US20220387631A1 - Near-infrared fluorescent contrast bioimaging agents and methods of use thereof - Google Patents

Near-infrared fluorescent contrast bioimaging agents and methods of use thereof Download PDF

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US20220387631A1
US20220387631A1 US17/371,663 US202117371663A US2022387631A1 US 20220387631 A1 US20220387631 A1 US 20220387631A1 US 202117371663 A US202117371663 A US 202117371663A US 2022387631 A1 US2022387631 A1 US 2022387631A1
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alkyl
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aryl
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John V. Frangioni
Hak Soo Choi
Maged M. Henary
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Georgia State University Research Foundation Inc
Beth Israel Deaconess Medical Center Inc
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Beth Israel Deaconess Medical Center Inc
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Definitions

  • NIR fluorescence Near infrared fluorescence has potential importance in the medical field, particularly in in vitro diagnostics, in vivo diagnostics, and image-guided surgery.
  • NIR fluorophores As imaging agents has been a primary hindrance.
  • ideal NIR fluorophores should have good optical properties as well as superior physicochemical properties with respect to solubility, biodistribution, targeting, and clearance.
  • Most current fluorophores contemplated for use as imaging agents fail in connection with their physicochemical properties.
  • known fluorophores suffer from failure to adequately accumulate at the target to be imaged (i.e., low signal), resulting in a low signal-to-background ratio (SBR), or exhibit significant non-specific background uptake in normal tissues (i.e., high background), also resulting in a low SBR.
  • SBR signal-to-background ratio
  • NIR fluorescent imaging agents particularly those that equilibrate rapidly between the intravascular and extravascular spaces, target various cells, tissues, or organs with high sensitivity and specificity, and are eliminated efficiently from the body if not targeted.
  • the imaging agents of the invention are directed toward these and other needs.
  • the present invention is directed, at least in part, to near-infrared fluorescent contrast agents and methods of using them.
  • the near-infrared fluorescent contrast is a compound of Formula (I):
  • Each R 1 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C 1 -C 6 alkyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or phenyl;
  • Each R 2 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C 1 -C 6 alkyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or phenyl;
  • R 1 and R 2 can be taken together with the carbon atoms to which they are attached to form a 5-6 membered aryl or heteroaryl ring, optionally substituted with halogen, alkyl, alkoxy, hydroxyl, —SO 2 OH, or —CO 2 H;
  • Each R 3 is independently H, OR′, halogen, sulfonato, substituted or un
  • the near-infrared fluorescent contrast is a compound of Formula (II):
  • Each R 1 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C 1 -C 6 alkyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or phenyl;
  • Each R 2 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C 1 -C 6 alkyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or phenyl;
  • R 1 and R 2 can be taken together with the carbon atoms to which they are attached to form a 5-6 membered aryl or heteroaryl ring, optionally substituted with halogen, alkyl, alkoxy, hydroxyl, —SO 2 OH, or —CO 2 H;
  • Each R 3 is independently H, OR′, halogen, sulfonato, substituted or un
  • the near-infrared fluorescent contrast is a compound of Formula (III):
  • Each R 1 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C 1 -C 6 alkyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or phenyl;
  • Each R 2 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C 1 -C 6 alkyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or phenyl;
  • R 1 and R 2 can be taken together with the carbon atoms to which they are attached to form a 5-6 membered aryl or heteroaryl ring, optionally substituted with halogen, alkyl, alkoxy, hydroxyl, —SO 2 OH, or —CO 2 H;
  • Each R 3 is independently H, OR′, halogen, sulfonato, substituted or
  • the near-infrared fluorescent contrast is a compound of Formula (IV):
  • R 1 , R 2 , R 3 , R 4 , R 6 and R 7 are each independently H or C 1 -C 6 alkyl;
  • R 5 , R 8 and R 9 are each independently H, CN, OH, or C 1 -C 6 alkyl; or
  • R 1 and R 3 taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring; or
  • R 2 and R 4 taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring;
  • R 5 and R 6 taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring;
  • R 7 and R 8 taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring; or
  • R 8 and R 9 taken together with the atoms to which they are connected, form an aryl or heteroaryl ring;
  • X is O
  • the near-infrared fluorescent contrast is a compound of Formula (V):
  • Q is —B(R 3 ) 2 —; Si(R 3 ) 2
  • Each R 1 is independently H, alkyl, aryl, or heteroaryl, wherein each alkyl, aryl, or heteroaryl is optionally substituted with alkoxy, alkoxy-N + (alkyl) 3 , alkoxy-OH, halogen, or COOH
  • Each R 2 is independently H, alkyl, aryl, or heteroaryl, wherein each alkyl, aryl, or heteroaryl is optionally substituted with alkoxy, alkoxy-N + (alkyl) 3 , alkoxy-OH, halogen, or COOH
  • Each R 3 is independently H, F, or alkyl; OH or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof.
  • the near-infrared fluorescent contrast is:
  • the imaging agent has peak absorbance at about 600 nm to 900 nm.
  • the tissue or cells is imaged ex vivo, e.g. for in vitro diagnostic applications.
  • the invention provides a method of imaging biological cells, the method comprising: (a) contacting the biological cells with a compound of the invention; (b) irradiating the cells at a wavelength absorbed by the compound; (c) and detecting a signal from the compound, thereby imaging the biological cells.
  • the biological cells could be a normal cell type in the body or its malignant counterpart, i.e., a tumor formed from a normal cell type.
  • the biological targets are found in biological tissues or organs.
  • the biological targets are blood vessel lumens, endothelial cells lining blood vessels, cartilage cells and/or their products, bone cells and/or their products, thyroid cells, thyroid glands, parathyroids cells, parathyroid glands, adrenal gland cells, adrenal glands, salivary gland cells, salivary glands, white adipose tissue, brown adipose tissue, ovarian cells, testicular cells, seminal vesicles, prostate cells, pancreas cells, spleen cells, gallbladder lumens, gallbladder cells, bile duct lumens, bile duct cells, Peyer's patches, brain grey matter, brain white matter, brain vasculature cells, choroid plexus tissue and fluid, cerebrospinal fluid, nerves, lymph nodes, sentinel lymph nodes, vulnerable plaque, stem cells, or neuroendocrine tumors.
  • the compounds of the invention accumulate in a lumen or other cavity in the body, thus highlighting the lumen's anatomical location and/or quantifying flow of the compound within the lumen.
  • a NIR fluorophore excreted by the kidney will accumulate in the ureters, thus identifying their location and also permitting direct visualization of pulsatile flow within the ureters.
  • blood vessels after intravenous injection of a compound or the thoracic duct after injection into the lower body.
  • the compounds of the invention accumulate in a tissue or organ but do so extracellularly.
  • a compound injected sub-dermally may enter the lymphatic channels and flow to a lymph node where it may be trapped in the extracellular space rather than, or in addition to, entrapment within cells of the lymph node.
  • the compounds of the invention may be modified to include a polyethylene glycol group.
  • PEGylated compounds may be branched or linear.
  • the linear PEGylated compounds are in the range of about 20 kDa to about 60 kDa.
  • the NIR fluorophores are conjugated covalently or non-covalently to other molecules, either to improve targeting of the NIR fluorophore or to co-localize other functional molecules.
  • the compounds of the invention can be conjugated to a metal chelator agent for use in single-photon emission computed tomography (SPECT) or positron emission tomography (PET) or in magnetic resonance imaging (MRI).
  • SPECT single-photon emission computed tomography
  • PET positron emission tomography
  • MRI magnetic resonance imaging
  • the metal cheltor agent is a DOTA, DTPA, hydrazinonicotinic acid (HYNIC), or desferoxime, or a derivative thereof.
  • the metal atom is selected from the group including, but not exclusively, Zr-89, Ga-68 and Rb-82, and the signal is detected by positron emission tomography; the metal atom is selected from the group including, but not exclusively, of Tc-99m, Lu-177, and In-111, and the signal is detected by single-photon emission computed tomography; or the metal atom is a lanthanide selected from the group including, but not exclusively, Gd, Eu, Y, Dy and Yb, and the signal is detected by magnetic resonance imaging.
  • the compounds of the invention can be conjugated to a therapeutic, such as a radioisotope, cytotoxin, or immune modulator, such that the targeting ability of the compound concentrates the therapeutic in the cell, tissue, organ, or lumen of interest.
  • a therapeutic such as a radioisotope, cytotoxin, or immune modulator
  • FIG. 1 depicts the imaging of Ureters at 800 nm using A71 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 2 depicts the imaging of Ureters at 700 nm using LN15 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 3 depicts the imaging of pan lymph node identification at 800 nm using MM25 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 4 depicts the imaging of Pan LN pan lymph node identification at 700 nm using A150 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 5 depicts the imaging of SLN identification at 800 nm using MM25 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 6 depicts the imaging of SLN identification at 700 nm using MHI86 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 7 depicts the imaging of Cartilage at 800 nm using LN50 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 8 depicts the imaging of Cartilage at 700 nm using SP56 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 9 depicts the imaging of neuroendocrine tumors at 800 nm using AL20 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 10 depicts the imaging of neuroendocrine tumors at 700 nm using ESS61 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 11 depicts the imaging of bone mineralization at 800 nm using P800SO3 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 12 depicts the imaging of bone mineralization at 700 nm using P700SO3 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 13 depicts the imaging of the thyroid gland at 800 nm using QBN14 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 14 depicts the imaging of the thyroid gland at 700 nm using T14 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 15 depicts the imaging of parathyroid gland at 800 nm using QBN14 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 16 depicts the imaging of parathyroid gland at 700 nm using T14 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 17 depicts the imaging of the adrenal gland at 800 nm using AL27 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 18 depicts the imaging of adrenal gland at 700 nm using E16 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 19 depicts the imaging of salivary glands at 800 nm using ZK211 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 20 depicts the imaging of salivary glands at 700 nm using NRB1 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 21 depicts the imaging of white adipose tissue at 800 nm using AH34 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 22 depicts the imaging of white adipose tissue at 700 nm using PS31 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 23 depicts the imaging of brown adipose tissue at 800 nm using QBN1 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 24 depicts the imaging of brown adipose tissue at 700 nm using SP60 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 25 depicts the imaging of ovaries at 800 nm using AL27 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 26 depicts the imaging of ovaries at 700 nm using PS62 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 27 depicts the imaging of the seminal vesicles at 800 nm using CNN2 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 28 depicts the imaging of the seminal vesicles at 700 nm using LN65 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 29 depicts the imaging of the prostate gland at 800 nm using LN66 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 30 depicts the imaging of the prostate gland at 700 nm using PS62 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 31 depicts the imaging of the pancreas at 800 nm using AL22 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 32 depicts the imaging of the pancreas at 700 nm using T14 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 33 depicts the imaging of the spleen at 800 nm using AL29 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 34 depicts the imaging of the spleen at 700 nm using E24 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 35 depicts the imaging of the gallbladder at 800 nm using ZK198 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 36 depicts the imaging of the gallbladder at 700 nm using PS62 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 37 depicts the imaging of the bile ducts at 800 nm using ZK198 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 38 depicts the imaging of the bile ducts at 700 nm using A106 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 39 depicts the imaging of Peyer's Patches at 800 nm using AL30 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 40 depicts the imaging of the brain vasculature at 700 nm using ZK214 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 41 depicts the imaging of brain grey matter at 800 nm using ZK189 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 42 depicts the imaging of brain grey matter at 700 nm using WuA96 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 43 depicts the imaging of the choroid plexus at 800 nm using ZK208 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 44 depicts the imaging of the choroid plexus at 700 nm using SP28 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 45 depicts the imaging of the cerebrospinal fluid at 800 nm using AL20 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 46 depicts the imaging of the cerebrospinal fluid at 700 nm using SP66 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 47 depicts the imaging of the thoracic duct at 800 nm using A71 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 48 depicts the imaging of thoracic duct at 700 nm using LN15 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 49 depicts the imaging of PEGylated agents at 800 nm using PEG60k-ZW800-1 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 50 depicts the imaging of PEGylated agents at 700 nm using PEG60k-LN15 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 51 depicts the imaging of the pituitary gland at 800 nm using AL22 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 52 depicts the imaging of pituitary gland at 700 nm using SP60 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 53 depicts the imaging of stem cells at 800 nm using PS126 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 54 depicts the imaging of stem cells at 700 nm using PS127 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 55 depicts the imaging of engineered tissue scaffolds and cells at 800 nm using A71-NHS (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 56 depicts the imaging of engineered tissue scaffolds and cells at 700 nm using LN15-NHS (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 57 depicts the imaging of intravital microscopy at 800 nm using Dex70k-ZW800-1 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 58 depicts the imaging of intravital microscopy at 700 nm using Dex70k-LN15 (Image without irradiation, NIR irradiated image, overlay of both)
  • NIR near infrared
  • compositions and methods include the recited elements, but do not exclude other elements.
  • Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.
  • the term “subject” or “patient” encompasses mammals and non-mammals.
  • mammals include, but are not limited to, humans, chimpanzees, apes monkeys, cattle, horses, sheep, goats, swine; rabbits, dogs, cats, rats, mice, guinea pigs, and the like.
  • non-mammals include, but are not limited to, birds, fish, parasites, microbes, and the like.
  • administering refers to providing a compound of the invention and/or prodrugs thereof to a subject in need of diagnosis or treatment.
  • carrier refers to chemical compounds or agents that facilitate the incorporation of a compound described herein into cells or tissues.
  • the term “acceptable” with respect to a formulation, composition or ingredient, as used herein, means having no persistent detrimental effect on the general health of the subject being treated.
  • the term “diluent” refers to chemical compounds that are used to dilute a compound described herein prior to delivery. Diluents can also be used to stabilize compounds described herein.
  • peripheral nerve tissue includes peripheral nerve tissue and cells, including myelinated nerves. Sensory nerves and motor nerves are examples of nerve tissue. Examples of specific nerves include the laryngeal nerve, femoral nerve, brachial plexus, sciatic nerve, pudendal nerves, penile nerves, and the like.
  • nerve tissue also includes brain grey matter and brain white matter.
  • alkyl refers to a straight or branched hydrocarbon radical having from 1-12 carbon atoms, from 1-8 carbon atoms, from 1-6 carbon atoms, or from 1-4 carbon atoms (unless stated otherwise) and includes, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl and the like.
  • An alkyl can be unsubstituted or substituted with one or more suitable substituents.
  • cycloalkyl refers to a monocyclic or polycyclic hydrocarbon ring group having from 3 to 6 carbon atoms, in the hydrocarbon ring (unless stated otherwise) and includes, for example, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, and the like.
  • a cycloalkyl group can be unsubstituted or substituted with one or more suitable substituents.
  • hetero refers to the replacement of at least one carbon atom member in a ring system with at least one heteroatom such as nitrogen, sulfur, and oxygen.
  • heterocyclic means a non-aromatic monocyclic ring having from 2 to 5 carbon atoms in the ring (unless stated otherwise) and at least one heteroatom, preferably, one heteroatom selected from nitrogen, sulfur (including oxidized sulfur such as sulfone or sulfoxide) and oxygen.
  • a heterocycloalkyl group can have one or more carbon-carbon double bonds or carbon-heteroatom double bonds in the ring group as long as the ring group is not rendered aromatic by their presence.
  • heterocyclic groups examples include pyrrolidinyl, piperidinyl, tetrahydropyranyl, and the like.
  • a heterocyclic group can be unsubstituted or substituted with one or more suitable substituents.
  • aryl refers to an unsubstituted or substituted carbocyclic aromatic monocyclic group such as a phenyl group.
  • aryl may be interchangeably used with “aryl ring”.
  • heteroaryl refers to an unsubstituted or substituted heterocyclic aromatic monocyclic group such as a pyridyl, furanyl, or thiophenyl group, and the like. Heteroaryl groups have 5 or 6 atoms in the heteroaromatic ring, 1 of which is independently selected from the group consisting of oxygen, sulfur and nitrogen. Typical heteroaryl groups include, for example, a pyridyl, furanyl, or thiophenyl group.
  • An aryl or heteroaryl can be unsubstituted or substituted with one or more suitable substituents.
  • a “substituent,” as used herein, refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest.
  • a ring substituent may be a moiety such as a halogen, alkyl group, haloalkyl group or other group that is covalently bonded to an atom (preferably a carbon or nitrogen atom) that is a ring member.
  • Substituents of aromatic groups are generally covalently bonded to a ring carbon atom.
  • substitution refers to replacing a hydrogen atom in a molecular structure with a substituent, such that the valence on the designated atom is not exceeded, and such that a chemically stable compound (i.e., a compound that can be isolated, characterized, and tested for biological activity) results from the substitution.
  • certain groups can be unsubstituted or substituted with one or more suitable substituents by other than hydrogen at one or more available positions, typically 1, 2, 3, 4 or 5 positions, by one or more suitable groups (which may be the same or different). Certain groups, when substituted, are substituted with 1, 2, 3 or 4 independently selected substituents. Suitable substituents include halo, alkyl, aryl, hydroxy, alkoxy, hydroxyalkyl, amino, and the like.
  • halogen refers to F, Cl, Br, At, and I.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • the near-infrared fluorescent contrast is a compound of Formula (I):
  • Each R 1 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C 1 -C 6 alkyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or phenyl;
  • Each R 2 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C 1 -C 6 alkyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or phenyl;
  • R 1 and R 2 can be taken together with the carbon atoms to which they are attached to form a 5-6 membered aryl or heteroaryl ring, optionally substituted with halogen, alkyl, alkoxy, hydroxyl, —SO 2 OH, or —CO 2 H;
  • Each R 3 is independently H, OR′, halogen, sulfonato, substituted or un
  • the near-infrared fluorescent contrast is a compound of Formula (II):
  • Each R 1 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C 1 -C 6 alkyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or phenyl;
  • Each R 2 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C 1 -C 6 alkyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or phenyl;
  • R 1 and R 2 can be taken together with the carbon atoms to which they are attached to form a 5-6 membered aryl or heteroaryl ring, optionally substituted with halogen, alkyl, alkoxy, hydroxyl, —SO 2 OH, or —CO 2 H;
  • Each R 3 is independently H, OR′, halogen, sulfonato, substituted or un
  • the near-infrared fluorescent contrast is a compound of Formula (III):
  • Each R 1 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C 1 -C 6 alkyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or phenyl;
  • Each R 2 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C 1 -C 6 alkyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or phenyl;
  • R 1 and R 2 can be taken together with the carbon atoms to which they are attached to form a 5-6 membered aryl or heteroaryl ring, optionally substituted with halogen, alkyl, alkoxy, hydroxyl, —SO 2 OH, or —CO 2 H;
  • Each R 3 is independently H, OR′, halogen, sulfonato, substituted or
  • the near-infrared fluorescent contrast is a compound of Formula (IV):
  • R 1 , R 2 , R 3 , R 4 , R 6 and R 7 are each independently H or C 1 -C 6 alkyl;
  • R 5 , R 8 and R 9 are each independently H, CN, OH, or C 1 -C 6 alkyl; or
  • R 1 and R 3 taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring; or
  • R 2 and R 4 taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring;
  • R 5 and R 6 taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring;
  • R 7 and R 8 taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring; or
  • R 8 and R 9 taken together with the atoms to which they are connected, form an aryl or heteroaryl ring;
  • X is O
  • the near-infrared fluorescent contrast is a compound of Formula (V):
  • Q is —B(R 3 ) 2 —; Si(R 3 ) 2
  • Each R 1 is independently H, alkyl, aryl, or heteroaryl, wherein each alkyl, aryl, or heteroaryl is optionally substituted with alkoxy, alkoxy-N + (alkyl) 3 , alkoxy-OH, halogen, or COOH
  • Each R 2 is independently H, alkyl, aryl, or heteroaryl, wherein each alkyl, aryl, or heteroaryl is optionally substituted with alkoxy, alkoxy-N + (alkyl) 3 , alkoxy-OH, halogen, or COOH
  • Each R 3 is independently H, F, or alkyl; OH or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof.
  • the near-infrared fluorescent biological contract agent is:
  • the compounds may be prepared using a suitable leaving group or reactive group.
  • suitable leaving groups include, but are not limited to N-hydroxysuccinimide (NHS) ester derivatives, sulfo-N-hydroxysuccinimide ester derivatives, and tetrafluorophenyl (TFP) esters.
  • An example of a reactive group would be an azide or an alkyne as used for click chemistry.
  • the near-infrared fluorescent biological contrast agent is: LN15, A104, TG42, or A71.
  • the compounds of the invention absorb light at different wavelengths in the near-infrared region. Specifically, in some embodiments, the compounds of the invention absorb light in the 660-720 nm range. In other embodiments, the compounds of the invention absorb light in the 760-820 nm range.
  • the near-infrared fluorescent biological contract agent is:
  • LN15, A104, or TG42 absorbs light in the 660-720 nm range.
  • the near-infrared fluorescent biological contract agent is A71 and absorbs light in the 760-820 nm range.
  • the compounds of the invention are cell-permeable. In preferred embodiments, the compounds of the invention are not significantly toxic to cells (e.g., to cells in culture or in vivo).
  • the imaging agent of the invention comprises a radioisotope having a single-photon or positron emission decay mode and suitable for detection by single-photon emission tomography (SPECT) or positron emission tomography (PET) in addition to its detection via optical properties (i.e., absorption and/or fluorescence).
  • SPECT single-photon emission tomography
  • PET positron emission tomography
  • suitable radioisotopes include C-11 and F-18.
  • isotopes can be incorporated into a compound of the invention, e.g., by use of appropriate isotopically-enriched reagents during synthesis of the compound.
  • radiotracers such as Ga-68 Zr-89, or Rb-82 (PET), or Tc-99m (SPECT)
  • PET Rb-82
  • SPECT Tc-99m
  • a radiometal chelator such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylene triamine pentaacetic acid (DTPA), hydrazinonicotinic acid (HYNIC), or desferoxime, respectively (or derivatives thereof).
  • DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
  • DTPA diethylene triamine pentaacetic acid
  • HYNIC hydrazinonicotinic acid
  • desferoxime desferoxime
  • Chelator moieties can be covalently attached to an oxazine compound, e.g., through a linking atom or group, e.g., by acylation of a hydroxyl group of a compound of Formula I-V with a carboxylate group of a chelator such as DOTA.
  • a compound according to the invention can be detected using SPECT or PET imaging (e.g., even when administered at a low dose), e.g., using a conventional SPECT or PET imaging system, while also being detectable optically (e.g., by fluorescence imaging), e.g., when administered at a higher dose.
  • Dual-mode optical and SPECT or PET imaging is also possible using such compounds.
  • imaging by magnetic resonance imaging (MRI), including dual-mode optical/MRI imaging can be performed by using a compound of the invention comprising a lanthanide (such as Yb 3+ , Dy 3+ or Gd 3+ ), e.g., by chelating the lanthanide ion using a suitable chelating moiety.
  • a lanthanide such as Yb 3+ , Dy 3+ or Gd 3+
  • Compounds of the invention can be prepared using a variety of methods, some of which are known in the art.
  • the compounds can be prepared using conventional methods of synthetic organic chemistry (see, e.g., Michael B. Smith, “March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th Edition”, Wiley (2013)).
  • compounds of the invention can be synthesized using the syntheses described in schemes 1-12 shown below.
  • General references for the syntheses described in the schemes 1-6, 7a-c, 7e, 8-10, 11, 11a, and 12 is found in Henary, M. et al. Bioorg. & Med. Chem. Let. 22, 242-1246, (2012), Henary, M. et al. J. Heterocycl. Chem. 46: 84-87, (2009), Henary, M. et al. Dyes and Pigments. 99, 1107-1116 (2013), Henary, M. et al. Heterocycl. Commun. 19 (1), 1-11 (2013), Mojzych, M. et al.
  • Scheme 1 outlines the synthesis of a series of 700 nm NIR emitting pentamethine cyanine dye derivatives 10-15.
  • the key step is the quaternization of the nitrogen atom in indolenine derivatives 1-3 in either refluxing acetonitrile or toluene for between 10 hours and 3 days, depending on the alkylating agent, to yield the cationic salts 4-9.
  • a majority of these dyes are constructed via a well-established S NR 1 mechanistic pathway where the meso chlorine atom of the polymethine dye is substituted with various nucleofugal functionalities. This route renders an array of fluorophores that contain highly functional aminoalkyl, hydroxyalkyl, hydroxyaryl, thioalkyl, and thioaryl substituents, which can be further conjugated to ligands or biomolecules.
  • heterocyclic-substituted pentamethine cyanine dyes originating from the indolenine only provides limited chemical space for modification; however, beginning with the 4-substituted phenylhydrazine hydrochloride and upon treatment with a single molar ratio of 3-methyl-2-butanone in acidic conditions (boiling glacial acetic acid) yields the corresponding indolene derivative which is then alkylated using aforementioned synthetic protocol to afford the quaternized nitrogen salts. These salts then react with substituted dianil-malononitrile analogs to yield the highly substituted pentamethine cyanines.
  • Scheme 4 outlines the synthesis of compounds with net charges between +2 to +4 (compound 6 in Scheme 4), which affords the potential for 4 quaternary ammonium cations.
  • the heterocyclic nitrogen of 2 is quaternized with trimethyl(3-bromopropyl)ammonium bromide or other alkylating agent with reaction conditions as described above to give intermediate product 2.
  • the carboxylic acid group of 2 is allowed to react with the free primary amine of (3-aminopropyl)trimethylammonium bromide in the presence of TSTU for 3-5 h under basic conditions to furnish amide 3.
  • the reaction of 3 with the Vilsmeier-Haack reagent 4 provides the chloro-substituted dye 5.
  • Scheme 5 summarizes the synthesis of analogs of NF800 (net charge+2) ether derivatives.
  • the phenolic nature of the Fischer salt heterocycle is modified further with quaternary salts via ether linkage under basic conditions in DMF at 80° C. and the heterocyclic ring nitrogens will be altered with various alkyl groups.
  • To synthesize analogs of NF800 with higher net charge (+4) the alkyl group of the heterocyclic nitrogens is terminated with additional quaternary ammonium salts. This is achieved by synthesizing the quaternary ammonium salt by reacting the 5-hydroxyindolenine compound with iodomethane for 3 days by heating 1,2-dichlorobenzene in a sealed tube.
  • the alcohol is deprotonated using 1.2 molar equivalents of sodium hydride in dry DMF followed by the addition of 3-bromopropyltrimethylammmonium bromide alkylating agent.
  • the reactive methyl group reacts as previously discussed to afford the final target compound 11.
  • the invention provides a method of preparing a compound, the method comprising reacting a decarboxylated bis-aldehyde with a quarternary salt to produce the compound.
  • the method comprises reacting a decarboxylated bis-aldehyde of the formula
  • the carbon-carbon bond coupling provides additional stability to the molecule and the compounds TG42 and A71 are synthesized using the palladium mediated Suzuki-coupling reaction.
  • the synthesis uses 5 molar percent of tetrakistriphenylphosophine palladium(O) for the coupling reaction and cesium carbonate (3 eq) as the base. This reaction proceeds satisfactorily in water or alcoholic water at elevated temperature using the 3-(4-boronophenyl)propanoic acid and meso-halogenated penta- or heptamethine precursors.
  • Pamidronate (PAM)-modified heptamethine fluorophores, PAM-ZW800-1 and PAM-CW800 are prepared originating with protected beta-alanine. Reacting the terminal acid of 3-[(benzyloxycarbonyl)amino]-propionic acid (20 mmol) with oxalyl chloride 100 mmol at 0° C. in dichloromethane for 30 min and at room temperature for 6 h yields the acyl chloride 2 which undergoes nucleophilic acyl substitution with trimethyl phosphite (22 mmol) which was added drop wise at 0° C. for 5 min.
  • the compounds presented in Scheme 10 contain a phosphonic acid group for molecular targeting.
  • (3-bromopropyl)phosphonic acid is used in boiling toluene for 18 h to synthesize the quaternized heterocyclic nitrogen in about 3 days.
  • the dye synthesis proceeds as previously discussed to yield the final Phosphonated 700 nm (P700H and P700SO3) and Phosphonated 800 nm (P800H and P800SO3) Fluorophores.
  • Synthesis of PEGylated 99mTc-MAS3 targeting compound begins by dissolving 1 eq.mol mPEG-NH 2 (2) in dry DMSO followed by the addition of 1.5 eq.mol Boc-Glu(OBzl)-OSu (5-benzyl-1-(2,5-dioxopyrrolidin-1-yl) (tert-butoxycarbonyl)glutamate) (1) and 0.5 eq.mol triethylamine, stirred at room temperature for 5 h, followed by catalytic hydrogenation to remove the Cbz group.
  • NHS ester (4) compound 3 was dissolved in dry THE followed by the addition of TBTU and NHS.
  • trimerized ligands (5) were taken in dry DMSO and added to the mPEG-NHS ester (4) followed by deprotection of the Boc-group. Finally, [ 99m Tc-MAS 3 ]-NHS was added to compound 6 in DMSO and reacted for 1 hr to yield compound 7.
  • ZW800-1 and 99mTc-MAS3 NHS ester can both be modified with various length units of polyethylene glycol modifications through the NHS-ester linkage.
  • the free primary amine on the polyethylene glycol group can easily react with the NHS-ester of the ZW800-1 NHS in PBS at pH 8.0 for 3 h and form the corresponding amide.
  • ZW800-1 must be modified to form the NHS-ester using our effective coupling method of HSPyU in a mixture of DIPEA and DMSO in 30 minutes. Further modifications with the NH 2 —PEG are performed in PBS, pH 8.0 for approximately 3 hours.
  • Pentamethine and heptamethine fluorophores bearing heterocyclic modifications are presented in Scheme 12. They have shown excellent promise in delineating the thyroid and parathyroid at 700 nm and 800 nm. They were prepared according to Scheme 12 using our developed methods as previously discussed in Schemes 1 and 2.
  • the invention provides pharmaceutical compositions of a compound of the invention.
  • compositions which comprise at least one compound provided herein, including at least one compound of the invention, pharmaceutically acceptable salts and/or solvates thereof, and one or more pharmaceutically acceptable carriers, diluents, adjuvant or excipients.
  • the methods of administration of such compounds and compositions include, but are not limited to, intravenous administration, inhalation, oral administration, rectal administration, parenteral, intravitreal administration, subcutaneous administration, intramuscular administration, intranasal administration, dermal administration, topical administration, ophthalmic administration, buccal administration, tracheal administration, bronchial administration, sublingual administration or optic administration.
  • Compounds provided herein are administered by way of known pharmaceutical formulations, including tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for parenteral or intramuscular administration, lotions, gels, ointments or creams for topical administration, and the like.
  • the amount administered will vary depending on, among others, the tissue or organ to be imaged, the age and relative health of the subject, the potency of the compound administered, the mode of administration and the like.
  • Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic/anionic or basic/cationic salts.
  • Pharmaceutically acceptable acidic/anionic salts include acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, malonate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphosphate, polygalactu
  • Pharmaceutically acceptable basic/cationic salts include, the sodium, potassium, calcium, magnesium, diethanolamine, N-methyl-D-glucamine, L-lysine, L-arginine, ammonium, ethanolamine, piperazine and triethanolamine salts.
  • a pharmaceutically acceptable acid salt is formed by reaction of the free base form of a compound of Formula I-V with a suitable inorganic or organic acid including, but not limited to, hydrobromic, hydrochloric, sulfuric, nitric, phosphoric, succinic, maleic, formic, acetic, propionic, fumaric, citric, tartaric, lactic, benzoic, salicylic, glutamic, aspartic, p-toluenesulfonic, benzenesulfonic, methanesulfonic, ethanesulfonic, naphthalenesulfonic such as 2-naphthalenesulfonic, or hexanoic acid.
  • a suitable inorganic or organic acid including, but not limited to, hydrobromic, hydrochloric, sulfuric, nitric, phosphoric, succinic, maleic, formic, acetic, propionic, fumaric, citric, tartaric, lactic, benzo
  • a pharmaceutically acceptable acid addition salt of a compound of Formula I-V can comprise or be, for example, a hydrobromide, hydrochloride, sulfate, nitrate, phosphate, succinate, maleate, formarate, acetate, propionate, fumarate, citrate, tartrate, lactate, benzoate, salicylate, glutamate, aspartate, p-toluenesulfonate, benzenesulfonate, methanesulfonate, ethanesulfonate, naphthalenesulfonate (e.g., 2-naphthalenesulfonate) or hexanoate salt.
  • the free acid or free base forms of the compounds of the invention may be prepared from the corresponding base addition salt or acid addition salt form, respectively.
  • a compound of the invention in an acid addition salt form may be converted to the corresponding free base form by treating with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, and the like).
  • a suitable base e.g., ammonium hydroxide solution, sodium hydroxide, and the like.
  • a compound of the invention in a base addition salt form may be converted to the corresponding free acid by treating with a suitable acid (e.g., hydrochloric acid, etc.).
  • Prodrug derivatives of the compounds of the invention may be prepared by methods known to those of ordinary skill in the art (e.g., for further details see Saulnier et al., Bioorg. Med. Chem. Letters, 1994, 4, 1985; the entire teachings of which are incorporated herein by reference).
  • Protected derivatives of the compounds of the invention may be prepared by means known to those of ordinary skill in the art. A detailed description of techniques applicable to the creation of protecting groups and their removal can be found in T. W. Greene, “Protecting Groups in Organic Chemistry,” 3rd edition, John Wiley and Sons, Inc., 1999, the entire teachings of which are incorporated herein by reference.
  • Compounds of the invention may be prepared as their individual stereoisomers by reaction of a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomers.
  • Resolution of enantiomers may be carried out using covalent diastereomeric derivatives of the compounds of the invention, or by using dissociable complexes (e.g., crystalline diastereomeric salts).
  • Diastereomers have distinct physical properties (e.g., melting points, boiling points, solubility, reactivity, etc.) and may be readily separated by taking advantage of these dissimilarities.
  • the diastereomers may be separated by chromatography, or by separation/resolution techniques based upon differences in solubility.
  • the optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization.
  • a more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture can be found in Jean Jacques, Andre Collet and Samuel H. Wilen, “Enantiomers, Racemates and Resolutions,” John Wiley And Sons, Inc., 1981, the entire teachings of which are incorporated herein by reference.
  • Suitable pharmaceutically acceptable carriers, diluents, adjuvants, or excipients for use in the pharmaceutical compositions of the invention include tablets (coated tablets) made of for example collidone or shellac, gum Arabic, talc, titanium dioxide or sugar, capsules (gelatin), solutions (aqueous or aqueous-ethanolic solution), syrups containing the active substances, emulsions or inhalable powders (of various saccharides such as lactose or glucose, salts and mixture of these excipients with one another) and aerosols (propellant-containing or -free inhale solutions).
  • tablets coated tablets
  • collidone or shellac made of for example collidone or shellac, gum Arabic, talc, titanium dioxide or sugar, capsules (gelatin), solutions (aqueous or aqueous-ethanolic solution), syrups containing the active substances, emulsions or inhalable powders (of various saccharides such as lactose or glucose, salts and mixture of
  • Excipients which may be used include, for example, water, pharmaceutically acceptable organic solvents such as paraffins (e.g., petroleum fractions), vegetable oils (e.g. groundnut or sesame oil), mono- or polyfunctional alcohols (e.g., ethanol or glycerol), carriers such as natural mineral powders (e.g., kaoline, clays, talc, chalk), synthetic mineral powders (e.g., highly dispersed silicic acid and silicates), sugars (e.g., cane sugar, lactose and glucose), emulsifiers (e.g., lignin, spent sulphite liquors, methylcellulose, starch and polyvinylpyrrolidone) and lubricants (e.g., magnesium stearate, talc, stearic acid and sodium lauryl sulphate).
  • paraffins e.g., petroleum fractions
  • vegetable oils e.g. groundnut or sesame oil
  • the present invention features various methods using the near-infrared fluorescent biological contrast agents described herein.
  • the invention provides a method of imaging biological tissue or cells, the method comprising:
  • the signal may be in the form of absorption, such as occurs during photoacoustic imaging.
  • the imaging agents can have a SBR suitable to permit fluorescence detection.
  • SBR is a measure of the intensity of the fluorescent signal obtained from a target (peak signal) over the measure of the intensity of the fluorescent signal obtained nearby the target (background signal), the target being the tissues, cells, space targeted by the imaging agent.
  • SBR measurements can be readily obtained through routine measurement procedures. For fluorescent imaging systems, and other optical-type systems, digital images recording optical signals of the target facilitate SBR measurement. Higher SBR values are more desirable, resulting in greater resolution of the imaged tissues.
  • the imaging agents achieve an SBR of at least about 1.1 (i.e., peak signal is at least 10% over background).
  • the imaging agents achieve an SBR of at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, or at least about 2.0. In yet further embodiments, the imaging agents achieve an SBR of about 1.1 to about 50, about 1.5 to about 30, about 2.0 to about 20, about 2.0 to about 5.0, or about 5.0 to about 10.
  • the imaging agents include one or more ionic groups.
  • the imaging agents include two or more, three or more, four or more, or five or more ionic groups.
  • Ionic groups serve to increase solubility of the generally hydrophobic dye portions of the imaging agent, thus improving biodistribution. They may also contribute to specific targeting.
  • the ionic groups can be located on any portion of the imaging agent.
  • the imaging agents are hydrophobic agents.
  • the hydrophobic agents are capable of conjugating to a hydrophobic compound for imaging, without altering the binding, biodistribution, cell permeation, and/or clearance of the hydrophobic compound.
  • hydrophobic agents include, but are not limited to L700-1A, L700-1C, L800-1A, and L800-1C.
  • the imaging agents are administered directly to a subject or biological system for the imaging of the targeted cells.
  • reactive derivates of the imaging agents of the invention are used to label chemical and biological molecules for further study.
  • Certain molecules which may be labeled using reactive derivatives of the imaging agents of the invention include small molecules (including pharmaceutical, neutraceutical, therapeutic and diagnostic compounds, proteins, peptides, peptidomimetics, antibodies, vaccines, and other chemical and biological molecules which may be of interest in studying by NIR imaging.
  • the imaging agent of the invention is reacted with the chemical or biological molecule to produce a labeled agent molecule which may then be administered to a subject or biological system for imaging as described herein.
  • the steps of irradiating the tissue or cells at a wavelength absorbed by the imaging agent, and detecting an optical signal from the irradiated tissue or cells, thereby imaging the tissue or cells can be performed using an imaging system such as the FLARETM Image-Guided Surgery System, which is a continuous-wave (CW) intraoperative imaging system that is capable of simultaneous, real-time acquisition and display of color video (i.e., surgical anatomy) and two channels of invisible NIR fluorescent (700 nm and 800 nm) light (see, e.g., Gioux et al., Mol. Imaging. 9(5): 237-255 (2010) and U.S. Pat. No. 8,473,035 to Frangioni, for a description of suitable systems).
  • FLARETM Image-Guided Surgery System which is a continuous-wave (CW) intraoperative imaging system that is capable of simultaneous, real-time acquisition and display of color video (i.e., surgical anatomy) and two channels of invisible NIR fluorescent (700 nm and 800 nm) light
  • contrast agent emission wavelength in the 800-850 nm range (Channel 2 of FLARETM) is preferred whenever possible because of lower autofluorescence and lower attenuation from both absorbance and scatter when compared to emission near 700 nm. Nevertheless, fluorophores emitting within Channel 1 ( ⁇ 700 nm) of the FLARETM imaging system still retain the benefits of NIR fluorescence imaging, including detection of nerves and other targets below the surface of blood and tissue.
  • the imaging agents can be formulated into pharmaceutically acceptable formulations and administered intravenously to an organism for imaging.
  • the dosed organism can be imaged using, for example, the FLARETM system.
  • the imaging system can irradiate the dosed organism with radiation absorbed by the imaging agent, and detect optical signals, such as NIR fluorescence, emanating from the targeted portions of the organism containing the imaging agent.
  • the detected signals can be recorded and analyzed by obtaining digital images or video of the subject organism, thereby facilitating diagnostic procedures and image-guided medical techniques.
  • the invention also provides methods of performing image-guided surgery, the methods comprising imaging cells, tissues, or organs according to a method described herein, and then performing surgery such that the targets are either removed or are preserved, depending on the goals of the surgical intervention.
  • the contrast agent is injected intravenously to ensure that all targets are labeled, and imaging is performed after sufficient time has passed for biodistribution to nerves and clearance of surrounding background signal.
  • the targets are biological tissues or organs.
  • the targets are lumens, such as the ureters, cartilage, bone cells, bone minerals, thyroid gland, parathyroid gland, adrenal gland, salivary gland, white adipose tissue, brown adipose tissue, ovaries, testes, seminal vesicles, prostate, pancreas, spleen, gallbladder, bile ducts, Peyer's patches, brain grey matter, brain white matter, brain vasculature, choroid plexus, cerebrospinal fluid, nerves, thoracic duct, pan lymph nodes, sentinel lymph nodes, vulnerable plaque, stem cells, or neuroendocrine tumor cells.
  • a NIR fluorophore injected into the bloodstream can act as an angiographic agent because during the first 8 seconds after intravenous injection there is a rapid arterial flush ( ⁇ 1 second), a rapid capillary flush (2-3 seconds), a rapid venous flush ( ⁇ 1-2 seconds), then minutes of clearance from the tissue. The first 8 seconds thus provides a “map” of the circulation in the tissue.
  • NIR angiography is important for imaging the perfusion of skin during plastic and reconstructive surgery and the anastomoses of bowel during gastrointestinal surgery. In general, NIR angiography agents are those that are cleared rapidly from the blood into either urine or bile.
  • the invention provides a method for imaging tissue perfusion, the method comprising:
  • the compound of the invention for angiography is LN15, A104, or TG42; and the irradiating wavelength is in the 660-700 nm range.
  • the compounds of the invention for use in angiography is A71 or WuA71; and the irradiating wavelength is in the 760-800 nm range.
  • Ureter mapping agents are those molecules that are rapidly cleared from the bloodstream by the kidney into urine. As the molecules traverse the ureters towards the bladder, the ureters become highly fluorescent and thus visible. This is useful during Caesarian section, where the ureters are sometimes damaged, as well as many abdominal cancer surgeries where finding the ureters and avoiding them can be difficult.
  • the invention provides a method for imaging the ureters, the method comprising:
  • the compound of the invention for use in ureter imaging is LN15, A104, or TG42; and the irradiating wavelength is in the 660-700 nm range.
  • the compounds of the invention for use in ureter imaging is A71 or WuA71; and the irradiating wavelength is in the 760-800 nm range.
  • Cartilage agents are useful in arthroscopic surgery, general surgery, and non-invasive assessment of neo-cartilage growth during tissue engineering.
  • the invention provides a method for imaging cartilage cells and/or their products, the method comprising:
  • the compound of the invention for use in cartilage imaging is SP56, E58, YY180, E59, E60, A196, E71, E72 or ZK15; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in cartilage imaging is LN50, A64, AL30, MM25, MM21, AL31, AL33, SP79, SP99, SP116, SP117, LN65, LN68, LN63, ZK48, CNN3, or CNN4; and the irradiating wavelength is in the 760-800 nm range.
  • Bone agents are useful in the detection of bone metastases, bone growth and tissue microcalcification.
  • the invention provides a method for imaging bone, the method comprising:
  • the compound of the invention for use in bone imaging is P700SO3, P700H, CMI24, E24, E37, E38, E44, or WuA110; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in bone imaging is P800SO3, P800H, ZK197, or WuA71; and the irradiating wavelength is in the 760-800 nm range.
  • the invention provides a method for imaging the thyroid gland, the method comprising:
  • the compound of the invention for use in thyroid imaging is T14, T27, T29, MHI84, T18, T20, T23, T24, T25, LO4, E27, E45, MHI106, MHI128, TP1, NRB3, SP28, SP29, SP30, SP33, SP34, SP51, SP59, SP60, SP72, PTN11, PTN12, ZK26, ZK143, ZK148, or ZK204; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in thyroid imaging is QBN14, QBN1, AL20, ZK172, ZK185, ZK190, ZK208, MDL17, CNN145, ZK154, or ZK159; and the irradiating wavelength is in the 760-800 nm range.
  • the invention provides a method for imaging the parathyroid gland, the method comprising:
  • the compound of the invention for use in parathyroid imaging is T14, T27, T29, or MHI84; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in parathyroid imaging is QBN14, MDL17, QBN1, or AL20; and the irradiating wavelength is in the 760-800 nm range.
  • Adrenal gland agents are useful to highlight the adrenal gland after an intravenous injection.
  • the invention provides a method for imaging adrenal medulla and/or adrenal cortex, the method comprising:
  • the compound of the invention for use in adrenal gland imaging is E16, MHI96, MHI97, EAO40, MHI186, LS1, LO3, LO4, E24, E27, E36, E37, E43, E45, E50, E51, E77, E79, E80, ZK50, ZK59, ZK106, SP29, SP30, SP33, SP51, SP53, SP60, SP64, YY161, YY163, or YY165; and the irradiating wavelength is in the 60-700 nm range.
  • the_compound of the invention for use in adrenal gland imaging is AL27, AL25, AL29, AL20, ZK190, ZK184, or MDL17; and the irradiating wavelength is in the 760-800 nm range.
  • Salivary gland agents are useful for targeting salivary gland tumors or for avoiding normal salivary glands during head and neck surgery.
  • the invention provides a method for imaging salivary glands, the method comprising:
  • the compound of the invention for use in salivary gland imaging is NRB1, ZK195, ZK135, NRB2, YY163, ZK195, YY161, E79, TP1, SP28, SP29, SP30, SP49, SP72, ZK101, ZK133, ZK134, ZK135, ZK143, ZK150, ZK155, ZK156, ZK159, ZK185, ZK204, T29, or CNN145; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in salivary gland imaging is ZK211, ZK172, MDL17, ZK198, ZK190, AL22, or AL20; and the irradiating wavelength is in the 760-800 nm range.
  • White adipose tissue agents are useful for highlighting white fat important for certain surgical procedures.
  • the invention provides a method for imaging white adipose tissue, the method comprising:
  • the compound of the invention for use in white adipose imaging is PS31, CMI26, E24, MHI86, ZK240, or ZK244; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in white adipose imaging is AH34, PS37, or ZK197; and the irradiating wavelength is in the 760-800 n, range.
  • Brown adipose tissue agents are useful for highlighting brown fat during surgery and for “imaging” perfusion of the tissue.
  • the invention provides a method for imaging brown adipose tissue, the method comprising:
  • the compound of the invention for use in brown adipose tissue imaging is SP60, PS39, SP30, SP29, SP33, SP34, E39, E44, E51, E81, ES17, ZK27, ZK26, SP28, SP27, SP67, PS31, LO1, LO3, YY165, or YY187; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in brown adipose tissue imaging is QBN1 or PS37; and the irradiating wavelength is in the 760-800 nm range.
  • Ovary-specific agents have two major functions. The first is to find and eradicate endometriosis, a painful condition of pre-menopausal women. The second is to find and eradicate ovarian carcinoma.
  • the invention provides a method for imaging ovaries, the method comprising:
  • the compound of the invention for use in ovarian imaging is PS62 or E43; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in ovarian imaging is AL27 or CNN5; and the irradiating wavelength is in the 760-800 nm range.
  • Testes-specific agents are useful for the highlighting of testicular tumor cells.
  • these agents can be used to aid aggressive metastasectomy treatments in advanced Stage IV patients prior to cytotoxic therapy.
  • the invention provides a method for imaging testicular cells, the method comprising:
  • Seminal vesicle agents are useful in assisting a urologist during robotic or open prostatectomy to ensure removal of all seminal vesicles.
  • the invention provides a method for imaging seminal vesicle cells, the method comprising:
  • the compound of the invention for use in seminal vesicle imaging is LN65, YY269, or Ox4; and the irradiating wavelength is in the 660-700 nm range.
  • the-compound of the invention for use in seminal vesicle imaging is CNN2, CNN4, ZK48, LN50, TG66, LN66, AL31, or AL30; and the irradiating wavelength is in the 760-800 nm range.
  • Prostate gland and/or prostate cancer agents are useful during robotic or open prostatectomy.
  • the invention provides a method for imaging prostate cells, the method comprising:
  • the compound of the invention for use in prostate imaging is PS62; and the irradiating wavelength is in the 660-700 nm range.
  • the compounds of the invention for use in prostate imaging is LN66, TG66, CNN4, or CNN10; and the irradiating wavelength is in the 760-800 nm range
  • the compounds of the invention can be conjugated to a prostate-specific membrane antigen (PSMA) targeting ligand.
  • PSMA prostate-specific membrane antigen
  • the invention provides a method for imaging pancreas, the method comprising:
  • the compound of the invention for use in pancreas imaging is T14, PS62, SRA94, SRA89, SP28, SP29, ESS61, or T27; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in pancreas imaging is AL22, CNN145, Rh800, Ox750, WuA96, or Ox170; and the irradiating wavelength is in the 760-800 nm range.
  • the invention provides a method for imaging the spleen and accessory splenic tissue, the method comprising:
  • the compound of the invention for use in spleen imaging is E24, E44, E37, E38, E39, E43, E50, E51, E78, LO1, PTN1, AL79, SP27, TG5, TP5, EAO42, PS31, SP34, TG115, MHI86, MHI96, or MHI97; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in spleen imaging is AL29, LS1, AH34, JM1, ZK166, ZK189, ZK197, ZK198, AL27, AL25, MDL16, ZK215, or ZK184; and the irradiating wavelength is in the 760-800 nm range.
  • Gallbladder contrast agents help localize the gallbladder during laparoscopic surgery and also help highlight the cystic duct.
  • the invention provides a method for imaging gallbladder, the method comprising:
  • the compound of the invention for use in gallbladder imaging is PS62, SRA94, SRA89, AC2, ESS61, A106, YY261, SP67, P700H, CNN13, ZK140, or ZK14; and the irradiating wavelength is in the 660-760 nm range.
  • the compound-of the invention for use in gallbladder imaging is ZK198, ZK208, ZK166, WuA71, or P800H; and the irradiating wavelength is in the 760-800 nm range.
  • the invention provides a method for imaging the bile ducts, the method comprising:
  • the compound of the invention for use in bile duct imaging is A106, CNN13, ZK140, SRA89, WuA96, Ox170, Ox750, Ox4, ESS61, ZK14, CNN16, CNN145, MHI84, P700H, CNN12, or CNN14; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in bile duct imaging is ZK198, ZK166, ZK208, P800H, MDL16, or WuA71; and the irradiating wavelength is in the 760-800 nm range.
  • Peyer's patches are small collections of lymphatic tissue that protect the mucosal membranes of the GI tract. Prior to the invention, they were extremely difficult to image in living organisms.
  • the invention provides a method for imaging Peyer's patches the method comprising:
  • the compound of the invention for use in imaging Peyer's patches is AL30; and the irradiating wavelength is in the 760-800 nm range.
  • Brain Grey Matter agents typically have the following features: 1) MW ⁇ 500 Da, 2) Log D at pH 7.4 between 0.5 and 3, and 3) retained by cell bodies of the brain (grey matter).
  • the low MW and lipophilic (but not too lipophilic) Log D permit crossing of the blood brain barrier. These molecules are important for various types of brain surgery, especially resection of tumors, where highlighting of normal brain is so important.
  • the invention provides a method for imaging brain grey matter cells, the method comprising:
  • the compound of the invention for use in brain grey matter imaging is WuA96, or ZK104; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in brain grey matter imaging is ZK189; and the irradiating wavelength is in the 760-800 nm range.
  • the invention provides a method for imaging brain white matter, comprised of nerve axons and associated glia, the method comprising:
  • Brain Vasculature Agents bind to either the arterial tree or vascular tree of the brain.
  • the invention provides a method for imaging brain vasculature, the method comprising:
  • the compound of the invention for use in brain vasculature imaging is ZK214, or ZK104; and the irradiating wavelength is in the 660-700 nm range.
  • the choroid plexus is the tissue that filters blood to produce cerebrospinal fluid (CSF). Several agents enter the choroid plexus but get trapped in this tissue while attempting to traverse the blood brain barrier.
  • CSF cerebrospinal fluid
  • the invention provides a method for imaging choroid plexus, the method comprising:
  • the compound of the invention for use in choroid plexus imaging is SP28, ZK135, SP66, ZK195, SP29, SP30, SP33, SP49, SP51, ZK78, ZK134, ZK135, ZK143, ZK140, ZK26, ZK78, ZK79, ZK133, ZK23, ZK101, SP66, SP72, MHI84, T14, T18, T20, or T23; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in choroid plexus imaging is ZK208, ZK185, AL22, ZK172, MDL16, ZK211, ZK153, ZK155, or ZK169; and the irradiating wavelength is in the 760-800 nm range.
  • Certain molecules of the invention can completely traverse the choroid plexus and enter the CSF. They are particularly useful for finding CSF before accidentally puncturing the meninges, or for finding and repairing tears in the meninges.
  • the invention provides a method for imaging cerebrospinal fluid, the method comprising:
  • the compound of the invention for use in cerebrospinal fluid imaging is SP66, SP43, SP72, SP28, MHI84, YY161, or YY163; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in cerebrospinal fluid imaging is AL20, ZK189, or ZK208; and the irradiating wavelength is in the 760-800 nm range.
  • the invention provides a method for imaging the pituitary gland, the method comprising:
  • the compound of the invention for use in pituitary imaging is SP60, SP64, SP28, SP29, SP30, SP33, SP34, SP43, SP51, SP53, ZK159, MHI84, YY187, SP59, SP67, ZK23, ZK204, ZK106, AL11, SP66, E79, E80, ES21, or LO3; and the irradiating wavelength is in the 660-700 nm range.
  • the compounds of the invention for use in pituitary imaging is AL22, ZK185, ZK208, ZK172, ZK190, QBN1, QBN14, ZK153, ZK156, AL25, AL29, AL20, MDL17, or MDL16; and the irradiating wavelength is in the 760-800 nm range.
  • the invention provides a method for imaging the thoracic duct, the method comprising:
  • the compound of the invention for use in thoracic duct imaging is LN15, A104, or TG42; and the irradiating wavelength is in the 660-700 nm range.
  • in thoracic duct imaging is A71, ZW800-1, or WuA71; and the irradiating wavelength is in the 760-800 nm range.
  • Sentinel lymph node mapping has revolutionized the treatment of breast cancer and melanoma.
  • 20-25% of patients are found to have tumor cells in their sentinel lymph node and therefore require a completion lymphadenectomy, i.e., removal of all the lymph nodes in the basin. Finding all lymph nodes in an area of the body is extremely difficult to do.
  • Pan-lymph node mapping agents that highlight all lymph nodes after a single intravenous injection are useful during many types of surgery. They can also be used in conjunction with a sentinel lymph node agent to find both sentinel lymph nodes and all lymph nodes in a particular basin.
  • the invention provides a method for imaging lymph nodes, the method comprising:
  • the compound of the invention for use in lymph node imaging is A150, A146, A149, A148, A160, A161, A20, SP27, SP43, SP117, ZK197, ZK134, ZK46, ZK101, AL11, AL12, EAO42, ZK148, E16, E50, E51, E77, E78, E58, E59, E60, E70, E72, LO1, LO2, PTN11, ZK143, ZK140, ZK29, WuA108, MHI86, MHI96, or MHI97; and the irradiating wavelength is in the 660-700 nm range.
  • the compounds of the invention for use in lymph node imaging is MM25, A64, AL30, AL33, PTN6, AH34, CNN10, MM21, CNN5, A71, MDL16, ZK172, LN63, ZK154, or ZK155; and the irradiating wavelength is in the 760-800 nm range.
  • Sentinal lymph node agents are injected in and around a tumor and quickly flow to the first lymph node that drains the tumor, called the sentinel lymph node (SLN).
  • SSN sentinel lymph node
  • the invention provides a method for imaging sentinel lymph nodes, the method comprising:
  • the compound of the invention for use in sentinel lymph imaging is MHI86, MHI96, MHI97, A150, A146, A149, A148, A160, A161, A20, E37, or E78; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in sentinel lymph node imaging is MM25, A64, AL30, or AL33; and the irradiating wavelength is in the 760-800 nm range.
  • certain compounds of the invention may be taken up by vulnerable plaque and therefore highlight areas of intima that are at a higher risk for rupture.
  • the invention provides a method for imaging vulnerable plaque cells, the method comprising:
  • the invention provides a method for imaging stem cells, the method comprising:
  • the compound of the invention for use in stem cell imaging is PS127, PS129, PS131, PS133, CNN12, CNN13, CNN14, CNN16, CNN17, Ox4, Ox170, ZK126, ZK211, ZK214, or EAO40; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in stem cell imaging is PS126, PS128, PS130, or PS132; and the irradiating wavelength is in the 760-800 nm range.
  • the compounds of the invention for use in stem cell imaging have primary or secondary amines as part of their structure, which permit covalent fixation in place after treatment with paraformaldehyde (Mannich reaction) or other amine-reactive fixatives.
  • Biodegradable scaffolds have been extensively used in the field of tissue engineering and regenerative medicine.
  • Tissue Engineering Agents provide noninvasive monitoring of in vivo scaffold degradation or product formation.
  • the invention provides a method for imaging biodegradable scaffolds, the method comprising:
  • the compound of the invention for use in imaging biodegradable scaffolds is A71-NHS ester, MHI103, CNN12, CNN13, CNN14, CNN16, CNN17, Ox4, Ox170, ZK126, ZK211, ZK214, EAO40, E59, EAO42, PTN12, E72, E24, E27, E50, E51, E79, E80, E81, ES17, ES21, LO1, LO2, T17, T23, T25, A106, A148, A150, A161, or AC8; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in imaging biodegradable scaffolds is LN15-NHS ester, LN68, LN50, CNN3, LN65, LN63, or ZK166; and the irradiating wavelength is in the 760-800 nm range.
  • the compounds of the invention are amine-containing or meso-brominated compounds which are conjugated to biodegradable scaffolds.
  • Neuroendocrine tumors are a group of rare tumors that show similar growth patterns and resistance to chemotherapy. They occur throughout the body and, although primary tumors are often curable by surgery, they are difficult to find when they are small.
  • a particularly vexing group of neuroendocrine tumors are the pancreatic endocrine tumors, comprised of gastrinoma, insulinoma, glucagonoma, VIPoma, and somatostatinoma.
  • Fluorophores targeted to pancreatic endocrine tumors provide surgeons with sensitive, specific and real-time image guidance after a single preoperative, intravenous injection.
  • the invention provides a method for imaging neuroendocrine tumors, the method comprising:
  • the compound of the invention for use in neuroendocrine tumor imaging is ESS61, SRA89, SRA94, CNN145, MHI84, Ox4, Ox170, Ox750, WuA96, CNN16, CNN12, or CNN14; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use in neuroendocrine tumor imaging is AL20, AL22, AL33, or AL30; and the irradiating wavelength is in the 760-800 nm range.
  • Hydrophobic molecules are often administered to a subject for various therapeutic and diagnostic purposes. Hydrophobic Fluorophores can conjugate to such molecules and allow for the imaging and study of the distribution of such molecules.
  • the invention provides a method for imaging a hydrophobic molecule in a biological system, the method comprising:
  • the compound of the invention for use with hydrophobic molecules is L700-1A and L700-1C; and the irradiating wavelength is in the 660-700 nm range.
  • the compound of the invention for use with hydrophobic molecules is L800-1A and L800-1C; and the irradiating wavelength is in the 760-800 nm range.
  • Vascular functions rely on the endothelial cells lining the vasculature to provide a semi-permeable barrier between blood contents and the tissue interstitium.
  • fluorophores should be large to circulate longer in the bloodstream.
  • PEGylation or bulky dextran conjugation may be used to increase the blood half-life of bioactive small molecules and peptides.
  • PEG polyethylene glycol
  • the compounds of the invention may be modified to include a polyethylene glycol group.
  • PEGylated compounds may be branched or linear.
  • the linear PEGylated compounds are in the range of about 20 kDa to about 60 kDa.
  • the compound of the invention is LN15, A104, or TG42; each of which is conjugated with linear or branched PEG of 60 kDa, 40 kDa, 20 kDa, or 100 kDa, or Dextran of 70 kDa, 100 kDa, or 150 kDa.
  • the compounds of the invention is ZW800-1-, A71-, or WuA71 each of which is conjugated with linear or branched PEG of 60 kDa, 40 kDa, 20 kDa, or 100 kDa, or Dextran of 70 kDa, 100 kDa, or 150 kDa.
  • the compounds of the invention can be conjugated to a metal chelator agent for use in single-photon emission computed tomography (SPECT), positron emission tomography (PET), and/or magnetic resonance imaging (MRI).
  • SPECT single-photon emission computed tomography
  • PET positron emission tomography
  • MRI magnetic resonance imaging
  • the metal chelator agent is a DOTA, DTPA, hydrazinonicotinic acid (HYNIC), or desferoxime, or a derivative thereof.
  • the metal atom is selected from the group consisting of Zr-89, Ga-68 and Rb-82, and the signal is detected by positron emission tomography; the metal atom is selected from the group consisting of Tc-99m and In-111, and the signal is detected by single-photon emission computed tomography; or the metal atom is a lanthanide selected from the group consisting of Gd, Dy and Yb, and the signal is detected by magnetic resonance imaging.
  • the compound of the invention is ZW800-1, A71, or LN15, conjugated to either DOTA, DTPA, or deferoxamine.
  • the dual-NIR channel FLARE a imaging system has been described in detail previously (26-28). It provides simultaneous illumination with white light (400-650 nm) at 40,000 lx, 660 nm NIR Channel 1 excitation at 4 mW/cm 2 and 760 bmm NIR Channel 2 excitation at 10 mW/cm 2 . Color and two independent NIR fluorescence emission images ( ⁇ 700 nm for Channel 1 and ⁇ 800 nm for Channel 2) were acquired simultaneously with custom software at rates up to 15 Hz over a 15 cm diameter field of view. NIR fluorescence from Channel 1 was pseudo-colored in red and NIR fluorescence from Channel 2 was pseudo-colored in lime green prior to merger with the color video image. The imaging system was positioned at a distance of 18 inches from the surgical field.
  • mice, rats, and pigs were used in the animal studies described below, either sex of 25 g CD-1 mice (Charles River Laboratories, Wilmington, Mass.) and either sex of 250 g Sprague-Dawley (SD) rats (Taconic Farms, Germantown, N.Y.) were used after anesthetizing with 100 mg/kg ketamine and 10 mg/kg xylazine intraperitoneally (Webster Veterinary, Fort Devens, Mass.). Either sex of Yorkshire pigs (E. M.
  • each NIR fluorophore was injected intravenously in CD-1 mice and sacrificed animals 1-4 h post-injection (n>3).
  • Target tissues/organs were observed at indicated time points such as 0, 5, 10, 15, 30, 60, 120, 180, and 240 min with the FLARETM imaging system.
  • animals were sacrificed, and the target tissue and other major organs including heart, lung, liver, spleen, pancreas, kidneys, duodenum, intestine, and muscle were resected to quantify biodistribution and clearance.
  • an optimized dose (10-1000 nmol) was injected depending on the targeting purpose, and targeting and biodistribution were observed 4 h post-injection (n>3).
  • the appropriate dose was calculated based on the previous dose dependence study in the small animal study.
  • 0.5-10 ⁇ mol of the NIR fluorescence was injected through the external jugular vein (n>3). Then the target tissue and surrounding organ were imaged at the indicated time points (0, 1, 5, 10, 15, 30, 60, 90, 120, 180, and 240 min).
  • Ureters A 35 kg female pig was injected intravenously at time zero with 5 ⁇ mol of compound LN15 (700 nm) or A71 (800 nm) dissolved in saline or D5W. After a waiting period of 30 min the animal was surgically exposed and the ureters were imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively over the next 4 hours. As shown in FIGS. 1 and 2 , the ureter is highlighted with high contrast using this compound.
  • Pan LN A 250 g male rat was injected intravenously at time zero with 20 nmol of compound A150 (700 nm) or MM25 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the pan lymph nodes were imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 3 and 4 , all lymph nodes are highlighted with high contrast using this compound.
  • SLN A 35 kg female pig was injected subcutaneously into bowel at time zero with 5 nmol of compound MHI86 (700 nm) or MM25 (800 nm) dissolved in saline or D5W. After a waiting period of 5 min, the target tissue was exposed and the SLN was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 5 and 6 , the SLN is highlighted with high contrast using this compound.
  • Cartilage A 35 kg female pig was injected intravenously at time zero with 5 ⁇ mol of compound SP56 (700 nm) or LN50 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the spinal cartilage was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 7 and 8 , the cartilage is highlighted with high contrast using this compound.
  • Neuroendocrine Tumors A 25 g male insulinoma-bearing mouse was injected intravenously at time zero with 140 nmol of compound ESS61 (700 nm) or AL20 (800 nm) dissolved in saline or D5W. After a waiting period of 1 hour, the animal was surgically exposed and the pancreas was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 9 and 10 , the tumors are highlighted with high contrast using this compound.
  • Bone A 35 kg female pig was injected intravenously at time zero with 5 ⁇ mol of compound P700SO3 (700 nm) or P800SO3 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the rib cage was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 10 and 11 , the bone is highlighted with high contrast using this compound.
  • Thyroid A 35 kg female pig was injected intravenously at time zero with 5 ⁇ mol of compound T14 (700 nm) or QBN14 (800 nm) dissolved in saline or D5W.
  • the animal was surgically exposed and the thyroid was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 12 and 13 , the thyroid is highlighted with high contrast using this compound.
  • Parathyroid A 35 kg female pig was injected intravenously at time zero with 5 ⁇ mol of compound T14 (700 nm) or QBN14 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the parathyroid was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively.
  • Adrenal A 35 kg female pig was injected intravenously at time zero with 5 ⁇ mol of compound E16 (700 nm) or AL27 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the adrenal was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in the FIGS. 17 and 18 , the adrenal is highlighted with high contrast using this compound.
  • Salivary glands A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound NRB1 (700 nm) or ZK211 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the salivary glands were imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 19 and 20 , the salivary glands are highlighted with high contrast using this compound.
  • White adipose tissue A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound PS31 (700 nm) or AH34 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the white adipose tissue was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 21 and 22 , the white adipose tissue is highlighted with high contrast using this compound.
  • Brown adipose tissue A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound SP30 (700 nm) or QBN1 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the brown fat was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 23 and 24 , the brown fat is highlighted with high contrast using this compound.
  • Ovaries A 25 g female mouse was injected intravenously at time zero with 25 nmol of compound PS62 (700 nm) or AL27 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the ovaries were imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 25 and 26 , the ovaries are highlighted with high contrast using this compound.
  • Seminal vesicles A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound LN65 (700 nm) or CNN2 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the seminal vesicle was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 27 and 28 , the seminal vesicle is highlighted with high contrast using this compound.
  • Prostate A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound PS62 (700 nm) or LN66 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the prostate was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 29 and 30 , the prostate is highlighted with high contrast using this compound.
  • Pancreas A 35 kg female pig was injected intravenously at time zero with 5 ⁇ mol of compound T14 (700 nm) or AL22 (800 nm) dissolved in saline or D5W.
  • Gallbladder A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound PS62 (700 nm) or ZK198 (800 nm) dissolved in saline or D5W.
  • Bile ducts A 35 kg female pig was injected intravenously at time zero with 5 ⁇ mol of compound A106 (700 nm) or ZK198 (800 nm) dissolved in saline or D5W.
  • Brain vasculature A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound ZK214 (700 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the brain vasculature was imaged for NIR fluorescence using Channel 1 (700 nm) of the FLARE imaging system. As shown in FIG. 40 , the brain vasculature is highlighted with high contrast using this compound.
  • Brain grey matter A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound WuA96 (700 nm) or ZK189 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the brain grey matter was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 41 and 42 , the brain grey matter is highlighted with high contrast using this compound.
  • Choroid plexus A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound SP28 (700 nm) or ZK208 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the choroid plexus was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 43 and 44 , the choroid plexus is highlighted with high contrast using this compound.
  • CSF A 35 kg female pig was injected intravenously at time zero with 5 ⁇ mol of compound SP66 (700 nm) or AL20 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the CSF was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 45 and 46 , the CSF is highlighted with high contrast using this compound.
  • Thoracic duct A 35 kg female pig was injected subcutaneously into the lower leg at time zero with 5 ⁇ mol of compound A106 (700 nm) or ZK198 (800 nm) dissolved in saline or D5W. After a waiting period of 30 minutes, the animal was surgically exposed and the thoracic duct was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 47 and 48 , the thoracic duct is highlighted with high contrast using this compound.
  • PEGylated agents A 25 g female xenograft tumor-bearing mouse was injected intravenously at time zero with 10 nmol of compound PEG60k-LN15 (700 nm) or PEG60k-ZW800-1 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hour, the tumor was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in the FIGS. 49 and 50 , the tumor is highlighted with high contrast using this compound.
  • Pituitary gland A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound SP60 (700 nm) or AL22 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the pituitary gland was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 51 and 52 , the pituitary gland is highlighted with high contrast using this compound.
  • Stem cell tracking Cells grown in 35 mm or 60 mm plates at 70% confluence were incubated in cell culture media with 2 ⁇ M compound PS127 (700 nm) or PS126 (800 nm) in the 37° C. incubator with 5% CO 2 . After an incubation time of 30 min, cells were washed 3 times with warm media, followed by fixing in 2% paraformaldehyde for 30 min. After centrifuge, cell pellets were frozen in OCT, and tested after washing with acetone. Cell pellets were cut to a 10 ⁇ m thickness and imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of fluorescence microscope, respectively. As shown in FIGS.
  • a biodegradable scaffold (1 cm ⁇ 1 cm ⁇ 0.5 cm) was conjugated with 50 nmol of LN15-NHS (700 nm) or A71-NHS (800 nm) dissolved in DMSO through the NHS ester-amine reaction.
  • the NIR scaffold was washed with water and ethanol 5 times, respectively, followed by freeze-drying.
  • the scaffold was implanted into the subcutaneous pocket of athymic nude mouse 30 days prior to imaging.
  • the extracted scaffold was frozen, cut to a 20 ⁇ m thickness, and imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of fluorescence microscope, respectively.
  • FIGS. 55 and 56 the cross-section of scaffold is highlighted with high contrast using this compound.
  • Intravital microscopy A 25 g male insulinoma-bearing mouse was injected intravenously at time zero with 25 nmol of compound Dex70k-LN15 (700 nm) or Dex70k-ZW800-1 (800 nm) dissolved in saline or D5W. After a waiting period of 1 min, the animal was surgically exposed and the tumor vasculature was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 57 and 58 , the tumor vasculature is highlighted with high contrast using this compound.

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Abstract

The instant invention provides near-infrared fluorescent biological contrast agents and methods of using them.

Description

    RELATED APPLICATIONS
  • This application is a Continuation of application Ser. No. 15/033,337 filed on Apr. 29, 2016; which application is a 35 U.S.C. § 371 National Phase Application of International PCT Patent Application no. PCT/US2014/063097, filed Oct. 30, 2014 which application claims the benefit of and priority to U.S. Provisional Patent Applications No. 61/929,916 filed Oct. 31, 2013 and 61/929,916 filed Jan. 21, 2014, the contents of which are incorporated herein by reference in their entirety.
    • GOVERNMENT SUPPORT
  • This invention was made with government support under CA115296, EB010022, and EB011523 awarded by NIH. The government has certain rights in the invention.
    • BACKGROUND
  • Near infrared (NIR) fluorescence has potential importance in the medical field, particularly in in vitro diagnostics, in vivo diagnostics, and image-guided surgery. However, the availability of suitable fluorophores as imaging agents has been a primary hindrance. To be viable, ideal NIR fluorophores should have good optical properties as well as superior physicochemical properties with respect to solubility, biodistribution, targeting, and clearance. Most current fluorophores contemplated for use as imaging agents fail in connection with their physicochemical properties. For example, known fluorophores suffer from failure to adequately accumulate at the target to be imaged (i.e., low signal), resulting in a low signal-to-background ratio (SBR), or exhibit significant non-specific background uptake in normal tissues (i.e., high background), also resulting in a low SBR.
  • Accordingly, there is a current need for new and improved NIR fluorescent imaging agents, particularly those that equilibrate rapidly between the intravascular and extravascular spaces, target various cells, tissues, or organs with high sensitivity and specificity, and are eliminated efficiently from the body if not targeted. The imaging agents of the invention are directed toward these and other needs.
    • SUMMARY
  • The present invention is directed, at least in part, to near-infrared fluorescent contrast agents and methods of using them.
  • In one aspect, the near-infrared fluorescent contrast is a compound of Formula (I):
  • Figure US20220387631A1-20221208-C00001
    • Wherein or Formula I
  • Each R1 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Each R2 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Or R1 and R2 can be taken together with the carbon atoms to which they are attached to form a 5-6 membered aryl or heteroaryl ring, optionally substituted with halogen, alkyl, alkoxy, hydroxyl, —SO2OH, or —CO2H;
    Each R3 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Q is H, alkyl optionally substituted with alkoxy, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, —N+(alkyl)3, —OCO-alkyl, —SO2OH, phenyl, sulfonato, phosphates, KUE, GPI,—or —NR3R4R5, wherein R3, R4 and R5 are each independently for each occurrence H or C1-C4 alkyl, or R4 and R5, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring;
    X and Y are each independently O, S, Se, C(R″)2, NR′″;
    Z is H, halogen, CN, R6, OR6, SR6, NHR6 or CH2R6, in which R6 is optionally substituted C1-C6 alkyl, optionally substituted aryl, or optionally substituted heteroaryl, alkyl-N3, aryl-N3, aryl-halogen;
    Each R′ is independently H, alkyl or aryl;
    Each R″ is independently H or alkyl;
    Each R′″ is independently H, alkyl, alkyl-SO3H, or alkyl-COOH;
    m and n are independently an integer from 0-3; and
    L is an anion;
    or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof.
  • In another aspect, the near-infrared fluorescent contrast is a compound of Formula (II):
  • Figure US20220387631A1-20221208-C00002
    • Wherein or Formula (II)
  • Each R1 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Each R2 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Or R1 and R2 can be taken together with the carbon atoms to which they are attached to form a 5-6 membered aryl or heteroaryl ring, optionally substituted with halogen, alkyl, alkoxy, hydroxyl, —SO2OH, or —CO2H;
    Each R3 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Q is H, alkyl optionally substituted with alkoxy, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, —N+(alkyl)3, —OCO-alkyl, —SO2OH, phenyl, sulfonato, phosphates, KUE, GPI,—or —NR3R4R5, wherein R3, R4 and R5 are each independently for each occurrence H or C1-C4 alkyl, or R4 and R5, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring;
    X and Y are each independently O, S, Se, C(R″)2, NR;
    Z is H, halogen, CN, R6, OR6, SR6, NHR6 or CH2R6, in which R6 is optionally substituted C1-C6 alkyl, optionally substituted aryl, or optionally substituted heteroaryl, alkyl-N3, aryl-N3, aryl-halogen;
    R is independently H, OR″″ (where R═H, alkyl, or aryl, NH2, NHR, alkyl NH2, alkyl COOH),
    L is an anion;
    Each R′ is independently H, alkyl or aryl;
    Each R″ is independently H or alkyl;
    Each R′″ is independently H, alkyl, alkyl-SO3H, or alkyl-COOH;
    Each R″″ is independently H, alkyl, or aryl, NH2, NHR, alkyl-NH2, or alkyl-COOH;
    m and n are independently an integer from 0-3; and
    L is an anion;
    or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof.
  • In still another aspect, the near-infrared fluorescent contrast is a compound of Formula (III):
  • Figure US20220387631A1-20221208-C00003
  • wherein For Formula (III)
    Each R1 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Each R2 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Or R1 and R2 can be taken together with the carbon atoms to which they are attached to form a 5-6 membered aryl or heteroaryl ring, optionally substituted with halogen, alkyl, alkoxy, hydroxyl, —SO2OH, or —CO2H;
    Each R3 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Q is H, alkyl optionally substituted with alkoxy, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, —N+(alkyl)3, —OCO-alkyl, —SO2OH, phenyl, sulfonato, phosphates, KUE, GPI,—or —NR3R4R5, wherein R3, R4 and R5 are each independently for each occurrence H or C1-C4 alkyl, or R4 and R5, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring;
    X and Y are each independently O, S, Se, C(R″)2, NR′″; Z is H, halogen, CN, R6, OR6, SR6, NHR6 or CH2R6, in which R6 is optionally substituted C1-C6 alkyl, optionally substituted aryl, or optionally substituted heteroaryl, alkyl-N3 (for click chemistry), aryl-N3 (for click chemistry), aryl-halogen (only for palladium catalyzed reactions);
    Each R′ is independently H, alkyl or aryl;
    Each R″ is independently H or alkyl;
    Each R′″ is independently H, alkyl, alkyl-SO3H, or alkyl-COOH;
    m and n are independently an integer from 0-3; and
    L is an anion;
    or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof.
  • In another aspect, the near-infrared fluorescent contrast is a compound of Formula (IV):
  • Figure US20220387631A1-20221208-C00004
  • wherein
    R1, R2, R3, R4, R6 and R7 are each independently H or C1-C6 alkyl;
    R5, R8 and R9 are each independently H, CN, OH, or C1-C6 alkyl; or R1 and R3, taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring;
    or R2 and R4, taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring;
    or R5 and R6, taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring;
    or R7 and R8, taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring;
    or R8 and R9, taken together with the atoms to which they are connected, form an aryl or heteroaryl ring;
    X is O, S, Se, N—R; where R═H or C1-C6 alkyl; and
    M− is an anion or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof.
  • In still another aspect, the near-infrared fluorescent contrast is a compound of Formula (V):
  • Figure US20220387631A1-20221208-C00005
    • Wherein:
  • Q is —B(R3)2—; Si(R3)2
    Each R1 is independently H, alkyl, aryl, or heteroaryl, wherein each alkyl, aryl, or heteroaryl is optionally substituted with alkoxy, alkoxy-N+(alkyl)3, alkoxy-OH, halogen, or COOH; and
    Each R2 is independently H, alkyl, aryl, or heteroaryl, wherein each alkyl, aryl, or heteroaryl is optionally substituted with alkoxy, alkoxy-N+(alkyl)3, alkoxy-OH, halogen, or COOH; and
    Each R3 is independently H, F, or alkyl; OH or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof.
  • In one aspect, the near-infrared fluorescent contrast is:
  • Name Structure
    AL22
    Figure US20220387631A1-20221208-C00006
    SP64
    Figure US20220387631A1-20221208-C00007
    SP60
    Figure US20220387631A1-20221208-C00008
    PTN1
    Figure US20220387631A1-20221208-C00009
    SP56
    Figure US20220387631A1-20221208-C00010
    QBN1
    Figure US20220387631A1-20221208-C00011
    TG18
    Figure US20220387631A1-20221208-C00012
    ZK195
    Figure US20220387631A1-20221208-C00013
    A71
    Figure US20220387631A1-20221208-C00014
    YY187
    Figure US20220387631A1-20221208-C00015
    CNN6
    Figure US20220387631A1-20221208-C00016
    TG42
    Figure US20220387631A1-20221208-C00017
    TG53
    Figure US20220387631A1-20221208-C00018
    CNN4
    Figure US20220387631A1-20221208-C00019
    CNN5
    Figure US20220387631A1-20221208-C00020
    LN24
    Figure US20220387631A1-20221208-C00021
    LN63
    Figure US20220387631A1-20221208-C00022
    LN66
    Figure US20220387631A1-20221208-C00023
    LN15
    Figure US20220387631A1-20221208-C00024
    YY180
    Figure US20220387631A1-20221208-C00025
    NRB1
    Figure US20220387631A1-20221208-C00026
    NRB2
    Figure US20220387631A1-20221208-C00027
    NRB3
    Figure US20220387631A1-20221208-C00028
    ZK190
    Figure US20220387631A1-20221208-C00029
    ZK189
    Figure US20220387631A1-20221208-C00030
    ZK50
    Figure US20220387631A1-20221208-C00031
    SP59
    Figure US20220387631A1-20221208-C00032
    JM1
    Figure US20220387631A1-20221208-C00033
    SP67
    Figure US20220387631A1-20221208-C00034
    MM21
    Figure US20220387631A1-20221208-C00035
    ZK38
    Figure US20220387631A1-20221208-C00036
    E60
    Figure US20220387631A1-20221208-C00037
    E58
    Figure US20220387631A1-20221208-C00038
    E59
    Figure US20220387631A1-20221208-C00039
    LN36
    Figure US20220387631A1-20221208-C00040
    AL27
    Figure US20220387631A1-20221208-C00041
    AL18
    Figure US20220387631A1-20221208-C00042
    AL16
    Figure US20220387631A1-20221208-C00043
    AL25
    Figure US20220387631A1-20221208-C00044
    AL29
    Figure US20220387631A1-20221208-C00045
    AL30
    Figure US20220387631A1-20221208-C00046
    AL33
    Figure US20220387631A1-20221208-C00047
    AL34
    Figure US20220387631A1-20221208-C00048
    AL35
    Figure US20220387631A1-20221208-C00049
    AL36
    Figure US20220387631A1-20221208-C00050
    AL14
    Figure US20220387631A1-20221208-C00051
    AL79
    Figure US20220387631A1-20221208-C00052
    SP27
    Figure US20220387631A1-20221208-C00053
    SP28
    Figure US20220387631A1-20221208-C00054
    SP29
    Figure US20220387631A1-20221208-C00055
    SP30
    Figure US20220387631A1-20221208-C00056
    SP33
    Figure US20220387631A1-20221208-C00057
    SP43
    Figure US20220387631A1-20221208-C00058
    SP49
    Figure US20220387631A1-20221208-C00059
    SP51
    Figure US20220387631A1-20221208-C00060
    SP53
    Figure US20220387631A1-20221208-C00061
    SP79
    Figure US20220387631A1-20221208-C00062
    SP99
    Figure US20220387631A1-20221208-C00063
    SP116
    Figure US20220387631A1-20221208-C00064
    SP117
    Figure US20220387631A1-20221208-C00065
    ZK184
    Figure US20220387631A1-20221208-C00066
    ZK185
    Figure US20220387631A1-20221208-C00067
    ZK197
    Figure US20220387631A1-20221208-C00068
    ZK198
    Figure US20220387631A1-20221208-C00069
    ZK134
    Figure US20220387631A1-20221208-C00070
    ZK135
    Figure US20220387631A1-20221208-C00071
    TG4
    Figure US20220387631A1-20221208-C00072
    TG5
    Figure US20220387631A1-20221208-C00073
    TG7
    Figure US20220387631A1-20221208-C00074
    TG8
    Figure US20220387631A1-20221208-C00075
    TG27
    Figure US20220387631A1-20221208-C00076
    TP5
    Figure US20220387631A1-20221208-C00077
    QBN14
    Figure US20220387631A1-20221208-C00078
    CNN154
    Figure US20220387631A1-20221208-C00079
    EAO42
    Figure US20220387631A1-20221208-C00080
    ZK166
    Figure US20220387631A1-20221208-C00081
    PTN13
    Figure US20220387631A1-20221208-C00082
    PTN12
    Figure US20220387631A1-20221208-C00083
    ZK148
    Figure US20220387631A1-20221208-C00084
    ZK154
    Figure US20220387631A1-20221208-C00085
    E72
    Figure US20220387631A1-20221208-C00086
    ZK159
    Figure US20220387631A1-20221208-C00087
    E70
    Figure US20220387631A1-20221208-C00088
    ZK153
    Figure US20220387631A1-20221208-C00089
    PTN11
    Figure US20220387631A1-20221208-C00090
    ZK155
    Figure US20220387631A1-20221208-C00091
    MHI103
    Figure US20220387631A1-20221208-C00092
    CNN13
    Figure US20220387631A1-20221208-C00093
    WuA71
    Figure US20220387631A1-20221208-C00094
    ZK143
    Figure US20220387631A1-20221208-C00095
    ZK140
    Figure US20220387631A1-20221208-C00096
    ZK29
    Figure US20220387631A1-20221208-C00097
    SP34
    Figure US20220387631A1-20221208-C00098
    ZK104
    Figure US20220387631A1-20221208-C00099
    TP1
    Figure US20220387631A1-20221208-C00100
    ZK14
    Figure US20220387631A1-20221208-C00101
    PTN6
    Figure US20220387631A1-20221208-C00102
    LN65
    Figure US20220387631A1-20221208-C00103
    LN79
    Figure US20220387631A1-20221208-C00104
    AH34
    Figure US20220387631A1-20221208-C00105
    TP4
    Figure US20220387631A1-20221208-C00106
    LS1
    Figure US20220387631A1-20221208-C00107
    YY163
    Figure US20220387631A1-20221208-C00108
    TP6
    Figure US20220387631A1-20221208-C00109
    ZK15
    Figure US20220387631A1-20221208-C00110
    WuA108
    Figure US20220387631A1-20221208-C00111
    ZK150
    Figure US20220387631A1-20221208-C00112
    ZK156
    Figure US20220387631A1-20221208-C00113
    ESS23
    Figure US20220387631A1-20221208-C00114
    CNN17
    Figure US20220387631A1-20221208-C00115
    TG56
    Figure US20220387631A1-20221208-C00116
    AL31
    Figure US20220387631A1-20221208-C00117
    AL43
    Figure US20220387631A1-20221208-C00118
    TG31
    Figure US20220387631A1-20221208-C00119
    CNN3
    Figure US20220387631A1-20221208-C00120
    AL20
    Figure US20220387631A1-20221208-C00121
    TG115
    Figure US20220387631A1-20221208-C00122
    CNN2
    Figure US20220387631A1-20221208-C00123
    TP04
    Figure US20220387631A1-20221208-C00124
    ZK26
    Figure US20220387631A1-20221208-C00125
    ZK48
    Figure US20220387631A1-20221208-C00126
    TG44
    Figure US20220387631A1-20221208-C00127
    ZK27
    Figure US20220387631A1-20221208-C00128
    ZK46
    Figure US20220387631A1-20221208-C00129
    CNN1
    Figure US20220387631A1-20221208-C00130
    ZK79
    Figure US20220387631A1-20221208-C00131
    WuA38
    Figure US20220387631A1-20221208-C00132
    LN68
    Figure US20220387631A1-20221208-C00133
    ESS13
    Figure US20220387631A1-20221208-C00134
    WuA110
    Figure US20220387631A1-20221208-C00135
    YY161
    Figure US20220387631A1-20221208-C00136
    E71
    Figure US20220387631A1-20221208-C00137
    CNN8
    Figure US20220387631A1-20221208-C00138
    CNN10
    Figure US20220387631A1-20221208-C00139
    LN68Boc
    Figure US20220387631A1-20221208-C00140
    TG60
    Figure US20220387631A1-20221208-C00141
    ZK78
    Figure US20220387631A1-20221208-C00142
    ZK133
    Figure US20220387631A1-20221208-C00143
    CNN7
    Figure US20220387631A1-20221208-C00144
    ZK23
    Figure US20220387631A1-20221208-C00145
    MDL17
    Figure US20220387631A1-20221208-C00146
    TG11A
    Figure US20220387631A1-20221208-C00147
    TG11B
    Figure US20220387631A1-20221208-C00148
    TP2
    Figure US20220387631A1-20221208-C00149
    LN50
    Figure US20220387631A1-20221208-C00150
    TG17
    Figure US20220387631A1-20221208-C00151
    TG22
    Figure US20220387631A1-20221208-C00152
    LN34
    Figure US20220387631A1-20221208-C00153
    CNN16
    Figure US20220387631A1-20221208-C00154
    CNN12
    Figure US20220387631A1-20221208-C00155
    CNN145
    Figure US20220387631A1-20221208-C00156
    ZK203
    Figure US20220387631A1-20221208-C00157
    ZK204
    Figure US20220387631A1-20221208-C00158
    ZK208
    Figure US20220387631A1-20221208-C00159
    ZK196
    Figure US20220387631A1-20221208-C00160
    WuA67
    Figure US20220387631A1-20221208-C00161
    WuA76
    Figure US20220387631A1-20221208-C00162
    EAO40
    Figure US20220387631A1-20221208-C00163
    ZK106
    Figure US20220387631A1-20221208-C00164
    ZK124
    Figure US20220387631A1-20221208-C00165
    ZK126
    Figure US20220387631A1-20221208-C00166
    ZK101
    Figure US20220387631A1-20221208-C00167
    ZK172
    Figure US20220387631A1-20221208-C00168
    TG16
    Figure US20220387631A1-20221208-C00169
    MDL16
    Figure US20220387631A1-20221208-C00170
    CNN14
    Figure US20220387631A1-20221208-C00171
    LN37
    Figure US20220387631A1-20221208-C00172
    TG20
    Figure US20220387631A1-20221208-C00173
    LO4
    Figure US20220387631A1-20221208-C00174
    ZK211
    Figure US20220387631A1-20221208-C00175
    ZK214
    Figure US20220387631A1-20221208-C00176
    ZK215
    Figure US20220387631A1-20221208-C00177
    ZK217
    Figure US20220387631A1-20221208-C00178
    SRA89
    Figure US20220387631A1-20221208-C00179
    YY190
    Figure US20220387631A1-20221208-C00180
    YY220
    Figure US20220387631A1-20221208-C00181
    YY229
    Figure US20220387631A1-20221208-C00182
    YY231
    Figure US20220387631A1-20221208-C00183
    YY233
    Figure US20220387631A1-20221208-C00184
    YY238
    Figure US20220387631A1-20221208-C00185
    SRA94
    Figure US20220387631A1-20221208-C00186
    PS31
    Figure US20220387631A1-20221208-C00187
    ZK239
    Figure US20220387631A1-20221208-C00188
    AL11
    Figure US20220387631A1-20221208-C00189
    AL12
    Figure US20220387631A1-20221208-C00190
    CMI24
    Figure US20220387631A1-20221208-C00191
    CMI26
    Figure US20220387631A1-20221208-C00192
    E16
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    E17
    Figure US20220387631A1-20221208-C00194
    E24
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    E27
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    E36
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    E37
    Figure US20220387631A1-20221208-C00198
    E38
    Figure US20220387631A1-20221208-C00199
    E39
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    E43
    Figure US20220387631A1-20221208-C00201
    E44
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    E45
    Figure US20220387631A1-20221208-C00203
    E50
    Figure US20220387631A1-20221208-C00204
    E51
    Figure US20220387631A1-20221208-C00205
    E77
    Figure US20220387631A1-20221208-C00206
    E78
    Figure US20220387631A1-20221208-C00207
    E79
    Figure US20220387631A1-20221208-C00208
    E80
    Figure US20220387631A1-20221208-C00209
    E81
    Figure US20220387631A1-20221208-C00210
    ES17
    Figure US20220387631A1-20221208-C00211
    ES21
    Figure US20220387631A1-20221208-C00212
    ESS61
    Figure US20220387631A1-20221208-C00213
    LO1
    Figure US20220387631A1-20221208-C00214
    LO2
    Figure US20220387631A1-20221208-C00215
    LO3
    Figure US20220387631A1-20221208-C00216
    MHI106
    Figure US20220387631A1-20221208-C00217
    MHI128
    Figure US20220387631A1-20221208-C00218
    MHI84
    Figure US20220387631A1-20221208-C00219
    MHI86
    Figure US20220387631A1-20221208-C00220
    MHI96
    Figure US20220387631A1-20221208-C00221
    MHI97
    Figure US20220387631A1-20221208-C00222
    P700H
    Figure US20220387631A1-20221208-C00223
    P700SO3
    Figure US20220387631A1-20221208-C00224
    P800H
    Figure US20220387631A1-20221208-C00225
    P800SO3
    Figure US20220387631A1-20221208-C00226
    T14
    Figure US20220387631A1-20221208-C00227
    T17
    Figure US20220387631A1-20221208-C00228
    T18
    Figure US20220387631A1-20221208-C00229
    T20
    Figure US20220387631A1-20221208-C00230
    T23
    Figure US20220387631A1-20221208-C00231
    T24
    Figure US20220387631A1-20221208-C00232
    T25
    Figure US20220387631A1-20221208-C00233
    T27
    Figure US20220387631A1-20221208-C00234
    T29
    Figure US20220387631A1-20221208-C00235
    A20
    Figure US20220387631A1-20221208-C00236
    A21
    Figure US20220387631A1-20221208-C00237
    A80
    Figure US20220387631A1-20221208-C00238
    A100
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    A104
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    A106
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    A134
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    A138
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    A146
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    A148
    Figure US20220387631A1-20221208-C00245
    A149
    Figure US20220387631A1-20221208-C00246
    A150
    Figure US20220387631A1-20221208-C00247
    A160
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    A161
    Figure US20220387631A1-20221208-C00249
    A24
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    AC2
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    AC3
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    AC8
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    AN3
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    WuA96
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    Ox4
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    Ox170
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    Ox750
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    Rh800
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    TG66
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    MM25
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    SP72
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    ZK138-2
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    ZK169
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    ZW800-1
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    A64
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    MHI85
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    SP66
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    YY113
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    YY142
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    PS37
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    PS53
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    ZK240
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    ZK244
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    EAO72
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    EAO76
    Figure US20220387631A1-20221208-C00276
    PS101A
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    PS35
    Figure US20220387631A1-20221208-C00278
    PS36
    Figure US20220387631A1-20221208-C00279
    PS39
    Figure US20220387631A1-20221208-C00280
    PS51
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    PS52
    Figure US20220387631A1-20221208-C00282
    PS62
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    PS73
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    PS76
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    YY255
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    YY260
    Figure US20220387631A1-20221208-C00287
    YY261
    Figure US20220387631A1-20221208-C00288
    YY269
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    ZK243
    Figure US20220387631A1-20221208-C00290
    ZK2515
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    ZK2525
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    ZK2565
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    ZK2566
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    ZK258
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    ZK2615
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    A71-NHS
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    LN15-NHS
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    PS126
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    PS127
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    PS128
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    PS129
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    PS130
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    PS131
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    PS132
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    PS133
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    YY283
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    YY284
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    YY285
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    YY294
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    YY295
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    YY2103
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    YY2106
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    YY2107
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    L700-1A
    Figure US20220387631A1-20221208-C00316
    L700-1C
    Figure US20220387631A1-20221208-C00317
    L800-1A
    Figure US20220387631A1-20221208-C00318
    L800-1C
    Figure US20220387631A1-20221208-C00319
  • In certain embodiments, the imaging agent has peak absorbance at about 600 nm to 900 nm.
  • In certain embodiments, the tissue or cells is imaged ex vivo, e.g. for in vitro diagnostic applications.
  • In another aspect, the invention provides a method of imaging biological cells, the method comprising: (a) contacting the biological cells with a compound of the invention; (b) irradiating the cells at a wavelength absorbed by the compound; (c) and detecting a signal from the compound, thereby imaging the biological cells. The biological cells could be a normal cell type in the body or its malignant counterpart, i.e., a tumor formed from a normal cell type.
  • In certain embodiments, the biological targets are found in biological tissues or organs. In specific embodiments, the biological targets are blood vessel lumens, endothelial cells lining blood vessels, cartilage cells and/or their products, bone cells and/or their products, thyroid cells, thyroid glands, parathyroids cells, parathyroid glands, adrenal gland cells, adrenal glands, salivary gland cells, salivary glands, white adipose tissue, brown adipose tissue, ovarian cells, testicular cells, seminal vesicles, prostate cells, pancreas cells, spleen cells, gallbladder lumens, gallbladder cells, bile duct lumens, bile duct cells, Peyer's patches, brain grey matter, brain white matter, brain vasculature cells, choroid plexus tissue and fluid, cerebrospinal fluid, nerves, lymph nodes, sentinel lymph nodes, vulnerable plaque, stem cells, or neuroendocrine tumors.
  • In certain embodiments, the compounds of the invention accumulate in a lumen or other cavity in the body, thus highlighting the lumen's anatomical location and/or quantifying flow of the compound within the lumen. For example, a NIR fluorophore excreted by the kidney will accumulate in the ureters, thus identifying their location and also permitting direct visualization of pulsatile flow within the ureters. The same is true for blood vessels after intravenous injection of a compound or the thoracic duct after injection into the lower body.
  • In certain embodiments, the compounds of the invention accumulate in a tissue or organ but do so extracellularly. For example, a compound injected sub-dermally may enter the lymphatic channels and flow to a lymph node where it may be trapped in the extracellular space rather than, or in addition to, entrapment within cells of the lymph node.
  • In certain embodiments, the compounds of the invention may be modified to include a polyethylene glycol group. Such PEGylated compounds may be branched or linear. In certain embodiments, the linear PEGylated compounds are in the range of about 20 kDa to about 60 kDa.
  • In certain embodiments, the NIR fluorophores are conjugated covalently or non-covalently to other molecules, either to improve targeting of the NIR fluorophore or to co-localize other functional molecules.
  • In some embodiments, the compounds of the invention can be conjugated to a metal chelator agent for use in single-photon emission computed tomography (SPECT) or positron emission tomography (PET) or in magnetic resonance imaging (MRI). In certain embodiments, the metal cheltor agent is a DOTA, DTPA, hydrazinonicotinic acid (HYNIC), or desferoxime, or a derivative thereof. In particular embodiments, the metal atom is selected from the group including, but not exclusively, Zr-89, Ga-68 and Rb-82, and the signal is detected by positron emission tomography; the metal atom is selected from the group including, but not exclusively, of Tc-99m, Lu-177, and In-111, and the signal is detected by single-photon emission computed tomography; or the metal atom is a lanthanide selected from the group including, but not exclusively, Gd, Eu, Y, Dy and Yb, and the signal is detected by magnetic resonance imaging.
  • In some embodiments, the compounds of the invention can be conjugated to a therapeutic, such as a radioisotope, cytotoxin, or immune modulator, such that the targeting ability of the compound concentrates the therapeutic in the cell, tissue, organ, or lumen of interest.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 —depicts the imaging of Ureters at 800 nm using A71 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 2 —depicts the imaging of Ureters at 700 nm using LN15 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 3 —depicts the imaging of pan lymph node identification at 800 nm using MM25 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 4 —depicts the imaging of Pan LN pan lymph node identification at 700 nm using A150 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 5 —depicts the imaging of SLN identification at 800 nm using MM25 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 6 —depicts the imaging of SLN identification at 700 nm using MHI86 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 7 —depicts the imaging of Cartilage at 800 nm using LN50 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 8 —depicts the imaging of Cartilage at 700 nm using SP56 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 9 —depicts the imaging of neuroendocrine tumors at 800 nm using AL20 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 10 —depicts the imaging of neuroendocrine tumors at 700 nm using ESS61 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 11 —depicts the imaging of bone mineralization at 800 nm using P800SO3 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 12 —depicts the imaging of bone mineralization at 700 nm using P700SO3 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 13 —depicts the imaging of the thyroid gland at 800 nm using QBN14 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 14 —depicts the imaging of the thyroid gland at 700 nm using T14 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 15 —depicts the imaging of parathyroid gland at 800 nm using QBN14 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 16 —depicts the imaging of parathyroid gland at 700 nm using T14 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 17 —depicts the imaging of the adrenal gland at 800 nm using AL27 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 18 —depicts the imaging of adrenal gland at 700 nm using E16 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 19 —depicts the imaging of salivary glands at 800 nm using ZK211 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 20 —depicts the imaging of salivary glands at 700 nm using NRB1 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 21 —depicts the imaging of white adipose tissue at 800 nm using AH34 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 22 —depicts the imaging of white adipose tissue at 700 nm using PS31 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 23 —depicts the imaging of brown adipose tissue at 800 nm using QBN1 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 24 —depicts the imaging of brown adipose tissue at 700 nm using SP60 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 25 —depicts the imaging of ovaries at 800 nm using AL27 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 26 —depicts the imaging of ovaries at 700 nm using PS62 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 27 —depicts the imaging of the seminal vesicles at 800 nm using CNN2 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 28 —depicts the imaging of the seminal vesicles at 700 nm using LN65 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 29 —depicts the imaging of the prostate gland at 800 nm using LN66 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 30 —depicts the imaging of the prostate gland at 700 nm using PS62 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 31 —depicts the imaging of the pancreas at 800 nm using AL22 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 32 —depicts the imaging of the pancreas at 700 nm using T14 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 33 —depicts the imaging of the spleen at 800 nm using AL29 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 34 —depicts the imaging of the spleen at 700 nm using E24 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 35 —depicts the imaging of the gallbladder at 800 nm using ZK198 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 36 —depicts the imaging of the gallbladder at 700 nm using PS62 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 37 —depicts the imaging of the bile ducts at 800 nm using ZK198 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 38 —depicts the imaging of the bile ducts at 700 nm using A106 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 39 —depicts the imaging of Peyer's Patches at 800 nm using AL30 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 40 —depicts the imaging of the brain vasculature at 700 nm using ZK214 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 41 —depicts the imaging of brain grey matter at 800 nm using ZK189 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 42 —depicts the imaging of brain grey matter at 700 nm using WuA96 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 43 —depicts the imaging of the choroid plexus at 800 nm using ZK208 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 44 —depicts the imaging of the choroid plexus at 700 nm using SP28 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 45 —depicts the imaging of the cerebrospinal fluid at 800 nm using AL20 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 46 —depicts the imaging of the cerebrospinal fluid at 700 nm using SP66 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 47 —depicts the imaging of the thoracic duct at 800 nm using A71 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 48 —depicts the imaging of thoracic duct at 700 nm using LN15 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 49 —depicts the imaging of PEGylated agents at 800 nm using PEG60k-ZW800-1 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 50 —depicts the imaging of PEGylated agents at 700 nm using PEG60k-LN15 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 51 —depicts the imaging of the pituitary gland at 800 nm using AL22 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 52 —depicts the imaging of pituitary gland at 700 nm using SP60 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 53 —depicts the imaging of stem cells at 800 nm using PS126 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 54 —depicts the imaging of stem cells at 700 nm using PS127 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 55 —depicts the imaging of engineered tissue scaffolds and cells at 800 nm using A71-NHS (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 56 —depicts the imaging of engineered tissue scaffolds and cells at 700 nm using LN15-NHS (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 57 —depicts the imaging of intravital microscopy at 800 nm using Dex70k-ZW800-1 (Image without irradiation, NIR irradiated image, overlay of both)
  • FIG. 58 —depicts the imaging of intravital microscopy at 700 nm using Dex70k-LN15 (Image without irradiation, NIR irradiated image, overlay of both)
    •  ABBREVIATIONS USED
    • Ad: white adipose
    • AG: adrenal gland
    • BG: brain grey matter (cells)
    • Bl: bladder
    • Bo: bone
    • BD: bile duct
    • BF: brown fat
    • BV: brain vasculature
    • BW: brain white matter (nerve axons and glia)
    • Ca: cartilage
    • CF: cerebrospinal fluid
    • CP: choroid plexus
    • Du: duodenum
    • GB: gall bladder
    • He: heart
    • HB: hepatobiliary clearance
    • In: intestine:
    • Ki: kidney
    • Li: liver
    • Lu: lung
    • LN: lymph node
    • Mu: muscle
    • Ne: nerve
    • NT: neuroendocrine tumor
    • Ov: ovary
    • Pa: pancreas
    • Pp: Peyer's patches
    • Pr: prostate
    • PG: pituitary gland
    • PT: parathyroid gland
    • RE: renal clearance
    • Sp: spleen
    • SG: salivary gland
    • SL: sentinel lymph node
    • SV: seminal vesicle
    • TG: thyroid gland
    • Ur: ureter
    DETAILED DESCRIPTION
  • It has now been found that compounds with absorption and/or emission in the near infrared (NIR) have desirable properties with respect to in vivo biodistribution and clearance, uptake and retention by cells, tissues, and/or organs of interest, and the imaging thereof. Such agents are compatible with Channel 1 (≈660 nm excitation; ≈700 nm emission) or Channel 2 (≈760 nm excitation; ≈800 nm emission) of the FLARE™ Imaging System, which permits color video and NIR fluorescence to be acquired simultaneously, thus providing real-time image-guidance to surgeons and others about target location.
    • Definitions
  • The following definitions will be useful in understanding the instant invention.
  • As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. “Consisting essentially of”, when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
  • As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.
  • Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.
  • Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive.
  • The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
  • As used herein, the term “subject” or “patient” encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes monkeys, cattle, horses, sheep, goats, swine; rabbits, dogs, cats, rats, mice, guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, parasites, microbes, and the like.
  • As used herein, the term “administration” or “administering” of the subject compound refers to providing a compound of the invention and/or prodrugs thereof to a subject in need of diagnosis or treatment.
  • As used herein, the term “carrier” refers to chemical compounds or agents that facilitate the incorporation of a compound described herein into cells or tissues.
  • As used herein, the term “acceptable” with respect to a formulation, composition or ingredient, as used herein, means having no persistent detrimental effect on the general health of the subject being treated.
  • As used herein, the term “diluent” refers to chemical compounds that are used to dilute a compound described herein prior to delivery. Diluents can also be used to stabilize compounds described herein.
  • The term “nerve” as used herein, includes peripheral nerve tissue and cells, including myelinated nerves. Sensory nerves and motor nerves are examples of nerve tissue. Examples of specific nerves include the laryngeal nerve, femoral nerve, brachial plexus, sciatic nerve, pudendal nerves, penile nerves, and the like. The term “nerve tissue” also includes brain grey matter and brain white matter.
  • The term “alkyl,” refers to a straight or branched hydrocarbon radical having from 1-12 carbon atoms, from 1-8 carbon atoms, from 1-6 carbon atoms, or from 1-4 carbon atoms (unless stated otherwise) and includes, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl and the like. An alkyl can be unsubstituted or substituted with one or more suitable substituents.
  • The term “cycloalkyl” refers to a monocyclic or polycyclic hydrocarbon ring group having from 3 to 6 carbon atoms, in the hydrocarbon ring (unless stated otherwise) and includes, for example, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, and the like. A cycloalkyl group can be unsubstituted or substituted with one or more suitable substituents.
  • The term “hetero” refers to the replacement of at least one carbon atom member in a ring system with at least one heteroatom such as nitrogen, sulfur, and oxygen.
  • The term “heterocyclic” means a non-aromatic monocyclic ring having from 2 to 5 carbon atoms in the ring (unless stated otherwise) and at least one heteroatom, preferably, one heteroatom selected from nitrogen, sulfur (including oxidized sulfur such as sulfone or sulfoxide) and oxygen. A heterocycloalkyl group can have one or more carbon-carbon double bonds or carbon-heteroatom double bonds in the ring group as long as the ring group is not rendered aromatic by their presence.
  • Examples of heterocyclic groups include pyrrolidinyl, piperidinyl, tetrahydropyranyl, and the like. A heterocyclic group can be unsubstituted or substituted with one or more suitable substituents.
  • As used herein, the term “aryl” refers to an unsubstituted or substituted carbocyclic aromatic monocyclic group such as a phenyl group. The term “aryl” may be interchangeably used with “aryl ring”.
  • As used herein, the term “heteroaryl: refers to an unsubstituted or substituted heterocyclic aromatic monocyclic group such as a pyridyl, furanyl, or thiophenyl group, and the like. Heteroaryl groups have 5 or 6 atoms in the heteroaromatic ring, 1 of which is independently selected from the group consisting of oxygen, sulfur and nitrogen. Typical heteroaryl groups include, for example, a pyridyl, furanyl, or thiophenyl group.
  • An aryl or heteroaryl can be unsubstituted or substituted with one or more suitable substituents.
  • A “substituent,” as used herein, refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest. For example, a ring substituent may be a moiety such as a halogen, alkyl group, haloalkyl group or other group that is covalently bonded to an atom (preferably a carbon or nitrogen atom) that is a ring member. Substituents of aromatic groups are generally covalently bonded to a ring carbon atom. The term “substitution” refers to replacing a hydrogen atom in a molecular structure with a substituent, such that the valence on the designated atom is not exceeded, and such that a chemically stable compound (i.e., a compound that can be isolated, characterized, and tested for biological activity) results from the substitution.
  • As described above, certain groups can be unsubstituted or substituted with one or more suitable substituents by other than hydrogen at one or more available positions, typically 1, 2, 3, 4 or 5 positions, by one or more suitable groups (which may be the same or different). Certain groups, when substituted, are substituted with 1, 2, 3 or 4 independently selected substituents. Suitable substituents include halo, alkyl, aryl, hydroxy, alkoxy, hydroxyalkyl, amino, and the like.
  • The term “halogen”, as used herein, refers to F, Cl, Br, At, and I.
  • Other definitions appear in context throughout the disclosure.
    • Compounds and Compositions
  • It has now been found that certain compounds are useful as near-infrared absorbing and/or fluorescing biological contrast agents.
  • Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • In one aspect, the near-infrared fluorescent contrast is a compound of Formula (I):
  • Figure US20220387631A1-20221208-C00320
    • Wherein for Formula (I)
  • Each R1 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Each R2 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Or R1 and R2 can be taken together with the carbon atoms to which they are attached to form a 5-6 membered aryl or heteroaryl ring, optionally substituted with halogen, alkyl, alkoxy, hydroxyl, —SO2OH, or —CO2H;
    Each R3 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Q is H, alkyl optionally substituted with alkoxy, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, —N+(alkyl)3, —OCO-alkyl, —SO2OH, phenyl, sulfonato, phosphates, KUE, GPI,—or —NR3R4R5, wherein R3, R4 and R5 are each independently for each occurrence H or C1-C4 alkyl, or R4 and R5, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring;
    X and Y are each independently O, S, Se, C(R″)2, NR′″;
    Z is H, halogen, CN, R6, OR6, SR6, NHR6 or CH2R6, in which R6 is optionally substituted C1-C6 alkyl, optionally substituted aryl, or optionally substituted heteroaryl, alkyl-N3, aryl-N3, aryl-halogen;
    Each R′ is independently H, alkyl or aryl;
    Each R″ is independently H or alkyl;
    Each R′″ is independently H, alkyl, alkyl-SO3H, or alkyl-COOH;
    m and n are independently an integer from 0-3; and
    L is an anion;
    or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof.
  • In another aspect, the near-infrared fluorescent contrast is a compound of Formula (II):
  • Figure US20220387631A1-20221208-C00321
    • Wherein for Formula (II)
  • Each R1 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Each R2 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Or R1 and R2 can be taken together with the carbon atoms to which they are attached to form a 5-6 membered aryl or heteroaryl ring, optionally substituted with halogen, alkyl, alkoxy, hydroxyl, —SO2OH, or —CO2H;
    Each R3 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Q is H, alkyl optionally substituted with alkoxy, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, —N+(alkyl)3, —OCO-alkyl, —SO2OH, phenyl, sulfonato, phosphates, KUE, GPI,—or —NR3R4R5, wherein R3, R4 and R5 are each independently for each occurrence H or C1-C4 alkyl, or R4 and R5, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring;
    X and Y are each independently O, S, Se, C(R″)2, NR;
    Z is H, halogen, CN, R6, OR6, SR6, NHR6 or CH2R6, in which R6 is optionally substituted C1-C6 alkyl, optionally substituted aryl, or optionally substituted heteroaryl, alkyl-N3, aryl-N3, aryl-halogen;
    R is independently H, OR″″ (where R═H, alkyl, or aryl, NH2, NHR, alkyl NH2, alkyl COOH),
    L is an anion;
    Each R′ is independently H, alkyl or aryl;
    Each R″ is independently H or alkyl;
    Each R′″ is independently H, alkyl, alkyl-SO3H, or alkyl-COOH;
    Each R″″ is independently H, alkyl, or aryl, NH2, NHR, alkyl-NH2, or alkyl-COOH;
    m and n are independently an integer from 0-3; and
    L is an anion;
    or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof.
  • In still another aspect, the near-infrared fluorescent contrast is a compound of Formula (III):
  • Figure US20220387631A1-20221208-C00322
  • wherein For Formula (III)
    Each R1 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Each R2 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Or R1 and R2 can be taken together with the carbon atoms to which they are attached to form a 5-6 membered aryl or heteroaryl ring, optionally substituted with halogen, alkyl, alkoxy, hydroxyl, —SO2OH, or —CO2H;
    Each R3 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
    Q is H, alkyl optionally substituted with alkoxy, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, —N+(alkyl)3, —OCO-alkyl, —SO2OH, phenyl, sulfonato, phosphates, KUE, GPI,—or —NR3R4R5, wherein R3, R4 and R5 are each independently for each occurrence H or C1-C4 alkyl, or R4 and R5, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring;
    X and Y are each independently O, S, Se, C(R″)2, NR′″; Z is H, halogen, CN, R6, OR6, SR6, NHR6 or CH2R6, in which R6 is optionally substituted C1-C6 alkyl, optionally substituted aryl, or optionally substituted heteroaryl, alkyl-N3 (for click chemistry), aryl-N3 (for click chemistry), aryl-halogen (only for palladium catalyzed reactions);
    Each R′ is independently H, alkyl or aryl;
    Each R″ is independently H or alkyl;
    Each R′″ is independently H, alkyl, alkyl-SO3H, or alkyl-COOH;
    m and n are independently an integer from 0-3; and
    L is an anion;
    or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof.
  • In another aspect, the near-infrared fluorescent contrast is a compound of Formula (IV):
  • Figure US20220387631A1-20221208-C00323
  • wherein
    R1, R2, R3, R4, R6 and R7 are each independently H or C1-C6 alkyl;
    R5, R8 and R9 are each independently H, CN, OH, or C1-C6 alkyl;
    or R1 and R3, taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring;
    or R2 and R4, taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring;
    or R5 and R6, taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring;
    or R7 and R8, taken together with the atoms to which they are connected, form a 5- to 6-membered heterocylic ring;
    or R8 and R9, taken together with the atoms to which they are connected, form an aryl or heteroaryl ring;
    X is O, S, Se, N—R; where R═H or C1-C6 alkyl; and
    M− is an anion
    or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof.
  • In still another aspect, the near-infrared fluorescent contrast is a compound of Formula (V):
  • Figure US20220387631A1-20221208-C00324
    • Wherein:
  • Q is —B(R3)2—; Si(R3)2
    Each R1 is independently H, alkyl, aryl, or heteroaryl, wherein each alkyl, aryl, or heteroaryl is optionally substituted with alkoxy, alkoxy-N+(alkyl)3, alkoxy-OH, halogen, or COOH; and
    Each R2 is independently H, alkyl, aryl, or heteroaryl, wherein each alkyl, aryl, or heteroaryl is optionally substituted with alkoxy, alkoxy-N+(alkyl)3, alkoxy-OH, halogen, or COOH; and
    Each R3 is independently H, F, or alkyl; OH
    or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof.
  • In certain aspects, the near-infrared fluorescent biological contract agent is:
  • Name Structure
    AL22
    Figure US20220387631A1-20221208-C00325
    SP64
    Figure US20220387631A1-20221208-C00326
    SP60
    Figure US20220387631A1-20221208-C00327
    PTN1
    Figure US20220387631A1-20221208-C00328
    SP56
    Figure US20220387631A1-20221208-C00329
    QBN1
    Figure US20220387631A1-20221208-C00330
    TG18
    Figure US20220387631A1-20221208-C00331
    ZK195
    Figure US20220387631A1-20221208-C00332
    A71
    Figure US20220387631A1-20221208-C00333
    YY187
    Figure US20220387631A1-20221208-C00334
    CNN6
    Figure US20220387631A1-20221208-C00335
    TG42
    Figure US20220387631A1-20221208-C00336
    TG53
    Figure US20220387631A1-20221208-C00337
    CNN4
    Figure US20220387631A1-20221208-C00338
    CNN5
    Figure US20220387631A1-20221208-C00339
    LN24
    Figure US20220387631A1-20221208-C00340
    LN63
    Figure US20220387631A1-20221208-C00341
    LN66
    Figure US20220387631A1-20221208-C00342
    LN15
    Figure US20220387631A1-20221208-C00343
    YY180
    Figure US20220387631A1-20221208-C00344
    NRB1
    Figure US20220387631A1-20221208-C00345
    NRB2
    Figure US20220387631A1-20221208-C00346
    NRB3
    Figure US20220387631A1-20221208-C00347
    ZK190
    Figure US20220387631A1-20221208-C00348
    ZK189
    Figure US20220387631A1-20221208-C00349
    ZK50
    Figure US20220387631A1-20221208-C00350
    SP59
    Figure US20220387631A1-20221208-C00351
    JM1
    Figure US20220387631A1-20221208-C00352
    SP67
    Figure US20220387631A1-20221208-C00353
    MM21
    Figure US20220387631A1-20221208-C00354
    ZK38
    Figure US20220387631A1-20221208-C00355
    E60
    Figure US20220387631A1-20221208-C00356
    E58
    Figure US20220387631A1-20221208-C00357
    E59
    Figure US20220387631A1-20221208-C00358
    LN36
    Figure US20220387631A1-20221208-C00359
    AL27
    Figure US20220387631A1-20221208-C00360
    AL18
    Figure US20220387631A1-20221208-C00361
    AL16
    Figure US20220387631A1-20221208-C00362
    AL25
    Figure US20220387631A1-20221208-C00363
    AL29
    Figure US20220387631A1-20221208-C00364
    AL30
    Figure US20220387631A1-20221208-C00365
    AL33
    Figure US20220387631A1-20221208-C00366
    AL34
    Figure US20220387631A1-20221208-C00367
    AL35
    Figure US20220387631A1-20221208-C00368
    AL36
    Figure US20220387631A1-20221208-C00369
    AL14
    Figure US20220387631A1-20221208-C00370
    AL79
    Figure US20220387631A1-20221208-C00371
    SP27
    Figure US20220387631A1-20221208-C00372
    SP28
    Figure US20220387631A1-20221208-C00373
    SP29
    Figure US20220387631A1-20221208-C00374
    SP30
    Figure US20220387631A1-20221208-C00375
    SP33
    Figure US20220387631A1-20221208-C00376
    SP43
    Figure US20220387631A1-20221208-C00377
    SP49
    Figure US20220387631A1-20221208-C00378
    SP51
    Figure US20220387631A1-20221208-C00379
    SP53
    Figure US20220387631A1-20221208-C00380
    SP79
    Figure US20220387631A1-20221208-C00381
    SP99
    Figure US20220387631A1-20221208-C00382
    SP116
    Figure US20220387631A1-20221208-C00383
    SP117
    Figure US20220387631A1-20221208-C00384
    ZK184
    Figure US20220387631A1-20221208-C00385
    ZK185
    Figure US20220387631A1-20221208-C00386
    ZK197
    Figure US20220387631A1-20221208-C00387
    ZK198
    Figure US20220387631A1-20221208-C00388
    ZK134
    Figure US20220387631A1-20221208-C00389
    ZK135
    Figure US20220387631A1-20221208-C00390
    TG4
    Figure US20220387631A1-20221208-C00391
    TG5
    Figure US20220387631A1-20221208-C00392
    TG7
    Figure US20220387631A1-20221208-C00393
    TG8
    Figure US20220387631A1-20221208-C00394
    TG27
    Figure US20220387631A1-20221208-C00395
    TP5
    Figure US20220387631A1-20221208-C00396
    QBN14
    Figure US20220387631A1-20221208-C00397
    CNN154
    Figure US20220387631A1-20221208-C00398
    EAO42
    Figure US20220387631A1-20221208-C00399
    ZK166
    Figure US20220387631A1-20221208-C00400
    PTN13
    Figure US20220387631A1-20221208-C00401
    PTN12
    Figure US20220387631A1-20221208-C00402
    ZK148
    Figure US20220387631A1-20221208-C00403
    ZK154
    Figure US20220387631A1-20221208-C00404
    E72
    Figure US20220387631A1-20221208-C00405
    ZK159
    Figure US20220387631A1-20221208-C00406
    E70
    Figure US20220387631A1-20221208-C00407
    ZK153
    Figure US20220387631A1-20221208-C00408
    PTN11
    Figure US20220387631A1-20221208-C00409
    ZK155
    Figure US20220387631A1-20221208-C00410
    MHI103
    Figure US20220387631A1-20221208-C00411
    CNN13
    Figure US20220387631A1-20221208-C00412
    WuA71
    Figure US20220387631A1-20221208-C00413
    ZK143
    Figure US20220387631A1-20221208-C00414
    ZK140
    Figure US20220387631A1-20221208-C00415
    ZK29
    Figure US20220387631A1-20221208-C00416
    SP34
    Figure US20220387631A1-20221208-C00417
    ZK104
    Figure US20220387631A1-20221208-C00418
    TP1
    Figure US20220387631A1-20221208-C00419
    ZK14
    Figure US20220387631A1-20221208-C00420
    PTN6
    Figure US20220387631A1-20221208-C00421
    LN65
    Figure US20220387631A1-20221208-C00422
    LN79
    Figure US20220387631A1-20221208-C00423
    AH34
    Figure US20220387631A1-20221208-C00424
    TP4
    Figure US20220387631A1-20221208-C00425
    LS1
    Figure US20220387631A1-20221208-C00426
    YY163
    Figure US20220387631A1-20221208-C00427
    TP6
    Figure US20220387631A1-20221208-C00428
    ZK15
    Figure US20220387631A1-20221208-C00429
    WuA108
    Figure US20220387631A1-20221208-C00430
    ZK150
    Figure US20220387631A1-20221208-C00431
    ZK156
    Figure US20220387631A1-20221208-C00432
    ESS23
    Figure US20220387631A1-20221208-C00433
    CNN17
    Figure US20220387631A1-20221208-C00434
    TG56
    Figure US20220387631A1-20221208-C00435
    AL31
    Figure US20220387631A1-20221208-C00436
    AL43
    Figure US20220387631A1-20221208-C00437
    TG31
    Figure US20220387631A1-20221208-C00438
    CNN3
    Figure US20220387631A1-20221208-C00439
    AL20
    Figure US20220387631A1-20221208-C00440
    TG115
    Figure US20220387631A1-20221208-C00441
    CNN2
    Figure US20220387631A1-20221208-C00442
    TP04
    Figure US20220387631A1-20221208-C00443
    ZK26
    Figure US20220387631A1-20221208-C00444
    ZK48
    Figure US20220387631A1-20221208-C00445
    TG44
    Figure US20220387631A1-20221208-C00446
    ZK27
    Figure US20220387631A1-20221208-C00447
    ZK46
    Figure US20220387631A1-20221208-C00448
    CNN1
    Figure US20220387631A1-20221208-C00449
    ZK79
    Figure US20220387631A1-20221208-C00450
    WuA38
    Figure US20220387631A1-20221208-C00451
    LN68
    Figure US20220387631A1-20221208-C00452
    ESS13
    Figure US20220387631A1-20221208-C00453
    WuA110
    Figure US20220387631A1-20221208-C00454
    YY161
    Figure US20220387631A1-20221208-C00455
    E71
    Figure US20220387631A1-20221208-C00456
    CNN8
    Figure US20220387631A1-20221208-C00457
    CNN10
    Figure US20220387631A1-20221208-C00458
    LN68Boc
    Figure US20220387631A1-20221208-C00459
    TG60
    Figure US20220387631A1-20221208-C00460
    ZK78
    Figure US20220387631A1-20221208-C00461
    ZK133
    Figure US20220387631A1-20221208-C00462
    CNN7
    Figure US20220387631A1-20221208-C00463
    ZK23
    Figure US20220387631A1-20221208-C00464
    MDL17
    Figure US20220387631A1-20221208-C00465
    TG11A
    Figure US20220387631A1-20221208-C00466
    TG11B
    Figure US20220387631A1-20221208-C00467
    TP2
    Figure US20220387631A1-20221208-C00468
    LN50
    Figure US20220387631A1-20221208-C00469
    TG17
    Figure US20220387631A1-20221208-C00470
    TG22
    Figure US20220387631A1-20221208-C00471
    LN34
    Figure US20220387631A1-20221208-C00472
    CNN16
    Figure US20220387631A1-20221208-C00473
    CNN12
    Figure US20220387631A1-20221208-C00474
    CNN145
    Figure US20220387631A1-20221208-C00475
    ZK203
    Figure US20220387631A1-20221208-C00476
    ZK204
    Figure US20220387631A1-20221208-C00477
    ZK208
    Figure US20220387631A1-20221208-C00478
    ZK196
    Figure US20220387631A1-20221208-C00479
    WuA67
    Figure US20220387631A1-20221208-C00480
    WuA76
    Figure US20220387631A1-20221208-C00481
    EAO40
    Figure US20220387631A1-20221208-C00482
    ZK106
    Figure US20220387631A1-20221208-C00483
    ZK124
    Figure US20220387631A1-20221208-C00484
    ZK126
    Figure US20220387631A1-20221208-C00485
    ZK101
    Figure US20220387631A1-20221208-C00486
    ZK172
    Figure US20220387631A1-20221208-C00487
    TG16
    Figure US20220387631A1-20221208-C00488
    MDL16
    Figure US20220387631A1-20221208-C00489
    CNN14
    Figure US20220387631A1-20221208-C00490
    LN37
    Figure US20220387631A1-20221208-C00491
    TG20
    Figure US20220387631A1-20221208-C00492
    LO4
    Figure US20220387631A1-20221208-C00493
    ZK211
    Figure US20220387631A1-20221208-C00494
    ZK214
    Figure US20220387631A1-20221208-C00495
    ZK215
    Figure US20220387631A1-20221208-C00496
    ZK217
    Figure US20220387631A1-20221208-C00497
    SRA89
    Figure US20220387631A1-20221208-C00498
    YY190
    Figure US20220387631A1-20221208-C00499
    YY220
    Figure US20220387631A1-20221208-C00500
    YY229
    Figure US20220387631A1-20221208-C00501
    YY231
    Figure US20220387631A1-20221208-C00502
    YY233
    Figure US20220387631A1-20221208-C00503
    YY238
    Figure US20220387631A1-20221208-C00504
    SRA94
    Figure US20220387631A1-20221208-C00505
    PS31
    Figure US20220387631A1-20221208-C00506
    ZK239
    Figure US20220387631A1-20221208-C00507
    AL11
    Figure US20220387631A1-20221208-C00508
    AL12
    Figure US20220387631A1-20221208-C00509
    CMI24
    Figure US20220387631A1-20221208-C00510
    CMI26
    Figure US20220387631A1-20221208-C00511
    El6
    Figure US20220387631A1-20221208-C00512
    El7
    Figure US20220387631A1-20221208-C00513
    E24
    Figure US20220387631A1-20221208-C00514
    E27
    Figure US20220387631A1-20221208-C00515
    E36
    Figure US20220387631A1-20221208-C00516
    E37
    Figure US20220387631A1-20221208-C00517
    E38
    Figure US20220387631A1-20221208-C00518
    E39
    Figure US20220387631A1-20221208-C00519
    E43
    Figure US20220387631A1-20221208-C00520
    E44
    Figure US20220387631A1-20221208-C00521
    E45
    Figure US20220387631A1-20221208-C00522
    E50
    Figure US20220387631A1-20221208-C00523
    E51
    Figure US20220387631A1-20221208-C00524
    E77
    Figure US20220387631A1-20221208-C00525
    E78
    Figure US20220387631A1-20221208-C00526
    E79
    Figure US20220387631A1-20221208-C00527
    E80
    Figure US20220387631A1-20221208-C00528
    E81
    Figure US20220387631A1-20221208-C00529
    ES17
    Figure US20220387631A1-20221208-C00530
    ES21
    Figure US20220387631A1-20221208-C00531
    ESS61
    Figure US20220387631A1-20221208-C00532
    LO1
    Figure US20220387631A1-20221208-C00533
    LO2
    Figure US20220387631A1-20221208-C00534
    LO3
    Figure US20220387631A1-20221208-C00535
    MHI106
    Figure US20220387631A1-20221208-C00536
    MHI128
    Figure US20220387631A1-20221208-C00537
    MHI84
    Figure US20220387631A1-20221208-C00538
    MHI86
    Figure US20220387631A1-20221208-C00539
    MHI96
    Figure US20220387631A1-20221208-C00540
    MHI97
    Figure US20220387631A1-20221208-C00541
    P700H
    Figure US20220387631A1-20221208-C00542
    P700SO3
    Figure US20220387631A1-20221208-C00543
    P800H
    Figure US20220387631A1-20221208-C00544
    P800SO3
    Figure US20220387631A1-20221208-C00545
    T14
    Figure US20220387631A1-20221208-C00546
    T17
    Figure US20220387631A1-20221208-C00547
    T18
    Figure US20220387631A1-20221208-C00548
    T20
    Figure US20220387631A1-20221208-C00549
    T23
    Figure US20220387631A1-20221208-C00550
    T24
    Figure US20220387631A1-20221208-C00551
    T25
    Figure US20220387631A1-20221208-C00552
    T27
    Figure US20220387631A1-20221208-C00553
    T29
    Figure US20220387631A1-20221208-C00554
    A20
    Figure US20220387631A1-20221208-C00555
    A21
    Figure US20220387631A1-20221208-C00556
    A80
    Figure US20220387631A1-20221208-C00557
    A100
    Figure US20220387631A1-20221208-C00558
    A104
    Figure US20220387631A1-20221208-C00559
    A106
    Figure US20220387631A1-20221208-C00560
    A134
    Figure US20220387631A1-20221208-C00561
    A138
    Figure US20220387631A1-20221208-C00562
    A146
    Figure US20220387631A1-20221208-C00563
    A148
    Figure US20220387631A1-20221208-C00564
    A149
    Figure US20220387631A1-20221208-C00565
    A150
    Figure US20220387631A1-20221208-C00566
    A160
    Figure US20220387631A1-20221208-C00567
    A161
    Figure US20220387631A1-20221208-C00568
    A24
    Figure US20220387631A1-20221208-C00569
    AC2
    Figure US20220387631A1-20221208-C00570
    AC3
    Figure US20220387631A1-20221208-C00571
    AC8
    Figure US20220387631A1-20221208-C00572
    AN3
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    WuA96
    Figure US20220387631A1-20221208-C00574
    Ox4
    Figure US20220387631A1-20221208-C00575
    Ox170
    Figure US20220387631A1-20221208-C00576
    Ox750
    Figure US20220387631A1-20221208-C00577
    Rh800
    Figure US20220387631A1-20221208-C00578
    TG66
    Figure US20220387631A1-20221208-C00579
    MM25
    Figure US20220387631A1-20221208-C00580
    SP72
    Figure US20220387631A1-20221208-C00581
    ZK138-2
    Figure US20220387631A1-20221208-C00582
    ZK169
    Figure US20220387631A1-20221208-C00583
    ZW800-1
    Figure US20220387631A1-20221208-C00584
    A64
    Figure US20220387631A1-20221208-C00585
    MHI85
    Figure US20220387631A1-20221208-C00586
    SP66
    Figure US20220387631A1-20221208-C00587
    YY113
    Figure US20220387631A1-20221208-C00588
    YY142
    Figure US20220387631A1-20221208-C00589
    PS37
    Figure US20220387631A1-20221208-C00590
    PS53
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    ZK240
    Figure US20220387631A1-20221208-C00592
    ZK244
    Figure US20220387631A1-20221208-C00593
    EAO72
    Figure US20220387631A1-20221208-C00594
    EAO76
    Figure US20220387631A1-20221208-C00595
    PS101A
    Figure US20220387631A1-20221208-C00596
    PS35
    Figure US20220387631A1-20221208-C00597
    PS36
    Figure US20220387631A1-20221208-C00598
    PS39
    Figure US20220387631A1-20221208-C00599
    PS51
    Figure US20220387631A1-20221208-C00600
    PS52
    Figure US20220387631A1-20221208-C00601
    PS62
    Figure US20220387631A1-20221208-C00602
    PS73
    Figure US20220387631A1-20221208-C00603
    PS76
    Figure US20220387631A1-20221208-C00604
    YY255
    Figure US20220387631A1-20221208-C00605
    YY260
    Figure US20220387631A1-20221208-C00606
    YY261
    Figure US20220387631A1-20221208-C00607
    YY269
    Figure US20220387631A1-20221208-C00608
    ZK243
    Figure US20220387631A1-20221208-C00609
    ZK2515
    Figure US20220387631A1-20221208-C00610
    ZK2525
    Figure US20220387631A1-20221208-C00611
    ZK2565
    Figure US20220387631A1-20221208-C00612
    ZK2566
    Figure US20220387631A1-20221208-C00613
    ZK258
    Figure US20220387631A1-20221208-C00614
    ZK2615
    Figure US20220387631A1-20221208-C00615
    A71-NHS
    Figure US20220387631A1-20221208-C00616
    LN15- NHS
    Figure US20220387631A1-20221208-C00617
    PS126
    Figure US20220387631A1-20221208-C00618
    PS127
    Figure US20220387631A1-20221208-C00619
    PS128
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    PS129
    Figure US20220387631A1-20221208-C00621
    PS130
    Figure US20220387631A1-20221208-C00622
    PS131
    Figure US20220387631A1-20221208-C00623
    PS132
    Figure US20220387631A1-20221208-C00624
    PS133
    Figure US20220387631A1-20221208-C00625
    YY283
    Figure US20220387631A1-20221208-C00626
    YY284
    Figure US20220387631A1-20221208-C00627
    YY285
    Figure US20220387631A1-20221208-C00628
    YY294
    Figure US20220387631A1-20221208-C00629
    YY295
    Figure US20220387631A1-20221208-C00630
    YY2102
    Figure US20220387631A1-20221208-C00631
    YY2103
    Figure US20220387631A1-20221208-C00632
    YY2106
    Figure US20220387631A1-20221208-C00633
    YY2107
    Figure US20220387631A1-20221208-C00634
    L700-1A
    Figure US20220387631A1-20221208-C00635
    L700-1C
    Figure US20220387631A1-20221208-C00636
    L800-1A
    Figure US20220387631A1-20221208-C00637
    L800-1C
    Figure US20220387631A1-20221208-C00638
  • Some of these molecules are shown in their carboxylic acid form, but are not limited thereto. In certain embodiments, such as those that will conjugate covalently to other molecules, the compounds may be prepared using a suitable leaving group or reactive group. Examples of preferred leaving groups include, but are not limited to N-hydroxysuccinimide (NHS) ester derivatives, sulfo-N-hydroxysuccinimide ester derivatives, and tetrafluorophenyl (TFP) esters. An example of a reactive group would be an azide or an alkyne as used for click chemistry.
  • In certain embodiments, the near-infrared fluorescent biological contrast agent is: LN15, A104, TG42, or A71.
  • In certain embodiments, the compounds of the invention absorb light at different wavelengths in the near-infrared region. Specifically, in some embodiments, the compounds of the invention absorb light in the 660-720 nm range. In other embodiments, the compounds of the invention absorb light in the 760-820 nm range.
  • In particular embodiments, the near-infrared fluorescent biological contract agent is:
  • LN15, A104, or TG42; and absorbs light in the 660-720 nm range.
  • In another particular embodiment, the near-infrared fluorescent biological contract agent is A71 and absorbs light in the 760-820 nm range.
  • In preferred embodiments, the compounds of the invention are cell-permeable. In preferred embodiments, the compounds of the invention are not significantly toxic to cells (e.g., to cells in culture or in vivo).
  • In certain embodiments, the imaging agent of the invention comprises a radioisotope having a single-photon or positron emission decay mode and suitable for detection by single-photon emission tomography (SPECT) or positron emission tomography (PET) in addition to its detection via optical properties (i.e., absorption and/or fluorescence). Examples of suitable radioisotopes include C-11 and F-18. Such isotopes can be incorporated into a compound of the invention, e.g., by use of appropriate isotopically-enriched reagents during synthesis of the compound. Additional useful radiotracers, such as Ga-68 Zr-89, or Rb-82 (PET), or Tc-99m (SPECT), can be attached to the compound through a radiometal chelator such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylene triamine pentaacetic acid (DTPA), hydrazinonicotinic acid (HYNIC), or desferoxime, respectively (or derivatives thereof). Chelator moieties can be covalently attached to an oxazine compound, e.g., through a linking atom or group, e.g., by acylation of a hydroxyl group of a compound of Formula I-V with a carboxylate group of a chelator such as DOTA. By incorporation of an appropriate PET- or SPECT-detectable isotope, a compound according to the invention can be detected using SPECT or PET imaging (e.g., even when administered at a low dose), e.g., using a conventional SPECT or PET imaging system, while also being detectable optically (e.g., by fluorescence imaging), e.g., when administered at a higher dose. Dual-mode optical and SPECT or PET imaging is also possible using such compounds. Similarly, imaging by magnetic resonance imaging (MRI), including dual-mode optical/MRI imaging, can be performed by using a compound of the invention comprising a lanthanide (such as Yb3+, Dy3+ or Gd3+), e.g., by chelating the lanthanide ion using a suitable chelating moiety.
  • Compounds of the invention can be prepared using a variety of methods, some of which are known in the art. For example, the compounds can be prepared using conventional methods of synthetic organic chemistry (see, e.g., Michael B. Smith, “March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th Edition”, Wiley (2013)).
  • For example, compounds of the invention can be synthesized using the syntheses described in schemes 1-12 shown below. General references for the syntheses described in the schemes 1-6, 7a-c, 7e, 8-10, 11, 11a, and 12 is found in Henary, M. et al. Bioorg. & Med. Chem. Let. 22, 242-1246, (2012), Henary, M. et al. J. Heterocycl. Chem. 46: 84-87, (2009), Henary, M. et al. Dyes and Pigments. 99, 1107-1116 (2013), Henary, M. et al. Heterocycl. Commun. 19 (1), 1-11 (2013), Mojzych, M. et al. Topics in Heterocyclic, Springer-Verlag Berlin Heidelberg. 14, 1-9 (2008), Strekowski, L. et al. J. Org. Chem. 57, 4578-80 (1992), Halder, S. et. al. Eur. J. Med. Chem. 54, 647-59 (2012), Sakiko, A. et al. Chem.-A Eur. J., 15, 9191-9200 (2009), Chang, Y-T. et al. Chem. Commun. 47, 3514-3516 (2011), Myochin, T. J. Am. Chem. Soc. 134, 13730-13737 (2012), Briza, T. et al. Chem. Comm. 16, 1901-1903 (2008), Chang, Y-T. et al. Chem. Commum. 46, 7406-7408 (2010), Zaheer, A., et al. Molecular Imag. 1 (4), 1536-0121 (2002), Misra, P. et al. J Nucl Med. 2007 August; 48(8):1379-89, and Humblet, V. et al, J Med Chem. 2009 Jan. 22; 52(2):544-50.
  • Figure US20220387631A1-20221208-C00639
  • Scheme 1 outlines the synthesis of a series of 700 nm NIR emitting pentamethine cyanine dye derivatives 10-15. The key step is the quaternization of the nitrogen atom in indolenine derivatives 1-3 in either refluxing acetonitrile or toluene for between 10 hours and 3 days, depending on the alkylating agent, to yield the cationic salts 4-9. Condensation of salts 4-9, in a two molar ratio, containing an activated methyl group with malonoaldehyde bisphenylamine hydrochloride under basic conditions, normally achieved through the addition of triethyl amine or sodium acetate, furnished the pentacyanines in 70 to 90% yield, as depicted in Scheme 1, in less than 6 hours.
  • A series of chloro derivatives of pentamethine cyanine dyes 16-20 (Scheme 1) have also been prepared. Using the same chemistry the chloro-substituted pentamethine cyanine dyes can readily undergo nucleophilic substitution at the meso position to replace the chlorine atom with versatile functionalities, although care has to be taken with charged substituents, which may complicate cross-coupling reactions. Chloro-substitution is also useful for introducing specific targeting ligands or biomolecules via covalent conjugation. These reactions require a precise choice of functionalities, as well as a feasible synthetic methodology. A majority of these dyes are constructed via a well-established S NR1 mechanistic pathway where the meso chlorine atom of the polymethine dye is substituted with various nucleofugal functionalities. This route renders an array of fluorophores that contain highly functional aminoalkyl, hydroxyalkyl, hydroxyaryl, thioalkyl, and thioaryl substituents, which can be further conjugated to ligands or biomolecules.
  • Figure US20220387631A1-20221208-C00640
  • Using similar chemistry as described in Scheme 1, including reaction conditions, the compounds depicted in Scheme 2 were synthesized. The compounds prepared contain cationic quaternary ammonium substituents on the heterocyclic nitrogen. Additionally, the heterocyclic character was varied by incorporating a methoxy group at the 5-position or by using a benz[e]indolenine core. The systematic set of 700 nm cyanines (24-26) was replicated using the heptamethine cyanine structure to achieve fluorescence wavelengths around 800 nm (32-34). Compared to the compounds depicted in Scheme 1, these final products (25, 26, 33 and 34) exhibit red-shifted optical properties (wavelength of maximum absorption and wavelength of maximum emission) of approximately 20 nm. These compounds also take slightly longer to form and an additional 2 hours are required to facilitate the completion of the condensation reaction in 60 to 80% yield after purification.
  • Figure US20220387631A1-20221208-C00641
  • In order to form heterocyclic-substituted pentamethine cyanine dyes originating from the indolenine only provides limited chemical space for modification; however, beginning with the 4-substituted phenylhydrazine hydrochloride and upon treatment with a single molar ratio of 3-methyl-2-butanone in acidic conditions (boiling glacial acetic acid) yields the corresponding indolene derivative which is then alkylated using aforementioned synthetic protocol to afford the quaternized nitrogen salts. These salts then react with substituted dianil-malononitrile analogs to yield the highly substituted pentamethine cyanines.
  • This synthetic route is necessary to prepare the ether, amide and highly charged analogs shown in Scheme 3. The amide-containing compounds require synthesizing the corresponding carboxylic acid which is then coupled to an amine to form the depicted compounds which would synthetically occur after amine quaternization. The compounds with ether linkages would begin with the aryl alcohol at R1 followed by treatment with sodium hydride and addition of the appropriate alkylating reagent.
  • Figure US20220387631A1-20221208-C00642
  • Scheme 4 outlines the synthesis of compounds with net charges between +2 to +4 (compound 6 in Scheme 4), which affords the potential for 4 quaternary ammonium cations. The heterocyclic nitrogen of 2 is quaternized with trimethyl(3-bromopropyl)ammonium bromide or other alkylating agent with reaction conditions as described above to give intermediate product 2. The carboxylic acid group of 2 is allowed to react with the free primary amine of (3-aminopropyl)trimethylammonium bromide in the presence of TSTU for 3-5 h under basic conditions to furnish amide 3. The reaction of 3 with the Vilsmeier-Haack reagent 4 provides the chloro-substituted dye 5. The treatment of 5 with a disodium derivative of 3-(4-hydroxyphenyl)propionic acid in DMF or in DW/DMSO at 65° C. for 6 hours produces a carboxylic derivative 6 (ZW-4) for effective coupling, via its single carboxylic acid, to targeting ligands with a +4 net charge. Other compounds carrying+2 can be synthesized using the same methodology as outlined in Scheme 4.
  • Figure US20220387631A1-20221208-C00643
  • Scheme 5 summarizes the synthesis of analogs of NF800 (net charge+2) ether derivatives. The phenolic nature of the Fischer salt heterocycle is modified further with quaternary salts via ether linkage under basic conditions in DMF at 80° C. and the heterocyclic ring nitrogens will be altered with various alkyl groups. To synthesize analogs of NF800 with higher net charge (+4) the alkyl group of the heterocyclic nitrogens is terminated with additional quaternary ammonium salts. This is achieved by synthesizing the quaternary ammonium salt by reacting the 5-hydroxyindolenine compound with iodomethane for 3 days by heating 1,2-dichlorobenzene in a sealed tube. After obtaining the desired compound 8 the alcohol is deprotonated using 1.2 molar equivalents of sodium hydride in dry DMF followed by the addition of 3-bromopropyltrimethylammmonium bromide alkylating agent. The reactive methyl group reacts as previously discussed to afford the final target compound 11.
  • Figure US20220387631A1-20221208-C00644
  • Using identical reaction conditions as outlined in Schemes 4 and 5 other synthetic routes are presented in Scheme 6 to synthesize various halogenated analogs of compounds type 5 (Scheme 4) and 10 (Scheme 5) at the central carbon of the heptamethine cyanine dye.
  • Figure US20220387631A1-20221208-C00645
  • As shown in Scheme 7, the replacement of the halogen at the meso carbon of the dye with various nucleophiles does not occur under various reaction conditions (with/without base and lower/higher reaction temperature) due to the steric effect of cis-trans photoisomerization. To overcome this problem, the synthetic procedures in Schemes 7a-c and 7d are optimized to synthesize large numbers of novel pentacyanine dyes substituted various nucleophiles at the meso carbon of the dye.
  • Figure US20220387631A1-20221208-C00646
  • Figure US20220387631A1-20221208-C00647
  • Figure US20220387631A1-20221208-C00648
  • As shown in Scheme 7a-c, the synthetic routes of 700 nm zwitterionic NIR fluorophore (LN15, A104, TG42) were successfully synthesized through the reaction between quaternary salt 3 and carboxylic acid reagents. LN15 was successfully synthesized by reacting the bromo reagent with disodium salt of 3-(4-hydroxyphenyl)propanoic acid in DMSO at 65° C. Without further purification, the crude product was used to react with the quaternary salt under basic conditions, and LN15 was obtained with very low yield (10%) after reverse phase chromatography purification. A104 was also synthesized with an ethanoic acid moiety added to the central carbon of the polymethine chain. The synthesis begins with methyl 5,5-dimethoxypentanoate reacting with oxalyl chloride followed by a basic workup and treatment with anilinium chloride to form reactive intermediate 4. Reaction with heterocyclic salt 3 proceeds with 4 hours of heating in a mixture of ethanol and acetic anhydride with sodium acetate to yield the final compound A104. TG42 was successfully synthesized through Suzuki-Miyaura cross coupling reaction with an effective 78% yield.
  • Another methodology as depicted in Scheme 7d may also be utilized
  • Figure US20220387631A1-20221208-C00649
  • As outlined in Scheme 7d, a novel methodology was applied to synthesis TG42 in fair yield 40% by reacting the phenolic compound 1 with 2-bromoacetic acid under basic conditions for 6 h to yield compound 2. Vilsmeier formylation using phosphorous oxychloride and DMF was conducted on 2 for 5 h followed by basic workup to form the decarboxylated bis-aldehyde 3. Compound 3 was allowed to react with the quaternary salt using sodium acetate in boiling ethanol for around 3-5 h to afford the desired compound.
  • As such, in another aspect, the invention provides a method of preparing a compound, the method comprising reacting a decarboxylated bis-aldehyde with a quarternary salt to produce the compound.
  • In certain embodiments, the method comprises reacting a decarboxylated bis-aldehyde of the formula
  • Figure US20220387631A1-20221208-C00650
  • with a quarternary salt of the formula
  • Figure US20220387631A1-20221208-C00651
  • Figure US20220387631A1-20221208-C00652
  • To synthesize pentamethine dyes with 5- or 6-membered rings at the dye central position, the chemistry outlined in Scheme 7e is applied to synthesize analogs of LN15, A104, and TG42. In particular, 2-halo 1,3-dicarbonyl pentane or hexane is used as the starting material under ethanol reflux using basic condition to react with activated indolenine derivatives under basic conditions to afford the desired compounds.\
  • Figure US20220387631A1-20221208-C00653
  • The carbon-carbon bond coupling provides additional stability to the molecule and the compounds TG42 and A71 are synthesized using the palladium mediated Suzuki-coupling reaction. In particular embodiments, the synthesis uses 5 molar percent of tetrakistriphenylphosophine palladium(O) for the coupling reaction and cesium carbonate (3 eq) as the base. This reaction proceeds satisfactorily in water or alcoholic water at elevated temperature using the 3-(4-boronophenyl)propanoic acid and meso-halogenated penta- or heptamethine precursors.
  • Figure US20220387631A1-20221208-C00654
  • Pamidronate (PAM)-modified heptamethine fluorophores, PAM-ZW800-1 and PAM-CW800 are prepared originating with protected beta-alanine. Reacting the terminal acid of 3-[(benzyloxycarbonyl)amino]-propionic acid (20 mmol) with oxalyl chloride 100 mmol at 0° C. in dichloromethane for 30 min and at room temperature for 6 h yields the acyl chloride 2 which undergoes nucleophilic acyl substitution with trimethyl phosphite (22 mmol) which was added drop wise at 0° C. for 5 min. The volatile organic solvent was evaporated under reduced pressure and washed with hexane to give dimethyl [3-(Benzyloxycarbonylamino)-1-(dimethoxyphosphoryl)-1-hydroxypropyl] phosphonate 3 as pale yellow oil (88% yield). Compound 3 is placed in a 500-mL Parr bottle under N2 and dissolved in 50 mL of cold denatured methanol. Palladium on activated carbon (10 wt. %) was added carefully and the Parr shaker apparatus assembled. The reaction is shaken at 50 psi H2 and room temperature until H2 uptake is complete (6-12 hours). The palladium on activated carbon was filtered over a Celite pad; the solvent was evaporated under reduced pressure to give dimethyl [3-amino-1-(dimethoxyphosphoryl)-1-hydroxypropyl] phosphonate as yellow solid (90% yield), the PAM-precursor 6 bearing a primary amine is synthesized and ready for further reactions with NHS-ester modified ZW800-1 and/or CW800. Reacting the precursor 6 with the NHS-ester dyes forms the amide bonds shown in the final compounds symmetrical PAM-ZW800-1 and asymmetrical PAM-CW800.
  • Figure US20220387631A1-20221208-C00655
  • Similar to compounds with a propyltrimethylammonium bromide moiety on the heterocyclic nitrogen, the compounds presented in Scheme 10 contain a phosphonic acid group for molecular targeting. Using identical reaction conditions as discussed above, (3-bromopropyl)phosphonic acid is used in boiling toluene for 18 h to synthesize the quaternized heterocyclic nitrogen in about 3 days. After this step, the dye synthesis proceeds as previously discussed to yield the final Phosphonated 700 nm (P700H and P700SO3) and Phosphonated 800 nm (P800H and P800SO3) Fluorophores.
  • Figure US20220387631A1-20221208-C00656
    Figure US20220387631A1-20221208-C00657
  • Synthesis of PEGylated 99mTc-MAS3 targeting compound begins by dissolving 1 eq.mol mPEG-NH2 (2) in dry DMSO followed by the addition of 1.5 eq.mol Boc-Glu(OBzl)-OSu (5-benzyl-1-(2,5-dioxopyrrolidin-1-yl) (tert-butoxycarbonyl)glutamate) (1) and 0.5 eq.mol triethylamine, stirred at room temperature for 5 h, followed by catalytic hydrogenation to remove the Cbz group. To synthesize the NHS ester (4), compound 3 was dissolved in dry THE followed by the addition of TBTU and NHS. To get compound 6, trimerized ligands (5) were taken in dry DMSO and added to the mPEG-NHS ester (4) followed by deprotection of the Boc-group. Finally, [99mTc-MAS3]-NHS was added to compound 6 in DMSO and reacted for 1 hr to yield compound 7.
  • Figure US20220387631A1-20221208-C00658
    Figure US20220387631A1-20221208-C00659
  • ZW800-1 and 99mTc-MAS3 NHS ester can both be modified with various length units of polyethylene glycol modifications through the NHS-ester linkage. The free primary amine on the polyethylene glycol group can easily react with the NHS-ester of the ZW800-1 NHS in PBS at pH 8.0 for 3 h and form the corresponding amide. ZW800-1 must be modified to form the NHS-ester using our effective coupling method of HSPyU in a mixture of DIPEA and DMSO in 30 minutes. Further modifications with the NH2—PEG are performed in PBS, pH 8.0 for approximately 3 hours.
  • Figure US20220387631A1-20221208-C00660
  • Pentamethine and heptamethine fluorophores bearing heterocyclic modifications (R═H, OMe, F, Cl, Br) are presented in Scheme 12. They have shown excellent promise in delineating the thyroid and parathyroid at 700 nm and 800 nm. They were prepared according to Scheme 12 using our developed methods as previously discussed in Schemes 1 and 2.
  • All products of Schemes 1-12 have been synthesized and were obtained in high purity (>97%) as indicated by HPLC and TLC analyses using silica gel or C-18 adsorbents. Their high-resolution 1HNMR and 13CNMR spectra are consistent with the indicated structures and confirmed high purity of the samples. Electron-spray mass spectroscopy (ES-MS) was also used to characterize the products and gave the expected peak M+1 as the only peak in the high molecular mass range in each case.
  • Each of the compounds of the invention can be synthesized using the methods outlined in Schemes 1-12 above upon modification of starting materials and other reagents as will be readily understood by one of ordinary skill in the art.
    • Compositions
  • In another aspect, the invention provides pharmaceutical compositions of a compound of the invention.
  • For the therapeutic uses of compounds provided herein, including compounds of the invention, or pharmaceutically acceptable salts, solvates, N-oxides, prodrugs, or isomers thereof, such compounds are administered in therapeutically effective amounts either alone or as part of a pharmaceutical composition. Accordingly, provided herein are pharmaceutical compositions, which comprise at least one compound provided herein, including at least one compound of the invention, pharmaceutically acceptable salts and/or solvates thereof, and one or more pharmaceutically acceptable carriers, diluents, adjuvant or excipients. The methods of administration of such compounds and compositions include, but are not limited to, intravenous administration, inhalation, oral administration, rectal administration, parenteral, intravitreal administration, subcutaneous administration, intramuscular administration, intranasal administration, dermal administration, topical administration, ophthalmic administration, buccal administration, tracheal administration, bronchial administration, sublingual administration or optic administration. Compounds provided herein are administered by way of known pharmaceutical formulations, including tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for parenteral or intramuscular administration, lotions, gels, ointments or creams for topical administration, and the like.
  • The amount administered will vary depending on, among others, the tissue or organ to be imaged, the age and relative health of the subject, the potency of the compound administered, the mode of administration and the like.
  • Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic/anionic or basic/cationic salts. Pharmaceutically acceptable acidic/anionic salts include acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, malonate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, hydrogensulfate, tannate, tartrate, teoclate, tosylate, and triethiodide salts. Pharmaceutically acceptable basic/cationic salts include, the sodium, potassium, calcium, magnesium, diethanolamine, N-methyl-D-glucamine, L-lysine, L-arginine, ammonium, ethanolamine, piperazine and triethanolamine salts.
  • A pharmaceutically acceptable acid salt is formed by reaction of the free base form of a compound of Formula I-V with a suitable inorganic or organic acid including, but not limited to, hydrobromic, hydrochloric, sulfuric, nitric, phosphoric, succinic, maleic, formic, acetic, propionic, fumaric, citric, tartaric, lactic, benzoic, salicylic, glutamic, aspartic, p-toluenesulfonic, benzenesulfonic, methanesulfonic, ethanesulfonic, naphthalenesulfonic such as 2-naphthalenesulfonic, or hexanoic acid. A pharmaceutically acceptable acid addition salt of a compound of Formula I-V can comprise or be, for example, a hydrobromide, hydrochloride, sulfate, nitrate, phosphate, succinate, maleate, formarate, acetate, propionate, fumarate, citrate, tartrate, lactate, benzoate, salicylate, glutamate, aspartate, p-toluenesulfonate, benzenesulfonate, methanesulfonate, ethanesulfonate, naphthalenesulfonate (e.g., 2-naphthalenesulfonate) or hexanoate salt.
  • The free acid or free base forms of the compounds of the invention may be prepared from the corresponding base addition salt or acid addition salt form, respectively. For example a compound of the invention in an acid addition salt form may be converted to the corresponding free base form by treating with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, and the like). A compound of the invention in a base addition salt form may be converted to the corresponding free acid by treating with a suitable acid (e.g., hydrochloric acid, etc.).
  • Prodrug derivatives of the compounds of the invention may be prepared by methods known to those of ordinary skill in the art (e.g., for further details see Saulnier et al., Bioorg. Med. Chem. Letters, 1994, 4, 1985; the entire teachings of which are incorporated herein by reference).
  • Protected derivatives of the compounds of the invention may be prepared by means known to those of ordinary skill in the art. A detailed description of techniques applicable to the creation of protecting groups and their removal can be found in T. W. Greene, “Protecting Groups in Organic Chemistry,” 3rd edition, John Wiley and Sons, Inc., 1999, the entire teachings of which are incorporated herein by reference.
  • Compounds of the invention may be prepared as their individual stereoisomers by reaction of a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomers. Resolution of enantiomers may be carried out using covalent diastereomeric derivatives of the compounds of the invention, or by using dissociable complexes (e.g., crystalline diastereomeric salts). Diastereomers have distinct physical properties (e.g., melting points, boiling points, solubility, reactivity, etc.) and may be readily separated by taking advantage of these dissimilarities. The diastereomers may be separated by chromatography, or by separation/resolution techniques based upon differences in solubility. The optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization. A more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture can be found in Jean Jacques, Andre Collet and Samuel H. Wilen, “Enantiomers, Racemates and Resolutions,” John Wiley And Sons, Inc., 1981, the entire teachings of which are incorporated herein by reference.
  • Suitable pharmaceutically acceptable carriers, diluents, adjuvants, or excipients for use in the pharmaceutical compositions of the invention include tablets (coated tablets) made of for example collidone or shellac, gum Arabic, talc, titanium dioxide or sugar, capsules (gelatin), solutions (aqueous or aqueous-ethanolic solution), syrups containing the active substances, emulsions or inhalable powders (of various saccharides such as lactose or glucose, salts and mixture of these excipients with one another) and aerosols (propellant-containing or -free inhale solutions).
  • Excipients which may be used include, for example, water, pharmaceutically acceptable organic solvents such as paraffins (e.g., petroleum fractions), vegetable oils (e.g. groundnut or sesame oil), mono- or polyfunctional alcohols (e.g., ethanol or glycerol), carriers such as natural mineral powders (e.g., kaoline, clays, talc, chalk), synthetic mineral powders (e.g., highly dispersed silicic acid and silicates), sugars (e.g., cane sugar, lactose and glucose), emulsifiers (e.g., lignin, spent sulphite liquors, methylcellulose, starch and polyvinylpyrrolidone) and lubricants (e.g., magnesium stearate, talc, stearic acid and sodium lauryl sulphate).
  • Exemplary methods for preparing the compounds of the invention are described herein, including in the Examples.
    • Methods
  • The present invention features various methods using the near-infrared fluorescent biological contrast agents described herein.
  • In one aspect, the invention provides a method of imaging biological tissue or cells, the method comprising:
  • (a) contacting the biological tissue cells with a compound of the invention;
  • (b) irradiating the cells at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the biological cells.
  • The signal may be in the form of absorption, such as occurs during photoacoustic imaging. Alternatively, the imaging agents can have a SBR suitable to permit fluorescence detection. SBR is a measure of the intensity of the fluorescent signal obtained from a target (peak signal) over the measure of the intensity of the fluorescent signal obtained nearby the target (background signal), the target being the tissues, cells, space targeted by the imaging agent. SBR measurements can be readily obtained through routine measurement procedures. For fluorescent imaging systems, and other optical-type systems, digital images recording optical signals of the target facilitate SBR measurement. Higher SBR values are more desirable, resulting in greater resolution of the imaged tissues. In some embodiments, the imaging agents achieve an SBR of at least about 1.1 (i.e., peak signal is at least 10% over background). In further embodiments, the imaging agents achieve an SBR of at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, or at least about 2.0. In yet further embodiments, the imaging agents achieve an SBR of about 1.1 to about 50, about 1.5 to about 30, about 2.0 to about 20, about 2.0 to about 5.0, or about 5.0 to about 10.
  • Some of the imaging agents include one or more ionic groups. In some embodiments, the imaging agents include two or more, three or more, four or more, or five or more ionic groups. Ionic groups serve to increase solubility of the generally hydrophobic dye portions of the imaging agent, thus improving biodistribution. They may also contribute to specific targeting. The ionic groups can be located on any portion of the imaging agent.
  • In certain instances, the imaging agents are hydrophobic agents. In such instances, the hydrophobic agents are capable of conjugating to a hydrophobic compound for imaging, without altering the binding, biodistribution, cell permeation, and/or clearance of the hydrophobic compound. Examples of hydrophobic agents include, but are not limited to L700-1A, L700-1C, L800-1A, and L800-1C.
  • In certain embodiments, the imaging agents are administered directly to a subject or biological system for the imaging of the targeted cells.
  • In other embodiments, reactive derivates of the imaging agents of the invention are used to label chemical and biological molecules for further study. Certain molecules which may be labeled using reactive derivatives of the imaging agents of the invention include small molecules (including pharmaceutical, neutraceutical, therapeutic and diagnostic compounds, proteins, peptides, peptidomimetics, antibodies, vaccines, and other chemical and biological molecules which may be of interest in studying by NIR imaging. In such embodiments, the imaging agent of the invention is reacted with the chemical or biological molecule to produce a labeled agent molecule which may then be administered to a subject or biological system for imaging as described herein.
  • The steps of irradiating the tissue or cells at a wavelength absorbed by the imaging agent, and detecting an optical signal from the irradiated tissue or cells, thereby imaging the tissue or cells, can be performed using an imaging system such as the FLARE™ Image-Guided Surgery System, which is a continuous-wave (CW) intraoperative imaging system that is capable of simultaneous, real-time acquisition and display of color video (i.e., surgical anatomy) and two channels of invisible NIR fluorescent (700 nm and 800 nm) light (see, e.g., Gioux et al., Mol. Imaging. 9(5): 237-255 (2010) and U.S. Pat. No. 8,473,035 to Frangioni, for a description of suitable systems). With FLARE™ and other NIR fluorescence imaging systems, contrast agent emission wavelength in the 800-850 nm range (Channel 2 of FLARE™) is preferred whenever possible because of lower autofluorescence and lower attenuation from both absorbance and scatter when compared to emission near 700 nm. Nevertheless, fluorophores emitting within Channel 1 (≈700 nm) of the FLARE™ imaging system still retain the benefits of NIR fluorescence imaging, including detection of nerves and other targets below the surface of blood and tissue.
  • In some embodiments, the imaging agents can be formulated into pharmaceutically acceptable formulations and administered intravenously to an organism for imaging. The dosed organism can be imaged using, for example, the FLARE™ system. The imaging system can irradiate the dosed organism with radiation absorbed by the imaging agent, and detect optical signals, such as NIR fluorescence, emanating from the targeted portions of the organism containing the imaging agent. The detected signals can be recorded and analyzed by obtaining digital images or video of the subject organism, thereby facilitating diagnostic procedures and image-guided medical techniques.
  • The invention also provides methods of performing image-guided surgery, the methods comprising imaging cells, tissues, or organs according to a method described herein, and then performing surgery such that the targets are either removed or are preserved, depending on the goals of the surgical intervention. In certain preferred embodiments, the contrast agent is injected intravenously to ensure that all targets are labeled, and imaging is performed after sufficient time has passed for biodistribution to nerves and clearance of surrounding background signal.
  • In certain embodiments, the targets are biological tissues or organs. In specific embodiments, the targets are lumens, such as the ureters, cartilage, bone cells, bone minerals, thyroid gland, parathyroid gland, adrenal gland, salivary gland, white adipose tissue, brown adipose tissue, ovaries, testes, seminal vesicles, prostate, pancreas, spleen, gallbladder, bile ducts, Peyer's patches, brain grey matter, brain white matter, brain vasculature, choroid plexus, cerebrospinal fluid, nerves, thoracic duct, pan lymph nodes, sentinel lymph nodes, vulnerable plaque, stem cells, or neuroendocrine tumor cells.
  • It should also be noted that although the examples given below are for in vivo imaging, which represents the most difficult situation because properties such as biodistribution and clearance are dictated in large part by the organism, those skilled in the art will recognize that these same contrast agents can be used for any type of in vitro assay, such as immunohistology, detection of targets in blood or bodily fluid samples, etc. using the same principles of contact with the medium, washout of unbound dose, and detection of a signal derived from absorption, fluorescence emission and/or radioactive emission.
    • NIR Angiography Agents
  • A NIR fluorophore injected into the bloodstream can act as an angiographic agent because during the first 8 seconds after intravenous injection there is a rapid arterial flush (≈1 second), a rapid capillary flush (2-3 seconds), a rapid venous flush (≈1-2 seconds), then minutes of clearance from the tissue. The first 8 seconds thus provides a “map” of the circulation in the tissue. NIR angiography is important for imaging the perfusion of skin during plastic and reconstructive surgery and the anastomoses of bowel during gastrointestinal surgery. In general, NIR angiography agents are those that are cleared rapidly from the blood into either urine or bile.
  • As such, in one aspect, the invention provides a method for imaging tissue perfusion, the method comprising:
  • (a) contacting the blood with a compound of the invention;
  • (b) irradiating the blood vessels and surrounding tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the distribution and clearance of fluorophore in the tissue over time.
  • In a particular embodiment, the compound of the invention for angiography is LN15, A104, or TG42; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compounds of the invention for use in angiography is A71 or WuA71; and the irradiating wavelength is in the 760-800 nm range.
    • Ureter Mapping Agents:
  • Ureter mapping agents are those molecules that are rapidly cleared from the bloodstream by the kidney into urine. As the molecules traverse the ureters towards the bladder, the ureters become highly fluorescent and thus visible. This is useful during Caesarian section, where the ureters are sometimes damaged, as well as many abdominal cancer surgeries where finding the ureters and avoiding them can be difficult.
  • As such, in one aspect, the invention provides a method for imaging the ureters, the method comprising:
  • (a) contacting the blood with a compound of the invention;
  • (b) irradiating the ureters at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the ureters.
  • In a particular embodiment, the compound of the invention for use in ureter imaging is LN15, A104, or TG42; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compounds of the invention for use in ureter imaging is A71 or WuA71; and the irradiating wavelength is in the 760-800 nm range.
    • Cartilage Agents:
  • Cartilage agents are useful in arthroscopic surgery, general surgery, and non-invasive assessment of neo-cartilage growth during tissue engineering.
  • As such, in one aspect, the invention provides a method for imaging cartilage cells and/or their products, the method comprising:
  • (a) contacting the cartilage cells and/or their products with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging cartilage cells and/or their products.
  • In a particular embodiment, the compound of the invention for use in cartilage imaging is SP56, E58, YY180, E59, E60, A196, E71, E72 or ZK15; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in cartilage imaging is LN50, A64, AL30, MM25, MM21, AL31, AL33, SP79, SP99, SP116, SP117, LN65, LN68, LN63, ZK48, CNN3, or CNN4; and the irradiating wavelength is in the 760-800 nm range.
    • Bone Agents:
  • Bone agents are useful in the detection of bone metastases, bone growth and tissue microcalcification.
  • As such, in one aspect, the invention provides a method for imaging bone, the method comprising:
  • (a) contacting bone cells and/or their products with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the bone cells and/or their products.
  • In a particular embodiment, the compound of the invention for use in bone imaging is P700SO3, P700H, CMI24, E24, E37, E38, E44, or WuA110; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in bone imaging is P800SO3, P800H, ZK197, or WuA71; and the irradiating wavelength is in the 760-800 nm range.
    • Thyroid Agents:
  • In one aspect, the invention provides a method for imaging the thyroid gland, the method comprising:
  • (a) contacting the thyroid cells and/or their products with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the thyroid cells and/or their products.
  • In a particular embodiment, the compound of the invention for use in thyroid imaging is T14, T27, T29, MHI84, T18, T20, T23, T24, T25, LO4, E27, E45, MHI106, MHI128, TP1, NRB3, SP28, SP29, SP30, SP33, SP34, SP51, SP59, SP60, SP72, PTN11, PTN12, ZK26, ZK143, ZK148, or ZK204; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in thyroid imaging is QBN14, QBN1, AL20, ZK172, ZK185, ZK190, ZK208, MDL17, CNN145, ZK154, or ZK159; and the irradiating wavelength is in the 760-800 nm range.
    • Parathyroid Agents:
  • In one aspect, the invention provides a method for imaging the parathyroid gland, the method comprising:
  • (a) contacting the parathyroid cells and/or their products with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the parathyroid cells and/or their products.
  • In a particular embodiment, the compound of the invention for use in parathyroid imaging is T14, T27, T29, or MHI84; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in parathyroid imaging is QBN14, MDL17, QBN1, or AL20; and the irradiating wavelength is in the 760-800 nm range.
    • Adrenal Gland Agents:
  • Adrenal gland agents are useful to highlight the adrenal gland after an intravenous injection.
  • As such, in one aspect, the invention provides a method for imaging adrenal medulla and/or adrenal cortex, the method comprising:
  • (a) contacting the adrenal tissue with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the adrenal medulla and/or adrenal cortex.
  • In a particular embodiment, the compound of the invention for use in adrenal gland imaging is E16, MHI96, MHI97, EAO40, MHI186, LS1, LO3, LO4, E24, E27, E36, E37, E43, E45, E50, E51, E77, E79, E80, ZK50, ZK59, ZK106, SP29, SP30, SP33, SP51, SP53, SP60, SP64, YY161, YY163, or YY165; and the irradiating wavelength is in the 60-700 nm range.
  • In another particular embodiment, the_compound of the invention for use in adrenal gland imaging is AL27, AL25, AL29, AL20, ZK190, ZK184, or MDL17; and the irradiating wavelength is in the 760-800 nm range.
    • Salivary Glands:
  • Salivary gland agents are useful for targeting salivary gland tumors or for avoiding normal salivary glands during head and neck surgery.
  • As such, in one aspect, the invention provides a method for imaging salivary glands, the method comprising:
  • (a) contacting the salivary glands with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the salivary glands.
  • In a particular embodiment, the compound of the invention for use in salivary gland imaging is NRB1, ZK195, ZK135, NRB2, YY163, ZK195, YY161, E79, TP1, SP28, SP29, SP30, SP49, SP72, ZK101, ZK133, ZK134, ZK135, ZK143, ZK150, ZK155, ZK156, ZK159, ZK185, ZK204, T29, or CNN145; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in salivary gland imaging is ZK211, ZK172, MDL17, ZK198, ZK190, AL22, or AL20; and the irradiating wavelength is in the 760-800 nm range.
    • White Adipose Tissue:
  • White adipose tissue agents are useful for highlighting white fat important for certain surgical procedures.
  • As such, in one aspect, the invention provides a method for imaging white adipose tissue, the method comprising:
  • (a) contacting the white adipose tissue with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the white adipose tissue.
  • In a particular embodiment, the compound of the invention for use in white adipose imaging is PS31, CMI26, E24, MHI86, ZK240, or ZK244; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in white adipose imaging is AH34, PS37, or ZK197; and the irradiating wavelength is in the 760-800 n, range.
    • Brown Adipose Tissue:
  • Brown adipose tissue agents are useful for highlighting brown fat during surgery and for “imaging” perfusion of the tissue.
  • As such, in one aspect, the invention provides a method for imaging brown adipose tissue, the method comprising:
  • (a) contacting the brown adipose tissue with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the brown adipose tissue.
  • In a particular embodiment, the compound of the invention for use in brown adipose tissue imaging is SP60, PS39, SP30, SP29, SP33, SP34, E39, E44, E51, E81, ES17, ZK27, ZK26, SP28, SP27, SP67, PS31, LO1, LO3, YY165, or YY187; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in brown adipose tissue imaging is QBN1 or PS37; and the irradiating wavelength is in the 760-800 nm range.
    • Ovaries:
  • Ovary-specific agents have two major functions. The first is to find and eradicate endometriosis, a painful condition of pre-menopausal women. The second is to find and eradicate ovarian carcinoma.
  • As such, in one aspect, the invention provides a method for imaging ovaries, the method comprising:
  • (a) contacting the ovarian cells with a compound of the invention;
  • (b) irradiating the cells at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the ovarian cells.
  • In a particular embodiment, the compound of the invention for use in ovarian imaging is PS62 or E43; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in ovarian imaging is AL27 or CNN5; and the irradiating wavelength is in the 760-800 nm range.
    • Testes:
  • Testes-specific agents are useful for the highlighting of testicular tumor cells. In certain embodiments, these agents can be used to aid aggressive metastasectomy treatments in advanced Stage IV patients prior to cytotoxic therapy.
  • As such, in one aspect, the invention provides a method for imaging testicular cells, the method comprising:
  • (a) contacting the testicular cells with a compound of the invention;
  • (b) irradiating the cells at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the testicular cells.
    • Seminal Vesicles:
  • Seminal vesicle agents are useful in assisting a urologist during robotic or open prostatectomy to ensure removal of all seminal vesicles.
  • As such, in one aspect, the invention provides a method for imaging seminal vesicle cells, the method comprising:
  • (a) contacting the seminal vesicles with a compound of the invention;
  • (b) irradiating the cells at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the seminal vesicles.
  • In a particular embodiment, the compound of the invention for use in seminal vesicle imaging is LN65, YY269, or Ox4; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the-compound of the invention for use in seminal vesicle imaging is CNN2, CNN4, ZK48, LN50, TG66, LN66, AL31, or AL30; and the irradiating wavelength is in the 760-800 nm range.
    • Prostate:
  • Prostate gland and/or prostate cancer agents are useful during robotic or open prostatectomy.
  • As such, in one aspect, the invention provides a method for imaging prostate cells, the method comprising:
  • (a) contacting the prostate cells with a compound of the invention;
  • (b) irradiating the cells at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the prostate cells.
  • In a particular embodiment, the compound of the invention for use in prostate imaging is PS62; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compounds of the invention for use in prostate imaging is LN66, TG66, CNN4, or CNN10; and the irradiating wavelength is in the 760-800 nm range
  • In certain instances the compounds of the invention can be conjugated to a prostate-specific membrane antigen (PSMA) targeting ligand.
    • Pancreas:
  • Prior to the invention, it was very difficult and unusual to find contrast agents that target cells of the exocrine pancreas.
  • Nevertheless, in one aspect, the invention provides a method for imaging pancreas, the method comprising:
  • (a) contacting the pancreas cells with a compound of the invention;
  • (b) irradiating the cells at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the pancreas.
  • In a particular embodiment, the compound of the invention for use in pancreas imaging is T14, PS62, SRA94, SRA89, SP28, SP29, ESS61, or T27; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in pancreas imaging is AL22, CNN145, Rh800, Ox750, WuA96, or Ox170; and the irradiating wavelength is in the 760-800 nm range.
    • Spleen:
  • In one aspect, the invention provides a method for imaging the spleen and accessory splenic tissue, the method comprising:
  • (a) contacting the spleen or accessory splenic tissue with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the spleen or accessory splenic tissue.
  • In a particular embodiment, the compound of the invention for use in spleen imaging is E24, E44, E37, E38, E39, E43, E50, E51, E78, LO1, PTN1, AL79, SP27, TG5, TP5, EAO42, PS31, SP34, TG115, MHI86, MHI96, or MHI97; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in spleen imaging is AL29, LS1, AH34, JM1, ZK166, ZK189, ZK197, ZK198, AL27, AL25, MDL16, ZK215, or ZK184; and the irradiating wavelength is in the 760-800 nm range.
    • Gallbladder:
  • Many agents that area cleared from blood by liver are excreted into bile and are then concentrated by the gallbladder. Gallbladder contrast agents help localize the gallbladder during laparoscopic surgery and also help highlight the cystic duct.
  • As such, in one aspect, the invention provides a method for imaging gallbladder, the method comprising:
  • (a) contacting the gallbladder with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the gallbladder.
  • In a particular embodiment, the compound of the invention for use in gallbladder imaging is PS62, SRA94, SRA89, AC2, ESS61, A106, YY261, SP67, P700H, CNN13, ZK140, or ZK14; and the irradiating wavelength is in the 660-760 nm range.
  • In another particular embodiment, the compound-of the invention for use in gallbladder imaging is ZK198, ZK208, ZK166, WuA71, or P800H; and the irradiating wavelength is in the 760-800 nm range.
    • Bile Ducts:
  • Similarly, in one aspect, the invention provides a method for imaging the bile ducts, the method comprising:
  • (a) contacting the bile ducts with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the bile ducts.
  • In a particular embodiment, the compound of the invention for use in bile duct imaging is A106, CNN13, ZK140, SRA89, WuA96, Ox170, Ox750, Ox4, ESS61, ZK14, CNN16, CNN145, MHI84, P700H, CNN12, or CNN14; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in bile duct imaging is ZK198, ZK166, ZK208, P800H, MDL16, or WuA71; and the irradiating wavelength is in the 760-800 nm range.
    • Peyer's Patches:
  • Peyer's patches are small collections of lymphatic tissue that protect the mucosal membranes of the GI tract. Prior to the invention, they were extremely difficult to image in living organisms.
  • As such, in one aspect, the invention provides a method for imaging Peyer's patches the method comprising:
  • (a) contacting Peyer's patches with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging Peyer's patches.
  • In another particular embodiment, the compound of the invention for use in imaging Peyer's patches is AL30; and the irradiating wavelength is in the 760-800 nm range.
    • Brain Grey Matter Agents:
  • Brain Grey Matter agents typically have the following features: 1) MW<500 Da, 2) Log D at pH 7.4 between 0.5 and 3, and 3) retained by cell bodies of the brain (grey matter). The low MW and lipophilic (but not too lipophilic) Log D permit crossing of the blood brain barrier. These molecules are important for various types of brain surgery, especially resection of tumors, where highlighting of normal brain is so important.
  • As such, in one aspect, the invention provides a method for imaging brain grey matter cells, the method comprising:
  • (a) contacting the brain grey matter with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the brain grey matter.
  • In a particular embodiment, the compound of the invention for use in brain grey matter imaging is WuA96, or ZK104; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in brain grey matter imaging is ZK189; and the irradiating wavelength is in the 760-800 nm range.
    • Brain White Matter Agents:
  • In another aspect, the invention provides a method for imaging brain white matter, comprised of nerve axons and associated glia, the method comprising:
  • (a) contacting the brain white matter with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the brain white matter.
    • Brain Vasculature Agents:
  • Brain Vasculature Agents bind to either the arterial tree or vascular tree of the brain.
  • As such, in one aspect, the invention provides a method for imaging brain vasculature, the method comprising:
  • (a) contacting the brain vasculature with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the brain vasculature.
  • In a particular embodiment, the compound of the invention for use in brain vasculature imaging is ZK214, or ZK104; and the irradiating wavelength is in the 660-700 nm range.
    • Choroid Plexus:
  • The choroid plexus is the tissue that filters blood to produce cerebrospinal fluid (CSF). Several agents enter the choroid plexus but get trapped in this tissue while attempting to traverse the blood brain barrier.
  • As such, in one aspect, the invention provides a method for imaging choroid plexus, the method comprising:
  • (a) contacting the choroid plexus with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the choroid plexus.
  • In a particular embodiment, the compound of the invention for use in choroid plexus imaging is SP28, ZK135, SP66, ZK195, SP29, SP30, SP33, SP49, SP51, ZK78, ZK134, ZK135, ZK143, ZK140, ZK26, ZK78, ZK79, ZK133, ZK23, ZK101, SP66, SP72, MHI84, T14, T18, T20, or T23; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in choroid plexus imaging is ZK208, ZK185, AL22, ZK172, MDL16, ZK211, ZK153, ZK155, or ZK169; and the irradiating wavelength is in the 760-800 nm range.
    • Cerebrospinal Fluid:
  • Certain molecules of the invention can completely traverse the choroid plexus and enter the CSF. They are particularly useful for finding CSF before accidentally puncturing the meninges, or for finding and repairing tears in the meninges.
  • As such, in one aspect, the invention provides a method for imaging cerebrospinal fluid, the method comprising:
  • (a) contacting the cerebrospinal fluid with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the cerebrospinal fluid.
  • In a particular embodiment, the compound of the invention for use in cerebrospinal fluid imaging is SP66, SP43, SP72, SP28, MHI84, YY161, or YY163; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in cerebrospinal fluid imaging is AL20, ZK189, or ZK208; and the irradiating wavelength is in the 760-800 nm range.
    • Pituitary Gland:
  • Several molecules of the invention appear to highlight either the anterior pituitary, posterior pituitary, or both after a single intravenous injection.
  • As such, in one aspect, the invention provides a method for imaging the pituitary gland, the method comprising:
  • (a) contacting the pituitary gland with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the pituitary gland.
  • In a particular embodiment, the compound of the invention for use in pituitary imaging is SP60, SP64, SP28, SP29, SP30, SP33, SP34, SP43, SP51, SP53, ZK159, MHI84, YY187, SP59, SP67, ZK23, ZK204, ZK106, AL11, SP66, E79, E80, ES21, or LO3; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compounds of the invention for use in pituitary imaging is AL22, ZK185, ZK208, ZK172, ZK190, QBN1, QBN14, ZK153, ZK156, AL25, AL29, AL20, MDL17, or MDL16; and the irradiating wavelength is in the 760-800 nm range.
    • Thoracic Duct Agents:
  • Agents injected into the lower lymphatics will eventually concentrate in the thoracic duct as lymph traverse from below the diaphragm to above the diaphragm, and prior to efflux into the left brachiocephalic vein. These agents are particularly valuable during several types of thoracic surgery because the thoracic duct is otherwise extremely difficult to find, and if lacerated, difficult to repair because lymph is clear.
  • As such, in one aspect, the invention provides a method for imaging the thoracic duct, the method comprising:
  • (a) contacting the thoracic duct with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the thoracic duct.
  • In a particular embodiment, the compound of the invention for use in thoracic duct imaging is LN15, A104, or TG42; and the irradiating wavelength is in the 660-700 nm range. in thoracic duct imaging is A71, ZW800-1, or WuA71; and the irradiating wavelength is in the 760-800 nm range.
    • Pan-Lymph Node Agents:
  • Sentinel lymph node mapping has revolutionized the treatment of breast cancer and melanoma. However, 20-25% of patients are found to have tumor cells in their sentinel lymph node and therefore require a completion lymphadenectomy, i.e., removal of all the lymph nodes in the basin. Finding all lymph nodes in an area of the body is extremely difficult to do.
  • Pan-lymph node mapping agents that highlight all lymph nodes after a single intravenous injection are useful during many types of surgery. They can also be used in conjunction with a sentinel lymph node agent to find both sentinel lymph nodes and all lymph nodes in a particular basin.
  • As such, in one aspect, the invention provides a method for imaging lymph nodes, the method comprising:
  • (a) contacting the lymph nodes with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the lymph nodes.
  • In a particular embodiment, the compound of the invention for use in lymph node imaging is A150, A146, A149, A148, A160, A161, A20, SP27, SP43, SP117, ZK197, ZK134, ZK46, ZK101, AL11, AL12, EAO42, ZK148, E16, E50, E51, E77, E78, E58, E59, E60, E70, E72, LO1, LO2, PTN11, ZK143, ZK140, ZK29, WuA108, MHI86, MHI96, or MHI97; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compounds of the invention for use in lymph node imaging is MM25, A64, AL30, AL33, PTN6, AH34, CNN10, MM21, CNN5, A71, MDL16, ZK172, LN63, ZK154, or ZK155; and the irradiating wavelength is in the 760-800 nm range.
    • Sentinel Lymph Node Agents:
  • Sentinal lymph node agents are injected in and around a tumor and quickly flow to the first lymph node that drains the tumor, called the sentinel lymph node (SLN).
  • As such, in one aspect, the invention provides a method for imaging sentinel lymph nodes, the method comprising:
  • (a) contacting the sentinel lymph node with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the sentinel lymph node.
  • In a particular embodiment, the compound of the invention for use in sentinel lymph imaging is MHI86, MHI96, MHI97, A150, A146, A149, A148, A160, A161, A20, E37, or E78; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in sentinel lymph node imaging is MM25, A64, AL30, or AL33; and the irradiating wavelength is in the 760-800 nm range.
    • Vulnerable Plaque Agents:
  • By virtue of their lipophilicity, certain compounds of the invention may be taken up by vulnerable plaque and therefore highlight areas of intima that are at a higher risk for rupture.
  • As such, in one aspect, the invention provides a method for imaging vulnerable plaque cells, the method comprising:
  • (a) contacting the vulnerable plaque with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the vulnerable plaque.
    • Stem Cell Tracking and Viability Agents:
  • Longitudinal monitoring of cell migration, division, and differentiation is of paramount importance in stem cell-based medical treatment. Many lipophilic cationic NIR fluorophores with a Log D at pH 7.4 within a narrow range will partition into living cells and thus serve as tracking and/or viability indicators.
  • As such, in one aspect, the invention provides a method for imaging stem cells, the method comprising:
  • (a) contacting the stem cells with a compound of the invention;
  • (b) irradiating the cells at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the stem cells.
  • In a particular embodiment, the compound of the invention for use in stem cell imaging is PS127, PS129, PS131, PS133, CNN12, CNN13, CNN14, CNN16, CNN17, Ox4, Ox170, ZK126, ZK211, ZK214, or EAO40; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in stem cell imaging is PS126, PS128, PS130, or PS132; and the irradiating wavelength is in the 760-800 nm range.
  • In certain embodiments, the compounds of the invention for use in stem cell imaging have primary or secondary amines as part of their structure, which permit covalent fixation in place after treatment with paraformaldehyde (Mannich reaction) or other amine-reactive fixatives.
    • Tissue Engineering Agents:
  • Biodegradable scaffolds have been extensively used in the field of tissue engineering and regenerative medicine. Tissue Engineering Agents provide noninvasive monitoring of in vivo scaffold degradation or product formation.
  • As such, in one aspect, the invention provides a method for imaging biodegradable scaffolds, the method comprising:
  • (a) contacting the biodegradable scaffold with a compound of the invention;
  • (b) irradiating the scaffold at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the biodegradable scaffold.
  • In a particular embodiment, the compound of the invention for use in imaging biodegradable scaffolds is A71-NHS ester, MHI103, CNN12, CNN13, CNN14, CNN16, CNN17, Ox4, Ox170, ZK126, ZK211, ZK214, EAO40, E59, EAO42, PTN12, E72, E24, E27, E50, E51, E79, E80, E81, ES17, ES21, LO1, LO2, T17, T23, T25, A106, A148, A150, A161, or AC8; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in imaging biodegradable scaffolds is LN15-NHS ester, LN68, LN50, CNN3, LN65, LN63, or ZK166; and the irradiating wavelength is in the 760-800 nm range.
  • In certain embodiments, the compounds of the invention are amine-containing or meso-brominated compounds which are conjugated to biodegradable scaffolds.
    • Neuroendocrine Tumors:
  • Neuroendocrine tumors are a group of rare tumors that show similar growth patterns and resistance to chemotherapy. They occur throughout the body and, although primary tumors are often curable by surgery, they are difficult to find when they are small. A particularly vexing group of neuroendocrine tumors are the pancreatic endocrine tumors, comprised of gastrinoma, insulinoma, glucagonoma, VIPoma, and somatostatinoma. Fluorophores targeted to pancreatic endocrine tumors provide surgeons with sensitive, specific and real-time image guidance after a single preoperative, intravenous injection.
  • As such, in one aspect, the invention provides a method for imaging neuroendocrine tumors, the method comprising:
  • (a) contacting the neuroendocrine tumor with a compound of the invention;
  • (b) irradiating the tissue at a wavelength absorbed by the compound;
  • (c) and detecting a signal from the compound, thereby imaging the Neuroendocrine tumor.
  • In a particular embodiment, the compound of the invention for use in neuroendocrine tumor imaging is ESS61, SRA89, SRA94, CNN145, MHI84, Ox4, Ox170, Ox750, WuA96, CNN16, CNN12, or CNN14; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use in neuroendocrine tumor imaging is AL20, AL22, AL33, or AL30; and the irradiating wavelength is in the 760-800 nm range.
    • Hydrophobic Molecules Tumors:
  • Hydrophobic molecules are often administered to a subject for various therapeutic and diagnostic purposes. Hydrophobic Fluorophores can conjugate to such molecules and allow for the imaging and study of the distribution of such molecules.
  • As such, in one aspect, the invention provides a method for imaging a hydrophobic molecule in a biological system, the method comprising:
  • (a) conjugating an imaging agent of the invention to a hydrophobic molecule to form a conjugated agent molecule;
  • (b) contacting a subject biological system with the conjugated agent molecule;
  • (c) irradiating the conjugated agent molecule at a wavelength absorbed by the imaging agent;
  • (c) and detecting a signal from the conjugated agent molecule, thereby imaging the hydrophobic molecule.
  • In a particular embodiment, the compound of the invention for use with hydrophobic molecules is L700-1A and L700-1C; and the irradiating wavelength is in the 660-700 nm range.
  • In another particular embodiment, the compound of the invention for use with hydrophobic molecules is L800-1A and L800-1C; and the irradiating wavelength is in the 760-800 nm range.
    • Agents for Intravital Microscopy and PEGylated Agents:
  • Vascular functions rely on the endothelial cells lining the vasculature to provide a semi-permeable barrier between blood contents and the tissue interstitium. For intravital microscopy, fluorophores should be large to circulate longer in the bloodstream. PEGylation or bulky dextran conjugation may be used to increase the blood half-life of bioactive small molecules and peptides.
  • Specifically, certain sized, linear polyethylene glycol (PEG) molecules, in the range of 20 kDa to 60 kDa, get retained at sites of abnormal vasculature, like tumors. Because PEG molecules show low non-specific binding to normal tissues and organs, the SBR is high. PEG molecules that are smaller than 20 kDa are filtered by the kidney and do not show uptakes in abnormal tissue/tumors while molecules larger than 60 kDa are not efficiently removed from the body by renal filtration and lead to high background.
  • As such, in certain embodiments, the compounds of the invention may be modified to include a polyethylene glycol group. Such PEGylated compounds may be branched or linear. In certain embodiments, the linear PEGylated compounds are in the range of about 20 kDa to about 60 kDa.
  • In a particular embodiment, the compound of the invention is LN15, A104, or TG42; each of which is conjugated with linear or branched PEG of 60 kDa, 40 kDa, 20 kDa, or 100 kDa, or Dextran of 70 kDa, 100 kDa, or 150 kDa.
  • In another particular embodiment, the compounds of the invention is ZW800-1-, A71-, or WuA71 each of which is conjugated with linear or branched PEG of 60 kDa, 40 kDa, 20 kDa, or 100 kDa, or Dextran of 70 kDa, 100 kDa, or 150 kDa.
    • Dual-Modality Optical/Nuclear Agents:
  • In some embodiments, the compounds of the invention can be conjugated to a metal chelator agent for use in single-photon emission computed tomography (SPECT), positron emission tomography (PET), and/or magnetic resonance imaging (MRI). In certain embodiments, the metal chelator agent is a DOTA, DTPA, hydrazinonicotinic acid (HYNIC), or desferoxime, or a derivative thereof. In particular embodiments, the metal atom is selected from the group consisting of Zr-89, Ga-68 and Rb-82, and the signal is detected by positron emission tomography; the metal atom is selected from the group consisting of Tc-99m and In-111, and the signal is detected by single-photon emission computed tomography; or the metal atom is a lanthanide selected from the group consisting of Gd, Dy and Yb, and the signal is detected by magnetic resonance imaging.
  • In a particular embodiment, the compound of the invention is ZW800-1, A71, or LN15, conjugated to either DOTA, DTPA, or deferoxamine.
  • The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
    • EXAMPLES Optical Property Measurements
  • Absorbance and fluorescence emission spectra were measured using fiber optic HR2000 absorbance (200-1100 nm) and USB2000FL fluorescence (350-1000 nm) spectrometers (Ocean Optics, Dunedin, Fla.). Excitation was provided by a 532 nm green laser pointer (Opcom Inc., Xiamen, China) set to 5 mW, a 655 nm red laser pointer (Opcom Inc., Xiamen, China) set to 5 mW, or a 770 nm NIR laser diode light source (Electro Optical Components, Santa Rosa, Calif.) set to 10 mW and coupled through a 300 μm core diameter, NA 0.22 fiber (Fiberguide Industries, Stirling, N.J.). In silico calculations of the partition coefficient (log D at pH 7.4) and surface molecular charge and hydrophobicity were calculated using MarvinSketch 5.2.1 by taking major microspecies at pH 7.4 (ChemAxon, Budapest, Hungary).
    • NIR Fluorescence Imaging System
  • The dual-NIR channel FLAREa imaging system has been described in detail previously (26-28). It provides simultaneous illumination with white light (400-650 nm) at 40,000 lx, 660 nm NIR Channel 1 excitation at 4 mW/cm2 and 760 bmm NIR Channel 2 excitation at 10 mW/cm2. Color and two independent NIR fluorescence emission images (≈700 nm for Channel 1 and ≈800 nm for Channel 2) were acquired simultaneously with custom software at rates up to 15 Hz over a 15 cm diameter field of view. NIR fluorescence from Channel 1 was pseudo-colored in red and NIR fluorescence from Channel 2 was pseudo-colored in lime green prior to merger with the color video image. The imaging system was positioned at a distance of 18 inches from the surgical field.
    • Animal Models and Intraoperative NIR Fluorescence Imaging
  • Animals were housed in an AAALAC-certified facility. Animal studies were performed under the supervision of Beth Israel Deaconess Medical Center's Institutional Animal Care and Use Committee (IACUC) in accordance with approved institutional protocols (#101-2011 for rodents and #046-2010 for pigs).
  • Initial in vivo screening occurred in mice, rats, and pigs In the animal studies described below, either sex of 25 g CD-1 mice (Charles River Laboratories, Wilmington, Mass.) and either sex of 250 g Sprague-Dawley (SD) rats (Taconic Farms, Germantown, N.Y.) were used after anesthetizing with 100 mg/kg ketamine and 10 mg/kg xylazine intraperitoneally (Webster Veterinary, Fort Devens, Mass.). Either sex of Yorkshire pigs (E. M. Parsons and Sons, Hadley, Mass.) averaging 35 kg were induced with 4.4 mg/kg intramuscular Telazol™ (Fort Dodge Labs, Fort Dodge, Iowa), intubated, and maintained with 2% isoflurane (Baxter Healthcare Corp., Deerfield, Ill.). Following anesthesia, a 16 G central venous catheter was inserted into the external jugular vein, and saline was administered as needed. Electrocardiogram, heart rate, pulse oximetry, and body temperature were monitored throughout surgery.
  • To screen the optimum targeted contrast agent, 2-200 nmol of each NIR fluorophore was injected intravenously in CD-1 mice and sacrificed animals 1-4 h post-injection (n>3). Target tissues/organs were observed at indicated time points such as 0, 5, 10, 15, 30, 60, 120, 180, and 240 min with the FLARE™ imaging system. After intraoperative imaging, animals were sacrificed, and the target tissue and other major organs including heart, lung, liver, spleen, pancreas, kidneys, duodenum, intestine, and muscle were resected to quantify biodistribution and clearance. For rats, an optimized dose (10-1000 nmol) was injected depending on the targeting purpose, and targeting and biodistribution were observed 4 h post-injection (n>3). For the large animal study, the appropriate dose was calculated based on the previous dose dependence study in the small animal study. To confirm the drug kinetics in large animals, 0.5-10 μmol of the NIR fluorescence was injected through the external jugular vein (n>3). Then the target tissue and surrounding organ were imaged at the indicated time points (0, 1, 5, 10, 15, 30, 60, 90, 120, 180, and 240 min).
    • Results
  • Ureters: A 35 kg female pig was injected intravenously at time zero with 5 μmol of compound LN15 (700 nm) or A71 (800 nm) dissolved in saline or D5W. After a waiting period of 30 min the animal was surgically exposed and the ureters were imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively over the next 4 hours. As shown in FIGS. 1 and 2 , the ureter is highlighted with high contrast using this compound.
    Pan LN: A 250 g male rat was injected intravenously at time zero with 20 nmol of compound A150 (700 nm) or MM25 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the pan lymph nodes were imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 3 and 4 , all lymph nodes are highlighted with high contrast using this compound.
    SLN: A 35 kg female pig was injected subcutaneously into bowel at time zero with 5 nmol of compound MHI86 (700 nm) or MM25 (800 nm) dissolved in saline or D5W. After a waiting period of 5 min, the target tissue was exposed and the SLN was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 5 and 6 , the SLN is highlighted with high contrast using this compound.
    Cartilage: A 35 kg female pig was injected intravenously at time zero with 5 μmol of compound SP56 (700 nm) or LN50 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the spinal cartilage was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 7 and 8 , the cartilage is highlighted with high contrast using this compound.
    Neuroendocrine Tumors: A 25 g male insulinoma-bearing mouse was injected intravenously at time zero with 140 nmol of compound ESS61 (700 nm) or AL20 (800 nm) dissolved in saline or D5W. After a waiting period of 1 hour, the animal was surgically exposed and the pancreas was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 9 and 10 , the tumors are highlighted with high contrast using this compound.
    Bone: A 35 kg female pig was injected intravenously at time zero with 5 μmol of compound P700SO3 (700 nm) or P800SO3 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the rib cage was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 10 and 11 , the bone is highlighted with high contrast using this compound.
    Thyroid: A 35 kg female pig was injected intravenously at time zero with 5 μmol of compound T14 (700 nm) or QBN14 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the thyroid was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 12 and 13 , the thyroid is highlighted with high contrast using this compound.
    Parathyroid: A 35 kg female pig was injected intravenously at time zero with 5 μmol of compound T14 (700 nm) or QBN14 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the parathyroid was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 14 and 16 ; the parathyroid is highlighted with high contrast using this compound.
    Adrenal: A 35 kg female pig was injected intravenously at time zero with 5 μmol of compound E16 (700 nm) or AL27 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the adrenal was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in the FIGS. 17 and 18 , the adrenal is highlighted with high contrast using this compound.
    Salivary glands: A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound NRB1 (700 nm) or ZK211 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the salivary glands were imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 19 and 20 , the salivary glands are highlighted with high contrast using this compound.
    White adipose tissue: A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound PS31 (700 nm) or AH34 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the white adipose tissue was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 21 and 22 , the white adipose tissue is highlighted with high contrast using this compound.
    Brown adipose tissue: A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound SP30 (700 nm) or QBN1 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the brown fat was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 23 and 24 , the brown fat is highlighted with high contrast using this compound.
    Ovaries: A 25 g female mouse was injected intravenously at time zero with 25 nmol of compound PS62 (700 nm) or AL27 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the ovaries were imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 25 and 26 , the ovaries are highlighted with high contrast using this compound.
    Seminal vesicles: A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound LN65 (700 nm) or CNN2 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the seminal vesicle was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 27 and 28 , the seminal vesicle is highlighted with high contrast using this compound.
    Prostate: A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound PS62 (700 nm) or LN66 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the prostate was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 29 and 30 , the prostate is highlighted with high contrast using this compound.
    Pancreas: A 35 kg female pig was injected intravenously at time zero with 5 μmol of compound T14 (700 nm) or AL22 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the pancreas was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 31 and 32 , the pancreas is highlighted with high contrast using this compound.
    Spleen: A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound E24 (700 nm) or AL29 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the spleen was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 33 and 34 , the spleen is highlighted with high contrast using this compound.
    Gallbladder: A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound PS62 (700 nm) or ZK198 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the gallbladder was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 35 and 36 , gallbladder is highlighted with high contrast using this compound.
    Bile ducts: A 35 kg female pig was injected intravenously at time zero with 5 μmol of compound A106 (700 nm) or ZK198 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the bile duct was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 37 and 38 , the bile duct is highlighted with high contrast using this compound.
    Peyer's patches: A 250 g male rat was injected intravenously at time zero with 100 nmol of compound AL30 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the Peyer's patches were imaged for NIR fluorescence using Channel 2 (800 nm) of the FLARE imaging system. As shown in FIG. 39 , the Peyer's patches are highlighted with high contrast using this compound.
    Brain vasculature: A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound ZK214 (700 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the brain vasculature was imaged for NIR fluorescence using Channel 1 (700 nm) of the FLARE imaging system. As shown in FIG. 40 , the brain vasculature is highlighted with high contrast using this compound.
    Brain grey matter: A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound WuA96 (700 nm) or ZK189 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the brain grey matter was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 41 and 42 , the brain grey matter is highlighted with high contrast using this compound.
    Choroid plexus: A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound SP28 (700 nm) or ZK208 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the choroid plexus was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 43 and 44 , the choroid plexus is highlighted with high contrast using this compound.
    CSF: A 35 kg female pig was injected intravenously at time zero with 5 μmol of compound SP66 (700 nm) or AL20 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the CSF was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 45 and 46 , the CSF is highlighted with high contrast using this compound.
    Thoracic duct: A 35 kg female pig was injected subcutaneously into the lower leg at time zero with 5 μmol of compound A106 (700 nm) or ZK198 (800 nm) dissolved in saline or D5W. After a waiting period of 30 minutes, the animal was surgically exposed and the thoracic duct was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 47 and 48 , the thoracic duct is highlighted with high contrast using this compound.
    PEGylated agents: A 25 g female xenograft tumor-bearing mouse was injected intravenously at time zero with 10 nmol of compound PEG60k-LN15 (700 nm) or PEG60k-ZW800-1 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hour, the tumor was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in the FIGS. 49 and 50 , the tumor is highlighted with high contrast using this compound.
    Pituitary gland: A 25 g male mouse was injected intravenously at time zero with 25 nmol of compound SP60 (700 nm) or AL22 (800 nm) dissolved in saline or D5W. After a waiting period of 4 hours, the animal was surgically exposed and the pituitary gland was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 51 and 52 , the pituitary gland is highlighted with high contrast using this compound.
    Stem cell tracking: Cells grown in 35 mm or 60 mm plates at 70% confluence were incubated in cell culture media with 2 μM compound PS127 (700 nm) or PS126 (800 nm) in the 37° C. incubator with 5% CO2. After an incubation time of 30 min, cells were washed 3 times with warm media, followed by fixing in 2% paraformaldehyde for 30 min. After centrifuge, cell pellets were frozen in OCT, and tested after washing with acetone. Cell pellets were cut to a 10 μm thickness and imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of fluorescence microscope, respectively. As shown in FIGS. 53 and 54 , the cytoplasm of cell is highlighted with high contrast using this compound.
    Tissue engineering: A biodegradable scaffold (1 cm×1 cm×0.5 cm) was conjugated with 50 nmol of LN15-NHS (700 nm) or A71-NHS (800 nm) dissolved in DMSO through the NHS ester-amine reaction. The NIR scaffold was washed with water and ethanol 5 times, respectively, followed by freeze-drying. The scaffold was implanted into the subcutaneous pocket of athymic nude mouse 30 days prior to imaging. The extracted scaffold was frozen, cut to a 20 μm thickness, and imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of fluorescence microscope, respectively. As shown in FIGS. 55 and 56 , the cross-section of scaffold is highlighted with high contrast using this compound.
    Intravital microscopy: A 25 g male insulinoma-bearing mouse was injected intravenously at time zero with 25 nmol of compound Dex70k-LN15 (700 nm) or Dex70k-ZW800-1 (800 nm) dissolved in saline or D5W. After a waiting period of 1 min, the animal was surgically exposed and the tumor vasculature was imaged for NIR fluorescence using Channel 1 (700 nm) or Channel 2 (800 nm) of the FLARE imaging system, respectively. As shown in FIGS. 57 and 58 , the tumor vasculature is highlighted with high contrast using this compound.
    • EQUIVALENTS
  • Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.
  • Such equivalents are intended to be encompassed by the following claims.
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Claims (19)

What is claimed is:
1. A near-infrared fluorescent contrast agent according to Formula (I):
Figure US20220387631A1-20221208-C00661
Wherein
Each R1 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
Each R2 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
Or R1 and R2 can be taken together with the carbon atoms to which they are attached to form a 5-6 membered aryl or heteroaryl ring, optionally substituted with halogen, alkyl, alkoxy, hydroxyl, —SO2OH, or —CO2H;
Each R3 is independently H, OR′, halogen, sulfonato, substituted or unsubstituted amino, C(O)NH—C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;
Q is H, alkyl optionally substituted with alkoxy, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, —N+(alkyl)3, —OCO-alkyl, —SO2OH, phenyl, sulfonato, phosphates, KUE, GPI,—or —NR3R4R5, wherein R3, R4 and R5 are each independently for each occurrence H or C1-C4 alkyl, or R4 and R5, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring;
X and Y are each independently O, S, Se, C(R″)2, NR′″;
Z is H, halogen, CN, R6, OR6, SR6, NHR6 or CH2R6, in which R6 is optionally substituted C1-C6 alkyl, optionally substituted aryl, or optionally substituted heteroaryl, alkyl-N3, aryl-N3, aryl-halogen;
Each R′ is independently H, alkyl or aryl;
Each R″ is independently H or alkyl;
Each R′″ is independently H, alkyl, alkyl-SO3H, or alkyl-COOH;
m and n are independently an integer from 0-3; and
L is an anion;
or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof.
2. The near-infrared imaging agent of claim 1 represented by the formula:
Name Structure AL22
Figure US20220387631A1-20221208-C00662
 SP64
Figure US20220387631A1-20221208-C00663
 SP60
Figure US20220387631A1-20221208-C00664
 PTN1
Figure US20220387631A1-20221208-C00665
 SP56
Figure US20220387631A1-20221208-C00666
 QBN1
Figure US20220387631A1-20221208-C00667
 ZK195
Figure US20220387631A1-20221208-C00668
 YY187
Figure US20220387631A1-20221208-C00669
 TG42
Figure US20220387631A1-20221208-C00670
 TG53
Figure US20220387631A1-20221208-C00671
 LN24
Figure US20220387631A1-20221208-C00672
 LN15
Figure US20220387631A1-20221208-C00673
 YY180
Figure US20220387631A1-20221208-C00674
 NRB1
Figure US20220387631A1-20221208-C00675
 NRB2
Figure US20220387631A1-20221208-C00676
 NRB3
Figure US20220387631A1-20221208-C00677
 ZK190
Figure US20220387631A1-20221208-C00678
 ZK189
Figure US20220387631A1-20221208-C00679
 SP59
Figure US20220387631A1-20221208-C00680
 JM1
Figure US20220387631A1-20221208-C00681
 SP67
Figure US20220387631A1-20221208-C00682
 E60
Figure US20220387631A1-20221208-C00683
 E58
Figure US20220387631A1-20221208-C00684
 E59
Figure US20220387631A1-20221208-C00685
 AL27
Figure US20220387631A1-20221208-C00686
 AL18
Figure US20220387631A1-20221208-C00687
 AL16
Figure US20220387631A1-20221208-C00688
 AL25
Figure US20220387631A1-20221208-C00689
 AL29
Figure US20220387631A1-20221208-C00690
 AL30
Figure US20220387631A1-20221208-C00691
 AL33
Figure US20220387631A1-20221208-C00692
 AL34
Figure US20220387631A1-20221208-C00693
 AL35
Figure US20220387631A1-20221208-C00694
 AL36
Figure US20220387631A1-20221208-C00695
 AL14
Figure US20220387631A1-20221208-C00696
 AL79
Figure US20220387631A1-20221208-C00697
 SP27
Figure US20220387631A1-20221208-C00698
 SP28
Figure US20220387631A1-20221208-C00699
 SP29
Figure US20220387631A1-20221208-C00700
 SP30
Figure US20220387631A1-20221208-C00701
 SP33
Figure US20220387631A1-20221208-C00702
 SP43
Figure US20220387631A1-20221208-C00703
 SP49
Figure US20220387631A1-20221208-C00704
 SP51
Figure US20220387631A1-20221208-C00705
 SP53
Figure US20220387631A1-20221208-C00706
 SP79
Figure US20220387631A1-20221208-C00707
 SP99
Figure US20220387631A1-20221208-C00708
 SP116
Figure US20220387631A1-20221208-C00709
 SP117
Figure US20220387631A1-20221208-C00710
 ZK185
Figure US20220387631A1-20221208-C00711
 ZK135
Figure US20220387631A1-20221208-C00712
 QBN14
Figure US20220387631A1-20221208-C00713
 EAO42
Figure US20220387631A1-20221208-C00714
 ZK166
Figure US20220387631A1-20221208-C00715
 PTN13
Figure US20220387631A1-20221208-C00716
 PTN12
Figure US20220387631A1-20221208-C00717
 ZK148
Figure US20220387631A1-20221208-C00718
 ZK154
Figure US20220387631A1-20221208-C00719
 E72
Figure US20220387631A1-20221208-C00720
 ZK159
Figure US20220387631A1-20221208-C00721
 E70
Figure US20220387631A1-20221208-C00722
 ZK153
Figure US20220387631A1-20221208-C00723
 PTN11
Figure US20220387631A1-20221208-C00724
 ZK155
Figure US20220387631A1-20221208-C00725
 ZK143
Figure US20220387631A1-20221208-C00726
 ZK140
Figure US20220387631A1-20221208-C00727
 ZK29
Figure US20220387631A1-20221208-C00728
 SP34
Figure US20220387631A1-20221208-C00729
 ZK104
Figure US20220387631A1-20221208-C00730
 TP1
Figure US20220387631A1-20221208-C00731
 ZK14
Figure US20220387631A1-20221208-C00732
 PTN6
Figure US20220387631A1-20221208-C00733
 LN79
Figure US20220387631A1-20221208-C00734
 AH34
Figure US20220387631A1-20221208-C00735
 TP4
Figure US20220387631A1-20221208-C00736
 LS1
Figure US20220387631A1-20221208-C00737
 YY163
Figure US20220387631A1-20221208-C00738
 ZK15
Figure US20220387631A1-20221208-C00739
 WuA108
Figure US20220387631A1-20221208-C00740
 ZK150
Figure US20220387631A1-20221208-C00741
 ZK156
Figure US20220387631A1-20221208-C00742
 ESS23
Figure US20220387631A1-20221208-C00743
 AL31
Figure US20220387631A1-20221208-C00744
 AL43
Figure US20220387631A1-20221208-C00745
 AL20
Figure US20220387631A1-20221208-C00746
 TG44
Figure US20220387631A1-20221208-C00747
 ZK79
Figure US20220387631A1-20221208-C00748
 ESS13
Figure US20220387631A1-20221208-C00749
 WuA110
Figure US20220387631A1-20221208-C00750
 YY161
Figure US20220387631A1-20221208-C00751
 E71
Figure US20220387631A1-20221208-C00752
 ZK78
Figure US20220387631A1-20221208-C00753
 ZK133
Figure US20220387631A1-20221208-C00754
 ZK23
Figure US20220387631A1-20221208-C00755
 MDL17
Figure US20220387631A1-20221208-C00756
 ZK203
Figure US20220387631A1-20221208-C00757
 ZK204
Figure US20220387631A1-20221208-C00758
 ZK208
Figure US20220387631A1-20221208-C00759
 ZK106
Figure US20220387631A1-20221208-C00760
 ZK124
Figure US20220387631A1-20221208-C00761
 ZK126
Figure US20220387631A1-20221208-C00762
 ZK101
Figure US20220387631A1-20221208-C00763
 ZK172
Figure US20220387631A1-20221208-C00764
 MDL16
Figure US20220387631A1-20221208-C00765
 LN37
Figure US20220387631A1-20221208-C00766
 TG20
Figure US20220387631A1-20221208-C00767
 LO4
Figure US20220387631A1-20221208-C00768
 ZK211
Figure US20220387631A1-20221208-C00769
 ZK214
Figure US20220387631A1-20221208-C00770
 ZK215
Figure US20220387631A1-20221208-C00771
 YY190
Figure US20220387631A1-20221208-C00772
 AL11
Figure US20220387631A1-20221208-C00773
 AL12
Figure US20220387631A1-20221208-C00774
 CMI24
Figure US20220387631A1-20221208-C00775
 CMI26
Figure US20220387631A1-20221208-C00776
 E16
Figure US20220387631A1-20221208-C00777
 E17
Figure US20220387631A1-20221208-C00778
 E24
Figure US20220387631A1-20221208-C00779
 E27
Figure US20220387631A1-20221208-C00780
 E36
Figure US20220387631A1-20221208-C00781
 E37
Figure US20220387631A1-20221208-C00782
 E38
Figure US20220387631A1-20221208-C00783
 E39
Figure US20220387631A1-20221208-C00784
 E43
Figure US20220387631A1-20221208-C00785
 E44
Figure US20220387631A1-20221208-C00786
 E45
Figure US20220387631A1-20221208-C00787
 E50
Figure US20220387631A1-20221208-C00788
 E51
Figure US20220387631A1-20221208-C00789
 E77
Figure US20220387631A1-20221208-C00790
 E78
Figure US20220387631A1-20221208-C00791
 E79
Figure US20220387631A1-20221208-C00792
 E80
Figure US20220387631A1-20221208-C00793
 E81
Figure US20220387631A1-20221208-C00794
 ES17
Figure US20220387631A1-20221208-C00795
 ES21
Figure US20220387631A1-20221208-C00796
 LO1
Figure US20220387631A1-20221208-C00797
 LO2
Figure US20220387631A1-20221208-C00798
 LO3
Figure US20220387631A1-20221208-C00799
 MHI106
Figure US20220387631A1-20221208-C00800
 MHI128
Figure US20220387631A1-20221208-C00801
 MHI84
Figure US20220387631A1-20221208-C00802
 MHI86
Figure US20220387631A1-20221208-C00803
 MHI96
Figure US20220387631A1-20221208-C00804
 MHI97
Figure US20220387631A1-20221208-C00805
 P700H
Figure US20220387631A1-20221208-C00806
 P700SO3
Figure US20220387631A1-20221208-C00807
 T14
Figure US20220387631A1-20221208-C00808
 T17
Figure US20220387631A1-20221208-C00809
 T18
Figure US20220387631A1-20221208-C00810
 T20
Figure US20220387631A1-20221208-C00811
 T23
Figure US20220387631A1-20221208-C00812
 T24
Figure US20220387631A1-20221208-C00813
 T25
Figure US20220387631A1-20221208-C00814
 T27
Figure US20220387631A1-20221208-C00815
 T29
Figure US20220387631A1-20221208-C00816
 A20
Figure US20220387631A1-20221208-C00817
 A21
Figure US20220387631A1-20221208-C00818
 A80
Figure US20220387631A1-20221208-C00819
 A100
Figure US20220387631A1-20221208-C00820
 A104
Figure US20220387631A1-20221208-C00821
 A106
Figure US20220387631A1-20221208-C00822
 A134
Figure US20220387631A1-20221208-C00823
 A138
Figure US20220387631A1-20221208-C00824
 A146
Figure US20220387631A1-20221208-C00825
 A148
Figure US20220387631A1-20221208-C00826
 A149
Figure US20220387631A1-20221208-C00827
 A150
Figure US20220387631A1-20221208-C00828
 A160
Figure US20220387631A1-20221208-C00829
 A161
Figure US20220387631A1-20221208-C00830
 A24
Figure US20220387631A1-20221208-C00831
 AC2
Figure US20220387631A1-20221208-C00832
 AC3
Figure US20220387631A1-20221208-C00833
 AC8
Figure US20220387631A1-20221208-C00834
 AN3
Figure US20220387631A1-20221208-C00835
 SP72
Figure US20220387631A1-20221208-C00836
 ZK138-2
Figure US20220387631A1-20221208-C00837
 ZK169
Figure US20220387631A1-20221208-C00838
 MHI85
Figure US20220387631A1-20221208-C00839
 SP66
Figure US20220387631A1-20221208-C00840
 EAO72
Figure US20220387631A1-20221208-C00841
 EAO76
Figure US20220387631A1-20221208-C00842
 ZK2565
Figure US20220387631A1-20221208-C00843
 ZK2615
Figure US20220387631A1-20221208-C00844
 LN15-NHS
Figure US20220387631A1-20221208-C00845
 PS127
Figure US20220387631A1-20221208-C00846
 PS128
Figure US20220387631A1-20221208-C00847
 PS129
Figure US20220387631A1-20221208-C00848
 PS130
Figure US20220387631A1-20221208-C00849
 PS131
Figure US20220387631A1-20221208-C00850
 PS132
Figure US20220387631A1-20221208-C00851
 PS133
Figure US20220387631A1-20221208-C00852
 L700-1A
Figure US20220387631A1-20221208-C00853
 L700-1C
Figure US20220387631A1-20221208-C00854
3. The near-infrared imaging agent of claim 2, represented by the formula:
Figure US20220387631A1-20221208-C00855
4. The near-infrared imaging agent of claim 2, represented by the formula:
Figure US20220387631A1-20221208-C00856
5. A method of imaging tissue, lumens, or cells, the method comprising:
(a) contacting the tissue, lumen, or cells with an imaging agent according to claim 1;
(b) irradiating the tissue, lumen, or cells at a wavelength absorbed by the compound;
{circle around (c)} and detecting a signal from the compound, thereby imaging the tissue, lumen, or cells.
6. The method of claim 5, wherein the imaging agent is:
Figure US20220387631A1-20221208-C00857
7. The method of claim 5, wherein the tissue or cells are blood vessels, lumens, ureters, cartilage, bone, thyroid, parathyroids, adrenal gland, salivary gland, white adipose tissue, brown adipose tissue, ovarian cells, testicular cells, seminal vesicles, prostate, pancreas, spleen, gallbladder, bile ducts, Peyer's patches, brain grey matter, brain white matter, brain vasculature, choroid plexus, cerebrospinal fluid, nerves, thoracic duct, pan lymph nodes, sentinel lymph nodes, vulnerable plaque, stem cells, or neuroendocrine tumors.
8. The method of claim 5, wherein the imaging agent is administered to an organism comprising the tissue, lumen, or cells.
9. The method of claim 8, wherein the organism is human.
10. The method of claim 5, wherein the imaging agent has peak absorbance at about 600 nm to 850 nm.
11. The method of claim 5, wherein the tissue or cells is imaged ex vivo.
12. The method of claim 5, where the imaging agent further comprises a PEG-moiety.
13. The method of claim 5, where the imaging agent further comprises a radioisotope for either single-photon emission computed tomography (SPECT) or positron emission tomography (PET).
14. The method of claim 5, wherein the compound comprises a reactive linking group, such as NHS ester, sulfo-NHS ester, or a TFP ester.
15. The method of claim 5, wherein the compound comprises a chelator moiety capable of chelating a metal atom.
16. The method of claim 5, wherein the chelator moiety is a DOTA, DTPA, HYNIC, or desferoxime, or a derivative thereof.
17. A method for imaging the sentinel lymph node, the method comprising:
(a) contacting the sentinel lymph node with an imaging agent according to claim 1;
(b) irradiating the tissue at a wavelength absorbed by the imaging agent;
(c) and detecting a signal from the imaging agent, thereby imaging the sentinel lymph node.
18. The method according to claim 17, wherein the imaging agent is
Figure US20220387631A1-20221208-C00858
and the irradiating wavelength is in the 660-760 nm range.
19. The method according to claim 17, wherein the imaging agent is
Figure US20220387631A1-20221208-C00859
and the irradiating wavelength is in the 760-800 nm range.
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