A bifunctional fluorescent molecule for optical imaging with receptor targeted biochemical constructs and methods for producing

Abstract

Disclosed are compositions, methods, and systems for imaging disease states using near-infrared fluorescent dyes conjugated to receptor-targeted molecules. In some aspects, a method for preparing an imaging agent includes covalently attaching a zwitterionic near-infrared fluorophore to a molecule containing a primary amino group, wherein the fluorophore comprises a sulfo-phenoxy substituent at the central methine carbon and sulfonic acid substituents at the 5-position of the indole nucleus. In some embodiments, the sulfo-phenoxy substituent imparts enhanced chemical stability to the dye, while the sulfonic acid substituents enable precise tuning of absorbance and emission maxima to desired near-infrared wavelengths. These features collectively facilitate the development of imaging agents with improved photostability, spectral properties, and conjugation efficiency for use in biomedical research and clinical diagnostics.

Description

PCT ApplicationAttorney Docket No. 009062.8553.WO00A BIFUNCTIONAL FLUORESCENT MOLECULE FOR OPTICAL IMAGING WITH RECEPTOR TARGETED BIOCHEMICAL CONSTRUCTS AND METHODS FOR PRODUCING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefits of U.S. Provisional Application No.63 / 694,729, filed on September 1 , 2024. The entire contents of the aforementioned patent application are incorporated by reference as part of the disclosure of this document.TECHNICAL FIELD

[0002] This patent document relates to fluorescent dyes, and in particular to fluorophores that are conjugatable to receptor-targeted molecules for use in the imaging and detection of disease states.BACKGROUND

[0003] Fluorescent dyes, or fluorophores, are chemical compounds that emit light upon excitation and are widely utilized in biomedical imaging, diagnostics, and research. These dyes can be conjugated to biomolecules, enabling visualization of cellular and molecular processes in vitro and in vivo. Near infra-red (NIR) fluorophores are particularly useful for imaging applications due to their ability to provide high-contrast images and deeper tissue penetration.SUMMARY

[0004] Bifunctional fluorescent molecules enable optical imaging of biological targets by covalently linking to receptor-targeted biochemical constructs. These advanced probes are essential for visualizing disease-related biomarkers with high specificity in vivo. However, many existing fluorescent dyes are limited by poor photostability, suboptimal water solubility, and insufficient functional groups for efficient conjugation, which can compromise imaging accuracy and versatility. Non-specific background signals and restricted spectral properties further hinder their effectiveness, especially in multiplexed or deep-tissue imaging applications. There is a clear need for fluorescent molecules that combine enhanced stability, tailored spectral characteristics, and reliable conjugation chemistry to improve the precision and utility of receptor-targeted imaging agents in both clinical diagnostics and biomedical research.PCT ApplicationAttorney Docket No. 009062.8553.WO00

[0005] The technology disclosed in this patent document relates to molecules, systems, and methods for optical imaging of disease states by conjugating fluorescent dyes to receptor- targeted biochemical constructs, wherein the dyes possess unique stability and spectral properties.

[0006] Tn some aspects, the present disclosure provides a drug conjugate comprising a targeting moiety conjugated to a fluorescent dye. The targeting moiety comprises tilmanocept (DTPA-mannosyl dextran), and the fluorescent dye comprises 2-((E)-2-((E)-3-(2-((E)-3,3- dimethyl-5-sulfonato-l-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethylidene)-5- (ethoxycarbonyl)-2-(4-sulfonatophenoxy)cyclohex-l-en-l-yl)vinyl)-3,3-dimethyl-l-(3 (trimethylammonio)propyl)-3H-indol-l-ium-5-sulfonate. This drug conjugate, referred to as lumanocept, is designed for targeted imaging and detection of disease states, including cancer, by leveraging the receptor-specific properties of tilmanocept and the unique spectral characteristics of the fluorescent dye.

[0007] The above and other aspects and implementations of the disclosed technology are described in more detail in the drawings, the detailed description, and the claims.BRIEF DESCRIPTION OF DRAWINGS

[0008] FIG. 1 shows the chemical structure of the fluorescent dye 2-((E)-2-((E)-3-(2-((E)-3.3-dimethyl-5-sulfonato-l-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethylidene)-5- (ethoxycarbonyl)-2-(4-sulfonatophenoxy)cyclohex-l-en-l-yl)vinyl)-3,3-dimethyl-l-(3- (trimethylammonio)propyl)-377-indol- 1 -ium-5-sulfonate.

[0009] FIG. 2 shows a schematic representation of the preparation of the fluorescent dye 2-((E)-2-((E)-3-(2-((.E)-3,3-dimethyl-5-sulfonato-l-(3-(trimethylammonio)propyl)indolin-2- ylidene)ethylidene)-5-(ethoxycarbonyl)-2-(4-sulfonatophenoxy)cyclohex-l-en- l-yl)vinyl)-3.3-dimethyl-l-(3-(trimethylammonio)propyl)-3H-indol-l-ium-5-sulfonate.

[0010] FIG. 3 shows the chemical structure of tilmanocept.

[0011] FIG. 4 shows the chemical structure of lumanocept.

[0012] FIG. 5 shows a schematic representation of the preparation of lumanocept using the fluorescent dye 2-((E)-2-((E)-3-(2-((E)-3,3-dimethyl-5-sulfonato-l-(3- (trimethylammonio)propyl)indolin-2-ylidene)ethylidene)-5-(ethoxycarbonyl)-2-(4- sulfonatophenoxyjcyclohex- 1 -en- 1 -yl)vinyl)-3 ,3-dimethyl- 1 -(3-(trimethylammonio)propyl)- 3H-indol-l-ium-5-sulfonate.PCT ApplicationAttorney Docket No. 009062.8553.WO00

[0013] FIG. 6 shows a schematic representation of the preparation of Ammonium 2,3,3- Trimethyl-3H-indole-5-sulfonate (Compound 2).

[0014] FIG. 7 shows a schematic representation of the preparation of Potassium 2,3,3- Trimethyl-3H-indole-5-sulfonate (Compound 3).

[0015] FIG. 8 shows a schematic representation of the preparation of 2,3,3-Trimethyl-l- [3-(trimethylammonio)propyl]-3H-indolium-5-sulfonic acid dibromide (Compound 4).

[0016] FIG. 9 shows a schematic representation of the preparation of A-((E)-((£')-2- chloro-5-(ethoxycarbonyl)-3-((phenylamino)methylene)cyclohex- 1 -en- 1 - yl)methylene)benzenaminium (Compound 6).

[0017] FIG. 10 shows a schematic representation of the preparation of 2-((E)-2-((E)-2- chloro-3-(2-((E)-3,3-dimethyl-5-sulfonato-l-(3-(trimethylammonio)propyl)indolin-2- ylidene)ethylidene)-5-(ethoxycarbonyl)cyclohex-l-en-l-yl)vinyl)-3,3-dimethyl-l-(3- (trimethylammonio)propyl)-377-indol-l-ium-5-sulfonate (Compound 7).

[0018] FIG. 11 shows a schematic representation of the preparation of the fluorescent dye 2-((£)-2-((£’)-3-(2-((£)-3,3-dimethyl-5-sulfonato-l-(3-(trimethylammonio)propyl)indolin-2- ylidene)ethylidene)-5-(ethoxycarbonyl)-2-(4-sulfonatophenoxy)cyclohex-l-en-l-yl)vinyl)- 3 ,3 -dimethyl- 1 (3 -(tri methylammonio (propyl )-37 / -i nd l - 1 -ium-5 -sulfonate (Compound 8) .

[0019] FIG. 12 shows an NMR spectrum obtained for Compound 3.

[0020] FIG. 13 shows an NMR spectrum obtained for Compound 2.

[0021] FIG. 14A shows an NMR spectrum obtained for Compound 4.

[0022] FIG. 14B shows an absorbance spectrum obtained for Compound 4.

[0023] FIG. 15 A shows an NMR spectrum obtained for Compound 4.

[0024] FIG. 15B shows an absorbance spectrum obtained for Compound 4.

[0025] FIG. 16A shows an NMR spectrum obtained for Compound 6.

[0026] FIG. 16B shows an absorbance spectrum obtained for Compound 6.

[0027] FIG. 17A shows an NMR spectrum obtained for Compound 7.

[0028] FIG. 17B shows an absorbance spectrum obtained for Compound 7.

[0029] FIG. 18A shows an NMR spectrum obtained for Compound 7.

[0030] FIG. 18B shows an absorbance spectrum obtained for Compound 7.PCT ApplicationAttorney Docket No. 009062.8553.WOOO

[0031] FIG. 18C shows an HPLC chromatogram obtained for Compound 7.

[0032] FIG. 19A shows an HPLC chromatogram obtained for Compound 8.

[0033] FIG. 19B shows an NMR spectrum obtained for Compound 8.

[0034] FIG. 19C shows an absorbance spectrum obtained for Compound 8.

[0035] FIG. 19D shows an NMR spectrum obtained for Compound 8.

[0036] FIG. 20 shows an absorbance spectrum obtained for purified Compound 8.

[0037] FIG. 21 shows an absorbance spectrum obtained for purified lumanocept.

[0038] FIG. 22 shows a fluorescence emission spectrum obtained for lumanocept.

[0039] FIG. 23 shows a ' l l NMR spectrum obtained for Compound 8.

[0040] FIG. 24 shows an HPLC chromatogram obtained for lumanocept.

[0041] FIG. 25 shows an Area % Report derived from the HPLC chromatogram of lumanocept.DETAILED DESCRIPTIONFluorescent Dye (Infra-Red Fluorophore)

[0042] Provided here is a fluorescent dye (or “fluorophore”) that can be conjugated to receptor-targeted molecules for use as an imaging agent for various disease states, including cancer. The fluorescent dye is a near infra-red fluorophore that can be covalently attached to molecules with primary amino groups, zwitterionic in nature, and unlike other commercial dyes, incorporates a sulfo-phenoxy substituent at the central methine carbon, which has been demonstrated to have unique stability properties. Further, the dye includes the sulfonic acid substituents in the 5-position on the indole nucleus, which are important to shift the absorbance and emission maxima to a desired wavelength range.

[0043] In some embodiments, the fluorophore is characterized by a symmetric chemical structure, which facilitates its synthesis with fewer steps and minimizes the generation of side products and impurities. The fluorophore is designed to react with primary amine groups, allowing for conjugation to a wide variety of biomolecules. For example, the fluorophore may be coupled to antibodies for immunofluorescence applications, peptide drugs for targeted delivery, receptor ligands for cell-specific imaging, organ- or tumor- specific peptides for disease localization, microbe- specific peptides for infection detection, or albumin forPCT ApplicationAttorney Docket No. 009062.8553.WO00 monitoring vascular barrier leakiness. The broad applicability of the fluorophore conjugates supports their use in diverse diagnostic, therapeutic, and research contexts.

[0044] In one embodiment, the fluorescent dye has the chemical structure depicted in FIG.1 and is named 2-((£)-2-((£)-3-(2-((£)-3,3-dimelhyl-5-sulfonato- l -(3- (trimethylammonio)propy])indolin-2-ylidene)ethylidene)-5-(ethoxycarbonyl)-2-(4- sulfonatophenoxyjcyclohex- 1 -en- 1 -yl)vinyl)-3 ,3-dimethyl- 1 -(3-(trimethylammonio)propyl)- 3 H-i ndol - 1 -ium- 5 - sulfonate.

[0045] In some embodiments, the fluorescent dye is produced using a method in accordance with the schematic depicted in FIG. 2, and as described in detail in the exemplary implementations below.Drug Conjugate: Lumanocept

[0046] In some embodiments, the fluorescent dye described and produced as disclosed above is conjugated to a drug or diagnostic agent for visualizing specific targets. In some embodiments, the dye is conjugated to tilmanocept to produce a drug conjugate referred to herein as lumanocept. Tilmanocept is DTPA-mannosyl-dextran, a macromolecule with an average diameter of 7 nm. It consists of multiple units of DTPA and mannose, each covalently attached to a dextran backbone. The mannose acts as a ligand for the CD206 receptor. Its small size allows rapid lymphatic uptake, and the targeted receptor binding on macrophages and dendritic cells in lymph nodes limits its migration into the distal nodes (Vera DR et al. I Nucl Med. 2001 ;42:951-959.9; see also AZAD AK et al. J. Immunology 2015; 195: 2019-2029). Tilmanocept has been used as a radiopharmaceutical for lymphatic mapping with sentinel lymph node (SLN) localization and can be used as a valuable prognostic tool in management of certain cancers such as melanoma and breast cancer. Tilmanocept has the chemical structure illustrated in FIG. 3. Lumanocept has the chemical structure illustrated in FIG. 4.

[0047] In certain embodiments, the DTPA chelator is not incorporated into the lumanocept compound. In certain embodiments, an alternative chelator to DTPA can be incorporated, such as EDTA, NOTA or DOTA. In certain embodiments, a combination of non-chelator bases and chelator-containing bases are incorporated into the lumanocept compound.

[0048] It is understood that the five bases in the lumanocept compound comprise a total chain length of 10 glucose moieties long to over 10 kb long. The order and length can be intentional, or the bases can be incorporated randomly or weighted to desired ratios.PCT ApplicationAttorney Docket No. 009062.8553.WO00

[0049] In some embodiments, the linkages for conjugating the mannose moieties is an amide or amidine linker. The amine terminated linkers / leashes do not have to be conjugated to the hydroxyl group on the second carbon of the glucose moieties. The linkers / leashes, with or without conjugated DTPA or dye, will be conjugated to the hydroxyl groups of the third or fourth carbons.

[0050] In certain embodiments, the conjugated dye is chosen from a group comprised of dyes with the absorption / emission spectra of the instrument used to visualize the lumanocept. In certain embodiments, the conjugation of the fluorescent dye to tilmanocept is accomplished by using a carboxylic acid derivative that acts as the synthetic handle. In certain embodiments, lumanocept is produced according to the procedures disclosed in the exemplary implementations below. In other embodiments, lumanocept is produced using the dye disclosed above in a method in accordance with the schematic depicted in FIG. 5.Methods of Using Lumanocept

[0051] In accordance with some embodiments, the lumanocept drug conjugate described above may be used in methods for fluorescently detecting and / or visualizing CD206 expressing macrophages and dendritic cells that are abundant in tumors (as tumor associated macrophages, TAMs) and in lymph nodes. Lumanocept and tilmanocept allow the detection of the first lymph nodes (the SLNs) in the lymphatic drainage of a tumor. SLNs may or may not contain tumor cells. Lumanocept thereby allows a pathologist to detect tumor cells, including benign tumor cells, malignant tumor cells, or metastatic cancer cells. In some embodiments, the tumor cells are located in sentinel lymph nodes, while in other embodiments, the tumor cells may be part of a tumor.

[0052] According to some embodiments, the fluorescent detection or visualization of CD206 expressing macrophages and dendritic cells occurs during a medical procedure such as surgery (including human and robotic surgery), an imaging procedure (e.g., SPECT, CT, MRI, PET, etc.), an endoscopy or colonoscopy, or any other investigative procedure. Lumanocept can be used for cystoscopy screens for bladder cancer (about 350k procedures / year in US) to help urologists identify “flat” tumors. Flat tumors are dangerous and more likely to metastasize and also challenging to see by cystoscopy. The visualization or fluorescent detection of the lymph nodes or any structure containing macrophages or dendritic cells in accordance to the embodiments described herein is advantageous because it can lead to a faster and / or more accurate staging of a cancer, a faster and / or more accurate di agnosis of a cancer or other diseasePCT ApplicationAttorney Docket No. 009062.8553.WO00 state, or a faster and / or more accurate identification of cancerous tissue for resection, thereby leading to longer survival of the patient.

[0053] In certain embodiments, the lumanocept drug conjugate may be used in methods of intraoperative tumor margin mapping for cancers such as glioma, lobular breast cancer, and pancreatic cancer. In such methods, lumanocept enables fluorescent visualization of tumor margins during surgery, assisting surgeons in determining whether all tumor tissue has been removed. This approach may improve surgical outcomes by reducing the likelihood of residual tumor tissue, thereby decreasing recurrence rates and enhancing patient prognosis.

[0054] In some embodiments, the methods using lumanocept disclosed herein may be used to guide a pathologist, technician, clinician, or surgeon to the microscopic location of metastatic cancer cells within a sentinel lymph node, including through intranodal mapping techniques that visualize and localize metastatic cells within the internal structure of the lymph node. In other embodiments, the methods disclosed herein may be used to guide a pathologist, technician, clinician, or surgeon to the microscopic location of tumor cells and / or other disease states during a diagnostic or surgical procedure. The following indications are non-limiting examples of methods for use of the lumanocept drug conjugate described above.

[0055] In other embodiments, the methods disclosed herein may be used in sentinel lymph node mapping, fluorescence imaging, robotic surgery, endoscopy, or colonoscopy. In some embodiments, lumanocept can be used in the operating room to visualize the sentinel lymph nodes using a hand-held imager or the camera system in a surgical robot.

[0056] In certain embodiments, the lumanocept drug conjugate may be used in methods of intra-nodal fluorescence imaging of sentinel lymph nodes for more efficient histo-pathologic examination of breast cancer metastases. In such methods, the lumanocept drug conjugate can be used to detect intra-nodal colocalization of metastatic cells in breast cancer patients and can result in a substantial reduction of pathology examination focused on the most probable landing site of the metastatic cells. Such methods may also provide a possible reduction in falsenegative rate compared to standard pathology examination, which requires sectioning of all SLNs to exhaustion.

[0057] In certain embodiments, the lumanocept drug conjugate may be used in methods of intra-operative fluorescence mapping of sentinel lymph nodes for gynecological cancer. In such methods, the lumanocept drug conjugate can be used to test the success and false negative rate of lumanocept compared to other sentinel lymph node (SLN) mapping methods, whichPCT ApplicationAttorney Docket No. 009062.8553.WOOO could result in an increased SLN mapping success rate and a possible reduction in falsenegative rate due to misidentification of SLN by other methods.

[0058] In certain embodiments, the lumanocept drug conjugate may be used in methods of intra-operative fluorescence mapping of sentinel lymph nodes for prostate cancer. In such methods, the lumanocept drug conjugate can be used during preoperative SPECT / CT and intraoperative imaging patients having or suspected of having prostate cancer, and may result in reduction of operative time by preoperative imaging and a possible reduction in recurrence rate by widening of SLN mapping sites by preoperative imaging.

[0059] In certain embodiments, the lumanocept drug conjugate may be used in methods of intra-operative fluorescence mapping of sentinel lymph nodes for head and neck cancer. In such methods, the lumanocept drug conjugate can be used during preoperative SPECT / CT and intraoperative imaging patients having or suspected of having head and neck cancer, and may result in reduction of operative time by preoperative imaging with an imager.

[0060] In certain embodiments, the lumanocept drug conjugate may be used in methods of fluorescence-enhancement for adenoma visualization during colonoscopic detection of colorectal cancer. In such methods, the lumanocept drug conjugate can be used during preoperative SPECT / CT and fluorescence-based colonoscopy in patients having or suspected of having colon cancer, and may result in reduction of procedure time by fluorescent contrastenhancement of polyps / adenomas / cancers. Such methods may also increase adenoma detection rates with possible reduction in cancer rates and possible SPECT / CT detection / staging of colon cancer and possible Al-based interpretation based on imaging cues from fluorescence images.

[0061] In certain embodiments, the lumanocept drug conjugate may be used in methods of intra-operative fluorescence mapping of sentinel lymph nodes for bladder wall cancer. In such methods, the lumanocept drug conjugate can be used to perform preoperative SPECT / CT and intraoperative imaging in patients having or suspected of having bladder wall cancer, and may result in a reduction of operative time by preoperative imaging and a possible reduction in recurrence rate by widening of SLN mapping sites by preoperative imaging.

[0062] In certain embodiments, the lumanocept drug conjugate may be used in methods of intra-operative fluorescence mapping of gastric cancer sentinel lymph nodes. In such methods, the lumanocept drug conjugate can be used to perform preoperative SPECT / CT and intraoperative in patients having or suspected of having gastric cancer, and may result in aPCT ApplicationAttorney Docket No. 009062.8553.WO00 reduction of operative time by preoperative imaging and a possible reduction in recurrence rate by widening of SLN mapping sites by preoperative imaging.

[0063] In certain embodiments, the lumanocept drug conjugate may be used in methods of intra-operative fluorescence mapping of rectal cancer sentinel lymph nodes. In such methods, the lumanocept drug conjugate can be used to perform preoperative SPECT / CT and intraoperative imaging in patients having or suspected of having rectal cancer, and may result in a reduction of operative time by preoperative imaging and a possible reduction in recurrence rate by widening of SLN mapping sites by preoperative imaging.

[0064] In certain embodiments, the lumanocept drug conjugate may be used in methods of fluorescence-contrast enhancement for adenoma visualization during capsule-based detection of GI cancers. In such methods, the lumanocept drug conjugate can be used to perform an endoscopy with a fluorescent-capable capsule in patients with prior history of colonic adenomas, or patients having or suspected of having a colonic adenoma. Such methods may result in a substantial reduction in time required for review of capsule images, and a possible Al-based interpretation based on imaging cues from fluorescence images.Exemplary Implementations

[0065] Example implementations were conducted for synthesis of a fluorescent dye (2- ((E)-2-((E)-3-(2-((E)-3,3-dimethyl-5-sulfonato-l-(3-(trimethylammonio)propyl)indolin-2- ylidene)ethylidene)-5-(ethoxycarbonyl)-2-(4-sulfonatophenoxy)cyclohex-l-en-l-yl)vinyl)- 3 ,3 -dimethyl- 1 -(3 -(tri methylammonio (propyl (-3 / -i nd l - 1 -ium-5 -sulfonate)

[0066] Starting with Compounds 1 (4-hydrazinobenzene sulfonic acid (FW = 188.2 g / mol)) and 5 (Ethyl 4-oxocyclohexanecarboxylate (FW 170.21 g / mol)), Compounds 2 (Ammonium 2,3,3-Trimethyl-3H-indole-5-sulfonate (C11H16N2O3S) (FW = 220.3 g / mol)), 3 (Potassium 2,3,3-Trimethyl-3H-indole-5-sulfonate (C11H12KNO3S) (FW = 277.4 g / mol)), 4 (2,3,3-Trimethyl-l -[3-(trimethylammonio)propyl]-3H-indolium-5-sulfonic acid dibromide (Ci7H27Br2KN2O3) (FW = 538.4 g / mol)), 6 (Keto ester (C23H24CI2N2O2) (FW = 378.2 g / mol)), 7 (Chloro Dye (C45H62C1N4O8S2+) (FW = 1168 g / mol)), and 8 (Acid Dye (C49H62N4O12S3) (FW = 1056 g / mol)) were synthesized according to the schematic depicted in FIG. 2.

[0067] Example implementations were conducted for preparation of Ammonium 2,3,3- Trimelhyl-3H-indole-5-sull'onale (Compound 2). Preparation of Compound 2 was carried out according to the schematic shown in FIG. 6 and methods below. See also Choi HS, et al.PCT ApplicationAttorney Docket No. 009062.8553.WO00Angew Chem Int Ed 2011; 50(28): 6258; Hyun H, et al. Contrast Media Mol Imaging 2012; 7(6): 516.

[0068] First, 9 mL 3-methyl-2-butanone was added to 12 g of 4- hydrazineylbenzenesulfonic acid (Structure 1) in a reaction flask (flask 1). To that mixture, 60 mL glacial acetic acid was added. That reaction mixture was stirred at 1 18°C for 18 hours under N2. After 18 hrs, the reaction solution in flask 1 was cooled down (RX temp probe: 37°C).

[0069] Crude product was obtained by precipitation with 75-80 mL ethyl acetate. The crude product Ammonium 2,3,3-Trimethyl-3H-indole-5-sulfonate (Compound 2) was filtered and collected as a pink solid and dried in an overnight before weighing the final product (10.9845g). The structure was verified by the NMR spectrum shown in FIG. 13.

[0070] Example implementations were conducted for preparation of Potassium 2,3,3- Trimethyl-3H-indole-5-sulfonate (Compound 3). Preparation of Compound 3 was carried out according to the schematic shown in FIG. 7 and methods below. See also Choi HS, et al. Angew Chem Int Ed 2011; 50(28): 6258; Hyun H, et al. Contrast Media Mol Imaging 2012; 7(6): 516.

[0071] First, 10 g of Ammonium 2,3,3-Trimethyl-3H-indole-5-sulfonate (Compound 2) was added to a reaction flask (flask 2). 150 mL CH3OH was added to the flask 2 to dissolve Compound 2.

[0072] Potassium hydroxide (85%, 3.0 g, 46 mmol) was separately dissolved in 2- propanol (31 mL) at 50°C. Once dissolved, the KOH solution was slowly added to flask 2 containing Compound 2 with a dropping funnel for 1 h.

[0073] The resulting brown / yellowish solid product (Potassium 2,3,3-Trimethyl-3H- indole-5-sulfonate, Compound 3) was filtered w / buchner’s funnel. The resulting product 3 was dried in an oven overnight and weighed (7.649 g). The structure was verified by the NMR spectrum shown in FIG. 12.

[0074] Example implementations were conducted for preparation of 2,3,3-Trimethyl- 1 - [3-(trimethylammonio)propyl]-3H-indolium-5-sulfonic acid dibromide (Compound 4). Preparation of Compound 4 was carried out according to the schematic shown in FIG. 8 and methods below. See also Choi HS, et al. Angew Chem Int Ed 2011 ; 50(28): 6258; Hyun H, et al. Contrast Media Mol Imaging 2012; 7(6): 516.PCT ApplicationAttorney Docket No. 009062.8553.WO00

[0075] First, 2.3 g of Potassium 2,3,3-trimethyl-3H-indole-5-sulfonate (Compound 3) and 2.5 g 3-bromopropyl)trimethylammonium bromide were added in a 250 mL round bottom flask. 60 mL toluene was added to the flask and the mixture was heated at 130°C for 72 h under a nitrogen atmosphere. The mixture was cooled to room temperature and the solvent was decanted. An aliquot of the crude mixture was saved for NMR and HPLC. The crude mixture was then washed with toluene (70 mL) and MTBE (100 mL) and purified by recrystallization in Methanol (10 mL) and MTBE (100 mL).

[0076] Next, the flask was placed in the freezer and the product (Compound 4) was collected through filtration. The product was dried in an oven overnight and weighed.

[0077] NMR and HPLC was performed for the purified product, as illustrated in FIG. 14A. The absorbance spectrum was performed as follows: (1) perform a blank scan with the same solvent used to dissolve the analyte; (2) use the wavelength scan method to obtain the absorbance spectrum. Through dilutions, acquire a spectrum where the peak of interest has an abs value less than 0.05, as shown in FIG. 14B.

[0078] Alternatively, preparation of Compound 4 can be carried out using Compound 2 as a starting material. In that case, 1.025 g of Ammonium 2,3,3-trimethyl-3H-indole-5- sulfonate (Compound 2) and 1.148 g 3-bromopropyl)trimethylammonium bromide were added in a 50 mL round bottom flask. 20 mL of toluene was added to the flask and the mixture was headed at 130°C for 72 h under a nitrogen atmosphere. The mixture was cooled to room temperature filtered with whatman filter paper and Buchner’s funnel. The retentate was washed with a small amount of Toluene (10 mL).

[0079] The crude product was air-dried and any trace amount of toluene was removed in high vacuum with the crude product. An aliquot of the crude mixture was saved for NMR and HPLC, with the NMR spectrum presented in FIG. 15A. (Crude wt.: 1.507 g). The crude product was washed with MTBE (lOmL) and purified by recrystallization in Methanol (5 mL) and MTBE (100 mL). The flask was then placed in the freezer and the product (Compound 4) was collected through filtration. The absorbance spectrum of the purified product is shown in FIG. 15B. The product was then dried in an oven overnight and weighed.

[0080] Example implementations were conducted for Preparation of A-((£ -((E)-2-chloro- 5-(ethoxycarbonyl)-3-((phenylamino)methylene)cyclohex-l-en-l- yl)methylene)benzenaminium (Compound 6). Preparation of Compound 6 was carried outPCT ApplicationAttorney Docket No. 009062.8553.WO00 according to the schematic shown in FIG. 9 and methods below. See also Strekowski L, et al. J Heterocyclic Chem 1996; 33: 1685; Strekowski L, et al. J Heterocyclic Chem 2004; 41 : 227.

[0081] The Vilsmeier reagent was obtained by dropwise addition of phosphorus oxychloride (POCL) (5.5 mL, 10 mmoles) to N,N-dimethylformamide (DMF) (6 mL) at 0°C, via a dropping funnel. That mixture was treated with a solution of Ethyl 4- oxocyclohexanecarboxylate (Compound 5) dissolved in 5 mL dichloromethane. The resultant mixture was heated to 100 °C under reflux for 1 hour.

[0082] Following removal of dichloromethane, the residue was cooled in an ice bath and slowly treated with a solution of 4 mL aniline in 10 mL methanol in such a rate that the temperature does not rise above 5 °C. Then the mixture was poured onto 50 g ice, followed by the addition of cold concentrated 5 mL hydrochloric acid and cooling in an ice bath for 5 hours. (RX temp probe: 0°C)

[0083] The resulting crystals of Compound 6 are collected and dried overnight and weighed (2.536 g). An NMR and HPLC were performed for the product. The NMR spectrum is shown in FIG. 16A, and the absorbance spectrum is shown in FIG. 16B.

[0084] Example implementations were conducted for preparation of 2-((E)-2-((E)-2- chloro-3-(2-((E)-3,3-dimethyl-5-sulfonato-l -(3-(trimethylammonio)propyl)indolin-2- ylidene)ethylidene)-5-(ethoxycarbonyl)cyclohex-l-en-l-yl)vinyl)-3,3-dimethyl-l-(3- (trimethylammonio)propyl)-377-indol-l-ium-5-sulfonate (Compound 7). Preparation of the Compound 7 Chloro Dye was carried out according to the schematic shown in FIG. 10 and methods below. See also Choi HS, et al. Angew Chem Int Ed 2011 ; 50(28): 6258.

[0085] The bromide salt (2,3,3-Trimethyl- l-[3-(trimethylammonio)propyl]-3H-indolium- 5-sulfonic acid dibromide, Compound 4, 538 mg, 1 mmol) was added to the Vilsmeier-Haack product (Compound 6) (215 mg, 0.5 mml), and anhydrous sodium acetate (246 mg, 3 mmol) in a lOOmL reaction flask. Absolute ethanol (25 mL) was added to the mixture and the reaction mixture was refluxed at 100°C for 6 hours under nitrogen. The reaction mixture was then cooled to room temperature and an aliquot was removed for NMR and HPLC analysis. The NMR spectrum shown in FIG. 17A, and the absorbance spectrum is shown in FIG. 17B. Then the rest of it was filtered, washed with ethanol and methanol.

[0086] Using rotavap, the solvent was removed in the filtrate and washed with CH2CI2 (20 mL) and MTBE (20 mL) (1 :1). The resulting product (Compound 7) was dried overnight andPCT ApplicationAttorney Docket No. 009062.8553.WO00 collected as a dark solid (Amt: 0.69 g). NMR and HPLC analyses were performed on the product.

[0087] In an alternative method, the bromide salt (Compound 4, 255 mg, 1 mmol) was added to compound 6 (102 mg, 0.5 mmol), and anhydrous sodium acetate (58 mg, 3 mmol) in a l OOmL reaction flask. Absolute ethanol (15 mL) was added to the mixture and the reaction mixture was refluxed at 100°C for 20 hours under nitrogen. The reaction mixture was then cooled to room temperature.

[0088] Using a rotovap, the solvent was removed followed by high vacuum. An aliquot of the dry solid was removed for NMR and HPLC analysis. The NMR spectrum is shown in FIG. 18A and the absorbance spectrum is shown in FIG. 18C. Then the rest of it is filtered, washed with ethanol (ImL) and methanol (2mL).

[0089] The product (Compound 7) was purified by HPLC using a flow rate of 2mL / min in a dual solvents system (Solvent A: 0.1% TFA in DI water; Solvent B: 95% ACN and 0.1% TFA). The solvent gradient was set as follows: 100% Solvent A to start, and ending with 55% Solvent B & 45% Solvent A, with a run time of 25 minutes. The result of the HPLC purification is shown in FIG. 18B.

[0090] For step 1, the HPLC method started with test runs, wherein a small amount of diluted sample of Compound 7 was injected and all fluorescent peaks were collected. Compound 7 was injected again and all peaks were collected to make sure of the purity. Finally, the absorbance spectra was obtained for all collections, and then it was determined which collection is the desired product.

[0091] For step 2, the injection port was cleaned with 2 mL DI H2O, and for step 3, 150 pL of concentrated sample of crude Compound 7 in DI H2O was injected. For step 4, the eluate was then collected from 20.2-21.2 min. These steps were repeated for step 5.

[0092] The purified product from step 5 was transferred to a round bottom flask for step 6. The solvent was removed by rotavap, and the dried product (Compound 7) was re-dissolved in DI water and then lyophilized and weighed.

[0093] Example implementations were conducted for preparation of 2-((E)-2-((E)-3-(2- ((E)-3,3-dimethyl-5-sulfonato-l-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethylidene)- 5-(ethoxycarbonyl)-2-(4-sulfonatophenoxy)cyclohex-l-en-l-yl)vinyl)-3,3-dimethyl-l-(3- (tri methy lam monio)propyl )-37f-i ndol- 1 -ium-5-sulfonate (Compound 8)PCT ApplicationAttorney Docket No. 009062.8553.WO00

[0094] Preparation of the Compound 8 NIR Acid Dye was carried out according to the schematic shown in FIG. 11 and methods below. See also Lugade A, et al. Cyanine Dyes. U.S. Patent 6,995,274; Cheung L, et al. Fluorescent Imaging with Substituted Cyanine Dyes. U.S. Patent Appl. Publication No. 2013 / 0039860 Al; Choi HS, Nasr K, Alyabyev S, et al. Synthesis and in vivo fate of zwitterionic near-infrared fluorophores, Angew Chem Int Ed Engl. 2011 ;50:6258-6263, Supporting Information.

[0095] First, a stock solution was made by dissolving 19.6 mg (0.08 mmol) of 4- hydroxybenzenesulfonate dihydrate with 0.52 mL d7-DMF. 95 pL (0.015 mmol) DMF was reserved in a small glass vial. 2.6 mg (60%, 0.06 mmol) sodium hydride was added to the mixture and stirred at room temperature for 30 min under nitrogen.

[0096] Next the Chloro dye (Compound 7) (9 mg, 0.01 mmol) was dissolved in 105 pL of dry d7-DMF, added to the reaction mixture, and stirred further for 50 min.

[0097] 10 pL of the reaction mixture was diluted with 400 pL of d7-DMF. The liquid was transferred to an NMR tube and a proton NMR was run. Evidence of phenoxide displacement was checked.

[0098] HPLC chromatographs with a gradient of 25% B to 40% B in 20 minute (A: 0.1% TFA in DI water; B: 95% ACN and 0.1% TFA) was acquired for compound 7, sodium phenoxide and the reaction mixture. All three analytes were diluted with 25% B in A with a concentration less than 1 mg / mL. The consumption of compound 7 and phenoxide was checked by monitoring the UV absorbance (280 nm) and fluorescence (Ex. 780 nml Em: 810 nm). 74 pL of 10% sulfuric acid solution was added to the reaction mixture and stirred at RT for 90 minutes.

[0099] An HPLC-MS was run to confirm the yield of Compound 8. (m / z: 497.9). The product was purified by HPLC equipped with a C18 column. Solvent gradient: 10% B to 40% B in 20 minutes (A:0.1% TFA in DI water; B: 0.1% TFA in ACN). The eluates were collected with the retention time ~13 min. (RT: 12.8-13.4 min). The purified product (Compound 8) was lyophilized and weighed.

[0100] FIG. 19A shows the HPLC chromatogram obtained for Compound 8. NMR spectra, presented in FIGS. 19B, 19D, and 23, further confirmed the structure of Compound 8, with FIG. 23 specifically depicting the proton nuclear magnetic resonance (1H NMR) spectrum. Absorbance spectra obtained for Compound 8 and purified Compound 8 are shown in FIGS. 19C and 20, respectively.PCT ApplicationAttorney Docket No. 009062.8553.WOOO

[0101] Example implementations were conducted for conjugation of NIR Acid Dye (Compound 8) to SN-DTPA-mannosyl dextran (Tilmanocept). Preparation of the fluorescent targeting moiety, lumanocept, produced from the eNIR Acid Dye (Compound 8) coupled to tilmanocept, was carried out according to the schematic shown in FIG. 5.

[0102] Two examples are provided to illustrate the coupling of the near-infrared (NIR) dye to macromolecules. Example 1 describes a procedure for coupling to macromolecules that are soluble only in aqueous solutions. Example 2 describes a procedure for coupling to macromolecules that are soluble in dimethyl sulfoxide (DMSO). In both examples, tilmanocept is used as the representative macromolecule, as it is soluble in both aqueous and DMSO solutions. Example 1 utilizes an aqueous buffer system, which is compatible with most proteins, enzymes, polypeptides, and complex carbohydrates. Example 2 utilizes DMSO as the solvent, which is compatible with small peptides and certain carbohydrates.

[0103] Example 1 : Aqueous Coupling Procedure - Reaction buffer (0.5 M sodium chloride, 0.05 M MES, pH 6.0), coupling buffer (0.1 M phosphate, pH 7.5), bicarbonate coupling buffer (0.55 M, pH 9.6), and purification buffers prepared from phosphate-buffered saline (PBS) and bicarbonate packs are prepared as specified. NIR Dye #8 is dissolved in reaction buffer. Freshly prepared sulfo-NHS and EDC solutions are added sequentially to activate the dye ester. The mixture is vortexed and incubated at room temperature (approximately 20-25 °C) for 15 minutes after each addition.

[0104] Tilmanocept is dissolved in bicarbonate coupling buffer to form a clear solution at pH approximately 7.6, then added to the activated dye solution for reaction. The conjugation proceeds for at least 4 hours at room temperature, with monitoring by absorbance scans. The reaction is quenched by addition of glycine solution, followed by purification using multiple rounds of centrifugation with AMICON filters and buffer exchanges between PBS and bicarbonate buffers.

[0105] The purified product is frozen at -80°C, lyophilized in pre-weighed tubes, and stored in a locked ultra-freezer at -80°C with appropriate labeling, including lot number, product date, and weight information. Samples are dissolved in deuterium oxide (DiO) or PBS for proton nuclear magnetic resonance (NMR) analysis to determine dye density using aromatic and on carbon proton peaks. Absorption (250-800 nm), emission (550-800 nm), and quantum yield measurements are performed using the absorption peak as the excitation wavelength. ThePCT ApplicationAttorney Docket No. 009062.8553.WO00 average molecular weight of the conjugate is calculated based on the dye-to-dextran ratio determined from NMR data.

[0106] Example 2: DMSO Coupling Procedure - Alternatively, Disuccinimidyl carbonate (DSC) is dissolved in anhydrous N,N-dimethylformamide (DMF) to prepare a stock solution, and a pyridine stock solution is also prepared. NIR Dye carboxylate #8 is dissolved in the pyridine stock solution with DMF, then activated by addition of the DSC stock solution. The mixture is stirred at 55-60°C under a nitrogen atmosphere for 90 minutes.

[0107] The activated dye is washed with ethyl acetate, centrifuged, and re-dissolved in dimethyl sulfoxide (DMSO) for further analysis. The dye mixture is purified by reverse phase Cl 8 high-performance liquid chromatography (HPLC), collecting fractions corresponding to retention times of approximately 10-12 minutes. The collected fractions are freeze-dried, weighed, dissolved in deuterated DMSO (de-DMSO), and analyzed by nuclear magnetic resonance (NMR) spectroscopy and ultraviolet (UV) absorbance to confirm purity and concentration.

[0108] Tilmanocept is dissolved in DMSO and combined with the activated dye at a specific molar ratio. After reaction at room temperature (approximately 20-25°C) with monitoring by UV absorbance, the conjugated product is purified using an Amicon filter, analyzed by HPLC with UV and fluorescence detection, lyophilized, weighed, and characterized by NMR spectroscopy to determine dye density through comparison of proton chemical shifts.

[0109] The resulting compound was subjected to analytical characterization to confirm its identity and purity. FIG. 21 presents the absorbance spectrum obtained for purified lumanocept, while FIG. 22 shows the fluorescence emission spectrum, both consistent with the expected optical properties of the conjugate. The purity of lumanocept was further assessed by HPLC, as shown in FIG. 24, and quantified using the Area % Report derived from the HPLC chromatogram, as depicted in FIG. 25.Conclusion

[0110] While this patent document contains many specifics, these should not be construed as limitations on the scope of any invention or of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments of particular inventions. Certain features that are described in this patent document in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely,PCT ApplicationAttorney Docket No. 009062.8553.WOOO various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.

[0111] Similarly, while operations are depicted in the drawings in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. Moreover, the separation of various system components in the embodiments described in this patent document should not be understood as requiring such separation in all embodiments.

[0112] Only a few implementations and examples are described and other implementations, enhancements and variations can be made based on what is described and illustrated in this patent document.

Claims

PCT ApplicationAttorney Docket No. 009062.8553.WOOOCLAIMSWhat is claimed is:

1. A fluorescent dye comprising a structure consistent with:

2. The fluorescent dye of claim 1 , wherein the carboxylic acid group is capable of being conjugated to a targeting moiety.

3. The fluorescent dye of claim 1 , wherein the absorbance and emission maxima are tunable by the number and position of sulfonic acid substituents.

4. A drug conjugate comprising a targeting moiety conjugated to a fluorescent dye, wherein the targeting moiety comprises tilmanocept and the fluorescent dye comprises 2-((E)-2-((£)-3-(2-((£)-3,3-dimethyl-5- sulfonato-l-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethylidene)-5-PCT ApplicationAttorney Docket No. 009062.8553.WOOO(ethoxycarbonyl)-2 (4-sulfonatophenoxy)cy clohex- 1 -en- 1 -yl)vinyl)-3 ,3 -dimethyl- 1 -(3-(trimethylammonio)propyl)-3H-indol- l-ium-5-sulfonate; and the drug conjugate has a structure consistent with:

5. The drug conjugate of claim 4, wherein tilmanocept comprises DTPA-mannosyl- dextran, having multiple units of DTPA and mannose covalently attached to a dextran backbone.

6. The drug conjugate of claim 5, wherein the mannose moieties are conjugated to the dextran backbone via an amide or amidine linker.

7. The drug conjugate of claim 5, wherein the DTPA chelator is replaced by an alternative chelator selected from the group consisting of EDTA, NOTA, and DOTA.

8. The drug conjugate of claim 4, wherein the fluorescent dye is conjugated to tilmanocept via a carboxylic acid derivative.

9. A method of producing the drug conjugate of any one of claims 4-8, comprising:(a) activating the fluorescent dye for conjugation;(b) dissolving tilmanocept in a suitable buffer or solvent;(c) reacting the activated dye with tilmanocept under conditions suitable for covalent attachment; and(d) purifying the conjugate by filtration and buffer exchange.PCT ApplicationAttorney Docket No. 009062.8553.WOOO10. The method of claim 9, wherein the conjugation is performed in an aqueous buffer system.

11. The method of claim 9, wherein the conjugation is performed in a dimethyl sulfoxide (DMSO) solvent system.

12. A method of detecting or visualizing CD206-expressing macrophages or dendritic cells in a subject, comprising:(a) administering to the subject an effective amount of the drug conjugate of any one of claims 4-8; and(b) detecting fluorescence in tissue or lymph nodes of the subject.

13. The method of claim 12, wherein the detection is performed during a medical procedure selected from the group consisting of surgery, imaging, endoscopy, colonoscopy, and cystoscopy.

14. The method of claim 12, wherein the drug conjugate is used for sentinel lymph node mapping in cancer patients.

15. The method of claim 12, wherein the drug conjugate is used for intraoperative tumor margin mapping.

16. The method of claim 12, wherein the drug conjugate is used for fluorescence- enhanced detection of adenomas during colonoscopy or capsule-based endoscopy.

17. The method of claim 12, wherein the drug conjugate is used for intra-nodal fluorescence imaging to localize metastatic cells within lymph nodes.

18. The method of claim 12, wherein the visualization of the sentinel lymph nodes or tumor containing macrophages leads to more accurate staging of a cancer, more accurate diagnosis of cancer, or more accurate identification of cancerous tissue for resection.

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