KR20210138429A - Compound for inhibiting PARP and medical use thereof - Google Patents

Abstract

The present invention provides a novel compound having a mechanism of decomposing poly (ADP-ribose) polymerase (PARP) or a pharmaceutically acceptable salt thereof. The present invention also provides a pharmaceutical composition for treating cancer comprising a compound or a pharmaceutically acceptable salt thereof according to the present invention as an active component. The present invention also provides a pharmaceutical use of the compound or the pharmaceutically acceptable salt thereof according to the present invention for the treatment of cancer.

Description

Compound for inhibiting PARP and medical use thereof

The present invention relates to a novel compound or a pharmaceutically acceptable salt thereof that exhibits an effect of decomposing PARP (Poly (ADP-ribose) polymerase). The present invention also relates to a pharmaceutical composition for treating cancer comprising the compound according to the present invention or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention also relates to the pharmaceutical use of the compound according to the present invention or a pharmaceutically acceptable salt thereof for the treatment of cancer. The present invention also relates to a process for the preparation of the compounds of the present invention.

PARP (Poly (ADP-ribose) polymerase) is involved in repairing damaged DNA in vivo. In the case of cancer cells, since the number of such damage and repair processes is hundreds of times higher, the number of times PARP is involved in this DNA repair process increases.

The DNA repair process is largely divided into DNA single-strand breaks (SSB) and double-strand breaks (DSB), and PARP is known as a key protein in the SSB process. In the case of PARP, above all, its activity varies depending on the presence or absence of BRCA, which plays the biggest role in the DSB process. This concept is called Synthetic lethality (J. Med. Chem. 2015, 58 3302), and shows the activity of PARP inhibitors against carcinomas accompanied by gene deficiency/mutations such as BRCAness.

Representative disease groups caused by such BRCA mutations include breast cancer, ovarian cancer, and prostate cancer. With this target, Olaparib was first approved as a treatment for breast and ovarian cancer in 2014, and the indication is currently being expanded to prostate cancer. Rucaparib, a second inhibitor, was approved for ovarian cancer patients with BRCA mutations in 2016. Since then, compounds such as Niraparib, Veliparib, Talazoparib, and Pamiparib are under development, but they are not yet effective. am.

US Patent Publication No. 7,151,102

Therefore, the problem to be solved by the present invention is to provide a compound for inhibiting PARP (Poly (ADP-ribose) polymerase) having advantages in various aspects as a pharmaceutical ingredient, and a pharmaceutical use thereof.

Another problem to be solved by the present invention is to provide a novel compound in which the effect of Olaparib is further improved based on Olaparib, and pharmaceutical uses thereof.

In order to solve the above problems, the present invention provides a compound of Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

Preferably, in the present invention, the hydrophobic tagging is any one of the following moieties.

The present inventors synthesized proteolysis inducers using the UPS system by linking various moieties to olaparib, and verified the degradation effect using the MDA-MB-231 cell line. However, among the compounds linking Olaparib and E3 ligase recruiter with a linker, a compound showing proteolytic identification could not be found.

However, when the cyclopropylcarbonyl moiety of olaparib is removed and a proteolytic pathway through hydrophobic tagging as in the present invention is introduced based on naked piperidine amine, it was confirmed that PARP is also capable of protein degradation according to the molecular dynamics pathway in vivo. The invention was completed.

The compound according to the present invention not only induces the degradation of PARP, but also induces intracellular ER stress to induce unfolded protein response (UPR), autophagy, and apoptosis, but the present invention is not limited to these theoretical mechanisms .

In the compound according to the present invention, the linker may be absent (direct linkage) or **-(R ₁ )-(C _1-10 alkyl)-(R ₂ )-*, wherein R ₁ and R ₂ are not present independently of each other or are NH, O, CO, SO ₂ O, or C(O)O. In another aspect of the present invention, the linker is a direct linkage or **-(C _1-10 alkyl)NH-*.

In the present specification, when described as “C _1-4 ” or “C1 to C6”, this means that the number of carbon atoms is 1 to 6 carbon atoms. For example, C _1-6 alkyl means alkyl having 1 to 6 carbon atoms.

In the present invention, the compound represented by Formula 1 may be used in the form of a salt derived from an inorganic acid or an organic acid, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid , glutaric acid, fumaric acid, malic acid, mandelic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid It may be used in the form of a salt derived from at least one acid selected from the group consisting of phonic acid, toluenesulfonic acid, and the like.

As used herein, the term "compound of the present invention" is meant to include compounds of Formula 1 as well as clathrates, hydrates, solvates, or polymorphs thereof. In addition, the term “compound of the present invention” is meant to include a pharmaceutically acceptable salt of the compound of the present invention unless a pharmaceutically acceptable salt thereof is mentioned.

In one embodiment, a compound of the invention is a stereoisomerically pure compound (e.g., substantially free of other stereoisomers (e.g., at least 85% ee, at least 90% ee, at least 95% ee, 97% ee or more, or 99% ee or more)). That is, when the compound of Formula 1 or a salt thereof according to the present invention is a tautomeric isomer and/or stereoisomer (eg, geometrical isomer and conformational isomers), their separated isomers and mixtures each are also included within the scope of the compounds of the present invention. When the compound of the present invention or a salt thereof has an asymmetric carbon in its structure, their optically active compounds and racemic mixtures are also included in the scope of the compound of the present invention.

As used herein, the term "polymorph" refers to a solid crystalline form of a compound of the present invention or a complex thereof. Different polymorphs of the same compound exhibit different physical, chemical and/or spectral properties. Differences in physical properties include, but are not limited to, stability (eg, thermal or light stability), compressibility and density (important for formulation and product manufacturing), and dissolution rate (which may affect bioavailability). doesn't happen Differences in stability may be due to changes in chemical reactivity (e.g., differential oxidation, such as a faster discoloration when composed of one polymorph than when composed of another polymorph) or mechanical properties (e.g., kinetically Tablet fragments stored as the preferred polymorph are converted to a thermodynamically more stable polymorph) or both (tablets of one polymorph are more susceptible to degradation at high humidity). Other physical properties of polymorphs can affect their processing. For example, one polymorph may be more likely to form a solvate than another polymorph, for example due to its shape or particle size distribution, or it may be more difficult to filter or wash.

As used herein, the term “solvate” refers to a compound of the present invention, or a pharmaceutically acceptable salt thereof, comprising a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents are volatile, non-toxic, and can be administered in trace amounts to humans.

As used herein, the term "hydrate" refers to a compound of the present invention or a pharmaceutically acceptable salt thereof comprising a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. .

As used herein, the term "clathrate" refers to a compound of the present invention in the form of a crystal lattice containing spaces (eg, channels) that confine guest molecules (eg, solvent or water). or salts thereof.

The term "purified" as used herein, when isolated, means that the isolate is at least 90% pure, in one embodiment at least 95% pure, in another embodiment at least 99% pure, and In another embodiment, it means at least 99.9% pure.

"Treatment" as used herein includes eradication, removal, modification, or control of primary, focal or metastatic cancerous tissue; Minimizing or delaying the expansion of cancer.

As used herein, an “effective amount” refers to destroying, modifying, controlling or eliminating primary, local or metastatic cancer cells or cancer tissue; slowing or minimizing the spread of cancer; or an amount of a compound of the invention sufficient to provide a therapeutic benefit in the treatment or management of cancer, neoplastic disease, or tumor. "Effective amount" also refers to an amount of a compound of the invention sufficient to cause cancer or neoplastic cell death. "Effective amount" also refers to an amount sufficient to inhibit or reduce mutagenic PARP, either in vitro or in vivo.

Non-limiting examples of preferred compounds according to the present invention include the following compounds and pharmaceutically acceptable salts thereof.

4-(3-(4-(2-(adamantan-1-yl)acetyl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1( 2H )-one,

4- (3- (4- (2- ( 9 H -fluoren-9-yl) acetyl) piperazine-1-carbonyl) -4-fluorobenzyl) phthalazin-1 (2 H) -one,

( S )-4-(4-fluoro-3-(4-(2-(4-isobutylphenyl)propanoyl)piperazine-1-carbonyl)benzyl)phthalazin-1( 2H )-one,

4-(3-(4-(2,2-diphenylacetyl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1( 2H )-one,

4-(3-(4-(2,2-dicyclohexylacetyl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1( 2H )-one,

2-(adamantan-1-yl) -N -(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl )ethyl)acetamide,

2-(adamantan-1-yl) -N -(4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl )butyl)acetamide,

2-(adamantan-1-yl) -N -(6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl )hexyl)acetamide,

2- (9 H -fluoren-9- yl) - N - (2- (4- (2-fluoro-5 - ((4-oxo-3,4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin- 1-yl)ethyl)acetamide,

2- (9 H -fluoren-9- yl) - N - (4- (4- (2-fluoro-5 - ((4-oxo-3,4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin- 1-yl)butyl)acetamide,

2- (9 H -fluoren-9- yl) - N - (6- (4- (2-fluoro-5 - ((4-oxo-3,4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin- 1-yl)hexyl)acetamide,

(S) - N - (2- (4- (2-fluoro-5 - ((4-oxo-3,4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) ethyl) -2- (4-isobutylphenyl)propanamide,

(S)- N -(4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)butyl)-2- (4-isobutylphenyl)propanamide,

(S) - N - (6- (4- (2-fluoro-5 - ((4-oxo-3,4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) hexyl) -2- (4-isobutylphenyl)propanamide,

N -(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)ethyl)-2,2-diphenylacetamide,

N -(4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)butyl)-2,2-diphenylacetamide,

N -(6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)hexyl)-2,2-diphenylacetamide;

2,2-dicyclohexyl- N -(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)ethyl)acetamide ,

2,2-dicyclohexyl- N -(4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)butyl)acetamide , or

2,2-dicyclohexyl- N -(6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)hexyl)acetamide .

Preferably, the compound according to the present invention is 4-(3-(4-(2-(9H-fluoren-9-yl)acetyl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1 represented by compound 3a. (2H)-one or a pharmaceutically acceptable salt thereof. Compound 3a according to the present invention exhibited a significantly superior effect than other compounds.

In another aspect, the present invention provides a (pharmaceutical) composition comprising a therapeutically effective amount of a compound of Formula 1 or a pharmaceutically acceptable salt thereof according to the present invention, and a pharmaceutically acceptable carrier.

In another embodiment, the present invention provides a therapeutically effective amount of the compound of Formula 1 according to the present invention, or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and an anticancer agent other than the compound of the present invention, cell proliferation An active pharmaceutical ingredient selected from the group consisting of a cytostatic drug, an angiogenesis inhibitor, a kinase inhibitor, a cytokine blocker, and an inhibitor of a cell adhesion molecule. A (pharmaceutical) composition comprising a therapeutically effective amount of a pharmaceutical ingredient) is provided.

In another aspect, the present invention provides a method for treating a disease or condition comprising administering to an individual in need thereof a therapeutically effective amount of a compound of Formula 1 or a pharmaceutically acceptable salt thereof. Methods are provided, wherein the disease or condition is a disease or condition that can be ameliorated by PARP inhibition. In another embodiment, the disease or condition is a disease or condition that can be ameliorated by PARP degradation. In another embodiment, the disease or condition is cancer. In another embodiment, the cancer is breast cancer, ovarian cancer, or prostate cancer. In another embodiment, the cancer is breast cancer. In another embodiment, the cancer is breast cancer that does not express three receptors: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2/neu).

In one aspect, the treatment is a preventative treatment. In another embodiment, the treatment is a palliative treatment. In another embodiment, the treatment is a restorative treatment.

That is, the present invention provides a pharmaceutical use, characterized in that the compound of Formula 1 or a pharmaceutically acceptable salt thereof according to the present invention is used as an active ingredient. In one aspect, the pharmaceutical use of the present invention is the use for the treatment or prophylaxis of a disease or condition described herein.

When the novel compounds according to the present invention are used as anticancer agents, the dosage is as follows. The compounds of the present invention may be administered by any suitable route, in the form of a pharmaceutical composition suitable for such route, and in an effective dosage for the intended treatment. An effective dosage is generally from about 0.001 to about 100 mg/kg body weight/day, preferably from about 0.01 to about 30 mg/kg/day, in single or divided doses. In another embodiment, the dosage of a compound of the present invention is from 10 to 2000 mg/day. In another embodiment, the dosage of a compound of the present invention is from 20 to 1000 mg/day. Dosage levels below the lower limit of this range may be suitable depending on the age, species, and disease or condition being treated. In other cases, still larger doses can be used without deleterious side effects. The larger dose may be divided into several smaller doses for administration throughout the day. Methods for determining the appropriate dosage are well known in the art.

For the treatment of the above-described disease or condition, the compound described herein or a pharmaceutically acceptable salt thereof may be administered as follows.

Oral administration

The compound of the present invention may be administered orally, and the oral cavity is a concept including swallowing. By oral administration, the compounds of the present invention may enter the gastrointestinal tract or may be absorbed directly into the bloodstream from the mouth, for example, by buccal or sublingual administration.

Suitable compositions for oral administration may be in solid, liquid, gel, or powder form, and may have dosage forms such as tablets, lozenges, capsules, granules, and powders. .

Compositions for oral administration may optionally be enteric coated and may exhibit delayed or sustained release through the enteric coating. That is, the composition for oral administration according to the present invention may be a formulation having an immediate or modified release pattern.

Liquid formulations may include solutions, syrups, and suspensions, and such liquid compositions may be contained in soft or hard capsules. Such formulations may contain a pharmaceutically acceptable carrier, for example, water, ethanol, polyethylene glycol, cellulose, or oil. The formulation may also contain one or more emulsifying and/or suspending agents.

In tablet formulations, the amount of drug as the active ingredient may be present in an amount of from about 0.05% to about 95% by weight relative to the total weight of the tablet, more typically from about 2% to about 50% by weight of the dosage form. Tablets may also contain from about 0.5% to about 35% by weight of a disintegrant, more typically from about 2% to about 25% by weight of the dosage form. Examples of the disintegrant include, but are not limited to, lactose, starch, sodium starch glycolate, crospovidone, croscarmellose sodium, maltodextrin, or mixtures thereof.

Suitable glidants included for the preparation of tablets may be present in an amount of from about 0.1% to about 5% by weight and include talc, silicon dioxide, stearic acid, calcium, zinc or magnesium stearate, sodium stearyl fumarate, and the like. It may be used as a lubricant, but the present invention is not limited to the types of these additives.

Gelatin, polyethylene glycol, sugar, gum, starch, polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, etc. may be used as a binder for manufacturing tablets. In addition, suitable diluents for manufacturing tablets include mannitol, xylitol, lactose, dextrose, sucrose, sorbitol, starch, microcrystalline cellulose, etc., but the present invention is not limited to the types of these additives. .

Optionally, the solubilizing agent that may be included in the tablet may be used in an amount of about 0.1% to about 3% by weight relative to the total weight of the tablet, for example, polysorbate, sodium lauryl sulfate, sodium dodecyl sulfate, propylene carbonate, Diethylene glycol monoethyl ether, dimethylisosorbide, polyoxyethylene glycolated natural or hydrogenated castor oil, HCOR ^™ (Nikkol), oleyl ester, Gelucire ^™ , caprylic/caprylic acid mono/ Diglyceride, sorbitan fatty acid ester, Solutol HS ^TM, etc. may be used in the pharmaceutical composition according to the present invention, but the present invention is not limited to the specific type of the solubilizing agent.

Parenteral Administration

The compounds of the present invention may be administered directly into the bloodstream, muscle, or intestine. Suitable methods for parenteral administration include intravenous, intra-muscular, subcutaneous intraarterial, intraperitoneal, intrathecal, intracranial injection, and the like. include Suitable devices for parenteral administration include injectors (including needle and needleless syringes) and infusion methods.

Compositions for parenteral administration may be formulations with an immediate or modified release pattern, and the modified release pattern may be a delayed or sustained release pattern.

Most parenteral formulations are liquid compositions, and the liquid composition is an aqueous solution containing the active ingredient according to the present invention, a salt, a buffer, an isotonic agent, and the like.

Parenteral formulations may also be prepared in dried form (eg, lyophilized) or as sterile non-aqueous solutions. These formulations may be used with a suitable vehicle such as sterile water. Solubility-enhancing agents may also be used in the preparation of parenteral solutions.

Topical Administration

The compounds of the present invention may be administered topically dermally or transdermally. Formulations for topical administration include lotions, solutions, creams, gels, hydrogels, ointments, foams, implants, patches, and the like. Pharmaceutically acceptable carriers for topical dosage forms may include water, alcohol, mineral oil, glycerin, polyethylene glycol, and the like. Topical administration may also be performed by electroporation, iontophoresis, phonophoresis, and the like.

Compositions for topical administration may be formulations with an immediate or modified release pattern, and the modified release pattern may be a delayed or sustained release pattern.

The novel compound of the present invention or a pharmaceutically acceptable salt thereof exhibits excellent effects in treating various cancers by decomposing PARP. In addition, the pharmaceutical composition for the treatment of cancer of the present invention comprising the compound or a pharmaceutically acceptable salt thereof provides excellent activity for the treatment of various carcinomas, particularly breast cancer, ovarian cancer, or prostate cancer.

The following drawings attached to the present specification, together with the contents of the above-described invention, serve to further understand the technical idea of the present invention, and the present invention is not limited to the matters described in the following drawings.
Fig. 1 shows the results of immunoblot analysis of MDA-MB-231 cells treated with the compound according to the present invention.
Fig. 2 is an immunoblot analysis result of HCC1937 cells treated with Compound 3a, which is an embodiment according to the present invention, and Olaparib, a comparative compound.
Fig. 3 is an experimental result showing that compound 3a according to the present invention causes apoptosis of TNBC cells through PARP degradation. (A) shows the results of immunoblot analysis on whole cell lysates of MDA-MB-231 cells after compound 3a treatment for the indicated times. (B and C) Quantitative RT-PCR analysis of proapoptotic (B) or antiapoptotic (C) target genes, values are mean ± SD; n=3. **p<0.01, ***p<0.001, ****p<0.001 (one-way ANOVA). (D) CCK-8 assay results after treatment with serially diluted compound 3a or olparib for 96 hours. Values are mean ± SD; n=3.
Fig. 4 shows experimental results showing that compound 3a treatment induces ER stress-related autophagy and apoptosis. (A) Shows signaling pathways in which unfolded or misfolded proteins induce apoptosis. (B and C) Results of immunoblot analysis performed on whole cell lysates of MDA-MB-231 cells after treatment with compound 3a (B and C) or olaparib (c) at the indicated times. (D) Quantitative RT-PCR analysis of ER stress and autophagy-related genes, performed using MDA-MB-231 cells treated with vehicle or compound 3a for the indicated time. Values are mean ± SD; n=3. *p<0.05, **p<0.01, ***p<0.001, ****p<0.001 (one-way ANOVA). (E) Representative TEM image of MDA-MB-231 cells. The scale bar is 2 μm. Red arrows indicate autophagomes, yellow arrows indicate apoptosis. Treatment with 5 μM compound 3a was performed at the times indicated. (F) Schematic model of PARP degradation by compound 3a.

Hereinafter, examples and the like will be described in detail to help the understanding of the present invention. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art to which the present invention pertains.

In the following examples, TLC was performed on precoated silica gel F254 plates (Merck, art. 5715). 5715). Column chromatography was performed on a silica cartridge using MPLC (Puchem Flash Column or Biotage ZIP KP-Sil). 1H spectra were recorded with a Bruker Avance 500 using TMS or residual solvent peak as internal standard. LC/MS analysis was performed on a Waters Acquity UPLC system with electrospray ionization in either positive or negative ion mode. The purity of all final compounds was 95% as determined by NMR and HPLC. Compound 1 was purchased from U-Chem and used.

Preparation of compounds 2a to 6a

[Scheme 1]

Reaction conditions: (a) HATU, DIPEA, DMF, rt, 12 hours

Acid (0.1 mmol, 1 equiv) was added to DMF (0.5 mL) followed by compound 1 (0.1 mmol, 1 equiv), HATU (0.11 mmol, 1.1 equiv) and DIPEA (0.4 mmol, 4 equiv). It was stirred at room temperature for 12 hours. After the reaction was complete, the reaction mixture was diluted with EtOAc and water, and the aqueous layer was extracted with EtOAc. The combined organic layers were _{dried over Na 2} SO ₄ , concentrated under reduced pressure, and purified by column chromatography using MeOH/DCM 8% as eluent to give the product (51-94%), respectively.

Each synthesized compound and its analysis results are shown below.

4-(3-(4-(2-(adamantan-1-yl)acetyl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1(2 H )-one (2a, Scheme 1).

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.22-10.18 (m, 1H), 8.50-8.48 (m, 1H), 7.83-7.76 (m, 2H), 7.75-7.69 (m, 1H), 7.42-7.31 (m, 2H), 7.09-7.04 (m, 1H), 4.31 (s, 2H), 3.80 (bs, 2H), 3.66-3.62 (m, 2H), 3.52 (t, J = 5.0 Hz, 1H), 3.36-3.25 (m, 2H), 2.22-2.15 (m, 2H), 2.00 (s, 3H), 1.78-1.59 (m, 13H); LC/MS (ESI) m/z [M+H] ⁺ : 543.1; [MH] ^- : 541.1

4-(3-(4-(2-(9) H -fluoren-9-yl)acetyl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1(2 H )-one (3a, Scheme 1).

¹ H NMR (500 MHz, CDCl ₃ ) δ 11.22 (s, 1H), 8.54-8.44 (m, 1H), 7.81-7.66 (m, 5H), 7.58-7.51 (m, 2H), 7.43-7.34 (m , 4H), 7.33-7.27 (m, 2H), 7.09-6.98 (m, 1H), 4.64 (t, J = 7.3 Hz, 1H), 4.32-4.28 (m, 2H), 3.88-3.68 (m, 4H) ), 3.52-3.13 (m, 4H), 2.81 (d, J = 7.2 Hz, 1H), 2.73 (d, J = 7.3 Hz, 1H); LC/MS (ESI) m/z [M+H] ⁺ :573.0 ; [MH] ^- :571.0

( S )-4-(4-fluoro-3-(4-(2-(4-isobutylphenyl)propanoyl)piperazine-1-carbonyl)benzyl)phthalazin-1(2 H )-one (4a, Scheme 1).

¹ H NMR (500 MHz, CDCl ₃ ) δ 11.11 (s, 1H), 8.52-8.45 (m, 1H), 7.76 (d, J = 3.3 Hz, 2H), 7.73-7.66 (m, 1H), 7.33 7.23 (m, 2H), 7.18-6.93 (m, 5H), 4.30-4.22 (m, 2H), 4.02 (bs, 1H), 3.86 (q, J = 7.0 Hz, 1H), 3.80-3.70 (m, 1H), 3.52-3.23 (m, 4H), 3.11 (bs, 1H), 2.46-2.38 (m, 2H), 1.47-1.38 (m, 3H), 1.30-1.18 (m, 2H), 0.89-0.84 ( m, 6H); LC/MS (ESI) m/z [M+H] ⁺ :555.2 ; [MH] ^- :553.1

4-(3-(4-(2,2-diphenylacetyl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1(2 H )-one (5a, Scheme 1).

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.35 (s, 1H), 8.50-8.44 (m, 1H), 7.77-7.75 (m, 2H), 7.70 (s, 1H), 7.35-7.28 (m, 7H) ), 7.25-7.17 (m, 5H), 7.07-6.95 (m, 1H), 5.22 (s, 1H), 5.13 (s, 1H), 4.28-4.21 (m, 2H), 3.77-3.23 (m, 8H) ); LC/MS (ESI) m/z [M+H] ⁺ : 561.0; [MH] ^- : 559.0

4-(3-(4-(2,2-dicyclohexylacetyl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1(2 H )-one (6a, Scheme 1).

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.35 (s, 1H), 8.50-8.44 (m, 1H), 7.77-7.75 (m, 2H), 7.70 (s, 1H), 7.35-7.28 (m, 7H) ), 7.25-7.17 (m, 5H), 7.07-6.95 (m, 1H), 5.30 (s, 1H), 4.28-4.21 (m, 2H), 3.77-3.23 (m, 8H); LC/MS (ESI) m/z [M+H] ⁺ : 561.0; [MH] ^- : 559.0

Preparation of compounds 2b to 6d

[Scheme 2]

Reaction conditions: (a) HATU, DIPEA, DMF, rt, 12 hours; (b) DIPEA, DMF, 70 °C, 12 hours; (c) 4N HCl in dioxane, DCM, rt, 30 min.

Step 1 (N-alkylation):

에서 12 시간 동안 교반하였다. 반응이 완결된 후, 물 및 EtOAc를 잔류물에 첨가하였다. 유기층을 분리하고, 여과하고, 증발시켰다. 반응 혼합물을 용출제로서 MeOH/DCM 8% 를 사용하여 플래쉬 실리카 겔 컬럼 크로마토그래피로 정제하여 목적하는 생성물을 수득하였다 To a solution of alkyl tosylate (0.5 mmol, 1.3 equiv) in DMF (1.5 mL) were added phthalazinone (0.37 mmol, 1 equiv) and DIPEA (0.75 mmol, 2.0 equiv), 70

was stirred for 12 hours. After the reaction was complete, water and EtOAc were added to the residue. The organic layer was separated, filtered and evaporated. The reaction mixture was purified by flash silica gel column chromatography using MeOH/DCM 8% as eluent to give the desired product

Step 2 (Boc-deprotection):

A solution of Boc-protected substrate in DCM was treated with 4 N HCl in 1,4-dioxane solution (1 mL). After the reaction mixture was stirred at room temperature for 30 min, the reaction mixture was concentrated under reduced pressure to give the product as a pale yellow solid. This product was used in the next step without purification.

Step 3 (amide coupling):

HATU (0.11 mmol, 1.1 equiv) and DIPEA (0.4 mmol, 4 equiv) were added to a solution of amine (0.1 mmol) and carboxylic acid (0.1 mmol, 1 equiv) in DMF and stirred at room temperature for 12 h. After the reaction was complete, water and EtOAc were added to the residue. The organic layer was separated, filtered and evaporated. The reaction mixture was purified by flash silica gel column chromatography using MeOH/DCM 8% as eluent to give the desired product.

Each synthesized compound and its analysis results are shown below.

2-(adamantan-1-yl)- N -(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)ethyl)acetamide (2b, Scheme 2).

¹ H NMR (500 MHz, CDCl ₃ ) δ 11.25 (s, 1H), 8.48 (d, J = 7.4 Hz, 1H), 7.83-7.71 (m, 3H), 7.38- 7.30 (m, 2H), 7.03 ( t, J = 8.7 Hz, 1H), 5.96 (s, 1H), 4.31 (s, 2H), 3.80 (s, 2H), 3.42-3.25 (m, 4H), 2.57-2.52 (m, 4H), 2.41 (s, 2H), 1.96-1.93 (m, 5H), 1.72-1.68 (m, 3H), 1.72-1.68 (m, 9H); LC/MS (ESI) m/z [M+H] ⁺ : 586.2; [MH] ^- : 584.2

2-(adamantan-1-yl)- N -(4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)butyl)acetamide (2c, Scheme 2).

¹ H NMR (500 MHz, CDCl ₃ ) δ 11.13 (s, 1H), 8.48 (d, J = 7.2 Hz, 1H), 7.80-7.72 (m, 3H), 7.33-7.28 (m, 2H), 7.03 ( t, J = 8.7 Hz, 1zH), 5.72 (s, 1H), 4.30 (s, 2H), 3.80 (s, 2H), 3.38-3.23 (m, 4H), 2.52-2.50 (m, 2H), 2.44 -2.30 (m, 4H), 1.96-1.90 (m, 5H), 1.72-1.68 (m, 3H), 1.64-1.59 (m, 9H), 1.53 (s, 4H); LC/MS (ESI) m/z [M+H] ⁺ : 614.1; [MH] ^- : 612.1

2-(adamantan-1-yl)- N -(6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)hexyl)acetamide (2d, Scheme 2).

¹ H NMR (500 MHz, MeOD) δ 8.39 (dd, J = 7.7, 1.5 Hz, 1H), 7.99-7.94 (m, 1H), 7.91-7.82 (m, 3H), 7.52-7.47 (m, 1H) , 7.35 (dd, J = 6.3, 2.3 Hz, 1H), 7.17 (t, J = 9.0 Hz, 1H), 4.40 (s, 2H), 3.80 (s, 2H), 3.24-3.14 (m, 2H), 2.63 (s, 2H), 2.51-2.38 (m, 4H), 1.98-1.91 (m, 5H), 1.76 (d, J = 12.4 Hz, 4H), 1.72-1.62 (m, 10H), 1.59-1.48 ( m, 5H), 1.44-1.36 (m, 4H); LC/MS (ESI) m/z [M+H] ⁺ : 642.2; [MH] ^- : 640.2

2-(9 H -fluoren-9-yl)- N -(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)ethyl)acetamide (3b, Scheme 2).

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.77 (s, 1H), 8.45 (d, J = 7.3 Hz, 1H), 7.74 (m, 5H), 7.50 (d, J = 7.5 Hz, 2H), 7.36 (t, J = 7.5 Hz, 2H), 7.33-7.26 (m, 4H), 7.02 (t, J = 8.7 Hz, 1H), 5.93 (s, 1H), 4.49 (t, J = 7.4 Hz, 1H) , 4.27 (s, 2H), 3.71 (s, 2H), 3.41 (q, J = 5.7 Hz, 2H), 3.23 (s, 2H), 2.65 (d, J = 7.3 Hz, 2H), 2.48-2.45 ( m, 4H), 2.34 (s, 2H); LC/MS (ESI) m/z [M+H] ⁺ :616.1 ; [MH] ^- :614.0

2-(9 H -fluoren-9-yl)- N -(4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)butyl)acetamide (3c, Scheme 2).

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.70 (s, 1H), 8.41 (d, J = 7.7 Hz, 1H), 7.77-7.69 (m, 5H), 7.49 (d, J = 7.5 Hz, 2H) , 7.36 (t, J = 7.5 Hz, 2H), 7.29-7.26 (m, 4H), 7.02 (t, J = 8.6 Hz, 1H), 5.91 (s, 1H), 4.50 (t, J = 7.4 Hz, 1H), 4.27 (s, 2H), 3.73 (s, 2H), 3.37-3.14 (m, 4H), 2.61 (d, J = 7.4 Hz, 2H), 2.52-2.24 (m, 6H), 2.04-2.02 (m, 2H), 1.27-1.24 (m, 2H); LC/MS (ESI) m/z [M+H] ⁺ :644.1 ; [MH] ^- :642.0

2-(9 H -fluoren-9-yl)- N -(6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)hexyl)acetamide (3d, Scheme 2).

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.65 (s, 1H), 8.45 (d, J = 7.2 Hz, 1H), 7.78-7.69 (m, 5H), 7.50 (d, J = 7.5 Hz, 2H) , 7.37 (t, J = 7.6 Hz, 2H), 7.29-7.26 (m, 4H), 7.02 (t, J = 8.7 Hz, 1H), 5.47 (s, 1H), 4.50 (t, J = 7.4 Hz, 1H), 4.27 (s, 2H), 3.80 (s, 2H), 3.31-3.25 (m, 4H), 2.61 (d, J = 7.3 Hz, 2H), 2.51 (s, 2H), 2.36 (s, 4H) ), 1.49-1.41 (m, 4H), 1.28 (s, 4H); LC/MS (ESI) m/z [M+H] ⁺ :672.1 ; [MH] ^- :670.0

( S )- N -(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)ethyl)-2-(4-isobutylphenyl) propanamide (4b, Scheme 2).

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.78 (s, 1H), 8.47 (d, J = 5.9 Hz, 1H), 7.82-7.74 (m, 2H), 7.72-7.70 (m, 1H), 7.34 7.25 (m, 2H), 7.19 (d, J = 7.7 Hz, 2H), 7.10 (d, J = 7.7 Hz, 2H), 7.02 (t, J = 8.8 Hz, 1H), 5.96 (s, 1H), 4.28 (s, 2H), 3.63 (bs, 1H), 3.54 (q, J = 7.3 Hz, 1H), 3.28 (s, 2H), 3.14 (s, 2H), 2.44-2.42 (m, 6H), 1.85 -1.79 (m, 1H), 1.51 (d, J = 7.3 Hz, 3H), 1.27-1.24 (m, 3H), 0.88 (d, J = 6.5 Hz, 6H); LC/MS (ESI) m/z [M+H] ⁺ :598.2 ; [MH] ^- :596.1

(S)- N -(4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)butyl)-2-(4-isobutylphenyl) propanamide (4c, Scheme 2).

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.53 (s, 1H), 8.48 (d, J = 7.2 Hz, 1H), 7.78-7.72(m, 3H), 7.33-7.30 (m, 2H), 7.20 ( d, J = 7.6 Hz, 2H), 7.12 (d, J = 7.7 Hz, 2H), 7.04 (t, J = 8.8 Hz, 1H), 5.55 (s, 1H), 4.30 (s, 2H), 3.79 ( s, 2H), 3.53 (q, J = 7.4 Hz, 1H), 3.30 (s, 2H), 3.22 (d, J = 6.4 Hz, 2H), 2.46-2.44 (m, 4H), 2.35-2.31 (m) , 4H), 2.05 (d, J = 10.9 Hz, 1H), 1.52 (d, J = 7.1 Hz, 3H), 1.45 (s, 4H), 0.91 (d, J = 6.3 Hz, 6H); LC/MS (ESI) m/z [M+H] ⁺ :626.2 ; [MH] ^- :624.1

( S )- N -(6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)hexyl)-2-(4-isobutylphenyl) propanamide (4d, Scheme 2).

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.60 (s, 1H), 8.46 (d, J = 7.1 Hz, 1H), 7.82 - 7.68 (m, 3H), 7.35 - 7.27 (m, 2H), 7.18 ( d, J = 7.7 Hz, 2H), 7.11 (d, J = 7.7 Hz, 2H), 7.02 (t, J = 8.7 Hz, 1H), 5.35 (s, 1H), 4.28 (s, 2H), 3.80 ( s, 2H), 3.52 (q, J = 7.2 Hz, 1H), 3.31 (s, 2H), 3.16 (q, J = 6.8 Hz, 2H), 2.56 - 2.41 (m, 4H), 2.41 - 2.26 (m) , 4H), 1.87 - 1.83 (m, 1H), 1.50 (d, J = 7.2 Hz, 3H), 1.40 (dd, J = 18.2, 10.9 Hz, 4H), 1.22 (d, J = 9.5 Hz, 4H) , 0.89 (dd, J = 6.6, 1.5 Hz, 6H); LC/MS (ESI) m/z [M+H] ⁺ :654.2 ; [MH] ^- :652.1

N -(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)ethyl)-2,2-diphenylacetamide (5b , Scheme 2).

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.97 (s, 1H), 8.47 (dd, J = 6.7, 2.6 Hz, 1H), 7.82-7.68 (m, 3H), 7.35-7.21 (m, 12H), 7.02 (t, J = 8.6 Hz, 1H), 6.26 (s, 1H), 5.29 (s, 2H), 4.95 (s, 1H), 4.28 (s, 2H), 3.62 (s, 2H), 3.38 (q) , J = 5.7 Hz, 2H), 3.12 (s, 2H), 2.46 (t, J = 6.0 Hz, 2H), 2.41 (s, 2H); LC/MS (ESI) m/z [M+H] ⁺ : 604.1; [MH] ^- : 602.1

N -(4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)butyl)-2,2-diphenylacetamide (5c , Scheme 2).

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.78 (s, 1H), 8.48 (d, J = 7.3 Hz, 1H), 7.8-7.72 (m, 3H), 7.36-7.25 (m, 12H), 7.05 ( t, J = 8.7 Hz, 1H), 5.91 (s, 1H), 5.32 (s, 2H), 4.93 (s, 1H), 4.30 (s, 2H), 3.78 (s, 2H), 3.34-3.27 (m) , 4H), 2.46-2.44 (m, 2H), 2.37-2.23 (m, 2H), 1.53-1.45 (m, 4H); LC/MS (ESI) m/z [M+H] ⁺ : 632.1; [MH] ^- : 630.1

N -(6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)hexyl)-2,2-diphenylacetamide (5d , Scheme 2).

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.31 (s, 1H), 8.52-8.44 (m, 1H), 7.84-7.71 (m, 3H), 7.39-7.31 (m, 5H), 7.30-7.26 (m , 7H), 7.09-7.00 (m, 1H), 5.70-5.58 (m, 1H), 4.95 (s, 1H), 4.30 (s, 2H), 3.89-3.77 (m, 2H), 3.34-3.26 (m) , 3H), 2.55-2.47 (m, 2H), 2.38-2.32 (m, 3H), 1.55-1.41 (m, 4H), 1.37-1.23 (m, 5H); LC/MS (ESI) m/z [M+H] ⁺ : 604.1; [MH] ^- : 602.1

2,2-dicyclohexyl- N -(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)ethyl)acetamide (6b, Scheme 2).

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.72 (s, 1H), 8.49 (d, J = 7.1 Hz, 1H), 7.84-7.72 (m, 3H), 7.37-7.30 (m, 2H), 7.04 ( t, J = 8.7 Hz, 1H), 5.92 (bs, 1H), 4.30 (s, 2H), 3.82 (s, 2H), 3.44-3.26 (m, 4H), 2.65-2.52 (m, 4H), 2.44 (s, 2H), 2.06-2.04 (m, 2H), 1.69 (s, 6H), 1.29-1.03 (m, 12H), 1.01-0.83 (m, 3H); LC/MS (ESI) m/z [M+H] ⁺ :616.2 ; [MH] ^- :614.1

2,2-dicyclohexyl- N -(4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)butyl)acetamide (6c, Scheme 2).

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.16 (s, 1H), 8.46 (d, J = 7.3 Hz, 1H), 7.77 (t, J = 6.4 Hz, 2H), 7.72 (d, J = 7.4 Hz) , 1H), 7.34-7.26 (m, 2H), 7.03 (t, J = 8.7 Hz, 1H), 5.62 (s, 1H), 4.28 (s, 2H), 3.83 (s, 2H), 3.33 (s, 2H), 3.25 (d, 2H, J =6.7 Hz), 2.58 (s, 2H), 2.47-2.43 (m, 4H), 2.08-1.95 (m, 4H), 1.55-1.48 (m, 8H), 1.18 -1.02 (m, 9H), 0.99-0.86 (m, 6H); LC/MS (ESI) m/z [M+H] ⁺ :644.2.

2,2-dicyclohexyl- N -(6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)hexyl)acetamide (6d, Scheme 2).

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.59 (s, 1H), 8.47 (d, J = 7.2 Hz, 1H), 7.81-7.70 (m, 3H), 7.35-7.28 (m, 2H), 7.02 ( t, J = 8.7 Hz, 1H), 5.39 (s, 1H), 4.28 (s, 2H), 3.81 (s, 2H), 3.32 (s, 2H), 3.23 (q, J = 6.7 Hz, 2H), 2.53 (s, 2H), 2.38-2.35 (m, 4H), 2.04-2.02 (m, 1H), 1.75-1.57 (m, 14H), 1.52-1.46 (m, 4H), 1.50-1.44 (m, 4H) ), 1.23-1.02 (m, 8H); LC/MS (ESI) m/z [M+H] ⁺ :672.2.

Evaluation of the activity of the compounds of the present invention

Experimental method

cell culture

MDA-MB-231 cells (ATCC) were cultured with ZellShield (Minerva Biolabs) in DMEM medium supplemented with 10% fetal bovine serum (Gibco). Cell lines were tested for mycoplasma contamination. MDA-MB-231 cells are a triple-negative breast cancer (TNBC) cell line that does not express three receptors: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2/neu). It is a breast cancer cell line that does not

Antibodies and Substances

Commercially available antibodies were used. Cell Signaling's anti-PARP (#9542), anti-Bax (#2772), anti-cytochrome C (#12959), anti-Bcl2 (#3498), anti-CHOP (#2895), anti-XBP-1s ( #12782), anti-LC3B (#2775); anti-b-actin from Sigma (A1978); and anti-p21 (sc-6246) from Santa Cruz Biotechnology. Cafilzomib (# 2385) was purchased from BioVision. Bortezomib (504314) was purchased from Calbiochem.

RNA Preparation and Quantitative RT-PCR

Total RNA was isolated using TRIzol (Invitrogen). RNA was reverse transcribed with oligo (dT) primers and SuPrimeScript Reverse Transcriptase (GeNetBio). The obtained cDNA was subjected to TOPreal?? for PCR. It was mixed with qPCR 2X PreMIX (SYBR Green with low ROX, Enzynomics) and gene-specific primers. The abundance of mRNA was detected by Rotor-Gene Q system (Qiagen) or 7500 Fast Real-Time PCR system (Applied Biosystems) with SYBR Green. The following primers were used.

human Bax Forward 5'-CGAGTGGCAGCTGACATGTTT -3',

human Bax Reverse 5'-AGGGCCTTGAGCACCAGTTT-3',

human p21 Forward 5'-GGCTTCATGCCAGGCTACTTC -3',

human p21 Reverse 5'-CCCTAGGCTGTGCTCACTTC -3',

human Puma Forward 5'-ATGCCTGCCTCACCTTCATC -3',

human Puma Reverse 5'-GGTCACACGTCGCTCTCTCTA A -3',

human Survivin Forward 5'-TGCAAAGGAAACCAACAATAAGAA-3',

human Survivin Reverse 5'-ATGGCACGGCGCACTT -3',

human Bcl2 Forward 5'-CTGCACCTGACGCCTTCACC -3',

human Bcl2 Reverse 5'-CACATGACCCCACCGAACTCAAAGA -3',

human LC3A Forward 5'-CGTCCTGGACAAGACCAAGT -3',

human LC3A Reverse 5'-CTCGTCTTTCTCCTGCTCGT -3',

human LC3B Forward 5'-GATGTCCGACTTATTCGAGAGC-3',

human LC3B Reverse 5'-TTGAGCTGTAAGCGCCTTCTA-3',

human ATF4 Forward 5'-GGGACAGATTGGATGTTGGAGA -3',

human ATF4 Reverse 5'-ACCCAACAGGGCATCCAAGT -3',

human CHOP Forward 5'-CAGAACCAGCAGAGGTCACA -3',

human CHOP Reverse 5'-AGCTGTGCCACTTTCCTTTC -3',

human XBP-1s Forward 5'-TGCTGAGTCCGCAGCAGGTG -3',

human XBP-1s Reverse 5'-GCTGGCAGGCTCTGGGGAAG -3'.

Whole-cell lysate preparation

All cells were rinsed briefly with ice-cold PBS before harvesting. For whole cell lysates, cells were resuspended in RIPA buffer to which inhibitor complete (Roche) had just been added. After vigorous shaking at 4° C. for 30 minutes, it was rotated at 13000 rpm at 4° C. for 15 minutes. The supernatant (whole cell lysate) was transferred to a new tube. All lysates were quantified by Pierce BCA protein assay kit (Thermo, 23227) and analyzed by SDS-PAGE.

SDS-PAGE and immunoblot analysis

4-20% Mini-PROTEAN TGX Precast Protein Gels (Bio-rad, #459-1096) were used for SDS-PAGE. Then, the protein was transferred from the gel to the PVDF membrane using the Mini Trans-Blot Cell system (Bio-Rad). For Western blot analysis, membranes were incubated in blocking solution (5% skim milk in TBS-T) for 30 min to block non-specific binding of antibodies. 3 in TBS-T (1:100 dilution, p21; 1:500 dilution, Bcl2; 1:1000 dilution, PARP, Bax, Cytochrome C, CHOP, XBP-1s, LC3B; 1:5000 dilution, b-actin) Incubated with primary antibody diluted with % BSA. After washing 6 times with TBS-T, the membrane was incubated with an appropriate secondary HRP-conjugated antibody (Thermo, 1:5000 dilutions in blocking solutions). After washing 6 times with TBS-T, the membranes were incubated on ECL substrate (Pierce, 34096) and imaged with ChemiDoc MP (Bio-Rad) or LAS-3000 (Fuji).

Cell viability assay

10,000 cells per well were seeded with complete medium onto 96-well clear bottom plates. The next day, the complete medium was replaced with serum-free. After 24 hours, compounds were added and the indicated concentrations of compounds were added and titrated for the indicated time points followed by incubation for the indicated time points. CCK8 (Dojindo) was added according to the manufacturer's instructions and the OD value was measured. Values were normalized by DMSO control and IC ₅₀ was calculated using GraphPad Prism 8.

Transmission electron microscopy (TEM)

에서 24 시간 동안 고정시키고, 실온에서 1 시간 동안 2% 오스뮴 테트로옥사이드로 후-고정시켰다. 세척 후, 세포를 50% 에탄올로부터 출발하여 100% 에탄올로 끝나는, 각 단계 20 분의 구배 시리즈의 에탄올을 통해 탈수시켰다. 그 후, 세포를 에탄올에 용해된 점진적으로 농축된 프로필렌 옥사이드와 함께 인큐베이션한 다음, Epon 812 수지의 농도를 증가시키면서 침투시켰다. 샘플을 60

오븐에서 48 시간 동안 베이킹한 다음, Ultra microtome (70 nm thickness, Leica EM UC7)을 사용하여 절단하였다. TEM (JEM-2100f, JOEL) 를 이용하여 측정되었다. Cells are treated with a test compound (eg compound 3a) for the indicated times. Cells were placed 4 in PBS containing 2% paraformaldehyde (EM grade) and 2% glutaraldehyde (EM grade).

was fixed for 24 h at room temperature and post-fixed with 2% osmium tetrooxide for 1 h at room temperature. After washing, cells were dehydrated through ethanol in a gradient series of 20 min each step, starting from 50% ethanol and ending with 100% ethanol. The cells were then incubated with progressively concentrated propylene oxide dissolved in ethanol and then permeabilized with increasing concentrations of Epon 812 resin. 60 samples

After baking in an oven for 48 hours, they were cut using an Ultra microtome (70 nm thickness, Leica EM UC7). It was measured using TEM (JEM-2100f, JOEL).

Experiment result

Fig. 1 shows the results of immunoblot analysis of MDA-MB-231 cells treated with the compound according to the present invention.

As shown in Fig. 1, the compounds according to the present invention exhibited PARP inhibitory activity, and among the various compounds according to the present invention, compounds 3a, 4d, and 6d were preferred. In particular, among the various compounds according to the present invention, the effect of compound 3a was excellent compared to other compounds.

Fig. 2 shows the results of immunoblot analysis of HCC1937 cells treated with Compound 3a, which is an embodiment according to the present invention, and Olaparib, a comparative compound.

As shown in Fig. 2, compound 3a according to the present invention was excellent in its effect compared to Olaparib, a comparative example.

Fig. 3 shows the results of compound 3a according to the present invention causing apoptosis of TNBC cells through PARP degradation. (A) shows the results of immunoblot analysis on whole cell lysates of MDA-MB-231 cells after compound 3a treatment for the indicated times. (B and C) Quantitative RT-PCR analysis of proapoptotic (B) or antiapoptotic (C) target genes, performed using MDA-MB-231 cells treated with vehicle or compound 3a for the indicated times. Values are mean ± SD; n=3. It is expressed as **p<0.01, ***p<0.001, ****p<0.001 (one-way ANOVA). (D) CCK-8 assay results after treatment with serially diluted compound 3a or olparib for 96 hours. Dose-response curve fitting was performed using non-linear regression. Values are mean ± SD; n=3.

Fig. 3 shows the results showing whether PARP1 degradation affects TNBC cell viability. As a result of treatment with the compound of the present invention, expression of apoptosis marker proteins (Bax, p21, cytochrome C) was induced along with PARP degradation (Fig. 3A). Bcl2, an anti-apoptotic protein, was gradually decreased during the treatment with the compound of the present invention (Fig. 3A). In addition, the compound of the present invention up-regulated the mRNA level of the apoptosis gene and down-regulated the level of the anti-apoptotic gene (Fig. 3B and 3C). Since the treatment with compound 3a causes PARP degradation and upregulates apoptosis signaling, it can be seen that compound 3a of the present invention provides an excellent anticancer effect in TNBC compared to olparib. Indeed, treatment with 3a at a concentration of 5 μM inhibited cell growth by approximately 80% after incubation for 96 h, but olaparib (olaparib, IC ₅₀ =32.36 ± 0.63 mM; compound 3a , IC ₅₀ =2.10 ± 0.31 mM) (Fig. 5D), only marginal inhibitory effects were observed.

Fig. 4 shows experimental results showing that compound 3a treatment induces ER stress-related autophagy and apoptosis. (A) Shows signaling pathways in which unfolded or misfolded proteins induce apoptosis. (B and C) Results of immunoblot analysis performed on whole cell lysates of MDA-MB-231 cells after treatment with compound 3a (B and C) or olaparib (c) at the indicated times. (D) Quantitative RT-PCR analysis of ER stress and autophagy-related genes was performed using MDA-MB-231 cells treated with vehicle or compound 3a for the indicated times. Values are mean ± SD; n=3. *p<0.05, **p<0.01, ***p<0.001, ****p<0.001 (one-way ANOVA). (E) Representative TEM image of MDA-MB-231 cells. The scale bar is 2 μm. Red arrows indicate autophagomes, yellow arrows indicate apoptosis. Treatment with 5 μM compound 3a was performed at the times indicated. (F) Schematic model of PARP degradation by compound 3a.

C/EBP homologous protein (CHOP) is a major proapoptotic transcription factor triggered by ER stress. CHOP has also been reported to induce transcriptional upregulation of LC3, autophagy and genes related to the apoptosis pathway. X box-binding protein 1 (XBP-1s) is spliced by ER stress from unspliced XBP-1u by ER stress and activates UPR target genes. CHOP and XBP-1s increased dramatically from 24 h after compound 3a treatment (Fig. 4B and C). In addition, protein expression of LC3B was induced after 48 hours. On the other hand, olaparib did not induce PARP degradation, UPR and ER stress, indicating that certain structures such as the compounds of the present invention are important for induction of UPR and ER stress. Transcriptional levels of UPR, ER stress and autophagy-related genes were also increased with compound 3a treatment (Fig. 4D). We evaluated the ultrastructure of the cells after compound 3a treatment by monitoring the cells by transmission electron microscopy (TEM). Large double membrane-bound vesicles surrounding the cytoplasmic component (red arrow) were detected in Compound 3a-treated cells. In addition, apoptotic bodies (yellow arrows) gradually increased with the incubation time of 3a. UPR and ER stress-mediated apoptosis triggered by 3a may be an important mechanism of action with superior antitumor effects than Olaparib in MDA-MB-231 cells.

Claims (10)

A compound of Formula 1 or a pharmaceutically acceptable salt thereof.
[Formula 1]

According to claim 1, wherein the hydrophobic tagging is any one of the following compound or a pharmaceutically acceptable salt thereof.

The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the linker is absent (direct connection) or **-(C _{1-10 alkyl)NH-*.} The method of claim 1, wherein the compound is
4-(3-(4-(2-(adamantan-1-yl)acetyl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1( 2H )-one,
4- (3- (4- (2- ( 9 H -fluoren-9-yl) acetyl) piperazine-1-carbonyl) -4-fluorobenzyl) phthalazin-1 (2 H) -one,
( S )-4-(4-fluoro-3-(4-(2-(4-isobutylphenyl)propanoyl)piperazine-1-carbonyl)benzyl)phthalazin-1( 2H )-one,
4-(3-(4-(2,2-diphenylacetyl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1( 2H )-one,
4-(3-(4-(2,2-dicyclohexylacetyl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1( 2H )-one,
2-(adamantan-1-yl) -N -(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl )ethyl)acetamide,
2-(adamantan-1-yl) -N -(4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl )butyl)acetamide,
2-(adamantan-1-yl) -N -(6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl )hexyl)acetamide,
2- (9 H -fluoren-9- yl) - N - (2- (4- (2-fluoro-5 - ((4-oxo-3,4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin- 1-yl)ethyl)acetamide,
2- (9 H -fluoren-9- yl) - N - (4- (4- (2-fluoro-5 - ((4-oxo-3,4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin- 1-yl)butyl)acetamide,
2- (9 H -fluoren-9- yl) - N - (6- (4- (2-fluoro-5 - ((4-oxo-3,4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin- 1-yl)hexyl)acetamide,
(S) - N - (2- (4- (2-fluoro-5 - ((4-oxo-3,4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) ethyl) -2- (4-isobutylphenyl)propanamide,
(S)- N -(4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)butyl)-2- (4-isobutylphenyl)propanamide,
(S) - N - (6- (4- (2-fluoro-5 - ((4-oxo-3,4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) hexyl) -2- (4-isobutylphenyl)propanamide,
N -(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)ethyl)-2,2-diphenylacetamide,
N -(4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)butyl)-2,2-diphenylacetamide,
N -(6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)hexyl)-2,2-diphenylacetamide;
2,2-dicyclohexyl- N -(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)ethyl)acetamide ,
2,2-dicyclohexyl- N -(4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)butyl)acetamide , or
2,2-dicyclohexyl- N -(6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)hexyl)acetamide sign
A compound or a pharmaceutically acceptable salt thereof. 5. The method of claim 4, wherein the compound is 4-(3-(4-(2-(9H-fluoren-9-yl)acetyl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one A phosphorus compound or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for inhibiting PARP comprising the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for the treatment of cancer comprising the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition according to claim 7, wherein the cancer is breast cancer, ovarian cancer, or prostate cancer. The pharmaceutical composition according to claim 8, wherein the cancer is breast cancer. The pharmaceutical composition according to claim 9, wherein the breast cancer is breast cancer that does not express three receptors: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2/neu).

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