Imaging agents for detecting CD206+ macrophages

Abstract

The present disclosure provides methods for detecting CD206 in vitro and in vivo. + For the detection of macrophages, the compound of formula (I): JPEG2025538444000053.jpg1640 wherein the variables are described herein, or a pharmaceutically acceptable salt thereof.

Description

[Technical Field]

[0001] Related application data This disclosure claims priority to U.S. Application No. 63 / 383,967, filed November 16, 2022, which is hereby incorporated by reference in its entirety.

[0002] Government support statement This invention was made with government support under grants K25 HL150305, R01 NS103998, RF1 AG075055 awarded by the National Institutes of Health, RG-1902-33633 from the National Multiple Sclerosis Society, and the National Academy of Medicine. The government has certain rights in this invention.

[0003] The present disclosure provides methods for detecting CD206 in vitro and in vivo. + Imaging agents for the detection of macrophages. [Background technology]

[0004] Macrophages are key drivers of innate immune responses, and various subtypes exist in vitro and in vivo. Two major subsets of activated macrophages are M1 and M2. Different populations of activated macrophages play distinct roles and can adapt to different phenotypes in response to novel microenvironmental stimuli in various pathophysiological processes, such as wound healing, cancer, and myocardial infarction, among others. Reprogramming to alter phenotype has become a promising strategy, especially in tumor-associated macrophage (TAM)-targeted immunotherapy. These newly emerging therapies targeting activated macrophages have been used to analyze CD206 to predict treatment outcomes. + This drives the need for non-invasive methods to map and track macrophages. Summary of the Invention

[0005] Activated macrophages play important and unique roles in response to different pathophysiological conditions. However, CD206 + Specific, non-invasive MRI agents for detecting macrophages are not available. We developed fluorescent (MR2-cy5) and MRI agents (Mann2-DTPA-Gd and MannGdFish) that target the mannose receptor (CD206), a surface marker expressed by anti-inflammatory / reparative macrophages, and demonstrated that these agents detect CD206 both in vitro and in vivo. + We demonstrated that Mann2-DTPA-Gd and MannGdFish are specific for macrophages. MRI of Mann2-DTPA-Gd and MannGdFish can track the progression of reparative inflammation in skin wound healing and detect tumor-associated macrophages (TAMs) in gliomas. Importantly, MannGdFish, with its high sensitivity, specificity, stability, and favorable biodistribution and pharmacokinetics, is a promising tool for the detection of CD206 in repair / regeneration and tumors in patients. + It is a promising transformation candidate for noninvasively monitoring macrophages.

[0006] Some embodiments are of formula (I)

[0007] [ka] (In the formula, P is a fluorescent imaging probe or a magnetic resonance imaging probe, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; Each X is independently X-1 or X-2:

[0008] [ka] and L is a C2-C20 alkylene; n is 1, 2, 3, 4, 5, or 6) or a pharmaceutically acceptable salt thereof.

[0009] Some embodiments provide a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0010] Some embodiments provide a method for detecting M2-like macrophages in vitro, comprising: (i) contacting a compound of formula (I) with a cell or tissue; (ii) waiting a period of time to allow the compound to accumulate in cells or tissues; and (iii) obtaining an image of a cell or tissue; The present invention provides a method comprising:

[0011] Some embodiments provide a method of monitoring M2-like macrophages in an organ or tissue of a subject, comprising: (i) administering to a subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein; (ii) waiting a sufficient time to allow the compound to accumulate in the organ or tissue to be imaged; and (iii) Obtaining images of the organ or tissue of interest Including, wherein observing the image resulting from the compound of formula (I) shows M2-like macrophages in the organ or tissue.

[0012] Some embodiments provide a method of monitoring treatment of a disease or condition in a subject, comprising: (i) administering to a subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein; (ii) waiting a sufficient time to allow the compound to accumulate in the organ or tissue of the subject affected by the disease or condition; (iii) obtaining a first image of the organ or tissue of interest; (iv) administering to the subject an effective amount of a therapeutic agent to treat the disease or condition; (v) administering to a subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein; (vi) waiting a sufficient time to allow the compound to accumulate in the organ or tissue of the subject affected by the disease or condition; (vii) obtaining a second image of the organ or tissue of interest; and (viii) comparing the first and second images. Including, The method provides a method wherein observing a difference between the first and second images attributable to the compound of formula (I) indicates progress in treating the disease or condition.

[0013] Some embodiments provide a method of diagnosing a disease or condition in a subject, comprising: (i) administering to a subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein; (ii) waiting a sufficient time to allow the compound to accumulate in the organ or tissue of the subject affected by the disease or condition; and (iii) Obtaining images of the organ or tissue of interest Including, The present invention provides a method wherein observing an image resulting from a compound of formula (I) is indicative of a disease or condition.

[0014] Some embodiments provide a method of intraoperative imaging in a subject, comprising: (i) administering to a subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein; (ii) waiting a sufficient time to allow the compound to accumulate in the organ or tissue of the subject affected by the disease or condition; and (iii) Obtaining images of the organ or tissue of interest Including, Observing the image resulting from the compound of formula (I) defines the boundary.

[0015] The details of one or more embodiments of the presently disclosed subject matter are set forth in the accompanying drawings and description. Other features, aspects, and advantages of the subject matter will become apparent from the description, drawings, and claims. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 shows the chemical structures of MR1-cy5, MR2-cy5, Mann2-DTPA-Gd, and MannGdFish. [Figure 2] Figure 2A shows that fluorescent imaging of MR1-cy5 incubated with M1 and M2 macrophages at 37°C showed low specificity for M2 macrophages, and Figure 2B shows fluorescent imaging of MR2-cy5 of M1 and M2 macrophages showing a much higher signal from M2 macrophages than M1 macrophages and higher specificity for M2 macrophages. [Figure 3] 1 is a graph showing that when incubated with Mann2-DTPA-Gd, the amount of gadolinium on M2 macrophages (as detected by inductively coupled plasma mass spectrometry (ICP-MS)) was much higher than on M1 macrophages and HEK293 cells (as a nonspecific control). (M2 vs. M1 with probe, one-way ANOVA. n=4 for M1 and M2 macrophages and n=3 for HEK293 cells. ****, p<0.0001). [Figure 4] MR images of Mann2-DTPA-Gd at 15 and 60 minutes in wild-type and mannose receptor knockout mice, respectively, in a mouse model of subcutaneous wound healing. [Figure 5] This graph shows that the MR contrast-to-noise ratio (CNR) of mannose receptor knockout mice was significantly reduced compared to wild-type mice at 15 minutes post-injection on day 7 in a mouse model of subcutaneous wound healing (n=3, Mann2-DTPA-Gd administered at 0.3 mmol / kg mice). [Figure 6]Figure 1 shows MR imaging of Mann2-DTPA-Gd in a mouse model of subcutaneous wound healing. Longitudinal MR imaging of Mann2-DTPA-Gd 60 minutes after injection showed that the CNR was much higher on day 7 compared to days 1 and 4. [Figure 7] 1 is a graph showing that the percentage of YFP+ cells (arginase-1 positive cells, a marker of M2-like cells) on day 7 was significantly higher than on days 1 and 4 in a mouse model of subcutaneous wound healing (n=3, one-way ANOVA. ****, p<0.0001). [Figure 8] MR imaging of a glioma with Mann2-DTPA-Gd. MR imaging of a glioma at 3 weeks before contrast and 30 and 60 minutes after injection of Mann2-DTPA-Gd. [Figure 9] Figure 1 is a graph showing the cytotoxicity of MannGdFish in an MTT assay. Cytotoxicity was not observed at concentrations as high as 5 mM (n=3). [Figure 10] 10 is a graph showing the biodistribution of MannGdFish at 3 hours and 7 days after MannGdFish injection (24 hours after wound induction) detected by ICP-MS in a mouse model of skin wound healing. [Figure 11] FIG. 1 is a graph showing that inductively coupled plasma mass spectrometry (ICP-MS) of wounded skin compared to normal skin showed accumulation of MannGdFish in wounded skin at 3 hours and 7 days (two-way ANOVA, ns: not significant, p=0.10; **, p=0.037). [Figure 12] Representative MR images of MannGdFish and DOTA-Gd before contrast and at 60 minutes. The signal of MannGdFish was much higher than that of DOTA-Gd in wound healing at 60 minutes. [Figure 13]Figure 1 shows that MR imaging of MannGdFish showed a slightly higher CNR and a similar dynamic profile than Mann2-DTPA-Gd in wild-type wound-healing mice at day 7, and both showed a slow decline after 45 minutes, while the CNR of DOTA-Gd imaging decreased significantly 15 minutes after injection. [Figure 14-1] High-resolution mass spectrum of MR1-cy5. [Figure 14-2] (As mentioned above.) [Figure 15-1] High-resolution mass spectrum of MR2-cy5. [Figure 15-2] (As mentioned above.) [Figure 16-1] High-resolution mass spectrum of Mann2-DTPA-Gd. [Figure 16-2] (As mentioned above.) [Figure 17] FIG. 17A is a graph showing the relaxivity (r1) of Mann2-DTPA-Gd, and FIG. 17B is a graph showing validation of the distinction between M1 and M2 macrophages. [Figure 18] Figure 18A is a graph showing that the contrast-to-noise ratio (CNR) of wild-type mice 45 minutes after injection was approximately doubled on day 7 compared to day 4 after wound injury, and Figure 18B is a graph showing that the percentage of CD86+ cells (a marker of M1-like cells) on day 4 was significantly higher than on days 1 and 7 (n=3, one-way ANOVA, **, p=0.005 and p=0.001 for days 1 and 7, respectively). [Figure 19-1] High-resolution mass spectrum of MannGdFish. [Figure 19-2] (As mentioned above.) [Figure 20] Figure 20A is a graph showing the relaxivity (r1) of MannGdFish, and Figure 20B is a graph showing that the blood half-life of MannGdFish, as detected by ICP-MS using a biexponential model, was 0.3 minutes for the fast phase and 6.1 minutes for the slow phase. [Figure 21]Figure 21A shows the conditions for the preparation of PET probes via chelation of

[68] Ga with MannGaFish. Figure 21B shows the radio-HPLC of

[68] Ga-MannGaFish, demonstrating clean labeling with high purity (>95%). Figure 21C shows the ex vivo biodistribution of

[68] Ga-MannGaFish by gamma counting. Figure 21D shows a representative image of

[68] Ga-MannGaFish PET imaging, demonstrating low uptake in the lung and heart. [Figure 22] FIG. 1 shows additional multi-mannose imaging agents of formula (I). DETAILED DESCRIPTION OF THE INVENTION

[0017] Activated macrophages play a key role in the innate immune response, and their diversity drives both damage and repair in many diseases. The development of noninvasive imaging techniques to distinguish different subtypes of activated macrophages is essential to better understand the function of these cells in disease and to develop novel therapies targeting macrophage subtypes. Here, we developed fluorescent and MRI agents to detect anti-inflammatory / repair macrophages by targeting the mannose receptor (CD206) and validated the specificity and efficacy of the agents in vitro in cellular assays and in vivo in animal models of skin wound healing and glioma.

[0018] Featured herein are fluorescent (MR2-cy5) and MRI agents (Mann2-DTPA-Gd and MannGdFish) that target the mannose receptor (CD206), a surface marker expressed by anti-inflammatory / reparative macrophages. In some embodiments, these agents inhibit CD206 both in vitro and in vivo. +In some embodiments, MRI of Mann2-DTPA-Gd and MannGdFish can track the progression of reparative inflammation in skin wound healing and detect tumor-associated macrophages (TAMs) in gliomas. In some embodiments, the MRI agent is MannGdFish, which exhibits high sensitivity, specificity, stability, and favorable biodistribution and pharmacokinetics. Therefore, MannGdFish can be used to detect CD206 in tumor repair / regeneration and tumor repair in patients. + It is a promising transformation candidate for noninvasively monitoring macrophages.

[0019] definition As used herein, "DOTA-Gd" has the structure:

[0020] [ka] It refers to a compound having the formula:

[0021] As used herein, the term "alkylene" refers to a straight or branched divalent hydrocarbon chain containing a fully saturated (no double or triple bonds) hydrocarbon group. The alkylene group may have 1 to 20 carbon atoms. (Whenever it appears herein, a numerical range such as "1 to 20" refers to each integer within the given range; for example, "1 to 12 carbon atoms" means that the alkylene group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 12 carbon atoms.) The alkylene group of the compound may be referred to as "C1-C3 alkylene" or a similar designation. By way of example only, "C1-C3 alkylene" indicates that there are 1 to 3 carbon atoms in the alkylene chain, i.e., the alkylene chain is selected from methylene, ethylene, propylene, and iso-propylene. An alkylene group optionally contains 1 to 6 oxo (C=O) groups, is optionally interrupted by 1 to 6 heteroatoms independently selected from N and O, where valence and chemical stability permit, and is optionally interrupted by up to one phenyl group. In some embodiments, the alkylene groups described herein are interrupted by one or two amide groups (-NH(C=O)- or -(C=O)NH-). In some embodiments, when an alkylene group is interrupted by a phenyl group, the phenyl functions as a branch point in the alkylene, as described herein.

[0022] As used herein, the terms "individual," "patient," or "subject," used interchangeably, refer to any animal, including a mammal, preferably a mouse, rat, other rodent, rabbit, dog, cat, pig, cow, sheep, horse, or primate, and most preferably a human.

[0023] As used herein, the phrase "effective amount" or "imaging effective amount" refers to an amount of active compound that enables imaging of a tissue, system, animal, individual, or human under standard imaging conditions as desired by a researcher, veterinarian, physician, or other clinician.

[0024] The terms "chelating ligand," "chelating scaffold," and "chelating moiety" are used interchangeably and refer to either a polydentate ligand that is capable of coordinating a metal ion directly or after removal of protecting groups, or a reagent, with or without appropriate protecting groups, used in the synthesis of an MR contrast agent and containing substantially all atoms that ultimately coordinate the metal ion of the final metal complex. The term "chelate" or "metal chelate" refers to the actual metal-ligand complex. It is understood that the polydentate ligand is capable of ultimately coordinating a medically useful or diagnostic metal ion.

[0025] The coordination of metal ions by water and other ligands is often considered in terms of coordination spheres (see, for example, D.T. Richens, The Chemistry of Aqua Ions, John Wiley and Sons, New York, 1997, Chapter 1). The first or primary coordination sphere represents all ligands directly attached to the metal ion and is defined by the ligands. There is a second coordination sphere, in which water molecules and counterions are bound to groups in the first coordination sphere through hydrogen bonding and electrostatic interactions. The third and subsequent coordination spheres are typically referred to as "bulk water" or "bulk solvent." The distinction between these spheres is spatial and temporal. The first coordination sphere is typically well-defined, and water or other ligands spend more time in the first coordination sphere than in other coordination spheres. The second sphere is less well-defined, but water therein has a lifetime longer than the typical diffusion time of water. Beyond the second sphere, water diffuses freely.

[0026] The term "fluorescent imaging probe," as used herein, refers to a moiety capable of fluorescent emission between 400 nm and 1000 nm, ideally bright, exhibiting a high signal-to-noise ratio, and reduced photobleaching.

[0027] The term "magnetic resonance imaging probe," as used herein, refers to a moiety that can create high intensity contrast (brightening of tissue of interest) through T1 (spin-lattice) relaxation time or T2 spin-spin relaxation, alter the relaxation rate of protons in water, and create a signal change in MRI.

[0028] The term "specific binding affinity," as used herein, refers to the ability of an imaging agent to be taken up by, retained by, or bind to a particular or target biological moiety to a greater extent than other non-target biological moieties. An imaging agent with this property is said to be "targeted" to the "target" moiety. An imaging agent lacking this property is said to be a "non-specific" or "non-targeted" agent. The binding affinity of a binding group for a target is determined by the equilibrium dissociation constant, "K d " is expressed in terms of

[0029] The term "relaxivity," as used herein, refers to the increase in either MR quantity 1 / T1 or 1 / T2 per millimolar (mM) concentration of a paramagnetic ion or imaging agent, which may be different if the imaging agent contains multiple paramagnetic ions, where T1 is the longitudinal or spin-lattice relaxation time of water protons or other imaging or spectroscopic nuclei containing protons found in molecules other than water, and T2 is the transverse or spin-spin relaxation time. Relaxivity is expressed in mM -1 s -1 It is expressed in units of .

[0030] The mannose receptor (CD206) is a well-established surface biomarker for anti-inflammatory / repair macrophages. CD206 is a 175 kDa transmembrane protein that recognizes pathogens by binding to glycoproteins terminated with mannose, fucose, or N-acetyl-glucosamine and mediates their endocytosis. Elevated expression of CD206 has been found on anti-inflammatory / repair macrophages in various pathophysiological conditions, making it an emerging therapeutic target. CD206-targeting imaging agents for detecting anti-inflammatory / repair macrophages have also been reported using either D-mannose-based agents or CD206-specific antibody / nanobody-based SPECT / PET imaging. However, in addition to radiation exposure concerns, these agents have specificity issues. D-mannose has a relatively low binding affinity for CD206 (IC) compared to its oligomeric and clustered analogs. 50 (5.5 mM vs. 18-23 μM or higher). Furthermore, studies to evaluate the efficacy of these clustered mannosides as MR imaging agents have not been conducted, in part due to challenges in synthesizing these carbohydrate-containing agents. Such studies are important for improving imaging efficacy and specificity, particularly in identifying anti-inflammatory / reparative macrophages using MRI with excellent spatial resolution and soft-tissue contrast. Single D-mannose-based imaging agents have been reported, but as demonstrated herein (Figure 2A), a single mannose moiety cannot sufficiently distinguish M1 from M2 macrophages. This specificity issue is overcome herein by adding more mannose moieties to the imaging agent, given that two mannose units have over 100-fold the binding affinity of D-mannose. Disclosed herein is the development of multiple mannose-based agents targeting CD206 and validation of their specificity and efficacy in detecting anti-inflammatory / reparative macrophages in valid animal models of wound healing and glioma.

[0031] Development of new drugs. MRI and fluorescence imaging agents containing one or two mannose moieties, MR1-cy5, MR2-cy5, and Mann2-DTPA-Gd (Figure 1), were designed to be specific for CD206 and anti-inflammatory / reparative macrophages. Detailed synthesis and characterization of these agents are described in the Examples. The relaxivity (r) of Mann2-DTPA-Gd was 3.6 mM s in PBS (0.47 T, 40°C) (Figure 17A), and verification of the differentiation of M1 and M2 macrophages is shown in Figure 17B.

[0032] In vitro validation of specificity for the mannose receptor (CD206). The specificity of MR1-cy5, MR2-cy5, and Mann2-DTPA-Gd for CD206 was verified in a cellular assay using M1 and M2 macrophages differentiated from Raw264.7 cells. The differentiation was confirmed by flow cytometry, with a higher percentage of CD206+ cells in M2 macrophages than in M1 macrophages (see Figure 17B). However, when incubated with MR1-cy5 containing only a single mannose moiety, fluorescence imaging showed similar signals from both M1 and M2 macrophages (Figure 2A), demonstrating the low specificity of MR1-cy5 for M2 macrophages. In contrast, fluorescence imaging from MR2-cy5, which has two mannose moieties in a cluster structure (Figure 1), showed a much higher signal in M2 macrophages compared to M1 and M2 macrophages incubated with MR1-cy5 (Figure 2B). The MRI agent Mann2-DTPA-Gd was incubated with M1 / M2 macrophages and human embryonic kidney cells (HEK293, a nonspecific binding control). As expected, the amount of gadolinium in M2 macrophages was significantly higher than in M1 macrophages and HEK293 cells (Figure 3). Taken together, these results confirmed the higher specificity of these agents containing two mannose units for M2 cells.

[0033] MR imaging of Mann2-DTPA-Gd in a mouse model of cutaneous wound healing Normal wound healing is a highly regulated process in which activated macrophages function uniquely at different stages. During the early inflammatory phase after injury (days 1–4), neutrophil- and monocyte-derived macrophages are pro-inflammatory, producing inflammatory cytokines, proteases, and reactive oxygen species (ROS). Between days 5–10 after injury, inflammation begins to resolve and the repair phase begins. Anti-inflammatory and repair macrophages become the most abundant cell type, peaking around day 7, and persist throughout the repair phase. Because wound healing has clearly defined inflammatory and repair phases in which pro- and anti-inflammatory macrophages play distinct roles, we investigated the role of CD206 in a mouse model of wound healing. + The specificity and ability of Mann2-DTPA-Gd to track macrophages was evaluated on days 1, 4, and 7. In wild-type mice on day 7 after wound induction, there was increased contrast enhancement at 60 minutes compared with 15 minutes after Mann2-DTPA-Gd administration (Figure 4), and the contrast-to-noise ratio (CNR) increased over time until 45 minutes after injection, after which it slowly decreased (Figure 5). In contrast, in mannose receptor-knockout (MR-KO) mice, signal intensity initially peaked 15 minutes after injection and then decreased over time (Figures 4 and 5), confirming the in vivo binding and specificity of Mann2-DTPA-Gd to the mannose receptor.

[0034] CD206 in wound healing + A longitudinal study was performed to track changes in macrophages. Using CNR at 60 min, we identified a subset of CD206 + It was found that CD206 cells were present on day 1 after wound induction, decreased on day 4, and then increased again on day 7 (Figure 6). There was approximately a 2-3 fold increase in CNR 45-75 minutes after injection on day 7 compared to day 4 (see Figure 18A), demonstrating the presence of CD206 cells at different stages of healing. +The development of macrophages was demonstrated. To validate the MRI data, flow cytometry studies were performed to distinguish between pro- and anti-inflammatory macrophages in the same model using transgenic YFP-labeled arginase I (YARG) mice at days 1, 4, and 7. Arginase I (Arg1) is another marker for M2-like macrophages. As expected, the flow cytometry data (Figure 7) mirrored the results from Mann2-DTPA-Gd MRI (Figure 6). To ensure that Mann2-DTPA-Gd imaging was not detecting pro-inflammatory macrophages, pro-inflammatory macrophages were also determined by flow cytometry using CD86 as a marker, which showed an opposite trend compared to those detected by Mann2-DTPA-Gd (peaking at day 4) (Figure 18B). Collectively, these data suggest that MR imaging of Mann2-DTPA-Gd supports the role of CD206 in wound healing. + We demonstrated that noninvasive mapping and tracking of dynamic changes in macrophages is possible.

[0035] MR imaging of experimental gliomas with Mann2-DTPA-Gd CD206 in wound healing + In contrast to the reparative role of macrophages, tumor-associated macrophages (TAMs) in cancer are the most abundant immune cells and are tumorigenic and immunosuppressive in the tumor microenvironment. TAMs facilitate tumor growth, immune evasion, and metastasis and are associated with poor prognosis. Therefore, despite these cells playing distinct roles in injury, we next determined whether Mann2-DTPA-Gd could detect these cells in experimental gliomas. Substantial contrast enhancement was observed after injection of the agent, increasing over time (Figure 8), indicating the presence of CD206 in the tumor microenvironment. +The CD206 MRI revealed the presence of TAMs. Interestingly, although there was a mild increase in signal with some more intense focal points within the tumor, the areas with the highest contrast enhancement formed a ring around the tumor, demonstrating that TAMs primarily surround the tumor. In even slower images (60 minutes), there were additional areas of increased enhancement seen within the surrounding brain parenchyma. The data presented herein demonstrate that CD206 MRI using Mann2-DTPA-Gd can effectively detect CD206 in a mouse model of glioma. + We show that TAMs can be detected and, as expected, these TAMs were distributed primarily around the tumor periphery.

[0036] Similar to wound healing, these pro-inflammatory and anti-inflammatory / repair phases also occur in myocardial infarction and acute kidney injury. Where M1-like macrophages / microglia exert inflammatory and damaging effects, such as in diabetic wound healing and many neurological diseases, repolarization from an inflammatory phenotype to an anti-inflammatory / repair phenotype favors repair and recovery. Conversely, CD206 + Conversion of TAMs into tumoricidal macrophages has become an important strategy for treating cancer and fibrosis. As in cancer, CD206 + Macrophages are strongly associated with renal fibrosis in both human and experimental diseases. In renal fibrosis, CD206 + Strategies that inhibit macrophage profibrotic processes while maintaining or enhancing tissue repair are promising therapeutic strategies. Furthermore, recent studies have demonstrated that CD206 + We have shown that macrophages are associated with diabetes and lymphoid clusters in adipose tissue in humans, and correlate with adverse patient outcomes in human laryngeal squamous cell carcinoma, oral squamous cell carcinoma, and renal fibrosis and other renal diseases. + Given the specificity and efficacy of the imaging agents disclosed herein for macrophages, they are expected to be valuable clinical tools. In some embodiments, the imaging agents disclosed herein provide a means to report healing and disease progression in patients and to monitor the effectiveness of treatment.

[0037] Development of the macrocyclic MRI agent MannGdFish MR imaging of the prototype Mann2-DTPA-Gd showed that CD206 + The feasibility of using MRI to track macrophages was demonstrated. However, Mann2-DTPA-Gd contains a linear chelator with low Gd stability and is therefore undesirable for conversion. Therefore, a thermodynamically more stable macrocyclic agent was designed, resulting in MannGdFish (Figure 1). The synthesis and characterization of MannGdFish are shown in Example 4. The relaxivity (r) of MannGdFish is 5.2 mmol / L. -1 s -1 (Fig. 20A), slightly higher than Mann2-DTPA-Gd (3.6 mmol s). MannGdFish showed no cytotoxic effect in an MTT assay using RAW264.7 cells up to 5 mM, a dose several thousand times higher than the expected first-pass concentration (µM) in blood (Fig. 9).

[0038] Biodistribution and pharmacokinetics of MannGdFish. The biodistribution of MannGdFish in mice with cutaneous wounds was evaluated. ICP-MS showed very little accumulation or retention of Gd in the body overall (<0.5 nmol Gd per gram of tissue), with the liver and spleen being the main organs containing gadolinium at both 3 hours and 7 days, followed by blood, urine, and kidneys (Figure 10). As expected, the amount of gadolinium in the injured wound 3 hours after MannGdFish administration (24 hours after injury) was low and not significantly different compared to normal skin, but the gadolinium content at 7 days was higher in the wound than in normal skin (Figure 11), again suggesting a role for CD206 during wound healing. + The ability of MannGdFish to track macrophages was highlighted. Using a biphasic exponential model, the blood half-life of MannGdFish was determined to be 0.3 minutes during the fast phase and 6.1 minutes during the slow phase (Figure 20B).

[0039] In vivo imaging of MannGdFish in wound healing MR imaging of MannGdFish on day 7 of wound healing was performed and compared with Mann2-DTPA-Gd and DOTA-Gd (Figure 12 and Figures 13A and 13B). The signal of MannGdFish at 60 minutes was CD206. + Consistent with the binding and retention of MannGdFish to cells, it was much higher than DOTA-Gd (Figure 12). Unlike the slow decline in CNR for MannGdFish and Mann2-DTPA-Gd after 45 min, the CNR of DOTA-Gd peaked 15 min after injection and then declined rapidly (Figure 13A). The CNR of MannGdFish was slightly higher than that of Mann2-DTPA-Gd, consistent with the higher r1 of MannGdFish than that of Mann2-DTPA-Gd (3.6 vs. 5.2 mmol). -1 s -1 ) showed a 75-minute kinetic profile similar to that of Mann2-DTPA-Gd. These results demonstrate that MannGdFish has similar efficacy to Mann2-DTPA-Gd while exhibiting a significantly better safety profile due to its macrocyclic chelating backbone, which is consistent with CD206. + Considering its high sensitivity, specificity, stability, and favorable biodistribution and pharmacokinetics, MannGdFish is a potential translational candidate for macrophage MR imaging. It is expected to be useful in patients with repair / regeneration and CD206 in tumors. + In some embodiments, the imaging agents disclosed herein are useful for monitoring repair / regeneration in patients and CD206 in tumors. + Provides a means for monitoring macrophages.

[0040] Compounds of formula (I) Some embodiments are of formula (I):

[0041] [ka] (In the formula, P is a fluorescent imaging probe or a magnetic resonance imaging probe, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; Each X is independently X-1 or X-2:

[0042] [ka] and L is a C2-C20 alkylene; n is 1, 2, 3, 4, 5, or 6) or a pharmaceutically acceptable salt thereof.

[0043] In some embodiments, L is a C2-C20 alkylene having 1-6 oxo groups and 1-6 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having 1-3 oxo groups and 1-3 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having 3-6 oxo groups and 3-6 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having 1 oxo group and 1 nitrogen atom. In some embodiments, L is a C2-C20 alkylene having 2 oxo groups and 2 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having 3 oxo groups and 3 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having 4 oxo groups and 4 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having 5 oxo groups and 5 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having 6 oxo groups and 6 nitrogen atoms.

[0044] In some embodiments, L is a C2-C20 alkylene having a phenyl group, 1-6 oxo groups, and 1-6 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having a phenyl group, 1-3 oxo groups, and 1-3 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having a phenyl group, 3-6 oxo groups, and 3-6 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having a phenyl group, 1 oxo group, and 1 nitrogen atom. In some embodiments, L is a C2-C20 alkylene having a phenyl group, 2 oxo groups, and 2 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having a phenyl group, 3 oxo groups, and 3 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having a phenyl group, 4 oxo groups, and 4 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having a phenyl group, five oxo groups, and five nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having a phenyl group, six oxo groups, and six nitrogen atoms.

[0045] In some embodiments, L is a C2-C20 alkylene having 1-2 oxo groups and 0-2 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having one oxo group and 0-2 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having one oxo group and 0-1 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having one oxo group. In some embodiments, L is a C2-C20 alkylene having one oxo group and one nitrogen atom. In some embodiments, L is a C2-C20 alkylene having one oxo group and two nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having two oxo groups and 0-1 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having two oxo groups. In some embodiments, L is a C2-C20 alkylene having two oxo groups and one nitrogen atom. In some embodiments, L is a C2-C20 alkylene having two oxo groups and two nitrogen atoms.

[0046] In some embodiments, L is a C2-C20 alkylene having a phenyl group, 1-2 oxo groups, and 0-2 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having a phenyl group, 1 oxo group, and 0-2 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having a phenyl group, 1 oxo group, and 0-1 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having a phenyl group and 1 oxo group. In some embodiments, L is a C2-C20 alkylene having a phenyl group, 1 oxo group, and 1 nitrogen atom. In some embodiments, L is a C2-C20 alkylene having a phenyl group, 1 oxo group, and 1 nitrogen atom. In some embodiments, L is a C2-C20 alkylene having a phenyl group, 1 oxo group, and 2 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having a phenyl group, 2 oxo groups, and 0-1 nitrogen atoms. In some embodiments, L is a C2-C20 alkylene having a phenyl group and two oxo groups. In some embodiments, L is a C2-C20 alkylene having a phenyl group, two oxo groups, and one nitrogen atom. In some embodiments, L is a C2-C20 alkylene having a phenyl group, two oxo groups, and two nitrogen atoms.

[0047] In some embodiments, L is a C2-C12 alkylene having 1-2 oxo groups and 0-2 nitrogen atoms. In some embodiments, L is a C2-C12 alkylene having one oxo group and 0-2 nitrogen atoms. In some embodiments, L is a C2-C12 alkylene having one oxo group and 0-1 nitrogen atoms. In some embodiments, L is a C2-C12 alkylene having one oxo group. In some embodiments, L is a C2-C12 alkylene having one oxo group and one nitrogen atom. In some embodiments, L is a C2-C12 alkylene having one oxo group and two nitrogen atoms. In some embodiments, L is a C2-C12 alkylene having two oxo groups and 0-1 nitrogen atoms. In some embodiments, L is a C2-C12 alkylene having two oxo groups. In some embodiments, L is a C2-C12 alkylene having two oxo groups and one nitrogen atom. In some embodiments, L is a C2-C12 alkylene having two oxo groups and two nitrogen atoms.

[0048] In some embodiments, L is a C2-C10 alkylene having 1-2 oxo groups and 0-2 nitrogen atoms. In some embodiments, L is a C2-C10 alkylene having one oxo group and 0-2 nitrogen atoms. In some embodiments, L is a C2-C10 alkylene having one oxo group and 0-1 nitrogen atoms. In some embodiments, L is a C2-C10 alkylene having one oxo group. In some embodiments, L is a C2-C10 alkylene having one oxo group and one nitrogen atom. In some embodiments, L is a C2-C10 alkylene having one oxo group and two nitrogen atoms. In some embodiments, L is a C2-C10 alkylene having two oxo groups and 0-1 nitrogen atoms. In some embodiments, L is a C2-C10 alkylene having two oxo groups. In some embodiments, L is a C2-C10 alkylene having two oxo groups and one nitrogen atom. In some embodiments, L is a C2-C10 alkylene having two oxo groups and two nitrogen atoms.

[0049] In some embodiments, L is a C2-C8 alkylene having 1-2 oxo groups and 0-2 nitrogen atoms. In some embodiments, L is a C2-C8 alkylene having one oxo group and 0-2 nitrogen atoms. In some embodiments, L is a C2-C8 alkylene having one oxo group and 0-1 nitrogen atom. In some embodiments, L is a C2-C8 alkylene having one oxo group. In some embodiments, L is a C2-C8 alkylene having one oxo group and one nitrogen atom. In some embodiments, L is a C2-C8 alkylene having one oxo group and two nitrogen atoms. In some embodiments, L is a C2-C8 alkylene having two oxo groups and 0-1 nitrogen atoms. In some embodiments, L is a C2-C8 alkylene having two oxo groups. In some embodiments, L is a C2-C8 alkylene having two oxo groups and one nitrogen atom. In some embodiments, L is a C2-C8 alkylene having two oxo groups and two nitrogen atoms.

[0050] In some embodiments, L comprises one or more polyethylene glycol units.

[0051] In some embodiments, L comprises one or more amide groups (-NH(C=O)- or -(C=O)NH-).

[0052] In some embodiments, L is

[0053] [ka] (In the formula, * indicates the point of attachment to X).

[0054] In some embodiments, L is

[0055] [ka] (In the formula, * indicates the point of attachment to X).

[0056] In some embodiments, L is

[0057] [ka] (In the formula, * indicates the point of attachment to X).

[0058] In some embodiments, L is

[0059] [ka] (In the formula, * indicates the point of attachment to X).

[0060] In some embodiments, X is X-1.

[0061] In some embodiments, X is X-2.

[0062] In some embodiments, each X is X-1. In some embodiments, each X is X-2. In some embodiments, when m≧2, one or more X are X-1 and one or more are X-2. In some embodiments, each X is the same. In some embodiments, each X is different.

[0063] In some embodiments, when m≧2, L is linked to multiple independently selected X groups described herein.

[0064] In some embodiments, L is

[0065] [ka] (In the formula, * indicates the point of attachment to X-1).

[0066] In some embodiments, L is

[0067] [ka] (In the formula, * indicates the point of attachment to X-1).

[0068] In some embodiments, L is

[0069] [ka] (In the formula, * indicates the point of attachment to X-1).

[0070] In some embodiments, L is

[0071] [ka] (In the formula, * indicates the point of attachment to X-1).

[0072] In some embodiments, L is

[0073] [ka] (In the formula, * indicates the point of attachment to X-2).

[0074] In some embodiments, L is

[0075] [ka] (In the formula, * indicates the point of attachment to X-2).

[0076] In some embodiments, L is

[0077] [ka] (In the formula,* indicates the point of attachment to X-2).

[0078] In some embodiments, L is

[0079] [ka] (In the formula, * indicates the point of attachment to X-2).

[0080] In some embodiments, L is

[0081] [ka] (In the formula, * indicates the point of attachment to X-2).

[0082] In some embodiments, L is

[0083] [ka] (In the formula, * indicates the point of attachment to X-2).

[0084] In some embodiments, L is

[0085] [ka] (In the formula, * indicates the point of attachment to X-2).

[0086] In some embodiments, L is

[0087] [ka] (In the formula, * indicates the point of attachment to X-2).

[0088] In some embodiments, L is

[0089] [ka] (In the formula, * indicates the point of attachment to X-2).

[0090] In some embodiments, L is

[0091] [ka] (In the formula, * indicates the point of attachment to X-2).

[0092] In some embodiments, L is

[0093] [ka] (In the formula, * indicates the point of attachment to X-2).

[0094] In some embodiments, P is a fluorescent imaging probe.

[0095] In some embodiments, P is cy3 or cy5. In some embodiments, P is cy3. In some embodiments, P is cy5.

[0096] In some embodiments, P is

[0097] [ka] is.

[0098] In some embodiments, P is a magnetic resonance imaging probe.

[0099] In some embodiments, P is

[0100] [ka] is.

[0101] In some embodiments, P is

[0102] [ka] is.

[0103] In some embodiments, P is

[0104] [ka] is.

[0105] In some embodiments, P is

[0106] [ka] is.

[0107] In some embodiments, P is

[0108] [ka] is.

[0109] In some embodiments, P is

[0110] [ka] is.

[0111] In some embodiments, P is

[0112] [ka] is.

[0113] In some embodiments, P is

[0114] [ka] is.

[0115] In some embodiments, the radioisotope M is selected from the group consisting of radioisotopes of Ga, In, Mn, and Cu.

[0116] In some embodiments, the radioisotope M is 68 Ga, 111 In, 52 Mn, and 64 Cu.

[0117] In some embodiments, the radioisotope M is 68 It's Ga.

[0118] In some embodiments, the radioisotope M is 111 In.

[0119] In some embodiments, the radioisotope M is 52 Mn.

[0120] In some embodiments, the radioisotope M is 64 It is Cu.

[0121] In some embodiments, n is 1 or 2. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3 or 4. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5 or 6. In some embodiments, n is 5. In some embodiments, n is 6.

[0122] In some embodiments, m is 1, 2, 3, 4, 5, or 6. In some embodiments, m is 1, 2, or 3. In some embodiments, m is 4, 5, or 6. In some embodiments, m is 7, 8, 9, 10, 11, or 12. 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. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 11. In some embodiments, m is 12.

[0123] In some embodiments, the compound of formula (I) is selected from the compounds of Figure 1, Figure 22, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is

[0124] [ka] or a pharmaceutically acceptable salt of any of the foregoing.

[0125] In some embodiments, the compound of formula (I) is

[0126] [ka] or a pharmaceutically acceptable salt of any of the foregoing.

[0127] In some embodiments, the compound of formula (I) is

[0128] [ka] or a pharmaceutically acceptable salt of any of the foregoing.

[0129] In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is MR1-cy5, or a pharmaceutically acceptable salt thereof.

[0130] In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is MR2-cy5, or a pharmaceutically acceptable salt thereof.

[0131] In some embodiments, the compound of formula (I), or a pharmaceutically acceptable salt thereof, is Mann2-DTPA-Gd, or a pharmaceutically acceptable salt thereof.

[0132] In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is MannGdFish, or a pharmaceutically acceptable salt thereof.

[0133] How to use Some embodiments provide a method for detecting M2-like macrophages in vitro, comprising: (i) contacting a compound of formula (I) with a cell or tissue; (ii) waiting a period of time to allow the compound to accumulate in cells or tissues; and (iii) obtaining an image of a cell or tissue; The present invention provides a method comprising:

[0134] In some embodiments, the cells are isolated from a tissue. In some embodiments, the tissue is animal tissue. In some embodiments, the tissue is from a human. In some embodiments, the cells comprise a biopsy sample.

[0135] In some embodiments, the cells or tissues are selected cells or tissues derived from arteries, veins, lymph nodes, lungs, liver, kidneys, skin, brain, eyes, bones, intestines, gallbladder, pancreas, trachea, bladder, bowel, biliary tract, adrenal glands, uterus, ovaries, spleen, cartilage, muscle, heart, cartilage, epithelium, tendons, and ligaments. In some embodiments, the cells or tissues are cells or tissues derived from organs. In some embodiments, the cells or tissues are cells and tissues derived from connective tissue.

[0136] Some embodiments provide a method of monitoring M2-like macrophages in an organ or tissue of a subject, comprising: (i) administering to a subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein; (ii) waiting a sufficient time to allow the compound to accumulate in the organ or tissue to be imaged; and (iii) Obtaining images of the organ or tissue of interest Including, wherein observing the image resulting from the compound of formula (I) shows M2-like macrophages in the organ or tissue.

[0137] In some embodiments, the organ or tissue comprises an area affected by injury, cardiovascular disease, inflammation, neurodegenerative disease, or cancer, or a combination of any of the foregoing. In some embodiments, the organ or tissue comprises an area affected by injury, cardiovascular disease, inflammation, neurodegenerative disease, or cancer.

[0138] Some embodiments provide a method of monitoring treatment of a disease or condition in a subject, comprising: (i) administering to a subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein; (ii) waiting a sufficient time to allow the compound to accumulate in the organ or tissue of the subject affected by the disease or condition; (iii) obtaining a first image of the organ or tissue of interest; (iv) administering to the subject an effective amount of a therapeutic agent to treat the disease or condition; (v) administering to a subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein; (vi) waiting a sufficient time to allow the compound to accumulate in the organ or tissue of the subject affected by the disease or condition; (vii) obtaining a second image of the organ or tissue of interest; and (viii) comparing the first and second images. Including, The method provides a method wherein observing a difference between the first and second images attributable to the compound of formula (I) indicates progress in treating the disease or condition.

[0139] Some embodiments provide a method of diagnosing a disease or condition in a subject, comprising: (i) administering to a subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein; (ii) waiting a sufficient time to allow the compound to accumulate in the organ or tissue of the subject affected by the disease or condition; and (iii) Obtaining images of the organ or tissue of interest Including, The present invention provides a method wherein observing an image resulting from a compound of formula (I) is indicative of a disease or condition.

[0140] In some embodiments, the organ or tissue comprises an area affected by injury. In some embodiments, the organ or tissue comprises an area affected by cardiovascular disease. In some embodiments, the organ or tissue comprises an area affected by inflammation. In some embodiments, the organ or tissue comprises an area affected by neurodegenerative disease. In some embodiments, the organ or tissue comprises an area affected by cancer.

[0141] In some embodiments, the organ or tissue is selected from arteries, veins, lymph nodes, lungs, liver, kidneys, skin, brain, eyes, bones, intestines, gallbladder, pancreas, trachea, urinary bladder, bowel, biliary tract, adrenal glands, uterus, ovaries, spleen, cartilage, muscle, heart, cartilage, epithelium, tendons, and ligaments.

[0142] In some embodiments, the disease or condition is an injury, a cardiovascular disease, inflammation, a neurodegenerative disease, or cancer, or a combination of any of the foregoing. In some embodiments, the disease or condition is an injury, a cardiovascular disease, inflammation, a neurodegenerative disease, or cancer.

[0143] In some embodiments, the disease or condition is an injury. In some embodiments, the disease or condition is a cardiovascular disease. In some embodiments, the disease or condition is inflammation. In some embodiments, the disease or condition is a neurodegenerative disease. In some embodiments, the disease or condition is cancer.

[0144] Some embodiments provide a method of intraoperative imaging in a subject, comprising: (i) administering to a subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein; (ii) waiting a sufficient time to allow the compound to accumulate in the organ or tissue of the subject affected by the disease or condition; and (iii) Obtaining images of the organ or tissue of interest Including, Observing the image resulting from the compound of formula (I) defines the boundary.

[0145] In some embodiments, the disease or condition comprises a tumor or a lesion. In some embodiments, the disease or condition is a tumor or a lesion. In some embodiments, the disease or condition is a tumor. In some embodiments, the disease or condition is a lesion.

[0146] In some embodiments, the image comprises a fluorescent image. In some embodiments, the image is a fluorescent image.

[0147] In some embodiments, the image comprises a magnetic resonance image. In some embodiments, the image is a magnetic resonance image.

[0148] Some embodiments provide a method for detecting M2-like macrophages in vitro, comprising: (i) contacting a cell or tissue with a compound selected from MR1-cy5, MR2-cy5, Mann2DTPA, and MannGDFish; (ii) waiting a period of time to allow the compound to accumulate in cells or tissues; and (iii) obtaining an image of a cell or tissue; The present invention provides a method comprising:

[0149] Some embodiments provide a method of monitoring M2-like macrophages in an organ or tissue of a subject, comprising: (i) administering to a subject an effective amount of a compound selected from MR1-cy5, MR2-cy5, Mann2DTPA, and MannGDFish, or a pharmaceutically acceptable salt of any of the foregoing, or a pharmaceutical composition, as described herein; (ii) waiting a sufficient time to allow the compound to accumulate in the organ or tissue to be imaged; and (iii) Obtaining images of the organ or tissue of interest Including, The method provides that observing the image resulting from the compound shows M2-like macrophages in the organ or tissue.

[0150] Some embodiments provide a method of monitoring treatment of a disease or condition in a subject, comprising: (i) administering to a subject an effective amount of a compound selected from MR1-cy5, MR2-cy5, Mann2DTPA, and MannGDFish, or a pharmaceutically acceptable salt of any of the foregoing, or a pharmaceutical composition, as described herein; (ii) waiting a sufficient time to allow the compound to accumulate in the organ or tissue of the subject affected by the disease or condition; (iii) obtaining a first image of the organ or tissue of interest; (iv) administering to the subject an effective amount of a therapeutic agent to treat the disease or condition; (v) administering to a subject an effective amount of a compound selected from MR1-cy5, MR2-cy5, Mann2DTPA, and MannGDFish, or a pharmaceutically acceptable salt of any of the foregoing, or a pharmaceutical composition, as described herein; (vi) waiting a sufficient time to allow the compound to accumulate in the organ or tissue of the subject affected by the disease or condition; (vii) obtaining a second image of the organ or tissue of interest; and (viii) comparing the first and second images. Including, Observing a difference between the first and second images due to the compound indicates progress in treating the disease or condition.

[0151] Some embodiments provide a method of diagnosing a disease or condition in a subject, comprising: (i) administering to a subject an effective amount of a compound selected from MR1-cy5, MR2-cy5, Mann2DTPA, and MannGDFish, or a pharmaceutically acceptable salt of any of the foregoing, or a pharmaceutical composition, as described herein; (ii) waiting a sufficient time to allow the compound to accumulate in the organ or tissue of the subject affected by the disease or condition; and (iii) Obtaining images of the organ or tissue of interest Including, A method is provided wherein observing an image resulting from the compound is indicative of a disease or condition.

[0152] Some embodiments provide a method of intraoperative imaging in a subject, comprising: (i) administering to a subject an effective amount of a compound selected from MR1-cy5, MR2-cy5, Mann2DTPA, and MannGDFish, or a pharmaceutically acceptable salt of any of the foregoing, or a pharmaceutical composition, as described herein; (ii) waiting a sufficient time to allow the compound to accumulate in the organ or tissue of the subject affected by the disease or condition; and (iii) Obtaining images of the organ or tissue of interest Including, Observing the image resulting from the compound defines the boundary, providing a method.

[0153] Compositions and Routes of Administration The present application also provides a pharmaceutical composition comprising an effective amount of a compound of the present disclosure (e.g., MannGdFish or a pharmaceutically acceptable salt thereof) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0154] The pharmaceutical compositions of the present application include those suitable for any acceptable route of administration, including oral, cutaneous, intracervical, intrasinus, intratracheal, intraintestinal, epidural, intrainterstitial, intraabdominal, intra-arterial, intrabronchial, intravesical, intracerebral, intracisternal, intracoronary, intradermal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralymphatic, intramedullary, intrameningeal, intramuscular, intranasal, intraovarian, intraperitoneal, ), intraprostatic, intrapulmonary, intrasinus, intraspinal, intrasynovial, intratesticular, intrathecal, intratubular, intratumoral, intrauterine, intravascular, intravenous, nasal, nasogastric, oral, parenteral, percutaneous, peridural, rectal, respiratory (inhalation), subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transtracheal, ureteral, urethral, ​​and vaginal.

[0155] In the pharmaceutical compositions of the present application, the compound of the present disclosure (eg, MannGdFish or Mann2-DTPA-Gd) is present in an effective amount (eg, an imaging effective amount). [Example]

[0156] material and method All chemicals were obtained from Sigma Chemical Co. unless otherwise stated. D-mannosamine hydrochloride was purchased from Biosynth Carbosynth (UK), DOTA-GA anhydride from CheMatech (Dijon, France), Z-Ser-OH from Ambeed (Illinois, USA), and 1,2,3,4,6-penta-O-benzoyl-α-D-mannopyranose from BOC Science (New York, USA). 1 H-NMR and 13 C-NMR was recorded on a JEOL 11.7 T NMR system equipped with a 5 mm broadband probe. Flash chromatography was performed on a Combiflash (Telydyne ISCO CombiFlash, CA) with UV detection at 220 and 254 nm. High-resolution mass spectrometry was performed on a Thermo Scientific™ Q-Exactive Plus Ultimate 3000 HPLC flow injection system. Inductively coupled plasma mass spectrometry (ICP-MS) was performed on an Agilent 8800-QQQ system. Flow cytometry data were acquired on an LSRII flow cytometer (BD Bioscience). All animal experiments were performed in accordance with the National Institutes of Health's 'Guide for the Care and Use of Laboratory Animals' and were approved by and followed by the Institutional Animal Care and Use Committee at Massachusetts General Hospital.

[0157] Statistical Analysis. All numerical data were first analyzed for normality using the Shapiro-Wilk normality test, with significance determining the appropriate parametric or nonparametric test to use. One-way and two-way analysis of variance were used for data analysis. All statistical analyses were performed in GraphPad Prism version 9 (GraphPad Software, La Jolla, California), and data are presented as mean ± SEM. A P value of <0.05 was considered significantly different.

[0158] Chemical Examples MR1-cy5 was synthesized by coupling D-mannosamine with cy5-NHS ester (Scheme 1). The prototype MRI agent, Mann2-DTPA-Gd, was synthesized by coupling D-mannosamine with DTPA anhydride under basic conditions, followed by chelation with GdCl3 (Scheme 2). Intermediate 8, obtained from the coupling of compounds 5 and 6 followed by hydrogenation, was coupled with cy5-NHS ester and subsequently deprotected to give MR2-cy5 (Scheme 3). Synthesis of MannGdFish: Intermediate 8 was reacted with compound 10 (DOTA-GA anhydride) to give compound 11, which was subsequently deprotected with sodium methoxide and chelated with GdCl3 to give the final product, MannGdFish (Scheme 4).

[0159] [ka]

[0160] [ka]

[0161] [ka]

[0162] [ka]

[0163] [Example 1] Preparation of MR1-cy5. To a solution of D-mannosamine 1 (5 mg) in DMSO (1 mL), triethylamine (10 μL) was added and stirred for 20 minutes, followed by the addition of a solution of compound 2 Cy5-NHS (10 mg, 1 equivalent) in DMSO (1 mL). The reaction was stirred at room temperature for an additional hour. The reaction was filtered to remove solids and subjected to HPLC separation to give MR1-cy5 (3.7 mg, 35%). High-resolution MS: 840.2803 (M+Na, calculated 840.2806). The high-resolution mass spectrum is shown in Figure 14.

[0164] [Example 2] Preparation of Mann2-DTPA-Gd. To a solution of D-mannosamine 1 (472 mg, 2.2 equiv.) in DMSO (6 mL), triethylamine (700 μL, 5.0 equiv.) was added and stirred for 30 min. DTPA dianhydride 3 (318 mg, 1.0 equiv.) was then added in small portions and stirred for an additional 3 h. The reaction mixture was passed through a reverse column to give compound Mann2-DTPA3 (460 mg, 64%). LCMS found m / z: 716.2 (M+H). A solution of the above compound 4 (286 mg, 1.0 equiv.) in water was mixed with a solution of GdCl3 (148 mg, 1.0 equiv.) in sodium ascorbate buffer (pH 5.5) and stirred for 1 h. The reaction mixture was filtered, and the desired compound Mann2-DTPA-Gd (87 mg, 50%) was obtained by HPLC separation. High resolution MS: 871.1849 (M+H, calculated 871.1845). The high resolution mass spectrum is shown in FIG.

[0165] [Example 3] Preparation of MR2-cy5. Step 1: Synthesis of compound 7. Compound 5 (360 mg, 1.0 equiv.), compound 6 (2.32 g, 2.2 equiv.), obtained from the bromination of 1,2,3,4,6-penta-O-benzoyl-alpha-D-mannopyranose, and activated 4 Å molecular sieves (1.0 g) in dichloromethane (12 mL) were stirred for 10 min. Silver triflate (946 mg, 2.3 equiv.) was then added to the reaction mixture at 0 °C. The reaction was allowed to warm to room temperature and stirred for an additional 2 h to reach completion. Saturated NaHCO3 (5 mL) was added to the reaction and filtered. The filtrate was washed with brine, concentrated, dried, and subjected to flash chromatography (hexane / ethyl acetate gradient: 100% to 40%) to give the desired compound 7 (1.43 g, 65%) as a white powder. 1 H-NMR (500 MHz, CDCl3) δ: 8.10 (d, 4 x 1H, J = 8 Hz), 7.99 (d, 4 x 1H, J = 7.5 Hz), 7.93 (d, 4 x 1H, J = 8 Hz), 7.80 (d, 2 x 1H, J = 7.0 Hz), 7.76 (d, 2 x 1H, J = 7.5 Hz), 7.59-7.52 (m, 4H), 7.45-7.30 (m, 17H), 7.29-7.20 (m, 9H), 6.15 (t, 2 x 1H, J = 10 Hz), 5.90 (dd, 2 x 1H, J1 = 10 Hz, J2 = 3 Hz), 5.75 (d, 2 x 1H, J = 12 Hz), 5.48 (d, 1H), 5.22-5.14 (m, 4H), 4.76 (d, 2 x 0.5H, J = 11.5 Hz), 4.69 (m, 2 x 0.5H, J = 10.5 Hz), 4.57 (d, 2 x 0.5H, J = 12 Hz), 4.54-4.48 (m, 3H), 4.29 (b, 1H), 4.10-4.07 (m, 1H), 3.94 (d, 2 x 0.5H, J = 11.5 Hz), 3.79-3.76 (m, 1H). 13C-NMR (125 MHz, CDCl3) δ: 166.11, 166.08, 165.44, 165.39, 165.23, 155.96, 136.15, 133.36, 133.32, 133.25, 133.07, 133.01, 132.96, 129.83,129.75, 129.71, 129.68, 129.20, 129.16, 128.89, 128.85, 128.82, 128.52, 128.48, 128.41, 128.40, 128.33, 128.29, 128.21, 98.26, 98.08, 70.16, 70.07, 69.93, 69.32, 69.25, 67.59, 67.14, 66.98, 66.59, 62.70, 49.99. LCMS observed m / z: 1382.4 (M+H). The high-resolution mass spectrum is shown in Figure 15.

[0166] Step 2. Synthesis of compound 8. To a solution of compound 7 (1.1 g, 1.0 equivalent) in ethyl acetate and ethanol (v / v: 1 / 1, 5 mL), Pd / C (palladium on carbon, 110 mg) was added and vigorously stirred overnight using a hydrogen balloon. The reaction mixture was filtered and concentrated to give compound 8 without further purification. 1H-NMR (500 MHz, CDCl3) δ 8.10 (d, 2 x 1H, J = 3.5 Hz), 8.09 (d, 2 x 1H, J = 3.5 Hz), 7.95 (d, 2 x 1H, J = 7.5 Hz), 7.91-7.89 (m, 6H), 7.73 (d, 2 x 1H, J = 7 Hz), 7.69 (d, 2 x 1H, J = 7.5 Hz), 7.53-7.46 (m, 4H), 7.40-7.35 (m, 6H), 7.34-7.29 (m, 4H), 7.27-7.22 (m, 4H), 7.19-7.16 (m, 4H), 7.14-7.10 (m, 2H), 6.21 (t, 2 x 1H), 5.96 (dd, 2 x 1H, J1 = 10 Hz, J2 = 3 Hz), 5.89 (m, 2H), 5.29 (d, 2 x 1H, J = 19.5 Hz), 4.83-4.80 (m, 2H), 4.64 (d, 2 x 1H, J = 10 Hz), 4.55-4.50 (m, 2H), 4.40 (d, 1H, J = 7.5 Hz), 4.34 (dd, 1H, J1 = 10 Hz, J2 = 4 Hz), 4.17-5.10 (m, 3H), 3.33 (b, 2H). 13 C-NMR (125 MHz, CDCl3) δ 166.11, 165.99, 165.92, 165.50, 165.30, 165.23, 133.29, 133.16, 133.08, 132.95, 129.85, 129.77, 129.73, 129.66, 129.00, 128.88, 128.85, 128.73, 128.68, 128.41, 128.33, 128.24, 128.16, 98.37, 98.31, 70.76, 70.56, 69.95, 69.73, 69.46, 66.02, 65.78, 65.48, 62.45, 62.36, 50.97. LCMS measurement value m / z: 1248.2 (M+H).

[0167] ステップ3,4. Synthesis of compound MR2-cy. To a solution of compound 8 (20 mg, 1.2 equiv.) in DMSO (0.6 mL) was added triethylamine (10 μL), followed by a solution of cy5-SE (10 mg, 1.0 equiv.) in DMSO (0.6 mL). The reaction was stirred at room temperature for 2 h and monitored by LC-MS to give the major product 9. Water (10 mL) was added to the reaction, and the precipitate was filtered, washed, and carried on to the next step without further purification. To a suspension of the above precipitate in methanol (10 mL) was added a solution of sodium methoxide (25 wt.% in methanol, 1.1 equiv. relative to compound 8). The reaction was stirred at room temperature for 4 h. A minimal amount of water was added to the reaction to dissolve the suspension, and the solvent was removed under vacuum. The residue was subjected to HPLC to give the desired compound MR2-cy5 in 31% yield over two steps. High-resolution MS: 1054.3878 (M+H, calculated 1054.3883).

[0168] [Example 4] Preparation of MannGdFish. Step 1. Preparation of compound 11. To a solution of compound 8 (374 mg, 1.0 equiv.) in DMSO (3 mL) was added N,N-diisopropylethylamine (130 μL, 2.5 equiv.), followed by compound 10 (138 mg, 1.0 equiv.). The reaction was stirred at 70 °C overnight. The reaction was passed through a reverse-phase column (water / acetonitrile gradient: 95 / 5 to 0 / 100) to give compound 11 (363 mg) as a white solid in 71% yield. 1H-NMR (500 MHz, DMSO) δ: 8.76 (b, 1H), 8.05 (d, 4 x 1H, J = 6Hz), 7.90 (d, 4 x 1H, J = 6.5 Hz), 7.85 (d, 4 x 1H, J = 7 Hz), 7.70 (b, 4 x 1H), 7.64-7.44 (m, 16H), 7.37-7.23 (m, 8H), 6.02 (t, 2 x 1H, J = 10 Hz), 5.85-5.70 (m, 4H), 5.36-5.32 (m, 2H), 4.73-4.57 (m, 6H), 4.41 (b, 1H), 4.03-4.01 (m, 2H), 3.90-3.77 (m, 3H), 3.53-3.36 (m, 12H), 3.04-2.82 (m, 10H), 2.69-2.63 (m, 2H), 2.39-2.35 (m, 1H), 1.98 (b, 1H), 1.81 (m, 1H). 13 C-NMR (125 MHz, DMSO) δ 172.80, 172.28, 170.52, 170.44, 165.28, 164.96, 164.70, 164.60, 133.94, 133.63, 133.55, 129.55, 129.29, 129.18, 128.93, 128.86, 128.63, 97.45, 97.36, 97.27, 97.20, 70.47, 69.91, 69.85, 68.44, 68.40, 67.04, 65.88, 62.62, 62.08, 55.37, 55.28, 54.37, 51.05, 50.94, 50.59, 50.40, 49.71, 49.63, 47.92, 47.80, 46.95, 32.58, 32.46. LCMS measurement value m / z: 1708.3 (M+H).

[0169] ステップ2,3. Modulation of MannGdFish. To a solution of compound 11 (256 mg, 1.0 equiv.) in methanol was added sodium methoxide (25 wt.% in methanol, 1.2 equiv.) and stirred at room temperature for 3 h. The reaction was adjusted to pH 7 with 1 M HCl, concentrated to remove methanol, and the liquid phase was washed with ethyl acetate and lyophilized to give the deprotected compound without further purification. LCMS observed m / z: 874.2 (M+H). A solution of the above compound was added to a solution of GdCl3 (56 mg, 1.05 equiv.) in sodium acetate buffer (pH 5.5, 4 mL). The reaction was stirred at room temperature for 1 h. The reaction was passed through a reverse-phase column to give the desired compound, MannGdFish (68 mg, 45% over two steps). High-resolution MS: 1029.2790 (M+H, calculated 1029.2791). The high-resolution mass spectrum of MannGdFish is shown in Figure 19.

[0170] An alternative procedure for the chelation step is to formulate the deprotected compound in 3 M sodium acetate buffer (pH 4.5), then add 68GaCl3 elution solution, heat the mixture to 90 °C for 10 min, and then cool to room temperature.

[0171] Biological Examples [Example 5] Fluorescence imaging. M1- and M2-differentiated macrophages were incubated with a 1 / 1000 dilution of MR2-cy5 stock solution (10 mM in DMSO) for 1 hour at 37°C and counterstained with DAPI (4',6-diamidino-2-phenylindole, Invitrogen). Cells were washed with PBS, and fluorescence images were acquired with a digital microscope (Nikon Eclipse TE2000-U).

[0172] [Example 6] ICP-MS. M1- and M2-differentiated macrophages were incubated with 1 mM Mann2-DTPA-Gd for 1 h at 4°C. After washing with PBS (x3), the cells were collected, digested overnight with nitric acid (70%, 200 μL), and subjected to ICP-MS to detect the amount of Gd.

[0173] [Example 7] Flow cytometry. M1 / M2 macrophages differentiated from RAW264.7 cells and M1 / M2-like macrophages obtained from wound tissue were stained, and data were acquired with an LSRII flow cytometer (BD Bioscience) and analyzed with BD FlowJo software (10.4).

[0174] [Example 8] MR imaging. Eight- to nine-week-old C57BL / 6J female mice were used for wound healing and glioma MRI (Jackson Laboratory, ME). Wound healing and glioma generation are described in Examples 14 and 15, respectively. Mice bearing subcutaneous wounds were imaged longitudinally on days 1, 4, and 7 or on day 7. Mice bearing gliomas were imaged at week 3. All mice were monitored before administration and 0, 15, 30, 45, and 60 minutes after intravenous administration of 0.3 mmol / kg Mann2-DTPA-Gd or MannGdFish via the tail vein with chemical fat suppression using a Hermitian pulse shape with an 8.253 ms pulse and a 701.19 Hz bandwidth 3.5 ppm below the peak of water, and continuous T1 rapid acquisition with relaxation enhancement (RARE) sequence (TR: 935.77 ms, TE: 13.59 ms, average: 12, rare factor: 4, 256 × 256 × 48 matrix size, 0.156 × 0.156 × 1 mm) with respiratory gating on a 4.7 T small animal MR scanner (Bruker, Cambridge, MA) with a 3 cm orthogonal volume coil (Rapid MR International, Germany) at 0, 15, 30, 45, and 60 minutes after administration. 3 Imaging was performed using a 300-megapixel (voxel size). Regions of interest (ROIs) were drawn, and contrast-to-noise ratios (CNRs, pre-contrast CNRs subtracted) were calculated by a radiologist with over 20 years of experience who was blinded to the identity of the imaging agent used and the mouse strain (n = 3 per group).

[0175] [Example 9] Cytotoxicity of MannGdFish. The cytotoxicity of MannGdFish was assessed by MTT assay as described by Wang et al. (J Med Chem 64, 5874-5885 (2021)) using RAW264.7 cells (passage 8 from the cell core of the Center for Systems Biology, Massachusetts General Hospital, Boston) (n=3).

[0176] [Example 10] Biodistribution, retention, and blood half-life of MannGdFish. MannGdFish was intravenously administered to 6-10 week-old C57BL / 6J mice 24 hours after wound injury. Three hours and seven days after administration, the mice were sacrificed, and major organs, including wound skin, and control skin were collected (n = 3 for each time point). Blood samples were collected (n = 6) before and at different time points after administration of MannGdFish and centrifuged to obtain plasma. The samples were weighed, treated with nitric acid, and subjected to inductively coupled plasma mass spectrometry (ICP-MS) to determine the amount of gadolinium, as described in Example 6.

[0177] [Example 11] In vitro relaxivity of Mann2-DTPA-Gd / MannGdFish. The relaxation times (T1) of Mann2-DTPA-Gd / MannGdFish at concentrations of 0.1, 0.2, 0.3, 0.5, 0.6, 0.75, and 1 mM in PBS were measured at 40°C and 0.47 T (20 MHz) using a Bruker Minispect (Bruker Analytics, MA). The slope of the linear function of 1 / T1 (seconds) for the corresponding concentrations was defined as the relaxivity (n = 3 for each concentration).

[0178] [Example 12] In vitro specificity. M1 and M2 Polarized Differentiation. Raw264.7 cells (passage 8 from the cell core at the Center for Systems Biology, Massachusetts General Hospital, Boston) were cultured in 10 mL Petri dishes in high-glucose Dulbecco's Modified Eagle's Medium (DMEM, Thermo Fisher Scientific, NY, USA) containing 10% fetal bovine serum (FBS, Sigma-Aldrich, MO, USA) and 0.5% streptomycin / penicillin (Cellgro) until they reached 80% confluence. Differentiation medium containing IFN-gamma (100 ng / mL) for M1 polarization and IL-4 (100 ng / mL) for M2 polarization was then added to the culture dish. After 24 h, M1- and M2-polarized macrophages were ready for the following experiments.

[0179] [Example 13] Flow cytometry. M1- and M2-differentiated macrophages were collected, centrifuged, and resuspended in PBS. For surface and intracellular staining, anti-CD16 / CD32, anti-CD11B APC / CY7, and anti-CD206 antibodies were obtained from Biolegend (San Diego, CA). Cells were spun, counted, resuspended in FACS buffer, and first incubated with anti-CD16 / CD32 to block Fc binding sites for 20 minutes, then washed three times with wash buffer. Cells were then incubated with antibodies against surface markers for 30 minutes at 4°C in the dark. For intracellular staining, cells were fixed and permeabilized using 1X Fix / Perm solution (BD Bioscience), washed in 1X permeabilization buffer (BD Bioscience), and stained with anti-CD206-conjugated Brilliant Violet 711 for 30 minutes at 4°C in the dark. Cells were then washed and resuspended in FCS buffer. M2-differentiated cells were identified as CD11B+, CD206+ cells. Data were acquired on an LSRII flow cytometer (BD Bioscience) and analyzed with BD FlowJo software (10.4).

[0180] [Example 14] Subcutaneous wound healing. Wound healing procedure. Seven- to 10-week-old C57BL / 6J female mice (The Jackson Laboratories, ME) and mannose receptor-deficient mice were used in this study. A circular contour on the back of the mouse was created with a 4 mm biopsy punch (Kai Medical, Japan) under isoflurane anesthesia. A full-thickness excision wound extending to the subcutaneous tissue was then generated. A silicone splint was placed over the wound, secured with 6-0 nylon sutures, and covered with a transparent occlusive dressing (OpSite). Xylazine / norepinephrine (100 μL) was injected intraperitoneally twice daily for 2 days, and the mice were monitored daily.

[0181] To isolate pro- and anti-inflammatory macrophages from wound tissue, 8- to 12-week-old B6.129S4-Arg1tm1.1Lky / J mice (Jackson laboratory) were used. Wound models were generated as described above. Wound tissue was excised on days 1, 4, and 7. Briefly, wound tissue was cut into small pieces and incubated in HBSS (Millipore Sigma) containing 0.7 mg / mL collagenase D at 37°C for 1 h. Skin tissue was then passed through a 70 μm (BD Biosciences, San Jose, CA) strainer, and single cells were isolated from the 44 / 67 Percoll (GE Healthcare, Boston, MA) gradient interface. For staining, anti-CD16 / CD32, anti-CD11B APC / CY7, and anti-CD86 APC were obtained from Biolegend (San Diego, CA). Cells were spun, counted, resuspended in FACS buffer, and first incubated with anti-CD16 / CD32 to block Fc binding sites for 20 minutes, then washed three times with wash buffer. Cells were then incubated with antibodies against those mentioned above for 30 minutes at 4°C in the dark. Cells were then washed and resuspended in FCS buffer. Anti-inflammatory macrophages were identified as CD11B+, Arg1YFP+, CD86- cells, and pro-inflammatory macrophages were identified as CD11B+, CD86+, Arg1YFP- cells. Data were acquired and analyzed as described.

[0182] [Example 15] Mouse models of glioma. CT-2A-luc tumor cell culture followed a previously published protocol (N. Jalali Motlagh et al., Cancers (Basel) 13 (2021)). Cells were incubated at 37°C with humidified air containing 5% CO2. Monolayer CT-2A-luc cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. To generate neurospheres, CT-2A monolayer cells were enzymatically dissociated with Actase (Stem Cell Technology, San Diego) and plated at a cell concentration of 1 × 10 cells / mL in serum-free medium consisting of advanced DMEM / F12 medium (Life Technologies, Carlsbad, CA) with L-glutamine (2 mM; Cellgro, Manassas, VA), 1% N2 supplement (Life Technologies), 1% penicillin-streptomycin, recombinant EGF (20 ng / mL; R&D Systems, Minneapolis, MN), and recombinant FGF2 (20 ng / mL; Peprotech, East Windsor, NJ) at 25 cm cells. 2 After 10–11 days, neurosphere CT-2A-luc (NS / CT-2A-luc) cells were collected, enzymatically dissociated with actinase, and prepared for intracranial injection.

[0183] Eight- to nine-week-old C57BL / 6J female mice were used in this study (Jackson laboratory, ME). Dissociated NS / CT-2A-luc cells (7-8 × 104) were stereotactically implanted into the brain (2.5 mm lateral and 1 mm posterior to bregma and 3 mm deep) to generate orthotopic intracranial tumors. Mice were then monitored daily for signs of discomfort or neurological symptoms.

[0184] Other embodiments While the present disclosure contains details of many specific embodiments, these should not be construed as limitations on the scope of the subject matter or on the scope of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments. Certain features described in this disclosure in the context of a separate embodiment can also be implemented in combination in a single embodiment. Conversely, various features described in the context of a single embodiment can also be implemented in multiple embodiments individually or in any suitable subcombination. Furthermore, although the features described hereinabove may be described as working in a particular combination and even initially claimed as such, one or more features from a claimed combination can, in some cases, be deleted from the combination, and the claimed combination may be directed to a subcombination or a variation of the subcombination.

[0185] Specific embodiments of the subject matter have been described. Other embodiments, modifications, and permutations of the described embodiments, as will be apparent to those skilled in the art, are within the scope of the following claims. Although operations are shown in the figures and claims in a particular order, this should not be understood as requiring such operations to be performed in the particular order or sequence shown, nor should it be understood that all described operations must be performed to achieve a desired result (some operations may be considered optional).

[0186] Accordingly, the example embodiments set forth above do not define or limit the present disclosure, and other changes, substitutions, and alterations are also possible without departing from the spirit and scope of the present disclosure.

Claims

1. Formula (I): 【Chemistry 1】 (In the formula, P is a fluorescent imaging probe or a magnetic resonance imaging probe; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; Each X is independently X-1 or X-2: 【Chemistry 2】 and L is a C2-C20 alkylene; n is 1, 2, 3, 4, 5, or 6. or a pharmaceutically acceptable salt thereof.

2. 2. The compound of claim 1, wherein m is 1, 2, 3, 4, 5, or 6.

3. 2. The compound of claim 1, wherein m is 1, 2, or 3.

4. The compound according to any one of claims 1 to 3, wherein L is C2 to C16 alkylene.

5. 5. The compound of any one of claims 1 to 4, wherein L is C2 to C12 alkylene.

6. L, 【Transformation 3】 (In the formula, ＊ indicates the point of attachment to X.

7. L, 【Chemistry 4】 (In the formula, ＊ indicates the point of attachment to X.

8. L, 【Transformation 5】 (In the formula, ＊ indicates the point of attachment to X.

9. L, 【Transformation 6】 (In the formula, ＊ indicates the point of attachment to X.

10. 10. The compound of any one of claims 1 to 9, wherein each X is X-1.

11. 10. The compound of any one of claims 1 to 9, wherein each X is X-2.

12. 12. The compound of claim 1, wherein P is a fluorescent imaging probe.

13. P, 【Transformation 7】 13. The compound of any one of claims 1 to 12, wherein

14. 12. The compound of any one of claims 1 to 11, wherein P is a magnetic resonance imaging probe.

15. P, 【Transformation 8】 15. The compound of any one of claims 1 to 11 and 14, wherein:

16. P, 【Chemistry 9】 15. The compound of any one of claims 1 to 11 and 14, wherein:

17. 17. The compound of any one of claims 1 to 16, wherein n is 1.

18. 17. The compound of any one of claims 1 to 16, wherein n is 2. 【Request Item 19】 【Chemistry 10】 (In the formula, 【Chemistry 11】 2. The compound of claim 1, wherein the compound is selected from the group consisting of:

20. 20. A pharmaceutical composition comprising a compound of any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

21. 1. A method for detecting M2-like macrophages in vitro, comprising: (i) contacting a compound of any one of claims 1 to 19 with a cell or tissue; (ii) waiting a period of time to allow the compound to accumulate in the cells or tissues; and (iii) obtaining an image of the cell or tissue. The method comprising:

22. 1. A method for monitoring M2-like macrophages in an organ or tissue of a subject, comprising: (i) administering to the subject an effective amount of a compound of any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 20; (ii) waiting a sufficient time to allow the compound to accumulate in the organ or tissue to be imaged; and (iii) obtaining an image of the organ or tissue of the subject; Including, The method, wherein observing the image resulting from the compound of formula (I) shows M2-like macrophages in an organ or tissue.

23. 1. A method for monitoring treatment of a disease or condition in a subject, comprising: (i) administering to the subject an effective amount of a compound of any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 20; (ii) waiting a sufficient time to allow the compound to accumulate in the organs or tissues of the subject affected by the disease or condition; (iii) obtaining a first image of the organ or tissue of the subject; (iv) administering to the subject an effective amount of a therapeutic agent to treat the disease or condition; (v) administering to the subject an effective amount of a compound of any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 20; (vi) waiting a sufficient time to allow the compound to accumulate in the organ or tissue of the subject affected by the disease or condition; (vii) acquiring a second image of the organ or tissue of the subject; and (viii) comparing the first and second images. Including, The method, wherein observing a difference between the first and second images attributable to the compound of formula (I) indicates progress in treating the disease or condition.

24. 1. A method of diagnosing a disease or condition in a subject, comprising: (i) administering to the subject an effective amount of a compound of any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 20; (ii) waiting a sufficient time to allow the compound to accumulate in the organ or tissue of the subject affected by the disease or condition; and (iii) obtaining an image of the organ or tissue of the subject; Including, The method, wherein observing the image resulting from the compound of formula (I) is indicative of the disease or condition.

25. 1. A method of intraoperative imaging in a subject, comprising: (i) administering to the subject an effective amount of a compound of any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 20; (ii) waiting a sufficient time to allow the compound to accumulate in the organ or tissue of the subject affected by the disease or condition; and (iii) obtaining an image of the organ or tissue of the subject; Including, The method wherein observing the image resulting from the compound of formula (I) defines a boundary.

26. 26. The method of any one of claims 21 to 25, wherein the image comprises a fluorescence image.

27. 26. The method of any one of claims 21 to 25, wherein the image comprises a magnetic resonance image.

Images