KR20120005107A - New arylsulfonylimidazolone derivatives and an anti-cancer pharmaceutical composition comprising the same - Google Patents

Abstract

The present invention provides an arylsulfonylimidazolone derivative compound or a pharmaceutically acceptable salt thereof having a high anticancer targeting potential and a shorter manufacturing process than the conventional one, and a method for preparing the same and an anticancer composition containing the same. It is about.

Description

New arylsulfonylimidazolone derivatives and an anticancer agent composition containing the same {New arylsulfonylimidazolone derivatives and an anti-cancer pharmaceutical composition comprising the same}

The present invention relates to an arylsulfonylimidazolone derivative compound or a pharmaceutically acceptable salt thereof, a method for preparing the same, and an anticancer agent composition containing the same, which has a high cancer cell target but a shorter manufacturing process.

Sulfonylurea compounds exhibit various physiological activities such as hypoglycemic action, preparations, and antifungal agents. In particular, diarylsulfonyl urea (diarylsulfonylurea (DSU) has been reported to exhibit anticancer activity. The compound also exhibits significant activity against solid cancer models that are not well treated with existing anticancer agents, which then modify the structure of the compound to further enhance sulofenur (Sulofenur, LY186641, J. Med. Chem., 1990, 33, 2393). Although these diarylsulfonyl urea derivative compounds showed excellent anticancer effects in transplantation of colorectal cancer xenografts, the development of anticancer drugs was stopped due to side effects such as methemoglobinemia and hemolytic anemia. Was found to be due to the ρ-chloroaniline derivative, a metabolite of the DSU material.

U.S. Patent No. 5,929,103 discloses a new arylsulfonylimidazolone-based compound that is superior to sulfofenur and does not produce aniline-based metabolites that cause the aforementioned side effects. These arylsulfonylimidazolone-based compounds are antimitotic agents that inhibit toxicity in tubule polymerization and express toxicity by resting the cell cycle at the G2 / M phase. According to the present invention, it shows excellent cytotoxicity against various cancer cells (HCT116, A-549, NCI-H460), and some derivatives have not been shown to express the multi-resistance of paclitaxel or vinca alkaloid anticancer drugs currently used in the clinic. (See Korean Patent Publication No. 2009-87697). The inventors of the present invention conducted a preclinical test on Compound 1 of Formula II, which exhibits particularly excellent anticancer effects, and found that a large portion of the administered drug is deposited in the intestine of the experimental animal, thereby causing toxicity. Therefore, development into a new drug has been stopped. In addition, the solubility in water was very low as 23μg / ㎖ not possible to use as an intravenous injection, the dissolution and absorption of intestinal drugs is not smooth, so that the intestinal toxicity was confirmed.

On the other hand, anti-cancer substances used for cancer treatment show cytotoxicity not only to cancer cells but also to normal cells, thereby showing side effects. Therefore, studies have been made to minimize side effects by increasing the selectivity of cancer cells for anticancer drugs, and further maximize the anticancer activity.

On the other hand, the present inventors have described a tripeptide group (D-Val-Leu-Lys- and D- as a linker with PABA (p-amino benzyl alcohol) to the arylsulfonyl imidazolone compound known from Korean Patent Publication No. 2009-87697. A new arylsulfonylimidazolone derivative compound has been prepared by synthesizing a prodrug which forms a bond of Ala-Phe-Lys-) (Patent Application No. 10-2010-0034507), and improved the linker. An arylsulfonylimidazolone derivative compound having a similar effect and shortening the manufacturing process without the compound was prepared.

An object of the present invention is to provide an arylsulfonylimidazolone derivative compound which can be administered intravenously, has no risk of enterotoxicity, and maximizes targeting to cancer cells, thereby minimizing side effects.

Another object of the present invention is to shorten the manufacturing process than the arylsulfonyl imidazolone derivative compound in the form of combined with the tripeptide group using a conventional p-amino benzyl alcohol (PABA) as a linker, while reducing the manufacturing cost and stability in the body It is to provide an increased novel arylsulfonylimidazolone derivative compounds.

The present invention relates to an arylsulfonylimidazolone derivative compound of formula (I) or a pharmaceutically acceptable salt thereof, a method for preparing the same, and an anticancer composition containing the same as an active ingredient.

[Formula I]

In Formula I,

R is CH (CH ₃ ) ₂ or CH ₃ and R ′ is CH ₂ CH (CH ₃ ) ₂ or CH ₂ Ph.

Compounds of the present invention are prepared according to Scheme I, specifically amides directly with a tripeptide group D-Val-Leu-Lys- or D-Ala-Phe-Lys- to compound 1 (the parent drug) of formula II It is prepared by synthesizing a prodrug (amide) to form a bond.

Thus prepared compound of formula (I) is efficiently converted to compound 1 of formula (II), a parent drug that exhibits anticancer activity by plasmin, which is one of proteases that are specifically expressed in cancer cells when administered in vivo. Targeting to cancer cells is increased.

≪ RTI ID = 0.0 &

Scheme I

The compound of the formula (I) of the present invention has a 70-150-fold increase in water solubility compared to the compound (1) of the formula (II), which is a parent drug, and exhibits high water solubility in water of 1.7 mg / ml or more, and thus can be easily formulated as an injection. .

The water solubility of the compounds is slightly lower than that of Patent Application No. 10-2010-0034507, but it has sufficient water solubility to be used as an intravenous injectable drug and the problem of enterotoxicity due to oral administration can be effectively solved. have.

Unlike patent application No. 10-2010-0034507, even though PABA (p-amino benzyl alcohol) is not used as a linker, it has similar effects in water solubility, cytotoxicity, and conversion rate to the parent drug, thereby shortening the drug manufacturing process. Can be.

As described above, the preparation in the form of prodrugs is a useful method for overcoming physicochemical limitations such as solubility and lipophilic in vivo, and biological limitations such as bioavailability. In the design of prodrugs, the desired prodrugs are easy to synthesize, have good half-life at room temperature or in vitro, and have good stability, so that they can be completely converted into parent drugs under appropriate conditions in vivo. Should be. In addition, prodrugs should normally be produced only by enzymatic reactions in vivo to satisfy all of the above conditions.

Compounds of formula (I) of the present invention are selectively degraded by plasmin, one of the proteases that specifically express cancer cells. Plasmin is present in the body, most of plasminogen inactive-type chair Imogen form, α ₂ present in vivo-anti-plasmin and α ₂ - is rapidly inhibited by macroglobulin, the u-PA that are overexpressed in cancer cells Is an enzyme that is activated locally.

Compounds of formula (I) of the present invention are more degraded by plasmin in cancer cells than in blood, so they are present in the form of relatively inactive prodrugs in the blood, and then converted into compounds of formula (II), the parent drug in cancer cells, Compounds of formula (II) exhibit potent anticancer activity in cancer cells, and as a result, the compounds of the present invention significantly increase the selectivity for cancer cells and reduce side effects.

Compounds of the present invention are lung cancer, colon cancer, rectal cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, fallopian tube carcinoma, ovarian cancer, vaginal carcinoma, vulvar carcinoma, liver cancer, gastric cancer, esophageal cancer, small intestine cancer, pancreatic cancer, gallbladder cancer, kidney cancer , Bladder cancer, urethral cancer, penile cancer, prostate cancer, testicular cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, non-small cell lung cancer, bone cancer, skin cancer, head or neck cancer, skin or ocular melanoma, hochekin disease, endocrine May be used for the treatment of cancers such as adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, central nervous system tumor, spinal cord tumor, brain stem glioma and pituitary adenoma, preferably lung cancer, colorectal cancer, rectal cancer, uterine cancer, ovarian cancer, or It can be used for the treatment of leukemia.

In addition, the compounds of the present invention can also be used for the treatment of all cancer diseases caused by cancer cells resistant to existing anticancer agents.

In addition, the compounds of the present invention are particularly effective in the treatment of cancer diseases caused by cancer cells expressing plasmin.

In the present invention, 'pharmaceutically acceptable salt' means hydrochloride, hydrobromide, hydroiodide, hydrogen fluoride, sulfate, sulfonate, citrate, camphorate, maleate, acetate, lactate, nikitinate Nitrate, Succinate, Phosphate, Malonate, Dried salt, Salicylate, Phenyl acetate, Stearate, Palmitate, Pyridine, Ammonium, Piperazine, Diethylamine, Nicotinamide, Formate, Fumarate, Urea, Sodium, Potassium, Calcium, Magnesium, Zinc, Lithium, Cinnamic Acid, Methylamino, Methanesulfonate, Picrinate, p-Toluenesulfonate, Naphthalenesulfonate, Tartarate, Triethylamino, Dimethylamino and Tri (hydroxymethyl Salts commonly used in the pharmaceutical arts, such as aminomethane, with hydrochloride particularly preferred.

Since one or more asymmetric carbon atoms are present in the compounds of the present invention, it is known to those skilled in the art to include enantiomers or diastereomers. Diastereomeric mixtures can be separated into individual diastereomers on the basis of their physicochemical differences by known methods, for example, by chromatography, recrystallization or fractional crystallization, and enantiomers can be used to form enantiomeric mixtures as appropriate. Reaction with the optically active compound to convert to the diastereomer mixture, separation of the diastereomers and the separation of each diastereomer into the corresponding pure enantiomers in conventional manner. Diastereomers, enantiomers and mixtures thereof are considered part of the present invention, wherein in the general formula (I), the position where phenyl is attached to the imidazolidinone is in the S configuration and the amino acids are all L-amino acids.

The composition of the present invention may be an aqueous solution or a powder itself, or may be a pharmaceutical composition of a suitable formulation prepared using known additives such as excipients, binders or lubricants as necessary.

In one embodiment of the present invention, when preparing an injection, a powder containing the compound of formula (I) as an active ingredient is easily dissolved in an aqueous isotonic solution prepared using distilled water or sodium chloride and sugars (eg, glucose, mannitol, inositol, etc.). After dissolution, the injection containing the active ingredient may be administered subcutaneously intravenously, intramuscularly, or directly to a lesion or a tumor cut such as a tumor at an effective drug concentration in vivo for the disease to be treated.

As another example of the present invention, when preparing oral preparations, it may be prepared in tablets, capsules, granules, granules, binders, liquids and the like. In the preparation of these formulations, it may further comprise inert ingredients, including pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” is a term that specifically refers to an ingredient except an active substance of a pharmaceutical composition. Examples of pharmaceutically acceptable carriers include binders, disintegrants, diluents, fillers, glidants, excipients, lubricants, dispersants, stabilizers, colorants, absorption enhancers, solubilizers or emulsifiers and salts.

In addition, sugars, preservatives, stabilizers, antistatic agents, and the like can be added to improve the properties of the powder obtained (such as sealing degree and capacity of the storage container).

The powder may also be formulated in preparations other than injections or oral preparations according to conventional methods. Examples of such agents are preparations, transdermal and implants administered to the mucous membranes of the nose, mouth, sublingual, rectum, vagina or uterus. Each of the above formulations may be molded into various controlled release or targeted therapeutic agents, and the compositions of the present invention may be used as raw materials for such formulations.

As described above, the compound of formula (I) of the present invention solves the poor solubility problem of water of the conventional arylsulfonylimidazolone-based compound, and can be administered intravenously. Therefore, it is possible to prompt drug delivery through the systemic circulatory system immediately after intravenous administration, and does not express enterotoxicity, and in particular, it is relatively safe for the living body because of increased targetability to cancer cells and minimizing side effects. Thus, various formulations such as injections, oral preparations, preparations administered to the oral cavity (troches, oral preparations, etc.), sublingual agents, eye drops, syrups, external preparations administered to the skin, nasal preparations, preparations administered through the lungs, Formulations applied to rectal suppositories or mucous membranes are very useful in pharmaceutical or veterinary compositions and as anticancer agents used in humans or non-human mammals (monkeys, small dogs, etc.).

The novel arylsulfonylimidazolone compounds of the present invention can be prepared as an injection that can be administered intravenously due to high water solubility, thereby avoiding the concern of enteric toxicity, which is a problem of conventional systemic anticancer drugs, and cancer-specific expression in the upper body. It is selectively converted by the enzyme plasmin, which is highly targeted against cancer cells, thereby minimizing side effects and exerting an excellent anticancer effect.

In addition, compared to the arylsulfonylimidazolone compounds linked to tripeptides by the PABA (p-amino benzyl alcohol) linker, which was previously manufactured, it is directly linked to tripeptides without the linker, which shortens the manufacturing process and reduces manufacturing costs. It can have the effect of increasing stability.

Figure 1 shows the conversion rate of the arylsulfonylimidazolone derivative compound 7 of the present invention is converted to compound 1, the parent drug in human plasma.
Figure 2 shows the conversion rate of the arylsulfonylimidazolone derivative compound 7 of the present invention is converted to compound 1 as a parent drug in human plasmin.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to ensure that the contents introduced herein are thorough and complete, and that the spirit of the present invention to those skilled in the art can fully convey.

Example 1 Synthesis of Arylsulfonylimidazolone Derivatives

1-1. Synthesis of Compound 4

Compound 1 is described in Bioorg. Med. Chem. Lett. 1988, 8, 1547-1550.

Compound 2 is [J. Med. Chem. 1999, 43, 3093-3102.

Under nitrogen gas, Compound 2 (Alloc-D-Val-Leu-Lys (Alloc) -OH, 360 mg, 0.68 mM) was dissolved in 30 ml of dry THF and stirred at -20 ° C for 30 minutes. Isobutyl chloroformate (98 µl, 0.75 mM) and N-methylmorpholine (N-methylmorpholine, 84 µl, 0.75 mM) were added dropwise to the mixed solution, followed by stirring at -20 ° C for 3 hours. To this mixed solution, Compound 1 (396 mg, 0.85 mM) and N-methylmorpholine (96 µl, 0.85 mM) were dissolved in 5 ml of dry THF, and then slowly added dropwise thereto. After dropping, the temperature was gradually raised and stirred at room temperature for 24 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, diluted with CH ₂ Cl ₂ (50 mL), and washed three times with saturated NaHCO ₃ (30 mL), 0.5N KHSO ₄ (30 mL), and brine (30 mL). Thereafter, the organic layer was dehydrated with anhydrous forget-me-not and concentrated under reduced pressure. The crude product thus obtained was separated in a Si-gel column with a mixed solvent of n-HX: EtOAc = 1: 1 → n-HX: EtOAc: MeOH = 10: 10: 1 to obtain a white solid (110 mg, 16.8%). .

mp 128.0-129.0 ° C .; FT-IR (cm- ¹ ,) 3313, 2957, 1653, 1539, 1374, 1219, 1157; 1 H-NMR (400 MHz, DMSO-d6) δ 0.92 -1.06 (m, 12H), 1.46-2.10 (m, 10H), 3.19 (m, 4H), 3.62 (q, J = 7.8 Hz, 9.2 Hz, 1H) , 3.80 (m, 1H), 4.15-4.54 (m, 10H), 4.74 (t, J = 8.0 Hz, 1H), 5.03-5.32 (m, 4H), 5.60 (m, 1H), 5.79 (m, 1H ), 5.91 (m, 1H), 6.42 (m, 1H), 7.23-7.40 (m, 7H), 7.52 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 9.2 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.88 (s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 8.39 (m, 1H), 8.89 (s, 1H).

1-2. Synthesis of Compound 5

Compound 3 is [J. Med. Chem. 2000, 42, 5277-5283.

Under nitrogen gas, Compound 3 (362 mg, 0.68 mM) was dissolved in 30 ml of dry THF, followed by stirring at −20 ° C. for 30 minutes. Isobutyl chloromate (98 µl, 0.75 mM) and N-methylmorpholine (84 µl, 0.75 mM) were added dropwise to this mixed solution, followed by stirring at -20 ° C for 3 hours. To this mixed solution, Compound 1 (396 mg, 0.85 mM) and N-methylmorpholine (96 µl, 0.85 mM) were dissolved in 5 ml of dry THF, and then slowly added dropwise thereto. After dropping, the temperature was gradually raised and stirred at room temperature for 24 hours. After completion of the reaction, the mixture was concentrated under reduced pressure, diluted with CH ₂ Cl ₂ (50 mL), and washed three times with saturated NaHCO ₃ (30 mL), 0.5N KHSO ₄ (30 mL), and brine (30 mL). The organic layer was then dehydrated with anhydrous forget-me-not and concentrated under reduced pressure. The crude product thus obtained was separated in a Si-gel column with a mixed solvent of n-HX: EtOAc = 1: 1 → n-HX: EtOAc: MeOH = 10: 10: 1 to obtain a white solid (188 mg, 28.7%). .

mp 152.0-153.0 ° C .; FT-IR (cm- ¹ ,) 3277, 1684, 1636, 1522, 1375, 1247; 1 H-NMR (400 MHz, DMSO-d6) δ0.97 (d, J = 7.2 Hz, 3H), 1.23-1.78 (m, 6H), 2.80-2.98 (m, 3H), 3.08-3.16 (m, 3H) , 3.50 (m, 1H), 4.02 (m, 2H), 4.14 (t, J = 8.0 Hz, 2H), 4.27 (t, J = 8.8 Hz, 1H), 4.38-4.56 (m, 6H), 4.80 ( t, J = 7.2 Hz, 1H), 5.13-5.26 (m, 4H), 5.86 (m, 3H), 7.18-7.25 (m, 8H), 7.32-7.39 (m, 4H), 7.61 (m, 2H) , 7.77 (m, 6 H), 8.20 (m, 2 H).

1-3. Synthesis of Compound 6

Under nitrogen gas, Compound 4 (100 mg, 0.1 mM) was dissolved in dry THF and stirred at room temperature. Acetic acid (acetic acid, 30 µl, 0.5 mM), tributyltinhydride (85 µl, 0.3 mM) and Pd (PPh ₃ ) ₄ (10 mg) were added dropwise to the mixed solution for 1 hour at room temperature. Stirred, tributyl hydride (85 μl, 0.3 mM) was further added dropwise and stirred for another 2 hours. To this mixed solution was slowly added dropwise 1.25M HCl (2 mL) using methanol as a solvent, stirred for 3 hours, and concentrated under reduced pressure. The material obtained by crystallization of the concentrated composition with ether (ether) was dissolved in distilled water again and centrifuged (3000rpm, 10 minutes) to freeze-dried the supernatant to give a pale yellow solid (38 mg, 41.5%).

mp 169.0-170.0 ° C .; FT-IR (cm- ¹ ,) 3269, 2957, 1652, 1521, 1375, 1218, 1158, 1074; ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ 0.85-0.96 (m, 12H), 1.10-1.64 (m, 10H), 2.09 (m, 1H), 2.78 (m, 2H), 3.17 (t, J = 8.4 Hz, 2H), 3.62 (q, J = 6.0 Hz, 9.6 Hz, 1H), 3.68 (m, 1H), 4.13 (t, J = 8.0 Hz, 1H), 4.28 (t, J = 8.8 Hz, 1H), 4.79 (m, 2H), 4.81 (t, J = 6.8 Hz, 1H), 7.24 (d, J = 6.8 Hz, 2H), 7.35 (m, 4H), 7.62 (m, 3H), 7.79 ( m, 4H), 8.25 (s, 1 H), 8.64 (m, 1 H).

1-4. Synthesis of Compound 7

Under nitrogen gas, Compound 5 (100 mg, 0.1 mM) was dissolved in dry THF and stirred at room temperature. Acetic acid (30 µl, 0.5 mM), tributyl tin hydride (85 µl, 0.3 mM) and Pd (PPh ₃ ) ₄ (10 mg) were added dropwise to this mixed solution, followed by stirring at room temperature for 1 hour. Tin hydride (85 μl, 0.3 mM) was further added dropwise and stirred for 2 hours. To this mixed solution was slowly added dropwise 1.25M HCl (2 mL) using methanol as a solvent, followed by stirring for 3 hours, followed by concentration under reduced pressure. The concentrated composition was crystallized with ether, the obtained material was dissolved in distilled water again, centrifuged (3000 rpm, 10 minutes) and the supernatant was lyophilized to give a pale yellow solid (61 mg, 67.1%).

mp 174.0-175.0 ° C .; FT-IR (cm- ¹ ,) 3271, 1653, 1521, 1375, 1244, 1156; ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ 0.99 (d, J = 6.8 Hz, 3H), 1.23-1.60 (m, 6H), 2.78 (m, 3H), 3.17 (m, 3H), 3.50 (m, 1H), 4.14 (m, 2H), 4.28 (m, 1H), 4.44 (m, 1H), 4.80 (m, 1H), 7.23-7.38 (m, 8H), 7.62 (m, 2H), 7.81 (m, 8 H), 8.25 (s, 1 H), 8.75 (m, 1 H).

<Example 2. Solubility test of arylsulfonylimidazolone derivative compound>

The arylsulfonylimidazolone derivative compounds of the present invention were measured for solubility in distilled water as compared to the parent compound, Compound 1. Solubility was measured by the method of Casinin et al. Intraocular Pressure-Lowering Agents with Long- Lasting Effects. J. Med. Chem. 2000, 43, 4884-92).

Dissolve 1 mg of each accurately weighed substance in MeOH (1 ml) for HPLC to make a standard sample solution.Then, the maximum UV absorbance (UV absorption maxium, λ _max in UV spectrophotometer Mini 1240, Shimadzu, Japan) was determined. After making a saturated aqueous solution for each material using a small amount of distilled water (250 μl), insoluble matter was removed by passing through a 0.45 μm syringe filter (Whatman, USA). The saturated aqueous solution thus prepared was scanned at the maximum absorption wavelength of each material determined using a standard sample to determine its absorbance. The aqueous solubility of each substance was determined by the following equation.

C ′ = (A ′ × C) ÷ A

Where C is the concentration of the standard solution (mg / ml), A is the absorbance of the standard solution, A 'is the absorbance of the saturated solution, and C' is the concentration of the aqueous solution (mg / ml).

compound
Solubility (mg / ml)
Compound 6
1.73
Compound 7
3.32
Compound 1 (Control)
0.024

As a result of Table 1, Compound 6 and Compound 7 of the present invention exhibited a significant solubility synergistic effect of about 70-150 times compared with Compound 1 (control), the parent drug, and through such experimental results plasmin Prodrugs activated by were found to be in a form that can be formulated into an injection.

<Example 3. Confirmation of cytotoxicity against human cancer cells in vitro-Confirmation of target by plasmin>

In particular, the arylsulfonylimidazolone derivative compounds of the present invention have a high plasmin expression of human breast cancer cell line (MDA-MB-231) and a low plasmin expression of human breast cancer cells (MCF-7). cell line) was confirmed for its cytotoxicity. The cell lines were distributed and used at the Institute of Biotechnology, and IC ₅₀ (μM) was measured by MTT assay.

The culture solution was a bag of DMEM medium containing L-glutamine in sterile injectable distilled water, 100 ml of bovine serum (FBS) inactivated by heat treatment for 30 minutes in a 60 ° C water bath, 33.7 g of NaHCO, penicillin (10000 units / ml), streptoco After dissolving mycin (10 mg / ml), the pH was adjusted to 0.1 L with 0.1 N hydrochloric acid to prepare 1 L and filtered. Cells were maintained while subcultured every three days, and a solution of trypsin (2.5 g / l) and EDTA (0.38 g / l) in PBS (phosphate buffered saline) solution was used to separate the cells from the adherent surface.

The cells to be used for the experiment were separated from the attachment surface with trypsin-EDTA solution and incubated for 24 hours in a 5% CO ₂ incubator (37 ° C.) with 5 × 10 ³ cells per well of a 96 well plate. The sample was dissolved in DMSO (dimethylsulfoxide) and diluted to the concentration required for the experiment by dilution with experimental medium or tertiary distilled water so that the final DMSO concentration was 0.2% or less. 2 ml of each diluted sample was added to each well of a 96 well plate, and then incubated for 72 hours in a 37 ° C., 5% CO ₂ incubator. After incubation, 20 ml of MTT solution (Thiazolyl blue tetrazolium bromide: 5 mg / ml in PBS, Sigma M2128) was added to the wells, followed by further 4 hours of incubation. To each well, 100 μl of a cell lysis buffer (10% SDS in 0.01 N HCl) was added again to completely dissolve the formazan produced by overnight cell lysis, followed by a microplate reader (570 nm). , Tecan A-5082, Salzburg, Austria) was used to measure the optical density (OD) of each well. This value was used to confirm the IC ₅₀ (50% inhibitory dose) of cancer cells of each substance. All experiments were performed at least three times with minor modifications to the MTT assay as described above. The cells were also cultured in the presence or absence of 400 KIU (kallikrein inhibitor units) of aprotinin (Sigma Aldrich, an inhibitor of plasmin's serine protease).

compound
Aprotinin
IC ₅₀ ([mu] M)
MDA-MB-231
MCF-7
Compound 6
-
0.274
7.021
+
5.253
8.959
Compound 7
-
0.316
5.991
+
4.329
7.314
Compound 1 (Control)
0.265
1.268

As can be seen from Table 2, the arylsulfonylimidazolone derivative compound of the present invention is a compound 1 (IC ₅₀ = 0.265 μM) as a control when aprotinin is not included in plasmin expressing MDA-MB-231 cells. It showed similar cytotoxicity as. On the other hand, when aprotinin is contained, its cytotoxicity (IC ₅₀ ) is 5.253 μM for compound 6 and 4.329 μM for compound 7, respectively, and it is 16-20 times more active than the parent compound due to the plasmin inhibitory effect of aprotinin. This decreased. In MCF-7 cells with low plasmin expression, compound 6 and compound 7 were 7.021 and 5.991 μM without aprotinin, respectively, and the activity was reduced by about 5 to 6 times compared to the control group, and compound 6 and compound 7 with aprotinin. Cytotoxicity of was 8.959μM, 7.314μM were all reduced by about 5-7 times compared to the control group. In MCF-7 cells, low plasmin expression, regardless of the presence of aprotinin, prevented the conversion of compounds 6 and 7 in prodrug form to compound 1, the parent drug. Through the results of the experiment, the arylsulfonylimidazolone derivative compound of the present invention selectively showed activity by plasmin of cancer cells, and showed cytotoxicity as strong as compound 1 (control) as a parent drug in plasmin-expressing cancer cells. You can check it.

Example 4 Mother Drug Conversion Experiments in Human Plasma and Human Plasmin

For the arylsulfonylimidazolone derivative compound 7 of the present invention, the conversion to the parent drug compound 1 in human plasma and plasmin was confirmed by kinetic experiments.

4-1. Human plasma

The human plasma used in the experiment was supplied from Chungnam National University Hospital, and the conversion test conditions in human plasma were as follows.

50 µl of a 1 mM solution of Compound 7 (0.1 M Tris / HCl buffer, pH 7.3) was previously mixed with 450 µl of cold human plasma (0 ° C.), and the plasma suspension was immediately transferred to a 37 ° C. incubator for reaction. Thereafter, 50 μl of this was collected at a predetermined time (0, 0.25, 0.5, 1, 2, 4, 8, 24 hours), and 450 μl of human plasma cooled in ice was added thereto. 250 μl of acetone was added thereto and then mixed vigorously and stored at -80 ° C. After 24 hours of reaction, samples were mixed with 50 µl of aqueous zinc sulfate solution (aqueous zinc sulfate 70%) and vortexed for 10 minutes and centrifuged (4000 g, 15 minutes). The separated supernatant was dried by evaporation with nitrogen and then diluted with 125 μl of distilled water to make 2 μM concentration, and the content of the parent drug was measured using HPLC. When the compound is completely converted into the parent drug, the parent drug content at each time period was calculated as a percentage, and the results are shown in Table 3 and FIG. 1.

compound
Time (hours)
Average peak area
Content (μM)
% Conversion




Compound 7
(Maximum concentration convertible to parent drug: 13.11 μM)
0.25
1507
0.27
2.03
0.5
3454
0.61
4.65
One
2929
0.52
3.94
2
3699
0.65
4.98
4
5015
0.89
6.75
8
4722
0.83
6.36
24
7906
1.40
10.64
Compound 1 (Mother Drug)
Average peak area
content
113325
20 μM

As can be seen in Table 3 and Figure 1, the arylsulfonylimidazolone derivative compound 7 of the present invention showed a conversion rate of about 10.64% by reaction for 24 hours. Because of this, compound 7 has a specificity that does not show a drug response in the environment that is not affected by cancer cells due to the low conversion rate from human plasma to the parent drug, which has a lower conversion rate than a compound with a previously applied linker. The compounds of the present invention were found to have stronger specificity for cancer cells.

4-2. Human plasmin

Human plasmin used in the experiment was purchased from Sigma (Plasmin from human plasma, EC 3. 4. 21. 7., ≥ 2.0 units / mg protein).

5 μl of a 1 mM (in 0.1 M Tris / HCl buffer, pH7.3) solution of Compound 7 and a plasmin solution (1 mg / l) in 983 μl (0 ° C.) of cold 0.1 M Tris / HCl buffer, pH7.3. in 0.1M Tris / HCl buffer, pH 7.3) was mixed and the plasmin mixed solution was immediately transferred to a 37 ° C. incubator for reaction. Thereafter, 50 μl of this was collected at predetermined times (0, 0.25, 0.5, 1, 2, 4, 8, 24 hours) and 450 μl of human plasma cooled on ice was added thereto. 250 μl of acetone was added thereto, followed by vigorous mixing and storage at −80 ° C. After 24 hours of reaction, samples were mixed with 50 µl of aqueous zinc sulfate solution (aqueous zinc sulfate 70%) and vortexed for 10 minutes and centrifuged (4000 g, 15 minutes). The separated supernatant was dried by evaporation with nitrogen and then diluted with 125 μl of distilled water to make 2 μM concentration, and the content of the parent compound was measured using HPLC. The results are shown in Table 4 and FIG.

compound
Time (minutes)
Average peak area
Content (μM)
% Conversion






Compound 7
(Maximum concentration that can be converted to parent drug: 1.05μM)
5
1828
0.32
30.75
15
2159
0.38
36.32
30
3108
0.55
52.29
45
2824
0.50
47.51
60
2476
0.44
41.66
120
3160
0.55
53.16
240
3331
0.588
56.04
480
4242
0.75
71.37
1440
4456
0.79
74.97
Compound 1 (Mother Drug)
Average peak area
content
11333
2 μM

As a result of Table 4 and FIG. 2, the arylsulfonylimidazolone derivative compound 7 of the present invention was found to have a high conversion rate of 74.97% to the parent compound 1 after 24 hours of mixing with human plasmin.

Through this, the arylsulfonylimidazolone derivative compound of the present invention can be confirmed that the human plasmin is very quickly converted to the compound 1, the parent drug.

In view of the comprehensive comparison with the results of the conversion in human plasma, the arylsulfonylimidazolone derivative compounds of the present invention are more selectively cleaved by human plasmin and rapidly converted into the parent drug, Compound 1, to give cancer cells. The target for is higher than before.

<Example 5. Confirmation of anticancer activity in vivo>

The arylsulfonyl imidazolone derivative compound 7 of the present invention was confirmed in vivo anticancer activity compared to the compound 1 as a parent drug.

As a tumor model, human colon adenocarcinoma cell SW620 (Human colorectal adenocarcinoma, SW620) was used, and the animals used in the experimental animals were 7-week-old female Balb / c nu / nu mice supplied by Orient Bio Co., Ltd. (Seongman, Korea). Was used. Animals were used for testing with a one-week acclimation period and were raised aseptically at 22 ° C.

Tumor cells SW620 were implanted subcutaneously into 2 × 10 ⁶ mice per mouse and grown until tumors grew to a certain size. When the tumor grew to 50 ~ 100mm ³ , the average tumor volume was separated so that the parent compound Compound 1 orally was administered orally, Compound 6 and Compound 7 was administered intravenously. Oral administration method was to administer a total of five times 70 mg / kg compound 1 once every two days. Intravenous administration was performed by dissolving Compound 6 and Compound 7 in sterile saline and intravenously administering 35 mg / kg once daily for 6 consecutive days.

When growth in weight of the tumor size and animal was weekly in duplicate measurement, tumor size calculation are as follows, based on the drug administration date primary (300mm ^3), second car (700mm ^{3), 3} car (1500mm ³⁾ The test was terminated.

Tumor volume (mm ³ ) = [length × (width ² )] ÷ 2

compound
Tumor growth inhibition rate 10 days after the start of administration
Compound 6 (Intravenous Administration)
90%
Compound 7 (Intravenous Administration)
87%
Compound 1 (Control, Oral Administration)
48%

Through Table 5, the arylsulfonylimidazolone derivative compounds of the present invention 10 days after the start of administration, the tumor growth inhibition rate of Compound 6 is 90%, Compound 7 is 87%, tumor growth inhibition rate of the orally administered control compound Compared with 48%, it showed very good inhibition rate.

In addition to this, the arylsulfonylimidazolone derivative compound 7 of the present invention is increased by more than three times the hydrolysis time by the enzyme in the body when administered intravenously, and is a stable prodrug until the drug is delivered to cancer cells. It is confirmed that it exists in the state and starts to be converted into the parent drug from reaching the cancer cells, and also increases the stability in the body. This is due to the lower stability of the linker linkages than the direct amide linkages.

Through this, the arylsulfonylimidazolone derivative compound of the present invention was administered through intravenous injection, which was more selectively targeted to cancer cells than the control compound, showed increased anticancer activity, and confirmed that the stability in the body was increased.

Claims (5)

Arylsulfonylimidazolone derivatives of formula (I)
(I)

In Formula I,
R is CH (CH ₃ ) ₂ or CH ₃
R ′ is CH ₂ CH (CH ₃ ) ₂ or CH ₂ Ph The method of claim 1,
R is CH (CH ₃ ) ₂ , R ′ is an arylsulfonylimidazolone derivative that is CH ₂ CH (CH ₃ ) ₂ . The method of claim 1,
R is CH ₃ , R ′ is an arylsulfonylimidazolone derivative that is CH ₂ Ph. Comprising a derivative of formula I according to claim 1,
Lung cancer, colorectal cancer, rectal cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, fallopian tube carcinoma, ovarian cancer, vaginal carcinoma, vulvar carcinoma, liver cancer, gastric cancer, esophageal cancer, small intestine cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, urethral cancer , Penile cancer, prostate cancer, testicular cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, non-small cell lung cancer, bone cancer, skin cancer, head or neck cancer, skin or ocular melanoma, Hodgkin's disease, endocrine adenocarcinoma, chronic or acute A pharmaceutical composition for the treatment of a disease selected from the group consisting of leukemia, lymphocytic lymphoma, central nervous system tumor, spinal cord tumor, brain stem glioma and pituitary adenoma. The method of claim 4, wherein
The cancer is a pharmaceutical composition, characterized in that due to cancer cells with existing anticancer drug resistance.

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