Novel double bond compound and near-infrared fluorescent contrast agent for elastin fiber imaging including the same

Abstract

Provided are a novel double bond compound or a pharmaceutically acceptable salt thereof, and a contrast agent composition including the same. The compound or the pharmaceutically acceptable salt thereof selectively binds to elastin present on a surface of a normal lung, and emits stable fluorescence for a long time in vivo, thereby being able to be used as an near-infrared (NIR) fluorescent contrast agent for determining in real time whether lung cancer has invaded a visceral pleural elastin layer.

Description

NOVEL DOUBLE BOND COMPOUND AND NEAR-INFRARED FLUORESCENT CONTRAST AGENT FOR ELASTIN FIBER IMAGING INCLUDING THE SAME

[0001] One or more embodiments relate to a novel double bond compound and a near-infrared fluorescent contrast agent composition for elastin fiber imaging including the same.

[0002] An imaging technology utilizing near-infrared (NIR) fluorescence has significant potential in the medical field, particularly in the areas of diagnosis and image-guided surgical procedures. However, a major obstacle to this imaging technology is the limited availability of suitable fluorescent contrast agents. In particular, at present, a fluorescent contrast agent and imaging method capable of imaging, during surgery, whether a lung cancer has invaded a visceral pleura elastin layer present on a surface of a lung have not yet been utilized in image-guided surgery.

[0003] Even when the size of a lung cancer of a patient is 2 centimeters (cm) or less, if lung cancer cells invade the visceral pleura elastin layer, the cancer stage is diagnosed as progressing from Stage 1A to Stage 1B, and a survival rate of the patient is significantly reduced from 90% to approximately 68%. In addition, when the size of lung cancer is 2 cm or less and a solidified part of the cancer is less than 25% based on radiologic characteristics, limited resection, such as segmentectomy, may be performed. However, even when the size of cancer is 2 cm or less, if lung cancer cells invade the visceral pleura elastin layer, lobectomy, in which the entire lung lobe is resected, must be performed.

[0004] However, it is difficult to determine the invasion of lung cancer into the visceral pleura elastin layer using preoperative computerized tomography (CT) or by visual inspection during surgery, and such invasion may be confirmed only through postoperative histopathological examination. Accordingly, insufficient resection frequently occurs, and problems arise in that a reoperation is performed after surgery due to the application of an inappropriate surgical method, or additional anticancer therapy is required due to poor prognosis.

[0005] Under these circumstances, the inventors of the disclosure have completed the disclosure by developing an NIR fluorescent contrast agent capable of enabling cancer targeting during minimally invasive surgeries such as laparoscopic, thoracoscopic, and robotic surgeries, and determining in real time whether lung cancer has invaded the visceral pleura elastin layer by imaging elastin present on a surface of the lung.

[0006] Embodiments provide a novel double bond compound or a pharmaceutically acceptable salt thereof and a method of preparing the same.

[0007] Embodiments provide a contrast agent composition including the compound or the pharmaceutically acceptable salt thereof as an active component.

[0008] Embodiments provide an imaging method including a step of treating a biological sample with the contrast agent composition.

[0009] Embodiments provide a method of diagnosing lung cancer invasion into a visceral pleura elastin layer or an information providing method for diagnosing lung cancer invasion into a visceral pleura elastin layer, the methods including a step of treating a lung with the contrast agent composition.

[0010] However, technical goals to be achieved are not limited to those described above, and other goals not mentioned above are clearly understood by one of ordinary skill in the art from the following description.

[0011] Additional aspects of embodiments will be set forth in part in the description which follows and, in part, will be apparent from the description, or may be learned by practice of the disclosure.

[0012] The double bond compound according to an embodiment of the disclosure has a complex chemical structure including sufficient double bonds, and thus has the effect of absorbing light in the NIR range and emitting light of a different wavelength (generally a longer wavelength) than the absorbed light. This allows it to emit stable fluorescence for 4 hours or longer in a target organ after spraying or injecting into the body, making it useful as an NIR fluorescent contrast agent.

[0013] The contrast agent composition according to an embodiment of the disclosure selectively binds to elastin after intravenous injection or spraying onto a target site, and targets cancer during minimally invasive surgeries such as laparoscopic, thoracoscopic, and robotic surgeries. Therefore, it is possible to determine in real time whether lung cancer has invaded a visceral pleural elastin layer present on a lung surface.

[0014] The effects of the double bond compound and the contrast agent composition including the same are not limited to the above-mentioned effects, and other unmentioned effects may be clearly understood from the above description by those having ordinary skill in the technical field to which the disclosure pertains.

[0015] According to an aspect, there is provided a compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[0016]

[0017] In Chemical Formula 1, R1and R2are the same as or different from each other, are each independently one or more selected from the group consisting of hydrogen, a hydroxyl group, a C1-C5alkoxy group, (Y)mX, (Y)mCOO-, (Y)mSO3-, (Y)mPO3H-, and combinations thereof, or may be desirably hydrogen or (Y)mSO3-, R3and R4are the same as or different from each other, are each independently one or more selected from the group consisting of (Y)mH, a C1-C5alkoxy group, a C1-C5alkenyl group, (Y)mNR5R6R7+, and combinations thereof, or may be desirably a C1-C5chain alkyl group or (Y)mNR5R6R7+, R5, R6, and R7are the same as or different from each other, are each independently hydrogen or an unsubstituted C1-C5chain alkyl group, or may be desirably hydrogen or a methyl group, X is a halogen group, Y is CH2or CH2CH2O, Z is Z is a halogen group, , , or , A is , , or , and a, b, c, d, e, and m are the same as or different from each other, and are each independently an integer from 0 to 5.

[0018] In an implementation example of the disclosure, in Chemical Formula 1, Z may be , , or , but is not limited thereto.

[0019] In another implementation example of the disclosure, the compound may be one or more selected from the group consisting of compounds represented by Chemical Formula 1-1 to Chemical Formula 1-12 below, but is not limited thereto.

[0020] ;

[0021] ;

[0022] ;

[0023] ;

[0024] ;

[0025] ;

[0026] ;

[0027] ;

[0028] ;

[0029] ;

[0030] ; and

[0031]

[0032] In another implementation example of the disclosure, the compound or the pharmaceutically acceptable salt thereof may selectively bind to elastin.

[0033] In another implementation example of the disclosure, the compound or the pharmaceutically acceptable salt thereof may absorb light in a near-infrared (NIR) range, and may desirably absorb light in a range of 800 nanometers (nm).

[0034] In another implementation example of the disclosure, the compound or the pharmaceutically acceptable salt thereof may emit light with a longer wavelength than a wavelength of the absorbed light.

[0035] According to another aspect, there is provided a contrast agent composition including the compound or the pharmaceutically acceptable salt thereof as an active component.

[0036] In an implementation example of the disclosure, the contrast agent composition may be an NIR fluorescent contrast agent and may be desirably a contrast agent that absorbs light in a range of 800 nm.

[0037] In another implementation example of the disclosure, the contrast agent composition may selectively bind to elastin.

[0038] In another implementation example of the disclosure, the contrast agent composition may be used in diagnosing lung cancer invasion into a visceral pleura elastin layer.

[0039] According to another aspect, there is provided an imaging method including the following steps:

[0040] (1) treating a biological sample with a contrast agent composition including the compound or the pharmaceutically acceptable salt thereof as an active component; and

[0041] (2) detecting a fluorescent signal emitted from the biological sample.

[0042] In an implementation example of the disclosure, the biological sample may be one or more selected from the group consisting of patient-derived cells, tissues, sputum, blood, plasma, urine, and combinations thereof, but is not limited thereto.

[0043] In another implementation example of the disclosure, the step (1) may include treating the biological sample with the contrast agent composition by administering the contrast agent composition to a subject.

[0044] According to another aspect, there is provided an information providing method for diagnosing lung cancer invasion into a visceral pleura elastin layer, the method including the following steps:

[0045] (1) treating a lung with a contrast agent composition including the compound or the pharmaceutically acceptable salt thereof as an active component;

[0046] (2) detecting a fluorescent signal emitted from the lung; and

[0047] (3) determining an area in which the fluorescent signal is not detected as a lung cancer invasion site.

[0048] In an implementation example of the disclosure, the step (1) may include treating a biological sample with the contrast agent composition by administering the contrast agent composition to a subject.

[0049] According to another aspect, there is provided a method of selecting a dye for imaging cells or tissues, the method including the following steps:

[0050] (1) selecting a compound having a maximum absorption wavelength of 500 to 1500 nm in an absorption spectrum and a maximum emission wavelength of 550 to 1700 nm in an emission spectrum; and

[0051] (2) confirming whether the compound maintains fluorescence properties for a long time when the compound is bound to elastin.

[0052] Here, the maximum absorption wavelength refers to a wavelength at which intensity is maximum in the absorption spectrum, and the maximum emission wavelength refers to a wavelength at which the intensity is maximum in the emission spectrum.

[0053] These and / or other aspects, features, and advantages of the disclosure will become apparent and more readily appreciated from the following description of embodiments, taken in conjunction with the accompanying drawings of which:

[0054] FIG. 1 illustrates a method of preparing a double bond compound according to an embodiment of the disclosure;

[0055] FIG. 2 illustrates results of imaging whether Double Bond Compound 19 specifically binds to elastin after being dissolved in saline and sprayed into a normal mouse lung according to an embodiment of the disclosure;

[0056] FIG. 3 illustrates results of observing fluorescence expression using a fluorescence microscope after sectioning lung tissue to have a thickness of 6 to 50 μm, the lung tissue obtained by dissolving Double Bond Compound 19 according to an embodiment of the disclosure in saline and spraying the mixture onto lungs of a normal mouse;

[0057] FIG. 4 illustrates results of imaging whether Double Bond Compound 19 specifically binds to elastin after being dissolved in saline and sprayed into a mouse lung with a tumor according to an embodiment of the disclosure;

[0058] FIG. 5 illustrates results of an optimization experiment comparing fluorescence intensity by concentration by treating lungs of normal mice with various concentrations (0.005 mM to 0.1 mM) of a compound according to an embodiment of the disclosure;

[0059] FIG. 6 illustrates results of optimizing a binding time by observing a change in fluorescence signal over time (15 seconds to 5 minutes) after treating lungs of normal mice with a compound according to an embodiment of the disclosure;

[0060] FIG. 7 illustrates results of imaging a fluorescence difference between a lesion area and a normal area after treating a rabbit lung model with a compound (0.05 mM), allowing it to react for 3 minutes, and then washing it once according to an embodiment of the disclosure; and

[0061] FIG. 8 illustrates results of observing a cross-section of rabbit lung tissue treated with a compound (0.05 mM) using a high-magnification (40X) fluorescence microscope (EVOS), and confirming that it is specifically bound to a visceral pleura layer according to an embodiment of the disclosure.

[0062] The inventors of the disclosure have developed a novel double bond compound or a pharmaceutically acceptable salt thereof that may absorb near-infrared (NIR) rays and emit fluorescence stably for a long time in vivo.

[0063] In addition, the inventors of the disclosure have confirmed that it is possible to enable cancer targeting during minimally invasive surgeries such as laparoscopic, thoracoscopic, and robotic surgeries, and determine in real time whether lung cancer has invaded a visceral pleura elastin layer as the compound or the pharmaceutically acceptable salt thereof images elastin present on a surface of the lung.

[0064] Accordingly, the disclosure provides a compound or a pharmaceutically acceptable salt thereof represented by Chemical Formula 1 below.

[0065]

[0066] In Chemical Formula 1, R1and R2are the same as or different from each other, and are each independently one or more selected from the group consisting of hydrogen, a hydroxyl group, a C1-C5alkoxy group, (Y)mX, (Y)mCOO-, (Y)mSO3-, (Y)mPO3H-, and combinations thereof, R3and R4are the same as or different from each other, and are each independently one or more selected from the group consisting of (Y)mH, a C1-C5alkoxy group, a C1-C5alkenyl group, (Y)mNR5R6R7+, and combinations thereof, R5, R6, and R7are the same as or different from each other, and are each independently hydrogen or an unsubstituted C1-C5chain alkyl group, X is a halogen group, Y is CH2or CH2CH2O, Z is a halogen group, , , or , A is , , or ,

[0067] and a, b, c, d, e, and m are the same as or different from each other, and are each independently an integer from 0 to 5.

[0068] In the disclosure, the term "substitution" refers to a reaction in which an atom or an atomic group included in a molecule of a compound is substituted with another atom or atomic group.

[0069] In the disclosure, the term "chain alkyl group" refers to a group derived from straight or branched chain saturated aliphatic hydrocarbon having a specific number of carbon atoms and having at least one valence. Examples of such alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, 2-butyl, 3-butyl, pentyl, n-hexyl, and the like, but are not limited thereto.

[0070] In the disclosure, the term "halogen group" refers to elements belonging to Group 17 of the periodic table, such as fluorine (F), chloride (Cl), bromine (Br), or iodine (I).

[0071] In the disclosure, the term "alkoxy group" refers to an atomic group CnH2n+1O- formed by an oxygen atom binding to an alkyl group, and examples of such alkoxy groups include methoxy, ethoxy, propoxy, or butoxy, but are not limited thereto.

[0072] In the disclosure, the term "pharmaceutically acceptable salt" refers to a formulation of a compound that does not cause serious irritation to an organism, to which the compound is administered, and does not impair biological activities and physical properties of the compound. The pharmaceutically acceptable salt may be obtained by reacting the compound of the disclosure with inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, and p-toluenesulfonic acid, and organic carbonic acid such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, and salicylic acid. In addition, the pharmaceutically acceptable salt may also be obtained by reacting the compound of the disclosure with a base to form an alkali metal salt such as an ammonium salt, sodium salt, or potassium salt, a salt such as an alkaline earth metal salt such as a calcium or magnesium salt, a alt of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, and tris(hydroxymethyl)methylamine, or an amino acid salt such as arginine and lysine.

[0073] In addition, the disclosure provides a contrast agent composition including the compound or the pharmaceutically acceptable salt thereof as an active component.

[0074] In the disclosure, the expression "including as an active component" refers to that the component is included in an amount necessary or sufficient to realize a desired biological effect. In actual application, the amount included as an active component may be determined as the amount for treating a target disease by considering the amount not causing other toxicity. The amount thereof may vary depending on various factors, for example, a disease or condition being treated, a form of a composition being administered, a size of a subject, or severity of a disease or condition. One of ordinary skill in the art to which the disclosure pertains may empirically determine an effective amount of an individual composition without undue experimentation.

[0075] In the disclosure, the term "contrast agent" refers to a substance administered into a body to strongly and specifically contrast or image cancer cells or the like in the body, and is currently widely used in the medical and diagnostic fields to enhance images of tissues and cells. The term "contrast agent" of the disclosure is not limited to the scope of contrast agents for magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET), and is used to encompass imaging agents for ultrasound image analysis, imaging agents for fluorescence image analysis, and the like.

[0076] In addition, the disclosure provides an imaging method including a step of treating a biological sample with the contrast agent composition. Also, the disclosure provides an imaging method including a step of administering the contrast agent composition to a subject.

[0077] In the disclosure, the term "imaging" refers to all methods of visualizing a target object. In the disclosure, the imaging is desirably optical imaging using light.

[0078] The "imaging" may be any one or more selected from the group consisting of fluorescence, bioluminescence, MRI, CT, PET, single photon emission computed tomography (SPECT), and combinations thereof, but is not limited thereto.

[0079] Also, the disclosure provides a method of diagnosing lung cancer invasion into a visceral pleura elastin layer or an information providing method for diagnosing lung cancer invasion into a visceral pleura elastin layer, the methods including a step of treating a lung with the contrast agent composition. Also, the disclosure provides a method of diagnosing lung cancer invasion into a visceral pleura elastin layer or an information providing method for diagnosing lung cancer invasion into a visceral pleura elastin layer, the methods including a step of administering the contrast agent composition to a subject.

[0080] In the disclosure, the term "diagnosis" includes determining the susceptibility of a subject to a specific disease or disorder, determining whether a subject currently has a specific disease or disorder, determining the prognosis of a subject suffering from a specific disease or disorder, therametrics (e.g., monitoring a status of a subject to provide information about treatment efficacy, or theranostics (e.g., diagnosing a disease using a substance that targets a lesion and simultaneously treating the disease by delivering a drug only to the relevant area). Specifically, in the disclosure, the term "diagnosis" refers to determining whether lung cancer cells have invaded a visceral pleural elastin layer in a subject.

[0081] In the disclosure, the term "subject" is not limited as long as the subject is a mammal such as livestock or human, but may be desirably a human.

[0082] The contrast agent composition according to the disclosure may be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular routes, and the dose of the active component may be appropriately selected according to various factors such as the route of administration, the patient's age, sex, weight, the patient's severity, and the like. In addition, the composition of the disclosure may be administered in parallel with a known compound that may enhance a desired effect.

[0083] The terminology used herein is for the purpose of describing particular embodiments only and is not to be limiting of the embodiments. The singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises / comprising" and / or "includes / including" when used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0084] Unless otherwise defined, all terms including technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments belong. Terms, such as those defined in commonly used dictionaries, are to be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the disclosure, and are not to be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0085] In the description of the components, terms such as first, second, A, B, (a), (b) or the like may be used herein when describing components of embodiments. These terms are used only for the purpose of discriminating one component from another component, and the nature, the sequences, or the orders of the components are not limited by the terms. When one component is described as being "connected," "coupled," or "attached" to another component, it should be understood that one component may be connected or attached directly to another component, and an intervening component may also be "connected," "coupled," or "attached" to the components.

[0086] Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, various alterations and modifications may be made to the embodiments. Here, the embodiments are not construed as limited to the disclosure. The embodiments should be understood to include all modifications, equivalents, and substitutions within the scope and spirit of the disclosure.

[0087] When describing the embodiments with reference to the accompanying drawings, like reference numerals refer to like components and a repeated description related thereto will be omitted. In the following description of the embodiments, a detailed description of known functions and configurations incorporated herein will be omitted when the same may make the subject matter of the embodiments disclosed in the present specification rather unclear.

[0088] Various modifications may be made to the embodiments in various forms. Thus, the embodiments will be exemplarily shown in the drawings and described in detail in the present specification. However, the embodiments are not construed as being limited to the disclosure and should be understood to include all changes, equivalents, and replacements within the technical scope of the disclosure. In the following description of the embodiments, a detailed description of known functions and configurations incorporated herein will be omitted when the same may make the subject matter of the embodiments disclosed in the present specification rather unclear.

[0089] Preparation Example 1. Preparation of Compounds 10 to 12

[0090] A method of preparing a double bond compound according to an embodiment of the disclosure is illustrated in FIG. 1. Compounds a to z below all refer to those described in FIG. 1. A mixture of an indole substrate (Compound a or b, 1.0 eq) and (3-bromopropyl)trimethylammonium bromide (1.2 eq) in toluene (0.3 M) was heated at 100ºC for 48 hours under a nitrogen atmosphere. The mixture was cooled to room temperature and a solvent was removed. A crude mixture was filtered, collected, and redissolved in a 1:1 (v / v) mixture of distilled water and methanol. Undissolved solids were removed by filtration. After that, the mixed solution was slowly added to acetonitrile (ACN) using a dropping funnel. A precipitate was filtered, dried, and identified by liquid chromatography-mass spectrometry (LC-MS). Products (Compounds c to e) were collected as red solids and used in a next step without further purification.

[0091] Next, phosphorus oxychloride (4.5 eq) was slowly added to dimethylformamide (DMF, 9 M) in a reaction flask in an ice bath, and after thorough mixing, a mixture of cyclohexanone or 4-phenylcyclohexan-1-one and dichloromethane (1 eq, total 5 M) was added dropwise under a nitrogen atmosphere. The reaction mixture was stirred in the ice bath for 1 hour, the ice bath was removed and replaced with an oil bath to heat the reaction mixture at 100 ± 5ºC with continuous stirring for 2 hours. Then, the reaction mixture was cooled to room temperature and the reaction flask was cooled to 0ºC using the ice bath again. Next, the mixture was added dropwise to 500 mL of ice water using a dropping funnel, and stirred for 4 hours once all the mixture was added. A yellow precipitate was observed in an orange solution. The product of this step was separated and dried under vacuum. After drying, DMF and 6 M hydrochloric acid (2 eq) were mixed and the product of Step 1 was dissolved in the ice bath. Reaction reagents of a next step, aniline (1.5 eq) and ethanol, were thoroughly mixed before adding to a previous product in the ice bath. Next, the ice bath was removed and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then poured into ice water while stirring for additional 30 minutes. A precipitate was filtered, dried, and identified by an HPLC-MS. The product (Compound f or g of FIG. 1) was collected as a dark brown solid.

[0092] Compounds h to j and Compounds 19 to 21 were synthesized as previously reported by the group of inventors of the disclosure. [1. Hyun, H., et al. cGMP-Compatible preparative scale synthesis of near-infrared fluorophores. Contrast Media & Molecular Imaging 7, 516-524 (2012). / 2.　Levitz, A., Marmarchi, F. & Henary, M. Introduction of various substitutions to the methine bridge of heptamethine cyanine dyes Via substituted dianil linkers. Photochemical & Photobiological Sciences 17, 1409-1416 (2018). / 3. Yang, C., et al. ZW800-PEG: A Renal Clearable Zwitterionic Near-Infrared Fluorophore for Potential Clinical Translation. Angewandte Chemie International Edition 60, 13847-13852 (2021).] Generally, a mixture of Compounds c to e (2 eq), Vilsmeier-Haack compounds f or g (1 eq), and anhydrous sodium acetate (2 eq) in anhydrous ethanol (0.5 M) was heated at 85ºC under reflux and stirred for 2 to 8 hours under a nitrogen atmosphere. After the reaction, the mixture was cooled to room temperature, filtered, and washed with ethanol and methanol, and brownish green solids were collected and dried. The dried solids were completely redissolved in a 1:1 mixture of dimethyl sulfoxide (DMSO) and water, and reprecipitated in ethyl acetate (EA) / methanol to remove excess Compounds c to e. The precipitate was filtered, dried under a vacuum state, and collected to obtain Compounds h to m as dark green solids.

[0093] A general synthesis process of Compounds 10 to 12 is as follows. Compounds h to j (1.0 eq) and N,N-diisopropylethylamine (DIEA, 2.0 eq) were well mixed in DMSO (1 M). 2-([1,1'-biphenyl]-4-yl)ethan-1-amine (1.2 molar equivalents) in DMSO was added to a reaction solution and heated overnight at 60ºC in an oil bath. The progress was monitored using the HPLC-MS system. After the reaction, the product was cooled to room temperature, and then, 0.5 mL (0.1 v / v%) of formic acid was slowly poured into 50 mL of EA and stirred at room temperature for 30 minutes. A suspension was filtered to collect the precipitate, and the precipitate was finally washed twice with 10 mL of EA. The precipitate was dried overnight at ambient temperature and was collected as a dark green solid. A final product was further purified using preparative HPLC and then lyophilized.

[0094] Preparation Example 1.1. Preparation confirmation of 2-((E)-2-((E)-2-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium (Compound 10)

[0095]

[0096] Compound 10 prepared (2-((E)-2-((E)-2-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium) is represented by Chemical Formula 1-1, and1H NMR and13C NMR data were as follows:

[0097] Accurate mass TOF MS m / z [M]+ calculated for [C46H50N3]+ 644.40, found [M+H]+ 644.6.1H NMR (500 MHz, DMSO-d6) δ = 8.17 (t, J = 5.8 Hz, 1H), 7.70 - 7.58 (m, 4H), 7.58 -7.32 (m, 8H), 7.28 (td, J = 7.4, 1.5 Hz, 1H), 7.27 - 7.19 (m, 4H), 7.04 (td, J = 7.5, 1.5 Hz, 1H), 6.89 (dt, J = 7.7, 0.9 Hz, 1H), 6.77 (dd, J = 7.5, 1.5 Hz, 1H), 6.50 (dt, J = 7.7, 1.0 Hz, 1H), 3.63 (s, 2H), 3.43 (td, J = 7.1, 5.7 Hz, 2H), 3.30 (s, 2H), 2.86 (dtt, J = 10.3, 7.1, 1.0 Hz, 4H), 2.66 (t, J = 7.1 Hz, 2H), 1.68 (s, 4H), 1.60 (s, 4H), 1.50 (p, J = 7.1 Hz, 2H).13C NMR (125 MHz, DMSO-d6) δ = 161.18, 159.76, 146.24, 145.90, 145.64, 142.59, 140.39, 139.32, 136.70, 135.82, 132.28, 129.50, 129.30, 128.80, 128.19, 127.90, 127.56, 127.46, 127.32, 127.30, 127.00, 125.86, 123.66, 121.40, 118.10, 113.77, 113.46, 104.90, 103.01, 45.73, 45.25, 34.70, 34.47, 32.35, 31.91, 31.68, 29.50, 28.69, 28.30, 23.08.

[0098] Preparation Example 1.2. Preparation confirmation of 2-((E)-2-((E)-2-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-(2-((E)-1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium-5-sulfonate (Compound 11)

[0099]

[0100] Compound 11 prepared (2-((E)-2-((E)-2-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-(2-((E)-1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium-5-sulfonate) is represented by Chemical Formula 1-2, and1H NMR and13C NMR data were as follows:

[0101] Accurate mass TOF MS m / z [M]+ calculated for [C46H50N3O6S2]+ 804.30, found [M+H]+ 804.2.1H NMR (500 MHz, DMSO-d6) δ = 8.16 (t, J = 5.8 Hz, 1H), 7.96 (d, J = 1.5 Hz, 1H), 7.90 (dd, J = 7.5, 1.6 Hz, 1H), 7.79 (d, J = 1.5 Hz, 1H), 7.66 (ddd, J = 7.1, 1.6, 0.9 Hz, 3H), 7.62 - 7.49 (m, 4H), 7.47 - 7.38 (m, 3H), 7.40 - 7.33 (m, 1H), 7.27 - 7.19 (m, 3H), 6.89 (dt, J = 7.7, 0.9 Hz, 1H), 6.47 (dt, J = 7.9, 1.1 Hz, 1H), 3.68 (s, 2H), 3.43 (td, J = 7.1, 5.7 Hz, 2H), 3.30 (s, 2H), 2.86 (dtt, J = 10.2, 7.1, 1.0 Hz, 4H), 2.66 (t, J = 7.1 Hz, 2H), 1.69 (s, 4H), 1.63 (s, 4H), 1.50 (p, J = 7.1 Hz, 2H).13C NMR (125 MHz, DMSO-d6) δ = 164.69, 161.55, 149.53, 145.90, 143.58, 142.59, 142.10, 142.04, 140.39, 139.33, 135.82, 133.75, 132.20, 129.50, 128.83, 128.80, 127.90, 127.57, 127.32, 127.00, 126.38, 124.14, 123.19, 121.58, 116.49, 115.24, 110.18, 92.50, 45.95, 45.47, 34.70, 34.46, 31.53, 31.25, 30.58, 29.50, 28.86, 28.30, 21.78.

[0102] Preparation Example 1.3. Preparation confirmation of 2-((E)-2-((E)-2-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate (Compound 12)

[0103]

[0104] Compound 12 prepared (2-((E)-2-((E)-2-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate) is represented by Chemical Formula 1-3, and1H NMR and13C NMR data were as follows:

[0105] Accurate mass TOF MS m / z [M]+ calculated for [C56H72N5O6S2]+ 974.49, found [M]+ 974.4.1H NMR (500 MHz, DMSO-d6) 1H NMR (500 MHz, DMSO-d6) δ = 8.16 (t, J = 5.8 Hz, 1H), 7.96 (d, J = 1.6 Hz, 1H), 7.91 (dd, J = 7.4, 1.6 Hz, 1H), 7.79 (d, J = 1.5 Hz, 1H), 7.68 - 7.62 (m, 3H), 7.59 (d, J = 15.0 Hz, 1H), 7.55 - 7.46 (m, 3H), 7.44 - 7.33 (m, 4H), 7.29 - 7.20 (m, 3H), 6.89 (dt, J = 8.2, 1.0 Hz, 1H), 6.41 (dt, J = 8.2, 1.0 Hz, 1H), 4.53 (t, J = 7.1 Hz, 2H), 4.00 (t, J = 7.1 Hz, 2H), 3.85 (t, J = 7.1 Hz, 2H), 3.54 - 3.47 (m, 2H), 3.43 (td, J = 7.1, 5.7 Hz, 2H), 3.27 (d, J = 1.0 Hz, 15H), 2.86 (dtt, J = 10.3, 7.2, 1.0 Hz, 4H), 2.66 (t, J = 7.1 Hz, 2H), 2.12 (ddq, J = 29.2, 14.0, 7.0 Hz, 2H), 1.97 (p, J = 7.1 Hz, 2H), 1.69 (s, 4H), 1.63 (s, 4H), 1.50 (p, J = 7.1 Hz, 2H).13C NMR (125 MHz, DMSO-d6) δ = 164.69, 161.55, 149.53, 145.90, 143.58, 142.59, 142.10, 142.04, 140.39, 139.33, 135.82, 133.75, 132.20, 129.50, 128.83, 128.80, 127.90, 127.57, 127.32, 127.00, 126.38, 124.14, 123.19, 121.58, 116.49, 115.24, 110.18, 92.50, 45.95, 45.47, 34.70, 34.46, 31.53, 31.25, 30.58, 29.50, 28.86, 28.30, 21.78.

[0106] Preparation Example 2. Preparation of Compounds 13 to 15

[0107] Generally, 3-(4-boronophenyl) propanoic acid (1.8 eq) and precursor chloro-dye compounds h to j (1.0 eq) in H2O were heated under reflux for 72 hours in the presence of Pd(PPh3)4 (0.2 eq). The reaction progress was monitored using the HPLC-MS system. The reaction mixture was then cooled to room temperature, and H2O was removed under reduced pressure. The solid was separated by precipitation with methanol / acetone, and the precipitate was further washed with acetone. Fluorophores k to m with analytical purity were obtained using open-reversed phase column chromatography (elution with ACN / water).

[0108] The fluorophores were added to anhydrous DMSO (0.1 M), triethylamine (TEA, 2.0 eq), and 2.0 eq of HSPyU to prepare NHS esters of Fluorophores k to m. The reaction mixture was stirred at room temperature for 1 to 2 hours, and the reaction progress was monitored by HPLC. The reaction solution was slowly poured into 10-fold EA at room temperature and stirred for 30 minutes to obtain precipitated green solids, and the precipitated green solids were filtered and washed with EA. Finally, the collected green solids were dried in a vacuum state for 1 hour and used in a next step without further purification. Next, the NHS esters were dissolved in DMSO (0.1 M) together with 1.5 eq of 2-([1,1'-biphenyl]-4-yl)ethan-1-amine, and 2.0 eq of TEA was added to the solution to adjust a final pH to about 10. The reaction mixture was stirred at room temperature for 1 hour, and the reaction progress was monitored by HPLC. The reaction solution was slowly poured into EA at room temperature (10X volume equivalent) and further stirred for 30 minutes to obtain precipitated green solids, and the precipitated green solids were filtered and further washed with EA. Finally, the collected solids were further purified using preparative HPLC and then lyophilized to obtain final green solid powders, Compounds 13 to 15.

[0109] Preparation Example 2.1. Preparation confirmation of 2-((E)-2-((E)-4'-(3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-oxopropyl)-6-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium (Compound 13)

[0110]

[0111] Compound 13 prepared (2-((E)-2-((E)-4'-(3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-oxopropyl)-6-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium) is represented by Chemical Formula 1-4, and1H NMR and13C NMR data were as follows:

[0112] Accurate mass TOF MS m / z [M]+ calculated for [C55H58N3O]+ 776.45, found [M]+ 776.4.1H NMR (500 MHz, DMSO-d6) 1H NMR (500 MHz, DMSO-d6) δ = 7.70 - 7.49 (m, 10H), 7.44 - 7.32 (m, 5H), 7.30 - 7.23 (m, 3H), 7.21 (dt, J = 7.5, 1.0 Hz, 2H), 7.12 (dt, J = 7.5, 1.0 Hz, 2H), 7.08 - 7.00 (m, 2H), 6.77 (dd, J = 7.5, 1.5 Hz, 1H), 6.47 (dt, J = 7.5, 1.0 Hz, 1H), 3.63 (s, 2H), 3.30 (d, J = 5.9 Hz, 5H), 2.98 - 2.86 (m, 6H), 2.77 (tt, J = 7.2, 1.1 Hz, 2H), 2.38 (t, J = 7.1 Hz, 2H), 1.70 (s, 4H), 1.62 (s, 4H), 1.53 (p, J = 7.1 Hz, 2H).13C NMR (125 MHz, DMSO-d6) δ = 174.16, 158.47, 157.06, 146.13, 145.90, 144.88, 142.59, 140.39, 139.39, 138.93, 136.70, 136.02, 135.82, 135.20, 134.04, 132.94, 130.14, 129.50, 128.80, 128.19, 127.90, 127.80, 127.56, 127.32, 127.30, 127.00, 126.69, 126.17, 121.40, 118.10, 108.08, 104.90, 103.72, 45.46, 44.99, 41.07, 36.51, 35.76, 31.92, 30.95, 29.50, 28.78, 28.30, 26.70, 26.25, 23.04.

[0113] Preparation Example 2.2. Preparation confirmation of 2-((E)-2-((E)-4'-(3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-oxopropyl)-6-(2-((E)-1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium-5-sulfonate (Compound 14)

[0114]

[0115] Compound 14 prepared (2-((E)-2-((E)-4'-(3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-oxopropyl)-6-(2-((E)-1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium-5-sulfonate) is represented by Chemical Formula 1-5, and1H NMR and13C NMR data were as follows:

[0116] Accurate mass TOF MS m / z [M]+ calculated for [C55H58N3O7S2]+ 936.36, found [M]+ 936.4.1H NMR (500 MHz, DMSO-d6) 1H NMR (500 MHz, DMSO-d6) δ = 7.96 (d, J = 1.5 Hz, 1H), 7.90 (dd, J = 7.5, 1.6 Hz, 1H), 7.79 (d, J = 1.5 Hz, 1H), 7.68 - 7.58 (m, 4H), 7.57 - 7.33 (m, 10H), 7.27 (t, J = 6.7 Hz, 1H), 7.21 (dt, J = 7.6, 1.0 Hz, 2H), 7.11 (dt, J = 7.5, 1.1 Hz, 2H), 7.03 (dt, J = 7.5, 0.9 Hz, 1H), 6.47 (dt, J = 7.5, 1.0 Hz, 1H), 3.68 (s, 2H), 3.32 - 3.25 (m, 5H), 2.98 - 2.86 (m, 6H), 2.77 (tt, J = 7.3, 1.0 Hz, 2H), 2.38 (t, J = 7.1 Hz, 2H), 1.69 (s, 4H), 1.63 (s, 4H), 1.53 (p, J = 7.1 Hz, 2H).13C NMR (125 MHz, DMSO-d6) δ = 174.16,161.98, 158.85, 149.42, 145.90, 143.07, 142.59, 142.10, 141.66, 141.43, 140.39, 139.39, 138.74, 135.82, 135.67, 135.20, 133.96, 133.24, 129.50, 128.83, 128.80, 127.90, 127.32, 127.00, 126.17, 124.06, 123.25, 122.55, 110.25, 109.86, 93.21, 45.68, 45.21, 41.07, 35.76, 35.49, 31.54, 30.95, 29.50, 28.95, 28.30, 26.70, 26.25, 21.74.

[0117] Preparation Example 2.3. Preparation confirmation of 2-((E)-2-((E)-4'-(3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-oxopropyl)-6-(2-((E)-3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate (Compound 15)

[0118]

[0119] Compound 15 prepared (2-((E)-2-((E)-4'-(3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-oxopropyl)-6-(2-((E)-3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate) is represented by Chemical Formula 1-6, and1H NMR and13C NMR data were as follows:

[0120] Accurate mass TOF MS m / z [M]+ calculated for [C65H80N5O7S2]+ 1106.55, found [M]+ 1106.8.1H NMR (500 MHz, DMSO-d6) 1H NMR (500 MHz, DMSO-d6) δ = 7.96 (d, J = 1.6 Hz, 1H), 7.91 (dd, J = 7.4, 1.6 Hz, 1H), 7.79 (d, J = 1.5 Hz, 1H), 7.68 - 7.62 (m, 3H), 7.62 - 7.32 (m, 10H), 7.27 (t, J = 6.7 Hz, 1H), 7.21 (dt, J = 7.5, 1.0 Hz, 2H), 7.11 (dt, J = 7.5, 1.1 Hz, 2H), 7.03 (dt, J = 8.1, 1.0 Hz, 1H), 6.47 (dt, J = 8.1, 1.0 Hz, 1H), 4.53 (t, J = 7.1 Hz, 2H), 4.00 (t, J = 7.1 Hz, 2H), 3.85 (t, J = 7.1 Hz, 2H), 3.54 - 3.47 (m, 2H), 3.27 (d, J = 0.9 Hz, 19H), 2.98 - 2.86 (m, 6H), 2.77 (tt, J = 7.3, 1.0 Hz, 2H), 2.38 (t, J = 7.2 Hz, 2H), 2.12 (ddq, J = 29.2, 14.0, 7.0 Hz, 2H), 1.97 (p, J = 7.1 Hz, 2H), 1.69 (s, 4H), 1.63 (s, 4H), 1.53 (p, J = 7.1 Hz, 2H).13C NMR (125 MHz, DMSO-d6) δ = 174.16, 165.15, 162.89, 150.67, 148.66, 145.86, 142.59, 142.14, 142.10, 141.36, 140.39, 139.39, 138.77, 135.82, 135.20, 134.82, 134.63, 131.73, 129.50, 128.83, 128.80, 127.90, 127.32, 127.00, 126.22, 126.17, 125.16, 122.24, 121.77, 121.24, 114.88, 88.01, 68.95, 63.04, 60.65, 52.96, 51.55, 45.32, 44.85, 41.07, 35.76, 34.36, 30.95, 29.50, 28.74, 28.31, 26.70, 26.25, 23.86, 21.74.

[0121] Preparation Example 3. Preparation of Compounds 16 to 18

[0122] Compounds h to j (1.0 eq) and DIEA (2.0 eq) were well mixed in DMSO (1 M). Then, thiol-PEG2-acid (1.2 molar equivalents) in DMSO was added to a reaction solution and heated at 60ºC in an oil bath for 0.5 to 2 hours. The progress was monitored using the HPLC-MS system. After the reaction, the product was cooled to room temperature, and then, 0.5 mL (0.1 v / v%) of formic acid was slowly poured into 50 mL of EA and stirred at room temperature for 30 minutes. A suspension was filtered to collect the precipitate, and the precipitate was finally washed twice with 10 mL of EA. The precipitate was dried overnight at ambient temperature and collected as a dark green solids. The products (Compounds n to p) were further purified using preparative HPLC and then lyophilized.

[0123] Similar to the synthesis of Compounds 13 to 15, HSPyU chemistry was applied to synthesize NHS esters of Fluorophores n to p. The reaction progress was monitored by HPLC. The collected green solid NHS esters were dried in a vacuum state and used in a next step without further purification. Next, the NHS esters were dissolved in DMSO (0.1 M) together with 1.5 eq of 2-([1,1'-biphenyl]-4-yl)ethan-1-amine, and 2.0 eq of TEA was added to the solution to adjust a final pH to about 10. The reaction mixture was stirred at room temperature for 1 hour, and the reaction progress was monitored by HPLC. The reaction solution was slowly poured into EA at room temperature (10X volume equivalent) and further stirred for 30 minutes to obtain precipitated green solids, and the precipitated green solids were filtered and further washed with EA. Finally, the collected solids were further purified using preparative HPLC and then lyophilized to obtain final green solid powders, Compounds 16 to 18.

[0124] Preparation Example 3.1. Preparation confirmation of 2-((E)-2-((E)-2-((2-(2-(3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-oxopropoxy)ethoxy)ethyl)thio)-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium (Compound 16)

[0125]

[0126] Compound 16 prepared (2-((E)-2-((E)-2-((2-(2-(3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-oxopropoxy)ethoxy)ethyl)thio)-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium) is represented by Chemical Formula 1-7, and1H NMR and13C NMR data were as follows:

[0127] Accurate mass TOF MS m / z [M]+ calculated for [C53H62N3O3S]+ 820.45, found [M]+ 820.8.1H NMR (500 MHz, DMSO-d6) 1H NMR (500 MHz, DMSO-d6) δ = 7.70 - 7.32 (m, 12H), 7.30 - 7.17 (m, 5H), 7.05 (td, J = 7.4, 1.5 Hz, 1H), 6.89 (dt, J = 7.7, 1.0 Hz, 1H), 6.77 (dd, J = 7.5, 1.5 Hz, 1H), 6.49 (dt, J = 7.7, 0.9 Hz, 1H), 3.72 (td, J = 7.1, 4.7 Hz, 4H), 3.63 (s, 2H), 3.63 - 3.52 (m, 4H), 3.30 (s, 3H), 3.27 (ddt, J = 22.5, 14.1, 7.1 Hz, 2H), 3.12 (t, J = 7.1 Hz, 2H), 2.89 (dt, J = 18.1, 7.1 Hz, 1H), 2.82 (tt, J = 7.1, 1.0 Hz, 2H), 2.77 (ddd, J = 7.3, 6.2, 1.1 Hz, 2H), 2.77 - 2.67 (m, 1H), 2.49 (t, J = 7.1 Hz, 2H), 1.70 (s, 4H), 1.62 (s, 4H), 1.55 (p, J = 7.1 Hz, 2H).13C NMR (125 MHz, DMSO-d6) δ = 171.47, 170.22, 163.57, 145.69, 141.72, 141.54, 141.27, 141.19, 140.45, 138.59, 138.47, 138.18, 137.66, 131.84, 131.01, 129.65, 129.22, 129.21, 128.98, 127.95, 127.71, 127.56, 127.19, 124.62, 122.65, 122.53, 122.29, 113.54, 111.37, 103.55, 69.33, 69.20, 68.60, 66.64, 48.72, 46.25, 40.58, 37.36, 35.61, 35.36, 33.40, 32.59, 29.54, 28.02, 27.97, 25.58, 24.26.

[0128] Preparation Example 3.2. Preparation confirmation of 2-((E)-2-((E)-2-((2-(2-(3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-oxopropoxy)ethoxy)ethyl)thio)-3-(2-((E)-1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium-5-sulfonate (Compound 17)

[0129]

[0130] Compound 17 prepared (2-((E)-2-((E)-2-((2-(2-(3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-oxopropoxy)ethoxy)ethyl)thio)-3-(2-((E)-1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium-5-sulfonate) is represented by Chemical Formula 1-8, and1H NMR and13C NMR data were as follows:

[0131] Accurate mass TOF MS m / z [M]+ calculated for [C53H62N3O9S3]+ 978.35, found [M]+ 978.7.1H NMR (500 MHz, DMSO-d6) 1H NMR (500 MHz, DMSO-d6) δ = 7.96 (d, J = 1.5 Hz, 1H), 7.90 (dd, J = 7.5, 1.6 Hz, 1H), 7.79 (d, J = 1.5 Hz, 1H), 7.68 - 7.60 (m, 3H), 7.60 - 7.49 (m, 3H), 7.48 - 7.33 (m, 5H), 7.26 - 7.18 (m, 3H), 6.89 (dt, J = 7.7, 1.0 Hz, 1H), 6.49 (dt, J = 7.7, 0.9 Hz, 1H), 3.76 - 3.66 (m, 6H), 3.63 - 3.52 (m, 4H), 3.30 (s, 3H), 3.27 (ddt, J = 22.5, 14.1, 7.1 Hz, 2H), 3.12 (t, J = 7.1 Hz, 2H), 2.89 (dt, J = 18.1, 7.1 Hz, 1H), 2.82 (tt, J = 7.1, 1.0 Hz, 2H), 2.77 (ddd, J = 7.3, 6.2, 1.1 Hz, 2H), 2.77 - 2.67 (m, 1H), 2.49 (t, J = 7.1 Hz, 2H), 1.69 (s, 4H), 1.63 (s, 4H), 1.50 (p, J = 7.1 Hz, 2H).13C NMR (125 MHz, DMSO-d6) δ = 171.70, 170.14, 163.57, 146.51, 142.76, 141.97, 141.54, 141.30, 141.19, 141.05, 140.33, 138.47, 138.18, 137.70, 135.56, 131.84, 129.22, 128.97, 128.91, 127.70, 127.56, 127.19, 125.24, 124.94, 123.63, 122.50, 122.29, 113.50, 111.81, 103.55, 69.34, 69.33, 68.77, 66.67, 48.84, 46.37, 40.58, 37.36, 35.61, 35.34, 33.24, 32.59, 29.85, 28.14, 27.85, 25.58, 24.53.

[0132] Preparation Example 3.3. Preparation confirmation of 2-((E)-2-((E)-2-((2-(2-(3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-oxopropoxy)ethoxy)ethyl)thio)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate (Compound 18)

[0133]

[0134] Compound 18 prepared (2-((E)-2-((E)-2-((2-(2-(3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-oxopropoxy)ethoxy)ethyl)thio)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate) is represented by Chemical Formula 1-9, and1H NMR and13C NMR data were as follows:

[0135] Accurate mass TOF MS m / z [M]+ calculated for [C63H85N5O9S3]+ 1150.54, found [M]+ 1150.3.1H NMR (500 MHz, DMSO-d6) 1H NMR (500 MHz, DMSO-d6) δ = 7.96 (d, J = 1.6 Hz, 1H), 7.91 (dd, J = 7.4, 1.6 Hz, 1H), 7.79 (d, J = 1.5 Hz, 1H), 7.68 - 7.62 (m, 3H), 7.58 (d, J = 15.0 Hz, 1H), 7.55 - 7.47 (m, 3H), 7.44 (t, J = 6.7 Hz, 1H), 7.41 (d, J = 7.5 Hz, 1H), 7.41 - 7.32 (m, 3H), 7.27 - 7.18 (m, 3H), 6.89 (dt, J = 8.2, 1.0 Hz, 1H), 6.44 (dt, J = 8.2, 1.0 Hz, 1H), 4.53 (t, J = 7.1 Hz, 2H), 4.00 (t, J = 7.1 Hz, 2H), 3.85 (t, J = 7.1 Hz, 2H), 3.72 (td, J = 7.1, 4.7 Hz, 4H), 3.63 - 3.53 (m, 4H), 3.51 (t, J = 7.0 Hz, 2H), 3.27 (d, J = 0.9 Hz, 19H), 3.12 (t, J = 7.1 Hz, 2H), 2.89 (dt, J = 18.1, 7.1 Hz, 1H), 2.82 (tt, J = 7.1, 1.0 Hz, 2H), 2.77 (ddd, J = 7.2, 6.2, 1.1 Hz, 2H), 2.77 - 2.67 (m, 1H), 2.49 (t, J = 7.1 Hz, 2H), 2.12 (ddq, J = 29.2, 14.0, 7.0 Hz, 2H), 1.97 (p, J = 7.1 Hz, 2H), 1.69 (s, 4H), 1.63 (s, 4H).13C NMR (125 MHz, DMSO-d6) δ = 172.03, 170.19, 164.30, 144.23, 143.46, 142.90, 142.41, 141.19, 140.84, 140.44, 140.30, 138.45, 138.18, 136.74, 136.20, 131.84, 130.07, 129.22, 129.04, 127.72, 127.62, 127.19, 125.10, 125.04, 123.61, 122.63, 120.87, 115.34, 115.15, 103.08, 69.34, 69.33, 68.77, 66.70, 64.36, 63.99, 53.13, 52.95, 50.27, 49.47, 48.21, 47.65, 40.38, 37.22, 35.34, 33.24, 29.85, 28.14, 27.93, 26.14, 25.58, 24.68, 24.30.

[0136] Preparation Example 4. Preparation of Compounds 19 to 21

[0137] The synthesis procedure for Compounds 19 to 21 was similar to the synthesis procedure for Compounds h to j. A mixture obtained by mixing Compounds c to e (2.0 eq), Vilsmeier-Haack reagent g (1.0 eq), and anhydrous sodium acetate (2.0 eq) into anhydrous ethanol (0.5 M) was heated under reflux at 85ºC for 2 to 8 hours under a nitrogen atmosphere. After the reaction, the mixture was cooled to room temperature, filtered, and washed with ethanol and methanol, and brownish green solids were collected and dried. The dried solids were completely redissolved in a 1:1 mixture of DMSO and water, and reprecipitated in EA / methanol to remove excess Compounds c to e. The precipitate was filtered, dried under a vacuum state, and collected to obtain Compounds h to m as green solids.

[0138] Preparation Example 4.1. Preparation confirmation of 2-((E)-2-((E)-4-chloro-5-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)-1,2,5,6-tetrahydro-[1,1'-biphenyl]-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium (Compound 19)

[0139]

[0140] Compound 19 prepared (2-((E)-2-((E)-4-chloro-5-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)-1,2,5,6-tetrahydro-[1,1'-biphenyl]-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium) is represented by Chemical Formula 1-10, and1H NMR and13C NMR data were as follows:

[0141] Accurate mass TOF MS m / z [M]+ calculated for [C38H40ClN2]+ 559.29, found [M]+ 559.5.1H NMR (500 MHz, DMSO-d6) 1H NMR (500 MHz, DMSO-d6) δ = 7.88 (d, J = 15.0 Hz, 1H), 7.67 (td, J = 7.5, 1.5 Hz, 1H), 7.55 (dd, J = 7.6, 1.6 Hz, 1H), 7.46 (td, J = 7.4, 1.5 Hz, 1H), 7.35 (dd, J = 7.5, 1.4 Hz, 1H), 7.33 - 7.27 (m, 2H), 7.30 - 7.25 (m, 1H), 7.27 - 7.19 (m, 5H), 7.09 - 7.02 (m, 2H), 6.77 (dd, J = 7.5, 1.5 Hz, 1H), 6.57 (dt, J = 7.9, 1.0 Hz, 1H), 3.63 (s, 2H), 3.31 (s, 3H), 3.35 - 3.26 (m, 1H), 3.16 - 3.05 (m, 4H), 1.69 (s, 2H), 1.63 (d, J = 11.0 Hz, 4H), 1.57 (s, 2H).13C NMR (125 MHz, DMSO-d6) δ 169.38, 163.93, 145.53, 144.26, 142.79, 141.50, 138.91, 138.43, 137.34, 136.77, 135.91, 133.93, 128.33, 128.19, 128.07, 127.55, 126.41, 124.43, 123.77, 123.74, 123.05, 122.52, 114.09, 113.39, 103.86, 48.97, 47.32, 38.33, 37.41, 36.25, 35.36, 33.36, 28.02, 25.92.

[0142] Preparation Example 4.2. Preparation confirmation of 2-((E)-2-((E)-4-chloro-5-(2-((E)-1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)ethylidene)-1,2,5,6-tetrahydro-[1,1'-biphenyl]-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium-5-sulfonate (Compound 20)

[0143]

[0144] Compound 20 prepared (2-((E)-2-((E)-4-chloro-5-(2-((E)-1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)ethylidene)-1,2,5,6-tetrahydro-[1,1'-biphenyl]-3-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium-5-sulfonate) is represented by Chemical Formula 1-11, and1H NMR and13C NMR data were as follows:

[0145] Accurate mass TOF MS m / z [M]+ calculated for [C38H40ClN2O6S2]+ 719.19, found [M]+ 719.3.1H NMR (500 MHz, DMSO-d6) 1H NMR (500 MHz, DMSO-d6) δ = 7.96 (d, J = 1.5 Hz, 1H), 7.90 (dd, J = 7.5, 1.6 Hz, 1H), 7.85 (d, J = 15.2 Hz, 1H), 7.79 (d, J = 1.5 Hz, 1H), 7.65 (dd, J = 7.4, 1.6 Hz, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.45 (d, J = 7.5 Hz, 1H), 7.31 (d, J = 15.0 Hz, 1H), 7.26 (s, 2H), 7.31 - 7.19 (m, 3H), 7.04 (dt, J = 7.9, 1.0 Hz, 1H), 6.58 (dt, J = 7.9, 1.0 Hz, 1H), 3.68 (s, 2H), 3.35 - 3.26 (m, 1H), 3.30 (s, 3H), 3.19 - 3.10 (m, 4H), 1.70 (s, 2H), 1.65 (s, 6H), 1.60 (s, 2H).13C NMR (125 MHz, DMSO-d6) δ = 169.33, 163.22, 146.17, 144.26, 142.79, 142.66, 141.87, 141.56, 138.08, 137.67, 137.07, 135.95, 135.91, 128.42, 128.26, 126.48, 125.24, 125.17, 125.10, 124.12, 122.70, 121.56, 113.51, 111.81, 103.86, 48.89, 46.83, 38.35, 37.45, 36.24, 35.36, 32.62, 27.85, 25.78.

[0146] Preparation Example 4.3. Preparation confirmation of 2-((E)-2-((E)-4-chloro-5-(2-((E)-3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethylidene)-1,2,5,6-tetrahydro-[1,1'-biphenyl]-3-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate (Compound 21)

[0147]

[0148] Compound 21 prepared (2-((E)-2-((E)-4-chloro-5-(2-((E)-3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethylidene)-1,2,5,6-tetrahydro-[1,1'-biphenyl]-3-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate) is represented by Chemical Formula 1-12, and1H NMR and13C NMR data were as follows:

[0149] Accurate mass TOF MS m / z [M]+ calculated for [C48H64ClN4O6S2]+ 891.38, found [M]+ 891.7.1H NMR (500 MHz, DMSO-d6) 1H NMR (500 MHz, DMSO-d6) δ = 7.96 (d, J = 1.6 Hz, 1H), 7.91 (dd, J = 7.4, 1.6 Hz, 1H), 7.87 - 7.77 (m, 2H), 7.65 (dd, J = 7.5, 1.6 Hz, 1H), 7.49 (d, J = 7.5 Hz, 1H), 7.41 (d, J = 7.5 Hz, 1H), 7.36 - 7.23 (m, 2H), 7.26 (s, 3H), 7.26 - 7.19 (m, 1H), 7.10 (dt, J = 8.6, 1.0 Hz, 1H), 6.58 (dt, J = 8.4, 1.0 Hz, 1H), 4.59 - 4.46 (m, 2H), 4.05 (dt, J = 12.3, 7.1 Hz, 1H), 3.95 (dt, J = 12.5, 7.1 Hz, 1H), 3.85 (qt, J = 12.3, 7.0 Hz, 2H), 3.49 (dt, J = 12.5, 7.1 Hz, 1H), 3.42 (dt, J = 12.3, 7.1 Hz, 1H), 3.27 (d, J = 1.0 Hz, 16H), 3.19 - 3.10 (m, 4H), 2.20 - 2.04 (m, 2H), 2.04 - 1.88 (m, 2H), 1.71 (s, 2H), 1.66 (d, J = 5.9 Hz, 6H), 1.60 (s, 2H).13C NMR (125 MHz, DMSO-d6) δ = 170.19, 163.89, 148.52, 144.20, 143.45, 143.03, 142.41, 140.49, 137.67, 137.25, 137.02, 136.12, 135.30, 128.70, 128.46, 126.46, 126.40, 125.07, 124.32, 123.75, 123.01, 121.17, 115.16, 115.14, 105.64, 64.32, 63.91, 53.64, 53.54, 49.83, 49.55, 48.27, 47.57, 38.35, 37.43, 36.20, 27.90, 26.25, 26.14, 25.78.

[0150] Experimental Example 1. Confirmation of specific binding of double bond compound of disclosure to elastin

[0151] The double bond compound according to an embodiment of the disclosure was dissolved in saline and sprayed to lungs of mice to determine whether the double bond compound specifically binds to elastin. It is an image which is captured 5 to 10 minutes after dissolving 5 to 50 μM of Compound 19 (Chemical Formula 1-10) in 5 to 10 mL of saline and spraying it to the lungs of 25 g CD-1 mice. A surface was washed with saline three times before imaging. All images were imaged using an NIR filter using FIAT-LTMimaging device. At this time, the filter used has Ex = 750 ± 25-nm; Em = 810 ± 20-nm.

[0152] FIG. 2 illustrates a case where Double Bond Compound 19 of the disclosure is sprayed to lungs of a normal mouse, the entire area of lungs, a case where a lobectomy is performed on the lungs, and a case where a segmentectomy is performed on the lungs. FIG. 3 illustrates results of observing fluorescence expression using a fluorescence microscope after sectioning lung tissue obtained in FIG. 2 to have a thickness of 6 to 50 μm. Since an elastin layer is present on a surface of a normal lung, it may be confirmed that Compound 19 is bound to the entire surface of the lung.

[0153] Meanwhile, FIG. 4 illustrates images of lungs when Double Bond Compound 19 of the disclosure was sprayed to the lungs of mice with tumors. Since lung cancer tissue has a destroyed elastin layer, it may be observed that Compound 19is not bound, does not show a fluorescence signal, and is thus shown dark. In other words, it may be seen that the double bond compound of the disclosure is specifically bound to elastin.

[0154] Experimental Example 2. Optimization of dosage concentration and binding time of double bond compound

[0155] To establish the conditions under which the double bond compound according to the disclosure binds to elastin in lung tissue and exhibits optimal imaging performance, concentration- and time-dependent fluorescence changes were observed using a mouse model.

[0156] First, as shown in FIG. 5, Compound 19 (Elastin 800) was diluted in saline at various concentrations (0 mM, 0.005 mM, 0.01 mM, 0.025 mM, 0.05 mM, and 0.1 mM) and sprayed to the lungs of normal mice. As a result, it was confirmed that the fluorescence signal intensity on the lung surface increased as the concentration increased. Particularly, sufficient fluorescence intensity for clearly distinguishing the elastin layer was shown at a concentration of 0.05 mM or higher, thereby confirming an optimal concentration range that may clearly secure a target signal with respect to a background signal during clinical application.

[0157] In addition, a binding reaction rate was determined for a rapid intraoperative diagnosis. FIG. 6 illustrates fluorescence images captured 15 seconds, 30 seconds, 1 minute, 3 minutes, and 5 minutes after the lungs of mice were treated with Compound 19. Fluorescent signals began to be observed within 1 minute after the treatment, and significantly clear and stable fluorescent images were able to be obtained between 3 and 5 minutes. This implies that the compound of the disclosure may detect a target site within minutes after application, making it highly suitable for real-time intraoperative imaging that requires rapidity.

[0158] Experimental Example 3. Validation using rabbit lung model

[0159] For validation in a medium- to large-sized animal model that is more similar to the human lung structure than the mouse model, an experiment was conducted using rabbit lungs. In the experiment, a compound with a concentration of 0.05 mM, which is the optimized condition in Experimental Example 2 above, was used and a reaction (incubation) was performed for 3 minutes.

[0160] FIG. 7 illustrates results of compound treatment on rabbit lungs with lesions in which an elastin layer was destroyed by artificial damage. As a result of imaging after the compound treatment and 1 wash, strong green fluorescence (based on merge images) was observed in areas with normal visceral pleura, whereas no fluorescence signal was observed in areas with damaged elastin layers (black arrows or dark areas) (negative contrast). This is a visual confirmation that the compound of the disclosure may clearly distinguish the area where the elastin layer is destroyed due to lung cancer invasion or the like.

[0161] Furthermore, to confirm the precision of the binding, the tissue was sectioned and observed at a high magnification. FIG. 8 is an image of rabbit lung tissue observed under an EVOS fluorescence microscope at 40x magnification. It may be confirmed that an NIR fluorescence signal, marked in red, is formed clearly and thinly along a line of the visceral pleura, which is a thin membrane of the lung surface. This shows that the compound of the disclosure does not unnecessarily penetrate into the lung parenchyma, but precisely binds only to the elastin layer of a target surface. Therefore, it was confirmed that this compound has excellent value as a precise diagnostic contrast agent for determining whether there is microscopic lung cancer invasion.

[0162] While the embodiments are described with reference to drawings, it will be apparent to one of ordinary skill in the art that various alterations and modifications in form and details may be made in these embodiments without departing from the spirit and scope of the claims and their equivalents. For example, suitable results may be achieved if the described techniques are performed in a different order, and / or if components in a described system, architecture, device, or circuitry are combined in a different manner, or replaced or supplemented by other components or their equivalents.

[0163] Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims

1.A compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof:in Chemical Formula 1,R1and R2are the same as or different from each other, and are each independently one or more selected from the group consisting of hydrogen, a hydroxyl group, a C1-C5alkoxy group, (Y)mX, (Y)mCOO-, (Y)mSO3-, (Y)mPO3H-, and combinations thereof,R3and R4are the same as or different from each other, and are each independently one or more selected from the group consisting of (Y)mH, a C1-C5alkoxy group, a C1-C5alkenyl group, (Y)mNR5R6R7+, and combinations thereof,R5, R6, and R7are the same as or different from each other, and are each independently hydrogen or an unsubstituted C1-C5chain alkyl group,X is a halogen group,Y is CH2or CH2CH2O,Z is a halogen group,,, or, A is,, or, and a, b, c, d, e, and m are the same as or different from each other, and are each independently an integer from 0 to 5.2.The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein, in Chemical Formula 1, R1and R2are each independently hydrogen or (Y)mSO3-.3.The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein, in Chemical Formula 1, R3and R4are each independently a C1-C5chain alkyl group or (Y)mNR5R6R7+, and R5, R6, and R7are each independently hydrogen or a methyl group.4.The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein, in Chemical Formula 1, Z is a halogen group, , , or .5.The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein the compound is one or more selected from the group consisting of compounds represented by Chemical Formula 1-1 to Chemical Formula 1-12.6.The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein the compound or the pharmaceutically acceptable salt thereof selectively binds to elastin.7.The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein the compound or the pharmaceutically acceptable salt thereof absorbs light in a near-infrared (NIR) range.8.A contrast agent composition comprising a compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active component:in Chemical Formula 1,R1and R2are the same as or different from each other, and are each independently one or more selected from the group consisting of hydrogen, a hydroxyl group, a C1-C5alkoxy group, (Y)mX, (Y)mCOO-, (Y)mSO3-, (Y)mPO3H-, and combinations thereof,R3and R4are the same as or different from each other, and are each independently one or more selected from the group consisting of (Y)mH, a C1-C5alkoxy group, a C1-C5alkenyl group, (Y)mNR5R6R7+, and combinations thereof,R5, R6, and R7are the same as or different from each other, and are each independently hydrogen or an unsubstituted C1-C5chain alkyl group,X is a halogen group,Y is CH2or CH2CH2O,Z is a halogen group,,, or, A is,, or,a, b, c, d, e, and m are the same as or different from each other, and are each independently an integer from 0 to 5, andthe compound or the pharmaceutically acceptable salt thereof selectively binds to elastin.9.The contrast agent composition of claim 8, wherein the contrast agent composition is a near-infrared (NIR) fluorescent contrast agent.10.The contrast agent composition of claim 8, wherein the contrast agent composition is used in diagnosing lung cancer invasion into a visceral pleura elastin layer.11.An imaging method comprising the following steps:(1) treating a biological sample with a contrast agent composition comprising a compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active component; and(2) detecting a fluorescent signal emitted from the biological sample,in Chemical Formula 1,R1and R2are the same as or different from each other, and are each independently one or more selected from the group consisting of hydrogen, a hydroxyl group, a C1-C5alkoxy group, (Y)mX, (Y)mCOO-, (Y)mSO3-, (Y)mPO3H-, and combinations thereof,R3and R4are the same as or different from each other, and are each independently one or more selected from the group consisting of (Y)mH, a C1-C5alkoxy group, a C1-C5alkenyl group, (Y)mNR5R6R7+, and combinations thereof,R5, R6, and R7are the same as or different from each other, and are each independently hydrogen or an unsubstituted C1-C5chain alkyl group,X is a halogen group,Y is CH2or CH2CH2O,Z is a halogen group,,, or, A is,, or,a, b, c, d, e, and m are the same as or different from each other, and are each independently an integer from 0 to 5, andthe compound or the pharmaceutically acceptable salt thereof selectively binds to elastin.12.An information providing method for diagnosing lung cancer invasion into a visceral pleura elastin layer, the method comprising the following steps:(1) treating a lung with a contrast agent composition comprising a compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active component;(2) detecting a fluorescent signal emitted from the lung; and(3) determining an area in which the fluorescent signal is not detected as a lung cancer invasion site,in Chemical Formula 1,R1and R2are the same as or different from each other, and are each independently one or more selected from the group consisting of hydrogen, a hydroxyl group, a C1-C5alkoxy group, (Y)mX, (Y)mCOO-, (Y)mSO3-, (Y)mPO3H-, and combinations thereof,R3and R4are the same as or different from each other, and are each independently one or more selected from the group consisting of (Y)mH, a C1-C5alkoxy group, a C1-C5alkenyl group, (Y)mNR5R6R7+, and combinations thereof,R5, R6, and R7are the same as or different from each other, and are each independently hydrogen or an unsubstituted C1-C5chain alkyl group,X is a halogen group,Y is CH2or CH2CH2O,Z is a halogen group,,, or, A is,, or, and a, b, c, d, e, and m are the same as or different from each other, and are each independently an integer from 0 to 5.

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