KR20200142215A - Novel compounds useful as fluorescent probes selectively binding to tau aggregates and preparation method thereof - Google Patents

Abstract

The present invention provides a compound having high selectivity for tau aggregates and a method for manufacturing the same, a tau target fluorescent probe comprising the compound, a composition for detecting tau fibroprotein comprising the fluorescent probe as an active component, and a use of the composition for diagnosing tauopathy. In particular, since the compound of the present invention does not bind to amyloid beta protein and has high selectivity to tau aggregates, which are specifically reported as the etiology of the initial state of tauopathy, the compound can be usefully used as a fluorescent detector for detecting tau fiberprotein for early diagnosis of tauopathy including Alzheimer′s disease.

Description

TECHNICAL FIELD The novel compounds useful as fluorescent probes selectively binding to tau aggregates and preparation method thereof are effective as fluorescent probes selectively binding to tau aggregates.

The present invention relates to a compound having high selectivity to tau aggregates and a method for preparing the same, a tau target fluorescent probe containing the compound, a composition for detecting tau fibroprotein comprising the fluorescent probe as an active ingredient, and diagnosis of tauopathy of the composition It is about the use.

As the population structure is aging, senile dementia, a neurodegenerative disease commonly observed in the elderly, is emerging as a serious issue as a public health problem inevitable.

On the other hand, medical imaging tests greatly contribute to the diagnosis and treatment of patients. Recently, with the introduction of reporter gene technology, molecular imaging that can image changes at the molecular and cellular levels in vivo has attracted attention. Molecular imaging is a non-invasive method of imaging life phenomena in cells or molecular units of living organisms.It can help diagnose diseases by imaging microscopic functional differences in an initial state where no anatomical changes have occurred due to disease. have. Therefore, molecular imaging detects and treats pre-disease conditions early, presents new possibilities in the development of therapeutic drugs, and evaluates the response after treatment early to minimize toxicity resulting from treatment, and tailored treatment appropriate to each patient. . Methods of obtaining such images include single photon emission computed tomography (SPECT) and positron emission tomography (PET) using radioactive elements. Molecular imaging using nuclear medicine techniques such as SPECT and PET has developed at a very rapid pace to evaluate the function of the central nervous system, and is actually a useful technique in basic medical research and clinical practice. In particular, research to develop radioactive probes for PET to image the accumulation of substances causing Alzheimer's disease is actively being conducted. To date, beta-amyloid is known as a major etiology of Alzheimer's disease, and the diagnosis of Alzheimer's disease is also made by positron emission tophography (PET) using a labeling compound that binds to beta-amyloid. However, PET imaging using a beta-amyloid probe is known to have a very weak correlation with Alzheimer's dementia, as it is observed in normal people, so there is a limit to early diagnosis of dementia patients. In addition, since the positron tomography method uses a probe labeled with radioactive isotopes, it may adversely affect the human body, and it has limitations that must be manufactured and used only in special facilities, and requires special equipment and requires high cost. As a biometric imaging method, it has many disadvantages.

On the other hand, the tau protein has a function of stabilizing the tubulin structure constituting the brain nervous system. The tau protein is composed of 352 to 441 amino acids, and there are six isoforms depending on the constitution form. Tau441 (2N4R), the longest form, is present in more than 85%, and phosphorylation occurs in serine, threonine, and tyrosine, and their hyperphosphorylated tau form is known to exist in many patients with brain diseases such as Alzheimer's disease. Accordingly, it is reported that there is a deep correlation between them and diseases, and compounds developed targeting them can be used as major biomarkers. The tau protein is positively charged and is water-soluble, but becomes neutralized when superphosphorylated and does not adhere well to the microtubules and falls off. It is known that the tau single molecules from which they are separated form a paired helical filament (PHF), and they exist in the form of a tau neurofibril tangle (NFT). Therefore, the degree of disease progression can be predicted according to the concentration of the abundance of each type of tau. Up to now, a large amount of amyloid beta protein entanglement has been found in the brains of dementia patients including Alzheimer's disease, and the presence or absence of amyloid beta protein is known as an important disease biomarker. However, since it has been found to exist in a considerable amount in the brain of normal people, there is a limit to diagnosing the disease because there is no discrimination from the initial state to the severe disease progression.

Under this background, the present inventors made diligent efforts to develop a novel compound that is effective as a fluorescent probe that selectively binds to tau aggregates in order to improve the existing problems of early diagnosis of tauopathy including Alzheimer's disease. By developing a new 5 H -pyrido [4,3-b] indole derivative compound and its preparation method, a tau-targeted fluorescent probe containing the compound and a composition for detecting tau fibroprotein comprising the same as an active ingredient, The invention was completed.

Korean Patent Registration No. 10-1802672 (2017.11.22) US Patent Publication No. 2016-0228586 (2016.08.11) US Patent Publication No. 2015-0030540 (2015.01.29) U.S. Patent No. 10004817 (2018.06.26)

An object of the present invention is to provide a novel 5 H -pyrido [4,3-b] indole derivative compound that selectively binds to tau aggregates.

Another object of the present invention is to provide a fluorescent probe for detecting tau fiber protein comprising the novel 5 H -pyrido [4,3-b] indole derivative compound.

Another object of the present invention is to provide a composition for diagnosing tauopathy comprising the fluorescent probe for detecting tau fibroprotein as an active ingredient.

Another object of the present invention is to provide a method for detecting tau aggregates in vitro or in vivo using the novel 5 H -pyrido [4,3-b] indole derivative compound.

Another object of the present invention is to provide a method for preparing the novel 5 H -pyrido [4,3-b] indole derivative compound.

In order to achieve the above object, the present invention provides a 5 H -pyrido [4,3-b] indole derivative compound represented by the following Formula 1:

[Formula 1]

In Formula 1,

R ₁ is hydrogen; C ₁ -C ₁₃ alkyl group; or C ₃ -C ₁₀ cyclyl group; and,

R ₂ is a C ₃ -C ₁₀ aryl group; Or a 5- to 9-membered heteroaryl group containing 1 to 4 heteroatoms selected from boron (B), nitrogen (N), oxygen (O) and sulfur (S) atoms; and,

n is an integer from 0 to 3,

Each of the C ₁ -C ₁₃ alkyl group or C ₃ -C ₁₀ cyclyl group is hydrogen; Hydroxy group; Halogen group; A carbonyl group (-(C=O)R ₃ R ₄ ); A C ₁ -C ₃ alkyl group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; A C ₁ -C ₃ alkoxy group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; C ₆ -C ₁₀ phenoxy; Amino group (-NR ₃ R ₄ ); An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; Including one or more substituents selected from the group consisting of a C ₃ -C ₁₀ heteroaryl group and a C ₃ -C ₁₀ heterocyclyl group,

Each of the C ₃ -C ₁₀ aryl group or 5 to 9 membered heteroaryl group is hydrogen; Hydroxy group; Halogen group; A carbonyl group (-(C=O)R ₃ R ₄ ); A C ₁ -C ₃ alkyl group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; A C ₁ -C ₃ alkoxy group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; C ₆ -C ₁₀ phenoxy; Amino group (-NR ₃ R ₄ ); An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; Including one or more substituents selected from the group consisting of a C ₃ -C ₁₀ heteroaryl group and a C ₃ -C ₁₀ heterocyclyl group,

Each of R ₃ and R ₄ is hydrogen; C ₁ -C ₆ alkyl group; C ₁ -C ₆ alkenyl group; C ₁ -C ₆ alkynyl group; C ₆ -C ₁₀ aryl group; C ₃ -C ₁₀ heteroaryl group; And one or more selected from the group consisting of a C ₃ -C ₁₀ heterocyclyl group.

In addition, the present invention provides a fluorescent probe for detecting tau fibrous protein comprising the 5 H -pyrido [4,3-b] indole derivative compound represented by Formula 1 above.

In addition, the present invention is used for the diagnosis of tauopathy selected from the group consisting of Alzheimer's disease, progressive supranuclear paralysis, cortical-basal ganglia degeneration, Argyrophilic Grain Disease, Peak disease, and hereditary frontotemporal dementia. It provides a composition comprising a fluorescent probe for detecting tau fiber protein as an active ingredient.

In addition, the present invention provides a method for detecting tau aggregates in vitro or in vivo using the 5 H -pyrido [4,3-b] indole derivative compound represented by Formula 1 above.

In addition, the present invention provides a method of preparing a 5 H -pyrido [4,3-b] indole derivative compound represented by Formula 1 above.

The novel 5 H -pyrido [4,3-b] indole derivative compound according to the present invention specifically binds to tau fibrous protein aggregates, so that conditions related to tau fibroprotein aggregates can be diagnosed by fluorescence change.

The novel compound according to the present invention is a fluorescent organic small molecule tracer compound that selectively binds to tau fibroprotein aggregates, Alzheimer's disease, progressive nuclear paralysis, cortical-basal nuclear degeneration, agiophyllic grain disease, peak disease, and hereditary anterior temporal lobe It can be usefully used for early diagnosis of tauopathies including dementia, that is, cranial and nervous system diseases caused by entanglement of tau fiber proteins.

The novel compound according to the present invention can be used to diagnose tauopathy early in a noninvasive method, thus contributing to the improvement of the in vivo imaging method, and the progression of a condition related to tau fibroprotein aggregates by quantifying tau fiber protein by fluorescence imaging. The degree can be easily analyzed.

Figure 1 is to confirm the degree of binding of the synthetic compound to tau aggregates and amyloid beta aggregates in vitro, when the target protein is lacking (PBS) and non-aggregated tau protein (Tau Mono.), tau fiber protein aggregates (Tau Agg. ), non-aggregated amyloid beta (Aβ Mono.), amyloid beta aggregate (Aβ Agg.), and bovine serum albumin (BSA) in the presence of changes in fluorescence spectrum and degree of binding. Specifically, when tau monomer, tau aggregate, Aβ monomer, Aβ aggregate and BSA (bovine serem albumin) in PBS (phosphate-buffered saline) are present at a concentration of 10 μM, the compound of the present invention KNSP001 (7-(6-fluoro) Lopyridin -3-yl)-5 H -pyrido[ 4,3 - b ]indole), KNSP004 (Compound No. 3), KNSP007 (Compound No. 6), KNSP009 (Compound No. 8), KNSP011 (Compound No. 10) , KNSP012 (Compound No. 11), KNSP013 (Compound No. 12), and KNSP014 (Compound No. 13) observed fluorescence spectra are shown.

Unless otherwise specified, all numbers, values, and/or expressions expressing ingredients, reaction conditions, and content of ingredients used herein are the variety of measurements that occur in obtaining such values, among other things, in which these numbers are essentially different. Since they are approximations that reflect uncertainty, they should be understood as being modified in all cases by the term "about". In addition, when numerical ranges are disclosed herein, these ranges are continuous and, unless otherwise indicated, include all values from the minimum value of this range to the maximum value including the maximum value. Furthermore, when this range refers to an integer, all integers from the minimum value to the maximum value including the maximum value are included, unless otherwise indicated.

In the present specification, when a range is described for a variable, it will be understood that the variable includes all values within the stated range, including the stated endpoints of the range. For example, a range of "5 to 10" includes values of 5, 6, 7, 8, 9, and 10, as well as any subranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc. Inclusive, and it will be understood to include any values between integers that are reasonable in the scope of the stated range, such as 5.5, 6.5, 7.5, 5.5 to 8.5 and 6.5 to 9, and the like. Also, for example, the range of "10% to 30%" is 10% to 15%, 12% to 10%, 11%, 12%, 13%, etc., as well as all integers including up to 30%. It will be understood to include any subranges such as 18%, 20% to 30%, and the like, and include any values between reasonable integers within the scope of the stated range, such as 10.5%, 15.5%, 25.5%, and the like.

In addition, terms and abbreviations used herein may be interpreted as meanings commonly understood by those skilled in the art to which the present invention belongs, unless otherwise defined.

Hereinafter, the present invention will be described in detail.

The present inventors continued research to develop a novel compound effective as a fluorescent probe that selectively binds to tau aggregates in order to improve the existing problems of early diagnosis of tauopathy including Alzheimer's disease. A 5 H -pyrido [4,3-b] indole derivative compound and a preparation method thereof, a tau-targeted fluorescent probe containing the compound, and a composition for detecting tau fibroprotein comprising the same as an active ingredient were developed.

One aspect of the present invention provides a 5 H -pyrido [4,3-b] indole derivative compound represented by the following Formula 1:

[Formula 1]

In Formula 1,

R ₁ is hydrogen; C ₁ -C ₁₃ alkyl group; or C ₃ -C ₁₀ cyclyl group; and,

R ₂ is a C ₃ -C ₁₀ aryl group; Or a 5- to 9-membered heteroaryl group containing 1 to 4 heteroatoms selected from boron (B), nitrogen (N), oxygen (O) and sulfur (S) atoms; and,

n is an integer from 0 to 3,

Each of the C ₁ -C ₁₃ alkyl group or C ₃ -C ₁₀ cyclyl group is hydrogen; Hydroxy group; Halogen group; A carbonyl group (-(C=O)R ₃ R ₄ ); A C ₁ -C ₃ alkyl group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; A C ₁ -C ₃ alkoxy group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; C ₆ -C ₁₀ phenoxy; Amino group (-NR ₃ R ₄ ); An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; Including one or more substituents selected from the group consisting of a C ₃ -C ₁₀ heteroaryl group and a C ₃ -C ₁₀ heterocyclyl group,

Each of the C ₃ -C ₁₀ aryl group or 5 to 9 membered heteroaryl group is hydrogen; Hydroxy group; Halogen group; A carbonyl group (-(C=O)R ₃ R ₄ ); A C ₁ -C ₃ alkyl group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; A C ₁ -C ₃ alkoxy group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; C ₆ -C ₁₀ phenoxy; Amino group (-NR ₃ R ₄ ); An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; Including one or more substituents selected from the group consisting of a C ₃ -C ₁₀ heteroaryl group and a C ₃ -C ₁₀ heterocyclyl group,

Each of R ₃ and R ₄ is hydrogen; C ₁ -C ₆ alkyl group; C ₁ -C ₆ alkenyl group; C ₁ -C ₆ alkynyl group; C ₆ -C ₁₀ aryl group; C ₃ -C ₁₀ heteroaryl group; And one or more selected from the group consisting of a C ₃ -C ₁₀ heterocyclyl group.

R ₁ is hydrogen; Or a C ₁ -C ₆ alkyl group;

R ₂ is substituted or unsubstituted benzene; Substituted or unsubstituted pyridazine; Substituted or unsubstituted pyrazine; Substituted or unsubstituted imidazole; Substituted or unsubstituted pyrazole; Substituted or unsubstituted furan; Substituted or unsubstituted pyrimidine; Substituted or unsubstituted pyrrole; Substituted or unsubstituted pyridine; Substituted or unsubstituted indole; Substituted or unsubstituted thiazole; Substituted or unsubstituted benzothiazole; and,

The C ₁ -C ₆ alkyl group, hydrogen; Hydroxy group; Halogen group; C ₁ -C ₁₃ alkyl group; C ₁ -C ₆ alkoxy group; An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; C ₃ -C ₁₀ heteroaryl group; And it may include one or more substituents selected from the group consisting of a C ₃ -C ₁₀ heterocyclyl group.

In one aspect of the present invention, the compound provides a 5 H -pyrido [4,3-b] indole derivative compound represented by Formula 1 selected from the group consisting of the following compound numbers 1 to 13:

(Compound No. 1) (E) -7- (2- (-1- methyl-5,5-difluoro -5H -4λ ^4, 5λ ⁴ - Diffie pyrrolo [1,2- c: 2 ', 1'- f ][1,3,2]diazavorinin-3-yl)vinyl) -5H -pyrido[ 4,3 - b ]indole;

(Compound No. 2) 7-(1 H -indole-6-yl)-5 H -pyrido[4,3- b ]indole;

(Compound No. 3) ( E )-7-(4-nitrostyryl) -5H -pyrido[ 4,3 - b ]indole;

(Compound No. 4) ( E )-2-(2-( 5H -pyrido[4,3- b ]indol-7-yl)vinyl)-6-methoxybenzo[ d ]thiazole;

(Compound No. 5) ( E )-2-(2-( 5H -pyrido[4,3- b ]indol-7-yl)vinyl)benzo[ d ]thiazol-6-ol;

(Compound No. 6) ( E )-4-(2-( 5H -pyrido[4,3- b ]indol-7-yl)vinyl)aniline;

(Compound No. 7) ( E )-7-(4- fluorostyryl ) -5H -pyrido[ 4,3 - b ]indole;

(Compound No. 8) ( E )-4-(2-(5 H -pyrido[4,3- b ]indol-7-yl)vinyl) -N -methylaniline;

(Compound No. 9) 2-((1 E ,3 E )-4-(5 H -pyrido[4,3- b ]indol-7-yl)buta-1,3-dien-1-yl)- 6-methoxybenzo[ d ]thiazole;

(Compound No. 10) 7-((1 E ,3 E )-4-(4-nitrophenyl)buta-1,3-dien-1-yl) -5H -pyrido[ 4,3 - b ]indole;

(Compound No. 11) 4-((1 E ,3 E )-4-(5 H -pyrido[4,3- b ]indol-7-yl)buta-1,3-dien-1-yl)aniline ;

(Compound No. 12) ( E )-7-(2-(6-fluoropyridin-3-yl)vinyl) -5H -pyrido[ 4,3 - b ]indole; And

(Compound No. 13) 4-((1 E ,3 E )-4-(5 H -pyrido[4,3- b ]indol-7-yl)buta-1,3-dien-1-yl)- N -methylaniline.

In the present invention, (Compound No. 1) is also expressed as ( E )-7-(2-(BODIPY-3-yl)vinyl) -5H -pyrido[ 4,3 - b ]indole, and BODIPY is boron -An abbreviation for dipyrromethene, which means an organoboron-based compound.

In the present invention, the term'substitution' refers to the introduction of a hydrogen atom in place of a hydrogen atom when a derivative is formed by replacing one or more hydrogen atoms in an organic compound with another atomic group, and the'substituent' refers to the introduced atomic group.

The substituent is, for example, a halogen atom, a C ₁ -C ₂₀ alkyl group substituted with a halogen atom (eg, CCF ₃ , CHCF ₂ , CH ₂ F, CCl _3, etc.), C ₁ -C ₂₀ alkoxy, C ₂ -C ₂₀ alkoxyalkyl, hydroxy group, nitro group, cyano group, amino group, amidino group, hydrazine, hydrazone, carboxyl group or salt thereof, sulfonyl group, sulfamoyl group, sulfonic acid group or salt thereof, phosphoric acid or salt thereof, or C ₁ -C ₂₀ alkyl group, C ₂ -C ₂₀ alkenyl group, C ₂ -C ₂₀ alkynyl group, C ₁ -C ₂₀ heteroalkyl group, C ₆ -C ₂₀ aryl group, C ₆ -C ₂₀ arylalkyl group, C _6- It may be a C ₂₀ heteroaryl group, a C ₇ -C ₂₀ heteroarylalkyl group, a C ₆ -C ₂₀ heteroaryloxy group, and a C ₆ -C ₂₀ heteroaryloxyalkyl group or a C ₆ -C ₂₀ heteroarylalkyl group.

In the definition of a substituent in the present invention, the term'alkyl' means an aliphatic hydrocarbon radical. Alkyl may be “saturated alkyl” that does not include an alkenyl or alkynyl moiety, or may be “unsaturated alkyl” that includes at least one alkenyl or alkynyl moiety. "Alkenyl" means a group containing at least one carbon-carbon double bond, and "alkynyl" means a group containing at least one carbon-carbon triple bond. When used alone or in combination, the alkyls may be cyclic, branched or straight, respectively.

The term'aryl', alone or in combination with other radicals, is a carbocyclic containing 6 carbon atoms which may be further fused with a second 5 or 6 membered carbocyclic group which may be aromatic, saturated or unsaturated. It means an aromatic monocyclic group. Examples of aryl include, but are not limited to, phenyl, indanyl, 1-naphthyl, 2-naphthyl, teprahiadronaphthyl, and the like. Aryl can be linked with other groups at appropriate positions on the aromatic ring.

The term'alkoxy' refers to an alkyl group (ie -O-alkyl) linked to another group via an oxygen atom. The alkoxy group may be unsubstituted or substituted with one or more suitable substituents. Examples of the alkoxy group include (C ₁ -C ₆ ) alkoxy groups such as -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O-2-methyl-1-propyl, -O -2-Methyl-2-propyl, -O-2-methyl-1-butyl, -O-3-methyl-1-butyl, -O-2-methyl-3-butyl, -O-2,2-dimethyl -1-propyl, -O-2-methyl-1-pentyl, 3-O-methyl-1-pentyl, -O-4-methyl-1-pentyl, -O-2-methyl-2-pentyl, -O -3-Methyl-2-pentyl, -O-4-methyl-2-pentyl, -O-2,2-dimethyl-1-butyl, -O-3,3-dimethyl-butyl, -O-2-ethyl -1-butyl, -O-butyl, -O-isobutyl, -Ot-butyl, -O-pentyl, -O-isopentyl, -O-neopentyl and -O-hexyl .

The term'phenoxy' refers to a phenyl group (ie -O-aryl) linked to another group through an oxygen atom. The phenoxy group is unsubstituted or one or more halogens; Alkyl group; It may be substituted with an aryl group and a hetero aryl group, but is not limited thereto.

The term'amino group' means an alkyl group (ie -NH- or -N-alkyl) linked to another group through a nitrogen atom. The amine group may be unsubstituted or substituted with one or more suitable substituents. Examples of alkoxy groups include (C1-C6) amino groups such as -NH-methyl, -NH-ethyl, -NH-propyl, -NH-isopropyl, -NH-2-methyl-1-propyl, -NH-2-methyl-2-propyl, -NH-2-methyl-1-butyl, -NH-3-methyl-1-butyl, -NH-2-methyl-3-butyl, -NH-2,2 -Dimethyl-1-propyl, -NH-2-methyl-1-pentyl, 3-NH-methyl-1-pentyl, -NH-4-methyl-1-pentyl, -NH-2-methyl-2-pentyl, -NH-3-methyl-2-pentyl, -NH-4-methyl-2-pentyl, -NH-2,2-dimethyl-1-butyl, -NH-3,3-dimethyl-butyl, -NH-2 -Ethyl-1-butyl, -NH-butyl, -NH-isobutyl, -NH-t-butyl, -NH-pentyl, -NH-isopentyl, -NH-neopentyl, -NH-hexyl, -N, N-dimethyl, -N-methyl-N-ethyl, -N-methyl-N-propyl, -N-methyl-isopropyl, -N-methyl-N-butyl, -N-methyl-N-isobutyl,- N-methyl-N-pentyl, -N-methyl-N-isopentyl, N-methyl-N-hexyl, N-methyl-N-isohexyl, -N,N-diethyl, -N-ethyl-N- Propyl, -N-ethyl-N-isopropyl, -N-ethyl-N-butyl, -N-ethyl-N-isobutyl, -N-ethyl-N-pentyl, -N-ethyl-N-isopentyl, -N-ethyl-N-hexyl,, -N-ethyl-N-isohexyl, -N,N-dipropyl, -N-propyl-N-isopropyl, -N-propyl-N-butyl, -N- Propyl-N-isobutyl, -N-propyl-N-pentyl, -N-propyl-N-isopentyl, -N-propyl-N-hexyl, -N-propyl-N-isohexyl, -N,N- Dibutyl, -N-butyl-N-isobutyl, -N-butyl-N-pentyl, -N-butyl-N-isopentyl, -N-butyl-N-hexyl, -N-butyl-N-isohexyl , -N,N-dipentyl, -N-pentyl-N-hexyl, -N-pentyl-N-isohexyl, -N,N-dihexyl, but are not limited thereto.

The term'halogen group' means fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

The term'carbonyl group' means -(C=O)-, and may be substituted with hydrogen, an alkyl group, an alkoxy group, and an amino group, but is not limited thereto.

The term'heterocycle group' means a heteroaromatic compound containing at least one hetero atom selected from the group consisting of N, O, and S unless otherwise stated. Preferably, the heterocyclyl group may include a pyrrolidine group, a furan group, a morpholine group, a piperazine and a piperidine group, more preferably a pyrrolidine group, a piperidine group, a piperazine group, and a mole It may include a foreign group, but is not limited thereto.

The term'heteroaryl group', unless otherwise stated, refers to a heteroaromatic compound containing at least one hetero atom selected from the group consisting of N, O, and S. Preferably, the heteroaryl group is a pyridine group, a pyrazine group, a pyrimidine group, a pyridazine group, a pyrazole group, an imidazole group, a triazole group, an indole group, an oxadiazole group, a thiadiazole group, a quinoline, an isoquinoline group, It may include isoxazole group, oxazole group, thiazole group, and pyrrole group, but is not limited thereto.

The term'derivative' refers to a compound obtained by substituting part of the structure of the compound with another atom or group of atoms.

Specific examples of the 5 H -pyrido [4,3-b] indole derivative compounds preferred as the compounds according to the present invention are as follows:

[Compound No. 1: (E) -7- (2- (-1- methyl -5H -4λ ^4, 5λ ⁴ to 5,5-difluoro-difficile pyrrolo [1,2- c: 2 ', 1'- f ][1,3,2]diazavorinin-3-yl)vinyl) -5H -pyrido[4,3- b ]indole (KNSP002)];

[Compound No. 2: 7-(1 H -indole-6-yl)-5 H -pyrido[4,3- b ]indole (KNSP003)];

[Compound No. 3: (E) -7- (4- nitro-styryl) -5H - pyrido [4, 3- b] indole (KNSP004)];

[Compound No. 4: ( E )-2-(2-(5 H -pyrido[4,3- b ]indol-7-yl)vinyl)-6-methoxybenzo[ d ]thiazole (KNSP005)] ;

[Compound No. 5: (E) -2- (2- (5 H - pyrido [4, 3- b] indol-7-yl) vinyl) benzo [d] thiazol-6-ol (KNSP006)];

[Compound No. 6: (E) -4- (2- (5 H - pyrido [4, 3- b] indol-7-yl) vinyl) aniline (KNSP007)];

[Compound No. 7: (E) -7- (4-fluoro-styryl) -5H - pyrido [4, 3- b] indole (KNSP008)];

[Compound No. 8: (E) -4- (2- (5 H - pyrido [4, 3- b] indol-7-yl) vinyl) - N - methylaniline (KNSP009)];

[Compound No. 9: 2-((1 E ,3 E )-4-(5 H -pyrido[4,3- b ]indole-7-yl)buta-1,3-dien-1-yl)- 6-methoxybenzo[ d ]thiazole (KNSP010)];

[Compound No. 10: 7-((1 E ,3 E )-4-(4-nitrophenyl)buta-1,3-dien-1-yl) -5H -pyrido[ 4,3 - b ]indole ( KNSP011)];

[Compound No. 11: 4-((1 E ,3 E )-4-(5 H -pyrido[4,3- b ]indol-7-yl)buta-1,3-dien-1-yl)aniline (KNSP012)];

[Compound No. 12: ( E )-7-(2-(6-fluoropyridin-3-yl)vinyl) -5H -pyrido[4, 3- b ]indole (KNSP013)]; And

[Compound No. 13: 4-((1 E ,3 E )-4-(5 H -pyrido[4,3- b ]indole-7-yl)buta-1,3-dien-1-yl)- N -methylaniline (KNSP014)];

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Another aspect of the present invention provides a fluorescent probe for detecting tau fibroprotein comprising a 5 H -pyrido [4,3-b] indole derivative compound represented by Chemical Formula 1 according to the present invention.

In the present invention, the term "tau fibroprotein" is a protein mainly involved in the entanglement of nerve fibers related to Alzheimer's disease, and a research result that the possibility of developing dementia is high when the blood concentration of this substance is higher than the normal value of the same age group has been recently reported. have.

Meanwhile, the 5 H -pyrido [4,3-b] indole derivative compound according to the present invention is an autofluorescent compound, and selectively binds to tau fiber protein, thereby exhibiting high tau fiber protein detection effect.

The fluorescent compound according to the present invention has excellent photophysical properties such as full-color tunable emission properties (fluorescence properties), stokes shifts, and drug-like lipid affinity for membrane permeability within a single molecule structure through changes in substituent groups. Properties and photochemical properties.

Therefore, the autofluorescent compound of the present invention can be usefully used in organic light emitting devices, bio-imaging and bio applications as a phosphor, a fluorescent dye or a fluorescent probe, and specifically label a protein of interest in the field of immunocytochemistry. It can be used as a fluorescent probe.

Another aspect of the present invention provides a composition comprising a fluorescent probe for detecting tau fiber protein as an active ingredient.

The composition comprising the fluorescent probe for detecting tau fibroprotein according to the present invention as an active ingredient is a composition for early diagnosis of tauopathy, including Alzheimer's disease, progressive nuclear paralysis, cortical-basal nucleus degeneration, agiophilic grain disease, It can be used in the diagnosis of tauopathy selected from the group consisting of Peak disease and hereditary anterior temporal dementia.

In the present invention, the term "tauopathy" refers to a family of neurodegenerative diseases which means a malfunction of tau protein (a family closely related to intracellular microtubule-related proteins). These neurodegenerative diseases (tauopathies) are, for example, Alzheimer's disease, Progressive Supranuclear Palsy, Corticobasal Degeneration, Argyrophilic Grain Disease, Pick's Disease), and genetic fronto-temporal dementia.

As described above, the fluorescent probe for detecting tau fibrous protein includes a 5 H -pyrido [4,3-b] indole derivative compound represented by Formula 1, and specific binding and fluorescence of the compound to tau aggregates Through its properties and excellent lipid affinity, it can be used for diagnosing related diseases caused by tau aggregates, that is, tauopathies.

In the present invention, the term "aggregate" may be used interchangeably with "aggregate", and refers to an aggregated state in which various insoluble fibrous proteins are deposited on tissues of a patient. In particular, "tau aggregate" means an aggregate formed by aggregation of tau fiber proteins mainly involved in nerve fiber entanglement.

"Tau fibroprotein detection" is achieved by "binding" between the compound according to the present invention and tau fibroprotein aggregates, and "binding" means a chemical interaction. Thus, examples of bonds include covalent bonds, ionic bonds, hydrophilic-hydrophilic interactions, hydrophobic-hydrophobic interactions, and complex compound conjugation.

The term "detection" includes not only ascertaining whether tau aggregates are present in the sample (qualitative analysis), but also to ascertaining the amount thereof (quantitative analysis).

The composition may further include a pharmaceutically acceptable carrier including the 5 H -pyrido [4,3-b] indole derivative compound represented by Formula 1 for diagnosing tauopathy. The composition may be administered orally or parenterally during clinical administration, and may be used in the form of a general pharmaceutical formulation.

That is, the composition of the present invention can be administered in various oral and parenteral dosage forms at the time of actual clinical administration. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants are used. It can be prepared using. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are mixed with at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Can be prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have. Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

Another aspect of the present invention is an in vitro cell or ex vivo tissue using a 5 H -pyrido [4,3-b] indole derivative compound represented by Formula 1 according to the present invention. Provides a method of detecting tau aggregates within.

The method for detecting tau aggregates in cells in vitro or tissues in vitro using the 5 H -pyrido [4,3-b] indole derivative compound represented by Formula 1 includes the following steps:

(a) administering a composition comprising the fluorescent probe for detecting tau fibroprotein as an active ingredient into cells in vitro or tissues in vitro;

(b) combining the 5 H -pyrido [4,3-b] indole derivative compound represented by Formula 1 contained in the probe with tau fiber protein;

(c) irradiating light having an excitation wavelength into the cells in vitro or tissues in vitro;

(d) detecting a fluorescence signal generated from the 5 H -pyrido [4,3-b] indole derivative compound represented by Chemical Formula 1;

(e) generating a graph or image indicating the location and amount of the fluorescent signal; And

(f) determining the distribution and extent of the tau aggregates in cells in vitro or tissues in vitro.

In the step (a), the step of administering the composition into cells in vitro or tissues outside the body includes a detectable amount of the composition containing the compound according to the present invention in cells in vitro or ex vivo. ). The introduction into cells in vitro or tissues ex vivo is administered into cells or tissues by methods known to those skilled in the art.

The "cell" may be, but is not limited to, cells separated from the brain, heart, blood vessels and arteries, and the "tissue" may be a brain, heart, blood vessel, and artery, but is not limited thereto. "Detectable amount" is the amount of composition required to be detected by the selected detection method. The amount of the composition introduced into cells in vitro or tissues ex vivo to be detected can be readily determined by a person skilled in the art. For example, the amount of the composition may be increased until the active ingredient in the composition is detected by the selected detection method, and administered into cells or tissues. Those skilled in the art can easily determine the time required for the compound according to the present invention to bind to tau aggregates by introducing the composition in a detectable amount into cells in vitro or tissues in vitro, and then detecting fluorescence signals at various times after administration. have.

After sufficient time has passed for the compound to bind to the tau fibrous protein in step (b), a fluorescent signal can be non-invasively detected in cells in vitro or tissues in vitro.

The excitation wavelength in step (c) may have a wavelength in the range of 300 to 600 nm, and the fluorescent signal generated in step (d) may have a wavelength in the range of 350 to 700 nm.

The fluorescence signal is not limited thereto, but may be quantified and imaged in real time using a commercial spectrophotometer or fluorescence microscopy equipped with a software program capable of analyzing fluorescence signal data.

In the step of "generating" in step (e), the obtained signal data is obtained through a software program mounted on the spectrophotometer or fluorescence microscope, and then the position of the fluorescence signal emitted by the compound from this data set and Create a graph or image representing the quantity.

The "determining" step in step (f) can be accomplished by evaluating the generated graph or image since the emitted signal is directly related to the amount of tau aggregates.

Imaging tau aggregates in the brain has several potential advantages. Imaging techniques can improve diagnostic methods by identifying potential patients who have accumulated excess tau aggregates in the brain and are therefore more likely to develop Alzheimer's disease, for example. In addition, the technique would be useful, for example, to monitor the progression of Alzheimer's disease. When drug treatments targeting the tau protein become possible, imaging of tau aggregates in the brain could provide an important means to monitor treatment.

The 5 H -pyrido [4,3-b] indole derivative compound represented by Chemical Formula 1 according to the present invention can be imaged using autofluorescence properties even without the use of radioactive isotopes, and exhibits high selective binding with tau aggregates. High sensitivity detection is possible.

Another aspect of the present invention can provide a method for diagnosing a patient with a disease related to tau aggregates.

The diagnostic method can also be used as a post-diagnosis method. The method of diagnosing a patient with a disease related to tau aggregates further comprises the step of setting diagnostic criteria by quantifying or imaging tau fiber protein detected through the method of detecting tau aggregates in the method for detecting tau aggregates, and diagnosing them. can do.

Another aspect of the invention, to 5 H represented by the following Formula 1 comprising the following, compounds represented by the formula (2) reacting a compound of the formula (3) -pyrido [4,3-b] indole A method for preparing a derivative compound is provided:

In Formulas 1 to 3,

R ₁ is hydrogen; C ₁ -C ₁₃ alkyl group; or C ₃ -C ₁₀ cyclyl group; and,

R ₂ is a C ₃ -C ₁₀ aryl group; Or a 5- to 9-membered heteroaryl group containing 1 to 4 heteroatoms selected from boron (B), nitrogen (N), oxygen (O) and sulfur (S) atoms; and,

n is an integer from 0 to 3,

m is an integer from 0 to 3,

Each of the C ₁ -C ₁₃ alkyl group or C ₃ -C ₁₀ cyclyl group is hydrogen; Hydroxy group; Halogen group; A carbonyl group (-(C=O)R ₃ R ₄ ); A C ₁ -C ₃ alkyl group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; A C ₁ -C ₃ alkoxy group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; C ₆ -C ₁₀ phenoxy; Amino group (-NR ₃ R ₄ ); An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; Including one or more substituents selected from the group consisting of a C ₃ -C ₁₀ heteroaryl group and a C ₃ -C ₁₀ heterocyclyl group,

Each of the C ₃ -C ₁₀ aryl group or 5 to 9 membered heteroaryl group is hydrogen; Hydroxy group; Halogen group; A carbonyl group (-(C=O)R ₃ R ₄ ); A C ₁ -C ₃ alkyl group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; A C ₁ -C ₃ alkoxy group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; C ₆ -C ₁₀ phenoxy; Amino group (-NR ₃ R ₄ ); An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; Including one or more substituents selected from the group consisting of a C ₃ -C ₁₀ heteroaryl group and a C ₃ -C ₁₀ heterocyclyl group,

Each of R ₃ and R ₄ is hydrogen; C ₁ -C ₆ alkyl group; C ₁ -C ₆ alkenyl group; C ₁ -C ₆ alkynyl group; C ₆ -C ₁₀ aryl group; C ₃ -C ₁₀ heteroaryl group; And one or more selected from the group consisting of a C ₃ -C ₁₀ heterocyclyl group.

Hereinafter, a specific example of a method of preparing the 5 H -pyrido [4,3-b] indole derivative compound represented by Chemical Formula 1 will be described.

Preparation Method 1: 5 of Formula 1 H -Preparation of pyrido [4,3-b] indole derivative compounds

As illustrated below, the method of preparing the 5 H -pyrido [4,3-b] indole derivative compound represented by Formula 1 may include reacting the compound of Formula 2 with the compound of Formula 3.

[Formula 1]

[Formula 2]

[Formula 3]

In Formulas 1 to 3,

R ₁ is hydrogen; C ₁ -C ₁₃ alkyl group; or C ₃ -C ₁₀ cyclyl group; and,

R ₂ is a C ₃ -C ₁₀ aryl group; Or a 5- to 9-membered heteroaryl group containing 1 to 4 heteroatoms selected from boron (B), nitrogen (N), oxygen (O) and sulfur (S) atoms; and,

n is an integer from 0 to 3,

m is an integer from 0 to 3,

Each of the C ₁ -C ₁₃ alkyl group or C ₃ -C ₁₀ cyclyl group is hydrogen; Hydroxy group; Halogen group; A carbonyl group (-(C=O)R ₃ R ₄ ); A C ₁ -C ₃ alkyl group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; A C ₁ -C ₃ alkoxy group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; C ₆ -C ₁₀ phenoxy; Amino group (-NR ₃ R ₄ ); An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; Including one or more substituents selected from the group consisting of a C ₃ -C ₁₀ heteroaryl group and a C ₃ -C ₁₀ heterocyclyl group,

Each of the C ₃ -C ₁₀ aryl group or 5 to 9 membered heteroaryl group is hydrogen; Hydroxy group; Halogen group; A carbonyl group (-(C=O)R ₃ R ₄ ); A C ₁ -C ₃ alkyl group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; A C ₁ -C ₃ alkoxy group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; C ₆ -C ₁₀ phenoxy; Amino group (-NR ₃ R ₄ ); An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; Including one or more substituents selected from the group consisting of a C ₃ -C ₁₀ heteroaryl group and a C ₃ -C ₁₀ heterocyclyl group,

Each of R ₃ and R ₄ is hydrogen; C ₁ -C ₆ alkyl group; C ₁ -C ₆ alkenyl group; C ₁ -C ₆ alkynyl group; C ₆ -C ₁₀ aryl group; C ₃ -C ₁₀ heteroaryl group; And one or more selected from the group consisting of a C ₃ -C ₁₀ heterocyclyl group.

The step of reacting the compound of Formula 2 with the compound of Formula 3 may be performed by the method of Reaction Scheme 1 below.

[Scheme 1]

In Formulas 1 to 3,

R ₁ is hydrogen; C ₁ -C ₁₃ alkyl group; or C ₃ -C ₁₀ cyclyl group; and,

R ₂ is a C ₃ -C ₁₀ aryl group; Or a 5- to 9-membered heteroaryl group containing 1 to 4 heteroatoms selected from boron (B), nitrogen (N), oxygen (O) and sulfur (S) atoms; and,

n is an integer from 0 to 3,

m is an integer from 0 to 3,

Each of the C ₁ -C ₁₃ alkyl group or C ₃ -C ₁₀ cyclyl group is hydrogen; Hydroxy group; Halogen group; A carbonyl group (-(C=O)R ₃ R ₄ ); A C ₁ -C ₃ alkyl group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; A C ₁ -C ₃ alkoxy group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; C ₆ -C ₁₀ phenoxy; Amino group (-NR ₃ R ₄ ); An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; Including one or more substituents selected from the group consisting of a C ₃ -C ₁₀ heteroaryl group and a C ₃ -C ₁₀ heterocyclyl group,

Each of the C ₃ -C ₁₀ aryl group or 5 to 9 membered heteroaryl group is hydrogen; Hydroxy group; Halogen group; A carbonyl group (-(C=O)R ₃ R ₄ ); A C ₁ -C ₃ alkyl group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; A C ₁ -C ₃ alkoxy group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; C ₆ -C ₁₀ phenoxy; Amino group (-NR ₃ R ₄ ); An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; Including one or more substituents selected from the group consisting of a C ₃ -C ₁₀ heteroaryl group and a C ₃ -C ₁₀ heterocyclyl group,

Each of R ₃ and R ₄ is hydrogen; C ₁ -C ₆ alkyl group; C ₁ -C ₆ alkenyl group; C ₁ -C ₆ alkynyl group; C ₆ -C ₁₀ aryl group; C ₃ -C ₁₀ heteroaryl group; And one or more selected from the group consisting of a C ₃ -C ₁₀ heterocyclyl group.

The manufacturing method of [Scheme 1] is the (E)-3-(5 H -pyrido [4,3-b] indol-7-yl) acrylaldehyde compound represented by the [Chemical Formula 2]. ] R ₂ substituted-2-(diethoxyphosphoryl) acetate and Knoevenagel condensation reaction (Knoevenagel condensation) or HWE reaction (Horner-Wadsworth-Emmons reaction) can be prepared by.

The aldehyde compound represented by [Chemical Formula 2] used as a reactant according to the present invention is specifically represented by Formulas (4) and (8), as shown in the following [Scheme 2], and 3-bro The Mo-4-nitro compound was prepared as a starting material.

Specifically, the 5 H -pyrido [4,3-b] indole-7-carboaldehyde compound represented by the formula (4) was prepared through 4 steps using 2-bromo-4-nitropyridine as a starting material. I did. Suzuki-linked reaction of 2-bromo-4-nitropyridine and (4-bromophenyl) boronic acid to obtain 3-(4-bromophenyl)-4-nitropyridine represented by formula (2) in high yield Then, an oxidation reaction was carried out to synthesize the aldehyde compound of formula (3). At this time, the oxidizing agent is not limited thereto, but PCC (pyridinium chlorochromate), PDC (pyridinium dichromate), DMP (Dess-Martin-Periodinane), TPAP (Tetrapropylammonium perruthenate), TEMPO (2,2,6,6,-tetramethyl1- Piperidinyloxy), Swern oxidation reagent, etc. organic oxidizing agent may be used, preferably DMP (Dess-Martin-Periodinane) may be used.

In addition, the compound represented by formula (4) is prepared by cyclizing the compound represented by compound (3) with triphenyl phosphine, and the compound represented by formula (2) is oxidized to The aldehyde compound was synthesized. After synthesis, ethyl-2-(diethoxyphosphoryl)acetate and ethyl(E)-3-(4-(4-nitropyridin-3-yl)phenyl represented by formula (6) by HWE reaction under basic conditions) The acrylate was prepared. Thereafter, it was reduced in the next step to convert to an alcohol represented by the formula (7), and then a 5 H -pyrido[4,3-b]indole ring was formed through a cyclization reaction. The cyclization reaction can be prepared by reacting triphenyl phosphine at high temperature in a solvent. The solvent is not limited thereto, but may be selected from 1,2-chlorobenzene, dimethylformamide, dimethylbenzene, tetrahydrofuran, and ethanol. The reaction temperature may be 100 ℃ to 200 ℃, preferably 180 ℃ ± 5 ℃. On the other hand, it is also possible to react triethoxy phosphine without a solvent.

[Scheme 2]

[Scheme 3]

The R ₂ substituted methylphosphite compound can be prepared from the corresponding R ₂ substituted brominated methane. Wherein R ₂ is a C ₃ -C ₁₀ aryl group; Or it may be a 5- to 9-membered heteroaryl group containing 1 to 4 heteroatoms selected from boron (B), nitrogen (N), oxygen (O) and sulfur (S) atoms.

Hereinafter, the present invention will be described in more detail through Examples, Formulation Examples and Experimental Examples. However, the following examples, formulation examples, and experimental examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples. It will be apparent to those of ordinary skill in the art that various changes and modifications can be made within the scope and scope of the present invention.

In addition, those of ordinary skill in the technical field to which the present invention belongs can prepare the target compound by various methods based on the structure of Formula 1, and all of these methods should be interpreted as being included in the scope of the present invention. do. That is, the compound of the present invention can be prepared by arbitrarily combining the synthesis methods specifically described in the following examples or various synthesis methods disclosed in the prior art, which is understood to be within the scope of the present invention, and the preparation of the compound of the present invention The method is not limited by the specific examples below.

[Example]

Reference Example 1: 3-bromo-4-nitropyridine

3-bromo-4-nitropyridine 1-oxide (5g, 22.83 mmol), Re(O)(Ph ₃ P) ₂ Cl ₃ (152 mg, 0.183 mmol) and triphenylphosphine (6.59 g, 25.1 mmol) The solution dissolved in dichloromethane (150 mL) was stirred at 70° C. for 8 hours under nitrogen conditions. The reaction mixture was cooled to room temperature, filtered through Celite, and distilled under reduced pressure to remove the solvent. The residue was triturated in n-hexane and the precipitate was filtered off. The organic layer was concentrated in vacuo and purified by column chromatography (elution solvent: ethyl acetate/n-hexane=1/7) to synthesize compound 1 as a yellow solid (4.53 g, 98%).

¹ H NMR (400 MHz CDCl ₃ ) δ 9.00 (s, 1H), 8.76 (d, J = 5.2 Hz, 1H), 7.70 (d, J = 5.2 Hz, 1H).

Reference Example 2: 3-(4-bromophenyl)-4-nitropyridine

Compound 1 (3-bromo-4-nitropyridine; 3.03g, 18.12mmol), (4-bromophenyl) boronic acid (4.37g, 21.74mmol) prepared in Reference Example 1, tetrakistriphenylphosphine A solution of palladium (1.06g, 0.91mmol) and sodium carbonate (4.8g, 45.29mmol) in a mixed solvent of 1,4-dioxane and water (1,4-dioxane/water = 6/1, 210 mL) was dissolved in nitrogen The mixture was stirred at 110° C. for 18 hours under conditions. The reaction mixture was cooled to room temperature, filtered through Celite, water was added to the reaction solution, and extracted with ethyl acetate. The extract was washed with brine, dried with sodium sulfate, and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: ethyl acetate/n-hexane=1/9), and a pale yellow solid compound 2a (3-(4-bromophenyl)-4-nitropyridine; 4.53 g, 98 %) was synthesized.

¹ H NMR (400 MHz CDCl ₃ ) δ 8.86 (d, J = 5.2 Hz, 1H), 8.78 (s, 1H), 7.69 (d, J = 5.2 Hz, 1H), 7.62 (dd, J = 1.8, 6.6 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H).

Reference Example 3: 7-Bromo-5 H -Pyrido[4,3- b ]Indol

A solution of compound 2a (3-(4-bromophenyl)-4-nitropyridine; 424.5mg, 1.52mmol) synthesized in Reference Example 2 in triethyl phosphite (30mL) was stirred at 110°C under nitrogen conditions And observed by thin layer chromatography every 1 hour. After confirming the completion of the reaction on thin layer chromatography, triethyl phosphite was removed under vacuum. The residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/50), and a dark yellow solid compound 3 (7-bromo-5 H -pyrido[4,3- b ]indole; 295 mg, 78%) was synthesized.

¹ H NMR (400 MHz acetone-d ₆ ) δ 9.36 (s, 1H), 8.48 (d, J = 5.6 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 1.6 Hz, 1H), 7.50 (d, J = 5.6 Hz, 1H), 7.44 (dd, J = 1.6 Hz, 8.4, 1H).

Reference Example 4: tert-butyl 7-bromo-5 H -Pyrido[4,3- b ]Indole-5-carboxylate (5)

Compound 3 synthesized in Reference Example 3 (7-bromo-5 H -pyrido[4, 3- b ]indole; 263.2 mg, 0.956 mmol) and DMAP (141.5 mg, 1.147 mmol) in dichloromethane (35 mL) ) Was stirred at 0° C. under nitrogen conditions, and (Boc) ₂ O (0.36 mL, 1.529 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/50), and a pale yellow solid compound 5 (tert-butyl 7-bromo-5 H -pyrido[4,3- b ]indole -5-carboxylate; 264.5 mg, 80%) was synthesized.

¹ H NMR (400 MHz CDCl ₃ ) δ 9.25 (s, 1H), 8.65 (d, J = 5.6 Hz, 1H), 8.56 (s, 1H), 8.10 (d, J = 5.6 Hz, 1H), 7.56 ( d, J = 1.6 Hz, 1H), 7.54 (d, J = 1.6 Hz, 1H), 1.78 (s, 9H).

Reference Example 5: tert-butyl 7-(6-fluoropyridin-3-yl)-5 H -Pyrido[4,3- b ]Indole-5-carboxylate (11)

Compound 4 synthesized in Reference Example 4 (tert-butyl 7-bromo-5 H -pyrido[4, 3- b ]indole-5-carboxylate; 264.5 mg, 0.762 mmol), (6-fluoropyridine -3-yl)boronic acid (153.4 mg, 1.066 mmol), 2-(dicyclohexylphosphino)biphenyl (30.3 mg, 0.084 mmol) and potassium carbonate (316 mg, 2.285 mmol) were added to 1,2-dimethoxy A solution dissolved in a mixed solvent of ethane (40 mL) and water (5 mL) was stirred, and palladium acetate (17.1 mg, 0.076 mmol) was added. The reaction mixture was stirred at 100° C. for 24 hours, then cooled to room temperature and filtered through Celite. The filtrate was diluted with water, extracted with dichloromethane, washed with brine, dried with sodium sulfate, and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (eluent: methanol / dichloromethane = 1/90) to yield a light ivory-white solid compound 11 (a tert-butyl 7- (pyridin-3-yl) -5-fluoro-6-H - Pyrido[4,3- b ]indole-5-carboxylate; 141.9 mg, 51%) was synthesized.

¹ H NMR (400 MHz acetone-d ₆ ) δ 9.43 (d, J = 0.8 Hz, 1H), 8.66 (d, J = 6 Hz, 1H), 8.64 (d, J = 1.2 Hz, 1H), 8.62- 8.61 (m, 1H), 8.39 (dd, J = 0.5, 8.1 Hz, 1H), 8.37-8.33 (m, 1H), 8.21 (dd, J = 1.0, 5.8 Hz, 1H), 7.81 (dd, J = 1.6, 8.0 Hz, 1H), 7.27-7.24 (m, 1H), 1.83 (s, 9H).

Reference Example 6: 7-(6-fluoropyridin-3-yl)-5 H -Pyrido[4,3- b ]Indol (13, KNSP001)

Reference Example 11 The compounds (tert synthesized from 5-butyl-7- (6-fluoro-3-yl) -5 H-pyrido [4, 3- b] indole-5-carboxylate; 141.9 mg, 0.39 mmol) in dichloromethane (15 mL) was stirred at 0° C. under nitrogen conditions, and trifluoroacetic acid (0.335 mL, 1.952 mmol) was added. The reaction mixture was stirred at room temperature for 6 hours and then neutralized at 0° C. with a saturated aqueous sodium carbonate solution. The reaction mixture was extracted with dichloromethane, washed with brine, dried with sodium sulfate, and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: methanol / dichloromethane = 1/50) to give compound as a light yellow solid 13, KNSP001 (7- (6- fluoro-pyridin-3-yl) -5 H - pyrido [4, 3- b ]indole; 93.15 mg, 91%) was synthesized.

¹ H NMR (400 MHz acetone-d ₆ ) δ 9.39 (s, 1H), 8.59 (d, J = 2.0 Hz, 1H), 8.48 (d, J = 5.6 Hz, 1H), 8.37 (d, J = 8.0 Hz, 1H), 8.33 (td, J = 2.4, 8.2 Hz, 1H), 7.90 (d, J = 0.8 Hz, 1H), 7.62 (dd, J = 1.6, 8.4 Hz, 1H), 7.51 (dd, J = 0.8, 5.6 Hz, 1H), 7.22 (dd, J = 2.8, 8.4 Hz, 1H); ¹³ C NMR (100 MHz acetone-d ₆ ) δ 166.10, 163.75, 147.78, 147.62, 146.97, 144.87, 142.22, 142.14, 137.26, 137.22, 137.04, 122.99, 121.41, 111.59, 111.34, 110.96, 108.00.

Example 1: tert-butyl 7-(1 H -Indol-6-work)-5 H -Pyrido[4,3- b ]Indole-5-carboxylate (12)

Compound 4 (tert-butyl 7-bromo-5 H -pyrido[4,3- b ]indole-5-carboxylate; 250 mg, 0.72 mmol), (1 H -indol-6-yl)boronic acid ( 167.3 mg, 1.008 mmol), 2-(dicyclohexylphosphino)biphenyl (29 mg, 0.079 mmol) and potassium carbonate (299 mg, 2.16 mmol) were added to 1,2-dimethoxyethane (40 mL) and water ( 5 mL) of the mixed solvent was stirred, and palladium acetate (16.2 mg, 0.072 mmol) was added. The reaction mixture was stirred at 100° C. for 24 hours, then cooled to room temperature and filtered through Celite. The filtrate was diluted with water, extracted with dichloromethane, washed with brine, dried with sodium sulfate, and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography to give (elution solvent: methanol / dichloromethane = 1/90), compound as a light yellow solid 12 (tert-butyl 7- (1 H-indol-6-yl) -5 H-pyrido [4, 3- b ]indole-5-carboxylate; 145.4 mg, 53%) was synthesized.

¹ H NMR (400 MHz CDCl ₃ ) δ 9.25 (s, 1H), 8.64 (s, 1H), 8.63 (d, J = 5.6 Hz, 1H), 8.57 (s, 1H), 8.13 (d, J = 5.6 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 1.6 Hz, 1H), 7.68 (s, 1H), 7.49 ( dd, J = 1.4, 8.2 Hz, 1H), 7.27-7.25 (m, 1H), 6.60 (s, 1H), 1.77 (s, 9H).

Example 2: 7-(1 H -Indol-6-work)-5 H -Pyrido[4,3- b ]Indol (14, KNSP003)

Example 1 The compound 12 (synthesized from the tert-butyl 7- (1 H-indol-6-yl) -5 H-pyrido [4, 3- b] indole-5-carboxylate; 145.4 mg, 0.38 mmol ) Was dissolved in dichloromethane (15 mL) and stirred at 0° C. under nitrogen conditions, and trifluoroacetic acid (0.1 mL, 1.14 mmol) was added. The reaction mixture was stirred at room temperature for 6 hours and then neutralized at 0° C. with a saturated aqueous sodium carbonate solution. The reaction mixture was extracted with dichloromethane, washed with brine, dried with sodium sulfate, and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/50), and a pale yellow solid compound 14, KNSP003 (7-(1 H -indol-6-yl)-5 H -pyrido[ 4, 3- b ]indole; 100 mg, 93%) was synthesized.

¹ H NMR (400 MHz MeOD) δ 9.22 (d, J = 0.4 Hz, 1H), 8.36 (d, J = 6.0 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 1.2 Hz, 1H), 7.23 (s, 1H), 7.65-7.62 (m, 2H), 7.49 (dd, J = 0.8, 6.0 Hz, 1H), 7.41 (dd, J = 1.6, 8.0 Hz, 1H) , 7.28 (d, J = 3.2 Hz, 1H), 6.48-6.48 (m, 1H); ¹³ C NMR (100 MHz MeOD) δ 144.97, 142.53, 142.33, 140.95, 140.88, 136.93, 134.79, 127.63, 125.09, 120.31, 120.24, 120.13, 119.47, 118.73, 109.50, 109.29, 106.20, 100.85.

Example 3: (4-(4-nitropidin-3-yl)phenyl)methanol (2b)

Compound 1 (3-bromo-4-nitropyridine; 1.51g, 7.44mmol), (4-(hydroxymethyl)phenyl) boronic acid (1.469g, 9.67mmol), tetrakistriphenylphosphine palladium (344mg, 0.298 mmol) and sodium carbonate (1.987 g, 18.75 mmol) in a mixed solvent of 1,4-dioxane and water (1,4-dioxane/water = 6/1, 210 mL) at 110° C. under nitrogen conditions Stir for 18 hours. The reaction mixture was cooled to room temperature, filtered through Celite, water was added to the reaction solution, and extracted with ethyl acetate. The extract was washed with brine, dried with sodium sulfate, and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: ethyl acetate/n-hexane=1/1), and a yellow solid compound 2b ((4-(4-nitropidin-3-yl)phenyl)methanol; 1.655 g, 97%) was synthesized.

¹ H NMR (400 MHz CDCl ₃ ) δ 8.82 (d, J = 5.6 Hz, 1H), 8.77 (s, 1H), 7.66 (d, J = 5.2 Hz, 1H), 7.49 (d, J = 8.0 Hz, 2H), 7.36-7.34 (m, 2H) 4.78 (d, J = 6.0 Hz, 2H).

Example 4: 4-(4-nitropyridin-3-yl)benzaldehyde (4)

A solution of compound 2b ((4-(4-nitropidin-3-yl)phenyl)methanol; 5.25 g, 22.80 mmol) in dichloromethane (200 mL) was stirred at 0°C under nitrogen conditions and DMP (14.51 g) , 34.2 mmol) was added. The reaction mixture was stirred at room temperature and observed by thin layer chromatography every 1 hour. After confirming the completion of the reaction by thin layer chromatography, the reaction mixture was filtered through celite and neutralized with a saturated aqueous sodium carbonate solution. The reaction mixture was extracted with ethyl acetate, washed with brine, dried with sodium sulfate, and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: ethyl acetate/n-hexane=1/2), and a yellow solid compound 4 (4-(4-nitropyridin-3-yl)benzaldehyde; 5 g, 96 %) was synthesized.

¹ H NMR (400 MHz CDCl ₃ ) δ 10.10 (s, 1H), 8.91 (d, J = 5.2 Hz, 1H), 8.83 (s, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.77 ( d, J = 5.2 Hz, 1H), 7.53 (d, J = 8.0 Hz, 2H).

Example 5: 5 H -Pyrido[4, 3- b ]Indole-7-carbaldehyde (6)

A solution of compound 4 (4-(4-nitropyridin-3-yl)benzaldehyde; 500 mg, 2.19 mmol) and triphenylphosphine (1.47 g, 5.48 mmol) in 1,2-dichlorobenzene (22 mL) Was stirred at 180° C. for 8 hours under nitrogen conditions. After cooling the reaction mixture to room temperature, 1,2-dichlorobenzene was removed under vacuum, and the residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/30), and a yellow solid compound 6 ( 5 H -pyrido[4,3- b ]indole-7-carbaldehyde; 352 mg, 82%) was synthesized.

¹ H NMR (400 MHz DMSO) δ12.12 (s, 1H), 10.15 (s, 1H), 9.47 (d, J = 0.8 Hz, 1H), 8.50 (d, J = 5.6 Hz, 1H), 8.45 ( d, J = 8.0 Hz, 1H), 8.12 (s, 1H), 7.82 (dd, J = 1.2, 8.0 Hz, 1H), 7.56 (dd, J = 0.8, 5.6 Hz, 1H).

Example 6: ( E )-7-(2-(5,5-difluoro-1-methyl -5H -4λ ⁴ ,5λ ⁴ -Dipyrrolo[1,2- c :2',1'- f ][1,3,2]diazaborinin-3-yl)vinyl) -5H -Pyrido[4, 3- b ]Indol (15, KNSP002)

Compound 29 (5,5-Difluoro-1,3-dimethyl -5H -4λ ^4, 5λ ⁴ - Diffie pyrrolo [1,2- c: 2 ', 1'- f] [1,3,2] Dia Zavorinine; 170 mg, 0.77 mmol), acetic acid (0.35 mL, 6.11 mmol) and piperidine (0.35 mL, 3.54 mmol) dissolved in toluene (10 mL) was stirred under nitrogen conditions, and compound 6 (5 H -Pyrido[4,3- b ]indole-7-carbaldehyde; 180.5 mg, 0.92 mmol) was added. The reaction mixture was stirred at 120° C. for 5 hours under Dean-Stark conditions and then cooled to room temperature. A saturated aqueous ammonium chloride solution was added to the reaction mixture, followed by extraction with ethyl acetate. Sodium sulfate was added to the extract, dried, and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: ethyl acetate/n-hexane=1/15), and a dark red solid compound 15, KNSP002 (( E )-7-(2-(5,5-difluoro 1-methyl -5H -4λ ^4, 5λ ⁴ - Diffie pyrrolo [1,2- c: 2 ', 1'- f] [1,3,2] diaza barley non-3-yl) vinyl) -5H -Pyrido[4, 3- b ]indole; 250.2 mg, 81%) was synthesized.

¹ H NMR (400 MHz DMSO) δ 11.88 (s, 1H), 9.37 (d, J = 6.0 Hz, 1H), 8.45 (d, J = 5.6 Hz, 1H), 8.33 (d, J = 8.0 Hz, 1H ), 8.01 (d, J = 16.0 Hz, 1H), 7.84 (s, 1H), 7.82 (s, 1H), 7.74 (s, 1H), 7.56 (d, J = 7.6 Hz, 1H), 7.51 (d , J = 6.0 Hz, 1H), 7.22 (s, 1H), 7.13 (d, J = 4.0 Hz, 1H), 6.54 (dd, J = 2.1, 3.6 Hz, 1H), 2.38 (s, 3H); ¹³ C NMR (100 MHz DMSO) δ 158.77, 145.96, 145.28, 145.10, 143.56, 142.14, 140.54, 139.07, 137.98, 134.54, 133.47, 126.96, 125.16, 122.94, 121.90, 120.22, 119.64, 118.36, 119.64, 118. , 107.11, 11.71.

Example 7: ( E )-7-(4-nitrostyryl) -5H -Pyrido[4, 3- b ]Indol (16, KNSP004)

A solution of compound 30a (diethyl (4-nitrobenzyl) phosphonate; 245 mg, 0.90 mmol) in anhydrous tetrahydrofuran (10 mL) was stirred at 0°C under nitrogen conditions and sodium hydride (48.9 mg, 1.22 mmol) ) Was added. After the reaction mixture was stirred at room temperature for 30 minutes, compound 6 (5 H -pyrido[4, 3- b ]indole-7-carbaldehyde; 160 mg, 0.82 mmol) was added, followed by stirring for 24 hours. The reaction mixture was cooled to 0° C. and saturated aqueous ammonium chloride solution was added. The reaction mixture was extracted with dichloromethane, dried by adding sodium sulfate, and then distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: methanol / dichloromethane = 1/30) to give a red solid of compound 16, KNSP004 ((E) -7- (4- nitro-styryl) -5H - pyrido [ 4, 3- b ]indole; 109.6 mg, 43%) was synthesized.

¹ H NMR (400 MHz DMSO) δ 11.84 (s, 1H), 9.34 (s, 1H), 8.43 (d, J = 5.6 Hz, 1H), 8.27-8.25 (m, 3H), 7.93 (d, J = 8.8 Hz, 2H), 7.81 (s, 1H), 7.75 (d, J = 16.4 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.54 (d, J = 16.4 Hz, 1H), 7.47 (d, J = 5.2 Hz, 1H); ¹³ C NMR (100 MHz DMSO) δ

Example 8: ( E )-4-(2-(5 H -Pyrido[4, 3- b ]Indole-7-yl)vinyl)aniline (21, KNSP007)

Compound 16 ((E) -7- (4- nitro-styryl) -5H - pyrido [4, 3- b] indole; 240.9 mg, 0.580 mmol) to a solution in ethanol (6 mL), 0.5 mL of Tin(II) chloride dihydrate (785 mg, 3.48 mmol) dissolved in concentrated hydrochloric acid was added. The reaction mixture was stirred at 106° C. for 6 hours under nitrogen conditions. The reaction mixture was cooled to 0° C., and the pH of the solution was adjusted to 8 using 1N aqueous sodium hydroxide solution. The reaction mixture was extracted with dichloromethane, dried with sodium sulfate, and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: methanol / dichloromethane = 1/20) to give a yellow solid compound 21, KNSP007 ((E) -4- (2- (5 H - pyrido [4, 3 -b ]Indole-7-yl)vinyl)aniline; 163 mg, 99%) was synthesized.

¹ H NMR (400 MHz DMSO) δ 11.79 (s, 1H), 9.27 (s, 1H), 8.37 (d, J = 5.6 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.61 (s , 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 5.6 Hz, 1H), 7.33 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 16.0 Hz, 1H ), 7.08 (d, J = 16.4 Hz, 1H), 6.58 (d, J = 8.4 Hz, 2H); ¹³ C NMR (100 MHz DMSO) δ148.10, 144.21, 143.77, 142.40, 139.60, 136.30, 130.33, 129.51, 127.54, 126.05, 123.80, 120.90, 120.17, 119.26, 113.26, 110.73, 106.13.

Example 9: ( E )-2-(2-(5 H -Pyrido[4, 3- b ]Indole-7-yl)vinyl)-6-methoxybenzo[ d ]Thyazole (17, KNSP005)

Compound 32 (diethyl ((6-methoxybenzo[d]thiazol-2-yl)methyl)phosphonate; 231 mg, 0.734 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL) under nitrogen conditions. While stirring at 0° C., sodium hydride (39.1 mg, 1.631 mmol) was added. After the reaction mixture was stirred at room temperature for 30 minutes, compound 6 (5 H -pyrido[4, 3- b ]indole-7-carbaldehyde; 80 mg, 0.408 mmol) was added, followed by stirring for 24 hours. The reaction mixture was cooled to 0° C. and saturated aqueous ammonium chloride solution was added. The reaction mixture was extracted with dichloromethane, dried by adding sodium sulfate, and then distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: methanol / dichloromethane = 1/30) to give a yellow solid compound 17, KNSP005 ((E) -2- (2- (5 H - pyrido [4, 3 -b ]indol-7-yl)vinyl)-6-methoxybenzo[ d ]thiazole; 105.7 mg, 73%) was synthesized.

¹ H NMR (400 MHz DMSO) δ 12.13 (s, 1H), 9.36 (s, 1H), 8.43 (d, J = 5.6 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H), 7.91 (s , 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.77- 7.70 (m, 3H), 7.65 (d, J = 16.4 Hz, 1H), 7.49 (d, J = 5.6 Hz, 1H), 7.12 (d, J = 8.0 Hz, 1H), 3.86 (s, 3H); ¹³ C NMR (100MHz DMSO) δ 163.83, 157.28, 147.69, 144.53, 144.13, 142.82, 139.67, 136.92, 135.35, 133.54, 122.78, 121.37, 120.74, 119.09, 118.93, 115.51, 110.68, 106.23, 104.56, 55.51

Example 10: ( E )-2-(2-(5 H -Pyrido[4, 3- b ]Indole-7-yl)vinyl)benzo[ d ]Thyazole-6-all (22, KNSP006)

Compound 17 (( E )-2-(2-(5 H -pyrido[4,3- b ]indol-7-yl)vinyl)-6-methoxybenzo[ d ]thiazole; 79.3 mg, 0.222 mmol ) Was dissolved in dichloromethane (2 mL) and stirred at -78°C under nitrogen conditions, and boron tribromide (2.22 mL, 2.22 mmol) was added. The reaction mixture was stirred at room temperature for 24 hours. Distilled water was added to the reaction mixture at 0°C, and the pH of the solution was adjusted to 7 using 1N aqueous sodium hydroxide solution. The reaction mixture was extracted with dichloromethane, dried with sodium sulfate, and then distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: methanol / dichloromethane = 1/20) to give a red solid of compound 22, KNSP006 ((E) -2- (2- (5 H - pyrido [4, 3 -b ]indol-7-yl)vinyl)benzo[ d ]thiazol-6-ol; 60 mg, 79%) was synthesized.

¹ H NMR (400 MHz DMSO) δ 9.32 (s, 1H), 8.39 (d, J = 5.6 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.82 (s, 1H), 7.63 (d , J = 8.4 Hz, 1H), 7.57 (d, J = 6.8 Hz, 1H), 7.54 (d, J = 14.0 Hz, 1H), 7.48 (d, J = 16.4 Hz, 1H), 7.45 (d, J = 5.6 Hz, 1H), 7.01 (s, 1H), 6.73 (dd, J = 2.0, 8.0 Hz, 1H); ¹³ C NMR (100 MHz DMSO) δ 144.02, 142.33, 139.67, 135.47, 134.45, 133.61, 122.30, 121.63, 120.70, 120.31, 118.75, 118.45, 117.34, 109.98, 106.07, 105.96.

Example 11: tert-butyl ( E )-(4-(2-(5 H -Pyrido[4, 3- b ]Indole-7-yl)vinyl)phenyl)(methyl)carbamate (18)

Compound 31 (tert-butyl (4-((diethoxyphosphoryl)methyl)phenyl)(methyl)carbamate; 182 mg, 0.510 mmol) was dissolved in anhydrous N,N -dimethylformamide (5 mL) under nitrogen conditions Under stirring at 0° C. sodium hydride (30.6 mg, 0.764 mmol) was added. After the reaction mixture was stirred at room temperature for 30 minutes, compound 6 (5 H -pyrido[4, 3- b ]indole-7-carbaldehyde; 50 mg, 0.225 mmol) was added, followed by stirring for 24 hours. The reaction mixture was cooled to 0° C. and saturated aqueous ammonium chloride solution was added. The reaction mixture was extracted with dichloromethane, dried by adding sodium sulfate, and then distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/30), and the ivory solid compound 18 (tert-butyl ( E )-(4-(2-( 5H -pyrido[4)) , 3- b ]indole-7-yl)vinyl)phenyl)(methyl)carbamate; 34.6 mg, 34%) was synthesized.

¹ H NMR (400 MHz CDCl ₃ ) δ 9.29 (s, 1H), 8.51 (d, J = 5.6 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.49-7.47 (m, 3H), 7.43 (s, 1H), 7.35 (d, J = 5.6 Hz, 1H), 7.27 (d, J = 7.2 Hz, 2H), 7.17 (d, J = 16.4 Hz, 1H), 7.12 (d, J = 16.0 Hz, 1H), 3.30 (s, 3H), 1.51 (s, 9H).

Example 12: ( E )-4-(2-(5 H -Pyrido[4, 3- b ]Indol-7-day)vinyl)- N -Methylaniline (23, KNSP009)

Compound 18 synthesized in Example 11 (tert-butyl ( E )-(4-(2-(5 H -pyrido[4,3- b ]indol-7-yl)vinyl)phenyl)(methyl)carba Mate; 34 mg, 0.085 mmol) was dissolved in dichloromethane (5 mL), and the solution was stirred at 0° C. under nitrogen conditions, and trifluoroacetic acid (0.1 mL, 1.14 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours and then neutralized at 0° C. with a saturated aqueous sodium carbonate solution. The reaction mixture was extracted with dichloromethane, washed with brine, dried with sodium sulfate, and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography to give (elution solvent: methanol / dichloromethane = 1/30), compound as a light yellow solid 23, KNSP009 ((E) -4- (2- (5 H - pyrido [4, 3 -b ]indol-7-yl)vinyl) -N -methylaniline; 14.6mg, 57%) was synthesized.

¹ H NMR (400 MHz DMSO) δ11.74 (s, 1H), 9.27 (s, 1H), 8.38 (d, J = 5.6 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.62 ( s, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.44-7.40 (m, 3H), 7.20 (d, J = 16.4 Hz, 1H), 7.10 (d, J = 16.4 Hz, 1H), 6.57 (d, J = 8.4 Hz, 2H), 5.93 (d, J = 4.4 Hz, 1H), 2.72 (d, J = 4.8 Hz, 3H); ¹³ C NMR (100 MHz DMSO) δ 150.23, 144.68, 144.45, 142.87, 140.74, 137.34, 129.46, 128.16, 125.13, 123.99, 121.14, 120.02, 119.88, 118.73, 112.14, 108.97, 106.72, 30.07.

Example 13: ( E )-7-(4-fluorostyryl) -5H -Pyrido[4, 3- b ]Indol (19, KNSP008)

A solution of compound 30c (diethyl (4-fluorobenzyl) phosphonate; 59.6 mg, 0.218 mmol) in dimethyl sulfoxide (5 mL) was stirred at 0° C. under nitrogen conditions, and potassium tert-butoxide (0.232 mL) , 0.232 mmol) was added. After the reaction mixture was stirred at room temperature for 30 minutes, compound 6 (5 H -pyrido[4, 3- b ]indole-7-carbaldehyde; 30 mg, 0.145 mmol) was added, followed by stirring for 24 hours. The reaction mixture was cooled to 0° C. and saturated aqueous ammonium chloride solution was added. The reaction mixture was extracted with dichloromethane, dried by adding sodium sulfate, and then distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography to give (elution solvent: methanol / dichloromethane = 1/30), the compound of ivory solid 19, KNSP008 ((E) -7- (4-fluoro-styryl) -5H - pyrido [4, 3- b ]indole; 16.9 mg, 40%) was synthesized.

¹ H NMR (400MHz MeOD) δ9.20 (s, 1H), 8.36 (d, J = 6.0 Hz, 1H), 8.16 (d, J = 8.4 Hz, 1H), 7.69 (s, 1H), 7.64-7.61 (m, 2H), 7.56 (dd, J = 1.4, 8.2 Hz, 1H), 7.49 (d, J = 6.4 Hz, 1H), 7.29 (s, 2H), 7.13-7.08 (m, 2H) -; ¹³ C NMR (100MHz DMSO) δ163.32, 160.89, 144.68, 144.60, 142.88, 140.62, 136.30, 134.29, 129.58, 128.86, 128.79, 127.59, 121.34, 120.79, 119.20, 116.17, 115.96, 110.08, 106.89.

Example 14: ( E )-7-(2-(6-fluoropyridin-3-yl)vinyl) -5H -Pyrido[4, 3- b ]Indol (20, KNSP013)

Compound 30d (diethyl ((6-fluoropyridin-3-yl) methyl) phosphonate; 376 mg, 1.522 mmol) was dissolved in anhydrous N,N -dimethylformamide (5 mL) to 0 under nitrogen conditions. While stirring at °C, sodium hydride (101 mg, 2.54 mmol) was added. After the reaction mixture was stirred at room temperature for 30 minutes, compound 6 (5 H -pyrido[4, 3- b ]indole-7-carbaldehyde; 174.6 mg, 0.845 mmol) was added, followed by stirring for 48 hours. The reaction mixture was cooled to 0° C. and saturated aqueous ammonium chloride solution was added. The reaction mixture was extracted with dichloromethane, dried by adding sodium sulfate, and then distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/30), and the ivory solid compound 20, KNSP013 (( E )-7-(2-(6-fluoropyridin-3-yl) )Vinyl) -5H -pyrido[ 4,3 - b ]indole; 94.4 mg, 39%) was synthesized.

¹ H NMR (400MHz DMSO) δ 11.84 (s, 1H), 9.28 (s, 1H), 8.39 (d, J = 5.6 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.96 (dd, J = 2.6, 9.8 Hz, 1H) 7.62 (s, 1H), 7.59 (d, J = 2.4 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 6.4 Hz, 1H ), 7.18 (d, J = 16.4 Hz, 1H), 7.12 (d, J = 16.4 Hz, 1H); ¹³ C NMR (100MHz DMSO) δ 161.22, 143.65, 143.46, 141.89, 139.60, 137.00, 135.51, 133.87, 124.99, 123.46, 120.12, 119.79, 119.21, 118.82, 117.65, 115.33, 108.29, 105.73.

Example 15: ethyl ( E )-3-(4-(4-nitropyridin-3-yl)phenyl)acrylate (7)

A solution of ethyl 2-(diethoxyphosphoryl)acetate (5.32 mL, 26.3 mmol) in anhydrous tetrahydrofuran (60 mL) was stirred at 0°C under nitrogen conditions, and sodium hydride (1.12 g, 27 mmol) was added. . After the reaction mixture was stirred at room temperature for 30 minutes, compound 4 (4-(4-nitropyridin-3-yl)benzaldehyde; 4 g, 17.53 mmol) was added and stirred for another 24 hours. The reaction mixture was cooled to 0° C. and saturated aqueous ammonium chloride solution was added. The reaction mixture was extracted with dichloromethane, dried by adding sodium sulfate, and then distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/30), and a yellow solid compound 7 (ethyl ( E )-3-(4-(4-nitropyridin-3-yl)phenyl) ) Acrylate; 4.3 g, 82%) was synthesized.

¹ H NMR (400MHz CDCl ₃ ) δ8.86 (d, J = 5.2 Hz, 1H), 8.82 (s, 1H), 7.72 (d, J = 16.0 Hz, 1H), 7.70 (d, J = 5.1 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 6.51 (d, J = 16.0 Hz, 1H), 4.29 (q, J = 7.2 Hz, 2H) , 1.36 (t, J = 7.0 Hz, 3H).

Example 16: ( E )-3-(4-(4-nitropyridin-3-yl)phenyl)prop-2-en-1-ol (8)

Compound 7 synthesized in Example 15 (ethyl ( E )-3-(4-(4-nitropyridin-3-yl)phenyl)acrylate; 2.03 mg, 6.82 mmol) was dissolved in dichloromethane (50 mL). The solution was stirred at -78°C under nitrogen conditions and a solution of DIBAL-H (1 M in toluene, 28.6 mL, 28.6 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 8 hours, and then neutralized at 0° C. with 3M NaOH solution (10 mL, 30 mmol). The reaction mixture was extracted with dichloromethane, and the residue was purified by column chromatography (elution solvent: ethyl acetate/n-hexane=1/2), and a yellow solid compound 8 (( E )-3-(4-( 4-nitropyridin-3-yl)phenyl)prop-2-en-1-ol; 1.205 g, 69%) was synthesized.

¹ H NMR (400MHz CDCl ₃ ) δ 8.83-8.81 (m, 2H), 7.66 (d, J = 5.2 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.0 Hz , 2H), 6.67 (d, J = 16.0 Hz, 1H), 6.46 (dt, J = 5.4, 16.0 Hz, 1H), 4.37 (t, J = 5.2 Hz, 2H).

Example 17: ( E )-3-(5 H -Pyrido[4, 3- b ]Indole-7-yl)prop-2-en-1-ol (9)

Compound 8 synthesized in Example 16 (( E )-3-(4-(4-nitropyridin-3-yl)phenyl)prop-2-en-1-ol; 1.42 g, 5.54 mmol) and tri A solution of phenylphosphine (3.63 g, 13.85 mmol) dissolved in 1,2-dichlorobenzene (30 mL) was stirred at 180° C. for 48 hours under nitrogen conditions. After cooling the reaction mixture to room temperature, 1,2-dichlorobenzene was removed under vacuum, and the residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/10), and a yellow solid compound 9 ( ( E )-3-( 5H -pyrido[4,3- b ]indol-7-yl)prop-2-en-1-ol; 903 mg, 73%) was synthesized.

¹ H NMR (400MHz MeOD) δ 9.17 (d, J = 1.2 Hz, 1H), 8.34 (d, J = 6.0 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.55 (s, 1H) , 7.45 (dd, J = 1.0, 5.8 Hz, 1H), 7.41 (d, J = 1.2, 8.4 Hz, 1H), 6.78 (d, J = 16.0 Hz, 1H), 6.49 (dt, J = 5.6, 16.0 Hz, 1H), 4.28 (dd, J = 1.6, 5.6 Hz, 2H).

Example 18: ( E )-3-(5 H -Pyrido[4, 3- b ]Indole-7-yl)acrylaldehyde (10)

Compound 9 synthesized in Example 17 (( E )-3-(5 H -pyrido[4,3- b ]indol-7-yl)prop-2-en-1-ol; 903 mg, 4.03 mmol) in dichloromethane (20 mL) was stirred at 0° C. under nitrogen conditions, and DMP (2.69 g, 6.04 mmol) was added. The reaction mixture was stirred at room temperature and observed by thin layer chromatography every 1 hour. After confirming the completion of the reaction by thin layer chromatography, the reaction mixture was filtered through celite and neutralized with a saturated aqueous sodium carbonate solution. The reaction mixture was extracted with ethyl acetate, washed with brine, dried with sodium sulfate, and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: ethyl acetate/n-hexane=1/1), and a yellow solid compound 10 (( E )-3-( 5H -pyrido[4,3- b ] Indole-7-yl)acrylaldehyde; 450 mg, 50%) was synthesized.

¹ H NMR (400MHz acetone-d ₆ ) δ 9.76 (d, J = 7.6 Hz, 1H), 9.40 (d, J = 0.8 Hz, 1H), 8.49 (d, J = 5.6 Hz, 1H), 8.34 (d , J = 8.0 Hz, 1H), 7.97 (s, 1H), 7.87 (d, J = 15.6 Hz, 1H), 7.71 (dd, J = 1.4, 8.2 Hz, 1H), 7.51 (dd, J = 0.8, 5.6 Hz, 1H), 6.88 (dd, J = 7.6, 16.0 Hz, 1H).

Example 19: 2-((1 E ,3 E )-4-(5 H -Pyrido[4, 3- b ]Indole-7-yl)buta-1,3-dien-1-yl)-6-methoxybenzo[ d ]Thyazole (24, KNSP010)

Compound 32 (diethyl ((6-methoxybenzo[d]thiazol-2-yl)methyl)phosphonate; 156 mg, 0.495 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL) under nitrogen conditions. While stirring at 0° C., sodium hydride (28.8 mg, 0.72 mmol) was added. After the reaction mixture was stirred at room temperature for 30 minutes, compound 10 (( E )-3-(5 H -pyrido[4,3- b ]indol-7-yl)acrylaldehyde; 100 mg, 0.45 mmol) was added thereto. It was added and stirred for another 24 hours. The reaction mixture was cooled to 0° C. and saturated aqueous ammonium chloride solution was added. The reaction mixture was extracted with dichloromethane, dried by adding sodium sulfate, and then distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/30), and a yellow solid compound 24, KNSP010 (2-((1 E ,3 E )-4-(5 H -pyrido [4, 3- b ]indol-7-yl)buta-1,3-dien-1-yl)-6-methoxybenzo[ d ]thiazole; 35 mg, 20%) was synthesized.

¹ H NMR (400MHz DMSO) δ 11.81 (s, 1H), 9.34 (s, 1H), 8.42 (d, J = 5.6 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.8 Hz, 1H), 7.70 (s, 1H), 7.66 (d, J = 2.8 Hz, 1H)-, 7.55 (dd, J = 1.0, 8.2 Hz, 1H), 7.48 (d, J = 5.2 Hz , 1H), 7.43-7.31 (m, 2H), 7.22 (d, J = 14.8 Hz, 1H), 7.10 (dd, J = 2.4, 8.8 Hz, 1H), 7.08 (d, J = 14.8 Hz, 1H) ; ¹³ C NMR (100MHz DMSO) δ164.33, 158.04, 148.51, 145.27, 144.75, 143.40, 140.49, 138.44, 137.94, 136.00, 135.57, 128.18, 125.65, 123.53, 121.56, 121.44, 119.79, 119.56, 116.26 , 105.27, 56.23.

Example 20: 7-((1 E ,3 E )-4-(4-nitrophenyl)buta-1,3-dien-1-yl) -5H -Pyrido[4, 3- b ]Indol (25, KNSP011)

A solution of compound 30a (diethyl (4-nitrobenzyl) phosphonate; 284 mg, 1.04 mmol) in anhydrous tetrahydrofuran (10 mL) was stirred at 0°C under nitrogen conditions and sodium hydride (60 mg, 1.49 mmol) ) Was added. After the reaction mixture was stirred at room temperature for 30 minutes, compound 10 (( E )-3-(5 H -pyrido[4,3- b ]indol-7-yl)acrylaldehyde; 165 mg, 0.74 mmol) was added. It was added and stirred for another 48 hours. The reaction mixture was cooled to 0° C. and saturated aqueous ammonium chloride solution was added. The reaction mixture was extracted with dichloromethane, dried by adding sodium sulfate, and then distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/30), and yellow solid compound 25, KNSP011 (7-((1 E ,3 E )-4-(4-nitrophenyl) Buta-1,3-dien-1-yl) -5H -pyrido[ 4,3 - b ]indole; 134 mg, 53%) was synthesized.

¹ H NMR (400MHz DMSO) δ 11.76 (s, 1H), 9.32 (d, J = 0.8 Hz, 1H), 8.41 (d, J = 5.6 Hz, 1H), 8.23-8.21 (m, 3H), 7.80 ( d, J = 8.8 Hz, 2H), 7.68 (s, 1H), 7.53 (dd, J = 1.0, 7.8 Hz, 1H), 7.46 (dd, J = 0.8, 5.6 Hz, 1H), 7.42 (dd, J = 10.8, 15.6 Hz, 1H), 7.29 (dd, J = 10.6, 15.0 Hz, 1H), 7.09 (d, J = 15.2 Hz, 1H), 6.93 (d, J = 15.2 Hz, 1H); ¹³ C NMR (100MHz DMSO) δ 146.41, 145.07, 144.73, 144.60, 143.31, 140.55, 137.09, 135.68, 134.99, 130.55, 129.17, 127.53, 124.56, 121.39, 121.33, 119.77, 119.42, 110.24, 106.

Example 21: 4-((1 E ,3 E )-4-(5 H -Pyrido[4, 3- b ]Indole-7-yl)buta-1,3-dien-1-yl)aniline (27, KNSP012)

Compound 25 synthesized in Example 20 (7-((1 E ,3 E )-4-(4-nitrophenyl)buta-1,3-dien-1-yl) -5H -pyrido[ 4,3 -b ]Indole; 110.3 mg, 0.323 mmol) was dissolved in ethanol (6 mL), and tin(II) chloride dihydrate (451 mg, 1.94 mmol) dissolved in 0.6 mL of concentrated hydrochloric acid was added. The reaction mixture was stirred at 106° C. for 8 hours under nitrogen conditions. The reaction mixture was cooled to 0° C., and the pH of the solution was adjusted to 8 using 1N aqueous sodium hydroxide solution. The reaction mixture was extracted with dichloromethane, dried with sodium sulfate, and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/20), and a yellow solid compound 27, KNSP012 (4-((1 E ,3 E )-4-(5 H -pyrido [4, 3- b ]indol-7-yl)buta-1,3-dien-1-yl)aniline; 31.8 mg, 32%) was synthesized.

¹ H NMR (400MHz DMSO) δ 11.77 (s, 1H), 9.27 (s, 1H), 8.38 (d, J = 5.6 Hz, 1H), 8.14 (d, J = 8.0 Hz, 1H), 7.57 (s, 1H), 7.44-7.42 (m, 2H), 7.21 (d, J = 8.4 Hz, 2H), 7.13 (dd, J = 10.5, 15.4 Hz, 1H), 6.81 (dd, J = 10.4, 15.0 Hz, 1H ), 6.75 (d, J = 15.2 Hz, 1H), 6.63 (d, J = 14.8 Hz, 1H), 6.55 (d, J = 8.0 Hz, 2H), 5.36 (s, 2H).

Example 22: tert-butyl (4-((1 E ,3 E )-4-(5 H -Pyrido[4, 3- b ]Indole-7-yl)buta-1,3-dien-1-yl)phenyl)(methyl)carbamate (26)

Compound 31 (tert-butyl (4-((diethoxyphosphoryl)methyl)phenyl)(methyl)carbamate; 281 mg, 0.79 mmol) was dissolved in anhydrous N,N -dimethylformamide (5 mL), and nitrogen While stirring at 0° C. under conditions, sodium hydride (76 mg, 1.89 mmol) was added. After the reaction mixture was stirred at room temperature for 30 minutes, compound 10 (( E )-3-(5 H -pyrido[4,3- b ]indol-7-yl)acrylaldehyde; 70 mg, 0.315 mmol) was added. It was added and stirred for another 48 hours. The reaction mixture was cooled to 0° C. and saturated aqueous ammonium chloride solution was added. The reaction mixture was extracted with dichloromethane, dried by adding sodium sulfate, and then distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/30), and compound 26 as an ivory solid (tert-butyl (4-((1 E ,3 E )-4-(5 H -)) Pyrido[4,3- b ]indol-7-yl)buta-1,3-dien-1-yl)phenyl)(methyl)carbamate; 38.5 mg, 29%) was synthesized.

¹ H NMR (400MHz acetone-d ₆ ) δ 9.31 (s, 1H), 8.43 (d, J = 5.6 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H), 7.69 (s, 1H), 7.52 -7.49 (m, 3H), 7.45 (d, J = 5.6 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.20 (dd, J = 10.6, 15.0 Hz, 1H), 7.11 (dd, J = 10.4, 15.2 Hz, 1H), 6.93 (d, J = 15.2 Hz, 1H), 6.78 (d, J = 15.2 Hz, 1H), 3.25 (s, 3H), 1.45 (s, 9H).

Example 23: 4-((1 E ,3 E )-4-(5 H -Pyrido[4, 3- b ]Indole-7-yl)buta-1,3-dien-1-yl)- N -Methylaniline (28, KNSP014)

Compound 26 synthesized in Example 22 (tert-butyl (4-((1 E ,3 E )-4-(5 H -pyrido[4,3- b ]indol-7-yl)buta-1, 3-dien-1-yl)phenyl)(methyl)carbamate; 39 mg, 0.092 mmol) was dissolved in dichloromethane (5 mL) and stirred at 0°C under nitrogen conditions, while trifluoroacetic acid (0.282 mL, 3.67) mmol) was added. The reaction mixture was stirred at room temperature for 24 hours and then neutralized at 0° C. with a saturated aqueous sodium carbonate solution. The reaction mixture was extracted with dichloromethane, washed with brine, dried with sodium sulfate, and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/30), and the ivory solid compound 28, KNSP014 (4-((1 E ,3 E )-4-(5 H -pyrido [4, 3- b ]indol-7-yl)buta-1,3-dien-1-yl) -N -methylaniline; 10 mg, 34%) was synthesized.

¹ H NMR (400MHz DMSO) δ11.84 (s, 1H), 9.27 (s, 1H), 8.38 (d, J = 5.6 Hz, 1H), 8.14 (d, J = 8.0 Hz, 1H), 7.57 (s , 1H), 7.44-7.42 (m, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.13 (dd, J = 10.2, 15.8 Hz, 1H), 6.83 (dd, J = 10.6, 15.4 Hz, 1H), 6.75 (d, J = 15.6 Hz, 1H), 6.65 (d, J = 15.6 Hz, 1H), 6.53 (d, J = 8.0 Hz, 2H), 5.97 (d, J = 5.2 Hz, 1H) , 2.70 (d, J = 4.8 Hz, 3H).

Example 24: 5,5-difluoro-1,3-dimethyl -5H -4λ ⁴ ,5λ ⁴ -Dipyrrolo[1,2- c :2',1'- f ][1,3,2] Diazaborinine (29)

A solution of pyrrole-2-carboxaldehyde (400 mg, 4.12 mmol) in dichloromethane (3 mL) was stirred at -5°C under nitrogen conditions, and 2,4-dimethylpyrrole (0.388 mL, 4.12 mmol) and chloride Phosphoryl (0.388 mL, 4.12 mmol) was slowly added dropwise. The reaction mixture was stirred at -5°C for 3 hours and at room temperature for 3 hours. After the starting material disappeared in thin layer chromatography, DIEA (2.176 mL, 12.37 mmol) and BF ₃ OEt ₂ (1.526 mL, 12.37 mmol) were added to the reaction mixture. When the reaction was completed, distilled water was added to the reaction mixture, extracted with ethyl acetate, dried with sodium sulfate, and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: ethyl acetate/n-hexane=1/5), and a dark red solid compound 29 (5,5-difluoro-1,3-dimethyl- 5H- 5λ ⁴ , 5λ ⁴ -dipyrrolo[1,2- c :2',1'- f ][1,3,2]diazaborinine; 899 mg, 99%) was synthesized.

¹ H NMR (400MHz CDCl ₃ ) δ 7.64 (s, 1H), 7.12 (s, 1H), 6.93 (d, J = 3.6 Hz, 1H), 6.43 (s, 1H), 6.16 (s, 1H), 2.59 (s, 3H), 2.28 (s, 3H).

Example 25: Diethyl (4-nitrobenzyl) phosphonate (30a)

A solution of 1-(bromomethyl)-4-nitrobenzene (1 g, 4.54 mmol) in triethylphosphite (3 mL) was stirred at 160° C. for 6 hours under nitrogen conditions. The volatile solvent was removed by distillation under reduced pressure, and the residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/50) to obtain an orange oily compound 30a (diethyl (4-nitrobenzyl) phosphonate; 1.23. g, 99%) was synthesized.

¹ H NMR (400 MHz CDCl ₃ ) δ 9.00 (s, 1H), 8.76 (d, J = 5.2 Hz, 1H), 7.70 (d, J = 5.2 Hz, 1H).

Example 26: tert-butyl (4-((diethoxyphosphoryl)methyl)phenyl)carbamate (30b)

A solution of tert-butyl (4-(bromomethyl)phenyl)carbamate (1 g, 3.39 mmol) in triethylphosphite (3 mL) was stirred at 160° C. for 6 hours under nitrogen conditions. The volatile solvent was removed by distillation under reduced pressure, and the residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/50) to obtain a white solid compound 30b (tert-butyl (4-((diethoxyphosphoryl)). Methyl)phenyl)carbamate; 1.07 g, 92%) was synthesized.

¹ H NMR (400 MHz CDCl ₃ ) δ9.00 (s, 1H), 8.76 (d, J = 5.2 Hz, 1H), 7.70 (d, J = 5.2 Hz, 1H).

Example 27: Diethyl (4-fluorobenzyl) phosphonate (30c)

A solution of 1-(bromomethyl)-4-fluorobenzene (0.659 mL, 5.13 mmol) in triethylphosphite (3 mL) was stirred at 160° C. for 6 hours under nitrogen conditions. The volatile solvent was removed by distillation under reduced pressure, and the residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/50) to obtain a clear oily compound 30c (diethyl (4-fluorobenzyl) phosphonate; 1.26 g, 100%) was synthesized.

¹ H NMR (400MHz MeOD) δ 9.00 (s, 1H), 8.76 (d, J = 5.2 Hz, 1H), 7.70 (d, J = 5.2 Hz, 1H).

Example 28: Diethyl ((6-fluoropyridin-3-yl)methyl)phosphonate (30d)

A solution of 5-(bromomethyl)-2-fluoropyridine (835 mg, 4.39 mmol) in triethylphosphite (3 mL) was stirred at 160° C. for 6 hours under nitrogen conditions. The volatile solvent was removed by distillation under reduced pressure, and the residue was purified by column chromatography (elution solvent: methanol/dichloromethane=1/50) to obtain a clear oily compound 30d (diethyl ((6-fluoropyridin-3-yl )Methyl)phosphonate; 670 mg, 62%) was synthesized.

¹ H NMR (400MHz CDCl ₃ ) δ 8.10 (s, 1H), 7.84 (tt, J = 2.3, 6.0 Hz, 1H), 6.91 (dd, J = 2.8, 8.4 Hz, 1H), 4.09-4.02 (m, 4H), 3.79 (d, J = 12.0 Hz, 2H), 1.27 (t, J = 7.0 Hz, 6H).

Example 29: Tert-butyl (4-((diethoxyphosphoryl)methyl)phenyl)(methyl)carbamate (31)

A solution of compound 30b (tert-butyl (4-((diethoxyphosphoryl)methyl)phenyl)carbamate; 306 mg, 0.89 mmol) in anhydrous tetrahydrofuran (8 mL) was stirred at 0°C under nitrogen conditions. Sodium hydride (71.3 mg, 1.78 mmol) was added. After the reaction mixture was stirred at room temperature for 30 minutes, methyl iodide (0.113 mL, 1.78 mmol) was added and stirred at room temperature for 24 hours. The reaction mixture was extracted with dichloromethane, dried with sodium sulfate, and distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: ethyl acetate/n-hexane=1/3), and a white solid compound 31 (tert-butyl (4-((diethoxyphosphoryl)methyl)phenyl)(methyl ) Carbamate; 304 mg, 95%) was synthesized.

¹ H NMR (400 MHz CDCl ₃ ) δ 9.00 (s, 1H), 8.76 (d, J = 5.2 Hz, 1H), 7.70 (d, J = 5.2 Hz, 1H).

Example 30: Diethyl ((6-methoxybenzo[d]thiazol-2-yl)methyl)phosphonate (32)

n-butyllithium (2.42 mL, 6.06 mmol), diisopropylamine (0.868 mL, 6.06 mmol) and 6-methoxy-2-methylbenzothiazole (0.415 mL, 2.71 mmol) were added to anhydrous tetrahydrofuran (25 mL). ) Was stirred for 30 minutes at -78 ℃ under nitrogen conditions. Diethyl phosphorochloridate (0.443 mL, 2.98 mmol) was added to the resulting red reaction mixture, followed by stirring at room temperature for 6 hours. A saturated aqueous ammonium chloride solution was added to the reaction mixture to complete the reaction. The reaction mixture was extracted with dichloromethane, dried by adding sodium sulfate, and then distilled under reduced pressure to remove the solvent. The residue was purified by column chromatography (elution solvent: ethyl acetate/n-hexane=1/1), and an orange oily compound 32 (diethyl ((6-methoxybenzo[d]thiazol-2-yl)) Methyl)phosphonate; 594.4 mg, 70%) was synthesized.

¹ H NMR (400MHz CDCl ₃ ) δ 7.87 (d, J = 9.2 Hz, 1H), 7.31 (d, J = 2.5 Hz, 1H), 7.06 (dd, J = 2.5, 8.9 Hz, 1H), 4.19-4.12 (m, 4H), 3.87 (s, 3H), 3.69 (d, J = 21.5 Hz, 2H), 1.32 (t, J = 7.1 Hz, 6H).

Test Example 1: Measurement of fluorescence for the synthesized compound

In order to investigate the optical properties of the compound synthesized in the present invention, the absorbance was measured using a V-730 UV-visible spectrophotometer manufactured by JASCO, and fluorescence was performed using a FlexStaion 3 multi-mode microplate reader manufactured by Molecular Devices. Was measured. Absorbance and fluorescence were measured three times in ethanol at a concentration of 10 μM, and are shown as average values in Table 1 below. At this time, as a control group, T807 (AV-1451), a positron emission tomography (PET) tracer, was used to confirm the production of tau in vivo.

As a result of measuring absorbance and fluorescence, it was confirmed that the compound newly synthesized in the present invention can emit light at a fluorescence wavelength of 400 nm to 600 nm, and it was confirmed to have a high Stokes shift of 90 nm to 140 nm.

[Table 1] Fluorescence measurement results of synthetic compounds

Λ _ex ^a : maximum excitation

Λ _em ^b : maximum emission

Stokes shift: The difference between the energy of the excitation light and the energy in Stokes' law

ClogP ^c : It means the partition coefficient of the compound and is a measure of lipophilicity

Test Example 2: In vitro for analysis of selectivity for tau and amyloid beta aggregates ( in vitro ) Screening

Test Example 2-1: Induction of tau and amyloid beta aggregation

For the induction of amyloid aggregation, the synthesized Abeta40 (Amyloid Beta 40) peptide was dissolved in PBS at a concentration of 0.5 mg/ml to induce aggregation for 7 days at 37°C and 200 rpm shaking conditions. In order to induce aggregation of tau protein, the concentration of Tau-K18 protein (0.5 mg/ml in PBS) separated and purified from E.coli was used with heparin (0.1 mg/ml) and DTT (100 μM) at 37°C, 200 rpm. Aggregation was induced for 7 days under shaking conditions.

Test Example 2-2: Measurement of labeling ability for tau and amyloid aggregated derivatives

Since the aggregation of tau and amyloid occurs only in the pathological environment, in order to confirm that the newly synthesized compound can label the tau aggregated protein, the composition and non-aggregated tau protein (pre-aggregate) and aggregated tau protein (aggregates) The reactivity of was compared and analyzed. At this time, as a control, thioflavin S (Th S), known to bind to a beta-sheet structure such as tau and amyloid aggregates, was used.

Specifically, in order to confirm the labeling ability of the newly synthesized compounds for tau aggregated protein, 25 μl of a 10 μM labeling material diluted in PBS was added to 0.125 mg/ml of tau or amyloid pre-aggregate and aggregate 25 diluted in PBS. After incubation with µl for 30 minutes each, the fluorescence spectrum reaction was measured using a spectrophotometer. At this time, the excitation (Ex) wavelength and the emission (Em) wavelength for each compound are as shown in Table 2 below.

Test Example 2-3: Measurement of non-specific binding using BSA

In order to exclude non-specific binding of the compound to serum, reactivity between bovine serum albumin (BSA) was further tested. After incubating 25 µl of 10 µM compound with 25 µl of 0.25 mg/ml BSA for 30 minutes each, the fluorescence spectrum reaction was measured using a spectrophotometer. At this time, the excitation (Ex) wavelength and the emission (Em) wavelength for each compound are as shown in Table 2 below. The BSA reaction is to confirm non-selective binding to other proteins in the blood when applied to a patient as an imaging agent.

As described above, the selectivity analysis for tau aggregates and amyloid beta aggregates of the novel synthetic compounds of the present invention was performed, and the results of in vitro fluorescence changes accordingly are summarized in Table 2 and FIG. 1 below.

In vitro screening tau / beta Table intelligence of new synthetic compounds of the present invention as the fluorescent probes in the result, the probes KNSP007 ((E) -4- (2- (5 H - pyrido [4, 3- b] indole -7-yl)vinyl)aniline), KNSP011 (7-((1 E ,3 E )-4-(4-nitrophenyl)buta-1,3-dien-1-yl) -5H -pyrido[4 , 3- b ]indole), KNSP012 (4-((1 E ,3 E )-4-(5 H -pyrido[4,3- b ]indole-7-yl)buta-1,3-diene- It was confirmed that 1-day)aniline) has high selectivity to tau aggregates compared to amyloid aggregates. KNSP007 showed 3.1 times, KNSP011 showed 4.1 times, and KNSP012 showed 3.8 times selectivity. While Thioflavin S showed high BSA reactivity, it was confirmed that the selected compound had relatively very low reactivity to BSA.

[Table 2] Fluorescence characteristics of synthesized probes according to binding to tau aggregates, amyloid beta aggregates and bovine serum albumin (BSA)

^a Fold Increase (FI) = fluorescence intensity of probe bound to tau aggregate (or Aβ aggregate or BSA)/fluorescence intensity of probe (free)

^b Selectivity Index (SI) = fluorescence intensity of probe bound to tau aggregate/fluorescence intensity of probe bound to Aβ aggregate (or BSA)

The novel 5 H -pyrido [4,3-b] indole derivative compound of the present invention exhibits autofluorescence and selectively binds to tau fiber protein to exhibit high tau fiber protein detection effect. Accordingly, the compound can be usefully used as a fluorescent probe composition for detecting tau fiber protein for early diagnosis of tauopathy caused by tau aggregates, and thus has industrial applicability.

Claims (8)

5 H -pyrido [4,3-b] indole derivative compound represented by the following formula (1):
[Formula 1]

In Formula 1,
R ₁ is hydrogen; C ₁ -C ₁₃ alkyl group; or C ₃ -C ₁₀ cyclyl group; and,
R ₂ is a C ₃ -C ₁₀ aryl group; Or a 5- to 9-membered heteroaryl group containing 1 to 4 heteroatoms selected from boron (B), nitrogen (N), oxygen (O) and sulfur (S) atoms; and,
n is an integer from 0 to 3,
Each of the C ₁ -C ₁₃ alkyl group or C ₃ -C ₁₀ cyclyl group is hydrogen; Hydroxy group; Halogen group; A carbonyl group (-(C=O)R ₃ R ₄ ); A C ₁ -C ₃ alkyl group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; A C ₁ -C ₃ alkoxy group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; C ₆ -C ₁₀ phenoxy; Amino group (-NR ₃ R ₄ ); An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; Including one or more substituents selected from the group consisting of a C ₃ -C ₁₀ heteroaryl group and a C ₃ -C ₁₀ heterocyclyl group,
Each of the C ₃ -C ₁₀ aryl group or 5 to 9 membered heteroaryl group is hydrogen; Hydroxy group; Halogen group; A carbonyl group (-(C=O)R ₃ R ₄ ); A C ₁ -C ₃ alkyl group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; A C ₁ -C ₃ alkoxy group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; C ₆ -C ₁₀ phenoxy; Amino group (-NR ₃ R ₄ ); An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; Including one or more substituents selected from the group consisting of a C ₃ -C ₁₀ heteroaryl group and a C ₃ -C ₁₀ heterocyclyl group,
Each of R ₃ and R ₄ is hydrogen; C ₁ -C ₆ alkyl group; C ₁ -C ₆ alkenyl group; C ₁ -C ₆ alkynyl group; C ₆ -C ₁₀ aryl group; C ₃ -C ₁₀ heteroaryl group; And one or more selected from the group consisting of a C ₃ -C ₁₀ heterocyclyl group.
The method of claim 1,
R ₁ is hydrogen; Or a C ₁ -C ₆ alkyl group;
R ₂ is substituted or unsubstituted benzene; Substituted or unsubstituted pyridazine; Substituted or unsubstituted pyrazine; Substituted or unsubstituted imidazole; Substituted or unsubstituted pyrazole; Substituted or unsubstituted furan; Substituted or unsubstituted pyrimidine; Substituted or unsubstituted pyrrole; Substituted or unsubstituted pyridine; Substituted or unsubstituted indole; Substituted or unsubstituted thiazole; Substituted or unsubstituted benzothiazole; and,
The C ₁ -C ₆ alkyl group, hydrogen; Hydroxy group; Halogen group; C ₁ -C ₁₃ alkyl group; C ₁ -C ₆ alkoxy group; An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; C ₃ -C ₁₀ heteroaryl group; And C ₃ -C ₁₀ containing one or more substituents selected from the group consisting of heterocyclyl group,
5 H represented by the following formula (1) -pyrido [4,3-b] indole derivatives.
The method of claim 1, wherein the compound is
(Compound No. 1) (E) -7- (2- (-1- methyl-5,5-difluoro -5H -4λ ^4, 5λ ⁴ - Diffie pyrrolo [1,2- c: 2 ', 1'- f ][1,3,2]diazavorinin-3-yl)vinyl) -5H -pyrido[ 4,3 - b ]indole;
(Compound No. 2) 7-(1 H -indole-6-yl)-5 H -pyrido[4,3- b ]indole;
(Compound No. 3) ( E )-7-(4-nitrostyryl) -5H -pyrido[ 4,3 - b ]indole;
(Compound No. 4) ( E )-2-(2-( 5H -pyrido[4,3- b ]indol-7-yl)vinyl)-6-methoxybenzo[ d ]thiazole;
(Compound No. 5) ( E )-2-(2-( 5H -pyrido[4,3- b ]indol-7-yl)vinyl)benzo[ d ]thiazol-6-ol;
(Compound No. 6) ( E )-4-(2-( 5H -pyrido[4,3- b ]indol-7-yl)vinyl)aniline;
(Compound No. 7) ( E )-7-(4- fluorostyryl ) -5H -pyrido[ 4,3 - b ]indole;
(Compound No. 8) ( E )-4-(2-(5 H -pyrido[4,3- b ]indol-7-yl)vinyl) -N -methylaniline;
(Compound No. 9) 2-((1 E ,3 E )-4-(5 H -pyrido[4,3- b ]indol-7-yl)buta-1,3-dien-1-yl)- 6-methoxybenzo[ d ]thiazole;
(Compound No. 10) 7-((1 E ,3 E )-4-(4-nitrophenyl)buta-1,3-dien-1-yl) -5H -pyrido[ 4,3 - b ]indole;
(Compound No. 11) 4-((1 E ,3 E )-4-(5 H -pyrido[4,3- b ]indol-7-yl)buta-1,3-dien-1-yl)aniline ;
(Compound No. 12) ( E )-7-(2-(6-fluoropyridin-3-yl)vinyl) -5H -pyrido[ 4,3 - b ]indole; And
(Compound No. 13) 4-((1 E ,3 E )-4-(5 H -pyrido[4,3- b ]indol-7-yl)buta-1,3-dien-1-yl)- A compound which is any one selected from the group consisting of N -methylaniline.
A fluorescent probe for detecting tau fibroprotein comprising the compound of any one of claims 1 to 3.
A composition comprising a fluorescent probe for detecting tau fiber protein according to claim 4 as an active ingredient.
The method of claim 5,
The composition is a composition used for the diagnosis of tauopathy selected from the group consisting of Alzheimer's disease, progressive supranuclear paralysis, cortical-basal ganglia degeneration, Argyrophilic Grain Disease, Peak disease and hereditary anterior temporal dementia.
A method for detecting tau aggregates in cells in vitro or in tissues ex vivo using the compound of any one of claims 1 to 3.
5 to H represented by the following formula (1) below and a compound represented by the general formula (II) comprises reacting a compound represented by General Formula (3) - process for producing a pyrido [4,3-b] indole derivative compounds:

In Formulas 1 to 3,
R ₁ is hydrogen; C ₁ -C ₁₃ alkyl group; or C ₃ -C ₁₀ cyclyl group; and,
R ₂ is a C ₃ -C ₁₀ aryl group; Or a 5- to 9-membered heteroaryl group containing 1 to 4 heteroatoms selected from boron (B), nitrogen (N), oxygen (O) and sulfur (S) atoms; and,
n is an integer from 0 to 3,
m is an integer from 0 to 3,
Each of the C ₁ -C ₁₃ alkyl group or C ₃ -C ₁₀ cyclyl group is hydrogen; Hydroxy group; Halogen group; A carbonyl group (-(C=O)R ₃ R ₄ ); A C ₁ -C ₃ alkyl group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; A C ₁ -C ₃ alkoxy group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; C ₆ -C ₁₀ phenoxy; Amino group (-NR ₃ R ₄ ); An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; Including one or more substituents selected from the group consisting of a C ₃ -C ₁₀ heteroaryl group and a C ₃ -C ₁₀ heterocyclyl group,
Each of the C ₃ -C ₁₀ aryl group or 5 to 9 membered heteroaryl group is hydrogen; Hydroxy group; Halogen group; A carbonyl group (-(C=O)R ₃ R ₄ ); A C ₁ -C ₃ alkyl group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; A C ₁ -C ₃ alkoxy group unsubstituted or substituted with a halogen or C ₃ -C ₁₀ heterocyclyl group; C ₆ -C ₁₀ phenoxy; Amino group (-NR ₃ R ₄ ); An amide group (-(C=O)NR ₃ R ₄ ); C ₆ -C ₁₀ aryl group; Including one or more substituents selected from the group consisting of a C ₃ -C ₁₀ heteroaryl group and a C ₃ -C ₁₀ heterocyclyl group,
Each of R ₃ and R ₄ is hydrogen; C ₁ -C ₆ alkyl group; C ₁ -C ₆ alkenyl group; C ₁ -C ₆ alkynyl group; C ₆ -C ₁₀ aryl group; C ₃ -C ₁₀ heteroaryl group; And one or more selected from the group consisting of a C ₃ -C ₁₀ heterocyclyl group.

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