Class of stable heptamethine cyanine fluorophores and biomedical applications thereof
Inventors
Schnermann, Martin John • Nani, Roger Rauhauser • Shaum, James Blaine
Assignees
US Department of Health and Human Services
Publication Number
US-10280307-B2
Publication Date
2019-05-07
Expiration Date
2034-11-05
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Abstract
Embodiments of C4′-alkyl-ether heptamethine cyanine fluorophores according to general formula I, and pharmaceutically acceptable salts thereof, are disclosed. Methods of making and using the C4′-alkyl-ether heptamethine cyanine fluorophores also are disclosed.
Core Innovation
This disclosure concerns a synthetic method for making cyanine fluorophores, particularly stable heptamethine cyanine (Cy7) fluorophores, embodiments of Cy7 fluorophores made by the disclosed method, and pharmaceutical salts thereof, and methods of using the Cy7 fluorophores. The disclosed Cy7 fluorophores are C4′-O-alkyl heptamethine cyanines demonstrating optical properties ideal for near-infrared imaging applications and excellent resistance to thiol nucleophiles. Some embodiments of the disclosed Cy7 fluorophores are suitable for bioconjugation.
Near-infrared fluorophores, especially heptamethine cyanines with emission maxima around 800 nm, are important for in vivo fluorescence imaging due to improved tissue penetration and low autofluorescence. However, many heptamethine cyanines suffer from chemical instability at the C4′ position and challenging synthesis methods. Cyanines modified at the C4′ position with an O-alkyl substituent are desirable because of their likely stability and potential for concise synthesis of symmetrical bioconjugatable variants. Such molecules have been rarely described and are unknown when functionalized for biomolecule conjugation due to difficulties in the standard preparative reactions for C4′-chloride exchange with alkoxide nucleophiles.
The invention addresses the need for stable, near-infrared heptamethine cyanine fluorophores and provides a synthetic method involving nitrogen quaternization by electrophiles to initiate an N- to O-transposition rearrangement from halogenated precursors and alkanolamines. The method allows preparation of C4′-alkyl-ether Cy7 fluorophores with defined substituents providing desirable properties such as aqueous solubility and reduced aggregation. The fluorophores show improved chemical stability, particularly resistance to biological thiols, and can be conjugated to biomolecules to create bioconjugates for imaging and detection.
Claims Coverage
The patent claims include one independent claim directed to a compound and one independent claim directed to a method of using the compound, featuring multiple inventive aspects related to chemical structure and usage.
Stable C4′-alkyl-ether heptamethine cyanine fluorophore compound
A compound of formula I or a pharmaceutically acceptable salt thereof, characterized by a heptamethine cyanine core with a C4′-alkyl-ether substitution and defined ranges and types of substituents (m, n, R1 to R17, Z) including biomolecule-containing groups, maleimidyl- and succinimidyl-containing groups, and sulfonate-containing groups, providing chemical stability and functionality for bioconjugation.
Use of C4′-alkyl-ether Cy7 fluorophores in biological imaging
A method comprising contacting a biological sample with the compound of formula I, irradiating with near-infrared light, and detecting fluorescence to indicate presence of the compound, with specified embodiments including fluorescence imaging, flow cytometry, and in vivo or ex vivo administration with biomolecule conjugates targeting specific antigens or tumors.
The claims cover the novel C4′-alkyl-ether Cy7 fluorophores with defined chemical structures and their use in fluorescence-based detection methods in biological samples, emphasizing stability and bioconjugation capabilities enabling imaging and therapeutic applications.
Stated Advantages
The C4′-alkyl-ether Cy7 fluorophores exhibit excellent chemical and biological stability, particularly resistance to thiol nucleophiles, preventing undesired substitution reactions common to other cyanine dyes.
They possess ideal near-infrared optical properties including high extinction coefficients, suitable absorbance and emission maxima, and small Stokes shifts, making them well suited for sensitive fluorescence imaging applications.
The synthetic method enables a concise and reliable preparation route for stable C4′-O-alkyl heptamethine cyanines, overcoming previous synthesis challenges and allowing incorporation of biomolecule conjugation functionalities.
The fluorophores are compatible with pharmaceutical formulations suitable for various routes of administration, enabling potential clinical diagnostic and therapeutic uses, including fluorescence-guided surgery.
Documented Applications
In vitro, in vivo, and ex vivo near-infrared fluorescence imaging for detection and visualization of drugs, antibodies, peptides, proteins, nucleic acids, receptors, and tumor cells.
Fluorescence-guided surgery, particularly cancer surgery, enabling tumor localization and excision with real-time fluorescence detection of tumor margins.
Flow cytometry applications where cells bound with fluorophore conjugates can be separated and identified based on near-infrared fluorescence.
Conjugation to drugs for tracking drug delivery and elimination in biological fluids and tissues.
Use as fluorescent tags for nucleic acids or proteins in sequencing, immunoassays, or other analytical techniques.
Potential use in clinical diagnostics and monitoring of biomarker presence in biological samples via specific biomolecule recognition and binding.
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