KR101836747B1 - 1,2,3-triazolobenzopyran derivatives for anticancer agent - Google Patents

Abstract

The present invention relates to 1,2,3-triazolo benzopyran derivatives for an anticancer agent, and to uses thereof, wherein the 1,2,3-triazolo benzopyran derivatives according to the present invention were confirmed to have an inhibitory effect on HIF-1α activity, and thus the 1,2,3-triazolo benzopyran derivatives can be usefully used as uses for prevention and/or treatment of angiogenesis-related diseases such as diseases associated with modulation of HIF-1 α, such as cancer, and diabetic retinopathy or rheumatoid arthritis and the like.

Description

Use of 1,2,3-triazolobenzopyran derivatives as anticancer agents {1,2,3-triazolobenzopyran derivatives for anticancer agent}

The present invention relates to anticancer applications of 1,2,3-triazolo benzopyran derivatives. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating an angiogenesis-related disease containing 1,2,3-triazolo-benzopyran derivatives and a pharmaceutically acceptable salt thereof having an inhibitory activity against HIF- To a pharmaceutical composition.

In general, a hypoxic state refers to a state in which the oxygen concentration in the tissue is less than normal, usually defined as ≤ 2% O ₂ and anoxic (severe hypoxic) state defined as ≤ 0.02% O ₂ . Most mammalian tissues have 2 to 9% O ₂ (average 40 mmHg partial pressure) and 21% O ₂ (150 mmHg partial pressure) ambient air. In the areas or cells where hypoxic conditions appear, the corresponding action is activated. In order for the cells to survive, a biological reaction is activated in which cell signaling such as cell proliferation, angiogenesis, and apoptosis are activated, and the expression of various growth factors is increased Promoting cell proliferation and angiogenesis (Muz B. Hypoxia (Auckl). 2015, 3, 83-92). Furthermore, the hypoxic condition is frequently found in acute or chronic vascular disease and various cancer tissues, and is formed by a rapid increase in cell proliferation during development. In cancer, angiogenesis into cancer cells mediates rapid cancer cell growth and metastasis (Wigerup C. Pharmacol. Ther. 2016, 164, 152-169).

Angiogenesis produced by the hypoxic condition acts as an important factor in the treatment of diseases and is applied to treatment through suppression of gene expression mediated by hypoxia. Because angiogenesis induced by hypoxic conditions is required for the growth of malignant tumors, hypoxic conditions play an important role in tumorigenesis. The extent of hypoxic conditions in malignant tumors is associated with increased expression of factors that cause angiogenesis and growth into more potent malignant tumors.

In this way, the low-oxygen study O ₂ level provides significant information about the molecular mechanisms affecting the properties and behavior of a tumor, including a change in the increase of cell metabolism, proliferation, and angiogenesis as well as resistance to chemotherapy and radiation, and have.

Hypoxia-inducible factors (HIFs) are known to be the main regulators of O ₂ homeostasis, and mediate many transgenic changes in response to low O ₂ tension. Here, HIF-1 is expressed by all existing metazoan organisms and consists of O ₂ -sensitive α-subunits and intrinsically expressed β-subunits. HIF-1α is synthesized endlessly, but degrades rapidly under normal oxygen conditions.

On the other hand, pro reel hydroxyl xylanase enzyme domain (Prolyl hydroxylases domain enzymes, PHDs) HIF-1α is the O ₂ - dependent degradation (ODD, the O ₂ -dependentdegradation) two proline residues within the domain (402 and / or 564 HIF-1α binds to the von Hippel-Lindau tumor suppressor protein (pVHL), and through the E3 ubiquitin ligase, ubiquitination and proteolytic degradation (proteasomal degradation). However, in the hypoxic state, the PHD activity is weakened to cause HIF-1α protein stabilization, and the stabilized HIF-1α dimerizes with HIF-1β and translocates to the nucleus. The HIF-1α / β heterodimer binds to the hypoxia response elements (HREs) of the target gene promoter and results in angiogenesis, metastasis, apoptosis and glycolysis Thereby activating the transcription of the downstream gene involved.

HIF-1α is highly expressed in lung cancer, inducing tumor size, invasion and angiogenesis, reducing the survival rate of animal models, and the deficiency of HIF-1α reduces tumor neoplasia, It has been reported that both resistant and non-resistant lung cancer cells inhibit tumor growth (Shimoda LA Am. J. Respir. Crit. Care. Med., 2011, 183, 152-156; Cancer 2004, 111, 43-50). Therefore, the reduction of hypoxic state and hypoxic induction in the treatment of these lung cancers is expected to be an important key. Lung cancer is one of the most common cancers worldwide, with approximately one million new cases diagnosed annually. Despite the encouraging advances in recent diagnostic modalities, the prognosis of advanced lung cancer is very poor, which is attributed to multiple cancer masses, vascular invasion and distant metastasis. Therefore, there is a great need for attention and attention for the treatment of lung cancer (Herbst RSJ Clin. Oncol. 2005, 23, 3243-3256), and the need for an effective inhibitor of HIF-1α activity , But the research has not been conducted until now.

DISCLOSURE OF THE INVENTION The present invention was conceived in order to solve the problems of the prior art as described above. As a result of searching for a compound having an inhibitory activity against HIF-1α, the 1,2,3-triazolobenzopyran derivative of the present invention was found to be HIF -1α, respectively. Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer, which comprises a 1,2,3-triazolo benzopyran derivative and a pharmaceutically acceptable salt thereof as an active ingredient.

It is still another object of the present invention to provide an anticancer adjuvant comprising 1,2,3-triazolo benzopyran derivative and a pharmaceutically acceptable salt thereof as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition comprising a 1,2,3-triazalobenzopyran derivative represented by the following formula 1 and a pharmaceutically acceptable salt thereof as an active ingredient: A pharmaceutical composition for preventing or treating:

[Chemical Formula 1]

In Formula 1,

R ¹ is H; OH; halogen; CN; NO ₂ ; A straight or branched or cyclic alkyl group of C _1-10 ; C _1-10 An alkoxy group; And C _1-10 haloalkyl groups of 1 to 4 substituents selected from the group consisting of a;

R ² is a C _1-10 linear or branched or cyclic alkyl group; An alkoxy group of C _1-10 ; An alkoxyalkyl group of C _2-10 ; A C _2-10 linear or branched alkenyl group; And - (CH ₂ ) n R ³ , wherein n is 0 or an integer from 1 to 3; Lt; / RTI > is any one selected from the group consisting of &

R ³ is selected from the group consisting of halo, cyano, nitro, hydroxy, C _1-5 alkyl, C _1-5 alkoxy, C _1-5 haloalkyl, and C _1-5 haloalkoxy. And the aromatic group is a furanyl group, a thiophenyl group, a phenyl group, a pyridinyl group, a piperazinyl group, a pyridazinyl group, a naphthyl group, a quinolinyl group, or an isoquinolyl group, wherein the four substituents are substituted or unsubstituted aromatic groups.

In one embodiment of the present invention, R ¹ is selected from the group consisting of H, Cl, F, Br, OH, CN, NO ₂ , methyl group, ethyl group, normal propyl group, isopropyl group, cyclopropyl group, A cyclohexyl group, a cyclohexylmethyl group, a cyclohexylethyl group, a methoxy group, an ethoxy group, a normal propoxy group, an isopropoxy group, an isopropoxy group, an isopropoxy group, , 1 to 3 substituents selected from the group consisting of a chloromethyl group, a chloroethyl group, a dichloroethyl group, a difluoromethyl group, a trifluoromethoxy group, and trifluoromethyl,

R ² is a group selected from the group consisting of a methyl group, an ethyl group, a normal propyl group, an isopropyl group, a cyclopropyl group, a cyclopropylmethyl group, a normal butyl group, a tert- butyl group, a tert- butylmethyl group, a cyclopentyl group, a cyclopropylethyl group, A cyclohexyl group, a cyclohexylmethyl group, a cyclohexylethyl group, and - (CH ₂ ) n R ⁴ (wherein n is an integer of 0, 1 or 2)

Wherein R ⁴ is selected from the group consisting of hydrogen, Cl, F, CN, NO ₂ , OH, methyl, ethyl, normal propyl, isopropyl, cyclopropyl, cyclopropylmethyl, normal butyl, tert- Methoxy, n-propoxy, isopropoxy, chloromethyl, chloroethyl, dichloroethyl, difluoromethyl, trifluoromethyl, chloromethoxy, chloromethoxy, dichloroethoxy, difluoromethoxy, trifluoromethoxy , And an amino group, or an amino group, or an amino group, an ethyl group, a normal propyl group, an isopropyl group, a cyclopropyl group, a cyclopropylmethyl group, a normal butyl group, a tert- butyl group, a cyclopentyl group, a cyclopropylethyl group, a cyclopentylmethyl group, A substituted or unsubstituted aromatic group having 1 to 4 substituents selected from the group consisting of a hexyl group, a cyclohexylmethyl group, and an amino group substituted with 1 to 2 cyclohexylethyl groups, The aromatic group is a furanyl group, a thiophenyl group, a phenyl group, a pyridinyl group, a piperazinyl group, a pyridazinyl group, a naphthyl group, a quinolyl group, or an isoquinolyl group.

In another embodiment of the present invention, R ¹ is 1 to 3 substituents selected from the group consisting of H, Cl, F, NO ₂ , methyl, methoxy, trifluoromethoxy and trifluoromethyl,

Wherein R ² is a group selected from the group consisting of a normal propyl group, an isopropyl group, a cyclopropyl group, a cyclopropylmethyl group, a normal butyl group, a tert-butyl group, a cyclohexyl group, a cyclohexylmethyl group and - (CH ₂ ) nR ⁵ , An integer of 1 or 2,

Wherein R ⁵ is selected from the group consisting of hydrogen, Cl, F, CN, NO ₂ , OH, methyl, ethyl, isopropyl, normal butyl, tert- butyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, difluoromethyl And an amino group substituted with one to two of an amino group or a methyl group, an ethyl group, a normal propyl group, an isopropyl group, a normal butyl group, a cyclopentyl group, a cyclopentylmethyl group, and a cyclohexylmethyl group. The aromatic group is a furanyl group, a thiophenyl group, a phenyl group, a pyridinyl group, a naphthyl group, a quinolyl group, or an isoquinolyl group, wherein the one to three substituents selected are a substituted or unsubstituted aromatic group.

In another embodiment of the present invention, R ¹ is NO ₂ or a methyl group,

Wherein R ² is a phenyl group, a 2-chlorophenyl group, a 3-chlorophenyl group, a 4-chlorophenyl group, a 2- fluorophenyl group, (Trifluoromethyl) phenyl group, a 4- (trifluoromethyl) phenyl group, a 3-methoxyphenyl group, a 4-methoxyphenyl group, or an isoquinolinyl group.

The 1,2,3-triazolo benzopyran derivative of the present invention is a 4- (4-methoxyphenyl) -1 - ((2-methyl-6-nitro- -1H-1,2,3-triazole, 4- (3-methoxyphenyl) -1 - (((2-methyl- , 2,3- triazole, 1 - ((2-methyl-6-nitro-2H-chromen-2- yl) methyl) -4- (p- Methyl-4- (3- (trifluoromethyl) phenyl) -1H-1,2,3-triazole, Yl) methyl) -1H-1,2,3-triazole, 4- (3-fluoro-phenyl) Yl) methyl) -1H-1,2,3-triazole, 4- (2-chlorophenyl) -1- (1 - ((2-methyl-6-nitro-2H-1,5-benzodiazepin- Methyl) -1H-1,2,3-triazol-4-yl) isoquinoline and 1 - ((2,6- -4-phenyl-1H-1,2,3-triazole from the group consisting of But is not limited thereto.

In another embodiment of the present invention, the composition is characterized by having HIF-1 alpha inhibitory activity.

The present invention also provides a chemotherapeutic adjuvant comprising, as an active ingredient, a 1,2,3-triazalobenzopyran derivative represented by the following formula (1) and a pharmaceutically acceptable salt thereof:

[Chemical Formula 1]

In Formula 1,

R ¹ is H; OH; halogen; CN; NO ₂ ; A straight or branched or cyclic alkyl group of C _1-10 ; C _1-10 An alkoxy group; And C _1-10 haloalkyl groups of 1 to 4 substituents selected from the group consisting of a;

R ² is a C _1-10 linear or branched or cyclic alkyl group; An alkoxy group of C _1-10 ; An alkoxyalkyl group of C _2-10 ; A C _2-10 linear or branched alkenyl group; And - (CH ₂ ) n R ³ , wherein n is 0 or an integer from 1 to 3; Lt; / RTI > is any one selected from the group consisting of &

R ³ is selected from the group consisting of halo, cyano, nitro, hydroxy, C _1-5 alkyl, C _1-5 alkoxy, C _1-5 haloalkyl, and C _1-5 haloalkoxy. And the aromatic group is a furanyl group, a thiophenyl group, a phenyl group, a pyridinyl group, a piperazinyl group, a pyridazinyl group, a naphthyl group, a quinolinyl group, or an isoquinolyl group, wherein the four substituents are substituted or unsubstituted aromatic groups.

In one embodiment of the present invention, the anti-cancer adjuvant may be in the form of a combination comprising other anti-cancer drugs or other anti-cancer adjuvants.

Furthermore, the present invention provides a method of preventing or treating cancer, comprising administering the pharmaceutical composition to a subject.

In addition, the present invention provides the use of the pharmaceutical composition for preventing or treating cancer.

Since the 1,2,3-triazolobenzopyran derivative of the present invention and its pharmaceutically acceptable salt have an inhibitory activity against HIF-1α (Hypoxia-inducible factor-1α) Derivatives are expected to be widely available for the prevention, control, or treatment of diseases associated with HIF-1α regulation, such as cancer, and angiogenesis-related diseases such as diabetic retinopathy or rheumatoid arthritis.

1 shows a 1,2,3-triazolobenzopyran derivative structure of the present invention.
FIG. 2 is a graph showing screening results for inhibiting HIF-1 activity of 144 compounds of the present invention. FIG.
Figure 3 is a graph showing the activity of HIF-1 alpha protein in hypoxic state by nine selected compounds of the present invention (compounds # 12, # 45, # 54, # 94, # 101, # 103, # 105, # 127, Lt; / RTI >
Figure 4 shows the chemical structure and the dose-dependent inhibitory effect of HIF-la expression on Compound # 12 in the compounds of the present invention. A: The chemical structure of Compound # 12 was determined by HEK-293 The degree of expression of HIF-1 alpha in the compound # 12 against cells (B) and A549 cells (C) was determined as the result of the maximum inhibitory concentration (IC ₅₀ ) value using the levels of HIF-1 alpha measured in D and E: B and C Fig.
FIG. 5 shows the effect of hydrolysis and proteasome-mediated degradation of HIF-1α of Compound # 12. The result of immunofluorescence analysis of A: A549 cells treated with Compound # 12 was compared with that of A549 The HIF-1 alpha half-life of the cells was determined by measuring the degree of expression of OH-HIF-la and PHD2 in A549 cells following treatment with C: -1α, E: the result of quantifying the relative level of ubiquitinated HIF-1α upon treatment of A549 cells with compound # 12.
FIG. 6 shows the effect of Compound # 12 on HIF-1.alpha. Target gene expression and in vivo angiogenesis. A: Expression level of HIF-1.alpha. Target gene in A549 cells according to treatment with Compound # 12 treated with Compound # 12 in the presence of VEGF, H & E staining results with VEGF and Compound # 12 treated with D: HUVEC, and H & E staining results with Compound # Matrigel plug analysis results of HUVEC treated with VEGF and compound # 12, and F: compound 12 and retinoic acid treated angiogenesis effects.
FIG. 7 shows the inhibitory effect of Compound # 12 on allograft tumor growth and its effect as a chemotherapeutic agent. A and B: C57BL / 6J mice were inoculated with LLC, a lung cancer cell line, (B) of the tumors were measured. C: Microvessel density of the tumor tissue was treated with Compound # 12 and / or gefitinib in the mouse, D: Dose was measured in the hypoxic region of the tumor with CA9 Protein and microvessel images and quantitative comparisons were obtained from E :. Figure 6 shows image and quantitative comparisons of Ki67 and proliferating cells in the central region of the tumor.
8 is a diagram showing body weights of allograft mice.

The present invention relates to a pharmaceutical composition comprising a 1,2,3-triazolobenzopyran derivative represented by the following formula (1) and a pharmaceutically acceptable salt thereof as an active ingredient, A pharmaceutical composition for therapeutic use is provided:

[Chemical Formula 1]

In Formula 1,

R ¹ is H; OH; halogen; CN; NO ₂ ; A straight or branched or cyclic alkyl group of C _1-10 ; C _1-10 An alkoxy group; And C _1-10 haloalkyl groups of 1 to 4 substituents selected from the group consisting of a;

R ² is a C _1-10 linear or branched or cyclic alkyl group; An alkoxy group of C _1-10 ; An alkoxyalkyl group of C _2-10 ; A C _2-10 linear or branched alkenyl group; And - (CH ₂ ) n R ³ , wherein n is 0 or an integer from 1 to 3; Lt; / RTI > is any one selected from the group consisting of &

R ³ is selected from the group consisting of halo, cyano, nitro, hydroxy, C _1-5 alkyl, C _1-5 alkoxy, C _1-5 haloalkyl, and C _1-5 haloalkoxy. And the aromatic group is a furanyl group, a thiophenyl group, a phenyl group, a pyridinyl group, a piperazinyl group, a pyridazinyl group, a naphthyl group, a quinolinyl group, or an isoquinolyl group, wherein the four substituents are substituted or unsubstituted aromatic groups.

As used herein, the term "prophylactic " means any act that inhibits cancer or delays the onset of cancer by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action that improves or alters the symptoms of cancer by the administration of the pharmaceutical composition according to the present invention.

The 1,2,3-triazalobenzopyran derivative represented by the formula (1) according to the present invention is more preferably as follows.

Wherein R ¹ is H, Cl, F, Br, OH, CN, NO ₂ , methyl, ethyl, A cyclohexyl group, a cyclohexylmethyl group, a cyclohexylethyl group, a methoxy group, an ethoxy group, a normal propoxy group, an isopropoxy group, a chloromethyl group, a chloroethyl group, a chloromethyl group, A trifluoromethyl group, a trifluoromethyl group, a dichloroethyl group, a difluoromethyl group, a trifluoromethoxy group, and trifluoromethyl,

R ² is a group selected from the group consisting of a methyl group, an ethyl group, a normal propyl group, an isopropyl group, a cyclopropyl group, a cyclopropylmethyl group, a normal butyl group, a tert- butyl group, a tert- butylmethyl group, a cyclopentyl group, a cyclopropylethyl group, A cyclohexyl group, a cyclohexylmethyl group, a cyclohexylethyl group, and - (CH ₂ ) n R ⁴ (wherein n is an integer of 0, 1 or 2)

Wherein R ⁴ is selected from the group consisting of hydrogen, Cl, F, CN, NO ₂ , OH, methyl, ethyl, normal propyl, isopropyl, cyclopropyl, cyclopropylmethyl, normal butyl, tert- Methoxy, n-propoxy, isopropoxy, chloromethyl, chloroethyl, dichloroethyl, difluoromethyl, trifluoromethyl, chloromethoxy, chloromethoxy, dichloroethoxy, difluoromethoxy, trifluoromethoxy , And an amino group, or an amino group, or an amino group, an ethyl group, a normal propyl group, an isopropyl group, a cyclopropyl group, a cyclopropylmethyl group, a normal butyl group, a tert- butyl group, a cyclopentyl group, a cyclopropylethyl group, a cyclopentylmethyl group, A substituted or unsubstituted aromatic group having 1 to 4 substituents selected from the group consisting of a hexyl group, a cyclohexylmethyl group, and an amino group substituted with 1 to 2 cyclohexylethyl groups, The aromatic group may be a furanyl group, a thiophenyl group, a phenyl group, a pyridinyl group, a piperazinyl group, a pyridazinyl group, a naphthyl group, a quinolinyl group, or an isoquinoline group, but is not limited thereto.

The 1,2,3-triazalobenzopyran derivative represented by the formula (1) according to the present invention is more preferably as follows.

Wherein R ¹ is 1 to 3 substituents selected from the group consisting of H, Cl, F, NO ₂ , methyl, methoxy, trifluoromethoxy and trifluoromethyl,

Wherein R ² is a group selected from the group consisting of a normal propyl group, an isopropyl group, a cyclopropyl group, a cyclopropylmethyl group, a normal butyl group, a tert-butyl group, a cyclohexyl group, a cyclohexylmethyl group and - (CH ₂ ) nR ⁵ , An integer of 1 or 2,

Wherein R ⁵ is selected from the group consisting of hydrogen, Cl, F, CN, NO ₂ , OH, methyl, ethyl, isopropyl, normal butyl, tert- butyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, difluoromethyl And an amino group substituted with one to two of an amino group or a methyl group, an ethyl group, a normal propyl group, an isopropyl group, a normal butyl group, a cyclopentyl group, a cyclopentylmethyl group, and a cyclohexylmethyl group. And the aromatic group may be a furanyl group, a thiophenyl group, a phenyl group, a pyridinyl group, a naphthyl group, a quinolinyl group, or an isoquinolinyl group, but the present invention is not limited thereto .

The 1,2,3-triazalobenzopyran derivative represented by the formula (1) according to the present invention is more preferably as follows.

Wherein R ¹ is NO _2, or a methyl group,

Wherein R ² is a phenyl group, a 2-chlorophenyl group, a 3-chlorophenyl group, a 4-chlorophenyl group, a 2- fluorophenyl group, - (trifluoromethyl) phenyl group, 4- (trifluoromethyl) phenyl group, 3-methoxyphenyl group, 4-methoxyphenyl group, or isoquinolinyl group.

The 1,2,3-triazolo benzopyran derivative of the present invention is a 4- (4-methoxyphenyl) -1 - ((2-methyl-6-nitro- -1H-1,2,3-triazole, 4- (3-methoxyphenyl) -1 - (((2-methyl- , 2,3- triazole, 1 - ((2-methyl-6-nitro-2H-chromen-2- yl) methyl) -4- (p- Methyl-4- (3- (trifluoromethyl) phenyl) -1H-1,2,3-triazole, Yl) methyl) -1H-1,2,3-triazole, 4- (3-fluoro-phenyl) Yl) methyl) -1H-1,2,3-triazole, 4- (2-chlorophenyl) -1- (1 - ((2-methyl-6-nitro-2H-1,5-benzodiazepin- Methyl) -1H-1,2,3-triazol-4-yl) isoquinoline and 1 - ((2,6- -4-phenyl-1H-1,2,3-triazole from the group consisting of But is not limited thereto.

As used herein, the term " pharmaceutically acceptable salt ", which can be prepared by conventional methods in the art, includes, for example, hydrochloric acid, hydrogen bromide, sulfuric acid, sodium hydrogen sulfate, phosphoric acid, With an inorganic acid or with an organic acid such as formic, acetic, oxalic, benzoic, citric, tartaric, gluconic, gestic, fumaric, Means forming a salt or reacting with an alkali metal ion such as sodium or potassium to form a metal salt thereof or reacting with an ammonium ion to form another form of a pharmaceutically acceptable salt.

The 1,2,3-triazolobenzopyran derivatives represented by Formula 1 according to the present invention can be crystallized or recrystallized from a solvent such as aqueous and organic solvents. In such cases, solvates (especially hydrates) may be formed. Thus, the compounds of the present invention may also contain stoichiometric solvates, including hydrates, in addition to varying amounts of water-containing compounds that can be prepared by methods such as lyophilization.

The 1,2,3-triazalobenzopyran derivative represented by Formula 1 according to the present invention has an optically active structure due to its structural characteristic including chiral carbon substituted with different substituents at the C-2 position of benzopyran As such, the derivatives of the present invention may be enantiomers, stereoisomers, or tautomers. In addition, the isomer can be separated or decomposed by a conventional method. In addition, any desired isomer can be obtained by conventional synthetic methods or by stereospecific or asymmetric synthesis.

Also, the 1,2,3-triazolobenzopyran derivative represented by the above formula (1) according to the present invention may include a radioactive derivative, and these radioactive compounds may be used in the field of biological research.

The present invention also provides a process for preparing 1,2,3-triazolo benzopyran derivatives represented by the above formula (1).

[Reaction Scheme 1]

(In the above Reaction Scheme 1, R ¹ and R ² are each as defined above.)

The method for preparing the 1,2,3-triazolo benzopyran derivative according to the present invention will be described in detail as follows.

The conventional process the benzopyran compound is R ¹ of formula (2) can be replaced obtained with an acid after switching the 2-position to the aldehyde group, by just using a reducing agent represented by the above formula (3) the alcohol groups are introduced (2-methyl-2H-chromen-2-yl) methanol; And

The compound represented by Chemical Formula 3 is reacted with tosyl chloride (TsCl), converted to sulfonate, and then reacted with sodium azide continuously to obtain 2- (azidomethyl) - 2-methyl-2H-chromene; And

Reacting the compound represented by Formula 4 with an alkane substituted with R ² under a copper catalyst to obtain 1,2,3-triazole-benzopyran And a third step of synthesizing the derivative, and the formula of the derivative is shown in FIG.

The manufacturing method according to the present invention will be described in more detail with respect to each manufacturing step.

In the first step, the reaction for converting the C-2 position to an aldehyde group can be carried out using dichloromethane (CH ₂ Cl ₂ ), acetonitrile (MeCN), dichloroethane (ClCH ₂ CH ₂ Cl), dioxane, Tetrahydropyran (THF), acetone, methanol, or ethanol can be used as a solvent, and dichloromethane can be preferably used as a solvent, but the present invention is not limited thereto. The acid used in this reaction may be used in an amount of 1 to 30 equivalents, preferably 3 to 10 equivalents, based on the compound represented by the formula (2), but is not limited thereto. At this time, trifluoroacetic acid (TFA), acetic acid (AcOH), hydrochloric acid (HCl), sulfuric acid (H ₂ SO ₄ ) and the like can be used as the acid to be used, and trifluoroacetic acid TFA) may be effective, but is not limited thereto.

In the first step, the reduction reaction may be performed using a solvent such as tetrahydrofuran, diethyl ether, 1,4-dioxane, dichloromethane, dichloroethane, toluene, methanol, or ethanol, preferably tetrahydrofuran And methanol may be used together, but the present invention is not limited thereto. The reducing agent used in this reaction may be used in an amount of 1 to 3 equivalents, preferably 1 to 1.5 equivalents, based on the compound represented by the formula (2), but the present invention is not limited thereto. Examples of the reducing agent include sodium borohydroride (NaBH ₄ ), sodium triacetoxyborohydride (NaBH (OAc) ₃ ), lithium aluminum hydride (LiAlH ₄ ) Or hydrogen / palladium (H ₂ / Pd), and preferably sodium borohydride (NaBH ₄ ) may be effective, but is not limited thereto.

In the second step, the reaction to convert to sulfonate can be carried out using a solvent such as dichloromethane, dichloroethane, tetrahydrofuran, diethyl ether, 1,4-dioxane, or pyridine, preferably dichloromethane But are not limited thereto. The base used in this reaction may be used in an amount of 1 to 3 equivalents relative to the compound represented by the formula (3), preferably 1 to 1.5 equivalents, but is not limited thereto. The base used may be triethylamine (Et ₃ N), N, N-diisopropylethylamine, DBU, pyridine, or lutidine, preferably triethylamine May be effective, but is not limited thereto.

In the second step, the reaction to convert to azide is carried out in a solvent such as N, N-dimethylformamide (DMF), N, N-dimethylacetamide (DMA), dimethylsulfoxide (DMSO), methanol, And N, N-dimethylformamide may be preferably used, but it is not limited thereto. The sodium azide used in this reaction may be used in an amount of 1 to 3 equivalents, preferably 1 to 1.5 equivalents, based on the compound represented by the formula (3), but it is not limited thereto no.

The third-step reaction can be carried out in a solvent such as t-butanol, n-butanol, i-propanol, ethanol, methanol, water, N, N-dimethylformamide, N, N-dimethylacetamide, dimethylsulfoxide or tetrahydrofuran And preferably t-butanol and water can be used together, but the present invention is not limited thereto. The R ² -substituted alkane used in this reaction may be used in an amount of 1 to 2 equivalents, preferably 1 to 1.2 equivalents, based on the compound represented by the formula (4). However, But is not limited thereto. The copper catalyst may be used in the range of 0.01 to 0.1 equivalent weight, preferably 0.02 to 0.07 equivalent weight, but it is not limited thereto. The oxidizing agent to be used with the copper catalyst may be used in a range of 2 to 4 equivalents of the amount of the copper catalyst, preferably 2 equivalents, but is not limited thereto. As the copper catalyst, copper sulfate (CuSO ₄ ), copper iodide (CuI), copper metal (Cu) or the like can be used, and it is effective to use copper sulfate, but the present invention is not limited thereto. Sodium ascorbate may be the most effective oxidizing agent for use with copper catalysts, but is not limited thereto.

According to an embodiment of the present invention, in the process of producing the compound represented by Formula 1, the progress of the reaction was confirmed by thin-layer chromatography (TLC) The compound was isolated and purified and analyzed for its structure by NMR or Mass spectroscopy (see Example 1).

The 1,2,3-triazalobenzopyran derivative compound represented by Formula 1, a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof, a racemate thereof, or a stereoisomer thereof may be used for HIF Inhibitory activity, the composition of the present invention can be used as a therapeutic agent for a disease caused by HIF. Diseases caused by HIF activity may specifically include cancer, angiogenesis-related diseases, and the like.

In the present invention, the term "cancer" is used to refer to any of cancer, colon cancer, liver cancer, gastric cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, And can be selected from the group consisting of endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, renal cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma and central nervous system tumor Is not limited to, but is not limited to, lung cancer.

In the present invention, the term "angiogenesis-related diseases" refers to diseases caused by diabetic retinopathy, retinopathy of prematurity, corneal transplant rejection, neovascular glaucoma, hypoxia, proliferative retinopathy, psoriasis, hemophilic joints, capillary proliferation in atherosclerotic plaque , Keloids, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune disease, Crohn's disease, recurrent stenosis, atherosclerosis, intestinal adhesion, cat scratch disease, ulcer, hepatopathy, glomerulonephritis, diabetic nephropathy, , Thrombotic microangiopathy, organ transplant rejection, gynecological complications, diabetes, inflammation or neurodegenerative diseases.

The pharmaceutical composition of the present invention is a 1,2,3-triazalobenzopyran derivative represented by the above formula (1), a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof, a racemate thereof, or a stereoisomer thereof For example, tablets, capsules, troches, solutions, suspensions, etc., by adding a conventional non-toxic pharmaceutically acceptable carrier, adjuvant, and excipient to the pharmaceutical preparation For oral administration, or for parenteral administration.

The excipient that can be used in the pharmaceutical composition of the present invention may include sweeteners, binders, solubilizers, solubilizers, wetting agents, emulsifiers, isotonic agents, adsorbents, disintegrants, antioxidants, preservatives, lubricants, fillers, . For example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, silica, talc, stearic acid, stearin, magnesium stearate, magnesium aluminum silicate, starch, gelatin, tragacanth gum, Water, ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, orange essence, strawberry essence, vanilla flavor, and the like.

The present invention also provides a chemotherapeutic adjuvant comprising, as an active ingredient, a 1,2,3-triazalobenzopyran derivative represented by the following formula (1) and a pharmaceutically acceptable salt thereof:

[Chemical Formula 1]

In Formula 1,

R ¹ is H; OH; halogen; CN; NO ₂ ; A straight or branched or cyclic alkyl group of C _1-10 ; C _1-10 An alkoxy group; And C _1-10 haloalkyl groups of 1 to 4 substituents selected from the group consisting of a;

R ² is a C _1-10 linear or branched or cyclic alkyl group; An alkoxy group of C _1-10 ; An alkoxyalkyl group of C _2-10 ; A C _2-10 linear or branched alkenyl group; And - (CH ₂ ) n R ³ , wherein n is 0 or an integer from 1 to 3; Lt; / RTI > is any one selected from the group consisting of &

R ³ is selected from the group consisting of halo, cyano, nitro, hydroxy, C _1-5 alkyl, C _1-5 alkoxy, C _1-5 haloalkyl, and C _1-5 haloalkoxy. And the aromatic group is a furanyl group, a thiophenyl group, a phenyl group, a pyridinyl group, a piperazinyl group, a pyridazinyl group, a naphthyl group, a quinolinyl group, or an isoquinolyl group, wherein the four substituents are substituted or unsubstituted aromatic groups.

In the present invention, "anticancer adjuvant" can be used to increase the anticancer effect of an anticancer drug and to suppress or improve side effects of the anticancer drug, and can be administered in combination with an anticancer drug or another kind of anticancer adjuvant. Further, May be in the form of a complex comprising an adjuvant.

The route of administration of the anticancer adjuvant may be administered through any conventional route as long as it can reach the target tissue. The anticancer adjuvant of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonarily or rectally, as desired, Do not. In addition, the anticancer adjuvant may be administered by any device capable of moving the active substance to the target cell.

Furthermore, the present invention provides a method of preventing or treating cancer, comprising administering the pharmaceutical composition to a subject.

In addition, the present invention provides the use of the pharmaceutical composition for preventing or treating cancer.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level is determined depending on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex, condition, body weight, absorbency, inactivation rate, excretion rate, type of disease, May be administered in an amount of 0.1 mg / kg to 100 mg / kg per day, preferably 1 to 30 mg / kg per day, and may be administered once or several times a day.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine, or intracerebral injection. The pharmaceutical composition of the present invention is determined depending on the kind of the active ingredient, along with various related factors such as the disease to be treated, the route of administration, the age, sex, weight, and disease severity of the patient.

As used herein, the term "individual" means a subject in need of treatment for a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, And the like.

In addition, the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response modifiers for the prevention and treatment of cancer.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Experimental Preparation and Experimental Method

1-1. sample

144 small molecules that have not been evaluated for pharmacological effects have been obtained from an in-house chemical library (Kyungpook National University, College of Pharmacy and Pharmaceutical Sciences), and the HIF- And screened. The chemical compound was prepared by dissolving in dimethyl sulfoxide (DMSO) at a stock concentration of 5 mM.

1-2. Compound synthesis

The compounds of the present invention were synthesized by a previously known procedure, and the structure and analytical results of the synthesized 1,2,3-triazolobenzopyran compounds are summarized in the following Table 1 (Korean Patent No. 10- 1666759).

(1)

(One) NO. R ¹ R ² Analytical data [ ¹ H NMR (500 MHz, CDCl ₃ ) and LC / MS (ESI)] [# 12] 6-NO ₂ 4-MeO-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 1.52 (s, 3H), 3.83 (s, 3H), 4.59 (d, J = 14.6 Hz, 1H), 4.68 (d, J = 14.5 Hz, 1H), 5.82 (d, J = 10.0 Hz, 1H), 6.47 (d, J = 10.0 Hz, 1H), 6.93 1H), 7.69 (d, J = 8.9 Hz, 2H), 7.88 (d, J = 2.7 Hz, 1H), 8.07 (dd, J = 2.7, 8.9 Hz, 1H); LC-MS (ESI) m / z 379 ([M + 1] < ⁺ >). [# 111] 6-NO ₂ Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 1.53 (s, 3H), 4.61 (d, J = 14.5 Hz, 1H), 4.70 (d, J = 14.5 Hz, 1H), 5.83 (d, J = 10.0 Hz 1H), 6.48 (d, J = 10.0 Hz, 1H), 6.94 (d, J = 8.9 Hz, 1H), 7.33 (m, 1H), 7.39-7.42 (m, 2H), 7.76-7.78 3H), 7.88 (d, J = 2.7 Hz, 1H), 8.08 (dd, J = 2.7, 8.9 Hz, 1H); LC-MS (ESI) m / z 349 ([M + 1] < ⁺ >). [# 97] 6-NO ₂ 3-MeO-Ph ¹ H NMR (500 MHz, CDCl ₃ )? 1.53 (s, 3H), 3.86 (s, 3H), 4.61 (d, J = 14.5 Hz, (d, J = 10.1 Hz, 1H), 6.48 (d, J = 10.0 Hz, 1H), 6.88 (ddd, J = 1.7, 2.5, 7.5 Hz, 1H) , 7.28-7.33 (m, 2H), 7.39 (m, 1H), 7.76 (s, 1H), 7.88 (d, J = 2.7 Hz, 1H), 8.08 (dd, J = 2.7, 9.0 Hz, 1H); LC-MS (ESI) m / z 379 ([M + 1] < ⁺ >). [# 99] 6-NO ₂ 2-MeO-Ph ¹ H NMR (500 MHz, CDCl ₃ )? 1.54 (s, 3H), 3.88 (s, 3H), 4.60 (d, J = 14.5 Hz, (d, J = 10.0 Hz, 1H), 6.47 (d, J = 10.0 Hz, 1H), 6.93 (Ddd, J = 1.6, 7.4, 8.3 Hz, 1H), 7.88 (d, J = 2.7 Hz, 1H), 8.04 J = 2.7, 8.9 Hz, 1H), 8.29 (dd, J = 1.7, 7.7 Hz, 1H); LC-MS (ESI) m / z 379 ([M + 1] < ⁺ >). [# 112] 6-NO ₂ 4-Me-Ph ¹ H NMR (500 MHz, CDCl ₃ )? 1.53 (s, 3H), 2.37 (s, 3H), 4.60 (d, J = 14.5 Hz, (d, J = 10.1 Hz, 1H), 6.47 (d, J = 10.0 Hz, 1H), 6.93 J = 8.1 Hz, 2H), 7.73 (s, 1H), 7.87 (d, J = 2.7 Hz, 1H), 8.07 (dd, J = 2.7, 8.9 Hz, 1H); LC-MS (ESI) m / z 363 ([M + 1] < ⁺ >). [# 113] 6-NO ₂ 3-Me-Ph ¹ H NMR (500 MHz, CDCl ₃ )? 1.53 (s, 3H), 2.39 (s, 3H), 4.60 (d, J = 14.5 Hz, (d, J = 10.0 Hz, 1H), 6.48 (d, J = 10.0 Hz, 1H), 6.94 (t, J = 7.6 Hz, 1 H), 7.53 (d, J = 7.7 Hz, 1 H), 7.61 (dd, J = 2.7, 8.9 Hz, 1 H); LC-MS (ESI) m / z 363 ([M + 1] < ⁺ >). [# 98] 6-NO ₂ 2-Me-Ph ¹ H NMR (500 MHz, CDCl ₃ )? 1.55 (s, 3H), 2.32 (s, 3H), 4.63 (d, J = 14.5 Hz, (d, J = 10.1 Hz, 1H), 6.47 (d, J = 10.0 Hz, 1H), 6.90 2H), 7.87 (d, J = 2.7 Hz, 1H), 8.06 (dd, J = 2.7, 8.9 Hz, 1H); LC-MS (ESI) m / z 363 ([M + 1] < ⁺ >). [# 100] 6-NO ₂ 4-CF3 _- Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 1.54 (s, 3H), 4.63 (d, J = 14.6 Hz, 1H), 4.72 (d, J = 14.5 Hz, 1H), 5.83 (d, J = 10.0 Hz (D, J = 8.1 Hz, 2H), 7.86 (s, 1H), 7.88 (d, J = J = 2.7 Hz, 1H), 7.89 (d, J = 8.1 Hz, 2H), 8.08 (dd, J = 2.7, 8.9 Hz, 1H); LC-MS (ESI) m / z 417 ([M + 1] < ⁺ >). [# 13] 6-NO ₂ 3-CF3 _- Ph ¹ H NMR (500 MHz, CDCl ₃ )? 1.54 (s, 3H), 4.63 (d, J = 14.5 Hz, 1H), 4.72 J = 7.8 Hz, 1H), 6.49 (d, J = 10.0 Hz, 1H), 6.95 J = 2.7 Hz, 1H), 7.85 (s, 1H), 7.88 (d, , 1H); LC-MS (ESI) m / z 417 ([M + 1] < ⁺ >). 6-NO ₂ 2-CF3 _- Ph ^{1 H NMR (500 MHz, DMSO-} d 6) δ 8.14 (s, 1H), 8.01 (dd, J = 8.9, 2.8 Hz, 1H), 7.97 (d, J = 2.8 Hz, 1H), 7.82 (d, (D, J = 7.8 Hz, 1H), 7.72 (t, J = 7.5 Hz, 1H), 7.62 J = 10.1 Hz, 1H), 6.00 (d, J = 10.1 Hz, 1H), 4.81 (q, J = 14.5 Hz, 2H), 1.50 (s, 3H); LC-MS (ESI) m / z 417 ([M + 1] < ⁺ >). 6-NO ₂ 4-F-Ph ^{1 H NMR (500 MHz, DMSO-} d 6) δ 8.42 (s, 1H), 8.02 (dd, J = 8.9, 2.8 Hz, 1H), 7.98 (d, J = 2.8 Hz, 1H), 7.85-7.79 ( J = 10.1 Hz, 1H), 6.69 (d, J = 10.0 Hz, 1H), 6.02 (d, J = 10.1 Hz, 1H), 7.29-7.22 4.76 (q, J = 14.6 Hz, 2H), 1.48 (s, 3H) LC-MS (ESI) m / z 367 ([M + 1] ⁺ ). 6-NO ₂ 3-F-Ph LC-MS (ESI) m / z 367 ([M + 1] < ⁺ >). 6-NO ₂ 2-F-Ph ^{1 H NMR (500 MHz, DMSO-} d 6) δ 8.25 (d, J = 3.7 Hz, 1H), 8.10-7.92 (m, 3H), 7.43-7.22 (m, 3H), 6.95 (d, J = 8.9 Hz, 1H), 6.68 (d, J = 10.1 Hz, 1H), 6.03 (d, J = 10.1 Hz, 1H), 4.82 (q, J = 14.4 Hz, 2H), 1.50 LC-MS (ESI) m / z 367 ([M + 1] < ⁺ >). 6-NO ₂ 4-CN-Ph ^{1 H NMR (500 MHz, DMSO-} d 6) δ 8.65 (s, 1H), 8.02 (d, J = 2.8 Hz, 1H), 8.00 (d, J = 1.9 Hz, 1H), 7.99 (d, J = J = 8.9 Hz, 1H), 6.69 (d, J = 10.1 Hz, 1H), 6.02 (d, J = 10.1 Hz, , 1 H), 4.79 (q, J = 14.6 Hz, 2 H), 1.49 (s, 3 H); LC-MS (ESI) m / z 374 ([M + 1] < ⁺ >). 6-NO ₂ 4-NO ₂ -Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.25 (td, J = 2.2, 9.1 Hz, 2H), 8.07-8.04 (m, 2H), 7.90 ((d, J = 2.7 Hz, 1H), 7.81 td, (D, J = 10.1 Hz, 1H), 4.77 (d, J = J = 14.3 Hz, 1H), 4.61 (d, J = 14.3 Hz, 1H), 1.52 (s, 3H); LC-MS (ESI) m / z 394 ([M + 1] < ⁺ >). 6-NO ₂ 3-NO ₂ -Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.25 (d, J = 7.6 Hz, 1H), 8.11 (s, 1H), 8.06 (dd, J = 8.9, 2.7 Hz, 1H), 7.94 (d, J = J = 10.0 Hz, 1H), 7.90-7.86 (m, 2H), 6.89 (d, J = , 4.67 (d, J = 14.3 Hz, 1H), 4.60 (d, J = 14.3 Hz, 1H), 1.55 (s, 3H); LC-MS (ESI) m / z 394 ([M + 1] < ⁺ >). 6-NO ₂ 2-NO ₂ -Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.10-8.05 (m, 3H), 7.88 (d, J = 2.7 Hz, 1H), 7.64-7.55 (m, 2H), 7.35 (dd, J = 1.5, 7.4 J = 10.0 Hz, 1H), 4.75 (d, J = 14.3 Hz, 1H), 6.89 (d, J = 1H), 4.63 (d, J = 14.3 Hz, 1H), 1.54 (s, 3H); LC-MS (ESI) m / z 394 ([M + 1] < ⁺ >). 6-NO ₂ 4-Cl-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.06 (s, 1H), 8.02 (dd, J = 9.0, 2.9 Hz, 1H), 7.89 (d, J = 2.9 Hz, 1H), 7.35-7.25 (m, J = 9.8 Hz, 1H), 6.85 (d, J = 9.0 Hz, 1H) , 4.59 (d, J = 14.2 Hz, 1 H), 1.54 (s, 3 H); LC-MS (ESI) m / z 383 ([M + 1] < ⁺ >). 6-NO ₂ 3-Cl-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.06 (dd, J = 8.9, 2.8 Hz, 1H), 7.92 (s, 1H), 7.89 (d, J = 2.2 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.30 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 10.1 Hz, 1H), 5.87 (d, J = 10.0 Hz, 1H), 4.71 , 3H); LC-MS (ESI) m / z 383 ([M + 1] < ⁺ >). 6-NO ₂ 2-Cl-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.02 (dd, J = 9.0, 2.5 Hz, 1H), 7.99 (s, 1H), 7.88 (d, J = 2.5 Hz, 1H), 7.36-7.31 (m, J = 10.2Hz, 1H), 4.71 (d, J = 10.2Hz, 2H), 7.26-7.19 d, J = 14.2 Hz, 1H), 4.56 (d, J = 14.2 Hz, 1H), 1.53 (s, 3H); LC-MS (ESI) m / z 383 ([M + 1] < ⁺ >). 6-NO ₂ 4-NMe ₂ -Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.07 (dd, J = 2.8, 8.9 Hz, 1H), 7.81 (s, 1H), 7.86 (d, J = 2.8 Hz, 1H), 7.51-7.53 (m, 2H), 6.89 (d, J = 9.0 Hz, 1H), 6.58-6.67 (m, 2H), 6.46 d, J = 14.3 Hz, 1H), 4.61 (d, J = 14.3 Hz, 1H), 2.99 (s, 6H), 1.51 (s, 3H); LC-MS (ESI) m / z 392 ([M + 1] < ⁺ >). 6-NO ₂ 3-NMe ₂ -Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.07 (dd, J = 2.7, 8.9 Hz, 1H), 7.87 (d, J = 2.7 Hz, 1H), 7.51 (s, 1H), 7.19 (dd, J = 7.8, 8.1 Hz, 1 H), 7.05 (m, 1H), 6.89 (d, J = 9.0 Hz, 1H), 6.80 (dd, J = 2.7,8.45 Hz, 1H) (d, J = 10.0 Hz, 1H), 5.82 (d, J = 10.0 Hz, 1H), 4.71 6H), 1.52 (s, 3H); LC-MS (ESI) m / z 392 ([M + 1] < ⁺ >). 6-NO ₂ 4-Ph-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.07 (dd, J = 9.0, 2.7 Hz, 1H), 7.89 (d, J = 2.7 Hz, 1H), 7.86 (s, 1H), 7.60-7.55 (m, J = 10.0 Hz, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.49 d, J = 14.4 Hz, 1H), 4.59 (d, J = 14.4 Hz, 1H), 1.57 (s, 3H); LC-MS (ESI) m / z 425 ([M + 1] < ⁺ >). 6-NO ₂ 4-PhO-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.06 (dd, J = 2.7, 8.9 Hz, 1H), 7.93 (s, 1H), 7.86 (d, J = 2.7 Hz, 1H), 7.72 (d, J = J = 7.6 Hz, 1H), 7.02 (d, J = 7.6 Hz, 2H), 6.95-6.89 (m, 3H) , 6.50 (d, J = 10.0 Hz, 1H), 5.83 (d, J = 10.0 Hz, 1H), 4.69 (s, 3 H); LC-MS (ESI) m / z 441 ([M + 1] < ⁺ >). 6-NO ₂ 4-Ac-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.08 (dd, J = 2.7, 8.9 Hz, 1H), 7.99 (s, 1H), 7.91-7.84 (m, 5H), 6.94 (d, J = 8.9 Hz, J = 10.0 Hz, 1 H), 4.73 (d, J = 14.5 Hz, 1 H), 4.57 (d, J = , 2.61 (s, 3 H), 1.60 (s, 3 H); LC-MS (ESI) m / z 391 ([M + 1] < ⁺ >). 6-NO ₂ 4-MeS-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.07 (dd, J = 8.7, 2.6 Hz, 1H), 7.89 (d, J = 2.6 Hz, 1H), 7.26-7.15 (m, 2H), 6.92 (d, J = 8.7 Hz, 1H), 6.57-6.47 (m, 3H), 5.84 (d, J = 10.2 Hz, 1H), 4.67 1H), 2.41 (s, 3H), 1.52 (s, 3H); LC-MS (ESI) m / z 395 ([M + 1] < ⁺ >). 6-NO _{2 3-CF} 3 O-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.06 (dd, J = 8.9, 2.7 Hz, 1H), 8.01 (s, 1H), 7.88 (d, J = 2.7 Hz, 1H), 7.40 (t, J = 7.9 Hz, 1H), 7.28-7.19 (m, 2H), 7.11 (s, 1H), 6.93 (d, J = 8.9 Hz, 1H) J = 10.0 Hz, 1H), 4.67 (d, J = 14.4 Hz, 1H), 4.58 (d, J = 14.4 Hz, 1H), 1.56 (s, 3H); LC-MS (ESI) m / z 433 ([M + 1] < ⁺ >). 6-NO ₂ 3,5-di-MeO-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.08 (dd, J = 2.8, 8.9 Hz, 1H), 7.88 (d, J = 2.7 Hz, 1H), 7.56 (s, 1H), 6.89 (d, J = (D, J = 10.1 Hz, 1H), 6.66 (d, J = 2.3 Hz, 2H), 6.53 1H), 4.79 (d, J = 14.3 Hz, 1H), 4.61 (d, J = 14.3 Hz, 1H), 3.78 (s, 6H), 1.52 (s, 3H); LC-MS (ESI) m / z 409 ([M + 1] < ⁺ >). 6-NO ₂ 3,4-di-MeO-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.07 (dd, J = 9.0, 2.8 Hz, 1H), 7.94 (s, 1H), 7.89 (d, J = 2.8 Hz, 1H), 6.86-6.78 (m, J = 14.2 Hz, 1H), 4.86 (d, J = 14.2 Hz, 1H) s, 3H), 3.81 (s, 3H), 1.52 (s, 3H); LC-MS (ESI) m / z 409 ([M + 1] < ⁺ >). 6-NO ₂ 2,4-di-MeO-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.12 (s, 1H), 8.10 (d, J = 8.8 Hz, 1H), 8.05 (dd, J = 2.8, 8.9 Hz, 1H), 7.84 (d, J = J = 8.9 Hz, 1H), 6.39 (d, J = 8.9 Hz, 1H), 6.85 (d, J = J = 14.1 Hz, 1H), 4.83 (d, J = 14.1 Hz, 1H), 3.83 (s, 3H), 3.69 s, 3H), 1.52 (s, 3H); LC-MS (ESI) m / z 409 ([M + 1] < ⁺ >). 6-NO ₂ 2,4-di-Me-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.08 ~ 8.05 (m, 2H), 7.88 (d, J = 2.5 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.00 (s, 1H) , 6.92 (d, J = 10.0 Hz, 1H), 4.68 (d, J = 14.8 Hz, 1H) J = 14.8 Hz, 1H), 2.50 (s, 3H), 2.30 (s, 3H), 1.53 (s, 3H); LC-MS (ESI) m / z 377 ([M + 1] < ⁺ >). 6-NO ₂ 3,5-di-Me-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.10 (s, 1H), 8.07 (dd, J = 8.9, 2.8 Hz, 1H), 7.90 (d, J = 2.8 Hz, 1H), 6.88 (d, J = 2H), 5.85 (d, J = 9.8Hz, 1H), 4.69-4.60 (m, 2H) 2H), 2.21 (s, 6 H), 1.51 (s, 3 H); LC-MS (ESI) m / z 377 ([M + 1] < ⁺ >). 6-NO ₂ 2,4,6-tri-Me-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.21 (s, 1H), 8.07 (dd, J = 2.6, 8.9 Hz, 1H), 7.87 (d, J = 2.6 Hz, 1H), 6.91-6.85 (m, J = 10.0 Hz, 1H), 4.69 (d, J = 14.8 Hz, 1H), 4.61 (d, J = , 2.40 (s, 3H), 2.25 (s, 6H), 1.55 (s, 3H); LC-MS (ESI) m / z 439 ([M + 1] < ⁺ >). 6-NO ₂ 3,4,5-tri-MeO-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.06 (dd, J = 2.7, 8.9 Hz, 1H), 7.92 (s, 1H), 7.87 (d, J = 2.7 Hz, 1H), 7.07 (s, 2H) , 6.92 (d, J = 8.9 Hz, 1H), 6.52 (d, J = 10.0 Hz, 1H) (d, J = 14.8 Hz, 1H), 3.87 (s, 3H), 3.76 (s, 6H), 1.54 (s, 3H); LC-MS (ESI) m / z 439 ([M + 1] < ⁺ >). 6-NO ₂

^{1 H NMR (500 MHz, CDCl} 3) δ 8.07 (dd, J = 2.7, 8.9 Hz, 1H), 7.91 (s, 1H), 7.88 (d, J = 2.7 Hz, 1H), 7.39 (dd, J = J = 8.9 Hz, 1H), 6.72 (d, J = 8.2 Hz, 1H), 6.50 (d, J = 10.0 Hz, 1H), 5.82 (d, J = 10.0 Hz, 1H), 4.68 (d, J = 14.8 Hz, 1H), 4.57 (d, J = 14.8 Hz, 1H). LC-MS (ESI) m / z 393 ([M + 1] < ⁺ >). 6-NO ₂ 3,4-di-F-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.05 (dd, J = 8.9, 2.7 Hz, 1H), 7.93 (s, 1H), 7.89 (d, J = 2.6 Hz, 1H), 7.15 (m, 1H) (D, J = 10.1 Hz, 1H), 7.05-6.94 (m, 2H), 6.88 J = 14.3 Hz, 1H), 4.58 (d, J = 14.3 Hz, 1H), 1.54 (s, 3H); LC-MS (ESI) m / z 385 ([M + 1] < ⁺ >). 6-NO ₂ 2,4-di-F-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.07 (dd, J = 8.9, 2.7 Hz, 1H), 7.92-7.89 (m, 2H), 7.29 (m, 1H), 6.92 (td, J = 8.6, 2.4 J = 10.1 Hz, 1H), 6.84 (m, 1H), 6.81 (d, J = 71 (d, J = 14.4 Hz, 1H), 4.62 (d, J = 14.4 Hz, 1H), 1.54 (s, 3H); LC-MS (ESI) m / z 385 ([M + 1] < ⁺ >). 6-NO ₂ 2,4-di-Cl-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.06 (dd, J = 8.9, 2.8 Hz, 1H), 7.92-7.89 (m, 2H), 7.41 (s, 1H), 7.28 (d, J = 8.0 Hz, (D, J = 10.1 Hz, 1H), 7.11 (d, J = 9.0 Hz, 1H), 6.81 , 4.68 (d, J = 14.5 Hz, 1H), 4.63 (d, J = 14.5 Hz, 1H), 1.54 (s, 3H); LC-MS (ESI) m / z 417 ([M + 1] < ⁺ >). 6-NO ₂ 3-F-4-Cl-Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.06 (dd, J = 8.9, 2.7 Hz, 1H), 7.95 (s, 1H), 7.89 (d, J = 2.7 Hz, 1H), 7.28 (s, 1H) , 7.15-7.13 (m, 2H), 6.89 (d, J = 8.9 Hz, 1H), 6.48 (d, J = 10.1 Hz, 1H) J = 14.3 Hz, 1H), 4.58 (d, J = 14.3 Hz, 1H), 1.54 (s, 3H); LC-MS (ESI) m / z 401 ([M + 1] < ⁺ >). 6-NO _{2 3-F-4-CF 3} -Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 8.06 (dd, J = 8.9, 2.7 Hz, 1H), 7.94 (s, 1H), 7.88 (d, J = 2.7 Hz, 1H), 7.53 (s, 1H) , 7.47 (d, J = 2.7 Hz, 1H), 7.22 (t, J = 9.3 Hz, 1H), 6.93 (d, J = 8.9 Hz, 1H) (d, J = 10.1 Hz, 1H), 4.67 (d, J = 14.3 Hz, 1H), 4.60 (d, J = 14.3 Hz, 1H), 1.55 (s, 3H); LC-MS (ESI) m / z 4357 ([M + 1] < ⁺ >). 6-NO ₂ 2-NO ₂ -5-MeO-Ph ¹ H NMR (500 MHz, CDCl ₃ )? 1.54 (s, 3H), 3.84 (s, 3H), 4.60 (d, J = 14.3 Hz, (d, J = 10.1 Hz, 1H), 6.51 (d, J = 10.0 Hz, 1H), 6.61 J = 2.8, 9.2 Hz, 1H), 7.90 (d, J = 2.7 Hz, IH), 8.04 (s, IH), 8.06 (dd, J = 2.7,8.9 Hz, 9.1 Hz, 1H); LC-MS (ESI) m / z 424 ([M + 1] < ⁺ >). 6-NO ₂ 3-NO ₂ -4-F-Ph ¹ H NMR (500 MHz, CDCl ₃ )? 1.52 (s, 3H), 4.60 (d, J = 14.3 Hz, 1H), 4.69 , 7.88 (d, J = 8.9 Hz, 1H), 7.38 (dd, J = 4.2, 8.8 Hz, 1H), 8.10 (s, 1H), 8.02 (dd, J = 2.8, 8.9 Hz, 1H), 8.30 (dd, J = 2.3, 6.9 Hz, 1H); LC-MS (ESI) m / z 412 ([M + 1] < ⁺ >). 6-NO ₂ 3-F-4-Me-Ph LC-MS (ESI) m / z 381 ([M + 1] < ⁺ >). 6-NO ₂ 3-Cl-4-Me-Ph C-MS (ESI) m / z 397 ([M + 1] < ⁺ >). 6-NO ₂ i-Pr ^{1 H NMR (500 MHz, CDCl} 3) δ 8.06 (dd, J = 2.7, 8.9 Hz, 1H), 7.88 (d, J = 2.7 Hz, 1H), 7.82 (s, 1H), 6.92 (d, J = J = 10.0 Hz, 1H), 4.72 (d, J = 14.8 Hz, 1H), 4.60 (d, J = 1H), 2.45 (m, 1H), 1.51 (s, 3H), 1.05 (t, J = 7.6 Hz, 6H); LC-MS (ESI) m / z 315 ([M + 1] < ⁺ >). 6-NO ₂ n-Bu ^{1 H NMR (500 MHz, CDCl} 3) δ 8.07 (dd, J = 9.0, 2.9 Hz, 1H), 7.90 (s, 1H), 7.86 (d, J = 2.9 Hz, 1H), 6.88 (d, J = J = 10.0 Hz, 1H), 4.65 (d, J = 14.4 Hz, 1H), 4.58 (d, J = 14.4 Hz, 1H) 1H), 2.30-2.22 (m, 2H), 1.63-1.47 (m, 2H), 1.53 (s, 3H), 1.37-1.26 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H); LC-MS (ESI) m / z 329 ([M + 1] < ⁺ >). 6-NO ₂ t-Bu ^{1 H NMR (500 MHz, CDCl} 3) δ 8.06 (dd, J = 9.0, 2.4 Hz, 1H), 7.91 (s, 1H), 7.88 (d, J = 2.4 Hz, 1H), 6.90 (d, J = J = 10.2 Hz, 1H), 4.68 (d, J = 14.2 Hz, 1H), 6.47 , ≪ / RTI > 1H), 1.54 (s, 3H), 1.31 (s, 9H); LC-MS (ESI) m / z 329 ([M + 1] < ⁺ >). 6-NO ₂ sec-Bu ^{1 H NMR (500 MHz, CDCl} 3) δ 8.04 (dd, J = 2.7, 8.9 Hz, 1H), 8.01 (s, 1H), 7.88 (d, J = 2.4 Hz, 1H), 6.89 (d, J = J = 10.1 Hz, 1H), 4.69 (d, J = 14.4 Hz, 1H), 3.59 (d, J = 14.1 Hz, J = 6.9 Hz, 3H), 0.84 (td, J = 1 Hz), 2.07 (m, 7.4, 9.7 Hz, 3H); LC-MS (ESI) m / z 329 ([M + 1] < ⁺ >). 6-NO ₂ iso-This ^{1 H NMR (500 MHz, CDCl} 3) δ 8.03 (dd, J = 2.8, 8.9 Hz, 1H), 7.99 (s, 1H), 7.89 (d, J = 2.7 Hz, 1H), 6.83 (d, J = J = 10.1 Hz, 1H), 4.68 (d, J = 14.3 Hz, 1H), 4.59 (d, J = 14.3 Hz, J = 5.0 Hz, 3H), 0.90 (d, J = 5.1 Hz, 3H); 1.97 (s, 3H). LC-MS (ESI) m / z 329 ([M + 1] < ⁺ >). 6-NO ₂ -CH ₂ -tBu ^{1 H NMR (500 MHz, CDCl} 3) δ 8.08 (dd, J = 9.0, 2.9 Hz, 1H), 7.91 (s, 1H), 7.86 (d, J = 2.9 Hz, 1H), 6.91 (d, J = J = 14.2 Hz, 1 H), 4.72 (d, J = 14.4 Hz, 1H), 4.59 (d, J = 14.2 Hz, , ≪ / RTI > 1H), 2.81 (s, 2H), 1.54 (s, 3H), 1.02 (s, 9H); LC-MS (ESI) m / z 343 ([M + 1] < ⁺ >). 6-NO ₂ n-pentyl LC-MS (ESI) m / z 343 ([M + 1] < ⁺ >). 6-NO _{2 4-F-Ph- (CH 2} ) 2 - ^{1 H NMR (500 MHz, CDCl} 3) δ δ 8.06 (dd, J = 2.7, 8.9 Hz, 1H), 7.91 (s, 1H), 7.88 (d, J = 2.7 Hz, 1H), 7.09-7.06 (m J = 10.0 Hz, 1H), 4.68 (d, J = 11.8 Hz, 1H), 6.81 (d, (d, J = 11.8 Hz, 1H), 2.83-2.79 (m, 2H), 2.53-2.49 (m, 2H), 1.52 (s, 3H); LC-MS (ESI) m / z 395 ([M + 1] < ⁺ >). 6-NO ₂ Ph- (CH _{2) 2} - ^{1 H NMR (500 MHz, CDCl} 3) δ 8.07 (dd, J = 2.7, 8.0 Hz, 1H), 7.87 (d, J = 2.8 Hz, 1H), 7.32 (s, 1H), 7.22-7.26 (m, (D, J = 10.1 Hz, 1H), 4.69 (d, J = (d, J = 14.3 Hz, 1H), 4.58 (d, J = 14.3 Hz, 1H), 2.91 (t, J = 7.5 Hz, 2H) 3H); LC-MS (ESI) m / z 377 ([M + 1] < ⁺ >). 6-NO _{2 4-MeO-Ph- (CH 2} ) 2 - LC-MS (ESI) m / z 407 ([M + 1] < ⁺ >). 6-NO ₂ cyclohexyl ^{1 H NMR (500 MHz, CDCl} 3) δ 8.06 (dd, J = 2.7, 8.9 Hz, 1H), 7.95 (s, 1H), 7.89 (d, J = 2.7 Hz, 1H), 6.87 (d, J = J = 14.3 Hz, 1H), 4.64 (d, J = 14.1 Hz, 1H) 1H), 1.63 (s, 3H), 1.29-1.39 (m, 2H), 2.03 (t, J = 3.3,11.7 Hz, 1H), 1.72-7.78 1.12-1.27 (m, 3 H); LC-MS (ESI) m / z 355 ([M + 1] < ⁺ >). 6-NO ₂ cyclopropyl ^{1 H NMR (500 MHz, CDCl} 3) δ 8.07 (dd, J = 2.8, 8.9 Hz, 1H), 7.94 (s, 1H), 7.90 (d, J = 2.7 Hz, 1H), 6.88 (d, J = J = 10.1 Hz, 1H), 4.71 (d, J = 14.3 Hz, 1H), 4.66 (d, J = , 1.45 (s, 3H), 1.24-1.31 (m, 1H), 0.92-0.94 (m, 2H), 0.69-0.73 (m, 2H); LC-MS (ESI) m / z 313 ([M + 1] < ⁺ >). 6-NO ₂

^{1 H NMR (500 MHz, CDCl} 3) δ 8.06 (dd, J = 9.0, 2.6 Hz, 1H), 7.94 (s, 1H), 7.88 (d, J = 2.6 Hz, 1H), 7.45 (d, J = J = 9.0 Hz, 1H), 6.48 (d, J = 10.2 Hz, 1H), 5.82 (d, J = 10.2 Hz, 1H), 4.71 (d, J = 14.4 Hz, 1H), 4.61 (d, J = 14.4 Hz, 1H), 1.52 (s, 3H); LC-MS (ESI) m / z 355 ([M + 1] < ⁺ >). 6-NO ₂

^{1 H NMR (500 MHz, CDCl} 3) δ 8.07 (dd, J = 9.0, 2.9 Hz, 1H), 7.99 (s, 1H), 7.88 (d, J = 2.9 Hz, 1H), 7.45 (d, J = J = 9.0 Hz, 1H), 6.49 (d, J = 9.8 Hz, 1H), 5.82 (d, J = 9.8 Hz, 1H), 4.72 (d, J = 14.5 Hz, 1H), 4.61 (d, J = 14.5 Hz, 1H), 1.51 (s, 3H); LC-MS (ESI) m / z 339 ([M + 1] < ⁺ >). 6-NO ₂

^{1 H NMR (500 MHz, CDCl} 3) δ 8.06-7.97 (m, 3H), 7.87-7.75 (m, 3H), 7.55-7.25 (m, 4H), 6.91 (d, J = 8.5 Hz, 1H), J = 9.8 Hz, 1H), 6.09 (d, J = 9.8 Hz, 1H), 4.73 (d, J = s, 3H); LC-MS (ESI) m / z 399 ([M + 1] < ⁺ >). 6-NO ₂

LC-MS (ESI) m / z 400 ([M + 1] < ⁺ >). 6-NO ₂

LC-MS (ESI) m / z 372 ([M + 1] < ⁺ >). 6-NO ₂

LC-MS (ESI) m / z 350 ([M + 1] < ⁺ >). 6-NO ₂ 4-Cl-Bn LC-MS (ESI) m / z 397 ([M + 1] < ⁺ >). 6-NO ₂ Bn LC-MS (ESI) m / z 363 ([M + 1] < ⁺ >). H 4-Me-Ph ¹ H NMR (500 MHz, CDCl ₃ )? 1.51 (s, 3H), 2.37 (s, 3H), 4.57 (d, J = 14.0 Hz, (d, J = 9.9 Hz, 1H), 6.49 (d, J = 9.9 Hz, 1H), 6.82 (dd, J = 1.6, 7.4 Hz, 1H), 7.13 (dt, J = 1.6,7.8 Hz, 1H), 7.17 (d, J = 7.9 Hz, 2H) , 7.89 (s, 1 H); LC-MS (ESI) m / z 318 ([M + 1] < ⁺ >). H _3-CF 3 -Ph ^{1 H NMR (500 MHz, CDCl} 3) δ 1.52 (s, 3H), 4.59 (d, J = 14.0 Hz, 1H), 4.72 (d, J = 14.0 Hz, 1H), 5.83 (d, J = 9.9 Hz J = 8.1 Hz, 1H), 6.87 (dt, J = 1.1, 7.4 Hz, 1H) J = 7.8 Hz, 1H), 7.97 (d, J = 1.6, 7.4 Hz, 1H), 7.13 ), 7.73 (d, J = 7.9 Hz, 1 H), 7.86 (s, 1 H); LC-MS (ESI) m / z 372 ([M + 1] < ⁺ >). 6-Me Ph LC-MS (ESI) m / z 318 ([M + 1] < ⁺ >). 6-Me 4-MeO-Ph LC-MS (ESI) m / z 348 ([M + 1] < ⁺ >). 6-Me 4-Me-Ph LC-MS (ESI) m / z 332 ([M + 1] < ⁺ >). 6-Me _3-CF 3 -Ph LC-MS (ESI) m / z 386 ([M + 1] < ⁺ >). 6-Me 4-CF ₃ -Ph LC-MS (ESI) m / z 386 ([M + 1] < ⁺ >). 6-Me 4-F-Ph LC-MS (ESI) m / z 336 ([M + 1] < ⁺ >). 6-Me 3-F-Ph LC-MS (ESI) m / z 336 ([M + 1] < ⁺ >). 6-Me 4-Cl-Ph LC-MS (ESI) m / z 352 ([M + 1] < ⁺ >). 6-Me 3-Cl-Ph LC-MS (ESI) m / z 352 ([M + 1] < ⁺ >). 6-Me 4-NO ₂ -Ph LC-MS (ESI) m / z 363 ([M + 1] < ⁺ >). 6-Me 3-NO ₂ -Ph LC-MS (ESI) m / z 363 ([M + 1] < ⁺ >). 6-Me _3-CF 3 O-Ph LC-MS (ESI) m / z 402 ([M + 1] < ⁺ >). 6-Me 4-CN-Ph LC-MS (ESI) m / z 343 ([M + 1] < ⁺ >). 6-Me 4-Cl-Bn LC-MS (ESI) m / z 366 ([M + 1] < ⁺ >). 7-Me 4-MeO-Ph LC-MS (ESI) m / z 348 ([M + 1] < ⁺ >). 7-Me 4-Me-Ph LC-MS (ESI) m / z 332 ([M + 1] < ⁺ >). 7-Me 4-F-Ph LC-MS (ESI) m / z 336 ([M + 1] < ⁺ >). 7-Me 4-Cl-Ph LC-MS (ESI) m / z 352 ([M + 1] < ⁺ >). 6-OMe _3-CF 3 -Ph LC-MS (ESI) m / z 402 ([M + 1] < ⁺ >). 6-OMe 4-Me-Ph LC-MS (ESI) m / z 348 ([M + 1] < ⁺ >). 6-F 4-Me-Ph LC-MS (ESI) m / z 336 ([M + 1] < ⁺ >). 6-Cl _3-CF 3 -Ph LC-MS (ESI) m / z 406 ([M + 1] < ⁺ >). 6-Cl 4-Me-Ph LC-MS (ESI) m / z 352 ([M + 1] < ⁺ >). 6-CF ₃ 4-Me-Ph LC-MS (ESI) m / z 386 ([M + 1] < ⁺ >).

1-3. mouse

Animal experiments were performed using C57BL / 6J mice (SLC, Japan) and strictly following laboratory animal management and use guidelines issued by the Animal Protection Committee of Kyungpook National University (Daegu, Korea). Mice were maintained under conditions free of specific pathogens.

1-4. Cell culture and hypoxic condition

HEK-293 human embryonic kidney epithelial cells were cultured in 10% fetal bovine serum (Hyclon, Logan, UT, USA) and 1% Were maintained in Dulbecco's modified Eagle's medium (DMEM, Hyclon, Logan, UT, USA) containing antibiotics (100 units / ml penicillin, 100 mg / ml streptomycin, Invitrogen, Carlsbad, CA, USA) A549 adenocarcinomic human alveolar basal epithelial cells were maintained in a Roswell Park Memorial Institute medium 1640 (RPMI 1640, Hyclon, Logan, UT, USA) containing 10% FBS and 1% antibiotics. Human umbilical vein endothelial cells (HUVECs, 4 to 10 passages) were treated with 20% FBS, 1% antibiotic, basic fibroblast growth factor (bFGF), 2 ng / (Ml 99, Hyclon, Logan, UT, USA) containing 1% fetal bovine serum albumin (Sigma, Sigma, St. Louis, MO) All cells were 5% CO ₂ and were kept in an incubator of 95% air conditions 37 ° C, of hypoxic cells are hypoxic chamber in 5% CO ₂ conditions with 1% O ₂ achieved a N ₂ and balance (Thermo Scientific , Waltham, MA, USA and Astec, Fukuoka, Japan).

1-5. MTT assay (MTT assay)

Cells per well at a density of 510 × 10 ³ cells, inoculated on a 96-well plate, and cultured for 24 hours. The medium was removed and the cells were treated with various concentrations of chemicals. After the cells were cultured for 24 hours, a solution of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, AMRESCO, Solon, OH, USA) Lt; / RTI > for 4 hours. The resulting formazan crystals were dissolved in DMSO (100 [mu] l / well) and the absorbance of the plate was read with a 540 nm microplate reader (Infinite M200 Pro, TECAN, Mannedorf, Switzerland). Three replicate wells were used for each analysis.

1-6. Dual luciferase assay

Inoculate HEK-293 cells in 96-well plates in a density of 5 × 10 ³ per well cell and allowed to attach for 24 hours. Next, the cells were transfected with pGL3-HRE-luciferase (VEGF) containing 5 HREs radiolabeled from vascular endothelial growth factor (VEGF) using Vivamagic Transfection Reagent (Vivagene, Sungnam, Korea) Plasmids and the pRL-SV40 plasmid encoding Renilla luciferase. After transfection, cells were treated with each chemical for 24 hours prior to reporter assay. Luciferase assays were performed according to the manufacturer's instructions using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA). Briefly, the Dual-Glo reagent was added in equal volumes to each well of the culture medium, and the wells were incubated for 10 minutes. Firefly luminescence was measured using a microplate reader (Infinite M200 Pro, TECAN, Mannedorf, Switzerland). Next, the Dual-Glo Stop & Glo reagent was added in equal volumes to each well of the culture medium, and the wells were incubated for 10 minutes. Renilla luminescence was measured using a microplate reader. Three replicate wells were used for each analysis and the results were normalized to the activity of Renilla luciferase.

1-7. Western blot analysis

Proteins extracted from cells by SDS-PAGE were separated using PRO-PREP (iNtRon Biotech, Korea) and transferred to a nitrocellulose membrane (Whatman, Maidstone, UK). Membranes were blocked with 5% skim milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 at room temperature for 30 minutes. After blocking, the membrane was incubated with primary antibody overnight at 4 ° C and then incubated with a secondary antibody (horseradish peroxidase-conjugated mouse- or rabbit-IgG) for 1 hour at room temperature. Bound antibodies were detected using an enhanced chemiluminescence (ECL) kit (BioRad, Hercules, Calif.) According to the manufacturer's instructions. The primary antibodies used in the assay are as follows:

HIF-1α (BD Biosciences, San Diego, Calif., USA), HIF-1β (Cell Signaling Technology, Danvers, MA, USA), β-actin (Santa Cruz Biotechnology, Santa Cruz, 1α (P564) (Cell Signaling Technology, Danvers, MA, USA) and PHD2 (Abcam, Cambridge, UK).

1-8. Immunofluorescence assay

Cells were incubated in 4-well plate coverslip per well at a density of 1 × 10 ⁵ cells and allowed to attach for 24 hours. The medium was removed; Cells were treated with various concentrations of chemicals and cells were cultured for 24 hours. The cells were then fixed with 4% paraformaldehyde solution and permeabilized with 1% Triton X-100. Immobilized cells were blocked with HIF-1α antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) At 4 ° C (50 ° C) with blocking with 0.5% bovine serum albumin / phosphate- buffered saline Lt; / RTI > Alexa Fluor 488-conjugated anti-mouse IgG was used as a secondary antibody and 4 ', 6-diamidino-2-phenylindole (DAPI) was used for nuclear labeling. The cover slips were mounted on slides and the cells were observed using a 400X magnification fluorescence microscope (Carl Zeiss, AG, Germany).

1-9. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted from the cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and analyzed using Moloney murine leukemia virus reverse transcriptase (MMLV) (Bioneer, Daejeon, Korea) CDNA was synthesized from RNA. Semiquantitative PCR conditions are 30 cycles of denaturation (94 ° C / 30 sec), annealing (50 ° C / 40 sec), extension (72 ° C / 40 sec) and final extension (72 ° C / 10 min) . Table 2 below summarizes the sequences of the primers used in this study. Amplification real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, CA, USA). Each PCR reaction contained 10-fold diluted cDNA and gene specific primers. The thermal cycle used is 40 cycles of 15 seconds denaturation at 95 ° C for 2 minutes at 50 ° C, 10 minutes at 95 ° C, and 1 minute annealing at 60 ° C. The mean cycle threshold (CT) value was calculated for gene expression with normalization to β-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. Each experiment was performed three times and the average was calculated.

name Primer sequence VEGF Forward 5-ACCATGAACTTTCTGCTC-3 Reverse 5-GGACGGCTTGAAGATATA-3 ALDOC Forward 5-TGAGCAGAAGAAGGAGTTGT-3 Reverse 5-GGTCTCATGGAAGAAAATGA-3 CA9 Forward 5GGAAGAAAACAGTGCCTATG-3 Reverse 5AGACCCCTCATATTGGAAGT-3 GLUT1 Forward 5TACCCTGGATGTCCTATCTG-3 Reverse 5CACACAGTTGCTCCACATAC-3 CXCR4 Forward 5GGCAGCAGGTAGCAAAGTGA-3 Reverse 5TGATGACAAAGAGGAGGTCG-3 β-actin Forward 5GACTACCTCATGAAGATC-3 Reverse 5GATCCACATCTGCTGGAA-3

1-10. Enzyme-linked immunosorbent assay (ELISA)

The amount of VEGF protein secreted by the cells in the culture medium was measured with a VEGF ELISA kit (# KHG0111, Invitrogen, Carlsbad, Calif., USA).

1-11. Bromodeoxyuridine (BrdU) cell proliferation assay

Human umbilical vein endothelial cells (HUVECs) were inoculated into 96-well plates at a density of 5 × 10 ³ cells per well and adhered for 24 hours. The medium was replaced with low serum medium (1% FBS in M199) for 16 hours and cells were treated with various concentrations of chemicals for 24 hours. After incubation, cell proliferation was measured using the Cell Proliferation ELISA, BrdU kit (Roche, Mannheim, Germany) according to the manufacturer's instructions.

1-12. ( In vitro ) Cell migration analysis

Endothelial cell migration was tested using a Transwell system (8 μm pore size and 6.5 mm diameter) in 24-well plates (Corning Costar, Lowell, MA, USA). The lower surface of the filter was coated with 10 [mu] l of collagen I (0.5 mg / ml). The chemicals were added to the lower chamber in the presence of VEGF (20 ng / ml) and HUVEC (5 x 10 ⁴ cells / well) was sprayed into the upper chamber of serum-free medium. And cultured at 37 ° C for 24 hours. Cells were fixed with methanol and stained with hematoxylin (Sigma, St Louis, MO, USA) and eosin (Sigma). Cells on the upper filter surface were removed and migration was measured by counting cells that migrated to the lower filter side using a 200X magnification microscope. Samples were analyzed twice for 3 times.

1-13. ( In vitro ) Tube Formation Analysis

The 96-well plate was coated with Matrigel (10 mg / ml, BD Biosciences, San Diego, Calif., USA) and polymerized at 37 ° C for 1 hour. HUVEC (3 x 10 ⁴ cells / well) was spiked with chemical substances at 37 ° C for 24 hours in the presence of VEGF (20 ng / ml) on the Matrigel surface. Changes in cell morphology were observed under a microscope and pictures were taken at 100X magnification.

1-14. Chick embryo chorioallantoic membrane (CAM) analysis

The fertilized eggs were kept in a humidifier (Eunjo incubator company, Korea) at 37 ° C for 3 days. 3-4 ml of chick skin and egg albumin were removed. On day 4.5, chemical-loaded Thermanox coverslips (NUNC, Rochester, NY, USA) were applied to the surface of chick embryo chorioallantoic membrane (CAM). Two days later, 10% fat emulsion (Intralipid) was injected under the CAM and observed under a microscope. Retinoic acid (RA) was used as a positive control.

1-15. Matrigel plug analysis

Matrigel (200 μl, BD Biosciences) containing DMSO, Compound 12 (100 nM) and gefitinib (5 μM) of the present invention was subcutaneously inoculated into C57BL / 6J mice. On the 10th day after inoculation, Matrigel plugs were removed and pictures were taken. To quantify the formation of functional blood vessels, hemoglobin content was measured using Drakin's reagent kit (Sigma).

1-16. ( In vivo ) Tumor allograft experiment and immunohistochemistry

LLC cells (1 x 10 ⁶ ) were subcutaneously injected into the side of 6-week-old C57BL / 6J mice (day 0, SLC, Japan). Tumor size and body weight were measured three times a week. Tumor tissues were removed 20 days after completion of vehicle or drug injection and fixed with 4% paraformaldehyde. After treatment with the frozen sample preparation protocol, the sample was inserted into the OCT compound and rapidly frozen by liquid nitrogen. The frozen block was cut into 10 μm sections and H & E stained for optical microscopy. Additional tissue sections for immunohistochemistry were blocked with 5% goat serum in PBST (0.03% Triton X-100 in PBS) and then incubated with primary anti-CD31 and anti-Ki67 And incubated at room temperature for 3 hours. Hypoxyprobe-1 ™ (60 mg / kg, Natural Pharmacia International) was intravenously injected 90 minutes before tissue fixation to detect hypoxic sites in tumors. Tumors were harvested, sectioned, and stained with FITC-conjugated anti-Hypoxyprobe antibody.

1-17. Statistical analysis

All data were expressed as mean ± standard deviation (SD) from at least three samples. Statistical comparisons were done using Student's t-test.

Example 2. Selection of HIF-1 alpha Inhibitor by HRE Reporter Assay

First, in order to confirm the cytotoxicity of the compounds of the present invention, MTT analysis was performed at various concentrations (0.5-5 μM) of 144 compounds (3-146). As a result, as shown in FIG. 2, Compound 22 was highly toxic at all doses and was excluded from further studies, and some compounds such as 51, 53, and 58 showed cytotoxicity at the highest dose (5 μM) , And this concentration was excluded from further investigation.

Next, a cell-based reporter assay was performed to identify the inhibitor of HIF-1α activity. HEK-293 cells were transfected with the pGL3-5xHRE-Luc plasmid carrying five hOR (hypoxia responsive elements) from the VEGF gene promoter and pRL-SV40 encoding the Renilla gene and then transfected into transfected cells Were treated with 1 μM of compound and exposed to the hypoxic condition (1% O ₂ ) for 24 hours before analysis. At this time, Echinomycin, which is well known as a HIF-1α inhibitor, was used as a positive control.

As a result, as shown in Table 3 below, the activity of the luciferase reporter was increased about 3 times in the hypoxic state as compared with the normal oxygen control condition, and 9 of the compounds (Compound # 12, (30-50% inhibition compared to the hypoxic control) of the reporter activity was observed in the compound of the present invention (# 45, # 54, # 94, # 101, # 103, # 105, # 127 and # 138) , Further experiments on these compounds were performed.

NO. HRE-Luc activity (%) at 1 μM chemical control 100 E (echinomycin) 78 # 12 50 # 111 - # 97 75 # 99 67 # 112 - # 113 - # 98 78 # 100 62 # 13 84

Example 3 Confirmation of the Effect of Compounds of the Present Invention on HIF-1α Expression

Next, by confirming the expression of hypoxic state-A549 cells through Western blot analysis, the inhibitory effect of the nine compounds on HIF-1 alpha protein accumulation was evaluated.

As a result, as shown in Fig. 3, when A549 cells were treated with 1 [mu] M of each compound for 16 hours in a hypoxic state, it was confirmed that all nine compounds significantly lowered HIF-1α expression. In particular, it was confirmed that Compound # 12 and # 45 exhibited the strongest inhibitory effect on HIF-1α accumulation. In the reporter analysis of Example 2, the inhibitory effect of Compound # 45 was smaller than that of Compound # 12 On the basis of this, further experiments were carried out using Compound # 12 below.

Compound # 12 was prepared by reacting Cu (I) of benzopyranyl 1,2,3-triazolo, 2- (azidomethyl) -2-methyl-6-nitro-2H- chromene and 1-ethynyl- (5'-hydroxymethyl-2'-furyl) -1-benzyl indazole], topotecan (S) -10 -1H-pyrano [3 ', 4': 6,7] indolizino [1,2-b] quinoline-3,14 (4H, 12H) -dione monohydrochloride, echinomycin, and manassantin, as shown in Fig. 4A.

Further, concentrations of less than 1 μM were tested to determine the minimum effective concentration required to achieve inhibition of HIF-1α. That is, HEK-293 cells and A549 cells were treated with 0.1-1000 nM concentration of Compound # 12 under hypoxic condition for 16 hours.

As a result, as shown in FIGS. 4B to 4E, Compound # 12 was found to downregulate HIF-1α protein expression by about 30% in HEK-293 cells and about 50% in A549 cells at a concentration of 1 nM 4B and 4C) and further calculated values of half maximal effective concentrations (EC ₅₀ ) using GraphPad Prism software showed that Compound # 12 required to inhibit HIF-1α protein levels in HEK-293 cells and A549 cells ₅₀ were 23.96 nM and 1.88 nM, respectively (Figs. 4D and 4E). This indicates that compound # 12 was more effective than YC-1 (2-50 μM), LW6 (2.5-3 μM), echinomycin (1.2 nM), bortezomib (0.6-30 nM), Apigenin (10-90 μM) 1 alpha protein inhibitory effect in comparison with other HIF-1 alpha inhibitors such as SYP-5 (10 [mu] M).

Example 4. Inhibitory effect of the compounds of the present invention on the stability of HIF-1 alpha protein by ubiquitination

To further confirm the effect of Compound # 12 on HIF-1 alpha protein expression inhibition, immunofluorescence analysis was performed on A549 cells. Cells were treated with Compound # 12 (1-100 nM) for 16 hours under hypoxic conditions, fixed with paraformaldehyde, and then treated to enable immunofluorescence of HIF-1α by HIF-1α antibody.

As a result, as shown in FIG. 5A, Compound # 12-untreated cells under hypoxic conditions were found to accumulate HIF-1α in the nucleus. In contrast, the cells treated with Compound # 12 under the same conditions showed HIF- 1α accumulation was dose-dependently inhibited, but did not affect the subcellular localization of HIF-1α.

These results suggest that Compound # 12 does not induce cytoplasmic migration of HIF-la, but rather decreases the stability of the protein.

The half-life of HIF-1 alpha was then measured in the presence or absence of compound # 12. A549 cells were treated with compound # 12 (10 nM) for 16 hours under hypoxic conditions and immediately treated with cycloheximide (CHX, protein synthesis inhibitor).

As a result, as shown in Figure 5B, the half-life (t _1/2) of HIF-1α protein is yieoteuna 12.4 minutes in the control cells, in 6.1 minutes in the presence of compound # 12, HIF-1α is twice that in the control cells , Indicating that Compound # 12 promotes HIF-1 alpha protein degradation.

Further, O ₂ - in the dependent degradation pathway PHD are O ₂ - because the dependent degradation (ODD, O ₂ -dependent degradation) hydroxide (hydroxylate) a proline residue in the HIF-1α in the domain, in addition hydroxide proline 402 and 564 ( OH) -HIF-1? And PHD2.

As a result, Compound # 12 increased the hydroxylation of HIF-1 alpha in a dose-dependent manner, and PHD2 levels were found to increase at the lowest dose (1 nM) of Compound # 12, as shown in Figure 5C. These results suggest that compound # 12 regulates the degradation of HIF-1α through increased hydroxylation of the HIF-1α ODD domain. Further, the relevance of PHD2 upon HIF-1α degradation by compound # 12 was further confirmed using two different siRNAs of PHD2, and as shown in FIG. 5D, the effect of HIF-1α The decrease in expression was recovered by knockdown of PHD2.

Next, the cells were treated with MG132 (protease inhibitor) in the presence or absence of compound # 12 and hypoxic conditions to confirm that compound # 12 alters the ubiquitination state of HIF-la. Cell lysates were immunoprecipitated (IP) with HIF-1α antibody and then immunoblotted with anti-ubiquitin antibody (IB).

As a result, as shown in FIG. 5E, the ubiquitination of HIF-1α under the hypoxic conditions and in the absence of Compound # 12 was decreased compared to the normal oxygen condition, while the presence of HIF-1α Of the ubiquitous ubiquitin was significantly increased compared with the normal oxygen control cells.

Taken together, this suggests that Compound # 12 inhibits HIF-1α protein stability by promoting HIF-1α proteolysis through an increase in proline hydroxylation and subsequent ubiquitination .

Example 5. Confirmation of HIF-1α Target Gene Expression and Angiogenesis Inhibitory Effect of Compounds of the Present Invention

First, HIF-1α was detected by using semiquantitative RT-PCR to regulate the transcription of target genes involved in angiogenesis, glucose metabolism and tumor progression, The effect of compound # 12 on gene expression was confirmed.

As a result, as shown in FIG. 6A, Compound # 12 was found to have a capacity-dependent effect on VEGF, aldolase C, CA9, carbonic anhydrase 9, glucose transporter GLUT1, glucose transporter 1) and chemokine receptor 4 (CXCR4, chemokine receptor type 4).

At this time, VEGF is a secretory protein and is one of the most powerful contributors to tumor angiogenesis and metastasis, further confirming the level of secreted VEGF protein in the culture medium through ELISA.

As a result, as shown in FIG. 6B, the level of VEGF secreted in the absence of Compound # 12 and cells under hypoxic conditions for 16 hours increased 1.7-fold compared to the normoxic condition, while Compound # 12 increased the secretion of hypoxia-induced VEGF Dose-dependently.

In addition, the inhibitory effect of Compound # 12 on VEGF secretion was also motivated to investigate whether it inhibits VEGF-induced angiogenesis. In the process of angiogenesis, vascular endothelial cells proliferate and migrate to the avascular (nonvascular) part and become mature into vascular tissue. To confirm this process, HUVECs were cultured and cell proliferation was measured using the BrdU uptake assay.

As a result, serum-deficient HUVECs were treated with Compound # 12 for 16 hours in the presence or absence of 20 ng / ml VEGF, as shown in Figure 6C. VEGF increased endothelial cell proliferation 1.8-fold, while compound # 12 significantly inhibited VEGF-induced proliferation and increased dose-dependently. In addition, as shown in Figures 6D and 6E, endothelial migration and tube formation ability induced by VEGF was also significantly inhibited by treating Compound # 12.

Further, in the living body (in vivo) a chick CAM assay was performed to confirm these in vitro (in vitro) results. As a result, Compound # 12 significantly inhibited CAM angiogenesis in a dose-dependent manner, similar to the positive control RA (retinoic acid), as shown in Figure 6F.

Example  6. Simultaneous treatment of a chemotherapeutic agent and a compound of the present invention, allograft ) Identification of inhibitory effect on tumor growth and angiogenesis

HIF-1α is synthesized by growth factor signaling pathways such as EGFR. Inhibition of HIF-1α in the degradation pathway as well as in the synthetic pathway seems to be able to produce a more effective anti-cancer / anti-angiogenic therapy.

To confirm this, allograft experiments were performed using Lewis lung carcinoma (LLC) cells. 1 × 10 ⁶ cells were subcutaneously sc injected and injected with Compound # 12 (5 mg / kg) and / or gefitinib (EGFR inhibitor, 50) via intraperitoneal (ip) mg / kg) treatment was performed every other day for 20 days. On day 20, mice were sacrificed and tumor tissues were fixed in formaldehyde for 1 hour to perform additional histological examination.

As a result, as shown in FIG. 7A, both the administration of only jenitib and the administration of Compound # 12 significantly inhibited tumor growth, and the combined administration of the combination of Compound # 12 and Compound # 12 more effectively inhibited tumor growth. At this time no noticeable change in mouse body weight over the entire treatment period was observed in all groups (Figure 8). In addition, as shown in Figure 7B, tumor weight was significantly reduced in the Compound # 12-treated group and to some extent similar or identical to the genitinib-treated group.

Furthermore, in order to confirm the effect of Compound # 12 on angiogenesis, angiogenesis was assayed using Matrigel plug assay and microvessel density (MSD) was measured by immunohistochemistry ).

As a result, as shown in FIG. 7C, the density in the vehicle-treated tumor mass was very high, but the density was significantly reduced in the compound # 12 or the genitinib-treated tumor. In particular, the co-treatment of Compound # 12 and genitinib further reduced MSD by synergistic action.

Finally, by confirming the expression levels of antibodies to hypoxic cell markers pimonidazole (PIMO, pimonidazole) and downstream target (CA9, carbonic anhydrase) of HIF-1, Respectively.

As a result, as shown in Figure 7D, both PIMO and CA9 were significantly decreased in Compound # 12 alone or in tumors treated with Jenitib nip alone, and PIMO / CA9 levels were significantly lower in Compound # 12 plus jenitib But decreased by synergy. In addition, as shown in Figure 7E, cells expressing the Ki-67 protein (a marker for cell proliferation) also significantly decreased in compound # 12 or zenithin-treated tumors.

As a result, the 1,2,3-triazalobenzopyran derivative of the present invention is a novel HIF-1 alpha inhibitor. It appears to suppress the stability and activity of HIF-1α in cancer cells and to inhibit tumor growth by synergy when used in combination with other chemotherapeutic agents.

Therefore, the 1,2,3-triazolo benzopyran derivative compound of the present invention has an effect of inhibiting angiogenesis in tumor growth and tumor, and can be used alone as an anticancer agent or can be used by combination therapy with other target chemotherapeutic agents And can be used in combination with other anticancer drugs or other anticancer drugs as anticancer drugs.

[Formulation Example]

Preparation Example 1. Preparation of tablets (pressurized system)

As the active ingredient, 5.0 mg of the compound represented by the formula (1) of the present invention was sieved, and 14.1 mg of lactose, 0.8 mg of crospovidone USNF and 0.1 mg of magnesium stearate were mixed and pressurized to prepare tablets.

Formulation Example 2. Preparation of tablets (wet granulation)

As an active ingredient, 5.0 mg of the compound represented by the formula (1) of the present invention was sieved, and 16.0 mg of lactose and 4.0 mg of starch were mixed. 0.3 mg of Polysorbate 80 was dissolved in pure water, and an appropriate amount of this solution was added, followed by atomization. After drying, the granules were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The granules were pressurized to make tablets.

Formulation Example 3. Preparation of powders and capsules

As an active ingredient, 5.0 mg of the compound represented by the formula (1) of the present invention was sieved and then mixed with 14.8 mg of lactose, 10.0 mg of polyvinylpyrrolidone and 0.2 mg of magnesium stearate. The mixture was filtered through a hard No. 5 gelatin capsules.

Formulation Example 4. Preparation of injection

Injection was prepared by adding 100 mg of the compound represented by the formula (1) of the present invention as an active ingredient, and further containing 180 mg of mannitol, 26 mg of Na ₂ HPO _{4 12} H ₂ O and 2,974 mg of distilled water.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (8)

A pharmaceutical composition for preventing or treating cancer, which comprises an effective amount of a 1,2,3-triazalobenzopyran derivative represented by the following formula (1) and a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

In Formula 1,
R ¹ is 1 to 3 substituents selected from the group consisting of H, Cl, F, NO ₂ , methyl, methoxy, trifluoromethoxy and trifluoromethyl,
Wherein R ² is a group selected from the group consisting of a normal propyl group, an isopropyl group, a cyclopropyl group, a cyclopropylmethyl group, a normal butyl group, a tert-butyl group, a cyclohexyl group, a cyclohexylmethyl group and - (CH ₂ ) nR ⁵ , An integer of 1 or 2,
Wherein R ⁵ is selected from the group consisting of hydrogen, Cl, F, CN, NO ₂ , OH, methyl, ethyl, isopropyl, normal butyl, tert- butyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, difluoromethyl And an amino group substituted with one to two of an amino group or a methyl group, an ethyl group, a normal propyl group, an isopropyl group, a normal butyl group, a cyclopentyl group, a cyclopentylmethyl group, and a cyclohexylmethyl group. The aromatic group is a furanyl group, a thiophenyl group, a phenyl group, a pyridinyl group, a naphthyl group, a quinolyl group, or an isoquinolyl group, wherein the one to three substituents selected are a substituted or unsubstituted aromatic group.
delete delete The method according to claim 1,
Wherein R ¹ is NO _2, or a methyl group,
Wherein R ² is a phenyl group, a 2-chlorophenyl group, a 3-chlorophenyl group, a 4-chlorophenyl group, a 2- fluorophenyl group, - (trifluoromethyl) phenyl group, 4- (trifluoromethyl) phenyl group, 3-methoxyphenyl group, 4-methoxyphenyl group, or isoquinolinyl group.

The method according to claim 1,
In Formula 1,
Methyl-6-nitro-2H-chromen-2-yl) methyl) -1H-1,2,3-triazole,
Methyl-6-nitro-2H-chromen-2-yl) methyl) -1H-1,2,3-triazole,
1 - ((2-methyl-6-nitro-2H-chromen-2- yl) methyl) -4- (p-
1 - ((2-methyl-6-nitro-2H-chromen-2- yl) methyl) -4- (3- (trifluoromethyl)
(2-methyl-6-nitro-2H-chromen-2-yl) methyl) -1H-1,2,3-triazole,
Methyl-6-nitro-2H-chromen-2-yl) methyl) -1H-1,2,3-triazole,
(2-methyl-6-nitro-2H-chromen-2-yl) methyl) -1 H- 1,2,3-triazole,
Methyl-1H-1,2,3-triazol-4-yl) isoquinoline, or
1 - ((2,6-dimethyl-2H-chromen-2-yl) methyl) -4-phenyl-1H-1,2,3-triazole.
The method according to claim 1,
Lt; RTI ID = 0.0 > HIF-la < / RTI > inhibitory activity.
1. An anticancer adjuvant comprising, as an active ingredient, a 1,2,3-triazalobenzopyran derivative represented by the following formula (1) and a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

In Formula 1,
R ¹ is 1 to 3 substituents selected from the group consisting of H, Cl, F, NO ₂ , methyl, methoxy, trifluoromethoxy and trifluoromethyl,
Wherein R ² is a group selected from the group consisting of a normal propyl group, an isopropyl group, a cyclopropyl group, a cyclopropylmethyl group, a normal butyl group, a tert-butyl group, a cyclohexyl group, a cyclohexylmethyl group and - (CH ₂ ) nR ⁵ , An integer of 1 or 2,
Wherein R ⁵ is selected from the group consisting of hydrogen, Cl, F, CN, NO ₂ , OH, methyl, ethyl, isopropyl, normal butyl, tert- butyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, difluoromethyl And an amino group substituted with one to two of an amino group or a methyl group, an ethyl group, a normal propyl group, an isopropyl group, a normal butyl group, a cyclopentyl group, a cyclopentylmethyl group, and a cyclohexylmethyl group. The aromatic group is a furanyl group, a thiophenyl group, a phenyl group, a pyridinyl group, a naphthyl group, a quinolyl group, or an isoquinolyl group, wherein the one to three substituents selected are a substituted or unsubstituted aromatic group.
8. The method of claim 7,
Wherein the anticancer adjuvant is in the form of a complex.

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