Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4192—1,2,3-Triazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
  • C07B59/002—Heterocyclic compounds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

• the present invention relates to novel radioactively labeled bioreducible tracers useful for detecting hypoxic cells in vivo.
• the present application relates to a novel hypoxia imaging agent that displays low background uptake leading to good tumor to background ratios.
• PET Positron Emission Tomography
• the isotopes are typically administered to a patient by injection of probe molecules that comprise a positron-emitting isotope, such as F-18, C-I l, N-13 or 0-15, covalently attached to a molecule that is readily metabolized or localized in cells (e.g., glucose) or that chemically binds to receptor sites within cells.
• the isotope is administered to the patient as an ionic solution or by inhalation.
• One of the most widely used positron- emitter labeled PET molecular imaging probes is 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]FDG).
• PET scanning using the glucose analog [ 18 F]FDG which primarily targets glucose transporters and hexokinase, is an accurate clinical tool for the early detection, staging, and restaging of cancer.
• PET-FDG imaging is also used to monitor cancer chemotherapy and chemoradiotherapy because early changes in glucose utilization have been shown to correlate with outcome predictions.
• a characteristic feature of tumor cells is their accelerated glycolysis rate, which results from the high metabolic demands of rapidly proliferating tumor tissue.
• FDG is taken up by cancer cells via glucose transporters and is phosphorylated by hexokinase to FDG-6 phosphate. FDG-6 phosphate cannot proceed any further in the glycolysis chain, or leave the cell due to its charge, allowing cells with high glycolysis rates to be detected.
• FDG-PET imaging for monitoring cancer
• Accumulation in inflammatory tissue limits the specificity of FDG-PET.
• nonspecific FDG uptake may also limit the sensitivity of PET for tumor response prediction.
• Therapy induced cellular stress reactions have been shown to cause a temporary increase in FDG-uptake in tumor cell lines treated by radiotherapy and chemotherapeutic drugs.
• physiologically high normal background activity e.g. in the brain
• PET imaging tracers are being developed to target other enzyme-mediated transformations in cancer tissue, such as 3'-[F-18]Fluoro-3'- deoxythymidine (FLT) for DNA replication, and [C-I l](methyl)choline for choline kinase, as well as ultra high-specific activity receptor-ligand binding (e.g., 16 ⁇ [F- 18]fluoroestradiol) and potentially gene expression (e.g., [F-18]fluoro-ganciclovir).
• FLT 3'-[F-18]Fluoro-3'- deoxythymidine
• [C-I l](methyl)choline for choline kinase
• ultra high-specific activity receptor-ligand binding e.g. 16 ⁇ [F- 18]fluoroestradiol
• gene expression e.g., [F-18]fluoro-ganciclovir
• hypoxia inducible factor- 1 leads to the up-regulation of several proteins necessary for tumor survival including vascular endothelial growth factor (VEGF), carbonic anhydrase-IX (CA-IX), and glycolysis enzymes.
• Hypoxic tumors are clinically problematic: they resist both the effects of radiation and cytotoxic therapy which can result in treatment failure.
• Adams, G. Hypoxia-mediated drugs for radiation and chemotherapy. Cancer, 1981. 48: p. 696-707; Moulder, J. and S. Rockwell, Hypoxic fractions of solid tumors: experimental techniques, methods of analysis, and a survey of existing data.
• hypoxic cancer cells have been linked to malignant cancers that are known to spread invasively throughout the patient.
• Tumour oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma. Cancer Res., 1996. 56: p. 941-943; H ⁇ ckel, M., et al., Association between tumor hypoxia and malignant progression in advanced cancer of the cervix. Cancer Res., 1996. 56: p. 941-943.
• Several types of human cancers are well known to become hypoxic including breast, cervical cancer and non-small cell lung cancer.
• Vaupel, P., et al. Oxygenation of human tumours: evaluation of tissue oxygen distribution in breast cancers by computerized O2 tension measurements. Cancer Res., 1991, 51 : p. 3316-3322.
• hypoxic tumors respond poorly to both traditional radiation and cytotoxic therapies, several alternate approaches exist for treating hypoxic cancer cells including hyperbaric O 2 , ARCON, radiosensitizers and bioreductive cytotoxic agents.
• a more general and less invasive method for detecting the hypoxic nature of cancer cells relies on radioactively labeled, bioreducible tracers that localize within hypoxic cells inversely proportional to their cellular p ⁇ 2 .
• [ 18 F]F-MISO the fluorine analog of the hypoxic cell radiosensitizer misonidazole.
• 18 F]F-MISO successfully identifies hypoxic tumors in patients, however, its slow diffusion into hypoxic tumors requires longer uptake times before imaging and, in addition, high background uptake leads to small tumor to background ratios.
• several groups have prepared nitroimidazoles with less lipophilic character in an attempt to increase the tumor to background ratio by increasing the tracer's washout from normoxic tissue.
• hypoxic imaging agents show clinical promise, there exists an unmet need for hypoxia tracers that possess enhanced pharmacokinetic profiles leading to peak signal to noise ratios in shorter periods for faster and potentially more accurate hypoxia assessments.
• the present invention relates to novel hypoxia imaging agents that display low background uptake leading to high tumor to background ratios. More specifically, the present invention relates to novel 2-nitroimidazole based hypoxia imaging agents that display rapid, predominantly renal clearance, leading to low background uptake, low abdominal uptake and generally high tumor to background ratios.
• the present invention relates to novel radioactively labeled bioreducible tracers useful for detecting hypoxic tumors in vivo.
• the tracers consist of a 2-nitroimidazole moiety, a triazole, metabolically stable linker with pharmacokinetics enhancing substituents, and a radioisotope suitable for single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging.
• SPECT single photon emission computed tomography
• PET positron emission tomography
• the preferred in vivo imaging modality is positron emission tomography.
• hypoxic cells resist cytotoxic and radiation therapies, and also possess an increased propensity of proliferation and propagation into nearby tissues
• accurate assessment of the hypoxic nature of a patient's cancer can guide and greatly affect the therapeutic regimen and outcome.
• X is a (C 1 -C 1 o)alkylenyl, unsubstituted or substituted with 1, 2, 3 or 4 X 1 , wherein one of the (C 1 -C 1 o)alkylenyl carbon atoms is optionally replaced by a group selected from -CO-, -CONR'-, -NR' CO-, -NR'-, -O- and -S-, or wherein 2, 3 or 4 contiguous atoms of the (C 1 -C 1 o)alkylenyl group form an unsubstituted or substituted (C 3 -Cg)cycloalkyl or a (C 3 -Cg)heterocycloalkyl ring, unsubstituted or substituted with 1, 2, 3 or 4 X 1 , or a combination thereof; each X 1 is independently hydroxyl, thiol, amino, alkyl, alkoxy, thioalkyl, or halo;
• Z is an (C 1 -C 1 o)alkylenyl group wherein one of the carbon atoms is optionally replaced by a group selected from -CO-, -CONR", -NR 11 CO-, -NR"-, -O- and -S-, and wherein the (C 1 -C 1 o)alkylenyl group is unsubstituted or substituted with 1, 2, 3 or 4 X 1 ;
• A is a radioactive element; and R' and R" are each independently H or are each independently selected from the group consisting of (C 1 -C 6 )alkyl, -CO(C 1 -C 3 )alkyl, -CONH(C 1 -C 3 )alkyl and -CO 2 (C 1 -C 3 )alkyl.
• X is a (Ci-C 4 )alkylenyl group optionally substituted with 1, 2 or 3 hydroxyl groups or 1, 2 or 3 -NH 2 or -NH(C 1 -C 4 ⁇ IkVl group.
• X is selected from the group consisting
• X is selected from the group consisting of -CONR'-, -(Ci-C 4 )alkylCONR'-, -CONR'(d-C 4 )alkyl-, -(Ci-C 4 )alkylCONR'(Ci-
• any one of X, Z or A comprises of a 11 C.
• X is a (C 1 -C 1 o)alkylenyl, unsubstituted or substituted with 1, 2, 3 or 4 X 1 , wherein one of the (C 1 -C 1 o)alkylenyl carbon atoms is optionally replaced by a group selected from -CO-, -CONR'-, -NR' CO-, -NR'-, -O- and -S-, or wherein 2, 3 or 4 contiguous atoms of the (C 1 -C 1 o)alkylenyl group form an unsubstituted or substituted (C 3 -Cg)cycloalkyl or a (C 3 -Cg)heterocycloalkyl ring, unsubstituted or substituted with 1, 2, 3 or 4 X 1 , or a combination thereof; each X 1 is independently selected from the group consisting of hydroxyl, thiol, amino, alkyl, alkoxy, thioalkyl
• A is a radioactive element; and R' and R" are each independently H or are each independently selected from the group consisting of (C 1 -C 6 )alkyl, -CO(C 1 -C 3 )alkyl, -CONH(C 1 -C 3 )alkyl and -CO 2 (C 1 - C 3 )alkyl.
• Z is an (C 1 -C 1 o)alkylenyl group wherein one of the carbon atoms of the
• (C 1 -C 1 o)alkylenyl group is optionally replaced by a group selected from -CO-, -CONR"-, -NR 11 CO-, -NR"-, -O- and -S-, and the (C 1 -C 10 )alkylenyl group is unsubstituted or substituted with 1, 2, 3 or 4 X 1 ;
• R" is H or is selected from the group consisting of
• each X 1 is independently hydroxyl, thiol, amino, alkyl, alkoxy, thioalkyl or halo; and A is a radioactive element.
• Z is an (C 1 -
• C 4 )alkylenyl group optionally substituted with 1 , 2 or 3 groups independently selected from the group consisting of hydroxyl, thiol, amino, Mo(C 1 -
• Z is selected from the group consisting of -CH 2 -, -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -, -CH 2 -CH(OH)-, -CH(OH)-C
• A is 18 F or 11 C-Me. In a particular variation of the above, A is F. In one variation of the above, there is provided a compound of the formula:
• X is a (C 1 -C 1 o)alkylenyl, unsubstituted or substituted with 1, 2, 3 or 4 X 1 , wherein one of the (C 1 -C 1 o)alkylenyl carbon atoms is optionally replaced by a group selected from -CO-, -CONR'-, -NR' CO-, -NR'-, -O- and -S-, or wherein 2, 3 or 4 contiguous atoms of the (C 1 -C 1 o)alkylenyl group form an unsubstituted or substituted (C 3 -C8)cycloalkyl or a (C 3 -Cg)heterocycloalkyl ring, unsubstituted or substituted with 1, 2, 3 or 4 X 1 , or a combination thereof;
• R' is H or is selected from the group consisting of (C 1 -C 6 )alkyl, -CO(C 1 -C 3 )alkyl, -CONH(C 1 -C 3 )alkyl and -CO 2 (C 1 -C 3 )alkyl; and each X 1 is independently selected from the group consisting of hydroxyl, thiol, amino, (C 1 -C 4 )alkyl, (Ci-C 4 )alkoxy, thio(Ci-C 4 )alkyl and halo.
• X is (Ci-Cs ⁇ lkylenyl, unsubstituted or substituted with 1 or 2 X 1 , or wherein one of the (C 1 -C 1 o)alkylenyl carbon atoms is optionally replaced by a group selected from -CONR'- or -NR' CO-, or wherein 2, 3 or 4 contiguous atoms of the (C 1 -C 1 o)alkylenyl group form an unsubstituted or substituted (C 3 -C 8 )cycloalkyl unsubstituted or substituted with 1, 2 or 3 X 1 , wherein X 1 is -OH or NH 2 .
• X is a (C 1 -C 1 o)alkylenyl, unsubstituted or substituted with 1, 2, 3 or 4 X 1 , wherein one of the (C 1 -C 1 o)alkylenyl carbon atoms is optionally replaced by a group selected from -CO-, -CONR'-, -NR' CO-, -NR'-, -O- and -S-, or wherein 2, 3 or 4 contiguous atoms of the (C 1 -C 1 o)alkylenyl group form an unsubstituted or substituted (C 3 -Cg)cycloalkyl or a (C 3 -Cg)heterocycloalkyl ring, unsubstituted or substituted with 1, 2, 3 or 4 X 1 , or a combination thereof;
• R' is H or is selected from the group consisting of (C 1 -C 6 )alkyl, -CO(C 1 -C 3 )alkyl, -CONH(C 1 -C 3 )alkyl and -CO 2 (C 1 -C 3 )alkyl; and each X 1 is independently selected from the group consisting of hydroxyl, thiol, amino, alkyl, alkoxy, thioalkyl and halo.
• a method for detecting hypoxia in cells comprising: a) administering to a mammal a compound of the formula I:
• X is a (C 1 -C 1 o)alkylenyl, unsubstituted or substituted with 1, 2, 3 or 4 X 1 , wherein one of the (C 1 -C 1 o)alkylenyl carbon atoms is optionally replaced by a group selected from -CO-, -CONR'-, -NR' CO-, -NR'-, -O- and -S-, or wherein 2, 3 or 4 contiguous atoms of the (C 1 -C 1 o)alkylenyl group form an unsubstituted or substituted (C 3 -Cg)cycloalkyl or a (C 3 -Cg)heterocycloalkyl ring, unsubstituted or substituted with 1, 2, 3 or 4 X 1 , or a combination thereof; each X 1 is independently selected from the group consisting of hydroxyl, thiol, amino, alkyl, alkoxy, thioalkyl
• Z is an (C 1 -C 1 o)alkylenyl group wherein one of the carbon atoms of the (C 1 -C 1 o)alkylenyl group is optionally replaced by a group selected from -CO-, -CONR'-, -NR 1 CO-, -NR'-, -O- and -S-, and the (C 1 -C 10 )alkylenyl group is unsubstituted or substituted with 1, 2, 3 or 4 X 1 ;
• A is a radioactive element;
• R' and R" are each independently H or are each independently selected from the group consisting of (C 1 -C 6 )alkyl, -CO(C 1 -C 3 )alkyl, -CONH(C 1 -C 3 )alkyl and -CO 2 (C 1 -
• X is a (Ci-C 4 )alkylenyl group optionally substituted with 1, 2 or 3 hydroxyl groups or 1, 2 or 3 -NH 2 or -NH(C 1 -C 4 ⁇ IkVl group.
• X is selected from the group consisting
• X is selected from the group consisting of -CONR'-, -(C 1 -C 4 MkVlCONR'-, -CONR'(Ci-C 4 )alkyl- and -(C 1 -C 4 MkVlCONRXC 1 -C 4 ) alkyl-, and wherein R' is H or (Ci-C 3 )alkyl.
• R' is H or (Ci-C 3 )alkyl.
• X is a (C 1 -C 1 o)alkylenyl, unsubstituted or substituted with 1, 2, 3 or 4 X 1 , wherein one of the (C 1 -C 1 o)alkylenyl carbon atoms is optionally replaced by a group selected from -CO-, -CONR'-, -NR' CO-, -NR'-, -O- and -S-, or wherein 2, 3 or 4 contiguous atoms of the (C 1 -C 1 o)alkylenyl group form an unsubstituted or substituted (C 3 -Cg)cycloalkyl or a (C 3 -Cg)heterocycloalkyl ring, unsubstituted or substituted with 1, 2, 3 or 4 X 1 , or a combination thereof; each X 1 is independently selected from the group consisting of hydroxyl, thiol, amino, alkyl, alkoxy, thioal
• Z is an (C 1 -C 1 o)alkylenyl group wherein one of the carbon atoms of the (C 1 -C 1 o)alkylenyl group is optionally replaced by a group selected from -CO-, -CONR"-, -NR 11 CO-, -NR"-, -O- and -S-, and the (Ci-Cio)alkylenyl group is unsubstituted or substituted with 1, 2, 3 or 4 X 1 ;
• A is a radioactive element; and R' and R" are each independently H or are each independently selected from the group consisting of (Ci-C 6 )alkyl, -C0(Ci-C 3 )alkyl, - CONH(C i_C 3 )alkyl and -CO 2 (C 1 -C 3 )alkyl.
• the compound is of the formula II.
• Z is (Ci-C 4 )alkylenyl group optionally substituted with 1, 2 or 3 hydroxyl groups or 1, 2 or 3 hydroxy(Ci-C 4 )alkyl groups.
• Z is selected from the group consisting Of -CH 2 -, -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -, -CH 2 -CH(OH)-, -CH(OH)-C H 2 -, -CH(OH)-CH 2 -CH 2 -, -CH(CH 2 OH)-CH 2 -, -CH 2 -CH(OH)-CH 2 -, -CH 2 -CH(OH)-CH 2 -, -CH 2 -CH 2 -CH(OH)-a nd -CH 2 -CH(OH)-CH(OH)-CH 2 -.
• A is 18 F or 11 C-Me.
• A is 18 F.
• the compound comprises the formula:
• hypoxic cells are tumor cells or ischemic cells.
• X is a (C 1 -C 1 o)alkylenyl, unsubstituted or substituted with 1, 2, 3 or 4 X 1 , wherein one of the (C 1 -C 1 o)alkylenyl carbon atoms is optionally replaced by a group selected from -CO-, -CONR'-, -NR' CO-, -NR'-, -O- and -S-, or wherein 2, 3 or 4 contiguous atoms of the (C 1 -C 1 o)alkylenyl group form an unsubstituted or substituted (C 3 -Cg)cycloalkyl or a (C 3 -Cg)heterocycloalkyl ring, unsubstituted or substituted with 1, 2, 3 or 4 X 1 , or a combination thereof each X 1 is independently selected from the group consisting of hydroxyl, thiol, amino, alkyl, alkoxy, thioalky
• Z is an (C 1 -C 1 o)alkylenyl group wherein one of the carbon atoms of the (C 1 -C 1 o)alkylenyl group is optionally replaced by a group selected from -CO-, -CONR"-, -NR 11 CO-, -NR"-, -O- and -S-, and the (C 1 -C 10 )alkylenyl group is unsubstituted or substituted with 1, 2, 3 or 4 X 1 ;
• A is a radioactive element; and R' and R" are each independently H or are each independently selected from the group consisting of (C 1 -C 6 MkVl, -CO(Ci-C 3 )alkyl, -CONH(Ci-C 3 )alkyl and -CO 2 (C 1 - C 3 )alkyl; the method comprising: a) treating an azide substituted compound A'-Z-N 3 with an acetylene substituted
• step b) radiolabeling the product of step a) by the displacement of A' with a radiolabeling agent to form compound II or III or a mixture thereof, wherein A is either 18 F or 11 C.
• A is either 18 F or 11 C.
• the radiolabeling agent is K F.
• Figure 1 Bio-distribution of Compound 1 in male white mouse 120 min post IV injection (200 ⁇ l of a 5 mM solution). [0039]
• novel compounds described herein contain the requisite 2-nitroimidazole core that is necessary for imaging hypoxic tumors.
• intracellular bioreduction under hypoxic conditions modifies the 2-nitroimidazole core which then undergoes covalently-mediated localization within the cell.
• the bioreduced nitroimidazole returns to its native state and diffuses freely into and out of the cell.
• novel compounds disclosed herein also display favorable imaging pharmacokinetic properties.
• the compounds possess favorable clearance properties via renal excretion, thus exhibiting lower uptake in the liver and gut regions. These compounds also clear quickly from both blood and muscle tissue, thus leading to desirable tumor to background ratios.
• these favorable clearance properties translate into peak tumor to background ratios at a timepoint earlier than the "gold standard" [ 18 F]F-MISO.
• These tracers are highly selective for hypoxic tumors over non-hypoxic tumors. In xenograph mice and rats, the tracer preferentially localizes within hypoxic tumors and, in most cases, with tumor to background ratios greater than 2:1.
• Alkyl refers to a hydrocarbon chain, typically ranging from about 1 to 20 atoms in length. Such hydrocarbon chains may be branched or straight chain, although typically straight chain is preferred. Exemplary alkyl groups include ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like. As used herein, “alkyl” includes cycloalkyl when three or more carbon atoms are referenced.
• Alkylenyl refers to a hydrocarbon chain, typically ranging from about 1 to 20 atoms in length, or 1 to 10 carbon atoms in length, that is divalently bonded to two different groups. Such hydrocarbon chains may be branched or straight chain.
• alkylenyl groups include ethylenyl, propylenyl, iso-propylenyl, butylenyl, iso-butylenyl, pentylenyl, 1-methylbutylenyl, 1-ethylpropyl enyl, 3-methylpentylenyl, and the like, wherein the divalent bonds may be at any of the carbon atoms of the alkylenyl group, or as specifically indicated.
• alkylenyl also includes cycloalkylenyl when three or more carbon atoms are referenced.
• Aryl means one or more aromatic rings, each of 5 or 6 core carbon atoms.
• Aryl includes multiple aryl rings that may be fused, as in naphthyl or unfused, as in biphenyl.
• Aryl rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings.
• aryl includes heteroaryl.
• a "biological target” can be any biological molecule involved in biological pathways associated with any of various diseases and conditions, including cancer (e.g., leukemia, lymphomas, brain tumors, breast cancer, lung cancer, prostate cancer, gastric cancer, as well as skin cancer, bladder cancer, bone cancer, cervical cancer, colon cancer, esophageal cancer, eye cancer, gallbladder cancer, liver cancer, kidney cancer, laryngeal cancer, oral cancer, ovarian cancer, pancreatic cancer, penile cancer, glandular tumors, rectal cancer, small intestine cancer, sarcoma, testicular cancer, urethral cancer, uterine cancer, and vaginal cancer), diabetes, neurodegenerative diseases, cardiovascular diseases, respiratory diseases, digestive system diseases, infectious diseases, inflammatory diseases, autoimmune diseases, and the like.
• cancer e.g., leukemia, lymphomas, brain tumors, breast cancer, lung cancer, prostate cancer, gastric cancer, as well as skin cancer, bladder cancer, bone cancer, cervical cancer, colon cancer, esoph
• Exemplary biological pathways include, for example, cell cycle regulation (e.g., cellular proliferation and apoptosis), angiogenesis, signaling pathways, tumor suppressor pathways, inflammation (COX-2), oncogenes, and growth factor receptors.
• the biological target may also be referred to as the "target biomacromolecule” or the “biomacromolecule.”
• the biological target can be a receptor, such as enzyme receptors, ligand-gated ion channels, G-protein-coupled receptors, and transcription factors.
• the biological target is preferably a protein or protein complex, such as enzymes, membrane transport proteins, hormones, and antibodies.
• Contiguous atoms means atoms that are adjacent to each other.
• the phrase "2, 3 or 4 contiguous atoms of the (C 1 -C 1 o)alkylenyl group form an unsubstituted or substituted (C 3 -Cg)cycloalkyl or a (C 3 -Cg)heterocycloalkyl ring” means that the 2, 3 or 4 adjacent atoms in an alkylenyl group or a linker, for example, form part of a (C 3 -Cg)cycloalkyl or a (C 3 -Cg)heterocycloalkyl ring.
• Fragment "A” when the atoms form an unsubstituted (C 3 -Cg)cycloalkyl group such as a cyclohexyl group, the fragment may form as shown in Fragment "B.”
• Cycloalkyl refers to a saturated, partially unsaturated or unsaturated cyclic hydrocarbon chain, including bridged, fused, or spiro cyclic rings, preferably made up of 3 to about 12 carbon atoms, more preferably 3 to 8 carbon atoms.
• Heteroaryl is an aryl group containing from one to four heteroatoms, preferably N, O, or S, or a combination thereof. Heteroaryl rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings.
• Heterocycle or “heterocyclic” means one or more rings of 3-12 atoms, preferably 5-7 atoms, with or without unsaturation or aromatic character, and having at least one ring atom which is not a carbon.
• Preferred heteroatoms include sulfur, oxygen, and nitrogen.
• leaving group refers to groups that are readily displaced, for example, by a nucleophile, such as an amine, a thiol or an alcohol nucleophile or its salt.
• a nucleophile such as an amine, a thiol or an alcohol nucleophile or its salt.
• Such leaving groups are well known and include, for example carboxylates, N- hydroxysuccinimide, N-hydroxybenzotriazole, halides, triflates, tosylates, nosylates, -OR and -SR and the like.
• Linker refers to a chain comprising 1 to 10 atoms and may comprise of 1, 2 or 3 adjacent or non-adjacent atoms or groups, such as C, -NR-, O, S, -S(O)-, -S(O) 2 -, CO, -C(NR)- and the like, and wherein R is H or is selected from the group consisting of (C 1 - I o)alkyl, (C 3 _g)cycloalkyl, aryl(C 1 - 5 )alkyl, heteroaryl(C 1 - 5 )alkyl, amino, aryl, heteroaryl, hydroxy, (C 1 - I o)alkoxy, aryloxy, heteroaryloxy, each substituted or unsubstituted.
• the linker may comprise of the groups: -CH 2 - , -CH 2 -CH 2 -, -CH 2 -O-CH 2 -, -CH 2 -CH 2 -O-CH 2 -CH 2 -, -CH 2 -NH-CH 2 -, -CH 2 -CH 2 - NH-CH 2 -CH 2 -, -CH 2 -NHC(O)-CH 2 -, -CH 2 -C(O)NH-CH 2 -, -CH 2 -C(O)-CH 2 - etc ...
• the linker chain may also comprise part of a saturated, unsaturated or aromatic ring, including polycyclic and heteroaromatic rings.
• variables X and Z may be the linker or linker chain.
• the representation of "(C 1 _ 3 )alkyl”, for example, is used interchangeably with “Ci ⁇ alkyl” to mean the same.
• patient and “subject” refer to any human or animal subject, particularly including all mammals.
• radiochemical is intended to encompass any organic, inorganic or organometallic compound comprising a covalently-attached radioactive isotope, any inorganic radioactive ionic solution (e.g., Na[ 18 F]F ionic solution), or any radioactive gas (e.g., [ 11 C]CO 2 ), particularly including radioactive molecular imaging probes intended for administration to a patient (e.g., by inhalation, ingestion or intravenous injection) for tissue imaging purposes, which are also referred to in the art as radiopharmaceuticals, radiotracers or radioligands.
• any inorganic radioactive ionic solution e.g., Na[ 18 F]F ionic solution
• radioactive gas e.g., [ 11 C]CO 2
• the present invention is primarily directed to synthesis of positron-emitting molecular imaging probes for use in PET imaging systems, the invention could be readily adapted for synthesis of any radioactive compound comprising a radionuclide, including radiochemicals useful in other imaging systems, such as single photon emission computed tomography (SPECT).
• SPECT single photon emission computed tomography
• radioactive isotope or “radioactive element” refers to isotopes exhibiting radioactive decay (i.e., emitting positrons) and radiolabeling agents comprising a radioactive isotope (e.g., [ ⁇ C]methane, [ l ⁇ Jcarbon monoxide, [ ⁇ C]carbon dioxide, [ l ⁇ Jphosgene, [ ⁇ C]urea, [ ⁇ C]cyanogen bromide, as well as various acid chlorides, carboxylic acids, alcohols, aldehydes, and ketones containing carbon-11).
• radioactive isotope e.g., [ ⁇ C]methane, [ l ⁇ Jcarbon monoxide, [ ⁇ C]carbon dioxide, [ l ⁇ Jphosgene, [ ⁇ C]urea, [ ⁇ C]cyanogen bromide, as well as various acid chlorides, carboxylic acids, alcohols, aldehydes, and ketones containing carbon-11).
• Radioactive isotopes are named herein using various commonly used combinations of the name or symbol of the element and its mass number (e.g., 18 F, F-18, or fluorine-18).
• Exemplary radioactive isotopes include 1-124, F-18 fluoride, C-I l, N-13, and 0-15, which have half-lives of 4.2 days, 110 minutes, 20 minutes, 10 minutes, and 2 minutes, respectively.
• the radioactive isotope is preferably dissolved in an organic solvent, such as a polar aprotic solvent.
• the radioactive isotopes used in the present method include F-18, C-I l, 1-123, 1-124, 1-127, 1-131, Br-76, Cu-64, Tc-99m, Y-90, Ga-67, Cr-51, Ir- 192, Mo-99, Sm- 153 and Tl-201.
• the radioactive isotope used in the present method is F-18.
• Other radioactive isotopes that may be employed include: As-72, As-74, Br-75, Co-55, Cu-61, Cu-67, Ga-68, Ge-68, 1-125, 1-132, In-H l, Mn-52, Pb-203 and Ru- 97.
• a substituent such as a -Ci-salkyl, C 2 _salkenyl, halogen or halo (chlorine, fluorine, bromine, iodine atom), -CF 3 , nitro, amino (-NH 2 , -NHR, -NR 2 , etc ...), oxo (i
• Triazole as used herein means either 1 ,3,4- or 1 ,2,3-triazole, or mixtures thereof.
• the "triazole” is a 1,2,3-triazole, substituted in the 1- and 5-positions ("syn") or in the 1- and 4-positions ("anti”) or mixtures thereof.
• the 1,2,3-triazole is substituted in the 1- and 4-positions.
• Click chemistry provides chemists an opportunity to rapidly produce libraries of candidate imaging agents, from which potential small molecule PET imaging tracers with optimal pharmacodynamic and pharmacokinetic properties may be identified.
• Click chemistry is a modular approach to chemical synthesis that utilizes only the most practical and reliable chemical transformations. Click chemistry techniques are described, for example, in the following references, which are incorporated herein by reference in their entirety:
• the triazole comprising a radioactive isotope or radioactive element may be prepared by the displacement or substitution of A with the radioactive isotope or the radioactive element, as disclosed herein.
• 2-Azido-3-hydroxypropyl acetate (24) 2-Azidopropane-l,3-diol 23 (10.46 g, 89.3 mmol) was dissolved in CH 2 Cl 2 (450 mL) and treated with a catalytic amount o ⁇ p- toluene sulfonic acid monohydrate (340 mg, 1.8 mmol) and triethylorthoacetate (24.4 mL, 134 mmol) at room temperature for 1 hr. After the formation of the orthoester was complete, a stoichiometric amount of water (2.4 mL, 134 mmol) was added to the mixture.
• a solution of the precursor 12, (20 ⁇ mol) in acetonitrile (0.9 mL) is added to the dried [F- 18] fluoride ion.
• the mixture is heated to approximately 110 0 C for 10 minutes to allow reaction with the [F-18]fluoride ion.
• the explora ® RN chemistry module is equipped with both UV and Geiger Mueller (GM) detectors.
• GM Geiger Mueller
• the product is collected from the column as monitored by flow- through radioactivity and UV (254 nm) detectors.
• the retention time of the [F-18]8 is approximately 8-10 minutes.
• the fraction containing the [F-18]-labeled tracer is passed through a sterile filter (0.22 ⁇ m) and collected into a sterile vial.
• the volume of the drug substance collected is typically 8-10 mL. Radiolabeling reaction for [F-18]8
• Radiochemical purity 100% [00107] Radiolabeling reaction for [F-18]3:
• a solution of the precursor 16, (15 ⁇ 5 mg, 20-40 ⁇ mol) dissolved in a mixture of acetonitrile (0.5 mL) and t-BuOH (0.5 mL) is added to the reaction vessel containing the anhydrous [F-18]Fluoride.
• the vessel is heated to 125 ⁇ 15 0 C for 7.5 ⁇ 2.5 minutes to induce displacement of the nosylate leaving group by [F- 18] fluoride.
• aqueous hydrochloric acid (1.0 N, 0.8 mL) is added and the mixture heated to 110 ⁇ 5 0 C for 7.5 ⁇ 2.5 minutes.
• the explora ® RN chemistry module is equipped with both UV and Geiger Mueller (GM) detectors.
• GM Geiger Mueller
• the product is collected from the column as monitored by flow- through radioactivity and UV (254 nm) detectors.
• the retention time of the [F-18]3 is approximately 8-10 minutes.
• the fraction containing the [F-18]-labeled tracer is passed through a sterile filter (0.22 ⁇ m) and collected into a sterile vial.
• the volume of the drug substance collected is typically 8-10 mL. min
• Radiolabeling reaction for [F-18]5 [00114] A solution of the precursor 21, (15 ⁇ 5 mg, 20-40 ⁇ mol) dissolved in a mixture of acetonitrile (0.5 mL) and t-BuOH (0.5 mL) is added to the reaction vessel containing the anhydrous [F-18]Fluoride. The vessel is heated to 125 ⁇ 15 0 C for 7.5 ⁇ 2.5 minutes to induce displacement of the nosylate leaving group by [F- 18] fluoride.
• the explora ® RN chemistry module is equipped with both UV and Geiger Mueller (GM) detectors.
• GM Geiger Mueller
• the product is collected from the column as monitored by flow- through radioactivity and UV (254 nm) detectors.
• the retention time of the [F-18]5 is approximately 7-9 minutes.
• the fraction containing the [F-18]-labeled tracer is passed through a sterile filter (0.22 ⁇ m) and collected into a sterile vial.
• the volume of the drug substance collected is typically 8-10 mL.
• Radiochemical yield 12.14 % decay corrected Radiochemical purity: 40%
• Radiolabeling reaction for [F-18]l [00119] A solution of the precursor 26, (15 ⁇ 5 mg, 20-40 ⁇ mol) dissolved in a mixture of acetonitrile (0.5 mL) and t-BuOH (0.5 rnL) is added to the reaction vessel containing the anhydrous [F-18]Fluoride. The vessel is heated to 125 ⁇ 15 0 C for 7.5 ⁇ 2.5 minutes to induce displacement of the nosylate leaving group by [F- 18] fluoride.
• aqueous hydrochloric acid (1.0 N, 0.8 mL) is added and the mixture heated to 110 ⁇ 5 0 C for 7.5 ⁇ 2.5 minutes. This hydrolyzes the acetate group converting it to a hydroxyl group. This hydrolysis reaction results in the formation of crude [F-18]l.
• the reaction mixture is cooled and neutralized by addition of sodium acetate (2.0 M, 0.4 mL).
• reaction mixture containing crude [F- 18] 1 is passed through a alumina sep-pak lite (to remove unreacted [F- 18]fluoride) and then transferred to the HPLC sample loop.
• the crude reaction material was purified via chromatographic separation using a semi-preparative HPLC column (Waters ACE AQ reverse phase semi-prep column (250 x 10 mm), p/n ACE-126-2510, 250 x 10 mm, 5% ethanol in 21 mM 95% Phosphate Buffer, 5.0 mL/min).
• the column effluent is monitored using UV (254 nm) and radiometric detectors connected in series.
• the explora ® RN chemistry module is equipped with both UV and Geiger Mueller (GM) detectors.
• the product is collected from the column as monitored by flow- through radioactivity and UV (254 nm) detectors.
• the retention time of the [F-18]l is approximately 17 ⁇ 5 minutes.
• a sterile empty collection vial is preloaded with 5-10 mL of sterile diluent containing 1-6% ethanol, 75 mg/mL of ascorbic acid and 95% 21 mM phosphate.
• the purified [F-18]l fraction eluted from the HPLC purification column is processed through a 0.2 ⁇ m sterile filter into the preloaded collection vial.
• Radiochemical yield 34 % decay corrected
• Radioactive [F- 18] fluoride is transferred into a 3 mL V-vial with 0.5 mL of K222/K 2 CO 3 solution (660 mg of K222 and 210 mg of K 2 CO 3 in 9 mL of MeCN and 9 mL OfH 2 O).
• the [F-18]Fluoride is azeotropically dried with 3 x 1 mL MeCN under Ar flow.
• a solution of the precursor 26, (16 ⁇ 5 mg, 20-40 ⁇ mol) dissolved in acetonitrile (1.0 mL) is added to the reaction vessel containing the anhydrous [F-18]Fluoride.
• the vessel is heated to 95 ⁇ 5 0 C for 7.5 ⁇ 2.5 minutes to induce displacement of the nosylate leaving group by [F-18]fluoride.
• GM Mueller (GM) detectors.
• the product is collected from the column as monitored by flow- through radioactivity and UV (254 nm) detectors. Under the described elution conditions, the retention time of the [F-18]l is approximately 17 ⁇ 5 minutes.
• a sterile empty collection vial is preloaded with 5-10 mL of sterile diluent containing 1-6% ethanol, 75 mg/mL of ascorbic acid and 95% 21 mM phosphate.
• the purified [F-18]l fraction eluted from the HPLC purification column is processed through a 0.2 ⁇ m sterile filter into the preloaded collection vial.
• Radiochemical yield 34 % decay corrected
• lysis buffer about 5 ml/gram tissue
• 50 ⁇ L of lysis buffer was added to the blood and urine samples, followed by briefly vortexing.
• a standard curve with a wide range of Compound 1 concentrations was prepared using the same method described above for tissue lysate treatment. The corresponding peak of the compound was identified first in the standards. The areas of the peaks were measured and converted into a standard correlation curve for estimation of
• Compound 1 concentration in the samples is expressed as % injected dose/gram tissue (%ID/g tissue).
• %ID/g at 120 minute was 88.69.
• the levels of Compound 1 in gall bladder and GI system are low.
• the blood levels of Compound 1 are 0.67 and 0.15 %ID/g at 30 and 120 minutes, respectively.
• the urine level was 5.3 %ID/g at 30 minute.
• the urine excretion index (averaged urine %ID/g divided by blood %ID/g at 120 minute) was 36.62.
• Compound 1 in rat urine excretion and blood clearance is fast given the high %ID/g in the urine and low %ID/g in the blood.
• Lsl74T human colorectal adenocarcinoma, A172 human brain glioblastoma, MRC5 human normal lung fibroblast, and ALM 12 mouse normal liver cell lines were selected for this study.
• Cells were cultured with different concentration of Compound 1 (0, 100, 500, 1000, 5000, 10000 nM) for 24 hours and then incubated with 0.5 mg/ml of 3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyl tetrazolium bromide for 1 hour. Cells were lysed and formazan crystals were dissolved with 2-propanol for 1 hour and the optical density of the resultant colored 2- propanol was measured. The percentages of cell survival were calculated based on the optical densities.

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