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

[0012]

[0013] 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 a C1-C5chain alkyl group, (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 , , 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.

[0014] In an implementation example of the disclosure, in Chemical Formula 1, Z may be , , or , but is not limited thereto.

[0015] 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 1-9 below, but is not limited thereto.

[0016] ;

[0017] ;

[0018] ;

[0019] ;

[0020] ;

[0021] ;

[0022] ;

[0023] ; and

[0024]

[0025] In another implementation example of the disclosure, the compound or the pharmaceutically acceptable salt thereof may selectively bind to elastin.

[0026] 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 700 nanometers (nm).

[0027] 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.

[0028] According to another aspect, there is provided a contrast agent composition including the compound or the pharmaceutically acceptable salt thereof as an active component.

[0029] 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 700 nm.

[0030] In another implementation example of the disclosure, the contrast agent composition may selectively bind to elastin.

[0031] 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.

[0032] According to another aspect, there is provided an imaging method including the following steps:

[0033] (1) treating a biological sample with a contrast agent composition including the compound or the pharmaceutically acceptable salt thereof as an active component; and

[0034] (2) detecting a fluorescent signal emitted from the biological sample.

[0035] 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.

[0036] 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.

[0037] 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:

[0038] (1) treating a lung with a contrast agent composition including the compound or the pharmaceutically acceptable salt thereof as an active component;

[0039] (2) detecting a fluorescent signal emitted from the lung; and

[0040] (3) determining an area in which the fluorescent signal is not detected as a lung cancer invasion site.

[0041] 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.

[0042] According to another aspect, there is provided a method of selecting a dye for imaging cells or tissues, the method including the following steps:

[0043] (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

[0044] (2) confirming whether the compound maintains fluorescence properties for a long time when the compound is bound to elastin.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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:

[0051] FIG. 1 illustrates a method of preparing a double bond compound according to an embodiment of the disclosure;

[0052] FIG. 2 illustrates results of imaging whether Double Bond Compound 6 specifically binds to elastin after being dissolved in saline and sprayed into a normal mouse lung according to an embodiment of the disclosure;

[0053] FIG. 3 illustrates results of imaging whether Double Bond Compound 6 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;

[0054] FIG. 4 illustrates results of a dose optimization experiment of Double Bond Compound 6 of the disclosure with respect to a mouse model;

[0055] FIG. 5 illustrates results of an incubation time optimization experiment of Double Bond Compound 6 of the disclosure with respect to a mouse model;

[0056] FIG. 6 illustrates whether Double Bond Compound 6 of the disclosure stains an elastin layer in mouse lung cancer tissue;

[0057] FIG. 7 illustrates results of co-administration of Double Bond Compound 6 of the disclosure and another contrast agent with a wavelength of about 800 nanometers (nm) in a rabbit tumor model;

[0058] FIG. 8 illustrates fluorescent staining results in which elastin staining is not observed in an area in which an elastin layer is broken in a rabbit tumor model; and

[0059] FIGS. 9a to 9b show the results of an in vivo toxicity evaluation of Compound 6 (0.1 mM) following intrathoracic injection in C57BL / 6 mice. FIG. 9a presents the serum biochemical analysis performed two weeks after injection. FIG. 9b shows representative H&E-stained histological images of major organs (liver, spleen, kidney, heart, and lungs) collected after the test period.

[0060] 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.

[0061] 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.

[0062] Accordingly, the disclosure provides a compound or a pharmaceutically acceptable salt thereof represented by Chemical Formula 1 below.

[0063]

[0064] 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 a C1-C5chain alkyl group, (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 , , or , A is , , or , an 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.

[0065] 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.

[0066] 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.

[0067] 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).

[0068] 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.

[0069] 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.

[0070] In addition, the disclosure provides a contrast agent composition including the compound or the pharmaceutically acceptable salt thereof as an active component.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] Preparation Example 1. Preparation of Compounds 1 to 3

[0087] 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 in 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 of FIG. 1, were collected as red solids and used in a next step without further purification.

[0088] Compound f (N-2-bromo-3-(phenylimino)prop-1-en-1-yl)aniline) was synthesized as follows. 3.54 mL of aniline was added to mucobromic acid (5.0 g, 19.38 mmol) dissolved in 40 mL of anhydrous ethanol. A reactant was stirred at 250 rpm for 15 minutes under nitrogen purging, then nitrogen was removed, and a flask was placed in an ice bath for 10 minutes. Then, 100 mL of diethyl ether was added dropwise to form a bright yellow precipitate. The solution was stirred for 35 minutes while settling. Yellow solids were collected by filtration using a Buchner funnel and dried overnight in a vacuum desiccator. N-2-bromo-3-(phenylimino)prop-1-en-1-yl)aniline (Compound f) was isolated as a light yellow solid (5.23 g, 89.6% yield) and used in a next step without further purification.

[0089] To synthesize Compound g, Compound f (1.0 eq) and N,N-diisopropylethylamine (DIEA, same as DIPEA, 2.0 eq) were mixed well in dimethyl sulfoxide (DMSO, 1 M). 2-([1,1'-biphenyl]-4-yl)ethan-1-amine (z, 1.2 molar equivalents) in DMSO was added to the reaction solution and heated overnight at 60ºC in an oil bath. The progress was monitored using an 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 ethyl acetate (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 Compound g was collected as a yellow solid for a next step without further purification.

[0090] To synthesize Compound h, Compound f (1.0 eq) and DIEA (2.0 eq) were mixed well in DMSO (1 M). Next, thiol-PEG2-acid (1.2 molar equivalents) in DMSO was added to the 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 Compound h was collected as a yellow solid for a next step without further purification.

[0091] A mixture obtained by mixing Compounds c to e (2.0 eq), Vilsmeier-Haack compound g (1.0 eq), and anhydrous sodium acetate (2.0 eq) into anhydrous ethanol (0.5 M) was heated under reflux at 85ºC and stirred for 2 to 8 hours in 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 1 to 3, respectively, as blue solids.

[0092] Preparation Example 1.1. Preparation confirmation of 2-((1E,3Z)-3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-5-((E)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium (Compound 1)

[0093]

[0094] Compound 1 prepared (2-((1E,3Z)-3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-5-((E)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium) is represented by Chemical Formula 1-1, and1H NMR and13C NMR data were as follows:

[0095] Accurate mass TOF MS m / z [M]+ calculated for [C41H44N3]+ 578.35, found [M+H]+ 578.7.1H NMR (500 MHz, DMSO-d6) δ 7.69 - 7.60 (m, 4H), 7.58 - 7.20 (m, 14H), 7.05 (td, J = 7.4, 1.5 Hz, 1H), 6.77 (dd, J = 7.5, 1.5 Hz, 1H), 6.22 (dd, J = 7.9, 1.5 Hz, 1H), 5.75 (dd, J = 7.9, 0.9 Hz, 1H), 3.68 (s, 2H), 3.39 (td, J = 7.1, 5.4 Hz, 2H), 3.30 (s, 2H), 2.86 (tt, J = 7.2, 1.0 Hz, 2H), 1.65 (s, 4H), 1.59 (s, 4H).13C NMR (125 MHz, DMSO-d6) δ 169.38, 159.82, 147.65, 145.53, 141.77, 141.21, 140.16, 138.93, 138.47, 138.43, 135.68, 133.44, 128.98, 128.88, 128.56, 128.48, 128.45, 127.93, 127.19, 124.89, 122.66, 122.34, 115.77, 114.09, 113.39, 105.97, 105.53, 48.97, 47.32, 42.45, 35.36, 34.77, 33.36, 27.85, 25.78.

[0096] Preparation Example 1.2. Preparation confirmation of 2-((1E,3Z)-3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-5-((E)-1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium-5-sulfonate (Compound 2)

[0097]

[0098] Compound 2 prepared (2-((1E,3Z)-3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-5-((E)-1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)penta-1,3-dien-1-yl)-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:

[0099] Accurate mass TOF MS m / z [M]+ calculated for [C41H44N3O6S2]+ 738.25, found [M+H]+ 738.2.1H 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 - 7.59 (m, 4H), 7.57 - 7.33 (m, 8H), 7.32 (dt, J = 15.0, 1.1 Hz, 1H), 7.23 (dt, J = 7.5, 1.0 Hz, 2H), 6.22 (dd, J = 7.9, 1.5 Hz, 1H), 5.75 (dd, J = 7.9, 0.9 Hz, 1H), 3.70 (s, 2H), 3.39 (td, J = 7.1, 5.4 Hz, 2H), 3.30 (s, 2H), 2.86 (tt, J = 7.2, 1.0 Hz, 2H), 1.69 (s, 4H), 1.63 (s, 4H).13C NMR (125 MHz, DMSO-d6) δ 169.38, 159.29, 147.65, 146.17, 142.66, 141.87, 141.56, 140.11, 140.08, 135.95, 135.68, 128.98, 128.90, 128.32, 127.77, 127.33, 125.24, 125.10, 122.70, 121.56, 115.77, 113.51, 111.81, 106.50, 106.32, 48.89, 46.50, 42.45, 35.36, 34.77, 32.62, 27.85, 25.78.

[0100] Preparation Example 1.3. Preparation confirmation of 2-((1E,3Z)-3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-5-((E)-3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate (Compound 3)

[0101]

[0102] Compound 3 prepared (2-((1E,3Z)-3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-5-((E)-3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)penta-1,3-dien-1-yl)-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:

[0103] Accurate mass TOF MS m / z [M]+ calculated for [C51H66N5O6S2]+ 908.44, found [M+H]+ 908.2.1H NMR (500 MHz, DMSO-d6) δ 7.99 (d, J = 1.6 Hz, 1H), 7.90 (dd, J = 7.5, 1.5 Hz, 1H), 7.79 (d, J = 1.5 Hz, 1H), 7.68 - 7.62 (m, 3H), 7.55 - 7.30 (m, 11H), 7.23 (dt, J = 7.5, 1.1 Hz, 2H), 6.23 (dd, J = 8.4, 1.4 Hz, 1H), 5.75 (dd, J = 8.4, 1.1 Hz, 1H), 4.50 (t, J = 7.1 Hz, 2H), 4.00 (t, J = 7.1 Hz, 2H), 3.85 (t, J = 7.1 Hz, 2H), 3.51 (t, J = 7.1 Hz, 2H), 3.39 (td, J = 7.1, 5.4 Hz, 2H), 3.27 (d, J = 1.0 Hz, 16H), 2.86 (tt, J = 7.3, 1.0 Hz, 2H), 2.21 - 2.10 (m, 1H), 2.08 (dt, J = 14.0, 7.0 Hz, 1H), 1.97 (p, J = 7.1 Hz, 2H), 1.69 (s, 5H), 1.63 (s, 5H).13C NMR (125 MHz, DMSO-d6) δ 170.58, 160.07, 148.29, 144.20, 142.44, 142.23, 140.76, 140.60, 140.16, 138.17, 137.02, 136.20, 135.30, 128.83, 128.73, 127.71, 127.56, 127.21, 125.89, 125.07, 123.01, 121.17, 115.77, 115.16, 115.14, 106.23, 104.67, 64.32, 63.91, 53.64, 53.54, 49.83, 49.55, 48.27, 47.57, 42.45, 34.59, 27.90, 26.25, 26.14, 25.93.

[0104] Preparation Example 2. Preparation of Compounds 4 to 6

[0105] A mixture obtained by mixing Compounds c to e (2.0 eq), Vilsmeier-Haack compound f (1.0 eq), and anhydrous sodium acetate (2.0 eq) into anhydrous ethanol (0.5 M) was heated under reflux at 85ºC and stirred for 1 hour in a nitrogen atmosphere. After the reaction for 2 to 8 hours, 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 i to k, respectively, as blue solids.

[0106] Bromo-dye Compounds i to k (1.0 eq) and 3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoic acid (1.5 eq) were heated under reflux in water in the presence of Pd(PPh3)4 (0.2 eq) for 72 hours. The reaction progress was monitored using the HPLC-MS system. The reaction mixture was then cooled to room temperature, and water was removed under reduced pressure. The solid was separated by precipitation with methanol / acetone, and the precipitate was further washed with acetone. Fluorophores l to n with analytical purity were obtained using open-reversed phase column chromatography (elution with ACN / water).

[0107] The fluorophores were added to anhydrous DMSO (0.1 M), DIPEA (2.0 eq), and 2.0 eq of HSPyU to prepare NHS esters of Fluorophores l to n. 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 (z), and 2.0 eq of DIPEA 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 blue solid powders, Compounds 4 to 6.

[0108] Preparation Example 2.1. Preparation confirmation of 2-((1E,3Z)-3-(4-(3-([1,1'-biphenyl]-4-ylamino)-3-oxopropyl)phenyl)-5-((E)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium (Compound 4)

[0109]

[0110] Compound 4 prepared (2-((1E,3Z)-3-(4-(3-([1,1'-biphenyl]-4-ylamino)-3-oxopropyl)phenyl)-5-((E)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium) is represented by Chemical Formula 1-4, and1H NMR and13C NMR data were as follows:

[0111] Accurate mass TOF MS m / z [M]+ calculated for [C48H48N3O]+ 682.38, found [M+H]+ 682.5.1H NMR (500 MHz, DMSO-d6) δ 9.75 (s, 1H), 7.78 (d, J = 15.2 Hz, 1H), 7.70 - 7.20 (m, 19H), 7.04 (td, J = 7.5, 1.6 Hz, 1H), 6.77 (dd, J = 7.5, 1.5 Hz, 1H), 6.69 (dd, J = 8.2, 1.3 Hz, 1H), 6.33 (dd, J = 8.1, 1.0 Hz, 1H), 3.66 (s, 2H), 3.30 (s, 2H), 2.91 (tt, J = 7.2, 1.0 Hz, 2H), 2.63 (t, J = 7.0 Hz, 2H), 1.68 (s, 4H), 1.61 (s, 4H).13C NMR (125 MHz, DMSO-d6) δ 171.44, 169.38, 165.18, 145.55, 142.38, 140.47, 140.09, 139.16, 138.60, 138.43, 138.28, 137.90, 136.25, 136.05, 132.75, 132.09, 128.98, 128.75, 128.55, 128.22, 128.07, 127.68, 127.33, 124.42, 122.61, 122.47, 120.44, 113.90, 113.46, 106.48, 103.59, 48.97, 46.68, 35.41, 35.36, 32.62, 30.61, 28.02, 25.78.

[0112] Preparation Example 2.2. Preparation confirmation of 2-((1E,3Z)-3-(4-(3-([1,1'-biphenyl]-4-ylamino)-3-oxopropyl)phenyl)-5-((E)-1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium-5-sulfonate (Compound 5)

[0113]

[0114] Compound 5 prepared (2-((1E,3Z)-3-(4-(3-([1,1'-biphenyl]-4-ylamino)-3-oxopropyl)phenyl)-5-((E)-1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)penta-1,3-dien-1-yl)-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:

[0115] Accurate mass TOF MS m / z [M]+ calculated for [C48H48N3O7S2]+ 840.28, found [M+H]+ 840.6.1H NMR (500 MHz, DMSO-d6) δ 9.75 (s, 1H), 7.96 (d, J = 1.5 Hz, 1H), 7.90 (dd, J = 7.5, 1.6 Hz, 1H), 7.81 - 7.75 (m, 2H), 7.69 - 7.58 (m, 8H), 7.54 (d, J = 7.5 Hz, 1H), 7.49 - 7.31 (m, 6H), 7.20 (dt, J = 7.5, 1.1 Hz, 2H), 6.73 (dd, J = 8.1, 1.4 Hz, 1H), 6.35 (dd, J = 8.2, 0.9 Hz, 1H), 3.68 (s, 2H), 3.30 (s, 2H), 2.91 (tt, J = 7.2, 1.0 Hz, 2H), 2.63 (t, J = 7.0 Hz, 2H), 1.69 (s, 4H), 1.63 (s, 4H).13C NMR (125 MHz, DMSO-d6) δ 171.68, 169.50, 164.84, 146.46, 143.19, 141.97, 141.56, 140.74, 140.09, 138.28, 137.90, 136.25, 135.74, 135.15, 132.07, 128.99, 128.68, 128.22, 127.74, 127.29, 126.29, 125.08, 122.70, 121.74, 120.27, 113.51, 111.81, 106.63, 103.09, 48.89, 46.50, 35.36, 35.26, 32.62, 30.66, 27.85, 25.78.

[0116] Preparation Example 2.3. Preparation confirmation of 2-((1E,3Z)-3-(4-(3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-oxopropyl)phenyl)-5-((E)-3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate (Compound 6)

[0117]

[0118] Compound 6 prepared (2-((1E,3Z)-3-(4-(3-((2-([1,1'-biphenyl]-4-yl)ethyl)amino)-3-oxopropyl)phenyl)-5-((E)-3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)penta-1,3-dien-1-yl)-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:

[0119] Accurate mass TOF MS m / z [M]+ calculated for [C60H74N5O7S2]+ 1040.50, found [M+H]+ 1040.2.1H 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 - 7.32 (m, 13H), 7.27 (t, J = 6.7 Hz, 1H), 7.21 (dt, J = 7.6, 1.0 Hz, 4H), 6.73 (dd, J = 8.1, 1.3 Hz, 1H), 6.33 (dd, J = 8.1, 0.9 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), 3.51 (t, J = 7.1 Hz, 2H), 3.27 (d, J = 0.9 Hz, 19H), 2.89 (tt, J = 7.1, 0.9 Hz, 2H), 2.77 (tt, J = 7.2, 1.0 Hz, 2H), 2.38 (t, J = 7.2 Hz, 2H), 2.12 (ddq, J = 29.1, 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 172.79, 168.98, 164.98, 144.14, 142.44, 142.23, 140.57, 140.49, 140.08, 138.91, 138.79, 138.35, 138.12, 137.87, 135.65, 135.54, 132.07, 128.98, 128.87, 128.17, 127.81, 127.67, 127.61, 127.37, 126.31, 125.23, 123.47, 121.24, 115.70, 115.15, 104.95, 101.67, 64.32, 63.91, 53.54, 53.25, 49.83, 49.55, 48.27, 47.57, 40.58, 35.40, 35.21, 30.56, 27.97, 26.15, 25.78, 25.49.

[0120] Preparation Example 3. Preparation of Compounds 7 to 9

[0121] A mixture obtained by mixing Compounds c to e (2.0 eq), Vilsmeier-Haack compound h (1.0 eq), and anhydrous sodium acetate (2.0 eq) into anhydrous ethanol (0.5 M) was heated under reflux at 85ºC and stirred for 2 to 8 hours in 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 o to q, respectively, as blue solids.

[0122] The fluorophores were added to anhydrous DMSO (0.1 M), DIPEA (2.0 eq), and 2.0 eq of HSPyU to prepare NHS esters of Fluorophores o to q. 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 a precipitated green solid, and the precipitated green solid was filtered and washed with EA. Finally, the collected green solid was 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 (z), and 2.0 eq of DIPEA was added 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 a precipitated green solid, and the precipitated green solid was filtered and further washed with EA. Finally, the collected solids were further purified using preparative HPLC and then lyophilized to obtain final blue solid powders, Compounds 7 to 9.

[0123] Preparation Example 3.1. Preparation confirmation of 2-((14Z,15E)-1-([1,1'-biphenyl]-4-yl)-4-oxo-14-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)-7,10-dioxa-13-thia-3-azahexadec-15-en-16-yl)-1,3,3-trimethyl-3H-indol-1-ium (Compound 7)

[0124]

[0125] Compound 7 prepared (2-((14Z,15E)-1-([1,1'-biphenyl]-4-yl)-4-oxo-14-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)-7,10-dioxa-13-thia-3-azahexadec-15-en-16-yl)-1,3,3-trimethyl-3H-indol-1-ium) is represented by Chemical Formula 1-7, and1H NMR and13C NMR data were as follows:

[0126] Accurate mass TOF MS m / z [M]+ calculated for [C48H56N3O3S]+ 754.40, found [M+H]+ 754.7.1H NMR (500 MHz, DMSO-d6) δ 7.75 - 7.62 (m, 4H), 7.58 - 7.47 (m, 4H), 7.44 (t, J = 6.7 Hz, 1H), 7.44 - 7.35 (m, 3H), 7.35 (dd, J = 7.6, 1.5 Hz, 1H), 7.26 (ddd, J = 14.5, 7.4, 1.5 Hz, 2H), 7.21 (dt, J = 7.5, 1.0 Hz, 2H), 7.05 (td, J = 7.5, 1.6 Hz, 1H), 6.81 (dt, J = 15.1, 1.1 Hz, 1H), 6.77 (dd, J = 7.5, 1.5 Hz, 1H), 6.62 (dd, J = 7.0, 1.4 Hz, 1H), 6.33 (dd, J = 7.1, 0.9 Hz, 1H), 3.75 - 3.64 (m, 6H), 3.63 - 3.52 (m, 4H), 3.30 (s, 3H), 3.28 (ddt, J = 29.5, 14.1, 7.0 Hz, 2H), 3.08 (t, J = 7.1 Hz, 2H), 2.77 (tt, J = 7.2, 1.1 Hz, 2H), 2.49 (t, J = 7.1 Hz, 2H), 1.70 (s, 4H), 1.62 (s, 4H).13C NMR (125 MHz, DMSO-d6) δ 170.91, 169.96, 165.54, 145.72, 145.55, 142.26, 140.11, 140.08, 138.99, 138.55, 138.34, 137.44, 135.20, 132.53, 129.25, 129.17, 128.98, 127.68, 127.60, 127.57, 127.33, 124.60, 122.61, 122.47, 113.74, 113.46, 108.00, 105.04, 69.33, 69.20, 68.08, 66.22, 48.97, 46.68, 40.66, 36.78, 35.36, 35.24, 32.80, 32.62, 28.02, 25.78.

[0127] Preparation Example 3.2. Preparation confirmation of 2-((14Z,15E)-1-([1,1'-biphenyl]-4-yl)-4-oxo-14-(2-((E)-1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)ethylidene)-7,10-dioxa-13-thia-3-azahexadec-15-en-16-yl)-1,3,3-trimethyl-3H-indol-1-ium-5-sulfonate (Compound 8)

[0128]

[0129] Compound 8 prepared (2-((14Z,15E)-1-([1,1'-biphenyl]-4-yl)-4-oxo-14-(2-((E)-1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)ethylidene)-7,10-dioxa-13-thia-3-azahexadec-15-en-16-yl)-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:

[0130] Accurate mass TOF MS m / z [M]+ calculated for [C48H56N3O9S3]+ 914.30, found [M+H]+ 914.9.1H 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), 7.71 - 7.62 (m, 4H), 7.57 - 7.33 (m, 8H), 7.21 (dt, J = 7.6, 1.0 Hz, 2H), 6.81 (dt, J = 15.1, 1.1 Hz, 1H), 6.62 (dd, J = 7.0, 1.4 Hz, 1H), 6.33 (dd, J = 7.1, 0.9 Hz, 1H), 3.75 - 3.52 (m, 11H), 3.30 (s, 3H), 3.27 (ddt, J = 22.5, 14.1, 7.1 Hz, 2H), 3.08 (t, J = 7.1 Hz, 2H), 2.77 (tt, J = 7.0, 1.0 Hz, 2H), 2.49 (t, J = 7.1 Hz, 2H), 1.69 (s, 4H), 1.63 (s, 4H).13C NMR (125 MHz, DMSO-d6) δ 171.16, 169.33, 165.19, 146.46, 145.49, 142.22, 141.79, 141.56, 140.60, 140.16, 138.65, 138.28, 138.17, 135.38, 135.15, 129.11, 128.73, 127.62, 127.56, 127.21, 125.74, 124.80, 123.04, 121.74, 113.51, 111.81, 108.00, 104.30, 69.33, 69.20, 68.08, 66.60, 48.89, 46.50, 40.58, 37.20, 35.36, 35.24, 32.96, 32.62, 27.90, 25.78.

[0131] Preparation Example 3.3. Preparation confirmation of 2-((14Z,15E)-1-([1,1'-biphenyl]-4-yl)-14-(2-((E)-3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethylidene)-4-oxo-7,10-dioxa-13-thia-3-azahexadec-15-en-16-yl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate (Compound 9)

[0132]

[0133] Compound 9 prepared (2-((14Z,15E)-1-([1,1'-biphenyl]-4-yl)-14-(2-((E)-3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethylidene)-4-oxo-7,10-dioxa-13-thia-3-azahexadec-15-en-16-yl)-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:

[0134] Accurate mass TOF MS m / z [M]+ calculated for [C58H78N5O9S3]+ 1084.50, found [M+H]+ 1084.8.1H NMR (500 MHz, DMSO-d6) δ 7.98 (d, J = 1.4 Hz, 1H), 7.91 (dd, J = 7.4, 1.6 Hz, 1H), 7.79 (d, J = 1.5 Hz, 1H), 7.68 - 7.32 (m, 12H), 7.21 (dt, J = 7.6, 1.0 Hz, 2H), 6.82 (dt, J = 15.0, 1.2 Hz, 1H), 6.62 (dd, J = 7.6, 1.4 Hz, 1H), 6.30 (dd, J = 7.6, 1.0 Hz, 1H), 4.50 (t, J = 7.1 Hz, 2H), 4.00 (t, J = 7.1 Hz, 2H), 3.85 (t, J = 7.1 Hz, 2H), 3.70 (dt, J = 9.7, 7.1 Hz, 4H), 3.63 - 3.53 (m, 4H), 3.51 (t, J = 7.1 Hz, 2H), 3.27 (d, J = 0.9 Hz, 19H), 3.08 (t, J = 7.1 Hz, 2H), 2.77 (tt, J = 7.1, 1.0 Hz, 2H), 2.49 (t, J = 7.1 Hz, 2H), 2.12 (ddq, J = 29.1, 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 171.16, 166.12, 166.06, 145.09, 144.14, 142.44, 142.23, 140.57, 140.49, 140.07, 138.35, 138.12, 137.27, 137.25, 135.65, 128.97, 128.87, 127.81, 127.67, 127.61, 126.31, 125.23, 123.47, 121.24, 115.70, 115.15, 107.04, 103.81, 69.37, 69.36, 68.18, 66.72, 64.32, 63.91, 53.54, 53.25, 49.83, 49.55, 48.27, 47.57, 40.79, 36.78, 35.21, 32.80, 27.94, 26.15, 25.78, 25.49.

[0135] Experimental Example 1. Confirmation of specific binding of double bond compound of disclosure to elastin

[0136] 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 6 (Chemical Formula 1-6) 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 = 650 ± 20-nm; Em = 680 ± 20-nm.

[0137] FIG. 2 illustrates a whole lung, a resected portion of lung tissue, and a cross-sectional image when Double Bond Compound 6 of the disclosure was sprayed to the lungs of normal mice. Since an elastin layer is present on a surface of a normal lung, it may be confirmed that Compound 6 is bound to the entire surface of the lung.

[0138] Meanwhile, FIG. 3 illustrates images of lungs when Double Bond Compound 6 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 6is 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.

[0139] Experimental Example 2. Optimization of dose and incubation time

[0140] C57BL / 6 mice (age: 6 weeks; weight: 20 to 25 g; Orient Biotech, Seoul, Korea) were used after a 1-week acclimation period to optimize the dose and time of administration of ELA700. During the experiment, the mice were anesthetized with a combination of isoflurane and oxygen. A chest wall was incised for imaging of the whole lung, and 1 to 0.005 mM of Compound 6 (Chemical Formula 1-6) was administered into the lungs for dose optimization. The incubation time of Compound 6was measured from 15 seconds to 300 seconds and then monitored. An optimal compound 6administration protocol was established by evaluating the dose and the incubation time in relation to a target signal-to-background ratio (SBR), and the experimental results are shown in FIGS. 4 and 5. As shown in FIG. 4, the optimal dose was confirmed to be 0.025 mM, and as shown in FIG. 5, the optimal incubation time was confirmed to be 60 seconds.

[0141] Experimental Example 3. Imaging in lung tumor mouse model

[0142] C57BL / 6 mice (age: 6 weeks) were used to establish a lung tumor mouse model after a 1-week acclimation period. The lung tumor mouse model was established 2 to 3 weeks after intravenous injection of LL / 2-Luc2 (100 μL, 2 Х 10^5 cells). To monitor lung tumors, mice with LL / 2-Luc2 lung tumors were injected intraperitoneally with luciferin (15 mg / mL), and the luminescence of the lung tumors was measured using IVIS. To evaluate visceral pleura invasion using Compound 6in the lung tumor mouse model, Compound 6(0.025 mM, 1 minute) was administered. The distribution in the visceral pleura of ELA700 was visualized by an NIR fluorescence imaging system (700 nm) and evaluated by the SBR. A resected specimen was prepared as a frozen section, observed under a confocal microscope to determine a cell distribution, and stained with hematoxylin and eosin (H&E). The results are shown in FIG. 6. It was confirmed that the elastin layer of mouse lung cancer tissue may be effectively stained.

[0143] Experimental Example 4. Imaging in rabbit lung tumor model

[0144] An intravenous injection protocol of cRGD-ZW800-PEG for detecting lung tumors was established through pilot studies. A VX2 lung cancer-induced rabbit model was anesthetized with xylazine and tiletamine-zolazepam, and cRGD-ZW800-PEG (0.1 mg / kg) was administered via the ear vein. A thoracotomy was performed 4 hours after the cRGD-ZW800-PEG injection, and lung tumors were identified during the surgery using an NIR imaging system (800 nm) in which color and fluorescence are integrated. After that, Compound 6(0.025 mM, 1 minute) was administered to evaluate the visceral pleural invasion in the rabbit model. The distribution in the visceral pleural of Compound 6 was visualized using the NIR imaging system (700 nm). The distribution of NIR fluorophores injected into the lungs was evaluated over time using the SBR for up to 30 minutes. The results are shown in FIGS. 7 and 8. As shown in FIG. 7, simultaneous use with a fluorescent contrast agent with a wavelength of 800 nm and distinction were possible, and as shown in FIG. 8, in a rabbit lung cancer model, it was confirmed that elastin staining did not occur in areas where the elastin layer was broken due to cancer invasion.

[0145] Experimental Example 5. Toxicity Test

[0146] C57BL / 6 mice (5 weeks old) were randomly divided into two groups and Compound 6(0.1 mM) was injected into the thoracic cavity. Blood samples were collected two weeks after the injection and a serum biochemical test was performed. After collection, major organs (liver, spleen, kidney, heart, and lungs) were fixed in 10% neutral buffered formalin, processed with paraffin, sectioned to have a thickness of 8 μm, stained with hematoxylin and eosin (H&E), and observed under a digital microscope. The body weight of the mouse was measured for 14 days. The results of the serum biochemical test are shown in FIG. 9a, and the staining images are shown in FIG. 9b. It was confirmed that no significant toxicity was observed.

[0147] 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.

[0148] Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims

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 a C1-C5chain alkyl group, (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,, or, A is,, or, anda, 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.The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein, in Chemical Formula 1, R1and R2are each independently hydrogen or (Y)mSO3-.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.The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein, in Chemical Formula 1, Z is,, or.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 1-9.The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein the compound or the pharmaceutically acceptable salt thereof selectively binds to elastin.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.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 a C1-C5chain alkyl group, (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,, or, A is,, or, anda, 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.The contrast agent composition of claim 8, wherein the contrast agent composition is a near-infrared (NIR) fluorescent contrast agent.The contrast agent composition of claim 8, wherein the compound or the pharmaceutically acceptable salt thereof selectively binds to elastin.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.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 a C1-C5chain alkyl group, (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,, or, A is,, or, anda, 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.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 a C1-C5chain alkyl group, (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,, or, A is,, or, anda, 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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