Near-infrared fluorescent imaging agents and methods of use thereof

Abstract

The present disclosure provides compounds of Formula (I) that are useful in imaging a tissue and / or in diagnosing diseases such as rheumatoid arthritis.

Description

[0001] NEAR-INFRARED FLUORESCENT IMAGING AGENTS AND METHODS OF USE THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 666,897, filed July 2, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0004] STATEMENT OF FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under Grant Nos.

[0006] CA280968 and EB022230 awarded by National Institutes of Health. The government has certain rights to this invention.

[0007] FIELD

[0008] The present disclosure relates to compounds useful for, e.g., imaging a tissue or for diagnosing a disease such as rheumatoid arthritis.

[0009] BACKGROUND

[0010] Rheumatoid arthritis (RA) is an autoimmune chronic inflammatory joint disease characterized by persistent synovitis, systemic inflammation, and autoantibodies, which can lead to irreversible joint damage and disability if left untreated. Therefore, early detection is critical to preventing disease progression and improving patient outcomes in RA treatment. Physical examinations, joint radiographs, serological tests, and imaging strategies are typically used to assess disease progression and treatment response. Careful clinical examination is a prerequisite but may miss subclinical inflammation in early-stage disease and clinical remission under treatment. In recent years, imaging has become an integral part of managing rheumatic diseases, enabling diagnosis, estimating prognoses, and evaluating therapeutic outcomes. However, conventional imaging modalities, such as radiographs, MRI, and ultrasonography, have limited sensitivity / specificity, lack the ability to detect inflammatory disease activities (inflammatory cell infiltration, synovitis, and bone destruction) in the joints, and often fail to detect early-stage disease. For instance, X-rays depend on relatively delayed indicators such as bone erosion and narrowing of joint spaces, which, in about 50% of cases, may not become apparent until six months after the onset of RA activity. Thus, imaging methods with high sensitivity / specificity are essential for detecting subclinical disease activity of RA before the appearance of clinical symptoms.

[0011] Cartilage is a critical component of joints, providing cushioning and shock absorption during joint movement. The articular cartilages in a synovial joint are protected by a synovial membrane (synovium) that produces synovial fluid, and synovitis (inflammation of the synovial membrane) is a hallmark of RA. To monitor this, the precise targeted molecular fluorescence imaging (MFI) of synovial joint tissue, such as cartilage lining and synovium, holds tremendous promise in specific molecular- level diagnosis prior to the permanent joint damage of RA. However, RA assessment of MFI mainly relies on the signal intensity changes in the joints, which may not always correlate with the severity of symptoms or disease activity. In addition, the probes emit fluorescence in the first NIR window (NIR-I, 700-1,000 nm), characterized by relatively high tissue absorption, light scattering, and autofluorescence. Especially in patients with skin pigmentation (i.e., melanin), fluorescence signals are largely mitigated and inconsistent with clinical and ultrasonographic findings. Recent advancements in fluorescence imaging using the second near-infrared window (NIR- II, wavelengths of 1,000-1,700 nm) spectral range have shown promise in improving tissue penetration and resolution due to reduced light scattering, minimal light absorption, and extremely low autofluorescence levels. Therefore, cartilage-specific contrast agents in the NIR-II window can enable selective visualization of articular tissues being able to serve as a distinct diagnostic indicator of the progressive course of RA.

[0012] SUMMARY

[0013] Some embodiments provide a compound of Formula (I): R15, and R18are as described herein. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound of Formula (I) and a pharmaceutically acceptable carrier.

[0014] In some embodiments, the present disclosure provides a method of imaging a tissue comprising contacting the tissue with a compound of Formula (I); irradiating the tissue at a wavelength absorbed by the compound; and detecting a signal from the compound, thereby imaging the tissue.

[0015] In some embodiments, the present disclosure provides a method of diagnosing rheumatoid arthritis, comprising administering to a subject identified as having or diagnosed with rheumatoid arthritis an imaging effective amount of a compound of Formula (I); irradiating a tissue at a wavelength absorbed by the compound; and determining the presence or absence of a signal from the compound, wherein the presence of a signal indicates a diagnosis of rheumatoid arthritis.

[0016] In some embodiments, the present disclosure provides a method of treating rheumatoid arthritis, comprising: administering to a subject identified as having or diagnosed with rheumatoid arthritis an imaging effective amount of a compound of Formula (I); irradiating a tissue at a wavelength absorbed by the compound; determining the presence or absence of a signal from the compound; and if a signal is detected, administering to the subject a therapeutically effective amount of one or more anti-inflammatory agents, one or more immunomodulatory agents, or a combination thereof.

[0017] In some embodiments, the present disclosure provides a method of treating rheumatoid arthritis, comprising: administering to a subject identified as having or diagnosed with rheumatoid arthritis an imaging effective amount of a compound of Formula (I); irradiating a tissue at a wavelength absorbed by the compound; determining the presence or absence of a signal from the compound; and if a signal is detected, administering to the subject a therapeutically effective amount of an antiinflammatory agent, an immunomodulatory agent, or a combination thereof.

[0018] In some embodiments, the present disclosure provides a method for detecting the presence of a signal generated the formation of a conjugate of a compound of Formula (I) and cartilage.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. Methods and materials are described herein for use in the present application; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0020] Other features and advantages of the present application will be apparent from the following detailed description and figures, and from the claims.

[0021] DESCRIPTION OF DRAWINGS

[0022] FIG. 1 (a) Illustration of the cartilage-targeting fluorophores incubating with cross-linked hyaluronic acid (xHA), a major component of cartilage extracellular matrix, in PBS (pH 7.4). Fluorescence images corresponding signal intensity of each fluorophore binding to xHA. (b) NIR-II fluorescence intensity enhancement of cartilage-targeting by substituting the / 7 / c.w-chloride of the heptamethine QuatCy skeleton with thiocholine, (c) absorption and NIR-I / II fluorescence emission spectra using silicon (left) and InGaAs (right) based spectrophotometers, (d) Schematic illustration of the NIR-II camera and laser setup for mice rib cartilage imaging. Ninox640 Vis-SWIR camera and zoom lens with SWIR coating were used with specific long pass filters and 808 nm excitation laser, (e) Representative images of rib cartilage from mice with skin (left) and skinless (right) injected with comparative example C700- OMe and compound CARF28 in obtained with NIR-I (exaction = 660 nm, power density = 2 mW cm'2; exposure time = 200 ms; optical filter = 710 / 50 nm bandpass) and NIR-II (excitation = 808 nm; power density = 35 mW cm'2; exposure time = 30-40 ms; optical filter = 1,070 nm long pass), respectively, showing higher contrast and resolution in the NIR-II window.

[0023] FIG. 2 shows the in vitro and in vivo toxicity test of CARF. (a) The cytotoxicity test result of cartilage-targeting fluorophores to chondrocyte, RAW264.7, and HEK293 cells treated with different concentrations ranging from 1-100 pM (n=5, mean ± s.e.m.) for 24 h. p values <0.05 were considered significant: ns; no significance, (b) Blood coagulation test; apparent blood viscosity after mixing with cartilage-targeting fluorophores, comparative example C800-OMe and new compound CARF28, and illustration for the cause of thrombosis (blood coagulation), (c) Photos and CT images of the lung from CD-I mice injected with C800-OMe (1 pmol / kg, immediately died) and CARF28 (3 pmol / kg, 2 h post-injection). After sacrificing mice, 500 pL of iopamidol (200 mg / mL) was perfused into the right ventricle of the heart. White arrowheads indicate pulmonary embolism, which is the cause of death, (d) Serum aspartate transferase (AST), alanine transferase (ALT), and AST / ALT ratio, (e) serum lactate dehydrogenase activity, and (f) blood urea nitrogen (BUN) and creatinine levels, (g) Body weights of mice for 14 days. CARF28 (3 pmol / kg) was injected at DO. (n = 4, mean ± s.e.m.). p values <0.05 were considered significant: ns; no significance, *p < 0.05.

[0024] FIG. 3 shows the influence of the functional group on CARF26 and 28-32. (a) Chemical structures and (b) absorption and emission spectra of CARF26 and 28-32 with variations in the functional groups, (c) NIR-II fluorescence images of rib cartilages from CD-I mice injected with the cartilage-targeting fluorophores and imaged 2 h postinjection. (d) Fluorescence intensity from rib cartilages from NIR fluorescence images in c. The data suggests that variations in functional groups can affect the in vivo targeting and imaging properties of the fluorophores. (e) Microscopic fluorescence and hematoxylin and eosin (H&E) images of the resected rib cage and (f) magnified images, including Safranin O staining, of red line square in (e).

[0025] FIG. 4 shows the metacarpophalangeal joint, (c) magnified images of the red- lined square in a. (d) Intensity profiling of ankle area from A to B in c. The numbers indicate full width at microstructural imaging of hind paw, ankle, finger joint, larynx, and trachea of less than 1 mm in NIR-II window, (a) Illustration and corresponding NIR-II image of mouse hind paw. (b) Microscopic fluorescence and H&E images from histological sections of the half maximum (FWHM). (e) Microscopic fluorescence and H&E images from the transverse section of the ankle joint, (f) Illustration and corresponding color and NIR-II images of mouse larynx and trachea, (g) Intensity profiling from trachea area from A to B in f. The numbers indicate the pitch between tracheal cartilage, (h) Microscopic fluorescence and H&E images of the sagittal section of the trachea.

[0026] FIG. 5 shows an analysis of cartilage junctions in the hind paw of a collagen antibody-induced arthritis (CAIA) mouse model for early detection of rheumatoid arthritis (RA). (a) Timeline depicting the making CAIA RA model, injections of cartilage targeting fluorophores. (b) Averaged clinical scores and paw thicknesses at imaging time points, along with color, X-ray CT, and NIR images of hind paw joints, (c) Clinical scores versus dates and (d) paw thickness versus dates, n = 10, mean ± SEM (e) Fluorescence signal profiling of the cartilage junction and full width at half maximum (FWHM) values in each signal profile. The broadening of cartilage junctions indicates early signs of RA. (f) Clinical scores and (g) paw thicknesses versus normalized FWHM. The number indicates how many data points overlapped, (h) NIR fluorescence and color images of metatarsophalangeal joints stained with safranin O / fast green and H&E.

[0027] FIG. 6 shows a single dose in vivo acute toxicity test of cartilage-targeting fluorophores. H&E staining of heart, liver, spleen, lung and kidney. Scale bar: 200 pm

[0028] FIG. 7 shows the cartilage targeting binding kinetics, pharmacokinetics, and biodistribution of CARF28. (a) Color and NIR fluorescence images of chest, knee, and hind paw. (b) signal intensity of rib cartilages and (c) intensity profiling of rib cartilages in a. (d) Color and NIR fluorescence images of mouse abdominal cavity and dissected organs (3 pmol kg ') treated mice at 48 h post-injection. Abbreviations used are: He, heart; Lu, lung; Li, liver; Pa / Sp, pancreas / spleen; Ki, kidney; Du, duodenum; In, intestine; Mu, muscle, (e) serum concentration and pharmacokinetic parameters. (n=5, mean ± SEM).

[0029] FIG. 8 shows the chemical structures, charge distribution, and absorbance and emission spectra of a) comparative example C800-OMe, b) 27, c) 21, and d) 28 (5 pM) in 5 wt / v° / o BSA / saline.

[0030] FIG. 9 shows the cartilage targeting in the rib cage and organ biodistribution of the fluorophores 21 and 27. Abbreviations used are Du, duodenum; He, heart; In, intestine; Ki, kidneys; Li, liver; Lu, lungs; Mu, muscle; Pa, pancreas; Sp, spleen.

[0031] FIG. 10 shows a) the Chemical structure of comparative example C700-OMe. b) longitudinal imaging of a mouse chest with intact skin injected intravenously with C700-OMe (Ipmol / kg, 5 n7 / v% BSA / saline) using NIR-I imaging system (excitation = 660 nm; power density = 5 mW cm'2; exposure time = 150 ms; optical filter = 710 / 40 nm bandpass), c) NIR images of abdomen, thorax, and organ. Abbreviations used are Du, duodenum; He, heart; In, intestine; Ki, kidneys; Li, liver; Lu, lungs; Mu, muscle; Pa, pancreas; Sp, spleen.

[0032] FIG. 11 shows the fluorescence imaging of live cells in CARF 28 cytotoxicity tests on a) chondrocytes, b) RAW264.7, and c) HEK293. Exposure time = 100 ms. Scale bar = 100 pm.

[0033] FIG. 12 shows the electron density calculation of quaternary ammoniums in C800-OMe, 28, 27, and 21. FIG. 13 shows the cartilage targeting in mouse rib cage of injected precursors a) 19, b) 21, c) 22, d) 23, e) 24, and f) 25. Organs from CD-I mice injected with 100 pL of each fluorophore solution (500 pM in 5 n7 / v% BSA saline) were resected and imaged at 4 h post-injection. Abbreviations used are Du, duodenum; He, heart; In, intestine; Ki, kidneys; Li, liver; Lu, lungs; Mu, muscle; Pa, pancreas; Sp, spleen.

[0034] FIG. 14 shows the color and NIR-II fluorescence images of CARF fluorophores 26 and 28-32 (5 pM in 5 wt% BSA saline), (excitation = 808 nm; power density = 35 mW cm'2; exposure time = 2 ms; optical filter = 1,070 nm LP).

[0035] FIG. 15 shows a comparison of the cartilage targetability of CARF a) 26, b) 28, c) 29, d) 30, e) 31, and f) 32. NIR-II images of CD-I mice injected with a 3 pmol / kg dose (in 5 wt / v° / o BSA / saline) were obtained at 4 h post-injection using the NIR-II imaging system (808 nm excitation, power density = 35 mW cm'2exposure time = 30- 150 ms, 1070 nm long-pass filter). Whole body and dissected organ images were obtained using the NIR-I fluorescence imaging system (FLARE). Abbreviations used are Du, duodenum; He, heart; In, intestine; Ki, kidneys; Li, liver; Lu, lungs; Mu, muscle; Pa, pancreas; Sp, spleen.

[0036] FIG. 16 shows the NIR-II images of cartilage tissues from the nose, ear, fore paw, and abdominal area with intact skin from CD-I mice injected with a 3 pmol / kg dose of CARF (in 5 wt / v° / o BSA / saline). (excitation = 808 nm; power density = 30 mW cm'2; exposure time = 20-150 ms; optical filter = 1,070 nm LP) Abbreviations used are In, intestine; Bl, bladder.

[0037] DETAILED DESCRIPTION

[0038] The present disclosure provides cartilage-targeting fluorophores (CARF), which employs the structure-inherent targeting (SIT) strategy by incorporating dyad quaternary ammonium groups (without considering the positive charge of N-atom in the indole ring). CARF enabled sensitive detection in the NIR-II window and demonstrated low potential in vivo and in vitro toxicities. The visualization of cartilage lining using CARF provide a reliable diagnostic indicator for the early detection of arthritis in preclinical mouse models. The molecular imaging of synovial joint tissue by CARF holds tremendous promise for specific molecular-level diagnosis by enabling earlier and more accurate detection, facilitating timely interventions to prevent permanent joint damage in RA. Compounds of Formula (I)

[0039] In some embodiments, the present disclosure provides a compound of e (CR6R7)mN(R19R20R21)+, -N(W)-(CR6R7)mN(R19R20R21)+. In some embodiments, m is 1, 2, 3, 4, 5, or 6. In some embodiments, W is selected from H, C1-C6 alkyl, and Cl- C6 haloalkyl.

[0040] In some embodiments, Z1is selected from O, S, Se, and C(R2R3). In some embodiments, Z2is selected from O, S, Se, and C(R16R17).

[0041] In some embodiments, R1, R10, R11, R18, R19, R20, and R21are independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R1, R10, R11, R18, R19, R20, and R21are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0042] In some embodiments, R2, R3, R6, R7, R8, R9, R12, R13, R16, and R17are independently selected from H, halogen, ORa, SRa, C1-C6 alkyl, C1-C6 haloalkyl, C3- C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R2, R3, R6, R7, R8, R9, R12, R13, R16, and R17are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0043] In some embodiments, R4, R5, R14, and R15are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4- 10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R4, R5, R14, and R15are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, or C1-C6 hydroxyalkyl.

[0044] In some embodiments, Ring A and Ring B are independently selected from monocyclic or bicyclic 6-12 membered aryl and monocyclic or bicyclic 5-10 membered heteroaryl, wherein the monocyclic or bicyclic 6-12 membered aryl and the monocyclic or bicyclic 5-10 membered heteroaryl are optionally substituted with 1-3 substituents independently selected from C1-C6 alkyl, C1-C6 thioalkyl, C1-C6 alkoxy, halogen, - S(O2)C1-C6 alkyl, and -(NH)-4-10 membered heterocyclyl optionally substituted with 1-3 substituents independently selected from halogen and C1-C6 alkyl;

[0045] In some embodiments, Rais selected from C1-C6 alkyl, C1-C6 haloalkyl, C3- C6 cycloalkyl, and 4-10 membered heterocyclyl.

[0046] In some embodiments, L is a pharmaceutically acceptable anion. In some embodiments, q is 1, 2, or 3.

[0047] In some embodiments, the formal charge of the compound of Formula (I) is 0.

[0048] In some embodiments, the compound of Formula (I) is a compound of Formula (II):

[0049] In some embodiments, X is CR10Rn, O, S, or (NR10Rn)+. In some embodiments, Y is halogen or -S-(CR6R7)mN(R19R20R21)+. In some embodiments, m is 1, 2, 3, 4, 5, or 6.

[0050] In some embodiments, R1, R10, R11, R18, R19, R20, and R21are independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R1, R10, R11, R18, R19, R20, and R21are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0051] In some embodiments, R2, R3, R16, and R17are independently selected from H, halogen, ORa, SRa, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R2, R3, R16, and R17are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxy alkyl.

[0052] In some embodiments, Ring A and Ring B are independently selected from monocyclic or bicyclic 6-12 membered aryl and monocyclic or bicyclic 5-10 membered heteroaryl, wherein the monocyclic or bicyclic 6-12 membered aryl and the monocyclic or bicyclic 5-10 membered heteroaryl are optionally substituted with 1-3 substituents independently selected from C1-C6 alkyl, C1-C6 thioalkyl, C1-C6 alkoxy, halogen, - S(O2)C1-C6 alkyl, and -(NH)-4-10 membered heterocyclyl optionally substituted with 1-3 substituents independently selected from halogen and C1-C6 alkyl;

[0053] In some embodiments, Rais selected from C1-C6 alkyl, C1-C6 haloalkyl, C3- C6 cycloalkyl, and 4-10 membered heterocyclyl.

[0054] In some embodiments, L is a pharmaceutically acceptable anion. In some embodiments, q is 1, 2, or 3.

[0055] In some embodiments, the formal charge of the compound of Formula (II) is 0.

[0056] In some embodiments, the compound of Formula (I) is a compound of Formula (III): In some embodiments, R1, R10, R11, R18, R19, R20, and R21are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R1, R10, R11, R18, R19, R20, and R21are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0057] In some embodiments, R2, R3, R6, R7, R16, and R17are independently selected from H, halogen, ORa, SRa, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R2, R3, R6, R7, R16, and R17are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0058] In some embodiments, m is 1, 2, 3, 4, 5, or 6.

[0059] In some embodiments, Ring A and Ring B are independently selected from monocyclic or bicyclic 6-12 membered aryl and monocyclic or bicyclic 5-10 membered heteroaryl, wherein the monocyclic or bicyclic 6-12 membered aryl and the monocyclic or bicyclic 5-10 membered heteroaryl are optionally substituted with 1-3 substituents independently selected from C1-C6 alkyl, C1-C6 thioalkyl, C1-C6 alkoxy, halogen, - S(O2)C1-C6 alkyl, and -(NH)-4-10 membered heterocyclyl optionally substituted with 1-3 substituents independently selected from halogen and C1-C6 alkyl.

[0060] In some embodiments, Rais selected from C1-C6 alkyl, C1-C6 haloalkyl, C3- C6 cycloalkyl, and 4-10 membered heterocyclyl.

[0061] In some embodiments, L is an anion. In some embodiments, q is 3.

[0062] In some embodiments, the formal charge of the compound of Formula (III) is 0.

[0063] In some embodiments, the compound of Formula (I) is a compound of Formula (IV):

[0064]

[0065] In some embodiments, R1, R10, R11, R18, R19, R20, and R21are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R1, R10, R11, R18, R19, R20, and R21are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0066] In some embodiments, R2, R3, R6, R7, R16, and R17are independently selected from H, halogen, ORa, SRa, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R2, R3, R6, R7, R16, and R17are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0067] In some embodiments, R22and R23are independently selected from C1-C6 alkyl, C1-C6 thioalkyl, C1-C6 alkoxy, halogen, OH, and -S(O2)C1-C6 alkyl; or two R22or two R23with the atoms to which they are attached together form a 6-12 membered aryl or 5-10 membered heteroaryl.

[0068] In some embodiments, m is 1, 2, 3, 4, 5, or 6. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

[0069] In some embodiments, n is 1, 2, 3, or 4. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, p is 1, 2, 3, or 4. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4.

[0070] In some embodiments, Rais selected from C1-C6 alkyl, C1-C6 haloalkyl, C3- C6 cycloalkyl, and 4-10 membered heterocyclyl.

[0071] In some embodiments, L is an anion. In some embodiments, q is 3.

[0072] In some embodiments, the formal charge of the compound of Formula (IV) is 0.

[0073] In some embodiments, the compound of Formula (I) is a compound of Formula (V):

[0074] (V).

[0075] In some embodiments, R1, R10, R11, R18, R19, R20, and R21are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R1, R10, R11, R18, R19, R20, and R21are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0076] In some embodiments, R2, R3, R6, R7, R8, R9, R12, R13, R16, and R17are independently selected from H, halogen, ORa, SRa, C1-C6 alkyl, C1-C6 haloalkyl, C3- C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R2, R3, R6, R7, R8, R9, R12, R13, R16, and R17are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0077] In some embodiments, R22and R23are independently selected from C1-C6 alkyl, C1-C6 thioalkyl, C1-C6 alkoxy, halogen, OH, and -S(O2)C1-C6 alkyl; or two R22or two R23with the atoms to which they are attached together form a 6-12 membered aryl or 5-10 membered heteroaryl.

[0078] In some embodiments, m is 1, 2, 3, 4, 5, or 6. In some embodiments, n is 1, 2, 3, or 4. In some embodiments, p is 1, 2, 3, or 4.

[0079] In some embodiments, Rais selected from C1-C6 alkyl, C1-C6 haloalkyl, C3- C6 cycloalkyl, and 4-10 membered heterocyclyl.

[0080] In some embodiments, L is an anion. In some embodiments, q is 1, 2, or 3.

[0081] In some embodiments, the formal charge of the compound of Formula (V) is 0.

[0082] In some embodiments, the compound of Formula (I) is a compound of Formula (VI):

[0083] In some embodiments, R1, R10, R11, R18, R19, R20, and R21are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R1, R10, R11, R18, R19, R20, and R21are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0084] In some embodiments, R2, R3, R6, R7, R16, and R17are independently selected from H, halogen, ORa, SRa, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R2, R3, R6, R7, R16, and R17are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0085] In some embodiments, R22and R23are independently selected from C1-C6 alkyl, C1-C6 thioalkyl, C1-C6 alkoxy, halogen, OH, and -S(O2)C1-C6 alkyl; or two R22or two R23with the atoms to which they are attached together form a 6-12 membered aryl or 5-10 membered heteroaryl.

[0086] In some embodiments, m is 1, 2, 3, 4, 5, or 6. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

[0087] In some embodiments, n is 1, 2, 3, or 4. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.

[0088] In some embodiments, p is 1, 2, 3, or 4. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4.

[0089] In some embodiments, Rais selected from C1-C6 alkyl, C1-C6 haloalkyl, C3- C6 cycloalkyl, and 4-10 membered heterocyclyl.

[0090] In some embodiments, L is an anion. In some embodiments, q is 3.

[0091] In some embodiments, the formal charge of the compound of Formula (VI) is 0.

[0092] In some embodiments, X is CR10R11+. In some embodiments, X is O. In some embodiments, X is S. In some embodiments, X is (NR10Rn)+.

[0093] In some embodiments, Y is selected from halogen and -S- (CR6R7)mN(R19R20R21)+. In some embodiments, Y is halogen. In some embodiments, Y is chloro. In some embodiments, Y is -S-(CR6R7)mN(R19R20R21)+. In some embodiments, Y is -S-(CH2)2N(CH 3+. In some embodiments, Y is -O- (CR6R7)mN(R19R20R21)+. In some embodiments, Y is selected from -N(W)- (CR6R7)mN(R19R20R21)+.

[0094] In some embodiments, W is H. In some embodiments, W is C1-C6 alkyl. In some embodiments, W is C1-C6 haloalkyl.

[0095] In some embodiments, Z1is O. In some embodiments, Z1is S. In some embodiments, Z1is Se. In some embodiments, Z1is C(R2R3). In some embodiments, Z1is O. In some embodiments, Z1is S. In some embodiments, Z1is Se. In some embodiments, Z1is C(R16R17).

[0096] In some embodiments, R1and R18are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl; wherein the C3-C6 cycloalkyl is optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0097] In some embodiments, R1and R18are independently selected from C1-C6 alkyl and C1-C6 haloalkyl. In some embodiments, wherein R1and R18are C1-C6 alkyl. In some embodiments, R1and R18are methyl.

[0098] In some embodiments, R2, R3, R16, and R17are independently selected from Cl- C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl; wherein the C3-C6 cycloalkyl is optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0099] In some embodiments, R2, R3, R16, and R17are independently selected from Cl- C6 alkyl and C1-C6 haloalkyl. In some embodiments, R2, R3, R16, and R17are C1-C6 alkyl. In some embodiments, R2, R3, R16, and R17are methyl.

[0100] In some embodiments, R10and R11are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl; wherein the C3-C6 cycloalkyl is optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0101] In some embodiments, R10and R11are independently selected from C1-C6 alkyl and C1-C6 haloalkyl. In some embodiments, R10and R11are C1-C6 alkyl. In some embodiments, R10and R11are methyl.

[0102] In some embodiments, R19, R20, and R21are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl; wherein the C3-C6 cycloalkyl is optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0103] In some embodiments, R19, R20, and R21are independently selected from C1-C6 alkyl and C1-C6 haloalkyl. In some embodiments, R19, R20, and R21are C1-C6 alkyl. In some embodiments, R19, R20, and R21are methyl.

[0104] In some embodiments, R8, R9, R12, R13are independently selected from H, Cl- C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl; wherein the C3-C6 cycloalkyl is optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl. In some embodiments, R8, R9, R12, R13are independently selected from H, Cl- C6 alkyl, and C1-C6 haloalkyl. In some embodiments, R8, R9, R12, R13are H.

[0105] In some embodiments, m is 1, 2, or 3. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3.

[0106] In some embodiments, each R6and each R7are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl; wherein the C3-C6 cycloalkyl is optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

[0107] In some embodiments, each R6and each R7are independently selected from H, C1-C6 alkyl, and C1-C6 haloalkyl.

[0108] In some embodiments, each R6and each R7are H.

[0109] In some embodiments, n is 1, 2, or 3. In some embodiments, n is 1 or 2.

[0110] In some embodiments, R22is selected from C1-C6 alkyl, C1-C6 thioalkyl, Cl- C6 alkoxy, halogen, OH, and -S(O2)C1-C6 alkyl.

[0111] In some embodiments, R22is C1-C6 alkyl.

[0112] In some embodiments, R22is C1-C6 alkoxy.

[0113] In some embodiments, R22is halogen.

[0114] In some embodiments, two R22with the atoms to which they are attached together form a 6-12 membered aryl or 5-10 membered heteroaryl.

[0115] In some embodiments, two R22with the atoms to which they are attached together form a 6-12 membered aryl.

[0116] In some embodiments, two R22with the atoms to which they are attached together form a phenyl.

[0117] In some embodiments, p is 1, 2, or 3.

[0118] In some embodiments, p is 1 or 2.

[0119] In some embodiments, R23is selected from C1-C6 alkyl, C1-C6 thioalkyl, Cl- C6 alkoxy, halogen, OH, and -S(O2)C1-C6 alkyl.

[0120] In some embodiments, R23is C1-C6 alkyl.

[0121] In some embodiments, R23is C1-C6 alkoxy.

[0122] In some embodiments, R23is halogen.

[0123] In some embodiments, two R23with the atoms to which they are attached together form a 6-12 membered aryl or 5-10 membered heteroaryl.

[0124] In some embodiments, two R23with the atoms to which they are attached together form a 6-12 membered aryl. In some embodiments, two R23with the atoms to which they are attached together form a phenyl.

[0125] The substitutions described throughout this section apply to anywhere that moiety or substitution is employed, e.g., across formulae (I), (II), (III), (IV), (V), or (VI).

[0126] In some embodiments, the compound is selected from:

[0127]

[0128] Pharmaceutically acceptable salts

[0129] In some embodiments, the compound of this disclosure are pharmaceutically acceptable salts that are formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the compound, such as a quaternary ammonium functional group.

[0130] In some embodiments, the compound is a pharmaceutically acceptable acid addition salt. In some embodiments, acids commonly employed to form pharmaceutically acceptable salts of the compounds of Formula (I), (II), (III), (IV), (V), or (VI) include a pharmaceutically acceptable anion L. In some embodiments, L is an anion selected from chloride, bromide, iodide, sulfate, phosphate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate, oxalate, succinate, benzoate and p- toluenesulfonate.

[0131] Compositions and Methods of use

[0132] The present application also provides pharmaceutical compositions comprising an effective amount of a compound of the present disclosure (e.g., Formula (I), (II), (III), (IV), (V), or (VI) disclosed herein and a pharmaceutically acceptable carrier. The carrier(s) are “acceptable” in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, not deleterious to the recipient thereof in an amount used in the medicament.

[0133] In some embodiments, the present disclosure provides a method of imaging a tissue comprising contacting the tissue with a compound of Formula (I), (II), (III), (IV), (V), or (VI); irradiating the tissue at a wavelength absorbed by the compound; and detecting a signal from the compound, thereby imaging the tissue.

[0134] In some embodiments, the tissue is cartilage. In some embodiments, the cartilage is hyaline, elastic, or fibrocartilage.

[0135] In some embodiments, the compound is administered to an organism comprising the tissue.

[0136] In some embodiments, wherein the organism is human.

[0137] In some embodiments, the compound of Formula (I), (II), (III), (IV), (V), or

[0138] (VI) has a peak absorbance at about 600 nm to about 850 nm. In some embodiments, the compound of Formula (I), (II), (III), (IV), (V), or (VI) has a peak absorbance at about 660 nm to about 850 nm. In some embodiments, the compound of Formula (I),

[0139] (II), (III), (IV), (V), or (VI) has a peak absorbance at about 760 nm to about 800 nm.

[0140] In some embodiments, the tissue is imaged ex vivo.

[0141] In some embodiments, the tissue is imaged in vivo.

[0142] In some embodiments, the present disclosure provides a method of diagnosing rheumatoid arthritis, comprising administering to a subject suspected of having rheumatoid arthritis an imaging effective amount of a compound of Formula (I), (II),

[0143] (III), (IV), (V), or (VI); irradiating a tissue at a wavelength absorbed by the compound; and determining the presence or absence of a signal from the compound, wherein the presence of a signal indicates a diagnosis of rheumatoid arthritis.

[0144] In some embodiments, the present disclosure provides a method of diagnosing rheumatoid arthritis, comprising administering to a subject identified as having or diagnosed with rheumatoid arthritis an imaging effective amount of a compound of Formula (I), (II), (III), (IV), (V), or (VI); irradiating a tissue at a wavelength absorbed by the compound; and determining the presence or absence of a signal from the compound, wherein the presence of a signal indicates a diagnosis of rheumatoid arthritis.

[0145] In some embodiments, the subject has joint pain.

[0146] In some embodiments, the subject was further diagnosed with rheumatoid arthritis from a serological test, a physical examination, and / or a joint radiographs.

[0147] In some embodiments, the tissue is cartilage.

[0148] In some embodiments, the compound of Formula (I), (II), (III), (IV), (V), or (VI) has a peak absorbance at about 600 nm to about 850 nm. In some embodiments, the compound of Formula (I), (II), (III), (IV), (V), or (VI) has a peak absorbance at about 660 nm to about 850 nm. In some embodiments, the compound of Formula (I),

[0149] (II), (III), (IV), (V), or (VI) has a peak absorbance at about 760 nm to about 800 nm.

[0150] In some embodiments, the present disclosure provides a compound a method of treating rheumatoid arthritis, comprising administering to a subject suspected of having rheumatoid arthritis an imaging effective amount of a compound of Formula (I), (II),

[0151] (III), (IV), (V), or (VI); irradiating a tissue at a wavelength absorbed by the compound; determining the presence or absence of a signal from the compound; and if a signal is detected, administering to the subject a therapeutically effective amount of one or more anti-inflammatory agents, one or more immunomodulatory agents, or a combination thereof.

[0152] In some embodiments, the present disclosure provides a compound a method of treating rheumatoid arthritis, comprising administering to a subject identified as having or diagnosed with rheumatoid arthritis an imaging effective amount of a compound of Formula (I), (II), (III), (IV), (V), or (VI); irradiating a tissue at a wavelength absorbed by the compound; determining the presence or absence of a signal from the compound; and if a signal is detected, administering to the subject a therapeutically effective amount of one or more anti-inflammatory agents, one or more immunomodulatory agents, or a combination thereof.

[0153] In some embodiments, the subject has joint pain.

[0154] In some embodiments, the subject was diagnosed with rheumatoid arthritis from a serological test, a physical examination, and / or a joint radiographs.

[0155] In some embodiments, the tissue is a biological tissue or organ. In specific embodiments, the tissue is a lumen, such as the ureter, cartilage, bone cell, bone mineral, 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, a nerve, thoracic duct, pan lymph nodes, sentinel lymph nodes, vulnerable plaque, stem cells, or neuroendocrine tumor cells. In some embodiments, the tissue is cartilage.

[0156] In some embodiments, the compound of Formula (I), (II), (III), (IV), (V), or (VI) has a peak absorbance at about 600 nm to about 850 nm. In some embodiments, the compound of Formula (I), (II), (III), (IV), (V), or (VI) has a peak absorbance at about 660 nm to about 850 nm. In some embodiments, the compound of Formula (I), (II), (III), (IV), (V), or (VI) has a peak absorbance at about 760 nm to about 800 nm. In some embodiments, the present disclosure provides a method for detecting the presence of a signal generated by the formation of a conjugate of a compound of Formula (I), (II), (III), (IV), (V), or (VI) and a tissue, thereby imaging the tissue. In some embodiments, the tissue is cartilage.

[0157] In some embodiments, the signal may be in the form of absorption, such as occurs during photoacoustic imaging. In some embodiments, the imaging agents can have a signal -to-background ratio (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.

[0158] In some embodiments, the present disclosure provides a method for detecting the presence of a signal generated by the formation of a conjugate of a compound of Formula ((I), (II), (III), (IV), (V), or (Vl)and glycosaminoglycans (GAG) from cartilage. In some embodiments, the glycosaminoglycans are sulfated and / or carboxylated.

[0159] In some embodiments, a compound of Formula (I), (II), (III), (IV), (V), or (VI) having two or more quaternary ammonium have a stronger binding affinity to GAG than a compound of Formula (I), (II), (III), (IV), (V), or (VI) having only one quaternary ammonium group. Definitions

[0160] As used herein, the term “about” means “approximately” (e.g., plus or minus 10% of the indicated value).

[0161] The term “halo” or “halogen” refers to any radical of fluorine, chlorine, bromine or iodine.

[0162] The term “alkyl” refers to a saturated hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, Ci-Ce alkyl indicates that the group may have from 1 to 6 (inclusive) carbon atoms in it. Any atom can be optionally substituted, e.g., by one or more substituents. Examples of alkyl groups include, without limitation, methyl, ethyl, w-propyl, isopropyl, and tert-butyl.

[0163] The term “alkenyl” refers to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms and having one or more carboncarbon double bonds. Any atom can be optionally substituted, e.g., by one or more substituents. Alkenyl groups can include, e.g., vinyl, allyl, 1-butenyl, and 2-hexenyl. One of the double bond carbons can optionally be the point of attachment of the alkenyl substituent.

[0164] The term “cycloalkyl” refers to a fully saturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon group. Any atom can be optionally substituted, e.g., by one or more substituents. A ring carbon serves as the point of attachment of a cycloalkyl group to another moiety. Cycloalkyl moieties can include groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbomyl (bicyclo[2.2.1]heptyl).

[0165] The term “heterocyclyl” refers to a fully saturated monocyclic, bicyclic, tricyclic or other polycyclic ring system having one or more constituent heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups may be present to complete the nitrogen valence and / or form a salt), or S. The heteroatom or ring carbon can be the point of attachment of the heterocyclyl substituent to another moiety. Any atom can be optionally substituted, e.g., by one or more substituents. Heterocyclyl groups can include groups such as tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl. By way of example, a phrase such as “heterocyclic ring containing from 5-6 ring atoms”, wherein from 1-2 of the ring atoms is independently selected from N, NH, N(Ci-Ce alkyl), NC(O)(Ci-Ce alkyl), O, and S; and wherein said heterocyclic ring is optionally substituted with from 1-3 independently selected Rawould include (but not be limited to) tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl.

[0166] The term “aryl” refers to an aromatic monocyclic, bicyclic (2 fused rings), tricyclic (3 fused rings), or polycyclic (> 3 fused rings) hydrocarbon ring system. One or more ring atoms can be optionally substituted by one or more substituents for example. Aryl moi eties include groups such as phenyl and naphthyl.

[0167] The term “heteroaryl” refers to an aromatic monocyclic, bicyclic (2 fused rings), tricyclic (3 fused rings), or polycyclic (> 3 fused rings) hydrocarbon groups having one or more heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups may be present to complete the nitrogen valence and / or form a salt), or S. One or more ring atoms can be optionally substituted, e.g., by one or more substituents. Examples of heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl, P-carbolinyl, carbazolyl, coumarinyl, chromenyl, cinnolinyl, dib enzo[b,d] furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, and xanthenyl.

[0168] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically inactive starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, N=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated or synthesized as a mixture of isomers or as separated isomeric forms. In some embodiments, the compound has the (^ / -configuration. In some embodiments, the compound has the (^-configuration. Compounds provided herein also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H- and 3H- imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H- pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

[0169] As used herein, the term “cell” is meant to refer to a cell that is in vitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal. In some embodiments, an in vitro cell can be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism such as a mammal.

[0170] As used herein, the term “subject” refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.

[0171] As used herein, the phrase “imaging effective amount” refers to the necessary amount of active compound or pharmaceutical agent that allows for imaging of a tissue in an animal, individual, or human to be detected by the detection method chosen. For example, a detectable quantity can be an administered amount sufficient to enable detection of binding of the labeled compound to a target of interest including, but not limited to cartilage.

[0172] As used herein, the term “conjugate” refers to the non-covalent association of a compound of Formula I with a tissue such as cartilage and thereby produces a signal that can be captured as a means for imaging the tissue.

[0173] As used herein, the phrase “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

[0174] As used herein the term “treating” or “treatment” refers to 1) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (z.e., arresting further development of the pathology and / or symptomatology), or 2) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (z.e., reversing the pathology and / or symptomatology).

[0175] As used herein, the term “preventing” or “prevention” of a disease, condition or disorder refers to decreasing the risk of occurrence of the disease, condition or disorder in a subject or group of subjects (e.g., a subject or group of subjects predisposed to or susceptible to the disease, condition or disorder). In some embodiments, preventing a disease, condition or disorder refers to decreasing the possibility of acquiring the disease, condition or disorder and / or its associated symptoms. In some embodiments, preventing a disease, condition or disorder refers to completely or almost completely stopping the disease, condition or disorder from occurring.

[0176] EXAMPLES

[0177] Reagents and materials

[0178] All chemicals and solvents were purchased from Fisher Scientific (Pittsburgh, PA, USA) and Sigma-Aldrich (Saint Louis, MO). TLC plates having silica gel 60 F254 (Merck EMD Millipore, Darmstadt, Germany) were used to guide the completion of the reaction. Flash column chromatographic technique was used to purify the final heptamethine dyes by using 60-200 pm, 60 A classic column silica gel (Dynamic Adsorbents, Norcross, GA). BothJH NMR and13C NMR spectra were obtained using high-quality Kontes NMR tubes (Kimble Chase, Vineland, NJ) rated to 500 MHz and were recorded on a Bruker Avance (400 MHz) spectrometer using Chloroform-t / or DMSO-t / e containing tetramethylsilane (TMS) as an internal calibration standard set to 0.0 ppm. UV-Vis / NIR absorption spectra were recorded on a Varian Cary 50 spectrophotometer. High-resolution accurate mass spectra (HRMS) were obtained at the GSU Mass Spectrometry Facility. Assay reagent kits (for aspartate aminotransferase, alanine aminotransferase, and serum creatinine) were purchased from Cayman Chemical (Ann Arbor, MI). General procedure for the preparation of heptamethine cyanine fluorophores

[0179] Scheme 1

[0180] As shown in Scheme 1, the Vilsmeier Haack linker (1 eq) containing a quaternary ammonium or containing a carbon in the cyclohexene ring was added to a dry round bottom flask and dissolved in 5 mL acetic anhydride. Individual indolium salts (2 eq) and sodium acetate (3 eq) were added to the round bottom flask. The reaction mixture was refluxed and stirred for 4 h at 70 °C. The reaction was monitored by UV-Vis spectroscopy and thin layer chromatography (TLC) in DCM and 2% methanol. After the reaction was completed, it was cooled to room temperature followed by the addition of diethyl ether to precipitate the desired crude product. The crude dye was purified by recrystallization with MeOH:EtOAc (1 : 10), yielding the pure product in high yields (70-85%).

[0181] General procedure for the preparation of meso modified heptamethine cyanine fluorophores

[0182] After the successful synthesis of heptamethine cyanine dyes, they were reacted for further meso modification. In a clean, dry vial, 20 mg of the individual heptamethine cyanine dye (1 eq) was dissolved in DMSO (2 mL). Thiocholine (1.5 eq) and diethylisopropyl amine (3 eq, DIEA) were dissolved in DMSO (2 mL) in a test tube and then added to the vial containing the dissolved dye. The reaction mixture was refluxed and stirred at 60 °C for 2 h. The reaction was monitored by UV-Vis spectroscopy. After the reaction was completed, the reaction mixture was transferred to a 15 mL centrifuge tube, and ethyl acetate (10 mL) was added. This mixture was vortexed and centrifuged, the supernatant was discarded, and additional ethyl acetate (10 mL) was added. This process was repeated a total of five times. After the final centrifugation and removal of supernatant, the precipitated dye pellet was transferred to a clean vial and dried under vacuum.

[0183] Intermediate 1

[0184] (E)-4-chloro-3-formyl-5-(hydroxymethylene)-l, 1-dimethylpiperidin-l-ium : 5 mL l-methylpiperidin-4-one was dissolved in 70 mL acetone placed in an ice bath. Methyl iodide (6 mL) was added dropwise to the solution for over 10 min. The ice bath was removed and left for stirring for 4 h at room temperature. After the reaction completed, white precipitate formed, and it was filtered and washed with cold acetone three times to yield 1,1 -dimethyl -4-oxopiperi din- 1-ium. After the successful synthesis of 2, the next step is synthesizing the bisaldehyde. DMF (9 mL) was cooled in an ice bath and POCh (5.5 mL) was added dropwise and stirred at 0 °C for 30 min. Compound 2 (5 g) was added to the reaction mixture and refluxed at 80 °C for 3 h. After the reaction was completed, it cooled down in an ice bath and dilute HC1 (0.1 M) solution was added dropwise until the precipitate forms. Then the solution was sonicated for 30 min and left in the fridge overnight to form bisaldehyde 4 as an orange precipitate brown solid, 'H NMR (400 MHz, DMSO-t / e): 6 (ppm) 3.15 (s, 6H), 4.33 (s, 4H), 8.96 (s, 2H);13C NMR (100 MHz, DMSO-t / e): 6 (ppm) 51.84, 59.29, 109.95, 146.19, 173.81.

[0185] Example 1

[0186] 2-( (E)-2-( (E) -4 -chlor o-l, 1 -dime thy 1-5 -(2 -( (E)-l, 3, 3-trimethylindolin-2- ylidene) ethylidene)-l,2,5,6-tetrahydropyridin-l-ium-3-yl) vinyl)- 1,3, 3-trimethyl-3H- indol-l-ium iodide (19): green solid Yield 75%. m.p. 165-167 °C, 'H NMR (400 MHz, DMSO-t / e) 6 (ppm): 1.75 (s, 12H), 3.90 (s, 6H), 4.07 (s, 6H), 5.40 (s, 4H), 6.72 (d, J = 14.4 Hz, 2H), 7.31 (m, 4H), 7.43 (m, 4H) 8.28 (d, J= 14.4 Hz, 2H).

[0187] Example 2

[0188] 20

[0189] 2-((E)-2-((E)-2-chloro-3-(2-((E)-5-methoxy-l, 3,3-trimethylindolin-2-ylidene) ethylidene) cyclohex- 1-en-l-y I) vinyl)-5-methoxy-l , 3, 3-trimethyl-3H-indol-l-ium iodide (20): green solid, Yield 70%. m.p. 177-179 °C, 'H NMR (400 MHz, DMSO- d6) 6 (ppm): 1.66 (s, 12H), 1.85 (s, 2H), 2.68 (s, 4H), 3.65 (s, 6H), 3.82 (s, 6H), 6.19 (d, J= 14.0 Hz, 2H), 6.98 (m, 2H), 7.30 (s, 2H), 7.36 (d, J= 8.4 Hz, 2H), 8.16 (d, J= 14.0 Hz, 2H).

[0190] Example 3

[0191] 21

[0192] 2-((E)-2-((E)-4-chloro-5-(2-((E)-5-methoxy-l, 3,3-trimethylindolin-2-ylidene) ethylidene)-!, 1 -dimethyl- 1, 2, 5, 6-tetrahydropyridin-l-ium-3-yl) vinyl)-5-methoxy- l,3,3-trimethyl-3H-indol-l-ium iodide (21): green solid, Yield 75%. m.p. 167-169 °C, ‘H NMR (400 MHz, DMSO-t / e) 6 (ppm): 1.69 (s, 12H), 3.35 (s, 6H), 3.78 (s, 6H), 3.84 (s, 6H), 4.80 (s, 4H), 6.32 (d, J= 14.8 Hz, 2H), 7.03 (d, J= 6.8 Hz, 2H), 7.37 (s, 2H), 7.45 (d, J= 6.8 Hz, 2H), 8.10 (d, J= 14.8 Hz, 2H).

[0193] Example 4 2-((E)-2-((E)-4-chloro-5-(2-((E)-5-fluoro-l,3, 3-trimethylindolin-2-ylidene) ethylidene)-!, 1 -dimethyl- 1, 2, 5, 6-tetrahydropyridin-l-ium-3-yl) vinyl) -5 -fluor o-l, 3, 3- trimethyl-3H-indol-l-ium iodide (22): green solid, Yield 70%. m.p. 167-169 °C 'H NMR (400 MHz, DMSO-t / e) 6 (ppm): 1.69 (s, 12H), 3.79 (s, 6H), 4.78 (s, 4H), 6.32 (d, J = 14.80 Hz, 2H), 7. 35 (m, 2H), 7.57 (m, 1H), 7.69 (m, 2H), 8.15 (d, J = 14.80 Hz, 2H).

[0194] Example 5

[0195] 5-chloro-2-( (E)-2-( (E)-4-chloro-5-(2-( (E) -5 -chlor o-l, 3, 3-trimethylindolin-2- ylidene) ethylidene)-!, 1-dimethyl-l, 2, 5, 6-tetrahydropyridin-l-ium-3-yl) vinyl)-!, 3, 3- trimethyl-3H-indol-l-ium iodide (23): green solid, Yield 69% m.p. 167-169 °C, 'H NMR (400 MHz, DMSO-t / e) 6 (ppm): 1.70 (s, 12H), 3.83 (s, 6H), 3.79 (s, 6H), 4.82 (s, 4H), 6.37 (d, J= 14.80 Hz, 2H), 7.56 (s, 4H), 7.88 (s, 2H), 8.16 (d, J = 14.80 Hz, 2H).

[0196] Example 6

[0197] 5-bromo-2-( (E)-2-( (E)-5-(2-( (E)-5-bromo-l, 3, 3-trimethylindolin-2- ylidene) ethylidene) -4-chlor o-l, 1-dimethyl-l, 2, 5, 6-tetrahydropyridin-l-ium-3-yl) vinyl)-l,3,3-trimethyl-3H-indol-l-ium iodide (24): green solid, Yield 85% m.p. 167- 169 °C, 'H NMR (400 MHz, DMSO-t / e) 6 (ppm): 1.69 (s, 12H), 3.30 (s, 4H), 3.75 (s, 6H), 4.74 (s, 6H), 6.31 (d, J = 12.00 Hz, 2H), 7.48 (d, J = 8.00 Hz, 2H), 7.68 (s, 2H), 7.98 (m, 2H), 8.16 (d, J= 12.00 Hz, 2H). Example 7

[0198] 25

[0199] 2-( (E)-2-( (E) -4 -chlor o-l, l-dimethyl-5-( (E)-2-( 1, 1, 3-trimethyl-l, 3-dihydro-2H- benzo[e]indol-2-ylidene)ethylidene)-l,2,5,6-tetrahydropyridin-l-ium-3-yl)vinyl)-

[0200] 1.1.3-trimethyl-lH-benzo [e]indol-3-ium iodide (25): green solid, Yield 82% m.p. 167- 169 °C, 'HNMR (400 MHz, DMSO-t / e) 6 (ppm): 1.97 (s, 12H), 3.94 (s, 6H), 4.85 (m, 4H), 6.42 (d, J = 14.00 Hz, 2H), 7.58 (t, J = 7.20 Hz, 2H), 7.71 (t, J = 7.20 Hz, 2H), 7.85 (d, J= 8.80 Hz, 2H), 8.14 (q, J= 8.00 Hz, 2H), 8.34 (d, J= 14.00 Hz, 2H).

[0201] Example 8 (Compound 26 or CARF26)

[0202] 26

[0203] 2-( (E)-2-( (E)-l, l-dimethyl-4-( (2-( trimethylammonio)ethyl)thio)-5-(2-( (E)-

[0204] 1.3.3-trimethyl-indolin-2-ylidene) ethylidene)-l,2,5,6-tetrahydropyridin-l-ium-3-yl) vinyl)-l,3,3-trimethyl-3H-indol-l-ium iodide (26): green solid, Yield 41% m.p. 169- 171 °C, 'H NMR (400 MHz, DMSO-ek) 6 (ppm): 1.72 (s, 12H), 3.11 (s, 9H), 3.32 (s, 6H), 3.59 (s, 4H), 3.83 (s, 6H), 5.00 (s, 4H), 6.50 (d, J= 14.80 Hz, 2H), 7.36 (t, J = 15.68 Hz, 2H), 7.50 (m, 4H), 7.67 (d, J= 6.8 Hz, 2H), 8.57 (d, J= 14.80 Hz, 2H).13C NMR (100 MHz, DMSO-ek) 6 (ppm): 27.62, 27.80, 29.39, 32.84, 49.77, 49.86, 52.08, 52.73, 60.03, 64.01, 102.83, 112.51, 120.96, 122.91, 126.28, 129.14, 141.74, 143.17, 144.42, 146.28. The exact mass of 26 without three T counter ions is 597.3974 Da, where the dye has a 3+ charge. The exact mass divided by two, which gives M2+ / 2, is calculated as 298.6987 and was seen in the mass spectrum at 298.1932. The mass divided by three, which gives M3+ / 3, is calculated as 199.1325 and was observed exactly in the spectrum at 199.1325.

[0205] Example 9 (Compound 27 or CARF27)

[0206] 27

[0207] 5-methoxy-2-( (E)-2-( (E)-3-(2-( (E) -5 -methoxy- 1, 3, 3-trimethylindolin-2- ylidene) ethylidene)-2-((2-(trimethylammonio) ethyl) thio) cyclohex- 1-en-l-y I) vinyl)- 1 ,3,3-trimethyl-3H-indol-l-ium (27): green solid, Yield 50% m.p. 170-172 °C, 'H NMR (400 MHz, DMSO-t / e) 6 (ppm): 1.69 (s, 12H), 2.62 (m, 2H), 3.04 (s, 9H), 3.13 (s, 4H), 3.66 (s, 6H), 3.82 (s, 6H), 6.21 (d, J = 14.0 Hz, 2H), 7.01 (m, 2H), 7.27 (s, 2H), 7.35 (d, J= 8.8 Hz, 2H), 8.53 (d, J= 14.0 Hz, 2H). The exact mass equals 627.3848 Da without two T counter ions. The molecular ion peak (m) equal to 627.3848 is observed at 628.3907 in the mass spectrum. The mass divided by two (m / 2) is calculated to be 313.6924 and was observed in the spectrum at 313.6924.

[0208] Example 10 (Compound 28 or CARF28)

[0209] 5-methoxy-2-( (E)-2-( (E)-5-(2-( (E) -5 -methoxy- 1, 3, 3-trimethylindolin-2- ylidene) ethylidene)-! , l-dimethyl-4-((2-(trimethylammonio)ethyl)thio)-l ,2,5,6- tetrahydropyridin-l-ium-3-yl) vinyl)-l,3,3-trimethyl-3H-indol-l-ium iodide (28): green solid, Yield 53% m.p. 169-171 °C, 'H NMR (400 MHz, DMSO-t / e) 6 (ppm): 1.72 (s, 12H), 3.11 (s, 9H), 3.30 (s, 6H), 3.58 (s, 4H), 3.79 (s, 6H), 3.85 (s, 6H), 4.84 (s, 6H), 6.32 (d, J = 14.00 Hz, 2H), 7.04 (d, J= 7.60 Hz, 2H), 7.31 (s, 1H), 7.43 (d, J = 8.00 Hz, 2H), 8.51 (d, J= 14.00 Hz, 2H).13C NMR (100 MHz, DMS0 ) 6 (ppm): 27.76, 29.37, 30.60, 32.97, 49.92, 52.26, 52.81, 52.95, 56.47, 60.29, 64.23, 64.67, 102.25, 109. 63, 113.31, 114.10, 119.63. The exact mass of 28 is 657.4186 Da without three T counter ions. The exact mass of the dye divided by three, notably m / 3, is calculated as 219.1395 and is seen at 219.1401 in the mass spectrum.

[0210] Example 11 (Compound 29 or CARF29)

[0211] 5-fluoro-2-( (E)-2-( (E)-5-(2-( (E)-5-fluoro-l, 3, 3-trimethylindolin-2- ylidene)ethylidene)-l, l-dimethyl-4-((2-( trimethylammonio) ethyl) thio)-l, 2, 5, 6- tetrahydropyridin-l-ium-3-yl) vinyl)-l,3,3-trimethyl-3H-indol-l-ium iodide (29): green solid, Yield 63% m.p. 167-169 °C, 'H NMR (400 MHz, DMSO-t / e) 6 (ppm): 1.72 (s, 12H), 3.08 (s, 9H), 3.13 (s, 2H), 3.27 (s, 4H), 3.52 (s, 4H), 3.80 (s, 6H), 4.78 (s, 6H), 6.33 (d, J= 14.8 Hz, 2H), 7.36 (m, 2H), 7.56 (m, 2H), 7.72 (m, 2H), 8.53 (d, J = 14.8 Hz, 2H).13C NMR (100 MHz, DMSO-t / e) 6 (ppm): 24.30, 27.55, 29.51, 30.35, 33.06, 50.11, 52.50, 52.84, 60.36, 64.29, 102.56, 110.96, 111.21, 113.83, 113.91, 115.72, 115.96, 120.28, 139.45, 143.88, 143.97, 144.37, 146.57, 159.99, 162.41, 174.92.19F NMR (100 MHz, DMSO-t / e) 6 (ppm): - 115.90. The exact mass of 29 is 633.3786 without three T counter ions. The mass divided by 2, which gives m / 2, is calculated to be 316.6893 and is seen in the mass spectrum at 316.1918. The mass divided by 3, which gives m / 3 is calculated to be 211.1262 and was observed at 211.1254 in the mass spectrum.

[0212] Example 12 (Compound 30 or CARF30)

[0213] 5-chloro-2-( (E)-2-( (E)-5-(2-( (E)-5-chloro-l, 3, 3-trimethylindolin-2- ylidene)ethylidene)-l , l-dimethyl-4-((2-(trimethylammonio)ethyl)thio)-l ,2,5,6- tetrahydropyridin-l-ium-3-yl)vinyl)-l,3,3-trimethyl-3H-indol-l-ium iodide (30): green solid, Yield 58% m.p. 167-169 °C, 'H NMR (400 MHz, DMSO-t / e) 6 (ppm): 1.73 (s, 12H), 3.07 (s, 9H), 3.27 (s, 6H), 3.35 (s, 4H), 3.78 (s, 6H), 4.75 (s, 2H), 4.81 (s, 2H),6.34 (d, J= 14.4 Hz, 2H), 7.55 (t, J= 9.20 Hz, 2H), 7.86 (s, 2H), 8.55 (d, J= 14.4 Hz, 2H).13C NMR (100 MHz, DMSO-t / e) 6 (ppm): 27.52, 29.54, 33.01, 50.02, 52.54, 52.86, 60.35, 64.31, 102.78, 114.00, 120.80, 123.41, 129.12, 130.72, 142.09, 143.68, 144.65, 147.08, 174.76. The exact mass of 30 is 665.3195 without three T counter ions. The exact mass of the dye divided by three charges, notably m / 3, is calculated as 221.7732 and was seen in the mass spectrum at 221.7794s.

[0214] Example 13 (Compound 31 or CARF31)

[0215] 5-bromo-2-( (E)-2-( (E)-5-(2-( (E)-5-bromo-l, 3, 3-trimethylindolin-2- ylidene)ethylidene)-l , l-dimethyl-4-((2-(trimethylammonio)ethyl)thio)-l ,2,5,6- tetrahydropyridin-l-ium-3-yl)vinyl)-l,3,3-trimethyl-3H-indol-l-ium iodide (31): green solid, Yield 65% m.p. 167-169 °C, 'H NMR (400 MHz, DMSO-t / e) 6 (ppm): 1.72 (s, 12H), 3.06 (s, 9H), 3.27 (s, 6H), 3.52 (s, 4H), 3.78 (s, 6H), 4.71 (s, 2H), 4.78 (s, 2H), 6.35 (d, J= 14.00 Hz, 2H), 7.48 (d, J= 8.00 Hz, 2H), 7.69 (d, J= 8.00 Hz, 2H ), 7.99 (s, 2H), 8.55 (d, J= 14.00 Hz, 2H).13C NMR (100 MHz, DMSO-t / e) 6 (ppm): 24.26, 27.52, 29.54, 32.98, 50.01, 52.87, 64.30, 102.81, 114.41, 118.85, 120.91, 126.18, 131.96, 142.52, 143.95, 144.66, 174.57. The exact mass of 31 is 753.2185 without three T counter ions. The exact mass divided by 3, which is m / 3, is calculated as 251.0728 and was observed at 251.7417 in the mass spectrum. Example 14 (Compound 32 or CARF32)

[0216] 32

[0217] 2-( (E)-2-( (E)-l, l-dimethyl-5-( (E)-2-( 1, 1, 3-trimethyl-l, 3-dihydro-2H- benzo[e]indol-2-ylidene) ethylidene)-4-((2-(trimethylammonio) ethyl) thio)-l, 2,5,6- tetrahydropyridin-l-ium-3-yl) vinyl)- 1, 1, 3-trimethyl-lH-benzo [ ]indol-3-ium iodide 32y. green solid, Yield 54% m.p. 167-169 °C, 'HNMR (400 MHz, DMSO-cf.) 6 (ppm): 2.01 (s, 12H), 3.14 (s, 9H), 3.35 (s, 6H), 3.63 (s, 4H), 3.95 (s, 6H), 4.91 (s, 4H), 6.45 (d, J = 14.8 Hz, 2H), 7.58 (m, 2H), 7.71 (m, 2H), 7,85 (d, J = 8.80 Hz, 2H), 8.15 (m, 2H), 8.30 (d, J= 8.80 Hz, 2H), 8.71 (d, J= 14.8 Hz, 2H).13C NMR(100 MHz, DMSO- d6) 6 (ppm): 27.40, 29.59, 30.51, 31.17, 33.26, 51.48, 52.42, 52.90, 52.98, 60.41, 64.33, 64.72, 102.12, 112.51, 120.44, 122.77, 125.86, 127.72, 128.38, 130.53, 130.96, 132.29, 134.61, 140.79, 143.83, 145.84, 176.12. The exact mass of 32 is 697.4287 without three T counter ions. The exact mass of the dye divided by 2, which gives m / 2, is calculated to be 348.7144 and is observed in the mass spectrum at 232.4766. The exact mass divided by 3, which gives m / 3, is calculated to be 232.4762 and was seen at 232.4766 in the mass spectrum.

[0218] Assays

[0219] Evaluation of in vitro binding affinity of CARE to cross-linked hyaluronic acid (HA) as model glycosaminoglycans.

[0220] The articular cartilage was composed of heavily sulfated and carboxylated glycosaminoglycans (GAGs, 5-10 wf / o) that grant cartilage a fixed negative charge.29Therefore, the binding affinity of CARF28 to cross-linked HA (xHA) was tested, the central structure of GAG, and compared the signal intensity with C800, 21, and 27 treated groups. The xHA was treated with CARF28, C800, 21, and 27 for 30 min and washed with PBS (pH 7.4) 3 times. And then, NIR fluorescence intensities were measured (Figure 1). Interestingly, fluorophores having only one quaternary ammonium group (21 and 27) did not show high bindings to xHA, while the fluorophores containing the two quaternary ammoniums (CARF28 and C800) exhibited high bindings. In vivo cartilage targeting data also showed that fluorophores having only one quaternary ammonium group (21 and 27) did not show high bindings to xHA (Figure 9). It is worth noting that dyad quaternary ammoniums are necessary to achieve sufficient binding strength and retention by charge-charge interactions between fluorophores and xHA.

[0221] NIR-II imaging capability of CARF28 compared with NIR-I imaging.

[0222] To verify the NIR-II capability, the signal intensity of 21, precursor for CARF28, before and after S- substitution in the meso position was compared (Figure 1). The substitution of the / 7 / c.w-chloride of the heptamethine skeleton with thiocholine enhances the fluorescence intensity 1.7-fold in the NIR-II window. Without wishing to be bound by theory, this enhancement may be attributed to a redshift in both absorbance and fluorescence emission (emission maximum is 813 nm in 5% BSA saline) which causes the tail emission in NIR-II window (Figure 1). This post-synthetic derivatization procedure of a cheap, commercially available, NIR-emitting cyanine dye promptly provides stable fluorescent bioconjugates suitable for in vivo imaging applications.

[0223] To demonstrate the improved fluorescence imaging capability, NIR-II images (>1,070 nm, using Ninox InGaAs camera) of mouse rib cartilage was compared with NIR-I (710 / 50 nm, using KFLARE system) as shown in Figures 1 and 10. CARF28 showed the ability to target cartilage selectively and has clear resolution and high contrast with reduced background signal. Skinless images showed both fluorophores successfully targeted cartilages indicating the blurring and low resolution in NIR-I are due to increased scattering from the skin. Overall, the thiocholine substitution not only gives dyad cartilage targetability to CARF28 but also superior fluorescence imaging ability in NIR-II.

[0224] Toxicity assessment of CARF28

[0225] Although dyad quaternary ammoniums are an essential component for cartilage targeting, a potential risk arises from highly positively charged molecules, which can interact with negatively charged biological components such as cell membranes, proteins, red blood cells, platelets, and endothelial cells. When strong charge-charge interactions significantly occur, they can trigger the formation of blood clots, which can block blood vessels in the lungs (a pulmonary embolism) and cause sudden death. To assess the potential toxicity of CARF28, a cell viability test, blood clotting test, pulmonary embolism test, and in vivo single dose acute toxicity test were performed. In Figure 2 and Figure 11, the results of the cytotoxicity tests showed that CARF28, even up to 100 pM, did not significantly affect the viability of primary chondrocytes from swine, macrophages (RAW264.7), and immortalized human embryonic kidney cells (HEK293), which is similar to the limited cytotoxicity of FDA-approved NIR fluorophores, ICG (data was not shown).

[0226] Next, the viscosity was measured to confirm that blood clotting occurs after mixing the fluorophores with whole blood (Figure 2). As a result, adding C800 to blood induced a significant viscosity increase (p>0.05) compared to control. Without wishing to be bound by theory, this may be due to blood clotting caused by charge-charge interactions. The viscosity did not change when CARF28 was added, despite containing two quaternary ammoniums. Without wishing to be bound by theory, this may be because the quaternary ammonium in the hexyl ring is not fully exposed, unlike those of C800, and has relatively low electron density (Figure 12).

[0227] In the animal study, all the mice injected with a single dose of 3 pmol / kg (n = 3) and 1 pmol / kg dose (n = 2) of C800 immediately died (Figure 2). It should be noted that dyes that contain more than two quaternary ammoniums in addition to C800 can cause mouse death. To reveal the cause of death, iopamidol as an X-ray / CT contrast agent was perfused into the right ventricle of the heart in the dead mouse, and pulmonary embolism was confirmed by X-ray / CT angiography. In contrast, no death nor pulmonary embolism was observed with the 3 pmol / kg dose for CARF28. Next, a single-dose acute toxicity test of CARF28 (3 pmol / kg) was conducted to determine short-term adverse effects. On the 14th day after the initial injection, blood and major organs (heart, liver, spleen, lung, and kidneys) were collected (Figure 6). And biochemical analyses (blood urea nitrogen; BUN, creatinine; CREA, lactate dehydrogenase; LDH, aspartate aminotransferase; AST, and aminotransferase; ALT) and histopathological examination by hematoxylin and eosin (H&E) staining were also performed. No abnormal behavior nor significant differences in biochemical analyses and histopathological examination on major organs (heart, liver, spleen, lung, and kidneys) were observed in the CARF28 injected group compared to the control group (Figure 2). These toxicity assessment results indicate that CARF28’s rational design mitigates the risk of pulmonary embolism and other adverse events associated with intravenous injection of highly positively charged molecules.

[0228] Structural influence of CARF28 in cartilage targeting ability In order to confirm the effect of side functional groups, several CARF28 derivatives (26 and 29-32) were synthesized with hydrogens, fluorides, chlorides, bromides, and phenyls instead of methoxy groups. A summary of optophysical properties, absorption and emission spectra, and NIR-II images are shown in Table 1, Figures 3 and 14, respectively. In the results of in vivo cartilage targeting with fluorophores, the only fluorophore with methoxy groups showed a high signal-to-background ratio (SBR, costal cartilage / neighboring muscle) and specific targeting to cartilage (Figures 3 and 15). It is likely due to the presence of the methoxy groups, an electron-donating group, which can increase the electron density on the phenyl ring and can alter the electrostatic interactions via hydrogen bonding between the dye molecule and the cartilage tissue. However, the influence of the methoxy group on cartilage binding may also depend on other factors, such as the steric hindrance and conformational flexibility of the dye molecule. Further studies would be needed to fully elucidate the mechanism of action for the methoxy group in cartilage targeting fluorophores. To determine whether CARF28 can target the other types of cartilage (hyaline, elastic, or fibrocartilage), all the major cartilage tissues, such as the nose, ear, and paw were imaged (Figure 16).

[0229] To further evaluate the binding components in cartilage, a histological analysis was conducted. As shown in Figure 3, CARF28 binds the extracellular matrix region but not the perichondrium area. It is likely that CARF28 binds to molecules released by chondrocytes, such as hyaluronic acid, collagen, proteoglycans, glycoproteins, and elastin.

[0230] Cartilage targeting binding kinetics and pharmacokinetics of cartilage targeting fluorophore.

[0231] To assess the binding kinetics, the rib, knee joint, and hind foot cartilages were observed longitudinally until 48 h post-injection (Figure 7). A peak of signal -to-background ratio (SBR) is observed at 1 h, and SBR gradually decreases over the following hours. The clearance route is both hepatobiliary and renal (Figures 15 and 16), and the signals in the liver, lung, and kidneys were observed (Figure 7). The plasma concentration-time profile was fitted to the conventional two-compartment pharmacokinetic model, and distribution and terminal half-lives were calculated to be 7.62 ± 5.24 and 43.26 ± 8.86, respectively, indicating that the background signal can be reduced after an injection of 1-2 hours (Figure 7). According to the result, the ideal imaging time point is 1 h following the injection.

[0232] Fine-scale NIR-II imaging of cartilage structures. An evaluation of the microstructural analysis capability of CARF28 was conducted on hind paw, larynx, and trachea cartilage structures. First, CARF28 was injected into a CD-I mouse and the hind paw was imaged at 1 h post-injection. The ankle bones, including fibulare, intermedium, tibiale, centrale, distal tarsal, and small joints such as the metatarsophalangeal (MTP) joints in the hind paw were clearly visualized by using the NIR-II fluorescence imaging system with zoom capability. Intensity profiling result in the MTP joint indicates the joint gap is about 300 pm and was compared to microscopic fluorescence and H&E images from histological sections (Figure 4). The spatial resolution was evaluated by characterizing the full width at half-maximum (FWHM) in the ankle highlighted from A to B in Figure 4. And FWHMs were calculated to be 164-218 pm which was confirmed by histological analyses (Figure 4). The larynx and trachea were also visualized, and tracheal cartilages were profiled, and the pitch in tracheal ring cartilages was calculated to be 398 pm corresponding to the microscopic analysis result. Using a cartilage-targeting dye could allow for detailed visualization and analysis of the microstructure and composition of these cartilaginous structures. Overall, microstructural analysis using a cartilage-targeting dye could be a powerful tool for studying the morphology and composition of cartilage in various anatomical structures, with potential applications in both research and clinical settings.

[0233] Early detection of RA using CARF28.

[0234] CARF28 was used to detect subclinical disease activity in the initial stages of RA. DBA / 1 J mice were injected with a collagen antibody cocktail and further induced arthritis by injecting lipopolysaccharides (LPS) on day 3 (D3) (Figure 5). From D-l, the mice were injected with CARF28 and the structural changes in cartilage joints in the NIR-II window were observed (Figure 5). X-ray CT imaging as a conventional diagnosis modality was also conducted to compare with NIR-II imaging. The clinical scores and paw thickness were assessed as additional evaluation methods (Figure 5). The MTP joint showed strong signals with subtle changes in signal intensity throughout the study (even at clinical score 4). However, the signal started broadening became noticeable on D4, preceding the onset of RA symptoms (Figure 5), by two days compared to the appearance of arthritis symptoms (increase in thickness or clinical score on D6), as shown in Figure 5. Notably, X-ray CT imaging did not reveal any signs of bone or cartilage destruction in the early stages of RA, suggesting the only subclinical disease activity may be present, which is challenging to detect. These results suggest that using CARF28 has the potential to detect the earlier stage of RA than currently possible.

[0235] Next, histological analysis was conducted to compare signal broadening with histopathological features in early RA progression (Figure 5). Focusing on the signal broadening of MTP joints, sagittal sections of those joint areas were stained with hematoxylin and eosin (H&E) and safranin O / fast green. Synovitis and synovial hyperplasia were rarely observed in the MTP joint areas in the H&E staining, likely due to the challenges in detecting histopathological features in early RA. Fluorescence signal was clearly observed in the cartilage, consistent with safranin O / fast green staining, although no definitive evidence was found for signal broadening in the histological analysis. Without wishing to be bound by theory, this could be due to potential targets, such as cells and macromolecules in the synovial fluid, being lost during histological sample preparation, particularly the decalcification step. Activated neutrophils within synovial fluids, which play a crucial role in RA initiation and progression, could be among these potential targets. Furthermore, the early stages of RA may result in the release of negatively charged proteoglycans into synovial fluids due to the reduced structural integrity of the cartilage matrix. This can lead the differential distribution of CARF28, particularly with more significant accumulation in synovial fluids. Nevertheless, further investigations are needed to identify the specific molecular targets responsible for signal broadening.

[0236] Determination of optophysical properties such as molar absorption coefficients and fluorescence quantum yields.

[0237] The absorption and emission spectra of fluorophores were measured in DMSO using UV / Vis / NIR spectrometers (USB2000+ VIS+NIR+ES and Flame-NIR, Ocean Insight, Dunedin, FL). Fluorescence emission spectra were collected by NIR excitation with an 808-nm laser. To calculate NIR-II fluorescence quantum yield, the following equation was used: where QYsampieand Le / are the QYs of CARF28 and ICG, respectively, / / sample and «ref are the refractive indices of CARF28 and ICG solutions, respectively, which are both DMSO (1.48) in this case. slopesampieand slopes represent values of line slope determined from the plot of the absorbance at 808 nm against the integrated fluorescence intensities (Xmax to 1,000 nm). These slopes could be obtained from fluorescence measurements. In silico calculations of molecular weight, the distribution coefficient (LogD at pH 7.4), surface molecular charge, polarizability, and 3D molecular surface area using MarvinSketch (ChemAxon, Budapest, Hungary) were

[0238] 5 performed (Table 1).

[0239] Table 1. Physicochemical and optical properties. In silico calculations of logD at pH 7.4 and 3D molecular surface area were calculated using the MarvinSketch calculator plug-in (ChemAxon). Optical properties were measured in 5% w / v BSA containing saline.

[0240] Fluorophores 26 28 29 30 31 32

[0241] Molecular weight (Da) 597.40 657.42 626.93 665.32 753.22 697.43

[0242] Log D at pH 7.4 -5.10 -5.41 -4.03 -3.89 -3.56 -3.12

[0243] 3D molecular surface area974 55j070.56 988.61 1,007.84 1,016.41 1,101.11

[0244] (A3)

[0245] Extinction coefficient (M164 400 1 13 834 139600 59,400 17,000 13,400 cm )

[0246] Absorbance maximum (nm) 753 777 750 766 773 810

[0247] Emission maximum (nm) 777 808 778 791 792 821

[0248] Stokes shift (nm) 24 31 28 25 19 11

[0249] •Quantum yield (%) (at ex 808n 74 2 ] ? 1 N / D 5 90 nm) a% fluorescence intensity >3 1 87 32 78 8 47 55 35 16 93 1926

[0250] 1070 nm 0 N / D: Not detectable aComparision with ICG as a standard. 26% was used for quantum yield of ICG in DMSO.

[0251] Blood coagulation test

[0252] To induce blood clots, 300 pL of blood from swine was mixed with 8 pL of5 solutions of AL31 or CARF28 (10 mM in DMSO). For the comparison, 8 pL of DMSO was added to the blood. The rheological properties of blood with fluorophores or DMSO were analyzed using Haake Viscotester IQ Rheometer (Thermo Scientific, Germany) with a Peltier temperature-controlled bottom plate and a 25.0 mm stainless steel parallel plate measuring system All measurements were performed at 37 °C with0 a 0.5 mm gap. The frequency sweep was conducted at an oscillating frequency of 0.1- 10 Hz and a y Strain of 0.5.

[0253] Cytotoxicity study The chondrocytes (passage 2) were cultured in DMEM media containing 10% FBS, 1% MEM non-essential amino acids, and 1% penicillin-streptomycin. RAW264.7 and HEK293 cells were cultured in DMEM media containing 10% FBS, and 1% penicillin-streptomycin. The culture media were changed every 3 days. The chondrocyte (passage 3), RAW264.7, and HEK293 cells were subsequently utilized for further experiments.

[0254] A 200 pL of cell suspension was added to 96-well plates in order to achieve a density of 5000 cells per well. The plate was pre-incubated for 24 hours in a humidified incubator to facilitate cell attachment. Stock solutions of CARF28, ICG, and DMSO were mixed with the cell culture media (DMEM containing 10% FBS), filtered through a 0.22 pm syringe filter, and then diluted to final concentrations ranging from 1-100 pM. Once the cells reached a density of 60-70% confluence, the existing media was removed and replaced with the above solutions at the specified concentrations. After further incubating for 24 hours, the cells were washed 3 times with fresh PBS, and 100 pL of fresh culture media was added. The morphology and fluorescent signals of the cells were observed using Cytation5 (BioTek, Winooski, VT). After adding 10 pL of CCK-8 solution to each well, the absorbance at 450 nm was measured using Cytation5 following an incubation time of 2 hours. All experiments were conducted with three replicates. The survival rate was calculated according to the equation below:

[0255] Survival rate

[0256] , where Asampie, Ab, and Acare absorption values from sample treatments, blank, and negative control, respectively.

[0257] In vivo toxicity of C800 and CARF28

[0258] To evaluate the potential long-term toxicity, blood samples were obtained by cardiac puncture at the end of the anti-tumor efficacy test (D14). These blood samples were stored without anticoagulant and then centrifuged at 15,00 rpm for 10 min. Serum was stored at -80°C until further assays. The biochemical parameters tested were a liver function panel (aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) and kidney function indication (blood urea nitrogen (BUN) and creatinine (CREA)). All parameters were tested using commercially available assay kits, and the absorbance was immediately measured by a plate reader. To conduct the pulmonary embolism test, CD-I mice were injected with C800 or CARF28 (100, 1000 or 3000 nmol / kg doses). If a mouse died within 4 h post injection, it was euthanized, and perfused with iopamidol as an X-ray / CT contrast agent (2mg / ml, 500 uL) into the right ventricle of the heart in the dead mouse, and pulmonary embolism was confirmed by X-ray / CT angiography.

[0259] General NIR-I and NIR-II fluorescence imaging systems: A 640 * 512-pixel InGaAs camera (Ninox640, Raptor Photonics) and macro zoom lens (0-1 Ox; Navitar Zoom 7000 with SWIR coating) along with 1,070 nm long-pass (LP) filters from Edmund optics were used to collect the NIR-II signal. InGaAs camera plays a key role as an open-air imaging system along with the support of the FLARE system [ref]. An 808 nm fiber-coupled diode laser (30-35 mW / cm2at the sample) was used as an excitation source. Data acquisition was performed using in-house developed software. The mice fur in the region of interest was shaved prior to imaging using a clipper and removed completely using a depilatory cream. 100 pL of CARF28 in 5w / v% BSA saline (500 pM) was injected through the tail vein. Mice were imaged with intact skin using an InGaAs camera. At least 3 mice were analyzed for each sample.

[0260] In vitro affinity test of cartilage targeting fluorophores to cross-linked hyaluronic acid (xHA)

[0261] The procedure described in our previous report was adapted for this study to prepare xHA48and will be briefly outlined here. An 800 mg of HA was dissolved in 2.4 mL of 0.3 M NaOH solution. 160 pL of 1,4-Butanediol diglycidyl ether (BDDE) was added to the HA solution. The reaction mixture was stirred for 24 h at 40 °C. The reactant was neutralized with 0.1 M HC1 to a pH of 7.0 and was dialyzed against deionized water (DW) using a 6-8kDa molecular weight cutoff (MWCO) cellulose dialysis membrane to remove residual reagents. After dialysis, the cross-linked HA (xHA) hydrogel was broken down to particle size using a syringe with a 23 -gauge needle, and xHA particles were then lyophilized. xHA solution is prepared by dissolving xHA particles in phosphate-buffered saline (PBS, pH 7.4) at 25 mg / mL concentration. Each stock solution of CARF28, C800, 21, and 27 was added to 1 mL of xHA solution at a final dye concentration of 10 mM each. The mixtures were incubated for 30 min at room temperature to allow for binding between the fluorophores and xHA. The treated xHA particles were washed with 5 mL of PBS (pH 7.4) three times using a centrifuge (3000 rpm, 10 min). And then, xHA particles were resuspended in 200 pL of PBS. The fluorescence images of xHA were taken using KFLARE and NIR-II imaging systems and were analyzed to determine the binding affinity between the cartilage-targeting NIR fluorophores and xHA.

[0262] Electron density calculation for quaternary ammonium

[0263] Geometry optimization and electron density calculations were performed with the Gaussian 16 program using the density functional theory (DFT) with Becke’s three- parameter hybrid exchange function with Lee-Yang-Parr gradient-corrected correlation functional (B3-LYP) and 6-31G(d) basis set. Solvent effects in water were calculated using the polarizable continuum model (PCM) method, no constraints to bonds / angles / dihedral angles were applied in the calculations, and all atoms were free to optimize. The Mulliken charge at each atom in the quaternary ammonium group was totaled for the given charges, excluding the joining atom for the exposed quaternary ammonium group (such that the value represents N+(CHs)3) and ignoring or averaging (shown in parenthesis) the contributions of the carbons on both sides of dimethyl quaternary ammonium in the hexyl ring.

[0264] 3D fluorescence tomography and X-ray CT imaging

[0265] The InSyTe FLECT / CT imaging system (TriFoil Imaging, CA, USA) is a preclinical imaging system capable of full 3D fluorescence tomography imaging and inline CT imaging. Animal subjects were anesthetized with isoflurane during imaging sessions. FLECT scans were performed with 116 projections per slice, using an 808 nm laser for excitation and an 853 / 45 bandpass fluorescence emission filter. The proprietary FLECT reconstruction engine was used to reconstruct the fluorescence images from the collected data. CT scans were acquired with 360 projections per slice, 35 kV tube voltage, 950 pA tube current, and 100 ms exposure, and CT images were reconstructed using the COBRA filtered back-projection algorithm (Exxim Computing Corporation, Pleasanton, CA, USA).

[0266] In vivo biodistribution and pharmacokinetics of cartilage targeting fluorophores.

[0267] Animals were housed in an AAALAC-certified facility and were studied under the supervision of MGH IACUC in accordance with the approved institutional protocol (2016N000136). To determine, six-week-old CD-I mice (male; 25-30 g from Charles River Laboratories (Wilmington, MA) were anesthetized with isoflurane & oxygen, and blood was sampled in capillary tubes (Fisher Scientific, Pittsburgh, PA) at the time point 0 min by slightly cutting the end of the tail. CARF28 in 5% BSA saline was intravenously injected at the same dose level as the imaging experiments. Blood samples were obtained at 1, 3, 5, 10, 30, 60, 120, 180, and 240 min post-injection, and the fluorescence intensities of serum samples in capillary tubes were measured to calculate distribution (Zi / 2a) and elimination (ti / 2p) half-life values (n = 5 for each group). After 4 h post-injection, mice were sacrificed to image organs (liver, lung, spleen, kidney, stomach, brain, intestine, and bladder). Results were presented as a biexponential decay curve using GraphPad Prism software version 9.0 (GraphPad, San Diego, CA).

[0268] Mouse model for rheumatoid arthritis

[0269] Six-week-old DBA / 1 J mice (male; 25-30 g) were purchased from Charles River Laboratories (Wilmington, MA). Mice were maintained under anesthesia with isoflurane and oxygen during the experiment. To generate CAIA models, DBA / 1 J mice were induced by a single intravenous injection of 1.5 mg of anti -type II collagen 5- clone monoclonal antibody cocktail (Chondrex, Redmond, WA, USA) on DO, followed by intraperitoneal injections of 25 pg lipopolysaccharide (LPS) on D3. Clinical development of arthritis in the paws was assessed by the arthritis score: 0, normal; 1, erythema; 2, erythema and mild swelling extending from the ankle to the tarsals; 3, erythema and moderate swelling extending from the ankle to the metatarsal joints; and 4, erythema and severe swelling encompassing the ankle, foot, and digits, or ankylosis of the limb.

[0270] Histopathological evaluation

[0271] Paws from normal and CAIA mice post-intraoperative imaging were placed in 10% formalin solution for 1 day, followed by 10 days incubation in 0.5 M EDTA solution for decalcification. For microscopic histological assessment, the fixed sections were counterstained with nuclear fast red, dehydrated by ethanol, transferred into xylene, and finally mounted according to the standard protocol. To determine the tissue distribution of the NIR fluorophore, the dissected tissues were embedded in Tissue-Tek optimum cutting temperature compound (Sakura Finetek, Torrance, CA) and flash- frozen in liquid nitrogen. Frozen sections were cut at a thickness of 10 pm by a cryostat (Leica, Germany). Then, the tissue sections were stained with hematoxylin and eosin (H&E) using BioTek Cytation 5 (Winooski, VT) for pathological fluorescence imaging and observation.

[0272] The tissue samples were also embedded in paraffin. After sectioning (15 pm) each sample, the slices were observed by fluorescence microscopy, and then they were separately stained with H&E, Safranin O / Fast green stain, and anti-CD68 antibody. Heart, liver, spleen, lung, kidney, and tumor tissues were fixed in 10% neutral buffered formalin for more than 8 h. Then, the tissues were dehydrated in ethanol, embedded in paraffin, and sectioned into slices (5 pm). After rinsing with PBS, the fixed sections were counterstained with nuclear fast red, dehydrated by ethanol, transferred into xylene, and finally mounted according to the standard protocol. Then, those sections were stained with H&E for pathology observation under optical microscopy.

[0273] Statistical analysis

[0274] The fluorescence and background intensities of a region of interest over each tissue were quantified using customized imaging software and ImageJ vl ,53e (National Institutes of Health, Bethesda, MD). The signal-to-background ratio (SBR) was calculated as SBR = fluorescence / background, where the background is the fluorescence intensity of the muscle.

[0275] Region-of-interest (ROI) was segmented via intensity thresholding, where the threshold limits were set to the local full-width-half-maximum (FWHM) intensity value for the lower threshold bound and local maximum intensity value for the upper threshold bound.

[0276] Data are reported as mean ± s.e.m. with a minimum of three biological replicates. Student’s / -test statistical analysis was performed to evaluate the significance of the experimental data. The differences among groups were determined using one-way ANOVA analysis to assess the statistical differences among more than two groups. A p value of less than 0.05 was considered significant. The data was indicated with *p <0.05, **p <0.01, ***p <0.001, and ****p <0.0001.

[0277] As demonstrated in the examples above, there are several key features that distinguish CARF28 from existing diagnostic and therapeutic tools for arthritis. First, it is shown to have specific / selective cartilage targeting without causing any harm. The CARF28 was designed to systematically demonstrate high target specificity and sensitivity. This is a significant improvement over current imaging modalities, which rely on nonspecific contrast agents or anatomical imaging. In addition, the fluorophores have more than two permanent positive charges, a prerequisite for the highly negatively charged GAGs inside cartilage that provides a unique opportunity to use electrostatic interactions. However, to mitigate adverse events associated with intravenous injection of highly positive molecules, the probes have been redesigned. Second, CARF28 has NIR-II imaging capabilities, enabling deeper tissue penetration and higher resolution imaging than traditional NIR dyes. NIR-II imaging has the potential to revolutionize diagnostic imaging by enabling noninvasive, high- resolution imaging of deep tissue structures such as joints, organs, and blood vessels. Although the development of NIR-II probes has been limited by their low stability and poor biocompatibility, CARF28 offers a minimally toxic NIR-II fluorophore with high quantum yield.

[0278] Lastly, CARF28 could enable detection of changes in cartilage structure and inflammatory status at an earlier stage of RA. Rheumatoid synovium is characterized by hyperplasia of the synovial lining layer and marked infiltration by lymphocytes, macrophages, and plasma cells. The pathogenesis of RA begins years before clinical diseases are evident, and early detection of arthritis can facilitate the initiation of disease-modifying treatments, such as disease-modifying antirheumatic drugs (DMARDs) or biologic therapies, which have been shown to improve outcomes and reduce joint damage. In this context, CARF28 could offer earlier diagnosis and monitoring of disease progression, allowing for more timely interventions and better patient outcomes. Furthermore, it can be used to detect subclinical joint inflammation in at-risk populations, such as patients with autoimmune diseases or a family history of arthritis.

[0279] OTHER EMBODIMENTS

[0280] It is to be understood that while the present application has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the present application, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:(I) wherein:X is CR10Rnor (NR10Rn)+;Y is selected from halogen, -S-(CR6R7)mN(R19R20R21)+, -O-(CR6R7)mN(R19R20R21)+, -N(W)-(CR6R7)mN(R19R20R21)+; wherein W is selected from H, C1-C6 alkyl, and C1-C6 haloalkyl; m is 1, 2, 3, 4, 5, or 6;Z1is selected from O, S, Se, and C(R2R3);Z2is selected from O, S, Se, and C(R16R17);R1, R10, R11, R18, R19, R20, and R21are independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4- 10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R1, R10, R11, R18, R19, R20, and R21are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxy alkyl;R2, R3, R6, R7, R8, R9, R12, R13, R16, and R17are independently selected from H, halogen, ORa, SRa, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R2, R3, R6, R7, R8, R9, R12, R13, R16, and R17are optionallysubstituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl;R4, R5, R14, and R15are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5- 10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R4, R5, R14, and R15are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, or C1-C6 hydroxyalkyl;Ring A and Ring B are independently selected from monocyclic or bicyclic 6- 12 membered aryl and monocyclic or bicyclic 5-10 membered heteroaryl, wherein the monocyclic or bicyclic 6-12 membered aryl and the monocyclic or bicyclic 5-10 membered heteroaryl are optionally substituted with 1-3 substituents independently selected from C1-C6 alkyl, C1-C6 thioalkyl, C1-C6 alkoxy, halogen, -S(O2)C1-C6 alkyl, and -(NH)-4-10 membered heterocyclyl optionally substituted with 1-3 substituents independently selected from halogen and C1-C6 alkyl;Rais selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, and 4-10 membered heterocyclyl;L is an anion; and q is 1, 2, or 3.m is 1, 2, 3, 4, 5, or 6;R1, R10, R11, R18, R19, R20, and R21are independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R1, R10, R11, R18, R19, R20, and R21are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxy alkyl;R2, R3, R16, and R17are independently selected from H, halogen, ORa, SRa, Cl- C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4- 10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R2, R3, R16, and R17are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl;Ring A and Ring B are independently selected from monocyclic or bicyclic 6- 12 membered aryl and monocyclic or bicyclic 5-10 membered heteroaryl, wherein the monocyclic or bicyclic 6-12 membered aryl and the monocyclic or bicyclic 5-10 membered heteroaryl are optionally substituted with 1-3 substituents independently selected from C1-C6 alkyl, C1-C6 thioalkyl, C1-C6 alkoxy, halogen, -S(O2)C1-C6 alkyl, and -(NH)-4-10 membered heterocyclyl optionally substituted with 1-3 substituents independently selected from halogen and C1-C6 alkyl;Rais selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, and 4-10 membered heterocyclyl;L is a pharmaceutically acceptable anion; and q is 1, 2, or 3.

3. A compound of Formula (III)wherein:R1, R10, R11, R18, R19, R20, and R21are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R1, R10, R11, R18, R19, R20, and R21are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl;R2, R3, R6, R7, R16, and R17are independently selected from H, halogen, ORa, SRa, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6- 12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R2, R3, R6, R7, R16, and R17are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxy alkyl; m is 1, 2, 3, 4, 5, or 6; ring A and ring B are independently selected from monocyclic or bicyclic 6-12 membered aryl and monocyclic or bicyclic 5-10 membered heteroaryl, wherein the monocyclic or bicyclic 6-12 membered aryl and the monocyclic or bicyclic 5-10 membered heteroaryl are optionally substituted with 1-3 substituents independently selected from C1-C6 alkyl, C1-C6 thioalkyl, C1-C6 alkoxy, halogen, -S(O2)C1-C6 alkyl, and -(NH)-4-10 membered heterocyclyl optionally substituted with 1-3 substituents independently selected from halogen and C1-C6 alkyl;Rais selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, and 4-10 membered heterocyclyl;L is an anion; and q is 3.

4. A compound of Formula (IV)wherein:R1, R10, R11, R18, R19, R20, and R21are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R1, R10, R11, R18, R19, R20, and R21are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl;R2, R3, R6, R7, R16, and R17are independently selected from H, halogen, ORa, SRa, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6- 12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R2, R3, R6, R7, R16, and R17are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxy alkyl;R22and R23are independently selected from C1-C6 alkyl, C1-C6 thioalkyl, Cl- C6 alkoxy, halogen, OH, and -S(O2)C1-C6 alkyl; or two R22or two R23with the atomsto which they are attached together form a 6-12 membered aryl or 5-10 membered heteroaryl; and m is 1, 2, 3, 4, 5, or 6; n is 1, 2, 3, or 4; p is 1, 2, 3, or 4; andRais selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, and 4-10 membered heterocyclyl;L is an anion; and q is 3.

5. A compound of Formula (V)R1, R10, R11, R18, R19, R20, and R21are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R1, R10, R11, R18, R19, R20, and R21are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl;R2, R3, R6, R7, R8, R9, R12, R13, R16, and R17are independently selected from H, halogen, ORa, SRa, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10membered heteroaryl of R2, R3, R6, R7, R8, R9, R12, R13, R16, and R17are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkylR22and R23are independently selected from C1-C6 alkyl, C1-C6 thioalkyl, Cl- C6 alkoxy, halogen, OH, and -S(O2)C1-C6 alkyl; or two R22or two R23with the atoms to which they are attached together form a 6-12 membered aryl or 5-10 membered heteroaryl; and m is 1, 2, 3, 4, 5, or 6; n is 1, 2, 3, or 4; p is 1, 2, 3, or 4;Rais selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, and 4-10 membered heterocyclyl;L is an anion; and q is 1, 2, or 3.

6. A compound of Formula (VI)wherein:R1, R10, R11, R18, R19, R20, and R21are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6-12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R1, R10, R11, R18, R19, R20, and R21are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl;R2, R3, R6, R7, R16, and R17are independently selected from H, halogen, ORa, SRa, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, 6- 12 membered aryl, and 5-10 membered heteroaryl; wherein the C3-C6 cycloalkyl, the 4-10 membered heterocyclyl, the 6-12 membered aryl, and the 5-10 membered heteroaryl of R2, R3, R6, R7, R16, and R17are optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxy alkyl;R22and R23are independently selected from C1-C6 alkyl, C1-C6 thioalkyl, Cl- C6 alkoxy, halogen, OH, and -S(O2)C1-C6 alkyl; or two R22or two R23with the atoms to which they are attached together form a 6-12 membered aryl or 5-10 membered heteroaryl; and m is 1, 2, 3, 4, 5, or 6; n is 1, 2, 3, or 4; p is 1, 2, 3, or 4; andRais selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, and 4-10 membered heterocyclyl;L is an anion; and q is 3.

7. The compound of any one of claims 1-5, wherein R1and R18are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl; wherein the C3-C6 cycloalkyl is optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

8. The compound of any one of claims 1-6, wherein R1and R18are independently selected from C1-C6 alkyl and C1-C6 haloalkyl.

9. The compound of any one of claims 1-7, wherein R1and R18are C1-C6 alkyl.

10. The compound of any one of claims 1-6, wherein R1and R18are methyl.

11. The compound of any one of claims 1-9, wherein R2, R3, R16, and R17are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl; whereinthe C3-C6 cycloalkyl is optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

12. The compound of any one of claims 1-10, wherein R2, R3, R16, and R17are independently selected from C1-C6 alkyl and C1-C6 haloalkyl.

13. The compound of any one of claims 1-11, wherein R2, R3, R16, and R17are Cl- C6 alkyl.

14. The compound of any one of claims 1-12, wherein R2, R3, R16, and R17are methyl.

15. The compound of any one of claims 1-13, wherein R10and R11are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl; wherein the C3-C6 cycloalkyl is optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

16. The compound of any one of claims 1-14, wherein R10and R11are independently selected from C1-C6 alkyl and C1-C6 haloalkyl.

17. The compound of any one of claims 1-15, wherein R10and R11are C1-C6 alkyl.

18. The compound of any one of claims 1-16, wherein R10and R11are methyl.

19. The compound of any one of claims 1-17, wherein R19, R20, and R21are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl; wherein the C3-C6 cycloalkyl is optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxyalkyl.

20. The compound of any one of claims 1-18, wherein R19, R20, and R21are independently selected from C1-C6 alkyl and C1-C6 haloalkyl.

21. The compound of any one of claims 1-19, wherein R19, R20, and R21are C1-C6 alkyl.

22. The compound of any one of claims 1-20, wherein R19, R20, and R21are methyl.

23. The compound of any one of claims 1 and 5-21, wherein R8, R9, R12, R13are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl; wherein the C3-C6 cycloalkyl is optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxy alkyl.

24. The compound of any one of claims 1, and 5-22, wherein R8, R9, R12, R13are independently selected from H, C1-C6 alkyl, and C1-C6 haloalkyl.

25. The compound of any one of claims 1, and 5-23, wherein R8, R9, R12, R13are H.

26. The compound of any one of claims 1-24, wherein m is 1, 2, or 3.

27. The compound of any one of claims 1-25, wherein m is 2.

28. The compound of any one of claims 1-26, wherein each R6and each R7are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl; wherein the C3-C6 cycloalkyl is optionally substituted with 1-3 substituents independently selected from halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 hydroxy alkyl.

29. The compound of any one of claims 1-27, wherein each R6and each R7are independently selected from H, C1-C6 alkyl, and C1-C6 haloalkyl.

30. The compound of any one of claims 1-28, wherein each R6and each R7are H.

31. The compound of any one of claims 4-27, wherein n is 1, 2, or 3.

32. The compound of any one of claims 4-28, wherein n is 1 or 2.

33. The compound of claim 30 or 31, wherein R22is selected from C1-C6 alkyl, Cl- C6 thioalkyl, C1-C6 alkoxy, halogen, OH, and -S(O2)C1-C6 alkyl.

34. The compound of any one of claims 30-32, wherein R22is C1-C6 alkyl.

35. The compound of any one of claims 30-32, wherein R22is C1-C6 alkoxy.

36. The compound of any one of claims 30-32, wherein R22is halogen.

37. The compound of any one of claims 30-32, wherein two R22with the atoms to which they are attached together form a 6-12 membered aryl or 5-10 membered heteroaryl.

38. The compound of any one of claims 30-32, wherein two R22with the atoms to which they are attached together form a 6-12 membered aryl.

39. The compound of any one of claims 30-32, wherein two R22with the atoms to which they are attached together form a phenyl.

40. The compound of any one of claims 4-37, wherein p is 1, 2, or 3.

41. The compound of any one of claims 4-38, wherein p is 1 or 2.

42. The compound of claim 39 or 40, wherein R23is selected from C1-C6 alkyl, Cl-C6 thioalkyl, C1-C6 alkoxy, halogen, OH, and -S(O2)C1-C6 alkyl.

43. The compound of any one of claims 39-41, wherein R23is C1-C6 alkyl.

44. The compound of any one of claims 39-41, wherein R23is C1-C6 alkoxy.

45. The compound of any one of claims 39-41, wherein R23is halogen.

46. The compound of any one of claims 39-41, wherein two R23with the atoms to which they are attached together form a 6-12 membered aryl or 5-10 membered heteroaryl.

47. The compound of any one of claims 39-41, wherein two R23with the atoms to which they are attached together form a 6-12 membered aryl.

48. The compound of any one of claims 39-41, wherein two R23with the atoms to which they are attached together form a phenyl.

49. A compound selected from:

50. A pharmaceutical composition comprising a compound of any one of claims 1- 48 and a pharmaceutically acceptable carrier.

51. A method of imaging a tissue comprising:(a) contacting the tissue with a compound according to any one of claims 1-48;(b) irradiating the tissue at a wavelength absorbed by the compound;(c) and detecting a signal from the compound, thereby imaging the tissue.

52. The method of claim 50, wherein the tissue is cartilage.

53. The method of claim 51, wherein the cartilage is hyaline, elastic, or fibrocartilage.

54. The method of any one of claims 50-52, wherein the compound is administered to an organism comprising the tissue.

55. The method of claim 53, wherein the organism is human.

56. The method of any one of claims 50-54, wherein the compound has a peak absorbance at about 600 nm to about 850 nm.

57. The method of any one of claims 50-55, wherein the compound has a peak absorbance at about 660 nm to about 850 nm.

58. The method of any one of claims 50-56, wherein the compound has a peak absorbance at about 760 nm to about 800 nm.

59. The method of any one of claims 50-57, wherein the tissue is imaged ex vivo.

60. The method of any one of claims 50-57, wherein the tissue is imaged in vivo.

61. A method of diagnosing rheumatoid arthritis, comprising(a) administering to a subject identified as having or diagnosed with rheumatoid arthritis an imaging effective amount of a compound according to any one of claims 1- 48;(b) irradiating a tissue at a wavelength absorbed by the compound; and(c) determining the presence or absence of a signal from the compound, wherein the presence of a signal indicates a diagnosis of rheumatoid arthritis.

62. The method of claim 60, wherein the subj ect has j oint pain.

63. The method of claim 60, wherein the subject was further diagnosed with rheumatoid arthritis from a serological test, a physical examination, and / or a joint radiographs.

64. The method of any one of claims 60-62, wherein the tissue is cartilage.

65. The method of any one of claims 60-63, wherein the compound has a peak absorbance at about 600 nm to about 850 nm.

66. The method of any one of claims 60-64, wherein the compound has a peak absorbance at about 660 nm to about 850 nm.

67. The method of any one of claims 60-65, wherein the compound has a peak absorbance at about 760 nm to about 800 nm.

68. A method of treating rheumatoid arthritis, comprising:(a) administering to a subject identified as having or diagnosed with rheumatoid arthritis an imaging effective amount of a compound according to any one of claims 1- 48;(b) irradiating a tissue at a wavelength absorbed by the compound;(c) determining the presence or absence of a signal from the compound; and(d) if a signal is detected, administering to the subject a therapeutically effective amount of one more anti-inflammatory agents, one or more immunomodulatory agents, or a combination thereof.

69. The method of claim 67, wherein the subject has joint pain.

70. The method of claim 67, wherein the subject was diagnosed with rheumatoid arthritis from a serological test, a physical examination, and / or a joint radiographs.

71. The method of any one of claims 67-69, wherein the tissue is cartilage.

72. The method of any one of claims 67-70, wherein the compound has a peak absorbance at about 600 nm to about 850 nm.

73. The method of any one of claims 67-71, wherein the compound has a peak absorbance at about 660 nm to about 850 nm.

74. The method of any one of claims 67-72, wherein the compound has a peak absorbance at about 760 nm to about 800 nm.

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