GB2579480A

GB2579480A - Composition for preventing or treating cancer containing Triazolopyridine-based derivative as active ingredient

Info

Publication number: GB2579480A
Application number: GB2001444.5A
Authority: GB
Inventors: Yeon Ann Ji; Young Song Jie; Nam Ky-Youb; Ni Ryu Hwa; Jung In-Sung; Gu Hwang Sang
Original assignee: Korea Inst Radiological & Medical Sciences; Korea Institute of Radiological and Medical Sciences
Current assignee: Korea Inst Radiological & Medical Sciences; Korea Institute of Radiological and Medical Sciences
Priority date: 2017-08-16
Filing date: 2018-02-28
Publication date: 2020-06-24
Anticipated expiration: 2038-02-28
Also published as: DE112018004197T5; GB2579480B; KR101924801B1; GB202001444D0; WO2019035522A1

Abstract

The present invention relates to a composition for preventing or treating cancer containing a triazolopyridine-based derivative or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the triazolopyridine-based derivative inhibits tankyrase enzyme activity to suppress the expression of β-catenin, which is a tumor factor, thereby increasing cancer cell death by apoptosis. Due to exhibiting excellent survival rate reduction when used in combination with anti-cancer drugs or irradiation, the triazolopyridine-based derivative can be usefully used as a pharmaceutical composition for preventing and treating cancer, a composition for increasing the effect of radiation therapy, or a health functional food for preventing or improving cancer.

Description

[DESCRIPTION]

[Invention Title] COMPOSITION FOR PREVENTING OR TREATING CANCER CONTAINING TRIAZOLOPYRIDINE-BASED DERIVATIVE AS ACTIVE 5 INGREDIENT

[Technical Field]

The present invention relates to a pharmaceutical composition for preventing or treating cancer, a composition for enhancing anticancer effect or a health food for preventing or improving cancer, comprising a triazolopyridine-based derivative as an active ingredient.

[Background Art]

Although many diseases are treated and prevented by the development of modern medicine, cancer is still one of the diseases difficult to treat. Cancer is currently the number one cause of death and is continuously increasing.

Cancer has been treated by chemotherapy, surgical therapy and radiation therapy. Among them, chemotherapy using an anticancer agent is most commonly used for the treatment of cancer. Today, about 60 different anticancer drugs are used in clinical practice, and as the knowledge regarding the cancer development and the characteristics of cancer cells is well known, new anticancer drugs continue to be developed. However, most of the anticancer drugs currently used in the clinical practice are often accompanied by side effects such as nausea, vomiting, ulcers in the oral cavity and small intestine, diarrhea, hair loss, and bone marrow suppression in which the production of blood effective ingredients is reduced. For example, mitomycin-C is known to have a side effect such as kidney failure and Adriamycin is known to have side effect such as myelosuppression. In particular, cisplatin, the most useful drug among the anticancer drugs developed so far, is widely used for the treatment of testicular cancer, ovarian cancer, lung cancer, head and neck cancer, bladder cancer, stomach cancer and cervical cancer, but since the occurrence of side effects including hematopoietic toxicity such as anemia, gastrointestinal toxicity such as vomiting, nausea, kidney toxicity such as kidney renal tubular damage, hearing loss, electrolyte abnormalities in the body, shock, peripheral nerve abnormalities, has become a big problem, the development of new anticancer drugs with excellent safety is urgently required.

In addition, as the number of cancer patients treating by radiation therapy increases every year, the importance of radiation therapy in cancer treatment is also increasing.

However, it has been pointed out that a problem of lowering the efficiency to of radiation therapy occurred due to the resistance of cancer cells against radiation and damage of normal tissues during high-dose radiation treatments. Therefore, researches on radiation therapy sensitizers have been attempted to improve the efficiency of radiation therapy.

Radiation therapy sensitizers reported to date are mainly anticancer 20 agents, for example Taxol and cisplatin have been reported to be used as radiation therapy sensitizers in solid cancers such as breast cancer, uterine cancer, lung cancer, gastric cancer and colorectal cancer.

In addition, as the radiation therapy effect enhancers, which does not have properties as an anticancer agent and is used only for radiation therapy, there is a tirapazamine, but it is effective only in tumor cells of hypoxia, and insufficient drug delivery into tumor tissue due to the hypoxia-specific tumor internal pressure has been known to be ineffective in clinical radiation therapy, and these radiation therapy sensitizers have high side effects and thus have a problem of their limited use.

Although there is a difference in sensitivity to radiation treatment for cancer cells, if the sensitivity can be improved, the same therapeutic effect can be obtained by irradiating the patient with a smaller dose of radiation and therefore, there is a need for the development of anticancer drugs that can treat cancer and increase the sensitivity of cancer cells to radiation.

[Disclosure]

[Technical Problem] The object of the present invention is to solve the side effects of the conventional anticancer drug or radiation therapy and to provide a composition 10 for enhancing the anticancer therapeutic effect that can improve the anticancer therapeutic effect.

[Technical Solution] The present invention provides a triazolopyridine-based derivative represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof: [Formula 1] in Chemical Formula 1, each of R1 and R2 can be the same or different and selected from hydrogen, CN, NO2, CF3, halogen and acetyl, and X is any one selected from nitrogen and carbon.

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising a triazolopyridine-based derivative represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] in Chemical Formula 1, each of R1 and R2 can be the same or different and selected from hydrogen, CN, NO2, CF3, halogen and acetyl, and X is any one selected from is nitrogen and carbon.

The present invention provides a composition for enhancing anticancer effect comprising a triazolopyridine-based derivative represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health food for preventing or improving cancer comprising a triazolopyridine-based derivative represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Advantageous Effects] According to the present invention, the triazolopyridine-based derivative represented by Chemical Formula 1 inhibits the activity of the tankyrase enzyme to suppress the expression of the tumor factor p-catenin, thereby increasing cancer cell death by apoptosis. When it is applied in combination with anticancer agents or radiation, excellent cell viability decreases and thus the triazolopyridine-to based derivative can be usefully used as a pharmaceutical composition for preventing and treating cancer, a composition for enhancing the effect of the anticancer agent or radiation treatment, or for a health functional food for preventing and improving cancer.

[Description of Drawings]

FIG. 1 shows the results of confirming the concentration-dependent inhibition of TNKS activity by treating the triazolopyridine derivative Compound 1 or Compound 2 at various concentrations in vitro.

FIG. 2 shows the results of confirming the inhibition of I3-catenin protein expression and the increase of tumor suppressor AXIN2 after the treatment of Compound 1 or Compound 2, which is a triazolopyridine derivative, to DLD-1 colorectal cancer cells.

FIG. 3 shows the results of confirming the inhibition of the expression of VEGF2 and Birc/Survivin, which are sub-genes of p-catenin after the treatment of Compound 1 or Compound 2, which is a triazolopyridine derivative, to DLD-1 colorectal cancer cells.

FIG. 4 shows the results of confirming the cell viability after the combination treatment of Compound 1 or Compound 2, which is a triazolopyridine derivative, respectively when irradiated to DLD-1 colorectal cancer cells in which 10 APC (adenomatous polyposis coli) gene is mutated.

FIG. 5 shows the results of confirming the decrease in cell viability after the combination treatment of the anticancer agent 5-FU and triazolopyridine derivative Compound 1 or Compound 2, respectively to two colorectal cancer cell lines in which APC gene was mutated.

to FIG. 6 shows the results of confirming the reduction of CYR61 and CTGF, which are sub-genes of tumor factor YAP/TAZ, after the treatment with Compound 1 or Compound 2, which is triazolopyridine derivative, to DLD-1 colorectal cancer cell lines, respectively.

FIG. 7 shows the results of confirming the decrease in cell viability after the treatment of Compound 1 or Compound 2, which is a triazolopyridine derivative at various concentrations, to SKMEL28 melanoma cell line and SKMEL28 cell line with the resistance of Vemurafenib, a BRAF Inhibitory anticancer agent.

[Best Mode] The present invention may provide a triazolopyridine-based derivative represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof: [Formula 1] in Chemical Formula 1, each of R1 and R2 can be the same or different and selected from hydrogen, CN, NO2, CF3, halogen and acetyl, and X can be any one selected from nitrogen and carbon.

Each of the R1 and R2 of the compound can be the same or different and 10 selected from the group consisting of CN, NO2, CF3 and halogen, and X can be carbon.

The triazolopyridine-based derivative may be selected from the group consisting of N-([1,2,4,]triazolo[4,3-a]pyridin-3-y1)-1-(2-cyanophenyl) piperidine-4-carboxamide, N-([1,2,4]triazolo[4,3-a]pyridin-3-y1)-1 -(2-nitrophenyppiperidine-4- carboxam ide, N-([1,2,4]triazolo[4,3-a]pyridin-3-y1)-1 -phenylpiperidine-4- carboxam ide, N-([1,2,4]triazolo[4,3-a]pyridin-3-y1)-1 -(4-nitrophenyppiperidine-4- carboxam ide, N-([1,2,4]triazolo[4,3-a]pyridin-3-y1)-1 -(2-nitro-4- (trifluoromethyl)phenyl)piperidine-4-carboxamide, N-al,2,4]triazolo[4,3-a] pyridin-3-yI)-1-(4-(trifluoromethyl)phenyl)piperidine-4-carboxam ide, N- ([1,2,4]triazolo[4,3-a]pyridin-3-yI)-1 -(4-fluoro-2-nitrophenyl)piperidine-4- carboxam ide, N-a1,2,4]triazolo[4,3-a]pyridin-3-y1)-1-(4-acetylphenyl)piperidine- 4-carboxam ide, N-([1,2,4]triazolo[4,3-a]pyridin-3-y1)-1-(4-chloro-2-nitrophenyl) piperidine-4-carboxam ide, N-([1,2,4]triazolo[4,3-a]pyridin-3-yI)-1-(4-cyanophenyl) piperidine-4-carboxamide, N-([1,2,4]triazolo[4,3-a]pyridin-3-yI)-1-(5-nitropyridin-2-yl) piperidine-4-carboxamide and N-([1,2,4]triazolo[4,3-a]pyridin3-y1)-1-(2-cyano 4 nitrophenyl)piperidine-4-carboxamide.

The present invention may provide a pharmaceutical composition for preventing or treating cancer comprising a triazolopyridine-based derivative represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] in Chemical Formula 1, each of R1 and R2 can be the same or different and selected from hydrogen, CN, NO2, CF3, halogen and acetyl, and X can be to any one selected from nitrogen and carbon.

More specifically, each of RI and R2 can be the same or different and selected from the group consisting of CN, NO2, CF3 and halogen, and X can be carbon.

The triazolopyridine-based derivative may be selected from the group consisting of N-([1,2,4,]triazolo[4,3-a]pyridin-3-yI)-1-(2-cyanophenyl) piperidine-4-carboxamide, N-al,2,4]triazolo[4, 3-a]pyridin-3-yI)-1 -(2-nitrophenyl)piperidine-4- carboxam ide, N-al,2,4]triazolo[4,3-a]pyridin-3-yI)-1-phenylpiperidine-4- carboxamide, N-al,2,4]triazolo[4, 3-a]pyridin-3-yI)-1 -(4-nitrophenyl)piperidine-4- carboxam ide, N-([1,2,4]triazolo[4,3-a]pyridin-3-y1)-1 -(2-nitro-4- (trifluoromethyl)phenyl)piperidine-4-carboxamide, N-al,2,4]triazolo[4,3-a] pyridin-3-yly1-(4-(trifluoromethypphenyl)piperidine-4-carboxam ide, N- ([1,2,4]triazolo[4,3-a]pyridin-3-yI)-1-(4-fluoro-2-nitrophenyl) piperidine-4-carboxamide, N-al,2,4]triazolo[4,3-a]pyridin-3-yI)-1 -(4-acetylphenyl)piperidine- 4-carboxam ide, N-([1,2,4]triazolo[4,3-a]pyridin-3-y1)-1-(4-chloro-2-nitrophenyl) piperidine-4-carboxam ide, N-a1,2,4]triazolo[4,3-a]pyridin-3-yI)-1-(4-cyanophenyl) piperidine-4-carboxamide, N-([1,2,4]triazolo[4,3-a]pyridin-3-y1)-1-(5-nitropyridin-2-yl) piperidine-4-carboxamide and N-([1,2,4]triazolo[4,3-a]pyridin3-y1)-1-(2-cyano 4 nitrophenyl)piperidine-4-carboxamide.

to The cancer may be any one selected from the group consisting of lung cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, breast cancer, stomach cancer, liver cancer, colon cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, brain cancer, laryngeal cancer, prostate cancer, bladder cancer, esophageal cancer, thyroid cancer, kidney cancer, blood cancer and rectal cancer.

More specifically, the triazolopyridine-based derivatives can be effectively used for the treatment of cancer cells in which p-catenin, a tumor factor is overexpressed, by inhibiting the activity of Tankyrase (TNKS) enzymes.

The TNKS has several binding protein partners including a double-stranded telomer repeat binding protein TRF1 (telomeric repeat binding factor 1); NuMA (NuclearMitotic associated protein), which is an essential protein in mitotic spindle assembly; IRAP (am inopeptidase), an intrinsic membrane protein involved in glucose uptake corresponding to insulin; and Mcl-1 (myeloid leukemia cell differentiation protein), which is an apoptosis-promoting protein, and exhibits biological function through their various interactions.

According to one embodiment of the present invention, it is confirmed that triazolopyridine-based compounds 1 to 12 as shown in Table 1 inhibits the activity of the TNKS-1 enzyme, but does not inhibit the activity of PARP-1, and thus the activity of the triazolopyridine-based derivatives is specific for the TNKS-1 enzyme.

From the above results of the treatment of DLD-1 colorectal cancer cells in which the APC gene was mutated, with the triazolopyridine-based compounds 1 and 2 identified as TNKS enzyme activity inhibitors, it was confirmed that as shown in FIG. 2, the expression of 13-catenin protein was decreased by Compound 1 and Compound 2, whereas the expression of AXIN2 protein was increased. Accordingly, the triazolopyridine derivatives inhibited the activity of Tankyrase (INKS) enzymes, which resulted in the expression of S-catenin and it can be provided as an anticancer agent to increase the cancer cell death by apoptosis.

In one embodiment of the present invention, the pharmaceutical composition for preventing or treating cancer comprising the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient may be used in any one of the formulations selected from the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or solutions according to a conventional method.

Another embodiment of the invention may further comprise at least one additive selected from the group consisting of carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, lubricants, slip modifiers, flavors, antioxidants, buffers, bacteristats, diluents, dispersants, surfactants, binders and lubricants, which is suitably used for the preparation of the pharmaceutical composition.

Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid formulations may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in addition to the composition. Furthermore, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid formulations for oral administration include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used as simple diluents.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories and the like. Examples of the non-aqueous solution and the suspension include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurinum, glycerogelatin and the like can be used.

According to one embodiment of the invention, the pharmaceutical composition may be administered intravenously, intraarterially, intraperitoneally, intramuscularly, intrasternally, transdermally, nasally, inhaled, topically, rectally, orally, intraocularlly or intradermally to the subject in a conventional manner. The preferred dosage of the compound represented by Chemical Formula 1 may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, the daily dosage may be, but is not limited to, 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or divided into several times, and the scope of the invention is not limited thereby.

In the present invention, the 'subject' may be a mammal including a human, but it is not limited thereto.

In addition, the present invention may provide a composition for enhancing anticancer effect comprising a triazolopyridine-based derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The composition for enhancing anticancer effect may be treated in combination with an anticancer agent or radiation.

The anticancer agent may be any one or more selected from the group consisting of cisplatin, 5-fluorouracil, paclitaxel, doxorubicin, daunorubicin, vinblastine, vincristine, actinomycin D, teniposide, etoposide, cyclophosphamide, epirubicin, adriamycin, daunomycin and mitomycin-C.

The composition may include 1 to 99 parts by weight of the anticancer agent and 1 to 99 parts by weight of the triazolopyridine-based derivative 10 represented by Chemical Formula 1 based on 100 parts by weight of the total composition.

The composition for enhancing anticancer effect may enhance the anticancer effect against any one cancer disease selected from the group consisting of lung cancer, acute myeloid leukemia, chronic myeloid leukemia, to acute lymphocytic leukemia, chronic lymphocytic leukemia, breast cancer, stomach cancer, liver cancer, colon cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, brain cancer, laryngeal cancer, prostate cancer, bladder cancer, esophageal cancer, thyroid cancer, kidney cancer, blood cancer and rectal cancer.

According to another embodiment of the present invention, the cultured DLD-1 colorectal cancer cells was treated with Compound 1 and Compound 2 at a concentration of 20 pM and then irradiated with gamma radiation 3 Gy and cultured for 3 days to determine cell viability and it was confirmed that the combination treatment of triazolopyridine-based compound 1 or compound 2 and radiation reduced the survival rate of cancer cells as shown in FIG. 4. In addition, DLD-1 cells and SVV480 cells were treated with the Compound 1 or Compound 2 at a concentration of 20 pM in combination with 5-FU (10 pM), a colorectal cancer treatment anticancer agent, for 48 hours and MTT assay was performed to confirm cell viability and as shown in FIG. 5, it was confirmed that the cell proliferation inhibitory effect of the experimental group administered with 5-FU in combination of Compound 1 and Compound 2 was increased by 2 times and 1.5 times, respectively, compared to the control treated with 5-FU as an anticancer agent.

From the above results that the triazolopyridine-based compound is confirmed to increase the anticancer therapeutic effect when used in combination with an anticancer agent or radiation, the triazolopyridine-based compound can be used as an anticancer adjuvant.

In addition, the present invention can provide a health food for preventing or improving cancer comprising a triazolopyridine-based derivative represented by the Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The cancer may be any one cancer disease selected from the group consisting of lung cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, breast cancer, stomach cancer, liver cancer, colon cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, brain cancer, laryngeal cancer, prostate cancer, bladder cancer, esophageal cancer, thyroid cancer, kidney cancer, blood cancer and rectal cancer.

The health food may be used with other foods or food additives in addition to the triazolopyridine derivatives represented by the Chemical Formula 1 or pharmaceutically acceptable salts thereof, and may be appropriately used according to conventional methods. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prophylactic, health or therapeutic treatment.

The effective dose of the compound contained in the health food may be used in accordance with the effective dose of the therapeutic agent, but may be less than the above range in the case of prolonged intake for the purpose of health and hygiene or health control. It is evident that the component can be used in an amount above the range because there is no problem in terms of safety. There is no particular limitation on the kind of the health food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, to beverages, tea, drinks, alcoholic drinks, vitamin complexes, etc. are included.

Furthermore, the present invention comprises a triazolopyridine-based derivative represented by the Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, and the triazolopyridine-based derivative or a pharmaceutically acceptable salt thereof can be provided as 20 reagent composition for inhibiting the activity of Tankyrase enzyme, characterized in that by inhibiting the activity of the Tankyrase (TN KS) enzyme of the cell in vitro.

In addition, the present invention may provide a method of inhibiting the activity of tankyrase enzyme in vitro comprising the step of treating cancer cell with triazolopyridine-based derivative represented by the Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

Hereinafter, the present invention will be described in detail with reference to the following examples. The examples are only for describing the present invention in more detail and it is obvious to those skilled in the art that that the scope of the present invention is not limited by these examples embodiments in accordance with the gist of the present invention.

<Synthesis Example 1> Synthesis of triazolopyridine derivatives Triazolopyridine-based derivative compounds 1 to 12 of Chemical Formulas 1 to 12 were synthesized as follows: 1. N-([1,2,41triazolo[4,3-a[pyridin-3-y1)-1-(2-cyanophenyl) piperidine-4-carboxamide (Compound 1) [Reaction Scheme 1] As shown in the above Reaction Scheme 1, 21 mg of Compound 1 (3) was obtained with a yield of about 25% by amide coupling reaction of a starting material (1) and carboxylic acid (2) using EDCI and HOBt in the presence of trimethylamine and solvent of dichloromethane.

1H NMR (DMSO-d6, 400 MHz): 5 10.96 (1H, s), 7.96 (1H, d, J = 8.0Hz), 7.73 (1H, m), 7.61 (1H, t, J = 8.0Hz), 7.4 (1H, ddd, J = 12.0, 4.0 and 0.8Hz), 7.22 (1H, d, J = 8.0Hz), 7.11 (1H, t, J = 8.0Hz), 6.99 (1H, t, J= 6.0Hz), 3.58 (2H, d, J=12.0Hz), 2.91 (2H, t, J = 12.0Hz), 2.50 (1H, m) 2.10 (2H, d, J = 12.0Hz), 1.91 (2H, t, J = 6.0Hz) N-([1,2,41triazolo[4,3-a]pyridin-3-yI)-1-(2-nitrophenyl) piperidine-4-carboxamide (Compound 2) [Reaction Scheme 2] As shown in the above Reaction Scheme 2, 25 mg of Compound 2 (3) was obtained with a yield of about 29% by amide coupling reaction of a starting material (1) and carboxylic acid (2) using EDCI and HOBt in the presence of trimethylamine and solvent of dichloromethane.

1H NMR (DMSO-d6, 400 MHz): 5 10.95 (1H, s), 7.95 (1H, d, J = 8.0Hz), 7.81 (1H, dd, J = 8.0 and 4.0Hz), 7.75 (1H, d, J = 8.0Hz), 7.59 (1H, td, J = 8.0, and 4.0Hz), 7.38 (2H, m), 7.13 (1H, t, J = 8.0Hz), 7.00 (1H, t, J = 4.0Hz), 3.27 (2H, d, J = 12.0Hz), 2.91 (2H, t, J = 12.0Hz), 2.71 (1H, m), 2.03 (2H, d, J = 12.0Hz), 1.85 (2H, m) 3. N-([1,2,41triazolo[4,3-alpyridin-3-y1)-1-phenylpiperidine-4-carboxamide (Compound 3) [Reaction Scheme 3] As shown in the above Reaction Scheme 3, 24 mg of Compound 3 (3) was obtained with a yield of about 28% by amide coupling reaction of a starting material (1) and carboxylic acid (2) using EDCI and HOBt in the presence of trimethylamine and solvent of dichloromethane.

1H NMR (CDCIs-d, 400 MHz): 5 11.65 (1H, s), 7.91 (1H, d, J = 4.0Hz), 7.67 (1 H, d, J = 8.0Hz), 7.27 (3H, m), 6.97 (2H, d, J = 8.0Hz), 6.9 (2H, m), 3.76 (2H, dt, J=11.0 and 3.1Hz), 2.9 (3H, td, J=10.0 and 3.2Hz), 2.19 (2H, dd, J=14.0 and 2.0Hz), 2.07(2H, ddd, J=24.0, 12.0 and 4.0Hz) 4. N-([1,2,41triazolo[4,3-alpvridin-3-yI)-1-(4-nitrophenyl) piperidine-4-carboxamide (Compound 4) [Reaction Scheme 4] As shown in the above Reaction Scheme 4, 22 mg of Compound 4 (3) was obtained with a yield of about 25% by amide coupling reaction of a starting material (1) and carboxylic acid (2) using EDCI and HOBt in the presence of trimethylamine and solvent of dichloromethane.

1H NMR (DMSO-d6, 400 MHz): 5 10.96 (1H, s), 8.06 (2H, d, J = 8.0Hz), 7.93 (1H, d, J = 4.0Hz), 7.74 (1H, d, J = 8.0Hz), 7.39 (1H, ddd, J = 8.4, 6.6 and 1.8Hz), 7.07 (2H, d, J = 8.0Hz), 6.97 (1H, t, J = 6.0Hz), 4.13 (2H, d, J = 12.0Hz), 3.14 (2H, t, J = 12.0Hz), 2.89 (1 H, m), 2.05 (2H, d, J = 12.0Hz), 1.75 (2H, m) N-([1,2,41triazolo[4,3-alpyridin-3-y1)-1-(2-nitro-4- (trifluoromethyl)phenvflpiperidine-4-carboxamide (Compound 5) [Reaction Scheme 5] As shown in the above Reaction Scheme 5, 28 mg of Compound 5 (3) was obtained with a yield of about 27% by amide coupling reaction of a starting material (1) and carboxylic acid (2) using EDCI and HOBt in the presence of trimethylamine and solvent of dichloromethane.

1H NMR (DMSO-d6, 400 MHz): 5 10.97 (1H, s), 8.16 (1H, d, J = 4.0Hz), 7.87 (1 H, d, J = 4.0Hz), 7.85 (1 H, d, J = 2.0Hz), 7.75 (1 H, dt, J = 10.6 and 0.4Hz), 7.49 (1H, d, J = 8.0Hz), 7.40 (1H, ddd, J = 10.2, 6.0 and 1.4Hz), 6.99 (1H, td, J = 7.5, 7.5 and 3.2Hz), 3.43 (2H, d, J = 16.0Hz), 3.07 (2H, t, J = 12.0Hz), 2.79 (1H, m), 2.05 (2H, d, J = 12.0Hz), 1.85 (2H, t. J = 12.0Hz) 6. N-(11,2,41triazolo[4,3-alpvridin-3-y1)-1-(4- (trifluoromethyl)phenyl)piperidine-4-carboxamide (Compound 6) [Reaction Scheme 6] As shown in the above Reaction Scheme 6, 24 mg of Compound 6 (3) was obtained with a yield of about 27% by amide coupling reaction of a starting material (1) and carboxylic acid (2) using EDCI and HOBt in the presence of trimethylamine and solvent of dichloromethane.

1H NMR (DMSO-d6, 400 MHz): 5 10.94 (1H, s), 7.93 (1H, d, J = 8.0Hz), 7.74 (1H, d, J = 12.0Hz), 7.50 (2H, d, J = 12.0Hz), 7.39 (1H, t, J = 8.0Hz), 7.10 (2H, d, J = 8.0Hz), 6.97 (1 H, t, J = 6.0Hz), 3.97 (2H, d, J = 16.0Hz), 2.95 (2H, t, J = 12.0Hz), 2.81 (1H, m), 2.02 (2H, d, J = 12.0Hz), 1.76 (2H, m) 7. N-([1,2,4]triazolo[4,3-a]pyridin-3-yI)-1-(4-fluoro-2-nitrophenyl) piperidine-4-carboxamide (Compound 7) [Reaction Scheme 7] Er, TEA, DC% As shown in the above Reaction Scheme 7, 28 mg of Compound 7 (3) was obtained with a yield of about 27% by amide coupling reaction of a starting material (1) and carboxylic acid (2) using EDCI and HOBt in the presence of trimethylamine and solvent of dichloromethane.

1H NMR (DMSO-d6, 400 MHz): 6 10.94 (1H, s), 7.94 (1H, d, J = 4.0Hz), 7.83 (1 H, dd, J = 8.0 and 4.0Hz), 7.75 (1 H, d, J = 8.0Hz), 7.52 (2H, m), 7.40 (1 H, dd, J = 12.0 and 6.0Hz), 6.99 (1H, t, J = 8.0Hz), 3.20 (2H, d, J = 12.0Hz), 2.88 (2H, t, J = 12.0Hz), 2.69 (1H, m), 2.03 (2H, d, J = 8.0Hz), 1.82 (2H, m) 8. N-([1,2,41triazolo[4,3-alpyridin-3-y1)-1-(4-acetylphenyl) piperidine-4-carboxamide (Compound 8) [Reaction Scheme 8] As shown in the above Reaction Scheme 8, 24 mg of Compound 8 (3) was obtained with a yield of about 28% by amide coupling reaction of a starting material (1) and carboxylic acid (2) using EDCI and HOBt in the presence of trimethylamine and solvent of dichloromethane.

1H NMR (DMSO-d6, 400 MHz): 6 10.95 (1H, s), 7.93 (1H, d, J = 8.0Hz), 7.81 (2H, d, J = 8.0Hz), 7.74 (1 H, d, J = 12.0Hz), 7.39 (1 H, ddd, J = 8.4, 6.4 and 1.6Hz), 6.99 (3H, m), 4.04 (2H, d, J = 12.0Hz), 3.00 (2H, t, J = 12.0Hz), 2.84 (1 H, m), 2.45 (3H, s), 2.02 (2H, d, J = 12.0Hz), 1.74 (2H, m) 9. N-([1,2,41triazolo[4,3-alpyridin-3-yI)-1-(4-chloro-2-nitrophenyl) piperidine-4-carboxamide (Compound 9) [Reaction Scheme 9]

N

I

As shown in the above Reaction Scheme 9, 28 mg of Compound 9 (3) was obtained with a yield of about 26% by amide coupling reaction of a starting o material (1) and carboxylic acid (2) using EDCI and HOBt in the presence of trimethylamine and solvent of dichloromethane.

1H NMR (DMSO-d6, 400 MHz): 6 10.95 (1H, s), 7.95 (2H, m), 7.75 (1H, d, J = 8.0Hz), 7.65 (1H, dd, J = 12.0 and 8.0Hz), 7.39 (2H, m), 6.99 (1H, t, J = 8.0Hz), 3.26 (2H, d, J = 12.0Hz), 2.92 (2H, t, J = 12.0Hz), 2.72 (1H, m), 2.03 (2H, d, J = 8.0Hz), 1.83 (2H, m) 10. N-([1,2,4]triazolo[4,3-a]pyridin-3-yI)-1-(4-cyanophenyl) piperidine-4-carboxamide (Compound 10) [Reaction Scheme 10] As shown in the above Reaction Scheme 10, 20 mg of Compound 10 (3) was obtained with a yield of about 23% by amide coupling reaction of starting material (1) and carboxylic acid (2) using EDCI and HOBt in the presence of trimethylamine and solvent of dichloromethane.

1H NMR (DMSO-d6, 400MHz): 6 10,94(1H, s), 7.93(1H, d, J=8.OHz), 7.74(1H, d, J=8.0Hz), 7.58(1H, d, J=8.0Hz), 7.39(1H, dd, J=8.0 and 8.0Hz), 7.06(2H, d, J=8.0Hz), 6.97(1 H, m), 4.03(2H, d, J=12.0Hz), 3.01(2H, m), 2.83(1 H, m), 2.02(2H, d, J=16Hz), 1.73(2H, m) 1 1. N-(11,2,41triazolo[4,3-alpyridin-3-y1)-1-(5-nitropyridin-2-yl) piperidine4-carboxamide (Compound 1 1) [Reaction Scheme 1 1] As shown in the above Reaction Scheme 11, 20 mg of Compound 11 (5) was obtained with a yield of about 26% by amide coupling reaction of starting material (1) and carboxylic acid (2) using EDCI and HOBt in the presence of trimethylamine and solvent of dichloromethane.

1H NMR (DMSO-d6, 400MHz): 5 10,97(1H, s), 8.98(1H, d, J=2.0Hz), 8.23(1H, dd, J=2.0 and 4.0Hz), 7.94(1H, d, J=8.0Hz), 7.74(1H, d, J=8.0Hz), 7.39(1H, dd, J=2.0 and 8.0Hz), 6.99(2H, m), 4.59(2H, d, J=12.0Hz), 3.22(2H, m), 2.94(1H, m), 2.08(2H, d, J=12.0Hz), 1.70(2H, m) 12. N-([1,2,4]triazolo[4,3-a]pyriclin-3-y1)-1-(2-cyano-4-nitrophenyl) piperidine-4-carboxam ide (Compound 12) [Reaction Scheme 12] As shown in the above Reaction Scheme 12, 20 mg of Compound 11 (3) was obtained with a yield of about 23% by amide coupling reaction of starting material (1) and carboxylic acid (2) using EDCI and HOBt in the presence of trimethylamine and solvent of dichloromethane.

1H NMR (DMSO-d6, 400 MHz): 5 11.00 (1H, s), 8.54 (1H, d, J = 4.0Hz), 8.3 (1H, dd, J = 10.0 and 2.0Hz), 7.96 (1H, d, J=4.0Hz), 7.75 (1H, d, J = 8.0Hz), 7.39 (1H, ddd, J=6.6, 4.0 and 1.4Hz), 7.30 (1H, d, J = 12.0Hz), 6.98 (1H, t, J= 6.0Hz), 4.05 (2H, d, J=12.0Hz), 3.25 (2H, t, J = 12.0Hz), 2.87 (1H, m), 2.13 (2H, d, J = 12.0Hz), 1.885 (2H, d, J = 12.0Hz) <Example 1> Confirmation of inhibitory effect of Tankyrase (TNKS) enzyme activity That whether the triazolopyridine-based derivative compounds 1 to 12 can inhibit the activity of Tankyrase (TNKS) enzyme was confirmed by using Tankyrase-1 colorimetric activity assay kit (Cat# 4500-192-K, Trevigen, RnD systems, USA), in accordance with manufacturer's instructions and {2-(4-(trifluoromethyl-pheny1)-3,5,7,8-tetradihydro-thiopyrano[4,3-d] pyrimidin-4-one (XAV939) as a positive control.

In addition, that whether Compound 1 to Compound 12 can inhibit PARP (poly-ADP ribose polymerase) enzyme activity was confirmed by measuring by PARP-1 colorimetric assay kit (Cat # 4677-096-K, Trevigen, RnD systmes) according to the manufacturer's instructions and Olaparib 1pM as a positive control.

As a result, TNKS and PARP inhibitory activity of Compounds 1 to 12 was confirmed as shown in Table 1.

[Table 1]

TNKS-1 inhibitory PARP inhibitory activity activity (Compared to control 0%) Concentration 10 10 (PM) Triazolopyridine 95.2±0.5 0 compound 1 Triazolopyridine 94.3±1.3 0 compound 2 Triazolopyridine 58.7±2.4 0 compound 3 Triazolopyridine 30.8±4.7 1.5±0.8 compound 4 Triazolopyridine 70.4±2.5 0 compound 5 Triazolopyridine 51.7±1.7 1.2±0.4 compound 6 Triazolopyridine 75.4±1.2 0 compound 7 Triazolopyridine 65.8±2.3 0.8±0.2 compound 8 Triazolopyridine 72.9±4.7 1.1±0.5 compound 9 Triazolopyridine 59.6±5.2 0.8±0.4 compound 10 Triazolopyridine 47.4±3.4 0.6±0.1 compound 11 Triazolopyridine 94.8±0.8 0 compound 12 The control group XAV939 (10pM) inhibited INKS-1 enzyme activity by about 96%, Olaparib (10pM) inhibited PARP enzyme activity by 98%. On the other hand, triazolopyridine-based compounds 1 to 12 showed the inhibition of TNKS-1 enzyme activity, as shown in the Table 1, whereas the PARP-1 activity was not inhibited.

From the above results, it was confirmed that the activity of the triazolopyridine-based derivatives were specific for the TNKS-1 enzyme.

In addition, 50% inhibitory concentration of TNKS-1 activity was confirmed in Table 2 with respect to Compound 1 and Compound 2, which were found to have the best activity.

[Table 2]

TNKS-1 activity 50% inhibitory concentration TNKS-1 (pM) (ICso) Triazolopyridine 0.760 compound 1 Triazolopyridine 0 863 compound 2 <Example 2> Confirmation of 13-catenin expression inhibition effect 1. Cell preparation to DLD-1, SW480 and SKMEL28 cell lines were purchased from ATCC (American Type Culture Collection). DLD-1 and SW480 cells were incubated in DMEM (Dulbecco's modified Eagle's medium) medium and SKMEL28 cells were incubated in MEM medium, to which 10% fetal bovine serum (FBS) and 100 fag/ml streptomycin and 100 unit/ml penicillin were added, respectively, under 5% CO2 and 37 °C.

The vemurafenib-resistant SKMEL28 cell line was treated with 5 pM of vemurafenib for 1 month continuously, followed by the treatment of vemurafenib using a cell line inhibiting 40% of cell proliferation.

2. Confirmation of 13-catenin expression inhibition It was confirmed by Western blot whether the triazolopyridine-based compound 1 and compound 2 can inhibit 13-catenin protein expression through INKS inhibition and maintain AXIN2 protein.

First, 10 pM of Compound 1 or Compound 2 was treated to ALD gene-mutated DLD-1 colorectal cancer cells, respectively and then cultured for 24 hours under 5% CO2 and 37 °C.

Thereafter, cells treated with Compound 1 or Compound 2 were obtained and cytoplasmic fractions were obtained by methods commonly used in the art. The fractions were separated by electrophoresis using 10% gradient to SDS-PAGE and transferred to nitrocellulose membranes (BioRad, Hercules, CA, USA). The membrane was blocked with 5% skim milk, and then reacted with anti13-catenin (Cell signaling, USA) and anti-AXIN2 antibody (Cell signaling, USA) as a primary antibody followed by the incubation of horseradish peroxidaseconjugated anti-mouse IgG antibody (Santa Cruz Biotechnology, USA) and 20 visualized using ECL system (GE, USA).

As a result, as shown in FIG. 2, the expression of B-catenin protein was decreased by the Compounds 1 and 2, which are TNKS inhibitors, while the AXIN2 protein was increased.

3. Confirmation of 6-catenin subgene expression inhibition DLD-1 colorectal cancer cells mutated with APC gene were aliquoted into 6 cm plates in a number of 5 x 105, and treated with 10 pM of Compound 1 and Compound 2, respectively, and cultured for 8 hours under conditions of 5% CO2 and 37 °C.

After obtaining cells treated with Compound 1 or Compound 2, total RNA was extracted using RNeasy kit (Qiagen, USA), and cDNA was synthesized using M-MLV reverse transcriptase (Enzynomics, Korea). Oligomers capable of binding 6-catenin sub-genes VEGF2 and Birc5/Survivin were in synthesized in Cosmogentec (Korea), labeled them with a SYBR-Green/fluorescein qPCR master mix (Thermo Scientific, USA), and real-time PCR was measured in a Lightcycler 96 Real-time PCR system (RocheDiagnositics, Germany) according to the manufacturer's guidelines.

As a result, it was confirmed that VEGF2 and Birc5/Survivin expression is to significantly reduced in Compound 1 or Compound 2 treated cells as shown in FIG. 3.

<Example 3> Confirmation of anticancer effect by combined treatment of radiation Cultured DLD-1 colorectal cancer cells were dispensed in a 96-well cell culture plate in the number of 3 x 103, treated with Compound 1 and Compound 2 at a concentration of 20 pM and irradiated with gamma radiation 3 Gy and Incubated at CO2 and 37 °C conditions for 3 days.

Thereafter, 0.5 mg/mL of MTT (3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide) solution was added to each well and further incubated for 3 hours at 37 °C. After incubation, the supernatant was removed and the formed formazan crystals were dissolved in DMSO and measured at 590 nm using a spectrometer (Labsystems, USA). The control group was treated with DMSO, and cell viability was expressed as a relative growth rate relative to 100%. As a result, as shown in FIG. 4, it was confirmed that the combined treatment of the triazolopyridine-based compound 1 or compound 2 at concentration of 20 pM with the radiation decreased the viability, respectively. From the above results, it was confirmed that the triazolopyridine-based 10 compound 1 and compound 2 can be used as a radiotherapy adjuvant that can enhance the effect in radiation therapy.

<Example 4> Confirmation of anticancer effect by combined treatment of anticancer drug to DLD-1 cells and SW480 cells cultured in the same manner as in Example 3 were treated with 20 pM of Compound 1 or Compound 2 in combination with 5-FU (10 pM), a colorectal cancer anticancer agent, for 48 hours, and then subjected to MTT assay for the analysis of the cell viability.

As a result, as shown in FIG. 5, the cell proliferation inhibitory effect of the 20 experimental group in which 5-FU and Compound 1 or Compound 2 were co-administered was increased by 2 times and 1.5 times higher than that of the control in which 5-FU, an anticancer agent was treated alone, respectively. From the above results, it was confirmed that the triazolopyridine-based compound can be used as an anticancer agent adjuvant capable of enhancing anticancer drug effects.

<Example 5> Confirmation of anticancer effect in resistant cancer cells by inhibition of tumor factor YAP activity 1. Confirmation of YAP/TAZ signaling inhibitory effect As TNKS was found to activate tumor factor YAP through Angiomotin, in order to confirm the YAP/TAZ signaling inhibitory effect by triazolopyridine compound which is the TNKS inhibitor, a real-time PCR was performed in the same manner as in Example 2 and the expression levels of the YAP/TAZ subgenes CYR61 and CTGF were confirmed.

As a result, as shown in FIG. 6, it was confirmed that the expression of CYR61 and CTGF was significantly reduced in the cells treated with Compound 1 or Compound 2.

From the above results, it was confirmed that the triazolopyridine to compound can target the YAP/TAZ tumor factor.

2. Confirmation of anticancer effect by combined treatment with anticancer agent in resistant cancer cells The B-RAF inhibitory anticancer drug, vemurafenib (PLX4032) has been reported to be involved in YAP/TAZ signaling (EMBO J. 2016;35(5):462), and thus SKMEL28 melanoma cells and vemurafenib-resistant SKMEL28 cells cultured in previous experiments were treated with Compound 1 at concentrations of 10 pM, 20 pM and 30 pM or 10 pM and 20 pM vemurafenib, respectively, and the cell viability was analyzed by MTT assay.

As a result, as shown in FIG. 7, the cell proliferation rate was reduced in in the SKMEL28 cell line and the vemurafenib-resistant SKMEL28 cells depending on the concentration of Compound 1. In particular, it was confirmed that the cell proliferation was further reduced in vemurafenib-resistant SKMEL28 cells than SKMEL28 cells and accordingly the triazolopyridine compound of the present invention can be used as a very effective anticancer agent.

While the present invention has been particularly described with reference to specific embodiments thereof, it is apparent that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby to those skilled in the art. That is, the practical scope of the present invention is defined by the appended claims and their equivalents.