , K , L and M is not -N-;
R2d is selected from: H, C1-C6 alkyl, (C1-C6
alkyl)carbonyl, (C1-C6 alkoxy)carbonyl, C~-C6
alkylaminocarbonyl, C3-C6 alkenyl, C3-C7
cycloalkyl, C4-C11 cycloalkylalkyl, aryl,
heteroaryl(C1-C6 alkyl)carbonyl,
heteroarylcarbonyl, aryl(C1-C6 alkyl)-, (C1-C6
alkyl)carbonyl, arylcarbonyl, alkylsulfonyl,
arylsulfonyl, aryl(C1-Cg alkyl)sulfonyl,
heteroarylsulfonyl, heteroaryl(C~-C6
alkyl)sulfonyl, aryloxycarbonyl, and aryl(C1-C6
alkoxy)carbonyl, wherein said aryl groups are
substituted with 0-2 substituents selected from the
group consisting of C1-C4 alkyl, C1-C4 alkoxy,
halo, CF3, and nitro;
R3d is selected from: H, C1-C~ alkyl, C3-C7 cycloalkyl,
Cg-C11 cycloalkylalkyl, aryl, aryl(C1-C6 alkyl)-,
and heteroaryl(C1-C6 alkyl)-;
R4d and R5d are independently selected from: H, C1-C~
alkoxy, NR2dR3d, halogen, N02, CN, CF3, C1-C6 alkyl,
C3-C6 alkenyl, C3-C7 cycloalkyl, C4-C11
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cycloalkylalkyl, aryl, aryl(C1-C6 alkyl)-~ C2-C7
alkylcarbonyl, and arylcarbonyl;
alternatively, when substituents on adjacent atoms, R4~
and R5d can be taken together with the carbon atoms
to which they are attached to form a 5-7 membered
carbocyclic or 5-7 membered heterocyclic aromatic
or non-aromatic ring system, said carbocyclic or
heterocyclic ring being optionally substituted with
0-2 groups selected from: C1-Cg alkyl, C1-C4
alkoxy, halo, cyano, amino, CF3, or NO~;
d
U is selected from:
d
-(CH2)n -,
- ( cH2 ) nd ( ~R~a=CR8d ) ( CHI ) md- ,
d d d
1~ -(CH2)t Q (CH2)m ".
d d
-(CH2)n 0(CH~)m -,
-(CH2)ndN(R6d)(CH2)md-~
d d
-(CH~)n C(W) (CH2)m -~ arid
d d d
-(CH2)n S(0)p (CH2)m -.
d
wherein one or more of the methylene groups in U is
optionally substituted with R7d;
d
Q is selected from 1,2-phenylene, 1,3-phenylene, 2,3-
pyridinylene, 3,4-pyridinylene, and 2,4-
pyridinylene;
R6~ is selected from: H, C1-C4 alkyl, and benzyl;
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R7d and R8d are independently selected from: H, C1-C6
alkyl, C3-C7 cycloalkyl, Cg-C11 cycloalkylalkyl,
aryl, aryl(C1-C6 alkyl)-, and
heteroaryl(Cp-Cg alkyl)-;
Wd is -C (=O) -N (Rl3d) _ (C (Rl2d) 2 ) qd-;
Xd 1S -C (Rl~d) (Rl4d) _C (Rl2d) (Rl5d) _;
d d
alternatively, W and X can be taken together to be
( CH ~ ) qdC ( =O ) -N N-R.l8d
Rl2d is H or C1-C6 alkyl;
d
Y is selected from:
-CORl9d~ _g03H,
N
'N N/ ~ CF3 0
\ \
H , H , and HO 0 ;
d is selected from 1, 2, 3, 4, and 5;
d' is 1-50;
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W is independently selected at each occurrence from the
group: 0, NH, NHC(=0), C(=0)NH, NR8C(=0), C(=0)N,
R8, C(=0), C(=0)0, OC(=0), NHC(=S)NH, NHC(=0)NH,
502, (OCH2CH2)S, (CH~CH20)S~, (OCH2CH~CH2)5~~,
(CH2CH2CH20)t, and (aa)t~;
as is independently at each occurrence an amino acid;
Z is selected from the group: aryl substituted with 0-1
Rlos C3-1o cycloalkyl substituted with 0-1. R10, 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, and Rg 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 Rlo,
NHC ( =O ) R11, C ( =0 ) NHR11, NHC ( =0 ) NHR11, NHR11, R11
and a bond to Ch;
k 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 R~-~, 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, -C02R18, -C(=0)R18, -C(=0)N(R18)2, -CH20R18,
-OC(=0)R18~ _OC(=0)ORl8a~ -OR18~ -OC(=0)N(R18)2,
-NR19C(=0)R18, -NR19C(=0)ORl8a~ _NR19C(=0)N(R18)2~
-NR19S02N(R18)2, -NR19S02R18a~ _s03H~ _g0~R18a~
-S(=0)Rl8a~ _S02N(R18)2, -N(R18)2, -NHC(=S)NHR18,
=NOR18, -C(=0)NHNR18R18a, -OCH~C02H, and
2-(1-morpholino)ethoxy;
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R18, RlBa, and R19 are independently selected at each
occurrence from the group: a bond to Ln, H, and ,.,-~;~
C1-C6 alkyl;
R2~ 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 O and substituted with 0-3 R23;
alternatively, R20 and R21, taken together with the
divalent carbon radical to which they are attached
form:
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: -C02R~5, -C(=0)N(R25)2, -CH20R~5,
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-OC(=0)R25, -ORBS, -S03H, -N(R25)~, and -OCH~C02H;
and,
R25 is independently selected at each occurrence from
the group: H and C1-C3 alkyl.
[34] In another embodiment, the present invention
provides a method according to Embodiment 30, wherein
the therapeutic radiopharmaceutical is selected from the
group consisting of:
99mTc ((((4-(4-(((3-(2-(2-(3-((6-(diazenido)(3-
pyridyl))carbonylamino)propoxy)-
ethoxy)ethoxy)propyl)amino)sulfonyl)-
phenyl)phenyl)sulfonyl)amino)-3-((1-(3-(imidazole-
2-ylamino)propyl)(1H-indazol-5-
yl))carbonylamino)propanoic acid) (tricine)(TPPTS);
99mTc (2-(2-( (5-(N-(1,3-bis(3-(2-(2-(3-( ( (4-(4-( ( (1-
carboxy-2-((1-(3-(imidazol-2-ylamino)propyl)(1H-
indazol-5-yl))carbonylamino)ethyl)amino)sulfonyl)-
phenyl)phenyl)sulfonyl)amino)propoxy)-
ethoxy)ethoxy)propyl)carbamoyl)propyl)carbamoyl)(2-
pyridyl))2-diazenido) (tricine)(TPPTS);
99mTc (2-((6-(diazenido)(3-pyridyl))carbonylamino)-4-(N-
(3-(2-(2-(3-( ( (4-(4-( ( (1-carboxy-2-( (1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))-
carbonylamino)ethyl)amino)sulfonyl)-
phenyl)phenyl)sulfonyl)amino)propoxy)-
ethoxy)ethoxy)propyl)carbamoyl)butanoic acid)
(tricine)(TPPTS);
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99mTc (2- (6- ( (6- (diazenido) (3-
r
pyridyl))carbonylamino)hexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonylamino)-propanoic acid)
(tricine)(TPPTS);
99mTc (2-((6-(diazenido)(3-pyridyl))carbonylamino)-3-
((1-(3-(imidazol-2-ylamino)propyl)(1H-indazol-5-
y1))carbonylamino)propanoic acid (tricine)(TPPTS);
99mTc [2-[[[5-[carbonyl]-2-pyridinyl]diazenido]-Glu(2-
{6-aminohexanoylamino)-3-{{1-(3-{imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)(2-(6-aminohexanoylamino)-3-
((1-(3-(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonyl-amino)propanoic acid))
(tricine)(TPPTS);
99mTc ([2-[[[5-[carbonyl]-2-pyridinyl]diazenido]-Glu-
bis-[Glu(2-(6-aminohexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonyl-amino)propanoic acid)(2-{6-
aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)]) (tricine)(TPPTS);
O O
N~OH
N ~ ° H NH _ H H O
~N~.N.~ O~ ~ ~ ~ ~ yN.~Ø~0-w.O.~.N~ ~-'~
H H (3~ - I ~O
~n.~O
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O O
~N~OH
H NH
N O-~ O
HRH ~ I S03H
O H
O~l .~ N O
H O Nl~' /'I
C3~ - n-O
~~~~0
O~''O
O O
OH
N O-~ O
HRH ~ I HO S
O I-~ ~ O
O'~N~N1~N~ H O
H O H i, N C
H O
O~f N '~ N N O ~~~, ~ ~~,~O
~ O H~ ~~~~0
H03S
O~$ O
N ~ I ~ N~OH
O O
O O
' ~ O
~ N ~ ~ i H H ~OH / / \ sN,~NO N
N~N~ 02 02 H~3S Q~~ n.-O
H H ~I~~O
O~-O
O O
H OH
N I N~ S02
H ~I
O~NwN
H
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O O
N ~ ~ ~ H HOH
N~N~ S02
H H \ ~ C02H
O.~N-w.N~NO NH ~-O-
H O H~ ~~ ~.N~O
C02H ~~I~~O
O O
~~N
H H
O O
t , H N OH
~N S02
H ~I
O
O.~N~.
H
5.
O O
N~OH
HO N H\ / ~ v ~N.~.O.~O~.O.~.N
N N
H H i
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p p
~- N~OH
H NH _ H H
~N N.J- N O° \ / / \ ~N.~,O.~p-w.O.~.N
N
H H i
0 0
N ~ I ~ H N OH
N~N~ O°~ O
H H ~ I ~S03H
O'~H~'N1~NH
N~.NIO
~~~~~0
O O
O O
/ N ~ I i H H OOH ~ / \ SN.~NO N
O~O
2 2 HC~3S O~ -~~O
~~~~~0
O O ,
and
O O
N ~ I ~ H N OH
N~N~ S02
H H \ ( C02H
O.~NwN~NO NH
H O H~ ~NY.N~O
C02H ~~I~~~O
O O
[35] In another embodiment, the present invention
provides a method according to Embodiment 30 wherein
administering the therapeutic radiopharmaceutical and
agent is concurrent.
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[36] In another embodiment, the present invention
provides a method according to Embodiment 30 wherein
administering the therapeutic radiopharmaceutical and
agent is sequential.
[37] In another embodiment, the present invention
provides a method according to Embodiment 30 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.
[38] In another embodiment, the present invention
provides a method according to Embodiment 30 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,
prednimustine, picibanil, levamisole, teniposide,
improsulfan, enocitabine, lisuride, oxymetholone,
tamoxifen, progesterone, mepitiostane, epitiostanol,
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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.
[39] In another embodiment, the present invention
provides a method according to Embodiment 30 wherein the
radiosensitizer agent is selected from the group
consisting of 2-(3-nitro-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.
[40] In another embodiment, the present invention
provides a method according to Embodiment 30 wherein the
anti-cancer agent is a anti-cancer agent agent.
[41] In another embodiment, the present invention
provides a method of treating cancer according to
Embodiment 30, wherein the administration is by
injection or infusion.
[42] In another embodiment, the present invention
provides a method of Embodiment 30, further comprising
treating the cancer by brachytherapy, external beam
radiation, laser therapy or surgical removal.
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[43] In another embodiment, the present invention
provides a kit comprising packaging material, and a
therapeutic radiopharmaceutical composition of
Embodiment 15, 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.
[44] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
of Embodiment 15, further comprising a photosensitizing
agent.
[45] In another embodiment, the present invention
provides a therapeutic radiopharmaceutical composition
according to Embodiment 44, 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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[46] In another embodiment, the present invention
provides a kit according to Embodiment 43, further
comprising a photosensitizing agent.
[47] In another embodiment, the present invention
provides a kit 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.
[48] In another embodiment, the present invention
provides a method of treating cancer according to
Embodiment 30, further comprising treating the patient
with photodynamic therapy.
[49] In another embodiment, the present invention
provides a method of treating cancer according to
Embodiment 48, 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
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agent having a characteristic light absorption waveband
and being preferentially absorbed by abnormal tissue;
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) determining 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.
[50] In another embodiment, the present invention
provides a method of treating cancer according to
Embodiment 55, 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.
[51] In another embodiment, the present invention
provides a method of treating cancer according to
Embodiment 49, wherein the photosensitizing agent is
selected from the group consisting of photofrin;
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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.
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
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be in the form of a sterile solution or a lyophilized
solid.
Another aspect of the present invention are
diagnostic kits for the preparation of
radiopharmaceuticals useful as imaging agents for
cancer. 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 a method of imaging cancer in a patient
involving: (1) synthesizing a diagnostic
radiopharmaceutical of the present invention, using a
reagent of the present invention, capable of localizing
in tumors; (2) administering said radiopharmaceutical to
a patient by injection or infusion; (3) imaging the
patient using planar or SPELT gamma scintigraphy, or
positron emission tomography.
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Another aspect of the present invention
contemplates a method of imaging cancer in a patient
involving: (1) administering a paramagnetic
metallopharmaceutical of the present invention capable
of localizing in tumors to a patient by injection or
infusion; and (2) imaging the patient using magnetic
resonance imaging.
Another aspect of the present invention
contemplates a method of imaging cancer in a patient
involving: (1) administering a X-ray contrast agent of
the present invention capable of localizing in tumors to
a patient by injection or infusion; and (2) imaging the
patient using X-ray computed tomography.
Another aspect of the present invention
contemplates a method of imaging cancer in a patient
involving: (1) administering a ultrasound contrast agent
of the present invention capable of localizing in tumors
to a patient by injection or infusion; and (2) imaging
the patient using sonography.
Another aspect of the present invention
contemplates a method of treating cancer in a patient
involving: (1) administering a therapeutic
radiopharmaceutical of the present invention capable of
localizing in tumors to a patient by injection or
infusion.
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
enhanced degree of tumor suppression relative to each
treatment modality alone without significant additive
toxicity.
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Another aspect of the present invention
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 indazole nonpeptide, and binds to a
receptor that is upregulated during angiogenesis and the
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,
etoposide, cladribine, mitobronitol, methotrexate,
doxorubicin, carboquone, pentostatin, nitracrine,
zinostatin, cetrorelix, letrozole, .raltitrexed,
daunorubicin, fadrozole, fotemustine, thymalfasin,
sobuzoxane, nedaplatin, cytarabine, bicalutamide,
vinoreTbine, vesnarinone, aminoglutethimide, amsacrine,
proglumide, elliptinium acetate, ketanserin,
doxifluridine, etretinate, isotretinoin, streptozocin,
nimustine, vindesine, flutamide, drogenil, butocin,
carmofur, razoxane, sizofilan, carboplatin, mitolactol,
tegafur, ifosfamide, prednimustine, picibanil,
levamisole, teniposide, improsulfan, enocitabine,
lisuride, oxymetholone, tamoxiferi, 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.
This combination therapy may further, optionally,
include a radiosensitizer agent, or a pharmaceutically
acceptable salt thereof, to enhance the radiotherapeutic
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-
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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
selected from the group consisting of an anti-cancer
agent and a radiosensitizer agent.
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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, 6,2.33,481,
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,
6,212,626, 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 embodiments, 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
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starting materials. Two distinct isomers (cis and
trans) 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 R52, then said group
may optionally be substituted with up to two R52, and R5~
at each occurrence is selected independently from the
defined list of possible R52. Also, by way of example,
for the group -N(R53)2, 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.
The term "nonpeptide" means preferably less than
three amide bonds in the backbone core of the targeting
moiety or preferably less than three amino acids or
amino acid mimetics in the targeting moiety.
The.term "metallopharmaceutical" means a
pharmaceutical comprising a metal. The metal is the
cause of the imageable signal in diagnostic applications
and the source of the cytotoxic radiation in
radiotherapeutic applications. Radiopharmaceuticals are
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metallopharmaceuticals in which the metal is a
radioisotope.
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 have Ki < 1000nM.
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., =O), 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.
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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 phrase "pharmaceutically acceptable" is
employed herein to refer to those compounds, materials,
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
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
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,
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
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
benzoyl), alkoxycarbonyl (such as ethoxycarbonyl),
trialkylsilyl (such as trimethyl- and triethysilyl),
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monoesters formed with dicarboxylic acids (such as
succinyl), and the like. Because of the ease with which
the metabolically cleavable groups of the compounds
useful according to this invention are cleaved in vivo,
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
and/or 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 En~ymology,
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
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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
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, examples of which include, but are not limited
to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,
sec-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl,
and decyl; "cycloalkyl" or "carbocycle" is intended to
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include saturated and partially unsaturated ring groups,
including mono-, bi- or poly-cyclic ring systems, such
as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, cyclooctyl and adamantyl; "bicycloalkyl" or
"bicyclic" is intended to include saturated bicyclic
ring groups such as [3.3.0]bicyclooctane,
[4.3.0]bicyclononane, [4.4.0]bicyclodecane (decalin),
[2.2.2]bicyclooctane, and so forth.
As used herein, the term "alkene" or "alkenyl" is
intended to include hydrocarbon chains having the
specified number of carbon atoms 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, propenyl, and the
like.
As used herein, the term "alkyne" or "alkynyl" is
intended to include hydrocarbon chains having the
specified number of carbon atoms of either a straight or
branched configuration and one or more unsaturated
carbon-carbon triple bonds which may occur in any stable
point along the chain, such as propargyl, and the like.
As used herein, "aryl" or "aromatic residue" is
intended to mean phenyl or naphthyl, which when
substituted, the substitution can be at any position.
As used herein, the term "heterocycle" or
"heterocyclic system" is intended to mean a stable 5- to
7- membered monocyclic or bicyclic or 7- to 10-membered
bicyclic heterocyclic ring which is saturated partially
unsaturated or unsaturated (aromatic), and which
consists of carbon atoms and from 1 to 4 heteroatoms
independently selected from the group consisting of N, 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
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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. If specifically noted, a nitrogen
in the heterocycle may optionally be quaternized. It is
preferred that when the total number of S and O 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" is intended to mean a stable 5- to
7- membered monocyclic or bicyclic or 7- to 10-membered
bicyclic heterocyclic aromatic ring which consists of
carbon atoms and from 1 to 4 heteroatoms independently
selected from the group consisting of N, 0 and S. It is
preferred that the 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, 1H-indazole, 2-pyrrolidonyl,
2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl,
4-piperidonyl, 4aH-carbazole, 4H-quinolizinyl,
6H-1,2,5-thiadiazinyl, acridinyl, azocinyl,
benzimidazolyl, benzofuranyl, benzothiofuranyl,
benzothiophenyl, benzoxazolyl, benzthiazolyl,
benztriazolyl, benztetrazolyl, benzisoxazolyl,
benzisothiazolyl, benzimidazalonyl, carbazolyl,
4aH-carbazolyl, ~3-carbolinyl, chromanyl, chromenyl,
cinnolinyl, decahydroquinolinyl,
2H,6H-1,5,2-dithiazinyl,
dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl,
imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl,
indolenyl, indolinyl, indolizinyl, indolyl,
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isobenzofuranyl, isochromanyl, isoindazolyl,
isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl,
isoxazolyl, morpholinyl, naphthyridinyl,
octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,
oxazolidinyl., oxazolyl, oxazolidinylperimidinyl,
phenanthridinyl, phenanthrolinyl, phenarsazinyl,
phenazinyl, phenothiazinyl, phenoxathiinyl,
phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl,
pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl,
purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,
pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole,
pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,
pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl,
quinolinyl, 4H-quinolizinyl, quinoxalinyl,
quinuclidinyl, carbolinyl, tetrahydrofuranyl,
tetrahydroisoquinolinyl, tetrahydroquinolinyl,
6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,
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, xanthenyl. Preferred
heterocycles include, but are not limited to, pyridinyl,
furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl,
indolyl, benzimidazolyl, 1H-indazolyl, oxazolidinyl,
benzotriazolyl, benzisoxazolyl, oxindolyl,
benzoxazolinyl, or isatinoyl. Also included are fused
ring and spiro compounds containing, for example, the
above heterocycles.
As used herein, the term "alkaryl" means an aryl
group bearing an alkyl group of 1-10 carbon atoms; the
term "aralkyl" means an alkyl group of 1-10 carbon atoms
bearing an aryl group; the term "arylalkaryl" means an
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aryl group bearing an alkyl group of 1-10 carbon atoms
bearing an aryl group; and the term "heterocycloalkyl"
means an alkyl group of 1-10 carbon atoms bearing a
heterocycle.
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, oc-cyclodextrin, hydroxyethyl-oc-cyclodextrin,
hydroxypropyl-OC-cyclodextrin, (3-cyclodextrin,
hydroxypropyl-~3-cyclodextrin,
carboxymethyl-(3-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
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.
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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 sulfinic 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.
".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
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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.
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
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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
diagnostic 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
just withdraws the dose from the aqueous solution
formulation as provided.
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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 lay 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 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
Cit citrulline
Dap 2,3-diaminopropionic acid
DCC dicyclohexylcarbodiimide
DIEA diisopropylethylamine
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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 MeArga-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
Gln - glutamine
Glu - glutamic acid
Gly - glycine
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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
As used herein, 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.
As used herein, the term "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.
As used herein, the term "lipid composition" refers
to a composition which comprises a lipid compound.
Exemplary lipid compositions include suspensions,
emulsions and vesicular compositions.
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As used. herein, the term "lipid formulation" refers
to a composition which comprises a lipid compound and a
bioactive agent.
As used herein, the term "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 described herein include such
entities commonly referred 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 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.
As used herein, the term "vesicular composition"
refers to a composition which is formulate from lipids
and which comprises vesicles.
As used herein, the term "vesicle formulation"
refers to a composition which comprises vesicles and a
bioactive agent.
As used herein, the term "lipsomes" refers to a
generally spherical cluster or aggregate of amphipathic
compounds, including lipid compounds, typically in the
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form of one or more concentric layers, for example,
bilayers. They may also be referred to herein as lipid
vesicles.
Angiogenesis is the process of formation of new
capillary blood vessels from existing vasculature. 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. The activated
endothelial cells secrete enzymes that degrade the
basement membrane of the vessels. The endothelial cells
then proliferate and migrate into the extracellular
matrix first forming tubules and subsequently new blood
vessels.
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
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.
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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.
The proliferation and migration of endothelial
cells in the extracellular matrix is mediated by
interaction with a variety of cell adhesion molecules.
Integrins are a diverse family of heterodimeric cell
surface receptors by which endothelial cells attach to
the extracellular matrix, each other and other cells.
Angiogenesis induced by bFGF or TNF-alpha depend on the
agency of the integrin avb3, while angiogenesis induced
by VEGF depends on the integrin avb5 (Cheresh et. al.,
Science, 1995, 270, 1500-2). Induction of expression of
the integrins albl and a2b1 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).
The pharmaceuticals of the present invention are
comprised of a non-peptide targeting moiety for the
vitronectin receptor that is expressed or upregulated in
angiogenic tumor vasculature.
The ultrasound contrast agents of the present
invention comprise a plurality of vitronectin receptor
targeting moieties attached to or incorporated into a
microbubble of a biocompatible gas, a liquid carrier,
and a surfactant microsphere, further comprising an
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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, 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, polypeptides or peptidomimetics.
X-ray contrast agents of the present invention are
comprised of one or more vit~onectin receptor 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
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disclosed as X-ray contrast agents (U.S. 5,804,161, PCT
W091/14460, and PCT W0 92/17215).
MRI contrast agents of the present invention are
comprised of one or more vitronectin receptor 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. U.S. 5,520,904, describes
particulate compositions comprised of paramagnetic metal
ions for use as MRT 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 by 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
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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 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
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 a anti-cancer agent and a radiosensitizer
agent (alone or in combination with diluent or carrier).
The pharmaceuticals of the present invention have
the formulae, (Q)d-Ln-(Ch-X), (Q)d-Ln-(Ch-X1)d
(Q)d-Ln-(X2)d~~, and (Q)d-Ln-(X3), wherein Q represents a
non-peptide that binds to a receptor expressed in
angiogenic tumor vasculature, d is 1-10, Ln represents
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an optional linking group, Ch represents a metal
chelator or bonding moiety, X represents a radioisotope,
X1 represents paramagnetic metal ion, X2 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. The
interaction of the non-peptide recognition sequences of
the vitronectin receptor binding portion of the
pharmaceuticals with the ocv(33 receptor results in
localization of the pharmaceuticals in angiogenic tumor
vasculature, which express the ocv(33 receptor.
The pharmaceuticals of the present invention can be
synthesized by several approaches. One approach
involves the synthesis of the targeting non-peptide
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 ion or
heavy atom containing solid particle, or to an echogenic
gas microbubble. Another approach involves the
synthesis of a non-peptide, 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,
or to a paramagnetic metal ion or heavy atom containing
solid particle, or to an echogenic gas microbubble.
The non-peptide vitronectin binding moieties, Q,
optionally bearing a linking group, Ln, or a fragment of
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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.
The attachment of linking groups, Ln, to the non-
peptides, Q; chelators or bonding units, Ch, to the non-
peptides, , Q, or to the linking groups, Ln; and non-
peptides, 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 non-
peptides, Q, to paramagnetic metal ion or heavy atom
containing solid particles, X~, 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 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 United States Patent
Application Serial No. 09/356,178, 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 non-peptides, Q to the surfactant microsphere, X3.
These are illustrated in following reaction. schemes
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where Sf represents a surfactant moiety that forms the
surfactant microsphere.
Acylation Reaction:
Sf-C(=0)-Y + Q-NH2 or __________-> Sf-C(=0)-NH-Q
Q-OH or Sf-C(=0)-O-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(=0)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.
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
non-peptides, Q, so as to minimize the possibility that
the moieties Ch-X, Ch-X1, X2, and X3, will interfere with
the interaction of the recognition sequences of Q with
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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, X~, and X3.
If 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 non-peptides, 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 vitronectin 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 moderate to fast blood clearance and enhanced
renal excretion.
The metal chelator or bonding moiety, Ch, is
selected to form stable complexes with the metal 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,
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such as 99mTc, 95TC~ 111In~ 62Cu~ 60Cu~ 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,
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
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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, O.,
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)
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
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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.
Preferred technetium radiopharmaceuticals of the
present invention are comprised of a hydrazido or
diazenido bonding unit and an ancillary ligand, AL1, or
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a bonding unit and two types of ancillary AL1 and AL2,
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 ALZ
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. 60/013360
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 AL2 are more stable compared to
radiopharmaceuticals that are not comprised of one er
more ancillary ligands, AL2; that is, they have a
minimal number of isomeric forms, the relative ratios of
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which do not change significantly with time, and that
remain substantially intact upon dilution.
The ligands AL2 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
(sp2 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 a~railable
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 AL2 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
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the radiopharmaceuticals, such as altering the
distribution into non-target tissues, cells or fluids,
and the mechanism and rate of elimination from the body.
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 lg6Re, 188Re, 153Sm~ 166Ho~ 177Lu,
149pm~ 90y~ 212Bi~ 103pd~ 109pd~ 159Gd~ 140La~ 198Au, 199Au,
169yb~ 175yb~ 165Dy~ 166Dy~ 67Cus 205Rh~ 111Ag, and 192Ir.
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-
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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
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
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a radionuclide, an ancillary ligand ALA, 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 ALA then adding a reagent of the
present invention and an ancillary ligand AL2 and
reacting further at temperatures from 0 to 100°C.
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 ration.
The pertechnetate salt form is preferably sodium
pertechneta~te 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 ug to 10 mg, or more preferably from 0.5 pg to
200 fig. 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.
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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
biologically active molecule or Say-products comprised of
technetium labeled biologically active molecules with
the ancillary ligand AL1 but without the ancillary
ligand AL2. Too small an amount of AL1 will result in
other by-products such as technetium labeled
biologically active molecules with the ancillary ligand
Av2 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 ALA 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 AL2 taut without the ancillary
ligand AL1. If the reagent bears one or more
substituents that are comprised of a soft donor atom, as
defined above, at least a ten-fold molar excess of the
ancillary ligand ALA to the reagent of formula 2 is
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required to prevent the substituent from interfering
with the coordination of the ancillary ligand ALA 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.
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 ALA, and the identity of the
radionuclide. Radiopharmaceuticals comprised of a
hydrazido or diazenido bonding unit synthesized using
concentrations of reagents of <100 ~~.g/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.
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The identities and amounts used of the ancillary
ligands Av1 and AL2 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 anal 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 AL2, z can be an integer
from 1 to 2; for bidentate or tridentate ancillary
ligands AL2, 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.
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
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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
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.
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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.
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.
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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 mmollkg 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
an amount of 10 to 30 uL of the echogenic gas per kg
body weight or by infusion at a rate of approximately 3
uL/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.
1-(3-((1-(triphenylmethyl)imidazol-2-yl)amino)propyl)-
1H-indazole-5-carboxylic acid was synthesized as
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described in U.S. 5,760,028. 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 Sachem Inc., Bachem
Biosciences Inc. (Philadelphia, PA), Advanced ChemTech
(Louisville, KY), Peninsula Laboratories (Belmont, CA),
or Sigma (St. Louis, MO). Boc-L-cysteic acid, Boc-L-
cysteic acid N-hydroxyphenyl ester, and Boc-L-cysteic
acid p-nitrophenyl ester were prepared as described in
Liebigs Ann. Chem. 1979, 776-783. 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, 1-(3-dimethylaminopropyl)-3-
"~ethylcarbodiimide hydrochloride (EDC), triethylsilane
(Et3SiH), and tris(3-sulfonatophenyl)phosphine 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)diphenylphosphine monosodium salt
(TPPMS)was purchased from TCI America, Inc. Tricine was
obtained from Research 0rganics, 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 (MURR). Yttrium-90 chloride was obtained from
the Pacific Northwest Research Laboratories.
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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. DOTA(OtBu)3-OH was prepared as
described or purchased from Macrocyclics, Inc (Texas).
Synthesis of Boc-Glu-(OTFP)-OTFP
F F
\ I F O O F \
F O O F
F Bo~~NH F
To a solution of Boc-Glu-OH (28.9 g, 117 mmol) in
DMF (500 mL) at room temperature, and under nitrogen,
was added a solution of 2,3,5,6-tetrafluorophenol (48.2
g, 290 mmol) in DMF (50 mL). After stirring for 10 min.
EDC (55.6 g, 290 mmol) was added and the reaction
mixture was stirred for about 96 h. The volatiles were
removed in vacuo and the residue was triturated in 0.1 N
HC1 (750 mL). To this mixture was added ethyl acetate
(600 mL), the layers separated. The aqueous layer was
extracted with ethyl acetate (3 x 500 mL), and all the
ethyl acetate fractions were combined, washed with water
(300 mL) and brine (300 mL), dried (MgS04), and
concentrated to give a tan solid (62 g). The tan solid
was washed with acetonitrile to give the title compound
(45.5 g, 73%) in purified form.
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ESMS: Calculated for CZ~H17F8N06, 543.09; found, 566.0
+1
[M+Na] .
Example 1
Synthesis of 2-(((4-(4-(((3-(2-(2-(3-((6-((1-Aza-2-(2-
sulfophenyl)vinyl)amino)(3-
pyridyl))carbonylamino)propoxy)-
ethoxy)ethoxy)propyl)amino)sulfonyl)-
phenyl)phenyl)sulfonyl)amino)-3-((1-(3-(imidazole-2-
ylamino)propyl)(1H-indazol-5-yl))carbonylamino)propanoic
Acid
O O H ~ SOH
N N. -
~N~OH H H ~ N
N ~ i H HN.S S'N~O~.O.~O~N~
N~N~ 02 \ / \ ~ 02 0
H H
Part A - Preparation of N- (3- (2- (2- (3
aminopropoxy)ethoxy)ethoxy)propyl)
(phenylmethoxy)formamide
i
H2N~O~..O~O~N~O w ~
O
2.0
A solution of 4,7,10-trioxa-1,13-tridecanediamine
(158 mL, 0.72 mol), TEA (16.7 mL, 0.12 mol), and MeOH
(300 mL) in peroxide-free THF (1,000 mL) was placed in a
3 liter 3-neck flask fitted with a mechanical stirrer, a
thermometer, and an addition funnel with nitrogen line.
The addition funnel was charged with a solution of
benzyl chloroformate (17.1 mL, 0.12 mol) in peroxide-
free THF (1,000 mL). The contents of the flask were
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cooled below 5 °C. The contents of the addition funnel
were added to the flask with rapid stirring over 4 h
while keeping the temperature below 5 °C. The solution
was stirred an additional 30 min and concentrated to
give a thick syrup. This syrup was taken up in
saturated NaCl (1800 mL) and 10% Na~C03 (200 mL) and
extracted with ether (3 x 1,000 mL). The combined ether
extracts were washed with saturated NaCl (500 mL), dried
(MgSOg), and concentrated to give a pale yellow oil
(36.74 g). Flash chromatography on a 7 x 29 cm silica
gel column (DCM/MeOH/TEA, 20/15/0.5) gave the title
compound as a colorless syrup (19.14 g, 45%). 1H NMR
(CDC13): 7.33-7.25 (m, 5H), 5.59 (s, 1H), 5.06 (s, 2H),
3.62-3.45 (m, 12H), 3.32-3.25 (m, 2H), 2.74 (t, J = 6.7
Hz, 2H), 1.75 (pentet, J = 6.0 Hz, 2H), 1.67 (pentet, J
- 6.4 Hz, 2H), 1.33 (s, 2H); MS: m/e 355.4 [M+H]; High
Resolution MS: Calcd for C1gH31N205 [M+H]: 355.2233,
Found: 355.2222.
Part B - Preparation of Methyl 3-((tert-Butoxy)-
carbonylamino)-2-(((4-(4-(((3-(2-(2-(3-((phenylmethoxy)-
carbonylamino)propoxy)ethoxy)ethoxy)propyl)-
amino)sulfonyl)phenyl)phenyl)sulfonyl)amino)propanoate
O
BoG ~
H HN O _N~O~~ ~O~N O w ~
p2 ~ / ~ / p2 O O
Biphenyl-4,4'-disulfonyl chloride (2.64 g, 7.5
mmol, freshly recrystallized from CHC13) and DCM (200
mL) were placed in a 500 mL 3-neck flask fitted with a
thermometer, an addition funnel, and a nitrogen line.
The addition funnel was charged with a solution of N-(3-
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(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-
(phenylmethoxy)formamide (1.77 g, 5.0 mmol) and DIEA
(0.87 mL, 5.0 mmol) in DCM (40 mL). The contents of the
flask were cooled below 5 °C. The contents of the
addition funnel were added to the flask with rapid
stirring over 3 h while keeping the temperature of the
flask below 5 °C. The addition funnel was charged with
a solution of N-(3-Boc-L-oc,(3, -diaminopropionic acid
methyl ester hydrochloride (2.55 g, 10 mmol) and DIEA
(3.8 mL, 22 mmol) in DCM (25 mL). This solution was
added to the flask with stirring at 5 °C over 15 min,
and stirred at ambient temperatures for an additional 20
h. The reaction solution was washed consecutively with
0.1 N HCl (100 mL) and water (2 x 100 mL), dried
(MgS04), and concentrated to give a viscous oil (5.79
g). Flash chromatography on a 5 x 21 cm silica gel
column (85/15 EtOAc/hexanes, followed by 100% EtOAc)
gave a colorless amorphous solid. Recrystallization
from toluene (85 mL) gave the title compound as a
colorless solid (2.52 g, 59%). MP: 104.5-106.5 °C; 1H
NMR (CDC13): 8.00-7.90 (m, 4H), 7.72-7.64 (m, 4H), 7.46-
7.24 (m, 5H), 5.96-5.88 (m, 1H), 5.86-5.73 (m, 1H), 5.41
(s, 1H), 5.16-5.00 (m, 3H), 4.15-4.02 (m, 1H), 3.68-3.39
(m, 17H), 3.34-3.22 (m, 2H), 3.13-3.03 (m, 2H), 1.80-
1.62 (m, 4H), 1.39 (s, 9H); 13C NMR (CDC13): 170.2,
156.5, 156.1, 143.9, 143.0, 140.4, 139.4, 136.7, 128.4,
128.1, 128.0, 127.9, 127.9, 127.8, 127.3, 80.1, 70.6,
70.5, 70.2, 70.1, 70.0, 69.6, 66.5, 56.1, 52.9, 43.2,
42.4, 39.3, 29.4, 28.5, 28.2; MS: m/e 868.3 [M+NHg];
High Resolution MS : Calcd for C39H55N4013S2 [M+H]
851.3207, Found: 851.3226.
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Part C - Preparation of Methyl 2-(((4-(4-(((3-(2-(2-(3-
((Phenylmethoxy)-
carbonylamino)propoxy)ethoxy)ethoxy)propyl)-
amino)sulfonyl)phenyl)phenyl)sulfonyl)amino)-3-((1-(3-
((1-(triphenylmethyl)imidazole-2-yl)amino)propyl)(1H-
indazol-5-yl))carbonylamino)propanoate.
O
N'~O~ H H
H HN.~ S,N~O~-.O~,O~NxO~
~~ N ~ 02 ~ / ~ ~ 02 O
Tr H
The product from Part B, above (141 mg, 0.166 mmol)
was dissolved in 25/75 TFA/DCM (5 mL) and allowed to
react at ambient temperatures for 15 min. The solution
was concentrated to give a viscous amber oil. This oil
was dissolved in anhydrous DMF (3 mL) and treated with
TEA until basic to pH paper. In a separate flask, 1-(3-
((1-(triphenylmethyl)imidazol-2-yl)amino)propyl)-1H-
indazole-5-carboxylic acid (76 mg, 0.141 mmol), TEA
(0.059 mL, 0.422 mmol), and HBTU (63.9 mg, 0.169 mmol)
were dissolved in anhydrous DMF (3 mL). The resulting
solution was stirred at ambient temperatures for 5 min
and combined with the DMF solution from the TFA
deprotection. The solution was concentrated after 2 h
to give a viscous amber oil. The oil was dissolved in
EtOAc (175 mL) and washed consecutively with water (50
mL), saturated NaHC03 (25 mL), and saturated NaCl (50
mL). The combined aqueous washings were back-extracted
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with EtOAc (50 mL). The combined EtOAc layers were
dried (MgS04) and concentrated to give a viscous amber
oil. Purification by flash chromatography on a 2 x 16
cm silica gel column using a EtOAc/MeOH step gradient
(95/5, 93/7, 85/15) gave the title compound as a pale
yellow foamy solid (86 mg, 48%). MS: m/e 1273.4 [M+H];
High Resolution MS: Calcd for C6gH73Ng013S2 [M+H]:
1273.4738, Found: 2273.4730.
Part D - Preparation of 2-(((4-(4-(((3-(2-(2-(3-
((Phenylmethoxy)-
carbonylamino)propoxy)ethoxy)ethoxy)propyl)-
amino)sulfonyl)phenyl)phenyl)sulfonyl)amino)-3-((1-(3-
((1-(triphenylmethyl)imidazole-2-yl)amino)propyl)(1H-
indazol-5-yl))carbonylamino)propanoic Acid
O
N~OH H H
H HN~ S,N~O~.O.~,O~N~O~
~~ N ~ 02 \ ! \ ! 02 O
H
The product from Part C, above (200 mg, 0.159 mmol)
was hydrolyzed in a mixture of peroxide-free THF (8.0
mL), 3 N LiOH (0.80 mL), and water (1.20 mL). The
mixture was stirred at ambient temperatures under an
atmosphere of nitrogen for 3 h. The THF was removed
under reduced pressure and the resulting yellow solution
was diluted with water (15 mL). The solution was
adjusted to pH 5.0, and the resulting yellow ppt was
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extracted into DCM (4 x 25 mL). The combined DCM
extracts were dried (MgS~4), and concentrated to give
the title compound as a yellow solid (174 mg, 88%). MS:
m/e 1246.4 [M+H]; High Resolution MS: Calcd for
S C6gH~~NgOl~S~ [M+H]: 1246.4741, Found: 1246.4730.
Part E - Preparation of 2-(((4-(4-(((3-(2-(2-(3-
Aminopropoxy)ethoxy)ethoxy)propyl)amino)sulfonyl)-
phenyl)phenyl)sulfonyl)amino)-3-((1-(3-(imidazole-2-
ylamino)propyl)(1H-indazol-5-yl))carbonylamino)propanoic
Acid.
O
N~OH
NN ~ / H HN O ~ / \ / p ~O~O~O~NH2
N~N~ 2 2
H H
The product from Part D, above (154 mg, 0.124 mmol)
was dissolved in degassed TFA (15 mL) and triethylsilane
(0.10 mL, 0.626 mmol), and heated at 70 °C under an
atmosphere of nitrogen for 1.5 h. The solution was
concentrated and the resulting oily solid was dissolved
in water (75 mL) and washed with ether (2 x 20 mL). The
combined ether washings were back-extracted with water
(10 mL). The two aqueous solutions were combined, and
lyophilized to give the title compound as a hygroscopic
off-white solid, (140 mg). MS: m/e 870.3 [M+H]; High
Resolution MS: Calcd for C3gH52Ng01pS2 [M+H] : 870.3278,
Found: 870.3301.
Part F - Preparation of 2-(((4-(4-(((3-(2-(2-(3-((6-((1-
Aza-2-(2-sulfophenyl)vinyl)amino)(3-
pyridyl))carbonylamino)propoxy)-
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ethoxy)ethoxy)propyl)amino)sulfonyl)phenyl)phenyl)-
sulfonyl)amino)-3-((1-{3-(imidazole-2-
ylamino)propyl)(1H-indazol-5-yl))carbonylamino)propanoic
Acid.
The product from Part E, above (15 mg, 0.0137 mmol)
was dissolved in anhydrous DMF {2.5 mL) and treated with
TEA until basic to pH paper. The solution was treated
with 2-(2-aza-2-((5-((2,5-dioxopyrrolidinyl)carbonyl)(2-
pyridyl))amino)vinyl)benzenesulfonic acid (9.0 mg, 0.020
mmol) and stirred at ambient temperatures under a
nitrogen atmosphere for 24 h. The DMF was removed under
vacuum, and the resulting oil was dissolved in 50% ACN
and purified by preparative HPLC on a Vydac C-1S column
(22 x 250 mm) using a 2.52o/min gradient of 0 to 63% ACN
containing 0.1% TFA at a flow rate of 20 mL/min. The
main product peak eluting at 21.9 min was collected and
lyophilized to give the title compound as a colorless
powder (9.0 mg, 51%). MS: m/e 1173.4 [M+H]; High
Resolution MS: Calcd for C52H61N12~1453 [M+H]: 1173.3592,
Found: 1173.360.
Example 2
Synthesis of 2-(2-Aza-2-((5-(N-(1,3-bis(3-(2-(2-(3-(((4
(4-(((1-carboxy-2-((1-(3-(imidazol-2-ylamino)propyl)(1H
indazol-5-yl))carbonylamino)ethyl)amino)sulfonyl)
phenyl)phenyl)sulfonyl)amino)propoxy)
ethoxy)ethoxy)propyl)carbamoyl)propyl)carbamoyl)(2
pyridyl))amino)vinyl)benzenesulfonic Acid
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N I H I °H
N NH _ O
°°,SO, \ / ~ ~ O-N~O~'O~O~N O
H H
O
O N ~
H H NH HJ~\~ N
'~N'~ o=~S /_\ ~ ~ ~'H'~'o'~'°'~"°'~ N H oS~OH
NH O
N NN I / N~OH
O ~ fIO
Part A - Preparation of N,N'-Bis(3-(2-(2-(3-(((4-(4-
(((1-carboxy-2-((1-(3-(imidazol-2-ylamino)propyl)(1H-
indazol-5-yl))carbonylamino)ethyl)amino)sulfonyl)-
phenyl)phenyl)sulfonyl)amino)propoxy)-
ethoxy)ethoxy)propyl)-2-((tert-
butoxy)carbonylamino)pentane-1,5-diamide.
0 0
N'~ OH
N ~ H ~H O
~'°~ O~°~ H O
H H
O N~O~t-Bu
H H O NH H
~~N~ °_~ / \
N I ~ H NH O
N0~ ~OH
O ~ tIO
The product from Example 1, Part D (44 mg, 0.04
mmol) was dissolved in anhydrous DMF (5 mL) and made
basic to pH paper with TEA. This solution was treated
with the bis-N-hydroxysuccinimide ester of Boc-Glu-OH
(7.9 mg, 0.018 mmol) and stirred at ambient temperatures
under a nitrogen atmosphere for 18 h. The DMF was
removed under vacuum and the resulting oil was dissolved
in 50~ ACN and purified by preparative HPLC on a Vydac
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C-18 column (22 x 250 mm) using a 2.1%/min gradient of 0
to 63% ACN containing 0.1% TFA at a flow rate of 20
mL/min. The peak eluting at 21.2 min was collected and
lyophilized to give the monomer 2-((tert-
butoxy)carbonylamino)-4-(N-(3-(2-(2-(3-(((4-(4-(((1-
carboxy-2-((1-(3-(imidazol-2-ylamino)propyl)(1H-indazol-
5-yl))carbonylamino)ethyl)amino)sulfonyl)-
phenyl)phenyl)sulfonyl)-
amino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)butanoic
acid as a colorless solid in 82% purity A second HPLC
purification using the above method gave 100% pure
monomer (3.4 mg, 7.0%). MS: m/e 1099.5 [M+H], 550.5
[M+2H].
0 0
fV~ OH
N~ I / H NH O
~0./'~.~H O
H H O
O N~O~t-Bu
OH H
The main peak eluting at 22.4 min was collected and
lyophilized to give the title compound as a colorless
solid (11 mg, 25%). MS: m/e 1952.1 [M+H]; 976.9 [M+2H];
651.6 [M+3H]; High Resolution MS: Calcd for
CggH116N19024s4= 1950.7323, Found: 1950.7340.
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Part B - Preparation of 2-(2-Aza-2-((5-(N-(1,3-bis(3-(2-
(2-(3-(((4-(4-(((1-carboxy-2-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonylamino)-
ethyl)amino)sulfonyl)phenyl)phenyl)sulfonyl)-
amino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)propyl)-
carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonic Acid
The dimeric product from Part A, above (11 mg.
0.0050 mmol) was dissolved in degassed TFA (2 mL) and
stirred at ambient temperatures under a nitrogen
atmosphere for 15 min and concentrated to a viscous
amber oil. This oil was dissolved in anhydrous DMF (2
mL) and made basic with TEA. The solution was treated
with 2-(2-aza-2-((5-((2,5-dioxopyrrolidinyl)carbonyl)(2-
pyridyl))amino)vinyl)benzenesulfonic acid (0.024 mmol)
and stirred at ambient temperatures under a nitrogen
atmosphere for 56 h. The DMF was removed under vacuum,
and the resulting oil was dissolved in 50o ACN and
purified by preparative HPLC on a Vydac C-18 column (22
x 250 mm) using a 2.1o/min gradient of 0 to 63% ACN
containing 0.1o TFA at a flow rate of 20 mL/min. The
main product peak eluting at 20.7 min was collected and
lyophilized to give the title compound as a colorless
powder (5 mg, 42%). MS: m/e 1077.6 [M+2H], 719.0
[M+3H]; High Resolution MS: Calcd for Cg6H117N22026s5~
2153.7112, Found: 2153.7140.
Example 3
Synthesis of 2-((6-((1-Aza-2
(sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)-4
(N-(3-(2-(2-(3-(((4-(4-(((1-carboxy-2-((1-(3-(imidazol
2-ylamino)propyl)(1H-indazol-5-yl))carbonylamino)
ethyl)amino)sulfonyl)phenyl)phenyl)sulfonyl)-
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amino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)butanoic
Acid
0 0
N ~ ~ HN'~OH
°= sH ~ / ~ ~ s- N~°~, o~°~ N o
H H O O
~ N O
~N, I H OH
N N
~.S,'-O H
O
The monomeric product from Example 2, Part A (3.4
mg, 0.0031 mmol) was dissolved. in TFA (1.5 mL) and
allowed to react for 15 min at ambient temperatures, and
concentrated to a viscous amber oil. This oil was
dissolved in anhydrous DMF (2 mL) and made basic to pH
paper with TEA. This solution was treated with 2-(2-
aza-2-((5-((2,5-dioxopyrrolidinyl)carbonyl)(2-pyridyl))-
amino)vinyl)benzenesulfonic acid (5.3 mg, 0.012 mmol)
and stirred at ambient temperatures under a nitrogen
atmosphere for 7 days. The DMF was removed under vacuum
and the resulting oil was dissolved in 50% ACN and
purified by preparative HPLC on a Vydac C-18 column (22
x 250 mm) using a 2.1%/min gradient of 0 to 63% ACN
containing 0.1% TFA at a flow rate of 20 mL/min. The
main product peak eluting at 18.1 min was collected and
lyophilized to give the title compound as a colorless
powder (1.8 mg, 410). MS: m/e 1302.5 [M+H], 651.9
[M+2H] ; High. Resolution MS: Calcd for C57H6gN1301753
[M+H]: 1302.4018, Found: 1302.4030.
Example 4
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Synthesis of 3-((1-(3-(Tmidazole-2-ylamino)propyl)(1H
indazol-5-yl))carbonylamino)-2-(((4-(4-(((3-(2-(2-(3-(2
(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)
cyclododecyl)-
acetylamino)propoxy)ethoxy)ethoxy)propyl)amino)sulfonyl)
phenyl)phenyl)sulfonyl)amino)propanoic Acid
Bis(trifluoroacetate) Salt
O
OH H02C-vN N~-C02H
N J~ NH - O H H C J
O- \ / ~ ~ $-N~O~O~O~N N~'-CO H
H H O O 2
~2TFA
Part A - Phenylmethyl 2-(1,4,7,10-Tetraaza-4,7,10-
tris(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)-
acetate
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
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].
t-Bu-02C~N ~-C02-t-Bu
i
O CN N
~ V C02-t-Bu
O
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Part B - 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 10% 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] .
t-Bu-02C~N~~C02-t-BU
HO CN N
~--r L.J C02-t Bu
O
Part C - Preparation of 3-((1-(3-(Imidazole-2-ylamino)
propyl)(1H-indazol-5-yl))carbonylamino)-2-(((4-(4-(((3
(2-(2-(3-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert
butyl)oxycarbonyl)methyl)cyclododecyl)-
acetylamino)propoxy)ethoxy)-
ethoxy)propyl)amino)sulfonyl)phenyl)phenyl)sulfonyl)-
amino)propanoic Acid Pentakis(trifluoroacetate) Salt
O O
N OH t-Bu-02C-~ N~--C02-t Bu
N.~ J i , H'
C J
NN~N ~N O'~ ~ / ~ ~ O-N~W /'O~O~N ~SN~/N'Cp2_t_ gu
H H O
~ 5 TFA
A solution of 2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-
butyl)oxycarbonyl)methyl)cyclododecyl)acetic acid (55
mg, 0.06 mmol), DIEA (0.063 mL, 0.36 mmol), and HBTU (17
mg, 0.045 mmol) in anhydrous DMF (3 mL) was stirred
under nitrogen at ambient temperatures for 15 min and
treated with the product of Example 1, Part E. Stirring
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was continued l h and the DMF was removed under vacuum.
The resulting amber oil was dissolved in 10o ACN and
purified by preparative HPLC on a ZTydaC C-18 column (22
x 250 mm) using a 2.1%/min gradient pof 0 to 63o ACN
containing 0.1% TFA at a flow rate of 20 mL/min. The
main product peak eluting at 23.0 min was collected and
lyophilized to give the title compound as a colorless,
hygroscopic solid (22 mg, 37%). MS: m/e 1424.8 [M+H];
713.2 [M+2H].
Part D - Preparation of 3-((1-(3-(Imidazole-2-ylamino)-
propyl)(1H-indazol-5-yl))Carbonylamino)-2-(((4-(4-(((3-
(2- (2- (3- (2- (1, 4, 7, 10-tetraaza-4, 7, 10-
tris(carboxymethyl)Cyclododecyl)-
acetylamino)propoxy)ethoxy)ethoxy)propyl)amino)-
sulfonyl)phenyl)phenyl)sulfonyl)amino)propanoiC Acid
Bis(trifluoroacetate) Salt
The product of Part C, above, (10 mg, 0.005 mmol) and
triethylsilane (0.10 mL) were dissolved in degassed TFA
(2.0 mL) and heated at 50 °C under nitrogen for 1 h.
The solution was concentrated under vacuum and the
resulting solid was dissolved in 7% ACN and purified by
preparative HPLC on a ZTydaC C-18 column (22 x 250 mm)
using a 1.5%/min gradient of 0 to 45% ACN containing
0.1% TFA at a flow rate of 20 mL/min. The main product
peak eluting at 19.3 min was collected and lyophilized
to give the title compound as a colorless solid (3.0 mg,
40%). MS: m/e 1256.5 [M+H]; 629.0 [M+2H]; 419.9 [M+3H].
The analytical HPLC methods utilized for examples 5
and 6 are described below:
Instrument: HP1050
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Column: Vydac 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)
Method A
Gradient: Time (min) %A oB
0 80 20
0 100
0 100
15 31 80 20
Method B
Gradient: Time (min) %A oB
20 0 9 8 2
16 63.2 36.8
18 0 100
28 0 100
30 98 2
Examsale 5
Synthesis of 2-(6-((6-((1-Aza-2-(2-
sulfophenyl)vinyl)-amino)(3-
pyridyl))carbonylamino)hexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonylamino)-propanoic acid
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/I o H o
H N H I
S03HN~ o ~ /
N N O OH
H
NH
~NH
Part A. Preparation of Methyl 2-((phenylmethoxy)
carbonylamino-3-((1-(3-((1-(triphenylmethyl)imidazol-2
yl)amino)propyl)(1H-indazol-5
yl))carbonylamino)propanoate
H O
I N
O ~H I / N -
O
q
NH
~, -N
1-[3-[N-(-Triphenylmethylimidazo-2-
yl)amino]propylyl]-5-carboxyindazole (0.950 g, 1.80
mmol), HBTU (0.751 g, 1.98 mmol), and methyl 3-amino-
2(S)-(benzyloxycarbonylamino)propionate (0.624 g, 2.16
mmol) were dissolved in N,N-dimethylformamide (10 mL).
Diisopropylethyl amine (94.1 ~L, 5.40 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 brought up in water.
The water layer was extracted with ethyl acetate. The
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organic layer was washed with brine, dried over
magnesium sulfate, filtered and concentrated to a small
volume. Product precipitated upon addition of hexane.
The product was filtered, washed with hexane and dried
under high vacuum to give 1.6128 g (1170) of product.
ESMS: Calcd. for C45H43N705, 761.33; Found, 762.2
[M+H]+1.
Analytical HPLC, Method A, Rt = 17.00 min, Purity = 90%
Part B. Preparation of 2-
((Phenylmethoxy)carbonylamino-3-((1-(3-((1-
(triphenylmethyl)imidazol-2-yl)amino)propyl)(1H-indazol-
5-yl))carbonylamino)propanoic acid
O
N
O ~~H ~ ~ _ ~N
O OH N
NH
Methyl 2-((phenylmethoxy)-carbonylamino-3-((1-(3-((1-
(triphenylmethyl)imidazol-2-yl)amino)propyl)(1H-indazol-
2,0 5-yl))carbonylamino)propanoate (1.55 g, 2.03 mmol) was
dissolved in tetrahydrofuran (20 mL). Lithium hydroxide
monohydrate (1.71 g, 40.6 mmol) was dissolved in water
and added to the reaction. The reaction was stirred
overnight under N2 for 18h. The tetrahydrofuran was
removed under high vacuum. The pH of the remaining
aqueous layer was adjusted to 5 with 1N HCl. The
aqueous layer was extracted with methylene chloride.
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The organic layer was washed with water, brine, dried
over magnesium sulfate, filtered, and concentrated to an
oil under high vacuum. The oil was recrystallized from
hexane: ethyl acetate to give 800.9 mgs (53%) of product.
ESMS: Calcd. for C44H41N7~5~ 747.32; Found, 748.3
[M+H]+1
Analytical HPLC, Method A, Rt = 15.66 min, Purity = 94%
Part C. Preparation of 2-Amino-3-((1-(3-((1-
(triphenylmethyl)imidazol-2-yl)amino)propyl)(1H-indazol-
5-yl))carbonylamino)propanoic acid
H
2-((Phenylmethoxy)carbonylamino-3-((1-(3-((1-
(triphenylmethyl)imidazol-2-yl)amino)propyl)(1H-indazol-
5-yl))carbonylamino)propanoic acid (0.750 g, 1.00 mmol)
was added to Pd/C (1.00 g) in ethanol (20 mL). The
reaction was evacuated and purged with nitrogen twice.
The reaction was then evacuated and purged with hydrogen
twice, and then maintained under an atmosphere of
hydrogen for 24 h. The reaction was filtered through
celite. The filtrate was concentrated to an oil. The
oil was recrystallized from hexane: ethyl acetate to give
215.6 mgs (35%) of product. ESMS: Calcd. for
C36H35N703~ 613.28; Found, 614.2 [M+H]+1
Analytical HPLC, Method A, Rt = 12.26 min, Purity = 900
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Part D. Preparation of 2-Amino-3-((1-(3-(imidazol-
2-ylamino)propyl)(1H-indazol-5-
yl))carbonylamino)propanoic acid
TFA~H
2-Amino-3-((1-(3-((1-(triphenylmethyl)imidazol-2-
yl)amino)propyl)(1H-indazol-5-
yl))carbonylamino)propanoic acid (0.203 g, 0.331 mmol)
was dissolved in trifluoroacetic acid (3 mL), and the
reaction was refluxed for 1 h. The reaction was
concentrated to an oil under high vacuum. The oil was
triturated with ether. The product was filtered, washed
with ether, dissolved in 50/50 acetonitrile/water, and
lyophilized to give 171.0 mgs (106%) of product. ESMS:
Calcd. for C17H21N703, 371.17; Found, 372.0 [M+H]+1
Analytical HPLC, Method B, Rt = 9.48 min, Purity = 95%
Part E. Preparation of 2-(6-((Tert-butoxy)-
carbonylamino)hexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)-propyl)(1H-indazol-5-
yl))carbonylamino)propanoic acid
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H O
O H N H I ~ \N
N
O O OH
NH
NH
2-Amino-3-((1-(3-(imidazol-2-ylamino)propyl)(1H-indazol-
5-yl))carbonylamino)propanoic acid (0.050 g, 0.103 mmol)
was dissolved in N,N-dimethylformamide (2 mL).
Triethylamine (43.1 ~zL, 0.309 mmol) was added and the
reaction was stirred for 5 minutes. A precipitate
formed so methyl sulfoxide (1 mL) was added.
Succinimidyl N-boc-6-aminohexanoate (0.0406 g, 0.124
mmol) was added anal the reaction was stirred under N2
for 18 h. The reaction was then concentrated to an oil
under high vacuum. The oil was purified by the
following method (Preparative HPLC Method A) to give
39.9 mgs {66%) of product. ESMS: Calcd. for
C2gH4pN806, 584.31; Found, 585.2 [M+H]+1.
Analytical HPLC, Method B, Rt = 18.72 min, Purity = 980
Preparative HPLC Method A:
Instrument: Rainin Rabbit; Dynamax software
Column: Vyadac 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 9 8 2
16 63.2 36.8
18 0 100
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28 0 100
30 98 2
Part F. Preparation of 2-(6-Aminohexanoylamino)-3-
((1-(3-(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonyl-amino)propanoic acid
TFA~H
2-(6-((Tert-butoxy)-carbonylamino)hexanoylamino)-3-((1-
(3-(imidazol-2-ylamino)-propyl)(1H-indazol-5-
yl))carbonylamino)-propanoic acid (0.0322 g, 0.0551
mmol) was dissolved in methylene chloride (1 mL).
Trifluoroacetic acid (1 mL) was added, and the reaction
was stirred for 2 h. The reaction was concentrated to
an oil under high vacuum. The oil was triturated with
ether. The product was filtered, washed with ether,
dissolved in 50/50 acetonitrile/water, and lyophilized
to give 29.9 mgs (91%) of product. ESMS: Calcd. for
C23H32N8~4~ 464.25; Found, 485.2 [M+H]+1
Analytical HPLC, Method B, Rt = 111.02 min, Purity = 97%
Part G. Preparation of 2-(6-((6-((1-Aza-2-(2-
sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)-
hexanoylamino)-3-((1-(3-(imidazol-2-ylamino)propyl)(1H-
indazol-5-yl))carbonylamino)propanoic acid
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il o H o
N N N
S03HN~ ~ H O ~~H I
N N O OH
H
NH
~NH
2-(6-Aminohexanoylamino)-3-((1-(3-(imidazol-2-ylamino)-
propyl)(1H-indazol-5-yl))carbonylamino)propanoic acid
(0.0265 g, 0.0443 mmol) was dissolved in N,N-
dimethylformamide (2 mL). Triethylamine (18.5 ~.L, 0.133
mmol) was added, and the reaction was stirred for 5 min.
2-[[[5-[[(2,5-Dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-
pyridinyl]hydrazono]-methyl]-benzenesulfonic acid,
monosodium salt (0.0234 g, 0.0532 mmol) was added, and
the reaction was stirred for 4 days. The reaction was
concentrated to an oil under high vacuum. The oil was
purified by Preparative HPLC Method A to give 33.7 mgs
(97%) of product. HRMS: Calcd. for C36H41N11~8S + H,
788.2938; Found, 788.2955.
Analytical HPLC, Method B, Rt = 14.06 min, Purity = 90%
Example 6
Synthesis of 2-((6-((1-Aza-2-(2-sulfophenyl)vinyl)-
amino)(3- pyridyl))carbonylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonylamino)propanoic
acid
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H
S03HN-N I N~ H O
/ I / N N I ~ N
I O ~H ~ N
O OH
NH
NH
N.J
2-Amino-3-((1-(3-(imidazol-2-ylamino)propyl)(1H-
indazol-5-yl))carbonylamino)propanoic acid (0.025 g,
0.051.5 mmol) was dissolved in N,N-dimethylformamide (2
mL). Triethylamine (21.5 uL, 0.154 mmol) was added, and
the reaction was stirred for 5 min. 2-[[[5-[[(2,5-
Dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-
pyridinyl]hydrazono]-methyl]-benzenesulfonic acid,
monosodium salt (0.0272 g, 0.0515 mmol) was added, and
the reaction was stirred under nitrogen for 18 h. The
reaction mixture was concentrated to an oil under high
vacuum. The oil was purified by preparative HPLC using
Preparative HPLC Method A to give 14.6 mgs (42%) of the
desired product. ESMS: Calcd. for C3pH30N10~7S,
674.20; Found, 697.1 [M+Na]+1.
Analytical HPLC, Method B, Rt = 13.48 min, Purity = 95%
Example 7
Synthesis of [2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-Glu(2-
(6-Aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)(2-(6-aminohexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)
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N
-NH
NH
H O O O H ~ N,N
O N ~,,,N I
N
H O
S03HN-N N~ H
/ ~ ~ / N O
H
00 H 0 N H I ' N
/ N
O OH
NH
NH
NJ
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
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
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90 % 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, 11H),
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 Glu{2-(6-aminohexanoylamino)-3-
((1-(3-(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonyl-amino)propanoic acid}{2-(6-
aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid}
N
NH
NH
O OH N
H O H ~ \ ~N
O N ~,~ N /
N
H O
H 2N
H O
O H N H f ~ N
O ~ / '
O OH N
NH
N~ NH
J
A solution of 2-(6-aminohexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-y1))carbonyl-
amino)propanoic acid (1 mmol), diisopropylethylamine (3
mmol), and Boc-Glu(OSu)OSu (0.5 mmol) is dissolved in
DMF (50 mL). The reaction mixture is stirred under
nitrogen and at room temperature for 18 h. The solvents
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are removed in vacuo and the crude material is
triturated in ethyl acetate, filtered and washed with
ethyl acetate. The crude product thus obtained is
dissolved in 50 mL of 50% TFA/DCM and the reaction
mixture is stirred for 3 h at room temperature under
nitrogen. TFA and DCM is then removed in vacuo and the
title compound isolated and purified by preparative RP-
HPLC.
Part C. Preparation of [2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-Glu(2-
(6-Aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)(2-(6-Aminohexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)
Glu(2-(6-Aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)(2-(6-Aminohexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid) (0.0481 mmol) is dissolved in DMF
(2 mL). Triethylamine (20.1 ~zL, 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 RP-HPLC
to obtain the desired product.
Example 8
Synthesis of [2-[[[5-[carbonyl]-2
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-Glu
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bis-[Glu(2-(6-Aminohexanoylamino)-3-((l-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)(2-(6-Aminohexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)]
S03H
HN"~N - NNH N~ H
HN~ ; ~N ~ NH
N / HO O O O O ~OH ~ N
I N~N~N O NH N~ N .,~~N I / ~N
O H O-, I -O H O
HN~HN
O H H O
N,/ / I H~'~~ N n H O O H~ N~ H I ~ ~ N
N ~ HO 00 00 OH / N
HN~ ~ NH
HN'~ . ~ NH
l' N NJ
Part A. Preparation of Glu-Bis[Glu{2-(6-
aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid}{2-(6-aminohexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid}]
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t' N N 'l
HN~ ~... NH
HN~ ~ NH
N HO O O OH
H O H NH2 H O ~ H ' N
Nv ~ I N~ ~, O O N~ .~~N I / /
O H O-' I -O H O
HN~1~HN
O ~. N ~ N O
N ~ I H ~ ~H O O H " ~H I /, N
N HO O OJ~OH
HN~ NH
HN/~ ~ NH
~N NJ
A solution of Glu{2-(6-aminohexanoylamino)-3-((1-
(3-(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonyl-amino)propanoic acid}{2-(6-
aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid} (1 mmol), diisopropylethylamine (3
mmol), and Boc-Glu(OSu)OSu (0.5 mmol) is dissolved in
DMF (50 mL). The reaction mixture is stirred under
nitrogen and at room temperature for 18 h. The solvents
are removed in vacuo and the crude material is
triturated in ethyl acetate, filtered and washed with
ethyl acetate. The crude product thus obtained is
dissolved in 50 mL of 50% TFA/DCM and the reaction
mixture is stirred for 3 h at room temperature under
nitrogen. TFA and DCM is then removed in vacuo and the
title compound isolated and purified by preparative RP-
HPLC.
Part B: Preparation of [2-[[[5-[carbonyl]-2-
pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-Glu-
bis-[Glu(2-(6-Aminohea~anoylamino)-3-((1-(3-(imidazol-2-
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ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)(2-(6-Aminohexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)]
Glu-bis-[Glu{2-(6-aminohexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid}{2-(6-aminohexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid}] (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 RP-HPLC
to obtain the desired product.
Example 9
Synthesis of of 2-(1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)-1-cyclododecyl)acetyl-{2-(6-
aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid}
O O
H
HOOC--~ ~N~H rN H I ~ ~ N
O ~ / '
O OH N
HOOC~N~,~,/N~COOH
NH
~NH
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Part A. Preparation of 2-(1,4,7,10-tetraaza-4,7,10-
tris(t-butoxycarbonylmethyl)-1-cyclododecyl)acetyl-{2-
(6-aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid}
O H O
tBu00C-~ ~ ~I.~ N
N N~H 'r H ~ ~ N
O
O OH N
N N
tBu00C--~ ~,-/ ~COOtBu NH
~NH
N.J
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) is added
HBTU (17 mg, 0.0456 mmol) and the mixture is stirred for
5 min. To this is added a solution of 2-(6-
aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid (0.0326 mmol) in DMF (1 mL) and the
reaction mixture is allowed to stir under nitrogen at
room temperature for 4 h. The solvent is removed in
vacuo and the residue is purified by preparative RP-HPLC
to obtain the product as a lyophilized solid.
Part B. Preparation of 2-(1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)-1-cyclododecyl)acetyl-{2-(6-
aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid}
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A solution of ~-(1,4,7,10-tetraaza-4,7,10-tris(t-
butoxycarbonylmethyl)-1-Cyclododecyl)acetyl-2-(6-
Aminohexanoylamino)-3-((1--(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoiC acid (8.71 mmol) in TFA (3 mL) is
stirred at room temperature under nitrogen for 5 h. The
solution is concentrated in vacuo and the residue is
purified by preparative RP-HPLC to obtain the desired
product as the lyophilized solid.
Example 10
Synthesis of 2-(1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)-1-Cyclododecyl)acetyl-Glu{2-(6-
Aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))Carbonyl-
amino)propanoiC acid}{2-(6-Aminohexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoiC acid}
N '1
~NH
-NH
O OH ~N
H O H ~ \ ~N
O N ~~ N
N
H O
HOOC-~ /-'~ H
N N~N
H O
~N N 00 H O N H I % N
N
HOOC ~/ COOH O OH
NH
~NH
NUJ
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Part A. Preparation of 2-(1,4,7,10-tetraaza-4,7,10-
tris(t-butoxycarbonylmethyl)-1-cyclododecyl)acetyl-
Glu{2-(6-Aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid}{2-(6-Aminohexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid}
N '1
NH
NH
O OH N
H O H ~ ~N
O N ~~ N
N ',,
H O
tBu00C-1N/~N N
H O
N O O H N H ~ ' N
O ~ / N
tBuOOC ~/ COOtBu O OH
NH
~' NH
N.J
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 uL) in DMF (2 mL) is added
HBTU (17 mg, 0.0456 mmol) and the mixture is stirred for
5 min. To this is added a solution of Glu{2-(6-
Aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid}{2-(6-Aminohexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid} (0.0326 mmol) in DMF (1 mL) and
the reaction mixture is allowed to stir under nitrogen
at room temperature for 4 h. The solvent is removed in
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vacuo and the residue is purified by preparative RP-HPLC
to obtain the product as a lyophilized solid.
Part B. Preparation of 2-(1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)-1.-cyclododecyl)acetyl-Glu{2-(6-
Aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid}{2-(6-Aminohexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid}.
A solution of 2-(1,4,7,10-tetraaza-4,7,10-tris(t-
butoxycarbonylmethyl)-1-cyclododecyl)acetyl-Glu{2-(6-
Aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid}{2-(6-Aminohexanoylamino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid} (8.71 mmol) in TFA (3 mL) is
stirred at room temperature under nitrogen for 5 h. The
solution is concentrated in vacuo and the residue is
purified by preparative RP-HPLC to obtain the desired
product as the lyophilized solid.
Example 11
Synthesis of DOTA/N,N'-Bis(3-(2-(2-(3-(((4-(4-(((1-
carboxy-2-((1-(3-(imidazol-2-ylamino)propyl)(1H-indazol
5-yl))carbonylamino)ethyl)amino)sulfonyl)phenyl)phenyl)
sulfonyl)amino)propoxy)ethoxy)ethoxy)propyl)-2
(amino)pentane-1,5-diamide Tris(trifluoroacetate) Salt
Conjugate
2os
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O
N~OH
~H NH p C02H
N - ~ ~ H H l n NCO H
N~..N~ O:O \ / O_N~O~O~O~.N O N N 2
H H ~ O CN NJ
O~ N ~ ~/ ~C02H
O O lNH H
/ \ ~ ~ ~_N~O~O~O~
N NN ~ H NH - OH
N~O H
~3TFA
O
Part A - Preparation of DOTA Tris-t-Butyl Ester/N,N'-
Bis(3-(2-(2-(3-(((4-(4-(((1-carboxy-2-((1-(3-(imidazol-
2-ylamino)propyl)(1H-indazol-5-yl))carbonylamino)-
ethyl)amino)sulfonyl)phenyl)phenyl)sulfonyl)-
amino)propoxy)ethoxy)ethoxy)propyl)-2-(amino)pentane-
1,5-diamide Hexakis(trifluoroacetate) Salt Conjugate
A solution of the product from Example 2, Part A in
degassed TFA is allowed to stand at ambient temperatures
under nitrogen for 15 min. The solution is concentrated
and the resulting oil is dissolved in 50% ACN. The TFA
salt is converted to the free base by treatment with an
ion exchange resin such as Bio-Rad AG-3x4A, hydroxide
form, until the pH of the solution is raised to 6.5.
The resin is removed by filtration and the filtrate is
lyophilised to give the free base of the deprotected
dimer.
A solution of DOTA tris-t-butyl ester and DIEA in
anhydrous DMF are treated with HBTU and allowed to react
15 min at ambient temperatures under nitrogen. The
deprotected dimer from above is added to this solution
and stirring is continued at ambient temperatures under
nitrogen for 18 h. The DMF is removed under vacuum and
the resulting oil is purified by preparative HPLC on a
C18 column using a water:ACN:0.1% TFA gradient. The
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product fraction is lyophilized to give the title
compound.
Part B - Preparation of DOTA/N,N'-Bis(3-(2-(2-(3-(((4-
(~-(((1-carboxy-2-((1-(3-(imidazol-2-ylamino)propyl)(1H-
indazol-5-yl))carbonylamino)ethyl)amino)sulfonyl)-
phenyl)phenyl)sulfonyl)amino)propoxy)-
ethoxy)ethoxy)propyl)-2-(amino)pentane-1,5-diamide
Tris(trifluoroacetate) Salt Conjugate
The product of Part A, above, and Et3SiH are
dissolved in degassed TFA and heated at 50 °C under
nitrogen for 1 h. The solution is concentrated and the
resulting residue is purified by preparative HPLC on a
C18 column using a water:ACN:0.1% TFA gradient. The
product fraction is lyophilized to give the title
compound.
Example 12
Synthesis of DOTA/2-Amino-4-(N-(3-(2-(2-(3-(((4-(4-(((1-
carboxy-2-.((1-(3-(imidazol-2-ylamino)propyl)(1H-indazol-
5-yl))carbonylamino)ethyl)amino)sulfonyl)phenyl)phenyl)-
sulfonyl)amino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)-
butanoic Acid Bis(trifluoroacetate) Salt
O
N'~OH
I ~ H NH O C02H
N ~ H H ~C02H
Ny~N~ O'~ ~ / ~ ' ~-N~Ow/'O~O~N lN~
HH ~ C J
O~.N~N~--/N'-C02H
~ 2 TFA OH H
The title compound is prepared by the procedure
described for Example 11 by substituting the monomeric
product of Example 2, Part A for the dimeric product of
Example 2, Part A.
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Example 13
Synthesis of DOTA/~-(((4-(3-(N-(3-(2-(2-(3-(2-Amino-3-
sulfopropyl)propoxy)ethoxy)ethoxy)propyl)carbamoyl)propo
xy)-2,6-dimethylphenyl)sulfonyl)amino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonylamino)propionic Acid Bis(trifluoroacetate)
Salt Conjugate
O O
N~OH
N' ~ / H NH
N S02 02H
N~N~ , ~~ ~C02H
H H ~ N N
OII H
O~ ~ ~O~ ~ ~N CN N
~ 2 TFA H O O H = ~ V C02H
H03S i O
Part A - Ethyl 4-(3,5-Dimethylphenoxy)butanoate
Sodium metal (17.12 g, 0.744 mol) was added to
anhydrous EtOH (350 mL) and stirred until dissolved.
3,5-Dimethylphenol was added and the solution was
stirred 15 min at ambient temperatures. Ethyl 4-
bromoacetate (58.7 mL, 0.41 mol) was added and the
solution was stirred at ambient temperatures under a
nitrogen atmosphere for 28 h. The EtOH was removed
under vacuum and the oily solid was partitioned between
water (1 L) and EtOAc (500 mL). The aqueous layer was
extracted with additional EtOAc (500 mL). The combined
EtOAc extracts were washed consecutively with saturated
NaHC03 (300 mL) and saturated NaCl (300 mL), dried
(MgS04), and concentrated to give an amber liquid. This
liquid was vacuum fractional distilled through a 15 cm
Vigreux column. The main fraction was collected from
91-117 °C/6 mm Hg to gave the title compound as a
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colorless liquid (77.77 g, 89%). 1H NMR (CDC13): 6.59
(s, 1H), 6.52 (s, 2H), 4.16 (q, J - 7.16 Hz, 2H), 3.98
(t, J = 6.14 Hz, 2H), 2.49 (t, J = 7.34 Hz, 2H), 2.28
(s, 6H), 2.11-2.07 (m, 2H), 1.26 (t, J = 7.16 Hz, 3H);
Anal. calcd for Cl4H~p03; C,71.16; H, 8.53, Found:
C,71.35; H, 8.59.
W
O
Part B - 4-(3,5-Dimethylphenoxy)butanoic Acid
The product of part A, above (75.52 g, 0.320 mol)
and KOH pellets (38.5 g, 0.584 mol) were dissolved in
absolute EtOH (1.50 L) and heated at reflux for 3 h.
The solution was concentrated to a colorless solid,
which was taken up in water (2.0 L) and washed with
ether (2 x 750 mL). The aqueous layer was adjusted to
pH 1 with concd HCl (55 mL) and the resulting oily ppt
was extracted into EtOAc (2 x 500 mL). The combined
EtOAc extracts were washed consecutively with water (300
mL) and saturated NaCl, dried (MgS04), and concentrated
to give a colorless solid (64.13 g). Recrystallization
from hexanes (500 mL) gave the title compound as a
colorless solid (59.51 g, 890). MP: 66-68.5 °C; 1H NMR
(CDC13): 11.70 (bs, 1H), 6.59 (s, 1H),6.52 (s, 2H),
3.99 (t, J = 6.06 Hz, 2H), 2.57 (t, = 7.29 Hz, 2H),
J
2.28 (s, 6H), 2.12-2.08 (m, 2H); Anal.calcd for
C12H1g03; C, 69.21; H, 7.74, Found: 69.23; H, 7.40.
C,
O
OOH
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Part C - 4-(4-(Chlorosulfon~l)-3,5-
dimethvlphenoxy)butanoic Acid
A solution of the product of Part B, above (20.8 g,
0.100 mol) in CHC13 (100 mL) was cooled to 0 °C and
treated with chlorosulfonic acid (36 mL, 0.54 mol)
dropwise and with rapid stirring while keeping the
temperature of the reaction at 0 °C. The resulting
gelatinous mixture was stirred an additional 10 min and
poured onto an ice/water mixture (600 mL). The
resulting solid ppt was collected by filtration, washed
with water (3 x 75 mL), and dried under vacuum to give a
colorless solid (12.52 g). MP: 114-115 °C (with
decomp); 1H NMR (CDC13): 13.84 (bs, 1H), 6.50 (s, 2H),
I5 3.91 (t, J = 6.48 Hz, 2H}, 2.48 (s, 6H), 2.32 (t, J =
7.32 Hz, 2H), 1.89-1.84 (m, 2H); IR (KBr cm-2): 1705
(s), 1370 (s), 1175 (s); MS: m/e 305.1 [M-H].
~I
S02
i~
O
o~OH
Part D - 4-(4-(((2-((tert-Butoxy)carbonylamino)-1-
(methoxycarbonyl)ethyl)amino)sulfonyl)-3,5-
dimethylphenoxy)butanoic Acid
A solution of N-(3-Boc-L-oc,(3, -diaminopropionic acid
methyl ester hydrochloride (568 mg, 2.10 mmol) and DIEA
(0.73 mL, 4.2 mmol) in DCM (5 mL) was cooled to 0 °C and
treated with a suspension of the product of Part C,
above (656 mg, 2.10 mmol) in DCM (20 mL) in small
portions over a 15 min period. The reaction was stirred
at ambient temperatures under a nitrogen atmosphere for
18 h. The reaction was diluted with DCM (100 mL) and
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washed with water {3 x 75 mL). The organic phase was
dried (MgS04), and concentrated to give crude product
(698 mg), which was purified by preparative HPLC on a
Vydac C-18 column {50 x 250 mm) using a 0.96%/min
gradient of 18 to 58.50 ACN containing 0.1o TFA at a
flow rate of 80 mL/min. The main product fraction
eluting at 23.8 min was collected adjusted to pH 3,
partially concentrated to remove ACN, and extracted with
DCM (2 x 100 mL). The DCM extracts were dried (MgS04)
and concentrated to give the title compound as a
colorless solid (297 mg, 29%). 1H NMR (CDCl3): S 6.61
(s, 2H), 5.66 (d, J = 7.2 Hz, 1H), 4.90 (s, 1H), 4.03
(bs, 2H), 3.86 (bs, 1H), 3.59 (s, 3H), 3.49 (bs, 2H),
2.62 (s, 6H), 2.58-2.51 (m, 2H), 2.18-2.07 (m, 2H), 1.41
(s, 9H); MS: m/e 489.4 [M+H]; High Resolution MS: Calcd
for C~1H33N~O9S [M+Na]: 511.1726, Found: 511.1747; Anal.
calcd for C~1H3~N~OgS: C, 51.62; H, 6.61; N, 5.74, Found:
C, 51.47; H, 6.27; N, 5.48.
O
Boa N,~y
H NH
02
~~OH
Part E - Methyl 3-((tert-Butoxy)carbonylamino)-2-(((2,6-
dimethyl-4- {3- {N- (3- {2- (2- (3-
((phenylmethoxy)carbonylamino)propoxy)ethoxy)-
ethoxy)propyl)carbamoyl)propoxy)phenyl)-
sulfonyl)amino)propanoate
A solution of the product from Part D, above (233
mg, 0.477 mmol), the product of Example 1, Part A (190
mg, 0.536 mmol), TEA (0.2 mL, 1.43 mmol), and HBTU (226
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mg, 0.701 mmol) in anhydrous DMF (8 mL) was stirred at
ambient temperatures under a nitrogen atmosphere for 1
h. The DMF was removed under vacuum and the oily
residue was taken up in EtOAc (50 mL) and washed
consecutively with 0.1 N HCl (35 mL), water (35 mL), and
saturated NaCl (35 mL), dried (MgS04), and concentrated
to give crude product as a yellow viscous oil. Flash
chromatography on a 3 x 18 cm silica gel column
(EtOAc/MeOH, 95/5) gave the title compound as a
colorless viscous oil (393 mg, 100%). 1H NMR (CDC13): 8
7.34-7.28 (m, 5H), 6.60 (s, 2H), 6.26 (bs, 1H), 5.67
(bs, 1H), 5.29 (bs, 1H), 5.08 (s, 2H), 4.88 (bs, 1H),
3.99 (t, J = 6.1 Hz, 2H), 3.88-3.84 (m, 1H), 3.62-3.40
(m, 17H), 3.37-3.26 (m, 4H), 2.62 (s, 6H), 2.32 (t, J =
7.2 Hz, 2H), 2.08 (t, J = 6.3 Hz, 2H), 1.79-1.70 (m,
4H), 1.41 (s, 9H); MS: m/e 825.5 [M+H]; High Resolution
MS: Calcd for C3gH61N4013S [M+H]: 825.3955, Found:
825.3940.
BoGN~O~ ,
H NH
~2
O~ N ~.i'O''\~Ow/'O~\/~N x0 i
H H
Part F - Methyl 3-Amino-2-(((2,6-dimethyl-4-(3-(N-(3-(2-
(2-(3-((phenylmethoxy)carbonylamino)propoxy)ethoxy)-
ethoxy)propyl)carbamoyl)propoxy)pheny1)-
sulfonyl)amino)propanoate
The product of Part E, above (750 mg, 0.91 mmol)
was dissolved in 4 M HC1/dioxane (25 mL) and stirred at
ambient temperatures for 1 h. The solution was diluted
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with ether (500 mL) and the resulting gummy ppt was
triturated with fresh ether (2 x 250 mL). The gummy
solid was dissolved in water (100 mL) and adjusted to pH
9 with NaHC03, causing an oily ppt to form. This ppt
was extracted into DCM (2 x 75 mL). The DCM extracts
were dried (MgS04) and concentrated to give the title
compound as a colorless oil (386 mg, 560). MS: m/e
725.5 [M+H].
H
~O~O~O~N~O i
H
Part G - Preparation of Methyl 2-(((2,6-Dimethyl-4-(3-
(N- (3- (2- (2- (3~-
((phenylmethoxy)Carbonylamino)propoxy)ethoxy)-
ethoxy)propyl)Carbamoyl)propoxy)phenyl)sulfonyl)amino)-
3-((1-(3-((1-(triphenylmethyl)imidazol-2-
yl)amino)propyl)(1H-indazol-5-
yl))Carbonylamino)propionate
N~O~
H NH
~~N~ 02
Tr H
Op
O~N ~O'~O~O~N
H H
A solution of 1-(3-((1-(triphenylmethyl)imidazol-2-
yl)amino)propyl)-1H-indazole-5-carboxylic acid, methyl
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3-amino-2- ( ( (2, 6-dimethyl-4- (3- (N- (3- (2- (2- (3-
((phenylmethoxy)carbonylamino)propoxy)ethoxy)ethoxy)-
propyl)carbamoyl)propoxy)phenyl)-
sulfonyl)amino)propanoate, DIEA, and HBTU in anhydrous
DMF are stirred at ambient temperatures under nitrogen
for 4 h. The DMF is removed under vacuum and the
resulting residue is dissolved in EtOAc and washed with
water, saturated NaHC03, and saturated NaCl. The EtOAc
layer is dried (MgS04) and concentrated to dryness. The
crude product is purified by flash chromatography on
silica gel using EtOAc/MeOH.
Part H - Preparation of 2-( ( (4-(3-(N-(3-(2-(2-(3-(2-
((tert-Butoxy)carbonylamino)-3-
sulfopropyl)propoxy)ethoxy)-
ethoxy)propyl)carbamoyl)propoxy)-2,6-dimethylphenyl)-
sulfonyl)amino)-3-((1-(3-(imidazol-2-ylamino)propyl)(1H-
indazol-5-yl))carbonylamino)propionic Acid
Trifluoroacetate Salt
O O
N~OH
H ~ TFA
~[ H
~N'Boc
H03S ~
The product from Part G, above is hydrolyzed in a
mixture of peroxide-free THF, water, and 3 N LiOH at
ambient temperatures under nitrogen for 6 h. The THF is
removed under vacuum and the resulting mixture is
diluted with water and adjusted to pH 3 using 0.1 N HCl.
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The mixture is extracted with EtOAc, and the combined
extracts are dried (MgS04) and concentrated.
A solution of the hydrolysis product from above and
Et3SiH in degassed TFA is heated at 70 °C under nitrogen
for 1 h. The solution is concentrated and the resulting
residue is dissolved in 50% ACN. The TFA salt is
converted to the free base by treatment with an ion
exchange resin such as Bio-Rad AG-3X4A, hydroxide form,
until the pH of the solution is raised to 6.5. The
resin is removed by filtration and the filtrate is
lyophilized to give the free base.
The above material is dissolved in anhydrous DMF,
and treated with the N-hydroxysuccinimide ester of Boc-
cysteic acid (as described in Liebigs Ann. Chem. 1979,
776-783) and DIEA. The solution is stirred at ambient
temperatures under nitrogen for 18 h, and the DMF is
removed under vacuum. The resulting residue is purified
by preparative HPLC on a C18 column using a
water:ACN:0.1% TFA gradient. The product fraction is
lyophilized to give the title compound.
Part I - Preparation of DOTA Tri-t-butyl Ester/2-(((4-
(3-(N-(3-(2-(2-(3-(2-Amino-3-
sulfopropyl)propoxy)ethoxy)ethoxy)-
propyl)carbamoyl)propoxy)-2,6-
dimethylphenyl)sulfonyl)amino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonylamino)propionic
Acid Pentakis(trifluoroacetate) Salt Conjugate
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O O
N~OH
H NH
02 02-t-Bu
/ ~ N~-C02-t-Bu
H C J
~ L~/ C02-t-BU
~ 5 TFA O~N'v"O'~O'~O~N ~N N N~
H H~~S~ O
The product of Part H, above is dissolved in
degassed TFA and stirred at ambient temperatures for 15
min. The solution is concentrated under vacuum, and the
resulting residue is dissolved in 50% ACN and
lyophilized to remove the last traces of TFA.
Tn a separate flask, a solution of DOTA tris-t-
butyl ester and DIEA in anhydrous DMF are treated with
HBTU and allowed to react 15 min at ambient temperatures
under nitrogen. The deprotected product from above is
added to this solution and stirring is continued at
ambient temperatures under nitrogen for 18 h. The DMF
is removed under vacuum and the resulting residue is
purified by preparative HPLC on a C18 column using a
water:ACN:0.1% TFA gradient. The product fraction is
lyophilized to give the title compound.
Part J - Preparation of DOTA/2-(((4-(3-(N-(3-(2-(2-(3-
(2-Amino-3-
sulfopropyl)propoxy)ethoxy)ethoxy)propyl)carbamoyl)-
propoxy)-2,6-dimethylphenyl)sulfonyl)amino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonylamino)propionic Acid Bis(trifluoroacetate)
Salt Conjugate
The product of Part I, above, and Et3SiH are
dissolved in degassed TFA and heated at 50 °C under
nitrogen for 1 h. The solution is concentrated and the
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resulting residue is purified by preparative HPLC on a
C18 column using a water:ACN:0.1% TFA gradient. The
product fraction is lyophilized to give the title
compound.
Example 14
Synthesis of DOTA/2-(((4-(3-(N-(3-(2-(2-(3-(2-Amino-3-
(4-
(phosphonooxy)phenyl)propanoylamino)propoxy)ethoxy)ethox
y)propyl)carbamoyl)propoxy)-2,6-
dimethylphenyl)sulfonyl)amino)-3-((1-(3-(imidazol-2
ylamino)propyl)(1H-indazol-5-yl))carbonylamino)propionic
Acid Trifluoroacetate Salt Conjugate
H
C02H
N~N l~~ ~-C02H
H H fV N
O O
TFA Hob O H ~t-J ~ C02H
O
H203 PO~
The title compound is prepared by the procedure
described for Example 13 by substituting Boc-Tyr(P03H~)
OSu for Boc-Cys(03H)-OSu.
Example 15
Synthesis of DOTA/2-(((4-(3-(N-(3-(2-(2-(3-(2-Amino-3-
(4-
(sulfooxy)phenyl)propanoylamino)propoxy)ethoxy)ethoxy)pr
opyl)carbamoyl)propoxy)-2,6-
dimethylphenyl)sulfonyl)amino)-3-((1-(3-(imidazol-2-
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ylamino)propyl)(1H-indazol-5-yl))carbonylamino)propionic
Acid Trifluoroacetate Salt Conjugate
The title compound is prepared by the procedure
described for Example 13 by substituting Boc-Tyr(S03H)-
OSu for Boc-Cys(03H)-OSu.
O O
~N'~OH
~N'~~ ~ H NH
J 02 C02H
N H ~ LN~~-C02H
H ,
O O H
O~ ~ ~O~ ~ ~.N CN N
TFA H O O H O V C02H
H 03S0
Example 16
Synthesis of DOTA/2-(((4-(3-(N-(3-(2-(2-(3-(2-Amino-4-
(N-(ethyl-3,6-0-disulfo-(3-D-galactopyranosyl)carbamoyl)-
butanoylamino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)pro
poxy)-2,6-dimethylphenyl)sulfonyl)amino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonylamino)propionic Acid Conjugate
N l~N l 2H
H N~~-C02H
H
CN N
V C02H
D
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Part A - Preparation of Boc-Glu(aminoethyl-3,6-O-
disulfo-(3-D-galactopyranosyl)-OSu
A solution of Boc-Glu-OMe, aminoethyl-3,6-0-
disulfo-~-D-galactopyranoside (as described in Tet.
Lett. 1997, 53, 11937-11952), DIEA, and HBTU in
anhydrous DMF is stirred at ambient temperatures under
nitrogen for 18 h. The DMF is removed under vacuum and
the resulting residue is hydrolyzed using aqueous NaOH.
The reaction solution is adjusted to pH 7 and purified
by preparative anion exchange chromatography using a
resin such as DEAE Cellulose and a Et3NH2C03 gradient.
The product fraction is treated with a cation exchange
resin, sodium form, to give the intermediate carboxylic
acid as the sodium salt.
The above compound, N-hydroxysuccinimide, and DCC
are dissolved in anhydrous DMF and stirred at ambient
temperatures under nitrogen for 18 h. The DMF is
removed under vacuum and the resulting residue is
purified by preparative anion exchange chromatography as
above to give the title compound as the triethylammonium
salt.
Part B - Preparation of DOTAJ2-(((4-(3-(N-(3-(2-(2-(3-
(2-Amino-4-(N-(ethyl-3,6-0-disulfo-(3-D-
galactopyranosyl)carbamoyl)-
butanoylamino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)-
propoxy)-2,6-dimethylphenyl)sulfonyl)amino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonylamino)propionic Acid Conjugate
The title compound is prepared by the procedure
described for Example 13 by substituting Boc-
Glu(aminoethyl-3,6-0-disulfo-(3-D-galactopyranosyl)-OSu
for Boc-Cys(03H)-OSu.
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Example 17
Synthesis of DOTA/2- ( ( (4- (3- (N- (3- (2- (2- (3- (2-Amino-4-
(N-(6-deoxy-(3-cyclodextryl)carbamoyl)-
butanoylamino)propoxy)ethoxy)-
ethoxy)propyl)carbamoyl)propoxy)-2,6-dimethylphenyl)-
sulfonyl)amino)-3-((1-(3-(imidazol-2-ylamino)propyl)(1H
indazol-5-yl))carbonylamino)propionic Acid
Bis(trifluoroacetate) Conjugate
O
N' Y -OH
~ i H NH
02 C02H
H H ~ I ~N~~C02H
O~ ~ ~.0~ ~ ~N CN N
~ 2 TFA H O O H ~ '~--' ~..._/ C02H
Cyclodextrin
Part A - Preparation of Boc-Glu(6-amino-6-deoxy-(3-
cyclodextryl)-OMe
A solution of Boc-Glu-OMe, 6-amino-6-deoxy-(3-
cyclodextrin (as described in J. Org. Chem. 1996, 61,
903-908), DIEA, and HBTU in anhydrous DMF is stirred at
ambient temperatures under nitrogen for 18 h. The DMF
is removed under vacuum and the resulting residue is
purified by preparative HPLC on a C18 column using a
water:ACN:0.1% TFA gradient. The product fraction is
lyophilized to give the title compound.
Part B - Preparation of Boc-Glu(6-amino-6-deoxy-(3-
cyclodextryl)-OSu
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The product of Part A, above, is hydrolyzed by
stirring in a mixture of LiOH, THF, and water at ambient
temperatures under nitrogen for 4 h. The THF is removed
under vacuum and the resulting mixture is diluted with
water and adjusted to pH 3 using 0.1 N HCl. The mixture
is extracted with EtOAc, and the combined extracts are
dried (MgSOg) and concentrated. The resulting material
is dissolved in anhydrous DMF along with N-
hydroxysuccinimide, and DCC, and stirred at ambient
temperatures under nitrogen for 18 h. The DMF is
removed under vacuum and the resulting residue is
purified by preparative HPLC on a C18 column using a
water:ACN:0.1~ TFA gradient. The product fraction is
lyophilized to give the title compound.
Part C - Preparation of DOTA/2-(((4-(3-{N-(3-(2-(2-
(3-(2-Amino-4-(N-(6-deoxy-(3-cyclodextryl)carbamoyl)-
butanoylamino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)-
propoxy)-2,6-dimethylphenyl)sulfonyl)amino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))-
carbonylamino)propionic Acid Bis(trifluoroacetate)
Conjugate
The title compound is prepared by the procedure
described for Example 13 by substituting Boc-Glu(6-
amino-6-deoxy-(3-cyclodextryl)-OSu for Boc-Cys(03H)-OSu.
Example 18
Synthesis of DOTA/2- { ( (4- (3- (N- (3- (2- (2- (3- (2-Amino-4-
(N-((~-methoxypolyethylene(5,000)glycoxyethyl)carbamoyl)-
butanoylamino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)-
propoxy)-2,6-dimethylphenyl)sulfonyl)amino)-3-((1-(3-
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(imidazol-2-ylamino)propyl)(1H-indazol-5
yl))carbonylamino)propionic Acid Bis(trifluoroacetate)
Conjugate
O
N~O H
~ ~~H NH
N~N~N 02 l 2H
H H , N~~-C02H
O O 1
H
O~ ~/'~ ~Ow/' ~,/~ ~.N CN N
~ 2 TFA H 0 0 H O V C02H
~~N~O
nH
Part A - Preparation of Boc-Glu(amino-(~J-
methoxypolyethylene glycol)-OMe
A solution of Boc-Glu-OMe, amino-(~-
methoxypolyethylene glycol, (MW = 5,000), DIEA, and HBTU
in anhydrous DMF is stirred at ambient temperatures
under nitrogen for 18 h. The DMF is removed under
vacuum and the resulting residue is purified lay
preparative HPLC on a C18 column using a water:ACN:0.1%
TFA gradient. The product fraction is lyophilized to
give the title compound.
Part B - Preparation of Boc-Glu(amino-(~-
methoxypolyethylene glycol)-OSu
The product of Part A, above, is hydrolyzed by
stirring in a mixture of LiOH, THF, and water at ambient
temperatures under nitrogen for 4 h. The THF is removed
under vacuum and the resulting solution is adjusted to
pH 7 using 0.1 N HCl. The solution is desalted using a
Sephadex PD-10 desalting column and the product eluant
is lyophilized. The resulting material is dissolved in
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anhydrous DMF along with N-hydroxysuccinimide, and DCC,
and stirred at ambient temperatures under nitrogen for
18 h. The DMF is removed under vacuum and the resulting
residue is purified by preparative HPLC on a C18 column
using a water:ACN:0.lo TFA gradient. The product
fraction is lyophilized to give the title compound.
Part C - Preparation of DOTA/2-(((4-(3-(N-(3-(2-(2-
(3- (2-Amino-4- (N- (C~-
methoxypolyethylene(5,000)glycoxyethyl)carbamoyl)-
butanoylamino)propoxy)ethoxy)ethoxy)propyl)carbamoyl)-
propoxy)-2,6-dimethylphenyl)sulfonyl)amino)-3-((1-(3-
(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))-
carbonylamino)propionic Acid Bis(trifluoroacetate) Salt
Conjugate
The title compound is prepared by the procedure
described for Example 13 by substituting Boc-Glu(amino-
~-methoxypolyethylene glycol)-OSu for Boc-Cys(03H)-OSu.
Example 19
Synthesis of 2-(((4-(3-(N-(3-(2-(2-(3-(2-(1,4,7,10-
Tetraaza-4,7,10-
tris(carboxymethyl)cyclododecylacetylamino)-6-
aminohexanoylamino)propoxy)ethoxy)ethoxy)propyl)-
carbamoyl)propoxy)-2,6-dimethylphenyl)sulfonyl)amino)-3-
((1-(3-(imidazol-2-ylamino)propyl)(1H-indazol-5-yl))-
carbonylamino)propionic Acid Tris(trifluoroacetate) Salt
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O O
~N~OH
~ i H NH
N~N ~ 02 ~ 2H
H H ~ ~ ni ~i~-C02H
O~ N ~O'~'O~/'O~ t
~3TFA H f
The title compound is prepared by the procedure
described for Example 13 by substituting BoC-Lys(Cbz)-
OSu for BoC-Cys(03H)-OSu.
Exams 1 a 2 0
Synthesis of the DOTA/2-(((4-(3-(N-(3-(2-(2-(3-(2-Amino-
(bis(phosphonomethyl)amino)acetylamino)hexanolylamino)pr
opoxy)ethoxy)ethoxy)propyl)Carbamoyl)propoxy)-2,6-
dimethylphenyl)sulfonyl)amino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))Carbonylamino)propioniC
Acid Trifluoroacetate Salt Conjugate
O O
'~ N~OH
N' ~ / H NH
~I N 02 C02H
N~N~ , l ~-C02H
H H ~ N
OI O H
O~ ~ ~O~ ~~ ~.N CN N
O O H O V CO2H
Oe,NH
lN'~P03H2
H203PJ
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A solution of bis(phosphonomethyl)glycine, DIEA,
and HBTU in anhydrous DMF is stirred at ambient
temperatures under nitrogen for 15 min, and treated with
the product of Example 19. Stirring is continued for 18
h and. the DMF is removed under vacuum. The resulting
residue is purified by ion exchange chromatography.
Example 21
Synthesis of DTPA adduct of 2-(6-Aminohexanoylamino)-3-
((1-(3-(imidazol-2-ylamino)propyl)(1H-indazol-5-.
yl))carbonyl-amino)propanoic acid
HOOC1 O H O
N~H N H I ' N
/~. ~ O ~ / N
HOOC N O OH
HOOC,~N~COOH NH
~NH
N~J
To a solution of DTPA dianhydride (3 mmol),
triethylamine (3 mmol) in DMF 20 mL is added a solution
of 2-(6-aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid (1 mmol) in DMF 5 mL dropwise. The
reaction mixture is stirred for 18 h at room temperature
under nitrogen, the volatiles are removed and the title
compound is obtained after purification and isolation
using preparative RP-HPLC.
The following procedure describe the synthesis of
radiopharmaceuticals of the present invention of the
formula 99mTc(VnA)(tricine)(phosphine), in which (VnA)
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represents a vitronectin receptor antagonist compound of
the present invention bonded to the Tc through a
diazenido (-N=N-) or hydrazido (=N-NH-) moiety. The
diazenido or 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 axe tricine and a phosphine.
Examples 22 - 26
Synthesis of Complexes [99mTc(HYNIC-
VnA)(tricine)(TPPTS)].
To a lyophilized vial containing 4.84 mg TPPTS, 6.3
mg tricine, 40 mg mannitol, succinic acid buffer, pH
4.8, and 0.1% Pluronic F-64 surfactant, was added 1.1 mL
sterile water for injection, 0.2 mL (20 ug) of the
appropriate HYNIC-conjugated vitronectin antagonist
(VnA) in deionized water or 50% aqueous ethanol, and 0.2
mL of 99mTc04- (50-1-5 mCi) in saline. The reconstituted
kit was heated in a 100 °C water bath for 15 minutes,
and was allowed to cool 10 minutes at room temperature.
A sample of the reaction mixture was analyzed by HPLC.
The RCP results are listed in the table 1.
Table 1. Analytical and Yield Data for
99mTc(VnA)(tricine)(TPPTS) Complexes
Example No. Reagent No. Ret. Time % Yield
(min)
22 1 18.6* 50
23 2 13.2** 55
24 3 17.0** 71
25 5 10.3*** 72
26 6 7.2* 64
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* The HPLC method using a reverse phase C1g Zorbax
column (4.6 mm x 25 cm, 80 A pore size) at a flow rate
of 1.0 mL/min with a gradient mobile phase from 100% A
(10 mM pH 6.0 phosphate buffer) to 75o B (acetonitrile)
at 20 min.
** The HPLC method using a reverse phase C1g Zorbax
column
(4.6 mm x 25 cm, 80 .A pore size) at a flow rate of 1.0
mL/min with a gradient mobile phase from 100% A (10 mM
pH 6.0 phosphate buffer) to 50% B (acetonitrile) at 20
min.
*** The HPLC method using a reverse phase C1g Zorbax
column
(4.6 mm x 25 cm, 80 A pore size) at a flow rate of 1.0
mL/min with a gradient mobile phase from 100% A (10 mM
pH 6.0 phosphate buffer) to 25% B (acetonitrile) at 20
min.
Example 27
Synthesis of the 177Lu Complex of 3-((1-(3-(Imidazole-2-
ylamino)propyl)(1H-indazol-5-yl))carbonylamino)-2-(((4-
(4-(((3-(2-(2-(3-(2-(1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)cyclododecyl)-
acetylamino)propoxy)ethoxy)ethoxy)propyl)amino)sulfonyl)
phenyl)phenyl)sulfonyl)amino)propanoic Acid
To a clean sealed 5 mL vial was added 0.5 mL of a
solution of the conjugate of Example 4 (200 ~.~.g/mL in
0.5 M ammonium acetate buffer, pH 6.9), followed by 0.05
mL of gentisic acid (sodium salt, 10 mg/mL in 0.5 M
ammonium acetate buffer, pH 6.9) solution, 0.3 mL of
0.25 M ammonium acetate buffer (pH 7.0), and 0.010 mL of
177LuC13 solution (1000 mCi/mL) in 0.05 N HCl. The
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resulting mixture was heated at 100 C for 30 min. After
cooling to room temperature, a sample of the resulting
solution was analyzed by radio-HPLC and ITLC. The
radiolabeling yield was 80%, and the retention time was
18.0 min.
HPLC Method
Column: Zorbax C18 , 25 cm x 4.6 mm
Flow rate . 1.0 mL/min
Solvent A: 25 mM sodium phosphate buffer, pH 6.0
Solvent B . 100 % CH3CN
t (min) 0 20 21 25 26 32
Solvent B 15 20 60 60 15 15
The instant thin layer chromatography (ITLC) method used
Gelman Sciences silica-gel strips and a 1:1 mixture of
acetone and saline as eluant.
Exam~ale 28
Synthesis of the 9~Y Complex of 3-((1-(3-(Imidazole-2-
ylamino)propyl)(1H-indazol-5-yl))carbonylamino)-2-(((4-
(4-(((3-(2-(2-(3-(2-(1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)cyclododecyl)-
acetylamino)propoxy)ethoxy)ethoxy)propyl)amino)sulfonyl)
phenyl)phenyl)sulfonyl)amino)propanoic Acid
To a clean sealed 5 mL vial was added 0.5 mL of a
solution of the conjugate of Example 4 (200 ug/mL in
0.5 M ammonium acetate buffer, pH 6.9), followed by 0.05
mL of gentisic acid (sodium salt, 10 mg/mL in 0.5 M
ammonium acetate buffer, pH 6.9) solution, 0.3 mL of
0.25 M ammonium acetate buffer (pH 7.0), and 0.010 mL of
90YC13 solution (1000 mCi/mL) in 0.05 N HCl. The
resulting mixture was heated at 100 C for 30 min. After
cooling to room temperature, a sample of the resulting
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solution was analyzed by radio-HPLC and ITLC. The
radiolabeling yield was 850, and the retention time was
18.2 min.
HPLC Method
Column: Zorbax C18 , 25 cm x 4.6 mm
Flow rate . 1.0 mL/min
Solvent A: 25 mM sodium phosphate buffer, pH 6.0
Solvent B . 100 % CH3CN
t (min) 0 20 21 25 26 32
Solvent B 15 20 60 60 15 15
The instant thin layer chromatography (ITLC) method used
Gelman Sciences silica-gel strips and a 1:1 mixture of
acetone and saline as eluant.
Example 29
Synthesis of the 111In Complex of 3-((1-(3-(Imidazole-2
ylamino)propyl)(1H-indazol-5-yl))-carbonylamino)-2-(((4
(4-(((3-(2-(2-(3-(2-.(1,4,7,10-tetraaza-4,7,10
tris(carboxymethyl)cyclododecyl)-
acetylamino)propoxy)ethoxy)ethoxy)propyl)amino)sulfonyl)
phenyl)phenyl)sulfonyl)amino)propanoic Acid
To a lead shielded and closed autosampler vial was
added: 80 ~.g of the conjugate of Example 4 dissolved in
160 ~.L 0.4 M ammonium acetate at pH 4.7 and 3 mCi In-
111-chloride in 12.5 uL 0.05 N HCl. The solution was
heated at 100 °C for 35-40 minutes. After cooling to
room temperature, a sample of the resulting solution was
analyzed by radio-HPLC and ITLC. The radiolabeling yield
was 95%, and the retention time was 9.5 min.
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HPLC Method
Column: Zorbax C18 , 25 cm x 4.6 mm
Flow rate . 1.0 mL/min
Solvent A: 25 mM sodium phosphate buffer, pH 6.0
Solvent B . 100 % CH3CN
t (min) 0 25 26 30 31 37
a Solvent B 16 18 60 60 16 16
The instant thin layer chromatography (ITLC) method used
Gelman Sciences silica-gel strips and a 1:1 mixture of
acetone and saline as eluant.
Example 30 --
Synthesis of the Gd Complex of 3-((1-(3-(Imidazole-2-
ylamino)propyl)(1H-indazol-5-yl))carbonylamino)-2-(((4-
(4-(((3-(2-(2-(3-(2-(1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)cyclododecyl)-
acetylamino)propoxy)ethoxy)ethoxy)propyl)amino)sulfonyl)
phenyl)phenyl)sulfonyl)amino)propanoic Acid
The gadolinium complex of the conjugate of Example
4 is prepared according to the following procedure. 3-
3.5 mg of the conjugate is 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) is added to it. The
reaction mixture is heated at 100 C for 30 minutes and
the product is isolated by preparative HPLC. The
fraction containing the complex is lyophilized. The
identity of the complex is confirmed by mass
spectroscopy.
The following examples describe the synthesis of
ultrasound contrast agents of the present invention.
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Example 31
Part A Synthesis of 1-(1,2-Dipalmitoyl-sn-glycero-3-
phosphoethanolamino)-12-(2-(6-aminohexanoylamino)-3-((1-
(3-(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonyl-amino)propanoic acid)-dodecane-1,12-dione
N '1
NH
NH
O OH
14 O N-"°ii N I ~ ~ N
~O
1O O°P O~N O N1 /5 O
OH OH
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 2-(6-
aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid 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 B Preparation of Contrast Agent Composition
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The 1-(1,2-Dipalmitoyl-sn-glycero-3-
phosphoethanolamino)-12-{2-(6-aminohexanoylamino)-3-((1-
(3-(imidazol-2-ylamino)propyl)(1H-indazol-5-
yl))carbonyl-amino)propanoiC acid)-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 composition is completed by agitating the
sealed vial in a dental amalgamator for 30 - 45 sec. to
form a milky white solution.
Example 32
Part A. Preparation of Preparation of ((~-amino-PEG3400-
oc-carbonyl)-2-(6-aminohexanoylamino)-3-((1-(3-(imidazol-
2-ylamino)propyl)(1H-indazol-5-yl))Carbonyl-
amino)propanoiC acid
N
NH
NH
O OH ~ N,
~ N
O O
H2N~0 O N' /5
~H
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To a solution of N-Boc-(~-amino-PEG34oo-a-
carboxylate sucinimidyl ester (1 mmol) and 2-(6-
aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid (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- ( ((~-amino-PEG3goo-a-
carbonyl)-(2-(6-aminohexanoylamino)-3-((1-(3-(imidazol-
2-ylamino)propyl)(1H-indazol-5-yl))carlaonyl-
amino)propanoic acid))-dodecane-1,12-dione
A solution of disuccinimidyl dodecane-1,12-dioate
(1 mmol), 1,2-dipalmitoyl-sn-glycero-3-
phosphoethanolamine (1 mmol) and (CO-amino-PEG34oo-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
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The 1-(1,2-Dipalmitoyl-sn-glycero-3-
phosphoethanolamino)-12-(((~-amino-PEG3400-a-carbonyl)-
(2-(6-aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid))-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 mLlmL), 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.
Example 33
Part A. Preparation of (C~-amino-PEG3400-a-carbonyl)-
Glu-(2-(6-aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)-propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)2
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-' NH
NH
O OH ~ N
H~
O N~ ~ N I / N
~ O
O NH' /5
O
H2N~O~O~ N O
m H HN.~ ~ O
'I 5
O~H~H I ~ ~N
O OH ~ N
NH
~H
~/N
To a solution of N-Boc-GJ-amino-PEG3400-a-
carboxylate sucinimidyl ester (1 mmol) and Glu-(2-(6-
aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)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 50o 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-((t~-amino-PEG34oo-a-carbonyl)-
(Glu-(2-(6-aminohexanoylamino)-3-((1-(3-(imidazol-2-
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ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)2))-dodecane-1,12-dione
A solution of disuccinimidyl dodecane-1,12-dioate (1
mmol), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine
or DPPE (1 mmol) and ((~-amino-PEG34oo-a-carbonyl) -Glu-
(2-(6-aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid) 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)-
(Glu-(2-(6-aminohexanoylamino)-3-((1-(3-(imidazol-2-
ylamino)propyl)(1H-indazol-5-yl))carbonyl-
amino)propanoic acid)2))-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. o. 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
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contrast agent composition is completed by agitating the
sealed vial in a dental amalgamator for 30 - 45 sec. to
form a milky white solution.
Example 34
Synthesis of 2-({[4-(3-{N-[2-((2R)-3-Sulfo-2-{2-
[1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)cyclododecyl]acetylamino}-
propyl)ethyl]carbamoyl}propoxy)-2,6-dimethylphenyl]-
sulfonyl}amino)(2S)-3-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)propanoic
Acid Bis(trifluoroacetate) Salt
O O
~ N~'OH
N I i H NH O
O=S=O
F3C OH
oS03H
O H . O
O~N~..NI~.N~--v N~--C02H O
H O H C , F3C~OH
H02C-' V '-C02H
Part A - Preparation of Methyl (2S)-3-[(tert-Butoxy)-
carbonylamino]-2-[({2,6-dimethyl-4-[3-(N-{2-
[(phenylmethoxy)carbonylamino]ethyl}carbamoyl)-
propoxy]phenyl}sulfonyl)amino]propanoate
O O
O~N~O~
H HN~SO
2
I
O i
O~IN~.N1~0 w I
H O
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A solution of the product from Example 13, Part D
(369 mg, 0.756 mmol), DIEA (0.52 mL, 3.0 mmol), and HBTU
(315 mg, 0.832 mmol) in anhydrous DMF (14 mL) was
stirred at ambient temperatures under nitrogen for 5
min, and treated with benzyl N-(2-aminoethyl)carbamate
hydrochloride (192 mg, 0.832 mmol), and stirred an
additional 1 h. The DMF was removed under vacuum, and
the oily residue was taken up in EtOAc (150 mL), washed
consecutively with 0.1 N HCl (40 mL), water (40 mL), and
saturated NaCl (40 mL), dried (MgS04), and concentrated
to give a colorless viscous oil. Flash chromatography
on a 3 x 16 cm silica gel column (EtOAc) gave the title
Compound as a colorless viscous oil (450 mg, 89.6%). 1H
NMR (CDC13) : 8 7.34-7.27 (m, 5H) , 6.58 (s, 2H) , 6.31
(bs, 1H), 5.86 (bs, 1H), 5.36 (bs, 1H), 5.14-5.03 (m,
3H), 3.96 (t, J = 6.0 Hz, 2H), 3.88-3.83 (m, 1H), 3.56
(s, 3H), 3.47-3.25 (m, 6H), 2.59 (s, 6H), 2.31 (t, J =
6.9 Hz, 2H) , 2. 05 (p, J = 6. 6 Hz, 2H) , 1.39 (s, 9H) ; 13C
NMR (CDC13): 8 172.9, 170.5, 160.6, 157.3, 155.9, 141.8,
136.3, 128.5, 128.2, 128.0, 116.6, 79.9, 66.9, 55.5,
52.8, 43.1, 40.9, 40.3, 32.4, 28.2, 24.9, 23.3; MS: m/e
665.4 [M+H]; 687.3 [M+Na]; High Resolution MS: Calcd for
C31H45N401oS [M+H]: 665.2856, Found: 665.2883.
Part B - Preparation of Methyl (2S)-3-Amino-2-[({2,6-
dimethyl-4-[3-(N-{2-[(phenylmethoxy)carbonylamino]ethyl}
carbamoyl)propoxy]phenyl}sulfonyl)amino]propanoate
Trifluoroacetate Salt
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O
H2N'~O'
HN.SO
2
w I O H ~ F3C~OH
O~IN~N~O w I
H O
The product of Part A, above (420 mg, 0.632 mmol)
was dissolved in 25/75 DCM/TFA (20 mL) and allowed to
stand at ambient temperatures under nitrogen for 10 min.
The solution was concentrated, and the resulting viscous
oil was dissolved in 50o ACN and lyophilized to give the
title compound as a colorless solid (437 mg, 1020). MS:
m/e 565.3 [M+H].
Part C - Preparation of Methyl (2S)-2-[({2,6-Dimethyl-4-
[3_(N_{2-
[(phenylmethoxy)carbonylamino]ethyl}carbamoyl)propoxy]
phenyl}sulfonyl)amino]-3-{[1-(3-{[1-(triphenylmethyl)-
imidazol-2-yl]amino}propyl)(1H-indazol-5-
yl)]carbonylamino}propanoate
O O
~ N'~O'
N ~H NH
O=S=O
N N
Trt H
O
O.~ N'~ N 1r0 w I
H O
A solution of 1-(3-((1-(triphenylmethyl)imidazol-2-
yl)amino)propyl)-1H-indazole-5-carboxylic acid (100 mg,
0.190 mmol), DIEA (0.099 mL, 0.57 mmol), and HBTU (91
mg, 0.24 mmol) in anhydrous DMF (2.0 mL) was stirred at
ambient temperatures under nitrogen for 5 min, treated
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with the product of Step B, above (142 mg, 0.21 mmol)
and additional DIEA (0.033 mL, 0.19 mmol), arid stirred
an additional 1 h. The DMF was removed under vacuum and
the amber oil was purified by HPLC on a ZTydac C-18
column (22 x 250 mm) using a 1.65%/min gradient of 18 to
67.50 ACN containing 0.1% TFA at a flow rate of 20
mL/min. The main product peak eluting at 23.2 min was
lyophilized to give the title compound as a colorless
powder (194 mg, 95.1%). 1H NMR (CDC13 + D20): 8 8.11
(s, 1H), 7.71 (s, 1H), 7.66 (d, J=8.75 Hz, 1H), 7.42-
7.24 (m, 16H), 7.17-7.13 (m, 6H), 6.93 (d, J=2.81 Hz,
1H), 6.52-6.47 (m, 2H), 5.04 (s, 2H), 4.07=4.00 (m, 3H),
3.93-3.78 (m, 3H), 3.69-3.64 (m, 4H), 3.37-3.27 (m, 4H),
3.14 (t, J=6.88 Hz, 2H), 2.57 (s, 6H), 2.29 (t, J=7.18),
2.01 (pentet, J=6.66, 2H), 1.73 (pentet, J=6.59, 2H);
MS: m/e 1074.4 [M+H], 537.9 [M+2H]; High Resolution MS:
Calcd for C5gH64NgOgS [M+H]: 1074.4548; found: 1074.452.
Part D - Preparation of (2S)-2-{[(4-{3-[N-(2-
Aminoethyl)carbamoyl]propoxy}-2,6-
dimethylphenyl)sulfonyl]amino}-3- ({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)propanoic
Acid
O O
~ N'~OH
I i H NH
O=S=O
H H I
O
O~N~NH2
H
f.
The product of Part C, above (194 mg, 0.180 mmol)
was dissolved in peroxide-free THF (8.0 mL) and water
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(1.2 mL), and treated with 3 N LiOH (0.80 mL). The
resulting mixture was stirred at ambient temperatures
under nitrogen for 2 h. The THF was removed under
vacuum and the resulting mixture was partitioned between
water (25 mL) and CHC13 (25 mL). The aqueous layer was
adjusted to pH 3 with 0.1 N HCl (22 mL) and extracted
with additional CHC13 (2 x 25 mL). The combined CHC13
extracts were washed with saturated NaCl (25 mL), dried
(MgS04), and concentrated to give the intermediate
carboxylic acid as a colorless amorphous solid (171 mg).
MS: m/e 1060.4 [M+H], 531.0 [M+2H].
The solid was dissolved in a solution of TFA (8.0
mL) and Et3SiH (0.40 mL), and heated at 70 °C under
nitrogen for 2 h. The solution was concentrated under
vacuum and the resulting oily solid was partitioned
between ether (20 mL) and water (20 mL). The aqueous
layer was washed with a second portion of ether (20 mL).
The combined ether washings were back-extracted with
water (20 mL). The combined aqueous layers were
lyophilized to give the title compound as a colorless
solid (139 mg, 84.8%). MS: m/e 684.3 [M+H], 343.0
[M+2H] .
Part E - Preparation of 2-{[(4-{3-[N-(2-{(2R)-2-[(tert-
Butoxy)carbonylamino]-3-sulfopropyl}ethyl)carbamoyl]-
propoxy}-2,6-dimethylphenyl)sulfonyl]amino}(2S)-3-({1-
[3-(imidazol-2-ylamino)propyl](1H-indazol-5-yl)}
carbonylamino)propanoic Acid
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O O
~ N~OH
~ i H NH
O=S=O
H H
~S03H
O
O~.~L. N.~ N 1~~ N.Boc
H O H
A solution of the product of Part D, above (91 mg,
0.10 mmol), the N-hydroxysuccinimide ester of Boc-L-
cysteic acid (103 mg, 0.25 mmol), and DIEA (0.104 mL,
0.60 mmo1) in anhydrous DMF (5.0 mL) was stirred at
ambient temperatures under nitrogen for 19 h. The DMF
was removed under vacuum and the resulting amber oil was
purified by HPLC on a Vydac C-18 column (22 x 250 mm)
using a 0.72%/min gradient of 0 to 36% ACN containing
0.1o TFA at a flow rate of 80 mLlmin. The main product
peak eluting at 40.0 min was lyophilized to give the
title compound as a colorless fluffy solid (69 mg,
74.0%). MS: m/e 935.3 [M+H].
Part F - Preparation of 2-({[4-(3-{N-[2-((2R)-2-Amino-3-
sulfopropyl)ethyl]carbamoyl}propoxy)-2,6-
dimethylphenyl]sulfonyl}amino)(2S)-3-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)propanoic
Acid Trifluoroacetate Salt
O O
~ N'~OH
~H NH
O-S-O O
H H ~ ~ DSO H FsC~OH
3
O H _'-
O~H~'N1~NH2
O
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A solution of the product of Part E, above (130 mg,
0.139 mmol) in 50/50 TFA/DCM (16.0 mL) and allowed to
stand at ambient temperatures under nitrogen for 10 min.
The solution was concentrated under vacuum, and the
resulting oily solid was purified by HPLC on a Vydac C-
18 column (50 x 250 mm) using a 0.90%/min gradient of 0
to 27% ACN containing 0.1% TFA at a flow rate of 80
mL/min. The main product peak eluting at 22.6 min was
lyophilized to give the title compound as a colorless
solid (117 mg, 88.8%). 1H NMR (D20): 8 8.09 (s, 1H),
7.75 (s (unresolved X portion of ABX system) 1H), 7.39
(B portion of ABX system, Jab = 8.9 Hz, Jbx = 1.6 Hz,
1H), 7.34 (A portion of ABX system, Jab = 8.9 Hz, 1H),
6.50 (s, 2H), 6.02 (s, 1H), 4.46 (t, J = 6.3 Hz, 2H),
4.31 (X' portion of A'B'X' system, Ja'x' - 7.8 Hz, J'x'
- 4.9 Hz, 1H), 4.16 (X portion of AMX system, Jax = 10.9
Hz, Jmx = 3.8 Hz, 1H), 3.70 (M portion of AMX system,
Jam = 14.1 Hz, Jmx = 3.8 Hz, 1H), 3.39-3.15 (m, 9H),
3.03 (t, J = 6.3 Hz, 2H), 2.34 (s, 6H), 2.14 (pentet, J
- 6.3 Hz, 2H), 2.07 (t, J = 7.4 Hz, 2H), 1.58 (pentet, J
- 7.4 Hz, 2H); MS: m/e 835.2 [M+H]; 857.3 [M+Na]; High
Resolution MS: Calcd for C34H47N1oG11S2 [M+H]: 835.2867,
found: 835.2888.
Part G - Preparation of 2-{[(4-{3-[N-(2-{(2R)-3-Sulfo-2-
[2-(1,4,7,10-tetraaza-4,7,10-tris{[(tert-
butyl)oxycarbonyl]-
methyl}cyclododecyl)acetylamino]propyl}ethyl)carbamoyl]-
propoxy}-2,6-dimethylphenyl)sulfonyl]amino}(2S)-3-({1-
[3-(imidazol-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)propanoic Acid Bis(trifluoroacetate)
Salt
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O O
~ N~'OH
~ i H NH
N ~~ N'~ O-S=O JL
H H \ ~ DSO H FsC OH
3
O H --_ O
O~IH~.N ~ H~--v N~-C02-t-Bu
F3C OH
CN N
t-Bu-02C V C02-t-Bu
A solution of the product of Example 4, Part B
(73.1 mg, 0.080 mmol), DIEA (0.083 mL, 0.480 mmol), and
HBTU (22.7 mg, 0.060 mmol) in anhydrous DMF (4.0 mL) was
stirred under nitrogen at ambient temperatures for 15
min and treated with the product of Part F, above (37.9
mg, 0.040 mmol). The DMF was removed under vacuum after
4.5 h and the resulting amber oil was purified by HPLC
in two steps. An initial HPLC purification was carried
out on a Vydac C-18 column (22 x 250 mm) using a
0.9%/min gradient of 9 to 45% ACN containing 0.1% TFA at
a flow rate of 20 mL/min. The main product peak eluting
at 26.4 min was lyophilized to give a colorless solid.
Final purification was accomplished on a ~orbax C-18
column (21.2 x 250 mm) under isocratic conditions using
33.30 ACN containing 0.1% TFA at a flow rate of 20
mL/min. The main product peak eluting at 5.2 min was
lyophilized to give the title compound as a colorless
fluffy solid {34.0 mg, 20.5%). MS: m/e 1389.6 [M+H];
High Resolution MS: Calcd for C62Hg7N1401gS2 [M+H]:
1389.6547, Found: 1389.655.
Part H - Preparation of 2-({[4-(3-{N-[2-((2R)-3-Sulfo-2-
{2-[1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)cyclododecyl]acetylamino}-
propyl)ethyl]carbamoyl}propoxy)-2,6-dimethylphenyl]-
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sulfonyl}amino)(2S)-3-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)propanoic
Acid Bis(trifluoroacetate) Salt
The product of Step G, above {32.0 mg, 0.0174 mmol)
was dissolved in a solution of TFA (4.0 mL) and Et3SiH
(0.20 mL), and heated at 50 ~C under nitrogen for 30
min. The solution was concentrated and the residue was
purified by HPLC on a Zorbax C-18 column (21.2 x 250 mm)
using a 0.90%/min gradient of 0 to 27% ACN containing
0.1% TFA at a flow rate of 20 mL/min. The main product
peak eluting at 23.5 min was lyophilized to give the
title compound as a colorless fluffy solid (22.2 mg,
88.1%). MS: m/e 1221.4 [M+H]; High Resolution MS: Calcd
for C5pH~3N14~1852 [M+H]: 1221.4669, Found: 1221.469.
Example 35
Synthesis of DOTA/2-{[(4-{3-[N-(2-{(2R)-2-[4-(N-{(1R)-1-
[N-(2-{4-[4-({[(1S)-1-carboxy-2-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)ethyl]-
amino}sulfonyl)-3,5
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-2
sulfoethyl}carbamoyl)(4S)-4-aminobutanoylamino]-3
sulfopropyl}ethyl)carbamoyl]propoxy}-2,6
dimethylphenyl)sulfonyl]amino}2S)-3-({1-[3-(imidazol-2
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)propanoic
Acid Bis(trifluoroacetate) Conjugate
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O O
N~OH
I i H NH
O=S=O
H H ~ I H03S~
O H . O H02C'-~N ~C02H
O~N~N1~N H C
H O H~,,, N N N~
O ~ '~-~ V C02H
O~N./..N N O O
O H~ O
I H03S F3C~OH
~N ~ \ OSO
N~H NH p
O N ~ OH F3C~OH
Part A - Preparation of Di-2,3,5,6-tetrafluorophenyl
(2S)-2-[(tart-Butoxy)carbonylamino]pentane-1,5-dioate
F F
FO OF
F O O F
F Boc~NH F
To a solution of Boc-L-Glu-OH (28.9 g, 117 mmol) in
DMF (500 mL) at ambient temperatures and under nitrogen,
was added a solution of 2,3,5,6-tetrafluorophenol (48.2
g, 290 mmol) in DMF (50 mL). Af tar stirring for 10 min,
EDC (55.6 g, 290 mmol) was added and the mixture was
stirred for 96 h. The volatiles were removed under
vacuum and the residue was triturated with 0.1 N HCl
(750 mL). To this mixture was added EtOAc (600 mL) and
the layers were separated. The aqueous layer was
extracted with EtOAc (3 x 500 mL), and all EtOAc
extracts were combined, washed consecutively with water
(300 mL) and saturated NaCl (300 mL), dried (MgS03), and
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concentrated to give a tan solid (62 g). The tan solid
was washed with ACN to give the title compound (45.5 g,
73.0%) in purified form. MS: m/e 566.0 [M+Na].
Part B - Preparation of 2-{[(4-{3-[N-(2-{(2R)-2-[4-(N-
{(1R)-1-[N-(2-{4-[4-({[(1S)-1-carboxy-2-({1-[3-
(imidazol-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-2-
sulfoethyl}carbamoyl)(4S)-4-[(tert-
butoxy)carbonylamino]butanoylamino]-3-
sulfopropyl}ethyl)carbamoyl]propoxy}-2,6-
dimethylphenyl)sulfonyl]amino}2S)-3-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)propanoic
Acid
O O
~ N~OH
~ i H NH
O=S=O
H H ~ I O H03S~ O
H -
O.~N~..N1'~N H
H O H~~,. N O
O
O'~~ N.~~ N N o O
o H
H H ~ ~ H03S
N N
~N N \ OSO
H NH
N~ ~ i N~OH
O O
A solution of the product of Example 34, Part F
(43,5 mg, 0.0459 mmol), the product of Part A, above
(10.8 mg, 0.020 mmol), and DIEA 0.015 mL, 0.084 mmol) in
anhydrous DMF (1.0 mL) was stirred at ambient
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temperatures under nitrogen for 23 h. The DMF was
removed under vacuum and the resulting amber oil was
purified by HPLC on a Vydac C-18 column (22 x 250 mm)
using a 0.90%/min gradient of 9 to 45% ACN containing
0.1% TFA at a flow rate of 20 mLJmin. The main product
peak eluting at 20.9 min was lyophilized to give the
title compound as a colorless fluffy solid (22.0 mg,
55.7%). MS: m/e 1880.7 [M+H], 941.4 [M+2H]; High
Resolution MS: Calcd for C7gH106N21~2654 [M+H]:
1880.6501; found: 1880.6530.
Part C - Preparation of 2-{[(4-{3-[N-(2-{(2R)-2-[4-(N-
{(1R)-1-[N-(2-{4-[4-({[(1S)-1-carboxy-2-({1-[3-
(imidazol-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-2-
sulfoethyl}carbamoyl)(4S)-4-aminobutanoylamino]-3-
sulfopropyl}ethyl)carbamoyl]propoxy}-2,6-
dimethylphenyl)sulfonyl]amino}2S)-3-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)propanoic
Acid
O O
~ N~OH
N ~ I i H NH
O=S=O
H H ~ I H03S~
O H . O
O~N~N1'~N~
H O H ~,. NH2
O ~
O N~~N N~O
H
O
H H \ I H03S
~N N N ~. O=S=O
N I H NH
i N~OH
O O
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A solution of the product of Part B, above (22.0
mg, 0.0117 mmol) in 50/50 TFA/DCM (8.0 mL) was allowed
to react at ambient temperatures under nitrogen for 10
min and concentrated to a pale amber oil. The oil was
dissolved in 50% ACN (20 mL) and lyophilized to give the
title compound as a colorless fluffy solid (21.2 mg,
95.6%). MS: m/e 1781.7 [M+H], 891.0 [M+2H], 594.4
[M+3H]; High Resolution MS: Calcd for C73HggN~1O24S4
[M+H]: 1780.5976; found: 1780.598.
Part D - Preparation of DOTA Tris-t-butyl Ester/2-{[(4-
{3- [N- (2-{ (2R) -2- [4- (N-{ (1R) -1- [N- (2-{4- [4- ( { [ (1S) -1-
carboxy-2-({1-[3-(imidazol-2-ylamino)propyl](1H-indazol-
5-yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-2-
sulfoethyl}carbamoyl)(4S)-4-aminobutanoylamino]-3-
sulfopropyl}ethyl)carbamoyl]propoxy}-2,6-
dimethylphenyl)sulfonyl]amino}2S)-3-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)propanoic
Acid Bis(trifluoroacetate) Salt Conjugate
O O
r ~ N~OH
I i H NH
O=S=O
N N
H H ~ I H03S~ C02-t-Bu
O~ ~N O ~ N~-C02-t-Bu
H O H~~.. N CN NJ
O ~ ~-' a '-C02-t-Bu
O~N~~N NH O O
O H~ O
I H03S F3C~OH
O=s=o
H ~H O
N ~ I i N~OH
O O F3C OH
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A solution of the product of Example 4, Part B
(21.4 mg, 0.0234 mmol), DIEA (0.024 mL, 0.14 mmol), and
HBTU (6.6 mg, 0.0176 mmol) in anhydrous DMF (1.0 mL) was
stirred under nitrogen at ambient temperatures for 15
min and treated with the product of Part C, above 21.0
mg, 0.0111 mmol). After 23 h the solution was diluted
with EtOH (5.0 mL) and water (3.0 mL) and treated with
0.5 N NaOH (0.30 mL). After 30 min the solution was
adjusted to pH 3 with 1 N HC1 (0.20 mL). The solution
was diluted with water (135 mL) and the resulting
solution was purified directly by HPLC on a Vydac C-18
column (22 x 250 mm) using a 0.90%/min gradient of 9 to
45% ACN containing 0.1% TFA at a flow rate of 20 mL/min.
The main product peak eluting at 27.0 min was
lyophilized to give the title compound as a colorless
fluffy solid (11.5 mg, 37.1%). MS: m/e 1168.1 [M+2H],
779.3 [M+3H]; High Resolution MS: Calcd for
C1o1H148N2503154 [M+H]: 2334.9656, found: 2334.967.
Part E - Preparation of DOTA/2-{[(4-{3-[N-(2-{(2R)-2-[4-
(N-{(1R)-1-[N-(2-{4-[4-({[(1S)-1-carboxy-2-({1-[3-
(imidazol-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-2-
sulfoethyl}carbamoyl)(4S)-4-aminobutanoylamino]-3-
sulfopropyl}ethyl)carbamoyl]propoxy}-2,6-
dimethylphenyl)sulfonyl]amino}2S)-3-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)propanoic
Acid Bis(trifluoroacetate) Conjugate
The product of Step D, above (11.5 mg, 0.00449
mmol) was dissolved in a solution of TFA (4.0 mL) and
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Et3SiH (0.20 mL) and heated at 50 °C under nitrogen for
30 min. The solution was concentrated under vacuum and
the residue was purified by HPLC on a ZTydac C-18 column
(22 x 250 mm) using a 0.90%/min gradient of 0 to 36% ACN
containing 0.1% TFA at a flow rate of 20 mL/min. The
main product peak eluting at 27.5 min was lyophilized to
give the title compound as a colorless fluffy solid (9.3
mg, 86.5%). MS: m/e 1084.1 [M+2H], 723.1 [M+3H]; High
Resolution MS: Calcd for CggH124N25O3~S4 [M+H]:
2166.7778; Found: 2166.778.
Example 3 6
Synthesis of 2-[({4-[4-({[2-((2R)-3-Sulfo-2-{2-
[1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)cyclododecyl]-
acetylamino}propyl)ethyl]amino}sulfonyl)phenyl]phenyl}
sulfonyl) amino] (2S) -3- ( {1- [3- (imidazol-2
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)propanoic
Acid Bis(trifluoroacetate) Salt
O O O
N~OH O O OH
~ N ~N I ~ H HN.S \ / / \ S.N~.N NH N '~O F3C OH
~~N~ O2 O2 H~O CN N' O
H H03S ~, U ~, O
HO OH F3C~OH
Part A - Preparation of Methyl (2S)-3-[(tert-
Butoxy)carbonylamino]-2-{[(4-{4-[({2-[(phenylmethoxy)-
carbonylamino]ethyl}amino)sulfonyl]phenyl}phenyl)sulfony
1]amino}propanoate
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O
Boc. N~'O~
H HN.S \ / / \ S.N~~N Cbz
02 02 H
Biphenyl-4,4'-disulfonyl chloride (5.30 g, 15.0
mmol, freshly recrystallized from CHC13) and DCM (400
mL) were placed in a 100 mL 3-neck flask fitted with a
thermometer, an addition funnel, and a nitrogen line.
The addition funnel was charged with a solution of
benzyl N-(2-aminoethyl)carbamate hydrochloride (2.30 g,
10.0 mural) and DIEA (1.80 mL, 10.0 mmol) in DCM (40 mL).
The contents of the flask were cooled below 5 °C, and
the contents of the addition funnel were added to the
flask with rapid stirring over 30 min while keeping the
temperature of the flask below 5 °C. The addition
funnel was then charged with a solution of N-(3-Boc-L-
oc,(3-diaminopropionic acid methyl ester hydrochloride
(5.10 g, 20.0 mmol) and DIEA (7.60 mL, 44.0 mmol) in DCM
(40 mL). This solution was added to the flask with
stirring at 5 °C over 15 min, and stirred at ambient
temperatures for an additional 4 days. The reaction was
concentrated and the resulting residue was partitioned
between EtOAc (6 L) and 0.1 N HCl (600 mL). The organic
solution was washed consecutively with water (3 L), and
saturated NaCl (2 L), dried (MgSOg), and concentrated to
give the title compound as a colorless solid (9.60 g).
MS: m/e 591.2.
Part B - Preparation of Methyl (2S)-3-Amino-2-{[(4-{4-
[({2-
[(phenylmethoxy)carbonylamino]ethyl}amino)sulfonyl]pheny
1}phenyl)sulfonyl]amino}propanoate Trifluoroacetate Salt
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O
H2N H'~O / \ .N~,~~ .Cbz JL
F3C OH
The product of Part A, above (8.80 g) was dissolved
in 50/50 TFA/DCM (200 mL) and allowed to react at
ambient temperatures under nitrogen for 1 h. The
solution was concentrated under vacuum and the resulting
viscous orange oil was purified by HPLC on a Vydac C-18
column (50 x 250 mm) using a 1.58%/min gradient of 0 to
63% ACN containing 0.1% TFA at a flow rate of 80 mLlmin.
The main. product peak eluting at 22.7 min was
lyophilized to give the title compound as a coloxless
solid (3.54 g, 54.9 for two steps from benzyl N-(2-
aminoethyl)carbamate hydrochloride). MS: m/e 591.2
[M+H]; High Resolution MS: Calcd for C26H31N40gS2 [M+H]:
591.1583; Found: 591.1585.
Part C - Preparation of Methyl (2S)-2-{[(4-{4-[({2-
[(Phenylmethoxy)earbonylamino]ethyl}amino)sulfonyl]pheny
1}phenyl)sulfonyl]amino}-3-{[1-(3-{[1-
(triphenylmethyl)imidazol-2-yl]amino}propyl)(1H-indazol-
5-yl)]carbonylamino}propanoate
O O
w N~O
N ~ ~ i H HN. / \ .N~~ .Cbz
NON
Trt H
A solution of 1-(3-((1-(triphenylmethyl)imidazol-2-
yl)amino)propyl)-1H-indazole-5-carboxylic acid (265 mg,
0.503 mmol), DIEA (0.099 mL, 0.42 mmol), and HBTU (158
mg, 0.417 mmol) in anhydrous DMF (10.2 mL) was stirred
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at ambient temperatures under nitrogen for 5 min,
treated with the product of Step B, above (246 mg, 0.417
mmol), and stirred an additional 1 h. The DMF was
removed under vacuum and the amber oil was purified by
HPLC on a Vydac C-18 column (50 x 250 mm) using a
1.8%/min gradient of 18 to 72% ACN containing 0.1o TFA
at a flow rate of 80 mL/min. The main product peak
eluting at 24.8 min was lyophilized to give a colorless
powder. This powder was repurified by HPLC using the
same column and gradient conditions. Product fractions
were lyophilized to give the title compound as a
colorless fluffy powder (245 mg, 53.50). MS: m/e 1100.3
[M-f-H] ; High Resolution MS: Calcd for C5gH57NgOgS2 [M+H]
1100.3799; Found: 1100.380.
Part D - Preparation of Methyl (2S)-2-({[4-(4-{[(2-
Aminoethyl)amino]sulfonyl}phenyl)phenyl]sulfonyl}amino)-
3-{[1-(3-{[1-(triphenylmethyl)imidazol-2-
yl]amino}propyl)(1H-indazol-5-
yl)]Carbonylamino}propanoate
O O
~ N~O' H
N ~ ~ ~ H HN. / \ N.~
S \ / S N Hz
N~.N'~ Oz Oz
l'rt H
A solution of the product of Part C, above (240 mg,
0.218 mmol) in MeOH (22 mL) was hydrogenolyzed over 10%
Pd/C at 55 psi for 3 h. The catalyst was removed by
filtration through Celite~ and the filtrate was
concentrated to give the title compound as a colorless,
viscous oil (240 mg). MS: m/e 966.3 [M+H], 724.2 [M+H
trityl].
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Part E - Preparation of (2R)-N-[2-({[4-(4-{[((1S)-1-
(Methoxycarbonyl)-2-{[1-(3-{[1-
(triphenylmethyl)imidazol-2-yl]amino}propyl)(1H-indazol-
5-yl)]carbonylamino}ethyl)-
amino]sulfonyl}phenyl)phenyl]sulfonyl}amino)ethyl]-2-
[(tert-butoxy)carbonylamino]propanesulfonic Acid
O O
~ N ~O~_ H O H
~ H HN.O \ / / \ ~ N'~N~N.Soc
N N z 2 H~3S
Trt H
A solution of the product of Part D, above (240 mg)
and DIEA (0.166 mL, 0.950 mmol) in anhydrous DMF (4.0
mL) was treated with the p-nitrophenyl ester of Boc-L-
cysteic acid (149 mg, 0.362 mmol) and stirred at ambient
temperatures under nitrogen for 18 h. Additional Boc-L-
cysteic acid p-nitrophenyl ester (50.0 mg, 0.121 mmol)
was added and stirring was continued an additional 24 h.
The DMF was removed under vacuum and the oily solid
residue was purified by HPLC on a Vydac C-18 column (22
x 250 mm) using a 1.12%/min gradient of 18 to 63% ACN
containing 0.1% TFA at a flow rate of 20 mL/min. The
main product peak was centered at 32.1 min. The
earliest eluting product fractions contained an impurity
which was removed by HPLC purification with the same
Column and flow conditions, but using a 1.0%/min
gradient of 18 to 58% ACN containing 0.1% TFA. The main
product peak eluted at 32.1 min. The product containing
fractions from these two runs were combined and
lyophilized to give the title compound as a colorless
solid (174 mg, 65.6% from the product of Part C). MS:
m/e 1217.3 [M+H], 1117.3 [M+H-Boc].
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Part F - Preparation of 2-[(~4-[4-({[2-((2R)-2-Amino-3-
sulfopropyl)ethyl]amino}sulfonyl)phenyl]phenyl}sulfonyl)
amino](2S)-3-(f1-[3-(imidazol-2-ylamino)propyl](1H-
indazol-5-yl)}carbonylamino)propanoic Acid
Trifluoroacetate Salt
O O
N~OH H O
I
.N~~ NH2 O
N i HN.S - S N
~~~N~ 02 ~ ' 02 H~ F3C~OH
H HO3S
A mixture of the product of Part E, above (21.4 mg,
0.0176 mmol), peroxide-free THF (0.70 mL), water (0.063
mL), and 3 N LiOH (0.043 mL, 0.129 mmol) was stirred at
ambient temperatures under nitrogen for 3 h, and
concentrated under vacuum to a colorless solid.
The above solid was dissolved in 95/5 TFA/Et3SiH
(1.20 mL) and heated at reflux under nitrogen for 1 h.
The solution was concentrated under vacuum and the oily
solid was purified by HPLC on a Vydac C-18 column {22 x
250 mm) using a 1.2o/min gradient of 0 to 36% ACN
containing 0.1o TFA at a flow rate of 20 mL/min. The
main product peak eluting at 19.2 min was lyophilized to
give the title compound as a colorless solid (11.0 mg,
64.20). MS: m/e 861.2 [M+H]; High Resolution MS: Calcd
for C34H41N10~1153 [M+H] : 861.21181; Found: 861.2132.
Part G - Preparation of 2-{[{4-{4-[({2-[{2R)-3-Sulfo-2-
(2-{1,4,7,10-tetraaza-4,7,10-tris{[(tert-
butyl)oxycarbonyl]-
methyl}cyclododecyl}acetylamino)propyl]ethyl}amino)sulfo
nyl]phenyl}phenyl)sulfonyl]amino}{2S)-3-({1-[3-
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(imidazol-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)propanoic Acid Bis(trifluoroacetate)
Salt
O O
N'~OH_ H O O~ n ~O-t-Bu ~L,
/ N i H HN.S \ / / \ S.N~N~NH CN N1 O F3C OH
N~N~ 02 02 HO S JO
3 V O O
H H t-Bu-O O-t-Bu F3C~OH
A solution of the product of Example 4, Part B
(15.9 mg, 0.0174 mmol), DIEA (0.012 mL, 0.070 mmol), and
HBTU (5.3 mg, 0.014 mmol) in anhydrous DMF (1.5 mL) was
stirred under nitrogen at ambient temperatures for 10
min and added to a solution of the product of Part F,
above (10.0 mg, 0.0116 mmol) and DIEA (0.012 mL, 0.070
mmol) in anhydrous DMF (1.0 mL). The resulting solution
was stirred at ambient temperatures under nitrogen for
18 h, and concentrated under vacuum. The resulting pale
amber oil was purified by HPLC on a Vydac C-18 column
(22 x 250 mm) using a 1.0%/min gradient of 9 to 49% ACN
containing 0.1% TFA at a flow rate of 20 mL/min. The
main product peak eluting at 30.0 min was lyophilized to
give the title compound as a colorless fluffy solid
(10.5 mg, 55.10) . MS: m/e 1415.4 [M+H] .
Part H - Preparation of 2-[({4-[4-({[2-((2R)-3-Sulfo-2-
{2-[1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)cyclododecyl]-
acetylamino}propyl)ethyl]amino}sulfonyl)phenyl]phenyl}-
sulfonyl)amino](2S)-3-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)propanoic
Acid Bis(trifluoroacetate) Salt
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A solution of the product of Part G, above (10.5
mg, 0.00639 mmol) in 95/5 TFA/Et3SiH (1.0 mL) was heated
at reflux under nitrogen for 3 h. The solution was
concentrated under vacuum and the resulting oily solid
was purified by HPLC on a Vydac C-18 column (22 x 250
mm) using a 0.90%/min gradient of 0 to 27% ACN
containing 0.1o TFA at a flow rate of 20 mL/min. The
main product peak eluting at 28.0 min was lyophilized to
give the title compound as a colorless fluffy solid (2.3
mg, 24.4%). MS: m/e 1247.3 [M+H]; High Resolution MS:
Calcd for C5pHg7N14~18s3 [M+H]: 1247.3919; Found:
1247.390.
Example 37
Synthesis of (4S)-4-(N-{1-[N-(2-{4-[4-({[(1S)-1-carboxy-
2-({1-[3-(2-pyridylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-3-
carboxypropyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)cyclododecyl]acetylamino}butanoic
acid
0
OH
O O O~ N
Ni I ~ NH~OH ~O OOH
'~ NH
'~' OH
~N
N
Step A: Synthesis of
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HZ
N-Boc-(1-[3-(2-pyridylamino)propyl]-1H-indazole)-5-
carboxylic acid (prepared as described in Jadhav et al,
US patent 5,760,028) (217 mg, 0.548 mmol) was added to
a solution of methyl (2S)-3-amino-2-[({2,6-dimethyl-4-
[3- (N-
{2[(phenylmethoxy)carbonylamino]ethyl}carbamoyl)propoxy]
ph.enyl}sulfonyl)amino]propanoate (Prepared as in Example
34, Step B) and HBTU (250 mg, 0.&58 mmol) in DMF (10
mL). Diisopropylethylamine (334 ~zL, 1.12 mmol) was
added dropwise. The reaction was stirred for 45 min,
the solvents concentrated, and the residue purified by
flash chromatography (EtOAc/MeOH, from 0% -> 6% MeOH).
The product fractions were combined and concentrated to
afford 526 mg (1020 of the product as a golden oil.
LRMS (ES): 943.5 [M+H]+; 843.4 [M-Boc+H]+.
Step B: Synthesis of
The product of step A (517 mg) in methanol (3 mL) was
added to 10o palladium on Carbon (200 mg) in methanol (7
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mL) under nitrogen in a Parr bottle. It was
hydrogenolyzed at 50psi for 90 min, filtered through
Celite, rinsed with methanol, and concentrated to afford
a viscous oil. This was redissolved in 1:1
water/acetonitrile containing 0.1o TFA ( mL) and
lyophilized (1:1 acetontrile/water/0.1%TFA) to afford
the product as a white powder (380 mg, 74 % yield).
LRMS (ES): 809.3 ([M+H]+, 450) 355.2 (100 %). 1HNMR
(600.1343 MHz, CDC13): 8.49 (t, 1H), 8.29 (m, 1H), 8.18
(d, 2H), 7.87 (t, 1H), 7.74 (m, 2H), 7.64 (d, 1H), 7.52
(d, 1H), 7.11 (t, 1H), 6.66 (d, 1H), 6.64 (s, 2H), 4.45
(t, 2H), 4.04 (t, 1H), 3.91 (t, 2H), 3.83 (t, 2H), 3.55
(m, 1H), 3.47 (m, 1H), 3.35 (s, 3H), 3.16 (m, underH20
peak, ), 2.50 (s, 3H), 2.21
2H), 2.52
2.71 (s,
(m, 3H)
2H
(t, 2H), 2.15 {t, 2H), 1.88 {t, 2H), 1.33,s 9H).
(
Step C: Synthesis of
~02tBu
H f:
H ~NHZ
O
C02tBU
Gamma-tert-butoxy-Z-glutamic acid succinimide ester (2.0
g, 4.75 mmol) was dissolved in dimethylformamide, and
gamma-tert-butoxyglutamic acid (0.98 g, 4.8 mmol) was
added, followed by diisopropylethylamine (1.75mL, 10.1
mmol). The solution was stirred 18 hr, concentrated,
and the residue partitioned into ethyl acetate/10o
citric acid. The aqueous fraction was extracted with
ethyl acetate and the combined organics were washed with
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water, 10% potassium hydrogen sulfate, and brine, and
then concentrated. The residual oil was purified by
flash chromatography on silica (CH2C12/EtOAc/EtOH,
1:1:0.50) and the product fractions combined and
evaporated to yield the product (1.3g, 53%) as a gummy
solid. LRMS (ES): 523.4 [M+H]~, 467.4; 1HNMR
(600.1330 MHz, CDC13) 7.30 (m, 6H), 5.80 (d,~lH), 5.09
(m, 2H), 4.53 (m, 1H), 4.29 (m, 1H), 2.36 (m, 4H), 1.88
- 2.16 (m, 4H), 1.42 (s, 9 H), 1.41 (s, 9H).
Step D: Synthesis of
0 0
r ~ NH~O O
N'N ~ / NH
N O;S; ZH
O
/ ~ O N NH O
Boc O~N~ O
O
O
In a flask under nitrogen were added
diisopropylethylamine (28 uL, 160 umol), the product XIC
(62 mg, 120 umol), and HBTU (130 umol, 49 mg). This was
stirred for 10 minutes and then the product of Step B
(100 mg, 108 umol) was added, followed by
diisopropylethylamine (50 uL, 288 umol). The reaction
was stirred for 60 minutes and concentrated. The
residue was purified by prep HPLC (Vydac C18, 21.2 mm x
cm, 90% acetonitrile/water/0.1%TFA; 20-75% B over 40
minutes). The product fractions were combined and
lyophilized to afford 135 mg (88%) of the product as a
25 white solid. The product was contaminated with ~15% of
the deBoc product after lyophilization, but this was not
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purified. LRMS (EI); 313.5 ([M+H]+~ 80%>, 1213.5 {[M-
Boc+H]+, 45%) 551.3 (100%).
Step E: Synthesis of
0 0
N' I \
,N /
~N
N
Boc
The product of step Step D {118 mg) was hydrogenolyzed
and isolated as in step B. The lyophilized solid (110
mg) was not purified, but used directly in the following
step. LRMS (EI); 1179.6 ([M+H]+~ 200, 1079.5 ([M-
Boc+H]+, 250) 540.3 (100%).
Step F: Synthesis of
O
o
N O
0 0 ~ C N~~
NH O ~ NJ
N I , I ~O
.N H O
\ /N O;S;O
N~ ~~ .. NH O
In dry glassware under nitrogen were mixed HBTU (35 mg,
90 ~Zmol) , DOTA(OtBu)3-OH (49 mg, 85 umol) , and
diisopropylethylamine (35 ~L, 200 ~mol) in dry DMF (7
mL). This was stirred for 10 minutes and then the
product of step E (100 mg, 77 }.lmol) was added, along
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with additional diisopropylethylamine (45 uL, 250 ~mol)
to bring solution pH>9. After stirring for 30 min, the
reaction was concentrated and purified by preparative
HPLC (Vydac C18, 21.2 mm x 25 cm, 90%
acetonitrile/water/0.1%TFA; 20-70% B over 50 minutes).
Four products were obtained after purification; a pair
of glutamic acid isomers (60 mg) and the corresponding
Boc deprotected compounds (29 mg) for a total yield of
66%.
Step G: Synthesis of 4-(N-{(1R)-1-[N-(2-{4-[4-({[(1S)-
1-carboxy-2-({1-[3-(2-pyridylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carlaamoyl]-3
carboxypropyl}carbamoyl)(4S)-4-{2-[1,4,7,10-tetraaza
4,7,10-
tris(carboxymethyl)cyclododecyl]acetylamino}butanoic
acid
0
~H
N~
~N .N ~H
N
The combined Boc and Boc-deprotected D-Glutamic acid
isomeric products of step F (45 mg, 23 umol) were
dissolved in THF/methanol (1:1, 4 mL) and lithium
hydroxide (3N in water, 75 uL, 225 umol) added with
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stirring. The solution was stirred for 4 hours,
concentrated under vacuum, and the residue treated with
dichloromethane (3 mL), trifluoroacetic acid (3 mL) and
triethylsilane (300 uL) under nitrogen. The solution
was stirred overnight, concentrated, and purified by
preparative HPLC (Zorbax C8, 21.2 mm x 25 cm, 50%
acetonitrile/water/ 0.1% formic acid; 15-30% B over 50
minutes). The product fractions were combined, frozen,
and lyophilized to afford the product as a white solid
(17.6 mg, 57 %) . LRMS (EI) ; 1339.5 ( [M+H]+~ 15%~,
670.4 ( [M+2H]+2, 1000) . HPLC (2 x (4.6 x 21.2 mm Zorbax
CN) Water190oacetonitrile/0.1% formic acid, 10-20%B over
180 min) Rt = 100.4 minutes.
Step H: The synthesis of (4S)-4-(N-{(1S)-1-[N-(2-{4-[4-
({[(1S)-1-carboxy-2-({1-[3-(2-pyridylamino)propyl](1H-
indazol-5-yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-3-
carboxypropyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)cyclododecyl]acetylamino}butanoic
acid
i
O
N~ ~ ~ OH
~N
/N ~ 7H
N
The L-glutamic acid isomeric products of Step F (46 mg,
23 umol) were combined with the corresponding Boc
deprotected analog and treated similarly to step G to
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afford the product as a white solid (15.5 mg, 50%).
LRMS (EI) ; 1339.5 ( [M+H]+~ 150> , 670.4 ( [M+2H]+2, 100%) .
HPLC (2 x (4.6 x 21.2 mm Zorbax CN)
Water/90%acetonitrile/0.1% formic acid, 10-20oB over 180
min) Rt = 101.3 minutes.
Example 38
Synthesis of (4S)-4-(N-{1-[N-(2-{4-[4-({[(1S)-1-carboxy-
2-({1-[3-(imidazol-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-3-
carboxypropyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)cyclododecyl]acetylamino}butanoic
acid
)H
H
Step A: Synthesis of tert-butyl 2,3,5,6-
tetrafluorophenyl (2S)-2-{(2S)-4-[(tert-
butyl)oxycarbonyl]-2-[(phenylmethoxy)
carbonylamino]butanoylamino}pentane-1,5-dioate
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The product of Example 37, Step C (640 mg, 1.23 mmol)
was dissolved in DMF (5 mL) with 2,3,5,6-
tetrafluorophenol (286 mg, 1.7 mmo1). To this was added
(3-dimethylaminopropyl)ethyl carbodiimide hydrochloride
(282 mg, 1.47 mmol) and the solution was stirred 18 hr.
The reaction was concentrated and the residue
partitioned between ethyl acetate and water. The
aqueous layer was extracted twice with ethyl acetate,
and the combined organic layer was washed with 0.1N HCl,
10o NaHC03, water, and brine. It was dried over sodium
sulfate, filtered, concentrated, and purified by flash
chromatography (5:1 hexane/ethyl acetate). The product
was obtained as a clear oil (385 mg, 480) LRMS (EI);
693.1 ([M+Na]~, 35%), 671.3 ([M+H]~, 100%), 615.2 ([(M-
tBu)+H]+, 20%) .
Step B: Synthesis of (2S) -2- ( f C4- (3-(N- [2- (2-{ (2S) -4-
[(tert-butyl)oxycarbonyl]-2-
[(phenylmethoxy)carbonylamino] butanoylamino}-4-[(tert-
butyl)oxycarbonyl]butanoylamino)
ethyl]carbamoyl}propoxy)-2,6-
dimethylphenyl]sulfonyl}amino)-3-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)propanoic
acid
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O I i
H~OH O O O
NH ~ O
O.i
~S~O
O N NH O
p~NH~ O
O
~O
The product of Example 34, Step D (45 mg, 50 umol) was
dissolved in DMF (1.5 mL) with the product of step A (44
mg, 65 umol) and diisopropylethylamine (30.5 uL, 175
umol) under nitrogen. The solution was stirred for 45
min, concentrated under vacuum, and purified by
preparative HPLC (Vydac C18, 21.2 mm x 25 cm, 900
acetonitrile/water/0.1%TFA; 20-70% B over 25 minutes).
The product fractions were frozen and lyophilized to
afford the product as a white powder (49 mg, 83%). LRMS
(EI); 693.1 ([M+Na]+, 35%), 1188.4 ([M+H]+, 45%), 595.3
( [M + 2H]+z, 100%) .
Step C: Synthesis of (2S) -2- ( { [4- (3-{N- [2- (2-{ (2S) -2-
amino-4-[(tert-butyl)oxycarbonyl]butanoylamino}-4-
[(tert-
butyl)oxycarbonyl]butanoylamino)ethyl]carbamoyl}propoxy)
-2,6-dimethylphenyl]sulfonyl}amino)-3-({1-[3-(imidazol-
2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)propanoic acid
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The product of step B (25 mg, 24 umol) in methanol (3
mL) was added to 10% palladium on carbon (14 mg) in
methanol (3 mL) under nitrogen in a Parr bottle. It was
hydrogenolyzed at 50psi for 180 min, filtered through
Celite, rinsed with methanol, and concentrated to afford
a viscous oil. This was redissolved in 1:1
water/acetonitrile containing 0.1% TFA ( mL) and
lyophilized (1:1 acetontrilelwater/0.loTFA) to afford
the product as a white powder (29 mg, 100 % yield) which
analyzes for 2 equal peaks by HPLC (4.6 x 150 mm Zorbax
C-18, 1 mL/min; Water/90%acetonitrile/0.1o
trifluoroacetic acid, 2-100% B over 14 min) Rt = 9.78
and 10.14 minutes. LRMS (ES): 1054.5 ([M+H]+, 10%)
527.8 ([M + 2H]+z, 100%); identical for each peak. This
was not further purified but taken into the next step as
a mixture of two diastereomers.
Step D: Synthesis of (2S)-2-({[4-(3-{N-[2-(2-{(2S)-4-
[(tert-butyl)oxycarbonyl]-2-[2-(1,4,7,10-tetraaza-
4,7,10-tris{[(tert-butyl)oxycarbonyl]
methyl}cyclododecyl) acetylamino]butanoylamino}-4-
[(tert-butyl)oxycarbonyl]
butanoylamino)ethyl]carbamoyl}propoxy)-2,6-
dimethylphenyl] sulfonyl}amino)-3-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)propanoic
acid
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0
N 7 O
O
\ p o ~ CN~ op
N~NH~OH ~ ~O
'N i NH H O
O~S;O
N / ~ NH O
_ O N
~NH O ~NH~
In dry glassware under nitrogen were mixed HBTU (16.4
mg, 43 umol), DOTA(OtBu)3-OH (36 mg, 52 umol), and
diisopropylethylamine (26 ~L, 85 ~.~.mol) in dry DMF (0.6
mL). This was stirred for 10 minutes and then the
product of step C (29 mg, 25 ~.mol) was added in DMF (0.8
mL), along with additional diisopropylethylamine (20 uL,
65 ~mol) to bring solution pH > 9. After stirring for
60 min, the reaction was concentrated and purified by
preparative HPLC (Vydac C18, 21.2 mm x 25 cm, 90%
acetonitrile/water/0.1%TFA; 20-70% B over 50 minutes).
Two products were obtained after purification, a pair of
glutamic acid stereoisomers, which were each frozen and
lyophilized to afford the products as white powders(8 mg
each, 400) with identical fragmentation patterns. LRMS.
(ES): 1609.0 ([M+H]+, 5%), 805.0 (IM + 2H]+2, 30%),
537.4 ([M + 3H]k3, 100%); Using the HPLC method in Step
X2C, Rt = 11.54 min and 11.78 min.
Step E: Synthesis of (4S)-4-(N-{1-[N-(2-{4-[4-({[(1S)-
1-carboxy-2-({1-[3-(imidazol-2-ylamino)propyl](1H-
indazol-5-yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy] butanoylamino}ethyl)carbamoyl]-3-
carboxypropyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-4,7,10-
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tris(carboxymethyl) cyclododecyl]acetylamino}butanoic
acid
0 0
Nr I ~ NH~OH
~N i NN
~ O~S~O
NFf
O
N'
~NH O~N
The products of step D were each individually dissolved
in a mixture of dichloromethane (1 mL), trifluoroacetic
acid (1 mL), and triethylsilane (0.2 mL) under nitrogen
and stirred 16 hours. The solutions were concentrated
and the residues purified by prep HPLC (Vydac C18, 21.2
mm x 25 cm, 90% acetonitrile/waterl0.1%TFA; 0-45% B
over 45 minutes). The product fractions were frozen and
lyophilized to afford the products as white solids (3.5
mg of each, ~50%) with identical fragmentation patterns
LRMS (ES) : 1328.5 ( [M+H]+, 5%) , 664.8 ( [M + 2H]+2,
1000), 372.2 (100%); Using the HPLC method in Step C,
Rt = 8.08 min and 8.09 min.
Example 39
Synthesis of (4S)-4-{N-[(1S)-1-(N-{1,3-bis[N-(2-{4-[4-
({[(1S)-1-carboxy-2-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]propyl}car
bamoyl)-3-carboxypropyl]carbamoyl}-4-(6-{2-[1,4,7,10-
tetraaza-4,7,10-
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tris(carboxymethyl)cyclododecyl]acetylamino}
hexanoylamino)butanoic acid
Step A: Synthesis of (2S) -2-{ [ (4-{3- [N- (2-{4- [N- (2-{4-
[4-({[(1S)-1-carboxy-2-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-4-[(tert-
butoxy)carbonylamino]butanoylamino}ethyl)carbamoyl]propo
xy}-2,6-dimethylphenyl)sulfonyl]amino}-3-({1-[3-
(imidazol-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)propanoic acid
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The product of Example 34, Step D (45 mg, 49.4 umol) was
added along with Boc-Glu-(OTFP)-OTFP (13 mg, 24 umol) to
DMF (1.5 mL) containing diisopropylethylamine (31 uL,
180 umol) and stirred for 18 hours. The solution was
concentrated and purified by prep HPLC (Vydac C18, 21.2
mm x 25 em, 90~ acetonitrile/water/ 0.1%TFA; 5-55o B
over 25 minutes). The product fractions were frozen and
lyophilized to afford the product as a white powder (31
mg, 82%) . LRMS (ES) : 1578.5 ( [M+H]+, 50) , 790.1 ( [M +
2H]~2, 100%) , 527.3 ( [M+3H]+3, 50%) .
Step B: Synthesis of tert-butyl (4S)-4-{(2S)-4-[(tert-
butyl)oxycarbonyl]-2-
[(phenylmethoxy)carbonylamino]butanoyl amino}-4-(N-{1,3-
bis[N-(2-{4-[4-({[(1S)-1-carboxy-2-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)
ethyl]amino}sulfonyl)-3,5-dimethylphenoxy]butanoylamino}
ethyl)carbamoyl]propyl}carbamoyl)butanoate
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The product of Step A {30 mg, 19 umol) was added to a
solution of trifluoroaCetiC acid (250 uL) in
dichloromethane (500 uL) and stirred for 30 minutes
under a nitrogen atmosphere. The solution was
concentrated and left under vacuum for 1 hour. The
residue was dissolved in DMF (800 uL) under nitrogen and
the product of Example 38, Step A {16 mg, 24 umol)
added, followed by diisopropylethylamine {75 uL, 730
umol) to adjust pH > 9. The solution was stirred for 60
minutes, concentrated, and purified by prep HPLC {Vydac
C18, 21.2 mm x 25 cm, 90o aCetonitrile/water/0.1%TFA;
20-60o B over 40 minutes). The product fractions were
frozen and lyophilized to afford the product as a white
powder (30 mg, 810). LRMS (ES): 1983.6 ([M+H]+, 10~),
992.0 ([M + 2H]+2, 100%), 661.8 ([M+3H]~3, 80%), 643.2
( [ (M-tBu) + 3H]+3, 40%) , 624.4 ( [ (M-2tBu) + 3H)+3, 30%) .
HRMS: Calculated for C93H124N21v29S2 ~ ~-982.857; Fourid
1982.55.
Step C: Synthesis of (4S)-4-((2S)-2-{6-[(tert-butoxy)
carbonylamino]hexanoylamino}-4-Carboxybutanoylamino)-4
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(N-{1,3-bis[N-(2-{4-[4-(f[(1S)-1-carboxy-2-(~1-[3-
(imidazol-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino} sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]
propyl}carbamoyl)butanoic acid
0 0'I
O N NH~O
O O
NH O
H
OH
NH~ O
O ,O
N ~ 'NH
N~ ~ / NH~OH
O O
The product of step B (29 mg, 14.6 umol) was dissolved
in neat trifluoroacetic acid (2 mL) and triethylsilane
(250 uL) added. The reaction was heated with stirring
under nitrogen to 70C for 3 hr, concentrated,
reconcentrated with toluene (5mL), dissolved in 1:1
water/acetonitrile, frozen, and lyophilized. The
resulting powder (27 mg) was dissolved in DMF~(0.8 mL)
with 2,3,5,6-tetrafluorophenyl 6-[(tert-
butoxy)carbonylamino]hexanoate (10 mg, 26 umol) and
diisopropylethylamine (18 uL, 100 umol) and stirred for
60 minutes. Additional 2,3,5,6-tetrafluorophenyl 6-
[(tert-butoxy)carbonylamino]hexanoate (20 mg, 52 umol)
was added and the reaction stirred 45 minutes. The
reaction, containing primarily the tris-hexanoyl
product, was concentrated, the residue dissolved in
ethanol (2 mL), and sodium hydroxide (5N solution, 200
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uL) added. The solution was stirred 25 minutes,
neutralized to pH < 5 with 1N HCl (~1.1 mL) and
concentrated. The residue was purified by preparative
HPLC (Vydac C18, 21.2 mm x 25 cm, 90%
acetonitrile/water/0.1%TFA; 15-55% B over 50 minutes).
The product fraction was frozen and lyophilized to
afford the product as a white powder (21 mg, 65%). LRMS
(ES): 1951.3 ([M+H]+, 5%), 975.5 ([M + 2H]+2, 90%),
617.5 ( [ (M-Boc)+3H]+3, 1000) .
Step D: Synthesis of (4S)-4-[(2S)-2-(6-aminohexanoyl -
amino)-4-carboxybutanoylamino]-4-(N-{1,3-bis[N-(2-{4-[4-
({[(1S)-1-carboxy-2-({1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]propyl}
carbamoyl)butanoic acid
The product of C (19 mg, 9.7 umol) was added to
trifluoroacetic acid (200 uL) and dichloromethane (600
uL) and stirred under nitrogen for 30 min, concentrated,
and purified by prep HPLC (Vydac C18, 21.2 znm x 25 cm,
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90% acetonitrile/water/0.1%TFA; 5-35% B over 40
minutes). The product fractions were frozen and
lyophilized to afford the product as a white powder (13
mg, 70%). LRMS (ES): 1850.3 ([M+H]+, 5a), 925.6 ([M +
2H]+2, 250) , 617.7 ( [M+3H]+3, 100%) .
Step E: Synthesis of 2,3,5,6-tetrafluorophenyl 2-
(1,4,7,10-tetraaza-4,7,10-tris{[(tert-butyl)oxycarbonyl]
methyl}cyclododecyl)acetate
DOTA(OtBu)3-OH (95 mg, 138 umol) was added to dry DMF (1
mL) along with HBTU (90 mg, 210 umol),
diisopropylethylamine (103 uL, 740 umol), and 2,3,5,6-
tetrafluorophenol (32 mg, 270 umol). The solution was
stirred under nitrogen for 18 hours, concentrated, and
purified by preparative HPLC (Vydac C18, 21.2 mm x 25
cm, 90% acetonitrile/water/0.loTFA; 20-80o B over 30
minutes). The product fractions were frozen arid
lyophilized to afford the product as a white powder (81
mg, 70%). LRMS (ES): 721.5 ([M+H]+, 1000), 665.5 ([(M-
tBu) + H]+, 70a) .
Step F: Synthesis of (4S)-4-((2S)-4-carboxy-2-{6-[2-
(1,4,7,10-tetraaza-4,7,10-tris{[(tert-butyl)oxycarbonyl]
methyl}cyclododecyl)acetylamino]hexanoylamino}butanoylam
ino)-4-(N-{1,3-bis[N-(2-{4-[4-({[(1S)-1-carboxy-2-({1-
[3-(imidazol-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino) ethyl]amino}sulfonyl)-3,5-
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dimethylphenoxy]butanoylamino}
ethyl)carbamoyl]propyl}carbamoyl)butanoic acid
The product of step D (12 mg, 6.1 umol) and the product
of step E (7.2 mg, 7.6 umol) were mixed together in dry
DMF (600 uL) with diisopropylethylamine (13.2 uL, 76
umol) and stirred under nitrogen. At 90 minutes and 4
hours, additional amounts of step E (5 mg, 5.1 umol)
were added. After 5 hours, the reaction was
concentrated, dissolved in ethanol (2 mL) and treated
with sodium hydroxide (540 uL of a 1N solution. After
45 minutes, the solution was acidified with 1N HCl (~-600
uL), concentrated and purified by preparative HPLC
(Vydac C18, 21.2 mm x 25 cm, 90%
acetonitrile/water/0.1%TFA; 10-55°~ B over 50 minutes).
The product fraction, which contained several impurities
by HPLC analysis, was frozen and lyophilized to afford
the product as a white powder (10.5 mg, 720). LRMS
(ES) : 802.2 ( [M+3H]+3, 1000) , 604.1 ( [M + 4H]+4, 90%) .
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Step G: Synthesis of (4S)-4-{N-[(1S)-1-(N-{1,3-bis[N-
(2-{4-[4-({[(1S)-1-carboxy-2-({1-[3-(imidazol-2-
ylamino)propyl] (1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]propyl}car
bamoyl)-3-carboxypropyl]carbamoyl}-4-(6-{2-[1,4,7,10
tetraaza-4,7,10-
tris(carboxymethyl)cyclododecyl]acetylamino}hexanoyl
amino)butanoic acid
The product of F (10 mg) was added to dichloromethane (1
mL) containing trifluoroacetic acid (1 mL) and
triethylsilane (200 uL) and stirred under nitrogen for
72 hours. The reaction was concentrated and purified by
preparative HPLC (Vydac C18, 21.2 mm x 25 cm, 90%
acetonitrile/water/0.1%TFA; 15-55% B over 50 minutes).
The product fraction was frozen and lyophilized to
afford the product as a white powder (1 mg, 15%). LRMS
(ES) : 1118.7 ( [M + 2H]+2, 100) , 746.3 ( [M+3H]+3, 40%)
560.0 ([M+4H]+4, 100%).
281
s.o
O
NH
N~ I / NH~OH
O O
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Example 40
Synthesis of (4S)-4-(N-{1-(N-(2-{4-[4-({[(1S)-1-carboxy-
2-({1-[3-(3,4,5,6-tetrahydropyrimidin-2-
ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyllamino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-3-carboxy
propyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-4,7,10-tris
(carboxymethyl)cyclododecyl]acetylamino}butanoic acid
0
N~ I ~ >H
~N
~NH H
~~ I-r
N
Step A: Synthesis of ethyl 1-[3-(pyrimidin-2-
ylamino)propyl]-1H-indazole-5-carboxylate
0
N, ~ w o~.
~N
NH~
Ethyl 1-(3-oxopropyl)-1H-indazole-5-carboxylate (1.0 g,
4.06 mmol, prepared as described in Jadhav et al, US
patent 5,760,028) was dissolved in toluene (15 mL) and
2-aminopyrimidine (463 mg, 4.9 mmol) added, along with
anhydrous magnesium sulfate (2.44 g, 20 mmol) under
nitrogen. The mixture was vigorously stirred for six
hours, filtered under nitrogen, the solids washed (10 mL
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toluene), and the filtrate treated with sodium
triacetoxyborohydride (8.6 g, 40 mmol). The reaction
was stirred under nitrogen for 18 hours, diluted with
toluene (25 mL), and poured into water (100 mL).
Saturated sodium bicarbonate solution (80 mL) was added
to adjust pH > 8. The layers were separated and the
aqueous layer extracted with three portions of ethyl
acetate. The combined organics were washed with
saturated bicarbonate solution, water, and brine, dried
over sodium sulfate, filtered, and concentrated under
vacuum to afford a golden oil (1:3 g). This purified by
preparative HPLC (Vydac C18, 21.2 mm x 25 cm, 90a
acetonitrile/water/0.loTFA; 10-70o B over 30 minutes).
The product fractions were frozen and lyophilized to
afford the desired product as a white powder (520 mg,
400). LRMS (ES): 326.2 ([M + H]+. HRMS: Calculated
f or Cl~HZON50z . 3 2 6 .1617 ; Found . 3 2 6 .16 0 5 . 1HNMR
(600.1343 MHz, CDC13): 9.68 (bs, 1H), 8.58 (m, 1H),
8.49 (s, 1H), 8.12 (s, 1H), 8.08 (m, 1H), 8.03, (t, 1H),
7.50 (d, 1H) 6.73 (t, 1H) 4.55 (m, 2H) , 4.39 (q, 2H)
, , ,
3.36 (m, 2H) 2.35 (m, 2H) 1.41 (t, 3H) .
, ,
Step B: Synthesis of 1-[3-(pyrimidin-2-ylamino)propyl]-
1H-indazole-5-carboxylic acid
0
Ni I ~ OH
~ N ,N i
~~NH~
The product of step A (510 mg, 1.16 mmol) was dissolved
in ethanol (50 mL) and sodium hydroxide (6.5 mL of a 1N
solution, 6.5 mmol) added. The solution was heated at
reflux for 1.5 hours, diluted with water (45 mL), and
the ethanol removed under vacuum. The solution was
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acidified to pH = 3 with 1N HCl (~7 mL) with stirring.
The resulting solids were filtered, washed with water,
and dried under vacuum to afford the product (308 mg,
89%). LRMS (ES): 298.1 ([M + H]+. HRMS: Calculated
for C15H16N5~2 ~ 298.1304; Found . 298.1320. 1HNMR
(600.1343 MHz, CDC13): 12.5 (b, H), 8.42 (s, 1H), 8.26
(d, 2H), 8.19 (s, 1H), 7.90 (d, 1H), 7.67, (d, 1H), 7.45
(m, 1H), 6.58 (s, 1H), 4.50 (t, 2H), 3.29 (m, 2H), 2.13
(t, 2H) .
Step C: Synthesis of methyl (2S)-2-[({2,6-dimethyl-4-
[3-(N-{2-
[(phenylmethoxy)earbonylamino]ethyl}carbamoyl)propoxy]
phenyl}sulfonyl)amino]-3-({1-[3-(pyrimidin-2-
ylamino)propyl] (1H-indazol-5-
yl)}carbonylamino)propanoate
0 0
Ni I ~ NH~O-
'N / NH
~ O~S~O
NI-f
O NH
~O
O~NH~ '/O
The product of step B (292 mg, 0.98 mmol) was treated as
in Example 37, Step A to afford the crude product which
was purified by preparative HPLC (Vydac C18, 21.2 mm x
cm, 90% acetonitrile/water/0.1%TFA; 10-70% B over 30
minutes). The product fractions were frozen and
lyophilized to afford the desired product as a white
powder (825 mg, 880). LRMS (ES): 844.3 ([M + H]+.
Step D: Synthesis of methyl (2S)-2-{[(4-{3-[N-(2-
aminoethyl)carbamoyl]propoxy}-2,6-
dimethylphenyl)sulfonyl] amino}-3-({1-[3-(3,4,5,6-
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tetrahydropyrimidin-2-ylamino) propyl](1H-indazol-5-
yl)}carbonylamino)propanoate
0 0
Ni I ~ NH~O-
'~ NH
~~ H O'S~O
NH~
O NH2
O~NH
The product of Step C (250 mg, 260 umol) was treated as
in Example 37, Step B to afford the product as a white
powder (220 mg, 89%). LRMS (ES): 714.3 ([M + H]+,
25%), 402.2 (30%), 357.1 ([M + 2H]+2,100%). HRMS:
Calculated for C33H48N7O7S . 714.3397; Found . 714.3374.
Step E: Synthesis of tent-butyl (4S)-4-[N-(2-{4-[4-
({[(1S)-1-(methoxycarbonyl)-2-({1-[3-(3,4,5,6-tetrahydro
pyrimidin-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino) ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino} ethyl)carbamoyl]-4-
[(phenylmethoxy)carbonylamino]butanoate
The product of step D {219 mg, 234 umol) was dissolved
in DMF (2 mL) and tert-butyl 2,5-dioxopyrrolidinyl (2S)-
2-[(phenylmethoxy)carbonylamino]pentane-1,5-dioate (108
mg, 250 umol) added, along with diisopropylamine (130
uL, 750 umol). The solution was stirred under nitrogen
for 90 minutes, concentrated, and purified by
preparative HPLC (Vydac C18, 21.2 mm x 25 cm, 900
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acetonitrile/water/0.1%TFA; 20-75% B over 40 minutes).
The product fractions were frozen and lyophilized to
afford the desired product as a white powder (242 mg,
81%). LRMS (ES): 1033.4 ([M + H]+, 100%), 489.2 ([(M-
tBu) + 2H]+2, 80%)
Step F: Synthesis of tart-butyl 4-[N-(2-{4-[4-({[(1S)-
1-(methoxycarbonyl)-2-({1-[3-(3,4,5,6-
tetrahydropyrimidin-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl] amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl) Carbamoyl]-4-
aminobutanoate
The product of C (228 mg, 198 umol) was treated as in
Step D to afford the product as a white powder (176 mg,
79%) . LRMS (ES) : 899.5 ( [M + H]+, 50%) , 450.2 ( [M +
2H]~2, 65%) , 422.4 ( [ (M-tBu) + 2H]+2, 100%) .
Step G: Synthesis of tart-butyl 4-{(2S)-4-[(tert-
butyl)oxycarbonyl]-2-[(phenylmethoxy)Carbonylamino]
butanoylamino}-4-[N-(2-{4-[4-({[(1S)-1-
(methoxycarbonyl)-2-({1-[3-(3,4,5,6-tetrahydropyrimidin-
2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]butanoate
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O O
Nr ~ ~ NH~O- O~O
y~ NH
~~ H O;g; NH
O
N I ~ O N NH O
O~NN~ p
O
~O
O
~O~
The product of step F (85 mg, 76 umol) was treated as in
step E to afford the product after lyophilization (87
mg, 87%). LRMS (ES): 1218.6 ([M + H]+, 100%), 610.0
([M + 2H]+2, 20%), 581.8 ([(M-tBu) + 2H]+z, 30%), 553.8
( [ (M-2tBu) + 2H]+2, 85%) .
Step H: Synthesis of tert-butyl (4S)-4-(N-{1-[N-(2-{4-
[4-({[(1S)-1-(methoxycarbonyl)-2-({1-[3-(3,4,5,6-
tetrahydro pyrimidin-2-ylamino)propyl](1H-inda~ol-5-
yl)}carbonylamino) ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino} ethyl)carbamoyl]-3-
[(tert-butyl)oxycarbonyl]propyl} carbamoyl)-4-
aminobutanoate
0 0
r ~ NH~O- O
i NH
~,~H .. ..
N O
NH~
The product of step G (75 mg, 56 umol) was treated as in
step F to afford the product as a white solid (72 mg,
97%). LRMS (ES): 1084.6 ([M + H]+, 20%), 542.8 ([M +
2H]+z, 1000) , 514.8 ( [ (M-tBu) + 2H]~2, 30%) , 486.9 ( [ (M-
2tBu) + 2H]+2, 20%) .
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Step I: Synthesis of tert-butyl (4S)-4-(N-{1-[N-(2-{4-
[4-({[(1S)-1-(methoxycarbonyl)-2-({1-[3-(3,4,5,6-
tetrahydropyrimidin-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-dimethyl
phenoxy]butanoylamino}ethyl)carbamoyl]-3-[(tert-butyl)
oxycarbonyl]propyl}carbamoyl)-4-[2-(1,4,7,10-tetraaza-
4,7,10-tris{[(tert-
butyl)oxycarbonyl]methyl}cyclododecyl)
acetylamino]butanoate
0
0
O O ~ CN N
Nr I ~ NH~O- ~ 00
'~ NH
~NH O~S; H ~O~
'~'O
~~NH~ ~ ~ " .. NH O
The product of step H (60 mg, 46 umol) was treated as in
Example 37, Step G to afford the product as a single
pure compound (40 mg, 54%) after lyophilization. LRMS
(ES) : 1638.7 ( [M + H]*, 10 0) , 820.1 ( [M + 2H]+2, 30%) ,
528.5 ( [ (M-tBu) + 3H]+3, 30%) , 509.8 ( [ (M-2tBu) + 3H]+3,
100%) , 491.1 ( [ (M-3tBu) + 3H]+3, 500) .
Step J: Synthesis of (4S)-4-(N-{1-[N-(2-{4-[4-({[(1S)-
1-carboxy-2-({1-[3-(3,4,5,6-tetrahydropyrimidin-2-
ylamino)propyl](1H-indazol-5-yl)}carbonylamino)ethyl]
amino}sulfonyl)-3,5-dimethylphenoxy]butanoylamino}ethyl)
carbamoyl]-3-carboxy propyl}carbamoyl)-4-{2-[1,4,7,10-
tetraaza-4,7,10-
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tris(carboxymethyl)cyclododecyl]acetylamino} butanoic
acid
0
N~ I ~ )H
~N
~NH
\~~NH~
The product of step I (25 mg, 14.3 umol) was dissolved
in THF (600 uL)jwater (100 uL) and lithium hydroxide (3N
in water, 60 uL, 180 umol) added with stirring. The
solution was stirred for 100 min, acidified to pH = 2
with trifluoroacetic acid (14 uL), and concentrated
under vacuum. The residue was treated with
dichloromethane (1 mL), trifluoroacetic acid (1 mL) and
triethylsilane (100 uL) under nitrogen. The solution
was stirred overnight, concentrated, and purified by
preparative HPLC (Zorbax CN, 21.2 mm x 25 cm, 50o
acetonitrilejwaterj 0.1% formic acid; 20-30o B over 50
minutes). The product fractions were combined, frozen,
and lyophilized to afford the product as a white solid
(13 mg, 57 0). LRMS (EI); 1344.5 ([M+H]+~ 15%~, 672.9
( [M+2H]*2, 100%) , 449.9 ( [M+3H]+3, 500) .
Example 41
Synthesis of (4S)-4-(N-~1-[N-(2-{4-[4-({[(1S)-1-carboxy-
2-({1-methyl-3-[3-(2-3,4,5,6-
tetrahydropyridylamino)propyl] (1H-indazol-6-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-3-
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carboxypropyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)cyclododecyl]acetylamino}butanoic
acid
N »h a
JF-f O
OH
O
Step A: Synthesis of methyl (2S)-2-[({2,6-dimethyl-4-
[3-(N-{2-
[(phenylmethoxy)carbonylamino]ethyl}carbamoyl)propoxy]
phenyl}sulfonyl)amino]-3-({1-methyl-3-[3-(2-
pyridylamino) propyl](1H-indazol-6-
yl)}carbonylamino)propanoate
/v
1-Methyl-3-[3-(2-pyridylamino)propyl]-1H-indazole-6
carboxylic acid (79 mg, 256 umol, prepared as described
in Jadhav et al, US patent 5,760,028) was treated with
the product of Example 34, Step B (223 mg 282 umol) as
in example 37, Step A to afford the crude product which
was purified by prep HPLC (Vydac C18, 21.2 mm x 25 cm,
90% acetonitrile/water/0.1%TFA; 10-60% B over 30
minutes). The product fractions were frozen and
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lyophilized to afford the desired product as a white
powder (122 mg, 49%). LRMS (ES): 857.3 ([M+H]+, 100%).
HRMS: Calculated for C$3H53NeO9 . 857.3656; Found
857.3676.
Step B: Synthesis of methyl (2S)-2-{[(4-{3-[N-(2-
aminoethyl)carbamoyl]propoxy}-2,6-
dimethylphenyl)sulfonyl] amino}-3-({1-methyl-3-[3-(2-
3,4,5,6-tetrahydropyridyl amino)propyl](1H-indazol-6-
yl)}carbonylamino)propanoate
0 0
N ~ NH~O-
N' , / NH
v i
O~S,O
N N _.i \
O NH2
O~NH
In a Parr bottle were added 10% Pd/C (50 mg) and
methanol (5 mL) under nitrogen. The product of Step A
(113 mg, 116 mmol) in methanol (,5 mL) was added, along
with 20 uL of trifluoroacetic acid. The mixture was
hydrogenated at 50 psi with shaking for 7.5 hours,
filtered through Celite, the Celite rinsed with
methanol, and the combined filtrates concentrated. The
residue was redissolved in 1:1 acetonitrile/water/0.1%
TFA, frozen and lyophilized to afford the product as a
white powder (98 mg, 880). LRMS (ES): 727.3 ([M+H]+,
25%), 364.2 ([M+2H]+~, 1000). HRMS: Calculated for
C35HS1Ns0~S . 727.3601; Found . 727.3613.
Step C: Synthesis of tent-butyl (4S)-4-[N-(2-{4-[4-
({[(1S)-1-(methoxycarbonyl)-2-({1-methyl-3-[3-(2-
3,4,5,6-tetrahydropyridylamino)propyl](1H-indazol-&-
yl)}carbonyl amino)ethyl]amino}sulfonyl)-3,5-
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dimethylphenoxy]butanoyl amino}ethyl)carbamoyl]-4-
[(phenylmethoxy)carbonylamino] butanoate
0 0II
N w NH~O- ~ /
N~ I , NH
O~S'O O
N
N ~ \ O NH NH
O~NN~ O
O
\O
The product of step B (98 mg, 102 umol) is reacted as in
Example 40, Step E cm, 90% acetonitrile/water/0.1%TF~;
10-70% B over 25 minutes). The product fractions were
frozen and lyophilized to afford the desired product as
a white powder (103 mg, 96%). LRMS (ES): 1046.5 ([M +
H]+, 100°x) , 495.9 ( [ (M-tBu) + 2H]+z, 60%) .
Step D: Synthesis of tart-butyl (4S)-4-{(2S)-4-[(tert-
butyl)oxycarbonyl]-2-
[(phenylmethoxy)carbonylamino]butanoyl amino}-4-[N-(2-
{4-[4-({[(1S)-1-(methoxycarbonyl)-2-({1-methyl-3-[3-(2-
3,4,5,6-tetrahydropyridylamino)propyl](1H-indazol-6-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]butanoate
0 0
N ~ NH~O- O O O
N~ I i NH NH O
i
O;g;O
N
N I ~ O N NH O
O. ~N~
The product of step C (97 mg, 83 umol) was treated as in
Example 37, StepB to afford the crude deprotected amine
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(87 mg). This was then reacted as in Example 40, Step E
and purified by preparative HPLC (Zorbax C8, 21.2 mm x
25 cm, 90o acetonitrile/water/0.1%TFA; 20-80% B over 30
minutes). The product fractions were frozen and
lyophilized to afford the desired product as a white
powder {77 mg, 76%). LRMS (ES): 1231.6 ([M + H]+,
90 0) , 616.4 ( [M + 2H]+2, 40%) , 588.4 ( [ (M-tBu) + 2H]+2,
50%) , 495.9 ( [ (M-2tBu) + 2H]+2, 1.00%) .
Step E: Synthesis of tert-butyl (4S)-4-(N-{(1S)-1-[N-
(2-{4-[4-({[(1S)-1-{methoxycarbonyl)-2-({1-methyl-3-[3-
(2-3,4,5,6-tetrahydropyridylamino)propyl](1H-indazol-6-
y1)} carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy] butanoylamino}ethyl)carbamoyl]-3-
[(tert-butyl)oxycarbonyl] propyl}carbamoyl)-4-[2-
(1,4,7,10-tetraaza-4,7,10-tris {[(tert-
butyl)oxycarbonyl]methyl}cyclododecyl)acetylamino]
butanoate
o i
NN I ~ v
N N
The product of step D (71 mg, 53 umol) was treated as in
Example 37, Step B to afford the crude deprotected amine
(64 mg). This was then reacted with DOTA(OtBu)3-OH
(29.5 mg, 52 umol) as in Example 40, Step z and the
crude product purified by preparative HPLC (Vydac C18,
21.2 mm x 25 cm, 90% acetonitrile/0.1%TFA; 20-75% B
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over 45 minutes). The product fractions were frozen and
lyophilized to afford the desired product as a white
powder (62 mg, 75%). LRMS (ES): 1651.9 ([M + H]+,
50) , 826.7 ( [M + 2H]+2, 30%) , 532.8 ( [ (M-tBu) + 3H]~3,
25%), 514.9 ([(M-2tBu) + 3H]+3, 100%), 495.4 ([(M-3tBu) +
3H]+3, 60 a) .
Step F: Synthesis of (4S)-4-(N-{1-[N-(2-{4-[4-({[(1S)-
1-carboxy-2-({1-methyl-3-[3-(2-3,4,5,6-
tetrahydropyridylamino) propyl] (1H-indazol-6-
yl)}carbonylamino)ethyl]amino} sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl] -3-
carboxypropyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)cyclododecyl]acetylamino}butanoic
acid
i
O
NN I ~ OH
~H
N N
The product of step E (42 mg, 25 umol) was treated as in
Example 40, Step J and the crude product purified by
preparative HPLC (Zorbax CN, 21.2 mm x 25 cm, 50%
acetonitrile/water/ 0.1% formic acid; 20-35% B over 60
minutes). The product fractions were combined, frozen,
and lyophilized to afford the product as a white solid
(11 mg, 48%). LRMS (EI); 1357.6 ([M+H]+. 15%~, 679.5
( [M+2H]t2, 100%) , 453.3 ( [M+3H]+3, 40%) .
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Example 42
Synthesis of (4S)-4-(N-{(1S)-1-[N-(2-{4-[4-({[(1S)-1-
carboxy-2-({1-[2-(2-3,4,5,6-
tetrahydropyridylamino)ethyl] (1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoylamino}ethyl)carbamoyl]-3-carboxy
propyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-4,7,10-tris
(carboxymethyl)cyclododecyl]acetylamino}butanoic acid
Step A: Synthesis of ethyl 1-[2-(1,3-dioxoisoindolin-2-
yl)ethyl]-1H-indazole-5-carboxylate and ethyl 2-[2-(1,3-
dioxoisoindolin-2-y1)ethyl]-1H-indazole-5-carboxylate
0
O
~N ~ ~ O
O ~ I / 'N~N~ I ~ O
O O ~N i
Ethyl 1H-indazole-5-carboxylate (1.5 g, 7.9 mmol) and
18-crown-6 (45 mg) were added to dry THF (45 mL) in
flame-dried glassware under nitrogen. Sodium
bis(trimethylsilyl)amide (8.7 mL of 1M solution in THF,
8.7 mmol) was added via syringe, followed by N-(2-
2,0 bromoethyl)phthalimide (2.5 g, 9.8 mmol). The reaction
was heated at reflux temperature for 22 hr, cooled, and
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concentrated under vacuum. The residue was partitioned
between toluene and water, separated, and the aqueous
layer extracted with ethyl acetate. The combined
organics were washed with water and brine, dried over
sodium sulfate, filtered and concentrated to afford 3.5
g of an oil. This was purified by flash chromatography
(toluene - ethyl acetate gradient), collecting two
separate products which were concentrated to yield the
products as oils which solidified on standing. The 1-
substituted indazole eluted first (980 mg), followed by
the 2-substituted analog (600 mg) for a combined yield
of 55%. Their mass spectra were identical. LRMS (ES):
364.1 ( [M + H]+, 100%) , 386.1 ( [M + Na]+, 150)
Step B: Synthesis of ethyl 1-(2-aminoethyl)-1H-
indazole-5-carboxylate
0
N~ I w o~
~N
H2N
Ethyl 1-[2-(1,3-dioxoisoindolin-2-yl)ethyl]-1H-indazole-
5-carboxylate (step A, 980 mg, 2.7 mmol) was dissolved
in ethanol/THF (1:1, 35 mL) under nitrogen. Hydrazine
(365 uL) was added and the reaction stirred 17 hours.
THF (75 mL) was added and the resulting solids were
filtered off. The filtrate was concentrated to an
orange solid, which was purified by flash chromatography
(dichoromethane/5% methanol/0.5% triethylamine). The
product fractions were combined and concentrated to an
orange solid (404 mg, 66%). LRMS (ES): 234.1 ([M +
H]+, 100%)
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Step C: Synthesis of ethyl 1-{2-[(1-hydroxy-2-
pyridyl)amino]ethyl}-1H-indazole-5-carboxylate
Ethyl 1-(2-aminoethyl)-1H-indazole-5-carboxylate (584
mg, 2.5 mmol, prepared as in step B), was added to dry
n-butanol along with 2-chloropyridine-N-oxide
hydrochloride (847 mg, 5.1 mmol) and anhydrous sodium
bicarbonate (850 mg, 10.1 mmol). The reaction mixture
was vigorously stirred and heated at 100C for 21 hr.
Additional aliquots of 2-chloropyridine-N-oxide
hydrochloride (847 mg, 5.1 mmol) and anhydrous sodium
bicarbonate (850 mg, 10.1 mmol) were added and heating
continued for 24 hours. The reaction was cooled and
filtered and the filtrate concentrated. The residue was
purified by flash chromatography (5% methanol-
dichloromethane) and the product fractions concentrated
to afford the product as an orange solid (358 mg, 44%).
LRMS (ES): 327.1 ([M + H]*, 100%), 653.3 ([2M + H]+,
40%) 1HNMR (600.1343 MHz, CDC13): 8.44 (s, 1H), 8.12
(s, 1H), 7.97 (d of t, 2H), 7.56 (bs, 1H), 7.45 (d, 1H),
7. 06, (m, 1H) , 6.45 (m, 1H) , 6.35 (t, 1H) , 4. 68 (t, 2H) ,
4.37 (q, 2H) , 3.90 (q, 2H) , 1.39 (t, 3H) .
Step D: Synthesis of 1-{2-[(1-oxy-2-
pyridyl)amino]ethyl}-1H-indazole-5-carboxylic acid
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The product of step C (349 mg, 1.07 mmol) was dissolved
in ethanol (35 mL) and 1N sodium hydroxide solution (6.0
mL, 6 mmol) added. The solution. was heated at reflux
for 75 min, the volume reduced by half, and water (30
mL) added. 1N hydrochloric acid was added to pH = 3 and
the remaining ethanol concentrated under vacuum. The
resulting solids were filtered and dried under vacuum to
afford the product as an off-white solid (163 mg, 510).
LRMS (ES) : 299.2 ( [M + H]+, 100%) .
Step E: Synthesis of methyl (2S)-2-[(~2,6-dimethyl-4-
[3- (N-~2-
[(phenylmethoxy)carbonylamino]ethyl}carbamoyl)propoxy]
phenyl}sulfonyl)amino]-3-[(1-~2-[(1-oxy(2-
pyridyl))amino] ethyl}(1H-indazol-5-
yl))carbonylamino]propanoate
0
r
N,N
NH
N,
O
The product of step D (137 mg, 460 umol) was dissolved
in DMF with the product of Example 34, Step B (312 mg,
460 umol), and HBTU (209 mg, 552 umol) under nitrogen.
Diisopropylethylamine (240 uL, 1.4 mmol) was added and
the reaction was stirred for 50 minutes. The solution
was concentrated and purified by preparative HPLC (Vydac
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C-18, 5 cm x 25 cm, 80mL/min, 90o acetonitrile/water/
0.1% trifluoroacetic acid; 20-55o B over 40 minutes).
The product fractions were combined, frozen, and
lyophilized to afford the product as a white solid (238
mg, 54%). LRMS (EI); 845.3 ([M+H]+~ 100%~, 1690.6
( [2M+H]+, 10%) , 711.3 ( [ (M-Z)+H]+, 30%) .
Step F: Synthesis of (2S)-2-{[(4-{3-[N-(2-aminoethyl)
carbamoyl]propoxy}-2,6-dimethylphenyl)sulfonyl]amino}-3-
({1-j2-(2-3,4,5,6-tetrahydropyridylamino)ethyl](1H-
indazol-5-yl)}carbonylamino)propanoic acid
Into a Parr bottle under nitrogen was placed 100
palladium on carbon (100 mg), followed lay methanol (10
mL). The product of step E (230 mg, 240 umol),
dissolved in methanol (30 mL) was added and the reaction
hydrogenated at 55 psi for 20 hours. Additional
catalyst (50 mg) and trifluoroacetic acid (60 uL) were
added and the hydrogenation continued for 34 hours. The
reaction was filtered through Celite, rinsed, and the
filtrates concentrated to yield 205 mg of an oil, which
still contained some deprotected N-oxide. This oil was
dissolved in water/THF (1:1, 1.5 mL) and 3N lithium
hydroxide solution (720 uL, 2.1 mmol) added. The
solution was stirred for 1 hour, acidified to pH = 2
with trifluoroacetic acid and concentrated. The residue
was purified by preparative HPLC (Vydac C-18, 21.2 mm x
25 cm, 90% acetonitrile/water/ 0.1% trifluoroacetic
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acid; 5-30o B over 50 minutes). The product fractions
were combined, frozen, and lyophilized to afford the
product as a white solid (38 mg, 26%). LRMS (EI);
685.3 ( [M+H]+~ 100%) .
Step G: Synthesis of (2S)-2-{[(4-{3-[N-(2-{(2S)-4-
[(tert-butyl)oxycarbonyl]-2-
[(phenylmethoxy)carbonylamino]butanoy1
amino}ethyl)carbamoyl]propoxy}-2,6-
dimethylphenyl)sulfonyl] amino}-3-({1-[2-(2-3,4,5,6-
tetrahydropyridylamino)ethyl](1H-indazol-5-
yl)}carbonylamino)propanoic acid
The product of Step F (125 mg, 183 umol) is treated as
in Example 40, Step E. The product is obtained as a
white solid after lyophilization.
Step H: Synthesis of (2S)-2-({[4-(3-{N-[2-((2S)-2-
{(2S)-4-[(tert-butyl)oxycarbonyl]-2-
[(phenylmethoxy)carbonylamino] butanoylamino}-4-[(tert-
butyl)oxycarbonyl]butanoylamino)
ethyl]carbamoyl}propoxy)-2,6-
dimethylphenyl]sulfonyl}amino)-3-({1-[2-(2-3,4,5,6-
tetrahydropyridylamino)ethyl](1H-indazol-5-
yl)}carbonylamino)propanoic acid
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The product of Step G is treated as in Example 40, Step
F. The residue is not purified but treated directly as
in Example 40, Step G. The product is obtained as a
white solid after lyophilization.
Step I: Synthesis of tert-butyl (4S)-4-(N-{(1S)-1-[N-
(2-{4-[4-({[(1S)-1-(methoxycarbonyl)-2-({1-[2-(2-
3,4,5,6-tetrahydropyridylamino)ethyl](1H-indazol-5-
yl)}carbonyl amino)ethyl]amino}sulfonyl)-3,5-
dimethylphenoxy]butanoyl amino}ethyl)carbamoyl]-3-
[(tert-butyl)oxycarbonyl]propyl} carbamoyl)-4-[2-
(1,4,7,10-tetraaza-4,7,10-tris{[(tert-butyl)
oxycarbonyl]methyl}cyclododecyl)acetylamino]butanoate
The product of Step H is treated as in Example 40, Step
H. The residue is not purified but coupled directly
with DOTA(OtBu)3-OH as in Example 40, step I. The
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product is obtained as a white solid after
lyophilization.
Step J: Synthesis of (4S)-4-(N-{(1S)-1-[N-(2-{4-[4-
({[(1S)-1-carboxy-2-({1-[2-(2-3,4,5,6-tetrahydropyridyl
amino)ethyl](1H-indazol-5-yl)}carbonylamino)ethyl]amino}
sulfonyl)-3,5-dimethylphenoxy]butanoylamino}ethyl)
carbamoyl]-3-carboxy propyl}carbamoyl)-4-{2-[1,4,7,10-
tetraaza-4,7,10-tris (carboxymethyl)cyclododecyl]
acetylamino}butanoic acid
i
OH
~H
The product of step I is treated as in Example 40, Step
J and the crude product is purified by preparative HPLC
(Zorbax CN, 21.2 mm x 25 cm, 50% acetonitrile/water/
0.1~ formic acid; 20-35o B over 60 minutes). The
product fractions are combined, frozen, and lyophilized
to afford the product as a white solid
Example 43
Synthesis of (2S)-2-{[(2,6-dimethyl-4-{3-[N-(2-{2-
[1,4,7,10-tetraaza-4,7,10-
tris(carboxymethyl)cyclododecyl]acetyl-
amino}ethyl)carbamoyl]propoxy}phenyl)sulfonyl]amino}-3-
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({2-[2-(2-3,4,5,6-tetrahydropyridylamino)ethyl](2-hydro-
1H-indazol-5-yl)}carbonylamino)propanoic acid
O O HO O
N~N '~ ~ NH~OH O~N OH
'~ NH C ~ O
~~ '~OH
\ O
O NH
O~NH
Step A: Synthesis of ethyl 2-(2-aminoethyl)-2-hydro-1H-
indazole-5-carboxylate
0
N
H2N~ , ,
N
The slower eluting product of Example 42, Step A (600
mg, 1.65 mmol) was treated as in Example 42, Step B to
afford the product as a single pure compound (164 mg,
43~) . LRMS (ES) : 234.2 ( [M ~- H]t, 100%) .
Step B: Synthesis of ethyl 2-{2-[(1-hydroxy-2-pyridyl)
amino]ethyl}-2-hydro-1H-indazole-5-carboxylate
/ \
N O
O NH-~ , %
N
N
The product of step A (249 mg, 1.07 mmol) was treated as
in Example 42, Step C to afford the product in 80a
purity after flash chromatography. This was purified by
preparative HPLC (ZTydac C18, 21.2 mm x 25 cm, 900
acetonitrile/0.1%TFA; 10-55% B over 25 minutes). The
product fractions were frozen and lyophilized to afford
the desired product as a white powder (204 mg, 430).
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LRMS (ES) : 327.2 ( [M + H]+, 100%) , 653 .3 ( [2M + H]+,
40%) .
Step C: Synthesis of 2-{2-[(1-hydroxy-2-pyridyl)amino]
ethyl}-2-hydro-1H-indazole-5-carboxylic acid
/ \
N O
O NH~N, % W OH
N
The product of step B (202 mg, 461 umol) was treated as
in Example 42, Step D to afford the product as a white
powder after filtration (138 mg, 100%). LRMS (ES):
299.2 ( [M + H]+, 100%) .
Step D: Synthesis of methyl (2S)-2-[({2,6-dimethyl-4-
[3- (N.-{2-
[(phenylmethoxy)carbonylamino]ethyl}carbamoyl)propoxy]
phenyl}sulfonyl)amino]-3-[(2-{2-[(1-oxy(2-
pyridyl))amino] ethyl}(2-hydro-1H-indazol-5-
yl))earbonylamino] propanoate
0 0
N
O NH~ ~ w NH~O
N' ' s NH
N I
O=S=O
O ~N~O
~~NH O//
O
The product of step C (35mg, 116 umol) was treated as in
Example 42, Step E to afford the product as a white
powder after lyophilization (64 mg, 58%). LRMS (ES):
845.3 ( [M + H]+, 100%) . HRMS: Calculated for CglH~9N801oS
845.3292; Found . 845.3264.
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Step E: Synthesis of methyl (2S)-2-{[(4-{3-[N-(2-
aminoethyl)carbamoyl]propoxy}-2,6-dimethylphenyl)
sulfonyl]amino}-3-({2-[2-(2-3,4,5,6-tetrahydropyridyl
amino)ethyl](2-hydro-1H-indazol-5-yl)}carbonylamino)
propanoate
The product of step D (25mg, 26 umol) was treated as in
Example 42, Step F to afford the product as a white
powder after lyophilization (11 mg, 52%). LRMS (ES):
685.3 ( [M + H]~, 100%) .
Step F: Synthesis of methyl (2S)-2-[({2,6-dimethyl-4-
[3-(N-{2-[2-(1,4,7,10-tetraaza-4,7,10-tris{[(tert-
butyl)oxy-
carbonyl]methyl}cyclododecyl)acetylamino]ethyl}carbamoy1
propoxy]phenyl}sulfonyl)amino]-3-({2-[2-(2-3,4,5,6-
tetrahydro pyridylamino)ethyl](2-hydro-1H-indazol-5-
yl)}carbonylamino) propanoate
0 0 0 0
N~ ~ w NH~OH o~ N O
N' ' / NH C ~ O
o'S=o NV ,~C
0 0
o
O~N
The product of Step E (11 mg, 15 umol) is reacted with
DOTA(OtBu)3-OH (10 mg, 17 umol) as in Example 37, Step
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F, to afford the product as a pure compound after
purification and lyophilization.
Step G: Synthesis of (2S)-2-{[(2,6-dimethyl-4-{3-[N-(2-
{2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclo-
dodecyl]acetylamino}ethyl)carbamoyl]propoxy}phenyl)sulfo
nyl]amino}-3-({2-[2-(2-3,4,5,6-
tetrahydropyridylamino)ethyl](2-hydro-1H-indazol-5-
yl)}carbonylamino)propanoic acid
i
The product of Step F (12 mg, 9.7 umol) is deprotected
as in Example 40, Step J, to afford the product as a
pure compound after preparative HPLC purification and
lyophilization of the product fractions.
Example 44
Synthesis of (4S)-4-{N-[(1S)-1-(N-{2-[({4-[4-({[(1S)-1-
carboxy-2-({1-[2-(2-3,4,5,6-
tetrahydropyridylamino)ethyl] (1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)phenyl]
phenyl}sulfonyl)amino]ethyl}carbamoyl)-3-carboxypropyl]
carbamoyl}-4-{2-[1,4,7,10-tetraaza-4,7,10-tris(carboxy-
methyl)cyclododecyl]acetylamino}butanoic acid
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NH
O~S
O
0
Step A: Synthesis of methyl (2S)-3-amino-2-{[(4-{4-
[(f2_
[(phenylmethoxy)carbonylamino]ethyl}amino)sulfonyl]pheny
1}phenyl)sulfanyl]amino}propanoate
Biphenyl-4,4'-disulfonyl chloride (5.3 g, 15 mmol) was
reacted with N-(2-aminoethyl)(phenylmethoxy)carboxamide
(2.3 g, 10 mmol) and methyl (2S)-2-amino-3-[(tert-
butoxy)carbonyl amino]propanoate (5.1 g, 20 mmol)
sequentially, in the same fashion as Step 1B, to afford
10.2 grams of the crude Boc-protected product after
concentration of the organic extracts. This was
directly dissolved in dichloromethane (100 mL) and
trifluoroacetic acid (100 mL) added under nitrogen. The
solution was stirred for 1 hour, concentrated and
dissolved in acetonitrile. Addition of 0.10
trifluoroacetic acid resulted in a solid precipitate,
which was filtered, and the filtrate was then purified
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by preparative HPLC (Vydac C-18, 5.5 cm x 25 cm, 90%
acetonitrile/water/ 0.1o trifluoroacetic acid, 80
mL/minute 0-70°s B over 40 minutes). The product
fractions were combined, frozen, and lyophilized to
afford the product as a white solid (3.6 g, 50% for two
steps). LRMS (ES): 591.1 ([M + H]+, 100%). 1HNMR
(600.1343 MHz, DMSO-d6): 8.1 (m, 3H), 7.97 (m, 4H),
7.91 (m, 4H), 7.83 (t, 1H), 7.30 (m, 5H), 4.98, (s, 2H),
4.25 (m, 1H), 3.35 (s, 3H), 3.15 (dd, 1H), 3.06 (m, 2H),
2.95 (dd, 1H), 2.83 (m, 2H).
Step B: Synthesis of methyl (2S)-3-[(1-{2-[(1-oxy(2-
pyridyl))amino]ethyl}(1H-indazol-5-yl))carbonylamino]-2-
{[(4-{4-[({2-[(phenylmethoxy)carbonylamino]ethyl}amino)
sulfonyl]phenyl}phenyl)sulfonyl]amino}propanoate
The product of Example 42, Step D (46 mg, 154 umol) was
dissolved in dry DMF (2.5 mL) with the product of Step A
(114 mg, 162 umol), HBTU (76 mg, 200 umol), and
diisopropylethylamine (81 uL, 462 umol). The reaction
was stirred for 1 hour, concentrated and the residue
purified by preparative HPLC (Zorbax C-8, 21.2 mm x 25
cm, 90% acetonitrile/water/ 0.1% trifluoroacetic acid;
20-60% B over 40 minutes). The product fractions were
combined, frozen, and lyophilized to afford the product
as a white solid (102 mg, 67%). LRMS (ES): 871.3 ([M +
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H]+, 100%) . HRMS: Calculated for C91H43N$OsoSz . 871.2544;
Found . 871.2540.
Step C: Synthesis of methyl (2S)-2-({[4-(4-{[(2-amino
ethyl)amino]sulfonyl}phenyl)phenyl]sulfonyl}amino)-3-
({1-[2-(2-3,4,5,6-tetrahydropyridylamino)ethyl](1H-
indazol-5-yl)}carbonylamino)propanoate
The product of Step B (75 mg, 76 umol) was treated as in
Example 41, Step B and purified by preparative HPLC
(Zorbax C-8, 21.2 mm x 25 cm, 90% acetonitrilejwaterl
0.1% trifluoroacetic acid; 15 - 25% B over 40 minutes).
The product fractions were combined, frozen, and
lyophilized to afford the product as a white solid (56
mg, 86%) . LRMS (ES) : 725.2 ( [M + H]+, 200) , 363.2 ( [M
+ 2H]+z, 100%) .
Step D: Synthesis of tert-butyl (4S)-4-(N-{2-[({4-[4-
({[(1S)-1-(methoxycarbonyl)-2-({1-[2-(2-3,4,5,6-
tetrahydro pyridylamino)ethyl](1H-indazol-5-
yl)}carbonylamino)ethyl]
amino}sulfonyl)phenyl]phenyl}sulfonyl)amino]ethyl}carbam
oyl)-4-[(phenylmethoxy)carbonylamino]butanoate
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O~S
O
y
0
The product of Step C was reacted as in Example 40, Step
E to afford the product as a white solid after
lyophilization. LRMS
Step E: Synthesis of tart-butyl (4S)-4-{(2S)-4-[(tert-
butyl)oxycarbonyl]-2-
[(phenylmethoxy)carbonylamino]butanoyl amino}-4-(N-{2-
[({4-[4-({[(1S)-1-(methoxycarbonyl)-2-({1-[2-(2-3,4,5,6-
tetrahydropyridylamino)ethyl](1H-indazol-5-yl)}
carbonylamino)ethyl]amino}sulfonyl)phenyl]phenyl}sulfony
1)amino]ethyl}carbamoyl)butanoate
0 0
Ni I ~ NH~'~'O~
i NH O
O~S
O ~ O O
~NH \ ~ ~NH
'O
\ /,S;0 ~NH~~O~
O~ NH O v ~O
The product of Step D is treated as in Example 40, Step
F to afford the product as a white solid after
lyophilization.
Step F: Synthesis of tart-butyl {4S)-4-{N-[(1S)-1-{N-
{2-[({4-[4-({[(1S)-1-(methoxycarbonyl)-2-({1-[2-(2-
3,4,5,6-tetrahydropyridylamino)ethyl](1H-indazol-5-
yl)}carbonyl amino)ethyl]amino}sulfonyl)phenyl]phenyl}
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sulfonyl)amino] ethyl}carbamoyl)-3-[(tert-
butyl)oxycarbonyl]propyl] carbamoyl}-4-[2-(1,4,7,10-
tetraaza-4,7,10-tris{[(tert-
butyl)oxycarbonyl]methyl}cyclododecyl)acetylamino]butano
ate
0
o~
NH
~=S
O
\ /
\ / .C
,S,
o n
The product of Step E is treated as in Example 40, step
G to afford the product as a white solid after
lyophilization.
Step G: Synthesis of (4S)-4-{N-[(1S)-1-(N-{2-[({4-[4-
({[(1S)-1-carboxy-2-({1-[2-(2-3,4,5,6-tetrahydropyridyl
amino)ethyl](1H-indazol-5-yl)}carbonylamino)ethyl]amino}
sulfonyl)phenyl]phenyl}sulfonyl)amino]ethyl}carbamoyl)-
3-carboxypropyl]carbamoyl}-4-{2-[1,4,7,10-tetraaza-
4,7,10-
tris(carboxymethyl)cyclododecyl]acetylamino}butanoic
acid
~=s
o
\ /
\ /
c
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The product of Step F is treated as in Example 40, Step
G to afford the product as a white solid after
lyophilization.
Example 45
Synthesis of (4S) -4-{N- [ (1S) -1- (N-{2- [ ( {4- [4- ( { [ (1S) -1-
carboxy-2-({1-[3-(3,4,5,6-tetrahydropyrimidin-2-ylamino)
propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)
phenyl]phenyl}sulfonyl)amino]ethyl}carbamoyl)-3-carboxy
propyl]carbamoyl}-4-{2-[1,4,7,10-tetraaza-4,7,10-tris
(carboxymethyl)cyclododecyl]acetylamino}butanoic acid
0
~OH
NH
)~S
O
S;
O
Step A: Synthesis of methyl (2S)-2-{[(4-{4-[({2-
[(phenylmethoxy)carbonylamino]ethyl}amino)sulfonyl]pheny
1}phenyl)sulfonyl]amino}-3-({1-[3-(pyrimidin-2-
ylamino)propyl] (1H-indazol-5-
yl)}carbonylamino)propanoate
s
/=O
H
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The product of Example 40, Step B (58 mg, 195 umol) was
reacted with the product of Example 44, Step A (144 mg,
205 umol) as in Example 44, Step B and the residue
purified by preparative HPLC (Vydac C-18, 21.2 mm x 25
cm, 90% acetonitrile/water/ 0.1% trifluoroacetic acid;
10-70% B over 30 minutes). The product fractions were
combined, frozen, and lyophilized to afford the product
as a white solid (102 mg, 54%). LRMS (ES): 870.3 ([M +
H]+, 100%). 1HNMR (600.1343 MHz, DMSO-d6): 8.52 (d,
1H), 8.51 (s, 1H), 8.29 (d, 1H), 8.17, (d, 1H), 7.82 (m,
9H), 7.74 (d, 1H), 7.64 (d, 1H), 7.49 (b, 1H), 7.31, (m,
6H), 6.61 (t, 1H), 4.98, (s, 2H), 4.46 (t, 2H), 4.19
(dd, 1H), 3.55 (m, 1H), 3.42 (m, 1H), 3.41 (s, 3H), 3.26
(t, 2H), 3.06 (t, 2H), 2.83 (m, 2H), 2.09 (m, 2H).
Step B: Synthesis of tert-butyl (4S)-4-(N-{2-[({4-[4-
({[(1S)-1-(methoxycarbonyl)-2-({1-[3-(3,4,5,6-tetrahydro
pyrimidin-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)
ethyl]amino}sulfonyl)phenyl]phenyl}sulfonyl)amino]ethyl}
carbamoyl)-4-[(phenylmethoxy)carbonylamino]butanoate
s
NH NH O
O~O
II \O
The product of Step A (100 mg, 102 umol) was treated as
in Example 41, Step B and the resulting solid (79 mg)
directly reacted as in Example 40, Step E to afford the
crude product as an oil, which was purified by
preparative HPLC (Vydac C-18, 21.2 mm x 25 cm, 90%
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acetonitrile/water/ 0.1% trifluoroacetic acid; 10-70% B
over 40 minutes). The product fractions were combined,
frozen, and lyophilized to afford the product as a white
solid (98 mg, 80%). LRMS (ES): 1059.3 ([M + H]+,
100%).
Step C: Synthesis of tart-butyl (4S)-4-{(2S)-4-[(tart-
butyl)oxycarbonyl]-2-
[(phenylmethoxy)carbonylamino]butanoyl amino}-4-(N-{2-
[({4-[4-({[(1S)-1-(methoxycarbonyl)-2-({1-[3-(3,4,5,6-
tetrahydropyrimidin-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}sulfonyl)phenyl]phenyl}sul
fonyl)amino]ethyl}carbamoyl)butanoate
Nr I W O NH~O- I i
NH
O'S
O
NH ~ !
N
~N ~ !
c
The product of Step B (96 mg, 82 umol) was treated as in
Example 41, Step D to afford the product as a white
solid (48 mg, 42%) after lyophilization. LRMS (ES):
1244.4 ( [M + H]+, 1000) , 566.8 ( [ (M-2tBu) + 2H]+2, 45%) .
Step D: Synthesis of tart-butyl (4S)-4-{N-[(1S)-1-(N-
{2-[({4-[4-({[(1S)-1-(methoxycarbonyl)-2-({1-[3-
(3,4,5,6-tetrahydropyrimidin-2-ylamino)propyl](1H-
indazol-5-yl)}
carbonylamino)ethyl]amino}sulfonyl)phenyl]phenyl}sulfony
1)amino]ethyl}carbamoyl)-3-[(tert-
butyl)oxycarbonyl]propyl] carbamoyl}-4-[2-(1,4,7,10-
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tetraa~a-4,7,10-tris{[(tert-butyl)
oxycarbonyl]methyl}cyclododecyl)acetylamino]butanoate
0
J1 /,
The product of Step C (47 mg, 35 umol) was treated as in
Example 41, Step E to afford the product as a white
solid (36 mg, 62%) after lyophilization. LRMS (ES):
1664. 6 ( [M + H]+, 5 %) , 833 .2 ( [ (M + 2H]+2, 60 0) , 518.4
( [ (M-2tBu) + 3H]+3, 100%) .
Step E: Synthesis of (4S)-4-{N-[(1S)-1-(N-{2-[({4-[4-
({[(1S)-1-carboxy-2-({1-[3-(3,4,5,6-tetrahydropyrimidin-
2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)ethyl]amino}
sulfonyl)phenyl]phenyl}sulfonyl)amino]ethyl}carbamoyl)-
3-carboxypropyl]carbamoyl}-4-{2-[1,4,7,10-tetraaza-
4,7,10-
tris(carboxymethyl)cyclododecyl]acetylamino}butanoic
acid
OyS
O
NH
N
~N ~ J
C
0 0
Ni I ~ NH~OH
'~ NH
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The product of Step D (29 mg, 15 umol) was treated as in
Example 40, Step J to afford the crude product which was
purified by preparative HPLC (Vydac C-18, 21.2 mm x 25
cm, 90% acetonitrile/water/ 0.1% trifluoroacetic acid;
5-35% B over 35 minutes). The product fractions were
combined, frozen, and lyophilized to afford the product
as a white solid (11 mg, 46%) after lyophilization.
LRMS (ES) : 1370.4 ( [M + H]+, 10 %) , 685.8 ( [ (M + 2H]+2,
90%) , 457 . 6 ( [M + 3H]+3, 100%) .
Example 46
Synthesis of (2S)-3-({3-[(imidazol-2-ylamino) methyl]-1-
methyl(1H-indazol-6-yl)}carbonylamino)-2-({[4-(4-{[(2-
{2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)
cyclododecyl]acetylamino}ethyl)amino]sulfonyl}phenyl)phe
nyl]sulfonyl}amino)propanoic acid
0 0
HON ~OH
H CNV '~
~O OH
,S O /NH
O N JH
Step A: Synthesis of methyl 3-formyl-1-methyl-1H-
indazole-6-carboxylate
0
NN I ~ Oi
i
O
In a dry flask under nitrogen is added dry DMF (1.6 mL)
and the solution cooled to -5C in an ice/ethanol bath.
Phosphorous oxychloride (530 uL, 5.7 mmol) is added via
syringe and the solution is allowed to stir for 30
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minutes in the bath. Methyl 1-methyl-1H-indazole-6-
carboxylate, (500 mg, 2.84 mmol, prepared as in Jadhav
et al, US patent 5,760,028) dissolved in DMF (3 mL) is
added slowly to the cold solution. The reaction is then
warmed to 35C, stirred for four hours, and then poured
onto crushed ice. The resulting slurry is neutralized
with 1N NaOH to pH = 7, heated rapidly to boiling for 1
minute, cooled quickly to room temperature, and the
solution extracted with ethyl acetate. The combined
organics are washed with water and brine, dried,
filtered and concentrated. The resulting oil is
purified by flash chromatography to afford the product.
Step B: Synthesis of methyl 1-methyl-3-({[1-(triphenyl
methyl)imidazol-2-yl]amino}methyl)-1H-indazole-6-
carboxylate
The product of Step A (204 mg, 1 mmol) is dissolved in
toluene (10 mL) with N-trityl-2-aminoimidazole (357 mg,
1.1 mmol) and heated to reflux with a Dean-Stark trap.
Four aliquots of toluene (3 mL each) are removed via
distillation at 1.5 hour intervals, being replaced by
dry toluene each time, and then the solution is left to
reflux for 18 hours. The reaction is cooled to room
temperature and sodium triacetoxyborohydride (1 gram, 5
mmol) is added in one portion. The reaction is stirred
at room temperature for 24 hours, poured into water, and
the layers separated. The aqueous layer is extracted
with ethyl acetate and the combined organic layers are
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washed with saturated bicarbonate, water, and brine,
dried over magnesium sulfate, concentrated, and purified
by flash chromatography to afford the product.
Step C: Synthesis of 1-({[1-(triphenylmethyl)imidazol-
2-yl]amino}methyl)-1H-indazole-5-carboxylic acid
The product of Step B (250 mg, 474 umol) is added to
THF/water (1:1, 15 mL) along with lithium hydroxide (3N,
0.8 mL, 2.4 mmol) and the solution stirred, following by
TLC until the .starting material has disappeared, when
the THF is removed under vacuum. The reaction is
acidified to pH = 2 with 1N HCl and the resulting solids
are filtered, washed with water, and dried under vacuum
to afford the product.
Step D: Synthesis of
0 0
N ~ NH~O~
NH
O~S
N
N'~ v
' ~N / _ oho
.O ~NH
eS;
0 NH
The product of Step C (190 mg, 370 umol) is reacted with
the product of Example 44, Step A (285 mg, 407 umol) as
in Example 44, Step B and the residue is purified by
preparative HPLC (Vydac C-18, 21.2 mm x 25 cm, 90%
acetonitrileJwater/ 0.1o trifluoroacetic acid; 10-70~ B
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over 30 minutes). The product fractions are combined,
frozen, and lyophilized to afford the product.
Step E: Synthesis of methyl (2S)-2-({[4-(4-{[(2-amino
ethyl)amino]sulfonyl}phenyl)phenyl]sulfonyl}amino)-3-
{[1-methyl-3-({[1-(triphenylmethyl)imidazol-2-
yl]amino}methyl) (1H-indazol-6-
yl)]carbonylamino}propanoate
The product of Step D (100 mg) in methanol (10 mL) is
added to a slurry of 10% palladium on carbon (50 mg) in
methanol (8 mL) in a Parr bottle under nitrogen. The
solution is hydrogenated at 50 psi for 1.5 hours,
filtered through Celite, washed with methanol, and the
combined filtrates concentrated. The resulting oil is
not purified but carried directly into the next step.
Step F: Synthesis of methyl (2S)-3-{[1-methyl-3-({[1-
(triphenylmethyl)imidazol-2-yl]amino}methyl)(1H-indazol-
6-yl)]carbonylamino}-2-{[(4-{4-[({2-[2-(1,4,7,10-
tetraaza-4,7,10-tris{[(tert-
butyl)oxycarbonyl]methyl}cyclododecyl)
acetylamino]ethyl}amino)sulfonyl]phenyl}phenyl)sulfonyl]
amino}propanoate
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O O
N ~ NH~O~ O
a
N\ I ~ O;SH ~O~N ~O
N o _ ~ o
N~N \ ~ ~N~% '~
\ ~ ,O /NH
,,S,
O NH
The product of Step E (73 mg, 77 umol) is reacted with
DOTA{OtBu)3-OH (49 mg, 85 umol) as in Example 37, Step
F, to afford the product as a pure compound after
purification and lyophilization.
Step G: Synthesis of (2S)-3-({3-[(imidazol-2-ylamino)
methyl]-1-methyl(1H-indazol-6-yl)}carbonylamino)-2-({[4-
{4-~[{2-~2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)
cyclododecyl]acetylamino}ethyl)amino]sulfonyl}phenyl)phe
nyl]sulfonyl}amino)propanoic acid
NH
p;S F
O
\ /.S,
O
The product of Step F (73 mg, 77 umol) is deprotected as
in Example 40, Step J, to afford the product as a pure
compound after preparative HPLC purification and
lyophilization of the product fractions.
Example 47
Synthesis of 3-[(7-{3-[(6-{[(1E)-1-aza-2-(2-
sul f ophenyl ) vinyl ] amino } ( 3 -
pyridyl))carbonylamino]propoxy}-1-[3-(imidazol-2-
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ylamino)propyl](1H-indazol-5-yl))-carbonylamino](2S)-2-
{[(2,4,6-trimethylphenyl)sulfonyl]-amino}propanoic acid
0
Nr I \ 'H
~N
O
HN
HN
~N HN O
N~ I
N,NH
I
I
S03H
~/COOH
NTH
I
02S
Part A. Preparation of ethyl 7-{3-[(tert-butoxy)-
carbonylamino]propoxy}-1-benzyl-1H-indazole-5-
carboxylate
N ~ I ~ COOC2H5
~N
PhJ p
NH-BOC
A solution of ethyl 7-hydroxy-1-benzyl-1H-indazole-5-
carboxylate (P. Baraldi et. al, Il. Farmaco, 52(12), 717
(1997)) in ethanol is treated with sodium ethoxide,
followed by commercially available boc-3-
aminopropylbromide, and refluxed for 2-5 hours. The
volatiles are removed and the crude residue is extracted
with ethyl acetate. The crude residue is obtained after
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removal of ethyl acetate and is purified by
chromatography to give the title compound.
Part B. Preparation of 7-{3-[(tert-
butoxy)carbonylamino]-propoxy}-1-(3-{[1-
(triphenylmethyl)imidazol-2-yl]amino}-propyl)-1H-
indazole-5-carboxylic acid
Try
I
Ethyl 7-{3-[(tent-butoxy)carbonylamino]propoxy}-1-
benzyl-1H-indazole-5-carboxylate is subjected to
hydrogenolysis to give the debenzylated derivative.
Using the procedure described in U.S. Patent 5,760,028,
Example 1050e, parts D, E, J, and K ethyl 7-{3-[(tert-
butoxy)carbonylamino]propoxy}-1H-indazole-5-carboxylate
is converted to the title compound in four steps.
Part C. Preparation of (2S)-3-({7-(3-aminopropoxy)-1-
[3-(imidazol-2-ylamino)propyl](1H-indazol-5-
yl)}carbonylamino)-2-{[(2,4,6-
trimethylphenyl)sulfonyl]amino}propanoic acid
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O
N ~ ~ N~ /COON
'N I / H ~N H
O 02S
HN ' /
HN
~N NH2
A solution of 7-{3-[(tert-butoxy)carbonylamino]-
propoxy}-1-(3-{[1-(triphenylmethyl)imidazol-2-yl]amino}-
propyl)-1H-indazole-5-carboxylic acid is treated with
Hunig's base and HBTU, then is stirred for about 10 min.
The reaction mixture is treated with methyl 3-amino-
2(S)-(2,4,6-trimethyl-benzenesulfonyl)aminopropionate.
The product is then isolated via chromatography. The
methyl ester is saponified using LiOH in THF, and the
trityl and boc protecting groups are removed by
treatment with trifluoroacetic acid. to give the title
compound. It is purified by reversed phase preparative
HPLC.
Part D. Preparation of 3-[(7-{3-[(6-{[(1E)-1-aza-2-(2-
sul f ophenyl ) vinyl ] amino } ( 3 -
pyridyl))carbonylamino]propoxy}-1-[3-(imidazol-2-
ylamino)propyl](1H-indazol-5-yl))carbonyl-amino](2S)-2-
{[(2,4,6-trimethylphenyl)sulfonyl]amino}-propanoic acid
(2S)-3-({7-(3-Aminopropoxy)-1-[3-(imidazol-2-ylamino)-
propyl](1H-indazol-5-yl)}carbonylamino)-2-{[(2,4,6-
trimethylphenyl)sulfonyl]amino}propanoic acid is
dissolved in N,N-dimethylformamide. Triethylamine (3
eq)is added, and the reaction is stirred for 5 min. 2-
[[[5-[[(2,5-Dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-
pyridinyl]hydrazono]-methyl]-benzenesulfonic acid,
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monosodium salt (1.1 eq.) is added, and the reaction is
stirred overnight. The reaction mixture is concentrated
under high vacuum and the crude is purified by reversed
phase preparative HPLC to give the title compound.
Example 48
Synthesis of 3-{[1-[3-(imidazol-2-ylamino)propyl]-7-(3
{2-[1,4,7,10-tetraaza-4,7,10
tris(carboxymethyl)cyclododecyl]
acetylamino}propoxy)(1H-in.dazol-5-yl)]carbonylamino}-2-
{[(2,4,6-trimethylphenyl)sulfonyl]amino}propanoic acid
N~ 'COOH
H TNH
I
O2S
NJ
Hooc
C ~ COOH
N~
HOOCJ
To a solution of tris(t-butyl)-1,4,7,10-tetra-
azacyclododecane-1,4,7,10-tetraacetic acid (1 eq) and
Hunig's base (3 eq.) in DMF is added HBTU (0.8 eq) and
the mixture is stirred for 5 min. To this is added a
solution of (2S)-3-({7-(3-Aminopropoxy)-1-[3-(imidazol-
2-ylamino)propyl](1H-indazol-5-yl)}carbonylamino)-2-
{[(2,4,6-trimethylphenyl)sulfonyl]-amino}propanoic acid
(0.75 eq) in DMF and the reaction mixture is allowed to
stir under nitrogen at room temperature for 4 h. The
solvent is removed in vacuo and the residue is purified
by preparative RP-HPLC to obtain the conjugate. A
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solution of the conjugate in TFA is stirred at room
temperature under nitrogen for 5 h. The solution is
concentrated in vacuo and the residue is purified by
preparative RP-HPLC to obtain the title compound as a
lyophilized solid.
Example 49 - 55
Synthesis of In-111 Complex of the Conjugate of
Example 34
To a lead shielded and crimped 1 cc autosampler
vial was added 40-50 ~,g of the conjugate of Example 34
dissolved in 100 ~.L ammonium citrate buffer (0.4 M, pH
4.7) followed by the addition of 2 mCi, (5 ~.L) In-111 in
0.05 N HCl (specific activity: 25 ~,g/mCi). The reaction
mixture was heated at 90-100 °C for 30 min and analyzed
by HPLC. Yield: 97.3%; Ret. Time: 8.1 min.
HPLC Method
Column: Zorbax Rx C18, 25 cm x 4.6 mm
Column Temperature: Ambient
Flow: 1.0 ml/min
Solvent A: 25 mM sodium phosphate buffer at pH~6
Solvent B: Acetonitrile
Detector: Sodium iodide (NaI) radiometric probe, and UV
at 220 nm wavelength.
Gradient
t (min) 0 25 26 35 36 45
%B 10 20 60 60 10 10
Example 50
Synthesis of In-111 Complex of the Conjugate of Example
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To a lead shielded and crimped 2 cc autosampler
vial was added 100 ~,g of the conjugate of Example 35
dissolved in 200 ~.L ammonium citrate buffer (0.4 M, pH
4.8) followed by the addition of 2.5 mCi, (7.5 ~,L) In-
111 (NEN) in 0.05 N HCl (specific activity: 40 ~,g/mCi).
The reaction mixture was heated at 100 °C for 30 min and
analyzed by HPLC. Yield: 74.7%, Ret. Time: 13.2 min.
HPLC Method
Column: Zorbax Rx C18, 25 cm x 4.6 mm
Column Temperature: Ambient
Flow: 1.0 ml/min
Solvent A: 25 mM sodium phosphate buffer at pH 6
Solvent B: Acetonitrile
Detector: Sodium iodide (NaI) radiometric probe, and UV
at 220 nm wavelength.
Gradient
t (min) 0 25 26 35 36 45
%B 14 16 60 60 14 14
Example 51
Synthesis of In-111 Complex of the Conjugate of Example
36
To a lead shielded and crimped 2 cc autosampler
vial was added 70 ~,g of the conjugate of Example 36
dissolved in 140 ~..~,L ammonium acetate buffer ( 0 . 5 M, pH
4.7) followed by the addition of 1 mg of gentisic acid
(sodium salt) dissolved in 10 ~.l,L of HzQ, and 1.7 mCi, (9
~.I,L) In-111 (NEN) in 0.05 N HC1 (specific activity: 41
~l,glmCi). The reaction mixture was heated at 100 °C for
20 min and analyzed by HPLC. Yield: 87 %, Ret. Time: 17-
18 min.
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HPLC Method
Column: Zorbax Rx C18, 25 cm x 4.6 mm
Column Temperature: Ambient
Flow: 1.0 ml/min
Solvent A: 10 mM ammonium acetate
Solvent B: Acetonitrile
Detector: IN-US (3-ram, and UV at 220 nm wavelength.
Gradient
t (min) 0 25 26 35 36 45
%B 7 7 60 60 7 7
Example 52
Synthesis of In-111 Complex of the Conjugate of Example
37
To a shielded and crimped 2 cc autosampler vial was
added 40-60 E.t,g of the conjugate of Example 37 dissolved
in 80-120 ~,l 0.5 M ammonium acetate buffer (pH 4.8)
followed by the addition of 1 mg gentisic acid sodium
salt and 1-1.3 mCi (6 ~,1) In-111 in 0.05M HCl. The
reaction mixture was heated at 100°C for 15 minutes and
analyzed by HPLC. yield: 75.3%; Ret. Time: 16.8 min.
HPLC Method
Column: Zorbax Rx C18, 25 cm x 4.6 mm
Column Temperature: Ambient
Flow: 1.0 ml/min
Solvent A: 10 mM ammonium acetate
Solvent B: Acetonitrile
Detector: IN-US (3-ram, and UV at 220 nm wavelength.
Gradient
t (min) 0 25 26 35 36 45
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%B 10 13 60 60 10 10
Example 53
Synthesis of In-111 Complex of the Conjugate of Example
38
To a lead shielded and crimped 1 cc autosampler
vial was added 40-50 ~l,g of the conjugate of Example 38
dissolved in 100 )..lL ammonium citrate buffer ( 0 . 4 M, pH
4.7) followed by the addition of 2 mCi, (5 ~.~.L) In-111 in
0.05 N HCl (specific activity: 25 ~,g/mCi). The reaction
mixture was heated at 90-100 °C for 30 min and analyzed
by HPLC. Each of the two diasteromers of the conjugate
of Example 38 forms an In-111 complex. Yield: 92.5 and
95.6%; Ret. Time: 13 and 14.7 min.
HPLC Method
Column: Zorbax Rx C18, 25 cm x 4.6 mm
Column Temperature: Ambient
Flow: 1.0 ml/min
Solvent A: 25 mM sodium phosphate buffer at pH b
Solvent B: Acetonitrile
Detector: Sodium iodide (NaI) radiometric probe, and UV
at 220 nm wavelength.
Gradient
t (min) 0 25 26 35 36 45
%B 9 9 60 60 9 9
Example 54
Synthesis of In-111 Complex of the Conjugate of Example
4 0
To a shielded and crimped 2 cc autosampler vial was
added 40-60 ~.g of the conjugate of Example 40 dissolved
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in 80-120 ~.l 0.5 M ammonium acetate buffer (pH 4.8)
followed by the addition of 1 mg gentisic acid sodium
salt and 1-1.3 mCi (6 ~,1) In-111 in 0.05M HCl. The
reaction mixture was heated at 100°C for 15 minutes and
analyzed by HPLC. Yield: 820; Ret. Time: 11 min.
HPLC Method
Column: Zorbax Rx C18, 25 cm x 4.6 mm
Column Temperature: Ambient
Flow: 1.0 ml/min
Solvent A: 10 mM ammonium acetate
Solvent B: Acetonitrile
Detector: IN-US (3-ram, and UV at 220 nm wavelength.
Gradient
t (min) 0 25 26 35 36 45
%B 9 10 60 60 9 9
Example 55
Synthesis of In-111 Complex of the Conjugate of Example
41
To a shielded and crimped 2 cc autosampler vial was
added 40-60 ~.l,g of the conjugate of Example 41 dissolved
in 80-120 ~,l 0.5 M ammonium acetate buffer (pH 4.8)
followed by the addition of 1 mg gentisic acid sodium
salt and 1-1 . 3 mCi ( 6 ~.l.l ) In-111 in 0 . 05M HCl . The
reaction mixture was heated at 100°C for 15 minutes and
analyzed by HPLC. Yield: 71.2%; Ret. Time: 12.2 min.
HPLC Method
Column: Zorbax Rx C18, 25 cm x 4.6 mm
Column Temperature: Ambient
Flow: 1.0 ml/min
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Solvent A: 10 mM ammonium acetate
Solvent B: Acetonitrile
Detector: IN-US (3-ram, and UV at 220 nm wavelength.
Gradient
t (min) 0 25 26 35 36 45
%B 10 10 60 60 10 10
Examples 56 - 58
Synthesis of Y-90 Complexes of the Conjugates of
Examples 34, 36, and 38
To a clean sealed 5 mL vial was added 0.5 -1.0 mL
of the appropriate conjugate solution (200 ~~.g/mL in 0.5
M ammonium acetate buffer, pH 7.0-8.0), followed by 0.05
mL of sodium gentisate (10 mg/mL in 0.5 M ammonium
acetate buffer, pH 7.0-8.0) solution, and 10 - 40 uL of.
9°YCl in 0.05 N HC1. The reaction mixture was heated at
3
100 °C for 5-10 min. After cooling to room temperature,
a sample of the resulting solution was analyzed by HPLC
and by ITLC.
Complex Conjugate Ret. Time % Yield HPLC
Ex # Ex. # (min) Method
56 34 14.0 90 C
57 36 15.5 70 E
58~ 38 9.5, 10.0 89, 68 B
* Example 5t5 is a mixture of two aiasteromers
HPLC Method B: The HPLC method using a reverse phase
C18 Zorbax column(4.6 mm x 25 cm, 80 A pore size) at a
flow rate of 1.0 mL/min with a gradient mobile phase
from 90% A (25 mM pH 6.0 phosphate buffer) and 10% B
(acetonitrile) to 80o A and 20% B at 20 min.
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HPLC Method C: The HPLC method using a reverse phase
C1g Zorbax column (4.6 mm x 25 cm, 80 .A pore size) at a
flow rate of 1.0 mL/min with a gradient mobile phase
from 92% A (25 mM pH 6.0 phosphate buffer) and 8% B
(acetonitrile) to 85o A and 15o B at 20 min.
HPLC Method E: The HPLC method using a reverse phase
C1g Zorbax column (4.6 mm x 25 cm, 80 A pore size) at a
flow rate of 1.0 mLlmin with a gradient mobile phase
from 90o A (25 mM ammonium acetate buffer, pH = 6.8) and
10% B (acetonitrile) to 850 A and 15% B at 20 min.
Utility
The pharmaceuticals of the present invention are
useful for imaging angiogenic tumor vasculature,
therapeutic cardiovascular angiogenesis, and cardiac
pathologies associated with the expression of
vitronectin receptors in a patient or for treating
cancer in a patient. The radiopharmaceuticals of the
present invention comprised of a gamma ray or positron
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, as well as atherosclerotic plaque, myocardial
reperfusion injury, and myocardial ischemia, stunning or
infarction. 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
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administration of the radiopharmaceuticals resulting in
a cytotoxic radiation dose to tumors.
The compounds of the present invention comprised of
one or more paramagnetic metal ions selected from
gadolinium, dysprosium, iron, arid manganese, are useful
as contrast agents for magnetic resonance imaging (MRI)
of pathological processes involving angiogenic
neovasculature, as well as atherosclerotic plaque,
myocardial reperfusion injury, and myocardial ischemia,
stunning or infarction.
The compounds of the present invention comprised of
one or more heavy atoms with atomic number of 20 or
greater are useful as X-ray contrast agents for X-ray
imaging of pathological processes involving angiogenic
neovasculature, as well as atherosclerotic plaque,
myocardial reperfusion injury, and myocardial ischemia,
stunning or infarction.
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, as well as atherosclerotic plaque,
myocardial reperfusion injury, and myocardial ischemia,
stunning or infarction.
Representative compounds of the present invention
were tested in the following in vitro assays and in vivo
models and were found to be active.
Immobilized Human Placental oc,~,(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
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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-avB5, 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
tissue debris removed via centrifugation. The lysate
was filtered. The receptors were isolated by affinity
chromatography using the immobilized ocv(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.
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
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
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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-
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
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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 ~g/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 ug 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
administered in appropriate amounts and the uptake at
the angiogenic sites can be quantified either non-
invasively by imaging for those isotopes with a
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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 imager 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 imager 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
animal can be imaging using an ultrasound probe held
proximate to the tumors. The effectiveness of the
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contrast agents can be readily seen by 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 ml/kg) 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 uCi/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
determined by taking the fraction of the total counts in
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an inscribed area for ROI/source and multiplying the
known ~.lCi . The result is ~.lCi 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 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.
Cardiovascular disease models that can be used to
assess the diagnostic radiopharmaceuticals, magnetic
resonance, X-ray and ultrasound contrast agents of the
present invention are reviewed in J. Nucl. Cardiol.,
1998, 5, 167-83. There are several well established
rabbit models of atherosclerosis; one model produces
predominantly proliferating smooth muscle cells by
balloon deendothelialization of infradiaphragmatic
abdominal aorta to simulate restenotic lesions; another
model that produces simulated advanced human
atherosclerotic plaque by balloon deendothelialization
followed by a high cholesterol diet.
A model of congestive heart failure is described in
Am. J. Physiol., 1998, 274, H1516-23. In general,
Yorkshire pigs are randomly assigned to undergo 3 wks of
rapid atrial pacing at 240 beats/min. or to be sham
controls. The pigs are chronically instrumented to
measure left ventricular function in the conscious
state. The pigs are anesthetized.
A shielded stimulating electrode is sutured onto the
left
atrium, connected to a modified programmable pace maker
and buried in a subcutaneous pocket. The pericardium is
closed loosely, the thoracotomy is closed, and the
pleural space is evacuated of air. After a recovery
period of 7-10 days, the pacemaker is activated in the
animals selected to undergo chronic rapid pacing. The
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animals are sedated, the pacemaker is deactivated
(pacing groups only. After a 30 min stabilization
period, indexes of LV function and geometry are
determined (by echocardiography as a control) by
injecting the radiolabeled compound. For
biodistribution, the animals are anesthetized, the heart
extirpate and the LV apex and midventricular regions are
evaluated.
A rat model of reversible coronary occlusion and
reperfusion is described in McNulty et al., J. Am.
Physiol., 1996, H2283-9.
All publications, patents, patent applications, and
patent documents are incorporated by reference herein,
as though individually incorporated by reference. The
invention has been described with reference to various
specific and preferred embodiments and techniques.
However, it should be understood that many variations
and modifications may be made while remaining within the
spirit and scope of the invention.
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
2.5 herein.
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