WO2021145641A1 - N-phenolbenzothiazol-2-amine compound having anti-angiogenetic effect, and pharmaceutical composition containing same - Google Patents

Abstract

The present invention relates to an N-phenolbenzothiazol-2-amine compound inhibiting angiogenesis and having structural stability, and to a pharmaceutical composition containing same as an active ingredient. The N-phenolbenzothiazol-2-amine compound of the present invention can be effectively used to treat or prevent diabetic retinopathy, cancer, duodenal ulcer, arthritis, or obesity.

Description

N-phenylbenzothiazol-2-amine compound having angiogenesis inhibitory effect and pharmaceutical composition comprising same

The present invention relates to an N-phenylbenzothiazol-2-amine compound having an angiogenesis inhibitory effect and a pharmaceutical composition comprising the same, and more particularly, to a vascular endothelial growth factor (VEGF) signaling system. It relates to an N-phenylbenzothiazol-2-amine compound having structural stability and inhibiting angiogenesis by inhibiting and mediating the transcription of HIF-1α to suppress the sub-signaling system, and a pharmaceutical composition comprising the same as an active ingredient .

VEGF is a signal protein that stimulates angiogenesis and is an important in vivo signal protein that is expressed when oxygen deficiency occurs in blood vessels and creates new blood vessels. In particular, VEGF is a protein highly associated with diseases such as diabetic retinopathy, cancer, duodenal ulcer, arthritis, and obesity, which are diseases in which blood vessels are unnecessarily generated.

On the other hand, HIF-1α (Hypoxia-inducible factor-1α) can be utilized as a target for aggravated diseases due to the activation of various angiogenesis by inhibiting angiogenesis. Diabetic retinopathy or rheumatoid arthritis may be exacerbated by increased expression of VEGF by HIF-1α under hypoxic conditions. Therefore, a compound that inhibits HIF-1α activated from hypoxia can be utilized as a new therapeutic agent for diseases such as diabetic retinopathy and rheumatoid arthritis.

Want FIG Nin (Wondonin) of formula (1) silanol stripe source in Fig N-Sys spongy PO A (Poecillastra wondoensis) and in Yasushi piece (Jaspis sp.) Structurally distinct from marine alkaloid with the identified clusters. Unlike conventional VEGF inhibitors, this does not inhibit human umbilical vascular endothelial cells (HUVEC), effectively inhibiting VEGF without cytotoxicity, thereby inhibiting angiogenesis [Korea Patent Publication No. 10-2012-0122705 See No.].

[Formula 1]

However, Wondonin has structural instability due to the benzodioxol moiety, so it has a disadvantage in that it is less stable in the manufacturing and storage process and it is difficult to achieve in vivo activity.

One object of the present invention is to provide an N-phenylbenzothiazol-2-amine compound of the following formula (I) or a pharmaceutically acceptable salt thereof, which inhibits angiogenesis through inhibition of the VEGF signaling system and has structural stability.

Another object of the present invention is to provide a pharmaceutical composition for inhibiting angiogenesis containing an N-phenylbenzothiazol-2-amine compound of the following formula (I) or a pharmaceutically acceptable salt thereof.

One embodiment of the present invention relates to an N-phenylbenzothiazol-2-amine compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.

[Formula I]

In the above formula,

R ¹ and R ² are each independently hydrogen, halogen, nitro, amino, a C ₁ -C ₆ alkoxy group or a C ₁ -C ₆ haloalkyl group,

R ³ is a C ₁ -C ₆ alkyl group,

L does not exist or

ego,

R ⁴ and R ⁵ are each independently hydrogen, a C ₁ -C ₆ alkyl group, a C ₂ -C ₆ alkynyl group, or a C ₃ -C ₁₀ cycloalkyl group,

R ⁴ and R ⁵ together with the nitrogen atom to which they are bonded form a 5 to 7 membered heterocycle,

n is an integer from 1 to 6,

m is an integer from 1 to 6.

As used herein, C ₁ -C ₆ Alkoxy group refers to a straight-chain or branched alkoxy group having 1 to 6 carbon atoms, and includes, but is not limited to, methoxy, ethoxy, n-propanoxy, and the like.

As used herein, a C ₁ -C ₆ haloalkyl group refers to a straight-chain or branched hydrocarbon having 1 to 6 carbon atoms substituted with one or more halogens selected from the group consisting of fluorine, chlorine, bromine and iodine, for example, trifluoromethyl, trichloromethyl, trifluoroethyl, and the like.

As used herein, C ₁ -C ₆ Alkyl group refers to a linear or branched monovalent hydrocarbon having 1 to 6 carbon atoms, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl , i-butyl, t-butyl, n-pentyl, n-hexyl, and the like.

As used herein, the C ₂ -C ₆ alkynyl group refers to a straight-chain or branched unsaturated hydrocarbon having 2 to 6 carbon atoms having at least one carbon-carbon triple bond, and acetylenyl, propynyl, butynyl, etc. included, but not limited thereto.

As used herein, a C ₃ -C ₁₀ cycloalkyl group refers to a simple or fused cyclic hydrocarbon consisting of 3 to 10 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. are included, but limited thereto it's not going to be

As used herein, a 5- to 7-membered heterocycle means a 5- to 7-membered ring having 1 to 3 heteroatoms selected from the group consisting of oxygen, sulfur and nitrogen, for example, piperidine, pyrrolidine , morpholine or pyrimidine, and the like, but are not limited thereto.

In one embodiment of the present invention, the N-phenylbenzothiazol-2-amine compound is

R ⁴ and R ⁵ are not hydrogen at the same time.

In one embodiment of the present invention, the N-phenylbenzothiazol-2-amine compound is

A compound in which L is absent.

In one embodiment of the present invention, the N-phenylbenzothiazol-2-amine compound is

R ¹ and R ² are each independently a C ₁ -C ₆ alkoxy group,

R ³ is a C ₁ -C ₆ alkyl group,

L does not exist,

R ⁴ and R ⁵ are each independently hydrogen or a C ₃ -C ₁₀ cycloalkyl group,

n is an integer from 1 to 6,

m is an integer from 1 to 6,

provided that R ⁴ and R ⁵ are not hydrogen at the same time.

In one embodiment of the present invention, the N-phenylbenzothiazol-2-amine compound is

R ¹ and R ² are methoxy groups,

R ³ is an ethyl group,

L does not exist,

R ⁴ and R ⁵ are each independently hydrogen or a cyclopentyl group,

n is an integer from 1 to 3,

m is an integer from 1 to 3,

provided that R ⁴ and R ⁵ are not hydrogen at the same time.

Pharmaceutically acceptable salts herein include both non-toxic inorganic and organic acid salts, for example, hydrochloride, sulfate, nitrate, phosphate, acetate, adipate, aspartate, benzoate, benzenesulfonate. Acid salt, citrate, camphorate, camphorsulfonate, diphosphate, ethanesulfonate, fumarate, glutamate, malate, lactate, methanesulfonate, succinate, tartrate acid salts, picrate salts, tosylate salts, and the like.

Representative compounds of the compounds of the present invention are selected from the following groups.

N-(4-(cyclopentylamino)butyl)-6-ethyl-N-(3,4-dimethoxyphenyl)benzo[d]thiazol-2-amine (I-1);

N1-cyclopentyl-N2-(3,4-dimethoxyphenyl)-N2-(6-ethylbenzo[d]thiazol-2-yl)ethane-1,2-diamine (I-2);

N-(3,4-dimethoxyphenyl)-6-ethyl-N-(5-(pyrrolidin-1-yl)pentyl)benzo[d]thiazol-2-amine (I-3);

N1-(3,4-dimethoxyphenyl)-N1-(6-ethylbenzo[d]thiazol-2-yl)-N5-(prop-2-yn-1-yl)pentane-1,5- diamine (I-4);

N1-(3,4-dimethoxyphenyl)-N1-(6-ethylbenzo[d]thiazol-2-yl)-N5-propylpentane-1,5-diamine (I-5);

N-(3,4-dimethoxyphenyl)-6-ethyl-N-(3-(piperidin-1-yl)propyl)benzo[d]thiazol-2-amine (I-6);

N-(3,4-dimethoxyphenyl)-6-ethyl-N-(4-(piperidin-1-yl)butyl)benzo[d]thiazol-2-amine (I-7);

N1-Cyclohexyl-N4-(3,4-dimethoxyphenyl)-N4-(6-ethylbenzo[d]thiazol-2-yl)butane-1,4-diamine (I-8);

N1-(3,4-dimethoxyphenyl)-N1-(6-ethylbenzo[d]thiazol-2-yl)-N4-propylbutane-1,4-diamine (I-9);

N-(3,4-dimethoxyphenyl)-6-ethyl-N-(4-(pyrrolidin-1-yl)butyl)benzo[d]thiazol-2-amine (I-10);

N1-(3,4-dimethoxyphenyl)-N1-(6-ethylbenzo[d]thiazol-2-yl)-N3-propylpropane-1,3-diamine (I-11);

N1-Cyclohexyl-N3-(3,4-dimethoxyphenyl)-N3-(6-ethylbenzo[d]thiazol-2-yl)propane-1,3-diamine (I-12);

N-(3,4-dimethoxyphenyl)-6-ethyl-N-(3-(pyrrolidin-1-yl)propyl)benzo[d]thiazol-2-amine (I-13);

N1-(3,4-dimethoxyphenyl)-N3,N3-diethyl-N1-(6-ethylbenzo[d]thiazol-2-yl)propane-1,3-diamine (I-14);

N-(3,4-dimethoxyphenyl)-6-ethyl-N-(3-morpholinopropyl)benzo[d]thiazol-2-amine (I-15);

N-(3,4-dimethoxyphenyl)-6-ethyl-N-(4-morpholinobutyl)benzo[d]thiazol-2-amine (I-16);

N-(3,4-dimethoxyphenyl)-6-ethyl-N-(2-(piperidin-1-yl)ethyl)benzo[d]thiazol-2-amine (I-17);

N-(3,4-dimethoxyphenyl)-6-ethyl-N-(2-(pyrrolidin-1-yl)ethyl)benzo[d]thiazol-2-amine (I-18);

N1-Cyclohexyl-N2-(3,4-dimethoxyphenyl)-N2-(6-ethylbenzo[d]thiazol-2-yl)ethane-1,2-diamine (I-19);

N-(3,4-dimethoxyphenyl)-6-ethyl-N-(2-morpholinoethyl)benzo[d]thiazol-2-amine (I-20);

N1-(3,4-dimethoxyphenyl)-N2,N2-diethyl-N1-(6-ethylbenzo[d]thiazol-2-yl)ethane-1,2-diamine (I-21);

N1-Cyclohexyl-N3-(3,4-dimethoxyphenyl)-N3-(6-ethylbenzo[d]thiazol-2-yl)-N1-methylpropane-1,3-diamine (I-22) ;

N-(3,4-difluorophenyl)-6-ethyl-N-(2-(piperidin-1-yl)ethyl)benzo[d]thiazol-2-amine (I-23);

6-ethyl-N-(2-(piperidin-1-yl)ethyl)-N-(4-(trifluoromethyl)phenyl)benzo[d]thiazol-2-amine (I-24);

6-ethyl-N-(4-nitrophenyl)-N-(2-(piperidin-1-yl)ethyl)benzo[d]thiazol-2-amine (I-25);

N1-(6-ethylbenzo[d]thiazol-2-yl)-N1-(2-(piperidin-1-yl)ethyl)benzene-1,4-diamine (I-26); and

N-(3,4-dimethoxyphenyl)-6-ethyl-N-((1-(2-(pyrrolidin-1-yl)ethyl)-1H-1,2,3-triazole-4- yl)methyl)benzo[d]thiazol-2-amine (I-27).

A method for preparing the N-phenylbenzothiazol-2-amine compound of Formula I of the present invention is shown in Schemes 1 and 2 below. The methods described in the following scheme are merely illustrative of the methods typically used, and the reaction reagents, reaction conditions, etc. may be changed according to the case.

[Scheme 1]

As shown in Scheme 1, the N-phenylbenzothiazol-2-amine compound of Formula I is prepared by reacting a compound of Formula II with a compound of Formula III under a copper catalyst by Chan-Lam coupling. one to give the compound of formula (IV) which is reacted with a compound of formula V a compound of formula IV in the presence of a base such as NaH to obtain a compound of formula VI, then the formula of the compound of formula VI in the presence of a base such as K ₂ CO ₃ It can be prepared by reaction with a compound of VII.

In this case, the Chan-Ram coupling reaction may be performed in the presence of _{pyridine as a ligand and Cs 2} CO _{3 as a base.}

[Scheme 2]

As shown in Scheme 2 above, L is

N-phenylbenzothiazol-2-amine compound of formula (I-1) is obtained by reacting a compound of formula (II) with a compound of formula (III) under a copper catalyst by Chan-Lam coupling to obtain a compound of formula (IV) and reacting a compound of formula IV with a compound _{of formula IX in the presence of a base such as K 2} CO ₃ to obtain a compound of formula X, and then reacting the compound of formula X with a compound of formula XI with an azide- It can be prepared by azide-alkyne cycloaddition.

In this case, the reducing agent used in the azide-alkyne cycloaddition reaction may include sodium ascorbate.

[Scheme 3]

As shown in Scheme 3, the compound of Formula II may be prepared by reacting the compound of Formula VIII with NH ₄ SCN, followed by oxidative cyclization using _{Br 2 .}

The compound of Formula I or a pharmaceutically acceptable salt thereof according to the present invention provides excellent blood vessels through inhibition of angiogenesis induced by vascular endothelial growth factor (VEGF) without cytotoxicity to human umbilical cord vascular endothelial cells (HUVEC). It exhibits angiogenesis inhibitory activity (see Test Examples 1 and 2).

The compound of Formula I or a pharmaceutically acceptable salt thereof according to the present invention exhibits excellent angiogenesis inhibitory activity by inhibiting the VEGF signaling system and mediating the transcription of HIF-1α to suppress the sub-signaling system (Test Example) see 3).

In addition, the compound of formula (I) or a pharmaceutically acceptable salt thereof according to the present invention has structural stability due to the tertiary amine structure in the molecule.

In addition, the compound of Formula I or a pharmaceutically acceptable salt thereof according to the present invention exhibits an effect of inhibiting angiogenesis in a cell model of D-glucose-induced diabetic retinopathy (see Test Example 4).

Accordingly, the present invention provides a pharmaceutical composition for inhibiting angiogenesis-mediated signal transduction comprising the compound of Formula I or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier, specifically, diabetic retinopathy, cancer, duodenum It relates to a pharmaceutical composition for the treatment or prevention of ulcers, arthritis or obesity, in particular diabetic retinopathy.

The pharmaceutical composition according to the present invention may be administered orally (eg, ingestion or inhalation) or parenterally (eg, injection, deposition, implantation, suppository), and injection is, for example, intravenous injection , may be subcutaneous injection, intramuscular injection or intraperitoneal injection. The pharmaceutical composition according to the present invention may be administered as a tablet, capsule, granule, fine subtilae, powder, sublingual tablet, suppository, ointment, injection, emulsion, suspension, syrup, spray, etc., depending on the route of administration. can be formulated. The various types of the pharmaceutical composition according to the present invention can be prepared by known techniques using a pharmaceutically acceptable carrier commonly used for each formulation. Examples of pharmaceutically acceptable carriers include excipients, binders, disintegrating agents, lubricants, preservatives, antioxidants, isotonic agents, buffers, coating agents, sweetening agents, solubilizing agents, bases, dispersing agents, wetting agents , suspending agents, stabilizing agents, coloring agents, and the like.

The pharmaceutical composition according to the present invention varies depending on the form of the drug, but contains about 0.01 to 95% by weight of the compound of the present invention or a pharmaceutically acceptable salt thereof.

The specific dosage of the pharmaceutical composition of the present invention may vary depending on the type of mammal, including the person to be treated, body weight, sex, degree of disease, judgment of a doctor, and the like. Preferably, in the case of oral administration, 0.01 to 50 mg of the active ingredient per 1 kg of body weight is administered, and in the case of parenteral administration, 0.01 to 10 mg of the active ingredient per 1 kg of body weight is administered. The total daily dose may be administered at one time or divided into several doses depending on the severity of the disease, the judgment of the doctor, and the like.

The compound of the present invention does not inhibit human umbilical cord vascular endothelial cells (HUVEC), thereby inhibiting the vascular endothelial growth factor (VEGF) signaling system without cytotoxicity, and mediating the transcription of HIF-1α to suppress the sub-signaling system, thereby providing excellent blood vessels. It exhibits angiogenesis inhibitory activity. In addition, the compound of the present invention has structural stability, so it has excellent stability during manufacturing and storage and can exhibit high plasma stability. Accordingly, the compound of the present invention can be effectively used in a pharmaceutical composition for the treatment or prevention of diabetic retinopathy, cancer, duodenal ulcer, arthritis or obesity.

1 is a graph showing the results of endothelial cell tubular structure formation analysis for the compound of Example 1, the compound of Example 2, and sunitinib.

Figure 2 is a photograph of the analysis of scratch wound movement for the compound of Example 1 and the compound of Example 2.

3 is a result of analysis of p-VEGFR2 protein expression level for the compound of Example 1 and the compound of Example 2.

4 is a result of analysis of the expression level of HIF-1α for the compound of Example 1 and the compound of Example 2.

5 is a result of evaluating the activity of the compound of Example 2 in a D-glucose-induced diabetic retinopathy model.

Hereinafter, the present invention will be described in more detail by way of Examples. These examples are for illustrative purposes only, and it is apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Preparation Example 1: Preparation of a compound of formula II

Preparation 1-1: 6-ethylbenzo [d] thiazol-2-amine (II-1)

4-aminoethylbenzene (leq) and NH ₄ SCN (leq) were put in AcOH (0.2M) and reacted at 10 ° C. for 10 minutes, and then bromine (Br ₂ ) in AcOH was added dropwise at 10 ° C. Stir by hand for 40 minutes. After the reaction was completed, _{it was quenched with saturated Na 2} S ₂ O ₃ and Na ₂ CO ₃ until pH 7 was reached. After extraction with ethyl acetate, _{water was removed with MgSO 4} , and column chromatography (hexane/EtOAc, 2.5:1) was performed to obtain the title compound (II-1) (yield: 43%) as a reddish-brown solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.43 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 1.2 Hz, 1H), 7.12 (dd, J = 1.2, 8.0 Hz, 1H), 2.68 (q, J = 7.73 Hz, 2H), 1.25 (t, J = 7.4 Hz, 3H)

Preparation Example 2: Preparation of a compound of formula IV

Preparation 2-1: 6-ethyl-N- (3,4-dimethoxyphenyl) benzo [d] thiazol-2-amine (IV-1)

6-ethylbenzo [d] thiazol-2-amine (1eq), 3,4-dimethoxyphenylboronic acid (1.5eq), Cu(OAc) ₂ (0.2eq), 2,2'-bipyridine (0.4 eq) and Cs ₂ CO ₃ (2eq) were added to DMSO (0.2M) and the reaction was carried out at 65° C. for 20 hours. After completion of the reaction, the reaction _{was quenched with H 2} O, extracted with ethyl acetate, _{water was removed with MgSO 4} , and column chromatography (hexane/EtOAc, 3:1) was performed to obtain the title compound (IV-1) as a pale yellow solid. (Yield: 47%) was obtained.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.36 (d, J = 1.6 Hz, 1H), 7.25-7.21 (m, 1H), 6.83 (s, 1H), 6.76 (d, J = 8.8 Hz, 1H) , 6.68-6.63 (m, 2H), 3.84 (s, 3H), 3.79 (s, 3H), 2.60 (q, J = 7.6 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H)

Preparation 2-2: 6-ethyl-N- (3,4-difluorophenyl) benzo [d] thiazol-2-amine (IV-2)

In the same manner as in Preparation Example 2-1, except for using 3,4-difluorophenylboronic acid instead of 3,4-dimethoxyphenylboronic acid, the title compound (IV-2) as a yellow solid (yield: 47%) was obtained.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.89 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 0.8 Hz, 1H), 7.51-7.47 (m, 1H), 7.05 (t, J = 8.8 Hz, 2H), 2.75 (q, J = 7.5 Hz, 2H), 1.26 (t, J = 7.6 Hz, 3H).

Preparation 2-3: 6-ethyl-N- (4- (trifluoromethyl) phenyl) benzo [d] thiazol-2-amine (IV-3)

In the same manner as in Preparation Example 2-1, except for using 4-(trifluoromethyl)phenylboronic acid instead of 3,4-dimethoxyphenylboronic acid, the title compound (IV-3) as a yellow solid (yield) : 46%) was obtained.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.47 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 2.0 Hz, 1H), 7.36 (dd, J = 1.8 Hz, 1H), 7.30 (t) , J = 9.0 Hz, 1H), 7.03 (d, J = 8.4 Hz, 2H), 2.63 (q, J = 7.6 Hz, 2H), 1.22 (t, J = 7.6 Hz, 3H)

Preparation 2-4: 6-ethyl-N- (4-nitrophenyl) benzo [d] thiazol-2-amine (IV-4)

The title compound (IV-4) (yield: 45%) was prepared as a yellow solid in the same manner as in Preparation Example 2-1, except that 4-nitrophenylboronic acid was used instead of 3,4-dimethoxyphenylboronic acid. got it

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.11 (td, J = 5.4, 11.7 Hz, 2H), 7.44-7.41 (m, 2H), 7.34 (t, J = 4.4 Hz, 1H), 7.10 (td, J = 2.6, 9.5 Hz, 2H), 2.66 (q, J = 7.7 Hz, 2H), 1.27 (t, J = 7.6 Hz, 3H)

Preparation Example 3: Preparation of a compound of formula VI

Preparation 3-1: N- (4-bromobutyl) -6-ethyl-N- (3,4-dimethoxyphenyl) benzo [d] thiazol-2-amine (VI-1)

Compound IV-1 obtained in Preparation Example 2-1 (leq) was added to DMF (0.2M), adjusted to 0 °C, and NaH (3eq) was added thereto. 1,4-dibromobutane (2eq) was slowly added and reacted at 0° C. for 10 minutes. After the reaction was completed at room temperature, the reaction was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine _{, water was removed with MgSO 4} , and column chromatography (hexane/EtOAc, 4:1) was performed to obtain the title compound (VI-1) as a yellow solid (yield: 67%). got

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.22 (d, J = 8.40 Hz, 1H), 7.08 (dd, J = 2.4, 8.4 Hz, 1H), 7.00-6.96 (m, 2H), 6.89 (d, J = 8.8 Hz, 1H), 6.70 (d, J = 1.6 Hz, 1H), 3.91 (s, 3H), 3.84 (s, 3H), 3.46-3.43 (m, 4H), 2.47 (q, J = 7.6) Hz, 2H), 2.04-1.92 (m, 4H), 1.08 (t, J = 7.60 Hz, 3H)

Preparation 3-2: N- (2-bromoethyl) -6-ethyl-N- (3,4-dimethoxyphenyl) benzo [d] thiazol-2-amine (VI-2)

The title compound (VI-2) as a yellow solid in the same manner as in Preparation Example 3-1, except that 1,2-dibromoethane was used instead of 1,4-dibromobutane (Yield: 72%) got

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.33 (d, J = 7.60 Hz, 1H), 7.092 (dd, J = 2.2, 8.2 Hz, 1H), 7.02-6.99 (m, 2H), 6.91 (d, J = 8.4 Hz, 1H), 6.73 (d, J = 2.0 Hz, 1H), 3.93 (s, 3H), 3.86 (s, 3H), 3.88-3.85 (m, 2H), 3.57 (t, J = 7.00) Hz, 2H), 2.50 (q, J = 7.5 Hz, 2H), 1.102 (t, J = 7.6 Hz, 3H).

Preparation 3-3: N- (3-bromopropyl) -6-ethyl-N- (3,4-dimethoxyphenyl) benzo [d] thiazol-2-amine (VI-3)

The title compound (VI-3) as a yellow solid in the same manner as in Preparation Example 3-1, except that 1,3-dibromopropane was used instead of 1,4-dibromobutane (Yield: 62%) got

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.22 (d, J = 7.60 Hz, 1H), 7.09 (dd, J = 2.2, 8.2 Hz, 1H), 7.01-7.00 (m, 2H), 6.90 (d, J = 8.4 Hz, 1H), 6.72 (d, J = 2.0 Hz, 1H), 3.92 (s, 3H), 3.85 (s, 3H), 3.64-3.52 (m, 4H), 2.49 (q, J = 7.6) Hz, 2H), 2.32 (q, J = 6.5 Hz, 2H), 1.11 (t, J = 7.6 Hz, 3H).

Preparation 3-4: N- (5-bromopentyl) -N- (3,4-dimethoxyphenyl) -6-ethylbenzo [d] thiazol-2-amine (VI-4)

The title compound (VI-4) as a yellow solid in the same manner as in Preparation Example 3-1, except that 1,3-dibromopentane was used instead of 1,4-dibromobutane (yield: 65%) got

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.21 (d, J = 8.40 Hz, 1H), 7.07 (dd, J = 2.2, 8.2 Hz, 1H), 6.99-6.96 (m, 2H), 6.88 (d, J = 8.0 Hz, 1H), 6.70 (d, J = 2.0 Hz, 1H), 3.90 (s, 3H), 3.84 (s, 3H), 3.42-3.38 (m, 4H), 2.47 (q, J = 7.6) Hz, 2H), 1.91-1.86 (m,2H), 2.04-1.76 (m, 4H), 1.65 1.53 (m, 2H), 1.08 (t, J = 7.60 Hz, 3H)

Preparation 3-5: N- (2-bromoethyl) -N- (3,4-difluorophenyl) -6-ethylbenzo [d] thiazol-2-amine (VI-5)

Using IV-2 obtained in Preparation Example 2-2 instead of Compound IV-1 obtained in Preparation Example 2-1 and using 1,2-dibromoethane instead of 1,4-dibromobutane Except, the title compound (VI-5) (yield: 62%) was obtained as a yellow solid in the same manner as in Preparation Example 3-1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.22 (d, J = 7.60 Hz, 1H), 7.09 (dd, J = 2.2, 8.2 Hz, 1H), 7.01-7.00 (m, 2H), 6.90 (d, J = 8.4 Hz, 1H), 6.72 (d, J = 2.0 Hz, 1H), 3.92 (s, 3H), 3.85 (s, 3H), 3.64-3.52 (m, 4H), 2.49 (q, J = 7.6) Hz, 2H), 2.32 (q, J = 6.5 Hz, 2H), 1.11 (t, J = 7.6 Hz, 3H).

Preparation 3-6: N- (2-bromoethyl) -6-ethyl-N- (4-nitrophenyl) benzo [d] thiazol-2-amine (VI-6)

Using compound IV-4 obtained in Preparation Example 2-4 instead of Compound IV-1 obtained in Preparation Example 2-1 and using 1,2-dibromoethane instead of 1,4-dibromobutane Except for that, the title compound (VI-6) (yield: 58%) was obtained as a yellow solid in the same manner as in Preparation Example 3-1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.13 (t, J = 2.2 Hz, 1H), 8.12 (t, J = 2.2 Hz, 1H), 7.41-7.34 (m, 2H), 7.22 (t, J = 2.2 Hz, 1H), 7.20 (t, J = 2.4 Hz, 1H) 3.59 (t, J = 6.8 Hz, 2H), 3.44 (t, J = 6.2 Hz, 2H), 2.67 (q, J = 7.6 Hz, 2H), 1.24 (t, J = 7.8 Hz, 3H).

Preparation 3-7: N- (2-bromoethyl) -6-ethyl-N- (4- (trifluoromethyl) phenyl) benzo [d] thiazol-2-amine (VI-7)

Using compound IV-3 obtained in Preparation Example 2-3 instead of Compound IV-1 obtained in Preparation Example 2-1 and using 1,2-dibromoethane instead of 1,4-dibromobutane Except for that, the title compound (VI-7) (yield: 60%) was obtained as a yellow solid in the same manner as in Preparation Example 3-1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.53 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 7.6 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H) 7.25-7.22 ( m, 2H), 3.808 (t, J = 7.0 Hz, 2H), 3.47 (t, J = 6.6 Hz, 2H), 2.60 (q, J = 7.6 Hz, 2H), 1.18 (t, J = 7.8 Hz, 3H).

Example 1: N-(4-(cyclopentylamino)butyl)-6-ethyl-N-(3,4-dimethoxyphenyl)benzo[d]thiazol-2-amine (I-1)

Compound VI-1 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-1 were placed in MeCN (0.2M) at room temperature, and then cyclopentanamine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 60 to 70° C. for 16 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 10:1) to obtain the title compound (I-1) (yield: 52%) as a white solid.

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.21 (d, J = 8.4 Hz, 1H), 7.07 (dd, J = 2.0, 8.4 Hz, 1H), 6.99-6.95 (m, 2H), 6.89 (d, J = 8.0 Hz, 1H), 6.66 (d, J = 1.6 Hz, 1H), 3.90 (s, 3H), 3.84 (s, 3H), 3.44 (t, J = 7.4 Hz, 2H), δ3.37- 3.30 (m, 1H), 2.92 (t, J = 8.0 Hz, 2H), 2.45 (q, J = 7.6 Hz, 2H), 2.04-2.00 (m, 4H), 1.87-1.80 (m, 6H), 1.55 (m, 2H), 1.07 (t, J = 7.6 Hz, 3H)

Example 2: N1-cyclopentyl-N2- (3,4-dimethoxyphenyl) -N2- (6-ethylbenzo [d] thiazol-2-yl) ethane-1,2-diamine (I-2)

Compound VI-2 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-2 were placed in MeCN (0.2M) at room temperature, and then cyclopentanamine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 60 to 70° C. for 16 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, _{water was removed with MgSO 4} , and column chromatography (DCM/MeOH, 15:1) was performed to obtain the title compound (I-2) (yield: 51%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.11-7.00 (m, 3H), 6.96 (d, J = 2.0 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.67 (d, J = 2.0 Hz, 1H), 4.93 (dq, J = 7.8, 7.8 Hz, 1H), 4.20-4.13 (m, 1H), 3.91 (s, 3H), 3.85 (s, 3H), 3.80-3.71 (m, 2H) ), 2.48 (q, J = 7.5 Hz, 2H), 2.24-2.11 (m, 2H), 1.73-1.58 (m, 7H), 1.08 (t, J = 7.4 Hz, 3H)

Example 3: N-(3,4-dimethoxyphenyl)-6-ethyl-N-(5-(pyrrolidin-1-yl)pentyl)benzo[d]thiazol-2-amine

Compound VI-4 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-4 were added to DMF/MeCN (0.2M) at room temperature, and then pyrrolidine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, _{water was removed with MgSO 4} , and column chromatography (DCM/MeOH, 10:1) was performed to obtain the title compound (I-3) (yield: 52%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.17 (d, J = 8.0 Hz, 1H), 7.05 (dd, J = 1.8, 8.2 Hz, 1H), 6.96 (dd, J = 1.6, 6.8 Hz, 2H) , 6.88 (d, J = 8.4 Hz, 1H ), 6.65 (d, J = 2 Hz, 1H), 3.88 (s, 3H), 3.82 (s, 3H), 3.40 (t, J = 7.2 Hz, 2H) , 3.10 (s, 4H), 2.90 (t, J = 8.0 Hz, 2H), 2.44 (q, J = 7.6 Hz, 2H), 2.02 (s, 4H), 1.89-1.74 (m, 4H), 1.51 ( dq, J = 7.6, 7.6 Hz, 2H), 1.05 (t, J = 7.6 Hz, 3H)

Example 4: N1- (3,4-dimethoxyphenyl) -N1- (6-ethylbenzo [d] thiazol-2-yl) -N5- (prop-2-yn-1-yl) pentane- 1,5-diamine (I-4)

After putting the compound VI-4 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation 3-4 in DMF/MeCN (0.2M) at room temperature, prop-2-yn-1-amine (3eq) was put in slowly. Thereafter, the reaction was carried out at 70° C. for 16 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, _{water was removed with MgSO 4} , and column chromatography (DCM/MeOH, 15:1) was performed to obtain the title compound (I-4) (yield: 58%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.20 (d, J = 8.0 Hz, 1H), 7.07 (dd, J = 2.4, 8.4 Hz, 1H), 6.97 (dd, J = 2.4, 9.6 Hz, 2H) , 6.88 (d, J = 8.4 Hz, 1H), 6.68 (d, J = 1.6 Hz, 1H), 3.90 (s, 3H), 3.84 (s, 3H), 3.40 (q, J = 2.4 Hz, 2H) , 2.69 (t, J = 7.0 Hz, 2H), 2.46 (q, J = 7.6 Hz, 2H), 2.18 (t, J = 2.4 Hz, 1H), 1.82-1.75 (m, 2H), 1.54-1.47 ( m, 6H), 1.08 (t, J = 8.2 Hz, 3H)

Example 5: N1-(3,4-dimethoxyphenyl)-N1-(6-ethylbenzo[d]thiazol-2-yl)-N5-propylpentane-1,5-diamine (I-5)

Compound VI-4 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-4 were added to DMF/MeCN (0.2M) at room temperature, and then propylamine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, _{water was removed with MgSO 4} , and column chromatography (DCM/MeOH, 15:1) was performed to obtain the title compound (I-5) (yield: 56%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.18 (d, J = 7.6 Hz, 1H), 7.06 (dd, J = 2.2, 8.2 Hz, 1H), 6.98-6.94 (m, 2H), 6.88 (d, J = 8.4 Hz, 1H ), 6.65 (d , J = 2 Hz, 1H), 3.89 (s, 3H), 3.83 (s, 3H), 3.40 (t, J = 7.0 Hz, 2H), 2.84 (td, J = 8.0, 12.7 Hz, 4H), 2.44 (q, J = 7.5 Hz, 2H), 1.91-1.74 (m, 6H), 1.55-1.47 (m, 2H), 1.06 (t, J = 7.6 Hz, 3H) ), 0.95 (t, J = 7.4 Hz, 3H)

Example 6: N-(3,4-dimethoxyphenyl)-6-ethyl-N-(3-(piperidin-1-yl)propyl)benzo[d]thiazol-2-amine (I-6 )

Compound VI-3 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-3 were added to DMF/MeCN (0.2M) at room temperature, and then piperidine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, _{water was removed with MgSO 4} , and column chromatography (DCM/MeOH, 15:1) was performed to obtain the title compound (I-6) (yield: 52%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.21 (d, J = 8.4 Hz, 1H), 7.07 (dd, J = 2.4, 8.4 Hz, 1H), 7.00-6.95 (m, 2H), 6.88 (d, J = 8.0 Hz, 1H ), 6.67 (dd, J = 1.8, 5.4 Hz, 1H), 3.90 (s, 3H), 3.84 (s, 3H), 3.48 (t, J = 7.2 Hz, 2H), 2.49- 2.39 (m, 8H), 2.01-1.92 (m, 2H), 1.56 (dq, J = 5.5, 5.5 Hz, 4H), 1.42 (d, J = 5.2 Hz, 2H), 1.07 (t, J = 7.8 Hz) , 3H)

Example 7: N-(3,4-dimethoxyphenyl)-6-ethyl-N-(4-(piperidin-1-yl)butyl)benzo[d]thiazol-2-amine (I-7 )

Compound VI-1 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-1 were added to DMF/MeCN (0.2M) at room temperature, and then piperidine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 15:1) to obtain the title compound (I-7) (yield: 53%) as a white solid.

Example 8: N1-Cyclohexyl-N4-(3,4-dimethoxyphenyl)-N4-(6-ethylbenzo[d]thiazol-2-yl)butane-1,4-diamine (I-8)

Compound VI-1 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-1 were added to DMF/MeCN (0.2M) at room temperature, and then cyclohexylamine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 12:1) to obtain the title compound (I-8) (yield: 47%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.19 (d, J = 7.6 Hz, 1H), 7.06 (dd, J = 2.4, 8.4 Hz, 1H), 6.98-6.94 (m, 2H), 6.88 (d, J = 8.4 Hz, 1H ), 6.65 (d, J = 1.6 Hz, 1H), 3.89 (s, 3H), 3.83 (s, 3H), 3.42 (t, J = 7.2 Hz, 2H), 2.89 (t, J = 7.8 Hz, 2H), 2.84-2.78 (m, 1H), 2.46 (q, J = 8.0 Hz, 2H), 2.15-1.67 (m, 8H), 1.48-1.39 (m, 2H), 1.25-1.13 (m, 5H), 1.07 (t, J = 7.6 Hz, 3H)

Example 9: N1-(3,4-dimethoxyphenyl)-N1-(6-ethylbenzo[d]thiazol-2-yl)-N4-propylbutane-1,4-diamine (I-9)

Compound VI-1 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-1 were added to DMF/MeCN (0.2M) at room temperature, and then propylamine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 60 to 70° C. for 16 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 15:1) to obtain the title compound (I-9) (yield: 54%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.22-7.20 (m, 1H), 7.10-7.06 (m, 1H), 7.02-6.96 (m, 2H), 6.92-6.88 (m, 1H ), 6.66 (d , J = 2.0 Hz, 1H), 3.91 (s, 3H), 3.85 (s, 3H), 3.44 (t, J = 7.0 Hz, 2H), 2.86 (t, J = 7.6 Hz, 2H), 2.74 (t) , J = 11.6 Hz, 2H), 2.50-2.43 (m, 2H), 1.94-1.71 (m, 7H), 1.10-1.04 (m, 3H), 0.93 (t, J = 7.6 Hz, 3H)

Example 10: N-(3,4-dimethoxyphenyl)-6-ethyl-N-(4-(pyrrolidin-1-yl)butyl)benzo[d]thiazol-2-amine (I-10 )

Compound VI-1 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-1 were added to DMF/MeCN (0.2M) at room temperature, and then pyrrolidine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 15:1) to obtain the title compound (I-10) (yield: 44%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.20 (d, J = 8.0 Hz, 1H), 7.07 (dd, J = 2.2, 8.2 Hz, 1H), 6.99-6.97 (m, 2H), 6.90 (d, J = 8.0 Hz, 1H ), 6.68 (d, J = 2.0 Hz, 1H), 3.91 (s, 3H), 3.85 (s, 3H), 3.77 (t, J = 4.6 Hz, 1H), 3.63 (t, J = 4.6 Hz, 1H), 3.45 (t, J = 6.8 Hz, 2H), 2.47 (q, J = 7.5 Hz, 2H), 2.04-1.84 (m, 6H), 1.24(s, 6H), 1.08 ( t, J = 7.6 Hz, 3H)

Example 11: N1-(3,4-dimethoxyphenyl)-N1-(6-ethylbenzo[d]thiazol-2-yl)-N3-propylpropane-1,3-diamine (I-11)

Compound VI-3 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-3 were added to DMF/MeCN (0.2M) at room temperature, and then propylamine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 15:1) to obtain the title compound (I-11) (yield: 55%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.12-7.07 (m, 2H), 7.00 (dd, J = 1.8, 7.8 Hz, 2H), 6.93 (d, J = 8.4 Hz, 1H), 6.59 (d, J = 2.0 Hz, 1H), 3.93 (s, 3H), 3.88 (s, 3H), 3.54-3.47 (m, 3H), 2.47 (q, J = 7.6 Hz, 2H), 2.36-2.15 (m, 2H) ), 1.85-1.68 (m, 5H), 1.08 (t, J = 7.6 Hz, 6H)

Example 12: N1-Cyclohexyl-N3-(3,4-dimethoxyphenyl)-N3-(6-ethylbenzo[d]thiazol-2-yl)propane-1,3-diamine (I-12)

Compound VI-3 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-3 were added to DMF/MeCN (0.2M) at room temperature, and then cyclohexylamine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 15:1) to obtain the title compound (I-12) (yield: 51%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.12-7.08 (m, 2H), 7.02 (d, J = 2.4 Hz, 1H), 6.99-6.97 (m, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.59 (d, J = 2.0 Hz, 1H), 4.52 (t, J = 10.8 Hz, 1H), 3.89 (d, J = 23.2 Hz, 6H), 3.75-3.55 (m, 2H), 3.48- 3.35 (m, 3H), 2.47 (q, J = 7.5 Hz, 2H), 2.20-2.00 (m, 3H), 1.92-1.87 (m, 2H), 1.76 (s, 2H), 1.69-1.58 (m, 3H), 1.48-1.40 (m, 2H), 1.08 (t, J = 7.6 Hz, 3H)

Example 13: N- (3,4-dimethoxyphenyl) -6-ethyl-N- (3- (pyrrolidin-1-yl) propyl) benzo [d] thiazol-2-amine (I-13 )

Compound VI-3 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-3 were added to DMF/MeCN (0.2M) at room temperature, and then pyrrolidine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 15:1) to obtain the title compound (I-13) (yield: 47%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.21 (d, J = 8.4 Hz, 1H), 7.07 (dd, J = 2.4, 8.0 Hz, 1H), 6.98-6.95 (m, 2H), 6.88 (d, J = 8.4 Hz, 1H ), 6.68 (d, J = 1.6 Hz, 1H), 3.87 (d, J = 26.8 Hz, 6H), 3.50 (t, J = 7.4 Hz, 2H), 2.64 (t, J = 6.6 Hz, 2H), 2.56 (s, 4H), 2.64 (q, J = 7.6 Hz, 2H), 2.04-1.94 (m, 2H), 1.78(s, 4H), 1.07 (t, J = 7.8 Hz, 3H)

Example 14: N1-(3,4-dimethoxyphenyl)-N3,N3-diethyl-N1-(6-ethylbenzo[d]thiazol-2-yl)propane-1,3-diamine (I- 14)

Compound VI-3 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-3 were added to DMF/MeCN (0.2M) at room temperature, and then diethylamine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 15:1) to obtain the title compound (I-14) (yield: 53%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.21 (d, J = 8.0 Hz, 1H), 7.07 (dd, J = 1.8, 8.2 Hz, 1H), 6.98-6.96 (m, 2H), 6.88 (d, J = 8.4 Hz, 1H), 6.69 (d, J = 1.6 Hz, 1H), 3.87 (d, J = 27.2 Hz, 6H), 3.67 (t, J = 4.8 Hz, 4H), 3.51 (t, J = 7.4 Hz, 2H), 2.90 (d, J = 29.6 Hz, 1H), 2.49-2.42 (m, 8H), 1.93 (dq, J = 7.1, 7.1 Hz, 2H), 1.68 (s, 1H), 1.07 ( t, J = 7.8 Hz, 3H)

Example 15: N- (3,4-dimethoxyphenyl) -6-ethyl-N- (3-morpholinopropyl) benzo [d] thiazol-2-amine (I-15)

Compound VI-3 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-3 were placed in DMF/MeCN (0.2M) at room temperature, and then morpholine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 10:1) to obtain the title compound (I-15) (yield: 59%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.21 (d, J = 7.6 Hz, 1H), 7.07 (dd, J = 2.2, 8.2 Hz, 1H), 6.98-6.95 (m, 2H), 6.88 (d, J = 8.0 Hz, 1H), 6.68 (d, J = 2.0 Hz, 1H), 3.87 (d, J = 26.8 Hz, 6H), 3.48 (t, J = 7.4 Hz, 2H), 2.59-2.52 (m, 6H), 2.46 (q, J = 7.5 Hz, 2H), 1.93 (dq, J = 7.0, 7.0 Hz, 2H), 1.24 (t, J = 7.0 Hz, 4H), 1.07 (t, J = 7.4 Hz, 3H)

Example 16: N- (3,4-dimethoxyphenyl) -6-ethyl-N- (4-morpholinobutyl) benzo [d] thiazol-2-amine (I-16)

Compound VI-1 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-1 were added to DMF/MeCN (0.2M) at room temperature, and then morpholine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 15:1) to obtain the title compound (I-16) (yield: 53%) as a white solid.

Example 17: N- (3,4-dimethoxyphenyl) -6-ethyl-N- (2- (piperidin-1-yl) ethyl) benzo [d] thiazol-2-amine (I-17 )

Compound VI-2 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-2 were added to DMF/MeCN (0.2M) at room temperature, and then piperidine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, _{water was removed with MgSO 4} , and column chromatography (DCM/MeOH, 15:1) was performed to obtain the title compound (I-17) (yield: 56%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.29 (d, J = 8.0 Hz, 1H), 7.06 (dd, J = 2.2, 8.6 Hz, 1H), 6.96 (dd, J = 1.8, 9.8 Hz, 2H) , 6.87 (d, J = 8.0 Hz, 1H), 6.68 (d, J = 1.6 Hz, 1H), 3.89 (s, 3H), 3.83 (s, 3H), 3.54 (t, J = 6.8 Hz, 2H) , 2.63-2.59 (m, 2H), 2.48-2.41 (m, 6H) 1.57-1.40 (m, 6H), 1.07 (t, J = 7.6 Hz, 3H)

Example 18: N-(3,4-dimethoxyphenyl)-6-ethyl-N-(2-(pyrrolidin-1-yl)ethyl)benzo[d]thiazol-2-amine (I-18 )

Compound VI-2 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-2 were added to DMF/MeCN (0.2M) at room temperature, and then pyrrolidine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 15:1) to obtain the title compound (I-18) (yield: 51%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.26 (d, J = 8.4 Hz, 1H), 7.06 (dd, J = 2.2, 8.2 Hz, 1H), 6.98-6.95 (m, 2H), 6.87 (d, J = 8.4 Hz, 1H), 6.68 (d, J = 2.0 Hz, 1H), 3.89 (s, 3H), 3.83 (s, 3H), 3.58 (t, J = 7.4 Hz, 2H), 2.84 (t, J = 7.2 Hz, 2H), 2.60-2.58 (m, 4H), 2.46 (q, J = 7.6 Hz, 2H), 1.79-1.77 (m, 4H), 1.07 (t, J = 7.6 Hz, 3H)

Example 19: N1-cyclohexyl-N2- (3,4-dimethoxyphenyl) -N2- (6-ethylbenzo [d] thiazol-2-yl) ethane-1,2-diamine (I-19)

Compound VI-2 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-2 were placed in DMF/MeCN (0.2M) at room temperature, and then cyclohexylamine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, _{water was removed with MgSO 4} , and column chromatography (DCM/MeOH, 10:1) was performed to obtain the title compound (I-19) (yield: 48%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.10 (d, J = 8.0 Hz, 1H), 7.07 (dd, J = 1.8, 8.2 Hz, 1H), 7.03 (d, J = 8.0 Hz, 1H), 6.98 (d, J = 7.4 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.68 (s, 1H), 3.93 (s, 3H), 3.87 (s, 3H), 3.75 (s, 3H) , 2.50 (q, J = 7.6 Hz, 2H), 1.78-1.69 (m, 5H), 1.57 (s, 6H), 1.43-1.31 (m, 2H), 1.10 (t, J = 7.6 Hz, 3H)

Example 20: N- (3,4-dimethoxyphenyl) -6-ethyl-N- (2-morpholinoethyl) benzo [d] thiazol-2-amine (I-20)

Compound VI-2 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-2 were placed in DMF/MeCN (0.2M) at room temperature, and then morpholine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, _{water was removed with MgSO 4} , and column chromatography (DCM/MeOH, 15:1) was performed to obtain the title compound (I-20) (yield: 60%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.30 (d, J = 8.4 Hz, 1H), 7.05 (dd, J = 2.2, 8.2 Hz, 1H), 6.97-6.94 (m, 2H), 6.87 (d, J = 8.0 Hz, 1H), 6.68 (d, J = 1.6 Hz, 1H), 3.89 (s, 3H), 3.82 (s, 3H), 3.68 (t, J = 4.6 Hz, 4H), 3.54 (t, J = 6.4 Hz, 2H), 2.63 (t, J = 6.4 Hz, 2H), 2.48-2.42 (m, 6H), 1.06 (t, J = 7.6 Hz, 3H).

Example 21: N1-(3,4-dimethoxyphenyl)-N2,N2-diethyl-N1-(6-ethylbenzo[d]thiazol-2-yl)ethane-1,2-diamine (I- 21)

Compound VI-2 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-2 were placed in DMF/MeCN (0.2M) at room temperature, and then diethylamine (3eq) was slowly added thereto. Thereafter, the reaction was carried out at 40° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 10:1) to obtain the title compound (I-21) (yield: 58%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.29 (d, J = 8.0 Hz, 1H), 7.08 (dd, J = 1.8, 8.2 Hz, 1H), 6.99-6.97 (m, 2H), 6.89 (d, J = 8.4 Hz, 1H), 6.72 (d, J = 1.6 Hz, 1H), 3.91 (s, 3H), 3.85 (s, 3H), 3.56 (t, J = 7.2 Hz, 2H), 2.84 (t, J = 7.2 Hz, 2H), 2.62 (q, J = 6.9 Hz, 4H), 2.48 (q, J = 7.7 Hz, 2H), 1.11-1.04 (m, 9H).

Example 22: N1-Cyclohexyl-N3-(3,4-dimethoxyphenyl)-N3-(6-ethylbenzo[d]thiazol-2-yl)-N1-methylpropane-1,3-diamine ( I-22)

Compound VI-3 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-3 were added to DMF/MeCN (0.2M) at room temperature, and then N-methylcyclohexylamine (3eq) was slowly added. . Thereafter, the reaction was carried out at 40° C. for 16 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 15:1) to obtain the title compound (I-22) (yield: 46%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.18 (d, J = 8.4 Hz, 1H), 7.04 (dd, J = 2.4, 8.4 Hz, 1H), 6.94 (dd, J = 1.8, 9.8 Hz, 2H) , 6.85 (d, J = 8.0 Hz, 1H), 6.66 (d, J = 1.6 Hz, 1H), 3.87 (s, 3H), 3.80 (s, 3H), 3.46 (t, J = 7.0 Hz, 2H) , 2.56 (t, J = 7.0 Hz, 2H), 2.43 (q, J = 7.6 Hz, 2H), 2.33-2.31 (m, 1H), 2.20 (s, 3H), 1.89 (td, J = 7.1, 14.3) Hz, 2H), 1.73-1.54 (m, 6H), 1.20-1.10 (m, 4H), 1.04 (t, J = 7.4 Hz, 3H).

Example 23: N- (3,4-difluorophenyl) -6-ethyl-N- (2- (piperidin-1-yl) ethyl) benzo [d] thiazol-2-amine (I- 23)

Compound VI-5 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-5 were added to DMF/MeCN (0.2M) at room temperature, and then piperidine (8eq) was slowly added thereto. Thereafter, the reaction was carried out at 50° C. for 16 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 15:1) to obtain the title compound (I-23) (yield: 46%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.38 (d, J = 8.4 Hz, 1H), 7.18-7.07 (m, 4H), 6.98 (d, J = 4.0 Hz, 1H), 4.07 (t, J = 5.8 Hz, 1H), 3.51 (t, J) = 6.6 Hz, 2H), 2.79 (t, J = 6.0 Hz, 1H), 2.55 (q, J = 4.4 Hz, 2H), 2.39-2.35 (m, 4H), 1.63 (td, J = 5.7, 11.3 Hz) , 2H), 1.57–1.52 (m, 4H), 1.15 (t, J = 7.6 Hz, 3H).

Example 24: 6-ethyl-N-(2-(piperidin-1-yl)ethyl)-N-(4-(trifluoromethyl)phenyl)benzo[d]thiazol-2-amine (I -24)

Compound VI-7 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-7 were added to DMF/MeCN (0.2M) at room temperature, and then piperidine (8eq) was slowly added thereto. Thereafter, the reaction was carried out at 50° C. for 16 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 15:1) to obtain the title compound (I-24) (yield: 53%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.50 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.8 Hz, 1H), 7.26 (d, J = 8.4 Hz, 2H), 7.21-7.20 (m, 2H), 3.48 (t, J) = 6.8 Hz, 2H), 2.59 (q, J = 7.7 Hz, 2H), 2.49 (t, J = 7.0 Hz, 2H), 2.32-2.30 (m, 4H), 1.52-1.33 (m, 6H), 1.17 (t, J = 7.6 Hz, 3H).

Example 25: 6-ethyl-N- (4-nitrophenyl) -N- (2- (piperidin-1-yl) ethyl) benzo [d] thiazol-2-amine (I-25)

Compound VI-6 (1eq) and K ₂ CO ₃ (4eq) obtained in Preparation Example 3-6 were added to DMF/MeCN (0.2M) at room temperature, and then piperidine (8eq) was slowly added. Thereafter, the reaction was carried out at 60° C. for 12 hours. After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, and _{water was removed with MgSO 4} , followed by column chromatography (DCM/MeOH, 15:1) to obtain the title compound (I-25) (yield: 42%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.12 (t, J = 2.4 Hz, 1H), 8.10 (t, J = 2.4 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.36 (d , J = 2.4 Hz, 1H), 7.32 (dd, J = 1.8, 8.2 Hz, 1H), 7.21 (t, J = 2.2 Hz, 1H), 7.19 (t, J = 2.4 Hz, 1H), 3.50 (t) , J = 6.6 Hz, 2H), 2.65 (q, J = 7.5 Hz, 2H), 2.51 (t, J = 6.4 Hz, 2H), 2.35-2.31 (m, 4H), 1.51 (td, J = 5.5, 10.9 Hz, 4H), 1.42-1.37 (m, 2H), 1.23 (t, J = 7.6 Hz, 3H)

Example 26: N1-(6-ethylbenzo[d]thiazol-2-yl)-N1-(2-(piperidin-1-yl)ethyl)benzene-1,4-diamine (I-26)

Compound I-25 (leq) and Pd/C (10%) obtained in Example 25 were added to MeOH (0.2M) at room temperature, and then the reaction was carried out under hydrogen conditions for 16 hours. After completion of the reaction, the mixture was filtered using celite and column chromatography (DCM/MeOH, 15:1) was performed to obtain the title compound (I-26) (yield: 78%) as a white solid.

Example 27: N-(3,4-dimethoxyphenyl)-6-ethyl-N-((1-(2-(pyrrolidin-1-yl)ethyl)-1H-1,2,3-tria zol-4-yl)methyl)benzo[d]thiazol-2-amine (I-27)

Compound IV-1 (1eq) obtained in Preparation Example 2-1, K ₂ CO ₃ (2eq), and propargyl bromide (1.2 eq) were added to DMF (0.3M) at room temperature, and then the reaction was carried out for 3 hours. . After the reaction was completed, it was quenched with _{H 2 O.} After extraction with ethyl acetate, the mixture was washed with brine, _{water was removed with MgSO 4} , and column chromatography (DCM/MeOH, 15:1) was performed to obtain an intermediate. The above intermediate (1 eq) and 1- (2- _{azidoethyl) pyrrolidine (1.2 eq), CuSO 4} ·5H ₂ O (0.05eq) and sodium ascorbate (0.1 eq) t -BuOH/H ₂ O (1:1, 0.5M) and stirred for 12 hours. After completion of the reaction, the mixture was extracted with ethyl acetate, washed with brine, _{water was removed with MgSO 4} , and column chromatography (DCM/MeOH, 20:1) was performed to obtain the title compound (I-27) as a yellow solid (yield: 88). %) was obtained.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.93 (s, 1H), 7.15 (d, J = 8.4 Hz, 1H), 7.09 (dd, J = 6.2, 12.2 Hz, 1H), 7.00 (d, J = 2.0 Hz, 1H), 6.96-6.89 (m, 2H), 6.72 (d, J = 1.6 Hz, 1H), 4.73 (s, 2H), 4.55 (t, J = 6.4 Hz, 2H), 3.92 (s, 3H), 3.86 (s, 3H), 3.02 (t, J = 6.4 Hz, 2H), 2.59-2.56 (m, 4H), 2.47 (q, J = 7.5 Hz, 2H), 1.81-1.77 (m, 4H) ), 1.08 (t, J = 7.6 Hz, 3H).

Test Example 1: Cytotoxicity test

Human umbilical vascular endothelial cells (HUVEC) were obtained from ATCC (Rockville, MD) _{at 37° C. under 5% CO 2} atmosphere with 10% FBS (Gibco) and 1% AA (antibiotic-antimyhotic, Gibco) was cultured in EGM-2 (Lonza) supplemented with

Cytotoxicity was assessed by MTT assay. HUVEC cells (8 × 10 ³ cells/well) were attached to a 96-well plate, cultured for 24 hours, all medium was removed, and serum starvation was given. After 12 hours, the medium was removed again and 2% VEGF (50 ng/ml) and the compound were treated in a medium supplemented with FBS. In the case of hypoxic conditions, VEGF was not added and cultured in _{1% O 2 conditions using a hypoxic chamber.} After compound treatment for 24 hours, live cells were stained with MTT reagent, dissolved in DMSO, and absorbance was measured at 570 nm using a VersaMax ELISA microplate reader (Molecular Devices, Sunnyvale, CA).

The results are shown in Table 1 below.

Test Example 2: in vitro Tube Formation Analysis

Coating was performed by putting Matrigel (70 μl/well) in a 96-well plate and incubating at 37° C. for 30 minutes. HUVECs (3×10 ⁴ cells/well) and various concentrations of compounds were seeded into each well of a Matrigel coated 96-well plate. The cells were then incubated at 37° C. for 8 hours. The formation of endothelial cell tubular structures was observed under an inverted microscope and photographed (Olympus Optical Co. Ltd., Tokyo, Japan). The inhibition rate of tube formation was determined using Equation 1 below.

[Equation 1]

Tube formation inhibition rate (%) = [1 - (average number of tubes _sample - average number of tubes _VEGF(-) ) / (average number of tubes _VEGF(+) - average number of tubes _VEGF(-) )] × 100

The same experiment was performed with sunitinib as a positive control.

The results are shown in Table 1 below.

In addition, to calculate the therapeutic index (therapeutic index, TI) is defined as the ratio of IC ₅₀ for the IC ₅₀ dae HUVEC cytotoxic for tube formation are shown in Table 1 below.

compound	Tube forming IC 50 (μM)	HUVEC cytotoxicity IC 50 (μM)	Therapeutic Index (TI)
I-1	2.34	31.66	13.53
I-2	3.78	>100	>26.5
I-4	11.81	49.5	4.19
I-5	8.37	16.55	1.98
I-6	2.31	49.95	21.62
I-7	3.28	55.06	16.79
I-8	1.82	19.45	10.69
I-9	6.44	57.04	8.86
I-10	20.43	48.81	2.39
I-11	44.03	>100	>2.27
I-12	19.64	41.95	2.14
I-13	14.93	>100	>6.70
I-14	15.66	>100	>6.39
I-15	12.28	>100	>8.14
I-16	29.86	>100	>3.35
I-17	9.04	>100	>11.06
I-18	6.33	94.83	14.98
I-19	17.69	>100	>5.65
I-20	5.36	>100	>18.66
I-21	13.9	>100	>7.19
I-22	5.51	17.51	3.18
I-23	12.75	>100	>7.84
I-24	9.56	71.71	7.50
I-27	2.36	44.91	19.03
Sunitinib	1.3	10	7.7

As can be seen in Table 1, the N-phenylbenzothiazol-2-amine compound according to the present invention showed excellent tube formation inhibitory efficacy without cytotoxicity to HUVEC. In particular, the compound (I-2) of Example 2 had a distinct inhibitory activity (IC ₅₀ = 3.78 μM) on tube formation and exhibited a TI higher than 26.5, showing the highest TI among the compounds of Examples. Under the same experimental conditions, sunitinib, a representative angiogenesis inhibitor, exhibited a TI of 7.7.

Test Example 3: Mechanism analysis

In order to understand the molecular mechanism of the N-phenylbenzothiazol-2-amine compound according to the present invention, endothelial cell tubular structure formation analysis and scratch wound migration analysis were additionally performed on the example compounds.

Endothelial cell tubular structure formation analysis was performed in the same manner as in Test Example 2 for Examples 1 and 2 and sunitinib.

Scratch wound migration analysis was performed as follows.

HUVECs were cultured in a 6-well plate coated with 0.1% gelatin at 5% CO ₂ , at 37° C., and then wounded by scratching using a 0.2 mL pipette tip. Thereafter, samples were treated by concentration in a medium containing 0.5% FBS/EGM-2 and incubated. After 18 hours, the number of migrated cells was counted by photographing under a microscope, and the inhibition rate for the control group was calculated.

The results are shown in FIGS. 1 and 2 .

1, at a concentration of 10 μM, Example 1, Example 2, and sunitinib showed high tube formation inhibitory activity in the order, and in consideration of toxicity, the compounds of Examples 1 and 2 showed excellent tube formation inhibitory activity. could see what

2, it was found that the compounds of Examples 1 and 2 effectively reduced cell mobility.

In addition, in order to evaluate the effect of regulating the VEGF and HIF-1α signaling system of the example compounds, the expression levels of p-VEGFR2 protein and HIF-1α were measured by Western blot analysis.

Western blot analysis was performed as follows.

The cell suspension was placed in a 100 mm dish, and the sample was processed after incubation at 37° C. for 24 hours to attach. After incubation for a certain period of time, the cells were lysed using a lysis buffer after washing 2-3 times with PBS. Proteins were quantified and 50 μg was separated using SDS-PAGE. The separated protein was transferred to a PVDF membrane and then blocked with 5% BSA in PBST at room temperature for 1 hour. The primary antibody was diluted and left at 4° C. for more than 12 hours, washed several times, and reacted with the HRP-conjugated secondary antibody at room temperature for 1 to 2 hours. After washing again with PBST, the expression level was confirmed using ImageQuant™ LAS 4000 (GE Healthcare Life Sciences) equipment.

The results of p-VEGFR2 protein expression analysis are shown in FIG. 3 .

3, it can be seen that the compounds of Examples 1 and 2 decrease the expression level of VEGF-induced p-VEGFR2 in HUVECs in a concentration-dependent manner.

The results of analysis of the expression level of HIF-1α are shown in FIG. 4 .

ARPE-19 retinal cell line was _{treated with MG132 and CoCl 2} , respectively, to induce the expression of HIF-1α, and the activity of the compound of Example 1 was measured. MG132 is a proteasome inhibitor that prevents HIF1-α from being degraded, and CoCl ₂ artificially induces transcription of HIF-1α.

4, when the compound of Example 1 was treated, the expression level of HIF-1α was not inhibited, and _{when the compound of Example 1 was treated with CoCl 2} , HIF-1α and the lower mechanism p-eNOS and It was confirmed that p-Akt was inhibited. Through this, it can be seen that the compound of Example 1 mediates the transcription of HIF-1α and suppresses the sub-signaling system.

Test Example 4: Activity evaluation in D-glucose-induced diabetic retinopathy model

Markers (HIF-1α, VEGFA, NOX4, ANGPT1, ANGPT2) involved in diabetic retinopathy and angiogenesis were identified through real-time PCR. Among them, ANGPT2 increased according to the concentration of D-glucose (5.5, 10, 20, 30 mM) was set as a biomarker of diabetic retinopathy.

Using ANGPT2 as a biomarker of diabetic retinopathy, the activity of the compound of Example 2 in a D-glucose-induced diabetic retinopathy model was evaluated as follows.

D-glucose-induced diabetic retinopathy model was constructed using HUVEC or HRMEC (Human Retinal Microvascular Endothelial Cell) cells.

After attaching the HUVEC or HRMEC cells to a 60 mm dish, the medium was removed 24 hours later, and the medium was replaced with a medium not containing FBS and cultured for half a day. The next day, the material was treated after changing to an EBM medium containing 2% FBS. After 30 minutes of substance treatment, the normal D-glucose group was treated with 5.5 mM D-glucose, and the high glucose group and the substance-treated group were treated with 30 mM D-glucose. After incubation for 48 hours, the cells were lysed using a lysis buffer after washing 2-3 times with PBS. Proteins were quantified and 20 μg was separated using SDS-PAGE. The separated protein was transferred to a PVDF membrane and then blocked with 5% BSA in PBST at room temperature for 1 hour. The primary antibody was diluted and left at 4° C. for more than 12 hours, washed several times, and reacted with the HRP-conjugated secondary antibody at room temperature for 1 to 2 hours. After washing again with PBST, the expression level was confirmed using ImageQuant™ LAS 4000 (GE Healthcare Life Sciences) equipment.

The results are shown in FIG. 5 .

5A, it can be confirmed that the expression level of ANGPT2 is increased by a high concentration of glucose (30 mM) in both HUVEC cells and HRMEC cells in the constructed D-glucose-induced diabetic retinopathy model. In addition, the binding of the compound of Example 2 to the ANGPT2 protein can be confirmed through molecular docking through FIG. 5B . 5C and 5D, it was verified that the compound of Example 2 inhibited the gene and protein expression levels of ANGPT2 increased by a high concentration of glucose in a concentration-dependent manner.

Claims (9)

1. N-phenylbenzothiazol-2-amine compound of formula (I) or a pharmaceutically acceptable salt thereof:

   [Formula I]

   

   In the above formula,

   R ¹ and R ² are each independently hydrogen, halogen, nitro, amino, a C ₁ -C ₆ alkoxy group or a C ₁ -C ₆ haloalkyl group,

   R ³ is a C ₁ -C ₆ alkyl group,

   L does not exist or

   

   ego,

   R ⁴ and R ⁵ are each independently hydrogen, a C ₁ -C ₆ alkyl group, a C ₂ -C ₆ alkynyl group, or a C ₃ -C ₁₀ cycloalkyl group,

   R ⁴ and R ⁵ together with the nitrogen atom to which they are bonded form a 5 to 7 membered heterocycle,

   n is an integer from 1 to 6,

   m is an integer from 1 to 6.
2. According to claim 1,

   R ⁴ and R ⁵ are not hydrogen at the same time N-phenylbenzothiazol-2-amine compound or a pharmaceutically acceptable salt thereof.
3. According to claim 1,

   L is an absent N-phenylbenzothiazol-2-amine compound or a pharmaceutically acceptable salt thereof.
4. According to claim 1,

   R ¹ and R ² are each independently a C ₁ -C ₆ alkoxy group,

   R ³ is a C ₁ -C ₆ alkyl group,

   L does not exist,

   R ⁴ and R ⁵ are each independently hydrogen or a C ₃ -C ₁₀ cycloalkyl group,

   n is an integer from 1 to 6,

   m is an integer from 1 to 6,

   With the proviso that R ⁴ and R ⁵ are not hydrogen at the same time, an N-phenylbenzothiazol-2-amine compound or a pharmaceutically acceptable salt thereof.
5. According to claim 1,

   R ¹ and R ² are methoxy groups,

   R ³ is an ethyl group,

   L does not exist,

   R ⁴ and R ⁵ are each independently hydrogen or a cyclopentyl group,

   n is an integer from 1 to 3,

   m is an integer from 1 to 3,

   With the proviso that R ⁴ and R ⁵ are not hydrogen at the same time, an N-phenylbenzothiazol-2-amine compound or a pharmaceutically acceptable salt thereof.
6. The N-phenylbenzothiazol-2-amine compound according to claim 1, characterized in that it is selected from the following compounds, or a pharmaceutically acceptable salt thereof:

   N-(4-(cyclopentylamino)butyl)-6-ethyl-N-(3,4-dimethoxyphenyl)benzo[d]thiazol-2-amine (I-1);

   N1-cyclopentyl-N2-(3,4-dimethoxyphenyl)-N2-(6-ethylbenzo[d]thiazol-2-yl)ethane-1,2-diamine (I-2);

   N-(3,4-dimethoxyphenyl)-6-ethyl-N-(5-(pyrrolidin-1-yl)pentyl)benzo[d]thiazol-2-amine (I-3);

   N1-(3,4-dimethoxyphenyl)-N1-(6-ethylbenzo[d]thiazol-2-yl)-N5-(prop-2-yn-1-yl)pentane-1,5- diamine (I-4);

   N1-(3,4-dimethoxyphenyl)-N1-(6-ethylbenzo[d]thiazol-2-yl)-N5-propylpentane-1,5-diamine (I-5);

   N-(3,4-dimethoxyphenyl)-6-ethyl-N-(3-(piperidin-1-yl)propyl)benzo[d]thiazol-2-amine (I-6);

   N-(3,4-dimethoxyphenyl)-6-ethyl-N-(4-(piperidin-1-yl)butyl)benzo[d]thiazol-2-amine (I-7);

   N1-Cyclohexyl-N4-(3,4-dimethoxyphenyl)-N4-(6-ethylbenzo[d]thiazol-2-yl)butane-1,4-diamine (I-8);

   N1-(3,4-dimethoxyphenyl)-N1-(6-ethylbenzo[d]thiazol-2-yl)-N4-propylbutane-1,4-diamine (I-9);

   N-(3,4-dimethoxyphenyl)-6-ethyl-N-(4-(pyrrolidin-1-yl)butyl)benzo[d]thiazol-2-amine (I-10);

   N1-(3,4-dimethoxyphenyl)-N1-(6-ethylbenzo[d]thiazol-2-yl)-N3-propylpropane-1,3-diamine (I-11);

   N1-Cyclohexyl-N3-(3,4-dimethoxyphenyl)-N3-(6-ethylbenzo[d]thiazol-2-yl)propane-1,3-diamine (I-12);

   N-(3,4-dimethoxyphenyl)-6-ethyl-N-(3-(pyrrolidin-1-yl)propyl)benzo[d]thiazol-2-amine (I-13);

   N1-(3,4-dimethoxyphenyl)-N3,N3-diethyl-N1-(6-ethylbenzo[d]thiazol-2-yl)propane-1,3-diamine (I-14);

   N-(3,4-dimethoxyphenyl)-6-ethyl-N-(3-morpholinopropyl)benzo[d]thiazol-2-amine (I-15);

   N-(3,4-dimethoxyphenyl)-6-ethyl-N-(4-morpholinobutyl)benzo[d]thiazol-2-amine (I-16);

   N-(3,4-dimethoxyphenyl)-6-ethyl-N-(2-(piperidin-1-yl)ethyl)benzo[d]thiazol-2-amine (I-17);

   N-(3,4-dimethoxyphenyl)-6-ethyl-N-(2-(pyrrolidin-1-yl)ethyl)benzo[d]thiazol-2-amine (I-18);

   N1-Cyclohexyl-N2-(3,4-dimethoxyphenyl)-N2-(6-ethylbenzo[d]thiazol-2-yl)ethane-1,2-diamine (I-19);

   N-(3,4-dimethoxyphenyl)-6-ethyl-N-(2-morpholinoethyl)benzo[d]thiazol-2-amine (I-20);

   N1-(3,4-dimethoxyphenyl)-N2,N2-diethyl-N1-(6-ethylbenzo[d]thiazol-2-yl)ethane-1,2-diamine (I-21);

   N1-Cyclohexyl-N3-(3,4-dimethoxyphenyl)-N3-(6-ethylbenzo[d]thiazol-2-yl)-N1-methylpropane-1,3-diamine (I-22) ;

   N-(3,4-difluorophenyl)-6-ethyl-N-(2-(piperidin-1-yl)ethyl)benzo[d]thiazol-2-amine (I-23);

   6-ethyl-N-(2-(piperidin-1-yl)ethyl)-N-(4-(trifluoromethyl)phenyl)benzo[d]thiazol-2-amine (I-24);

   6-ethyl-N-(4-nitrophenyl)-N-(2-(piperidin-1-yl)ethyl)benzo[d]thiazol-2-amine (I-25);

   N1-(6-ethylbenzo[d]thiazol-2-yl)-N1-(2-(piperidin-1-yl)ethyl)benzene-1,4-diamine (I-26); and

   N-(3,4-dimethoxyphenyl)-6-ethyl-N-((1-(2-(pyrrolidin-1-yl)ethyl)-1H-1,2,3-triazole-4- yl)methyl)benzo[d]thiazol-2-amine (I-27).
7. A pharmaceutical composition for inhibiting angiogenesis comprising the N-phenylbenzothiazol-2-amine compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier .
8. The pharmaceutical composition according to claim 7, for the treatment or prevention of diabetic retinopathy, cancer, duodenal ulcer, arthritis or obesity.
9. The pharmaceutical composition according to claim 8, for the treatment or prevention of diabetic retinopathy.

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