WO2017191893A1 - Conjugate of primary amine and diosgenin, preparation method therefor, and anticancer composition comprising same - Google Patents

Abstract

The present invention relates to a conjugate in which a primary amine is coupled to diosgenin via a linker, a preparation method therefor, and an anticancer composition comprising the same. With the introduction of a linear hydrophilic primary amine into the 3-OH position of diosgenin, the conjugate has the effect of exhibiting improved penetrability into a cancer cell membrane and thus significantly increasing in anticancer activity compared to diosgenin itself.

Description

Primary amines and diosgenin conjugates, preparation methods thereof, and anticancer compositions comprising the same

The present invention relates to a conjugate in which a primary amine and a diosgenin are linked with a linker, a method for preparing the same, and an anticancer composition comprising the same, and according to the introduction of a hydrophilic linear primary amine at the 3-OH position of the diosgenin, Since cancer cell membrane permeability is improved, anticancer activity is remarkably improved compared to dioxenin itself.

Cancer is the most common disease with circulatory disease in Korea. Normally, cells divide, grow and die by intracellular regulation, and live in balance of cell numbers. When these cells are damaged, abnormal growth and suppression of abnormal cells become uncontrolled and excessive proliferation, as well as invading surrounding tissues and organs, resulting in mass formation and destruction of normal tissues. This condition is defined as cancer or cancer.

Tumors include benign and malignant tumors, and benign tumors grow relatively slowly and are tumors that do not spread, metastasize, or can be removed and healed in many parts of the body. It does not cause it. Malignant tumors, on the other hand, are tumors that are life-threatening by rapid growth, invasive (digging or spreading) growth, and spreading metastases (moving away from their original location) to each part of the body.

The mechanism by which cancer cells develop is that the cells undergo growth, differentiation, or programmed apoptosis, or the growth is stopped, but some of the genes in the cells are abnormal. It is thought that the characteristics of protein, a product of genes, are changed, and as a result, abnormality in cell growth regulation occurs and cancer cells are produced.

In drug development, natural ingredients are a major source of new anticancer drugs. 17% of new drugs approved annually by the FDA between 2000 and 2013 are new drugs derived from natural materials.

Phytosterols, in their natural form, are gaining worldwide attention in the fields of medicinal chemistry and biochemistry. The use of phytosterols for the development of new drugs has shown that biocompatibility, biodegradability, various physiological activities, high cell membrane permeability, and simple modifications. This is because the process has the advantage of high potential as a therapeutic agent.

Diosgenin is a naturally occurring steroidal saponin, mainly isolated from legumes and yams, and exhibits high biocompatibility and various pharmacological activities.

Over the years, diosgenin has been studied for therapeutic effects such as cancer, stroke, Alzheimer's disease, diabetes, osteoporosis, and anti-inflammatory. However, clinical results show that diosgenin has shown weak activity, short half-life and low pharmacokinetics, despite various beneficial properties. Diosgenin has also been shown to be ineffective in treating resistant cancers, as it has been proven to be a p-glycoprotein (extracting drugs from intestinal cells).

Therefore, a situation that requires a simple but effective modification of the diosgenin to increase the activity.

Accordingly, the present inventors have an effect of introducing a hydrophilic linear primary amine at the 3-OH substituent position of diosgenin (DG) to improve cancer cell membrane permeability, thereby significantly improving anticancer activity compared to diosgenin itself. It was found that the present invention was completed.

[Preceding technical literature]

(Non-Patent Document 1) Nutrients 2015, 7, 4938-4954

It is an object of the present invention to provide a conjugate in which a hydrophilic linear primary amine and a dioxenin are linked by a linker, which has a markedly improved anticancer activity compared to diosgenin.

In order to achieve the above object,

The present invention provides a conjugate wherein a linear primary amine and Diosgenin are linked with a linker, or a pharmaceutically acceptable salt thereof.

The conjugate in which the linear primary amine and diosgenin are linked with a linker is represented by the following Chemical Formula 4 or Chemical Formula 6.

[Formula 4]

In Chemical Formula 4,

n is an integer of 0-2.

[Formula 6]

In Chemical Formula 6,

R is

,

,

,

,

or

to be.

In addition, the present invention as shown in Scheme 1,

Reacting diosgenin (DG) with p -4-toluenesulfonyl chloride in an organic solvent to obtain compound 1 (step 1);

Reacting compound 1 with ethylene glycol in an organic solvent to obtain compound 2 (step 2);

Reacting compound 2 with p -4-toluenesulfonyl chloride in an organic solvent to obtain compound 3 (step 3); And

Compound 3 is reacted with a primary amine in an organic solvent to obtain compound 4 (step 4).

Scheme 1

In Scheme 1, n is an integer of 0-2.

Furthermore, the present invention, as shown in Scheme 2 below,

Reacting diosgenin with triphosgene in an organic solvent to obtain compound 5 (step 1); And

Compound 5 is reacted with a primary amine in an organic solvent to produce compound 6 (Step 2).

Scheme 2

In Scheme 2,

R is

,

,

,

,

or

to be.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, containing the conjugate, or a pharmaceutically acceptable salt thereof as an active ingredient.

Conjugates in which the hydrophilic linear primary amine and the diosgenin are linked with a linker according to the present invention, as the hydrophilic linear primary amine is introduced at the 3-OH position of the diosgenin, the cancer cell membrane permeability is improved and anticancer activity is increased. Compared with diosgenin itself, there is a markedly improved effect.

Hereinafter, the present invention will be described in detail.

The present invention provides a conjugate wherein a linear primary amine and Diosgenin are linked with a linker, or a pharmaceutically acceptable salt thereof.

The linear primary amines are ethylenediamine (EDA), diethylenetriamine (DETA), amine, 2,2'-dithio (ethylamine), tetramethylenediamine, N1, N2, N3-tris ( tert-butoxycarbonyl) -triethylenetetraamine, triethylenetetraamine (TETA) and the like can be used.

The linker is

or

Wherein X is a linear primary amine and DG is diosgenin.

Preferably, the conjugate in which the linear primary amine and Diosgenin are linked with a linker is a compound represented by the following Chemical Formula 4 or Chemical Formula 6.

[Formula 4]

In Chemical Formula 4,

n is an integer of 0-2.

[Formula 6]

In Chemical Formula 6,

R is

,

,

,

,

or

to be.

Conjugates in which the hydrophilic linear primary amine and the diosgenin are linked with a linker according to the present invention, as the hydrophilic linear primary amine is introduced at the 3-OH position of the diosgenin, the cancer cell membrane permeability is improved and anticancer activity is increased. Compared with diosgenin itself, there is a remarkably improved effect (Experimental Example 1 and Table 1).

Conjugates in which the linear primary amine and the diosgenin of the present invention are linked with a linker may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid moiety formed by a pharmaceutically acceptable free acid. Salting is useful. The expression pharmaceutically acceptable salts is a concentration that is relatively nontoxic and harmless to the patient, and the side effects caused by these salts do not diminish the beneficial efficacy of conjugates with linear primary amines and diosgenin-linked linkers. Non linear or any organic or inorganic addition salt of a conjugate wherein the primary amine and diosgenin are linked with a linker.

These salts may include inorganic acids and organic acids as free acids, hydrochloric acid, bromic acid, nitric acid, sulfuric acid, perchloric acid, phosphoric acid, and the like, and citric acid, acetic acid, lactic acid, maleic acid, and fumarine as organic acids. Acids, Gluconic Acid, Methanesulfonic Acid, Glyconic Acid, Succinic Acid, Tartaric Acid, Galluturonic Acid, Embonic Acid, Glutamic Acid, Aspartic Acid, Oxalic Acid, (D) or (L) Malic Acid, Maleic Acid, Methanesulphonic Acid, Ethene Sulfur Phonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid or malonic acid and the like can be used.

These salts also include alkali metal salts (sodium salts, potassium salts, and the like), alkaline earth metal salts (calcium salts, magnesium salts, and the like) and the like. For example, acid addition salts include acetates, aspartates, benzates, besylates, bicarbonates / carbonates, bisulfates / sulfates, borates, camsylates, citrates, disylates, ecylates, formates, fumarates, Gluceptate, Gluconate, Glucuronate, Hexafluorophosphate, Hibenzate, Hydrochloride / Chloride, Hydrobromide / Bromide, Hydroiodide / Iodide, Isetionate, Lactate, Maleate, Mali Eate, malonate, mesylate, methylsulfate, naphthylate, 2-naphsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, saccha Laterate, stearate, succinate, tartrate, tosylate, trifluoroacete , Aluminum, arginine, benzatin, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, zinc salts, and the like. Heavy hydrochloride or trifluoroacetate are preferred.

The acid addition salt according to the present invention can be prepared by conventional methods, for example, a conjugate of a linear primary amine and a diosgenin conjugated with a linker, using an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, or the like. The precipitate produced by dissolving and adding an organic or inorganic acid may be prepared by filtration and drying, or may be prepared by distillation under reduced pressure of a solvent and an excess of acid and drying or crystallization under an organic solvent.

Bases can also be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding silver salts are also obtained by reacting alkali or alkaline earth metal salts with a suitable negative salt (eg, silver nitrate).

Furthermore, the present invention encompasses not only the conjugates in which the linear primary amines and diosgenin are linked with linkers and pharmaceutically acceptable salts thereof, but also all possible solvates, hydrates, isomers and the like that can be prepared therefrom. .

Preparation method 1

As the present invention is shown in Scheme 1,

By reaction with p-toluenesulfonyl chloride, the DIOS -4- angiogenin (DG) in an organic solvent to afford a compound 1 (step 1);

Reacting compound 1 with ethylene glycol in an organic solvent to obtain compound 2 (step 2);

Reacting compound 2 with p -4-toluenesulfonyl chloride in an organic solvent to obtain compound 3 (step 3); And

Compound 3 is reacted with a primary amine in an organic solvent to obtain compound 4 (step 4).

Scheme 1

In Scheme 1,

n is an integer of 0-2.

In Preparation 1 according to the present invention, Step 1 is a step of obtaining Compound 1 by reacting diosgenin (DG) with p -4-toluenesulfonyl chloride in an organic solvent.

Specifically, as the solvent usable in step 1, pyridine, dichloromethane (DCM), 1,4-dioxane, dimethylformamide (DMF), tetrahydrofuran (THF), etc. may be used alone or in combination. Preferably, pyridine and dichloromethane (DCM) can be mixed and used. The reaction temperature may be -10 to 25 ℃, preferably 0-5 ℃. The reaction time may be 10 minutes to 3 hours.

In Preparation 1 of the present invention, Step 2 is a step of obtaining Compound 2 by reacting Compound 1 with ethylene glycol in an organic solvent.

Specifically, as the solvent usable in step 2, pyridine, dichloromethane (DCM), 1,4-dioxane, dimethylformamide (DMF), tetrahydrofuran (THF), etc. may be used alone or in combination. Preferably, 1, 4- dioxane can be used. The reaction temperature may be 70-110 ° C., preferably 80-100 ° C. The reaction time may be 1-5 hours, preferably 2-4 hours.

In Preparation 1 according to the present invention, Step 3 may be carried out in the same manner as in Step 1 to obtain Compound 3 by reacting Compound 2 with p -4-toluenesulfonyl chloride in an organic solvent.

In Preparation 1 of the present invention, Step 4 is a step of obtaining Compound 4 by reacting Compound 3 with a primary amine in an organic solvent.

Specifically, as the solvent usable in step 4, pyridine, dichloromethane (DCM), 1,4-dioxane, dimethylformamide (DMF), tetrahydrofuran (THF), etc. may be used alone or in combination. Preferably, dimethylformamide (DMF) can be used. The reaction temperature may be 10-35 ° C., preferably 20-25 ° C. The reaction time may be 10-30 hours, preferably 20-26 hours.

Preparation method 2

As the present invention is shown in Scheme 2,

Reacting diosgenin with triphosgene in an organic solvent to obtain compound 5 (step 1); And

Compound 5 is reacted with a primary amine in an organic solvent to produce compound 6 (Step 2).

Scheme 2

In Scheme 2,

R is

,

,

,

,

or

to be.

In Preparation 2 according to the present invention, Step 1 is a step of obtaining compound 5 by reacting diosgenin with triphosgene in an organic solvent.

Specifically, solvents usable in step 1 include pyridine, dichloromethane (DCM), 1,4-dioxane, dimethylformamide (DMF), tetrahydrofuran (THF), triethyleneamine (TEA), and the like. Or may be used in combination, preferably pyridine and tetrahydrofuran (THF) may be mixed and used. The reaction temperature may be 1-30 ° C., preferably 15-25 ° C. The reaction time may be 10-24 hours, preferably 14-18 hours.

In Preparation 2 according to the present invention, Step 2 is a step of preparing Compound 6 by reacting Compound 5 with a primary amine in an organic solvent.

Specifically, solvents usable in step 2 include pyridine, dichloromethane (DCM), 1,4-dioxane, dimethylformamide (DMF), tetrahydrofuran (THF), triethyleneamine (TEA), and the like. Or may be used in combination, preferably dichloromethane (DCM) may be used. The reaction temperature may be -10 to 25 ℃, preferably 0-5 ℃. The reaction time may be 0.5-5 hours, preferably 1-2 hours.

Usage

The present invention provides a pharmaceutical composition for preventing or treating cancer, containing the conjugate of the linear primary amine and diosgenin as a linker, or a pharmaceutically acceptable salt thereof as an active ingredient. The cancer may be prostate cancer, cervical cancer, breast cancer, colon cancer, blood cancer, liver cancer, and the like.

In addition, the present invention provides a method for administering a conjugate in which the linear primary amine and Diosgenin are linked with a linker, or a pharmaceutically acceptable salt thereof, to a patient in need of cancer treatment in a therapeutically effective amount. It provides a method for treating cancer comprising the step.

Furthermore, the present invention provides the use of a conjugate in which the linear primary amine and Diosgenin are linked with a linker, or a pharmaceutically acceptable salt thereof, in the manufacture of a cancer therapeutic agent.

In the conjugate according to the present invention, the introduction of a hydrophilic linear primary amine at the 3-OH position of the diosgenin improves cancer cell membrane permeability, thereby significantly improving anticancer activity.

The conjugate according to the present invention may be administered in various formulations orally and parenterally during clinical administration, and when formulated, a diluent or excipient such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used, may be used. Are prepared using.

Solid form preparations for oral administration include tablets, patients, powders, granules, capsules, troches, and the like, which form at least one excipient such as starch, calcium carbonate, water, or the like. It is prepared by mixing cross, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, or syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.

In addition, the effective dosage of the conjugate according to the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, generally about 0.001 to 100 mg / kg / day It is preferably 0.01 to 35 mg / kg / day. Based on an adult patient with a weight of 70 kg, it is generally 0.07 ~ 7000 mg / day, preferably 0.7 ~ 2500 mg / day, once a day at regular intervals depending on the judgment of the doctor or pharmacist Multiple doses may be administered.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited by the following examples.

The compound of Example 1-3 was prepared using the following scheme.

<Example 1> (3α, 25R) - Preparation of ethylenediamine (4a) - spy roast-5-en-3-oxy-ethane - N1, N3

Step 1: Preparation of Compound 1

Anhydrous dichloromethane (DCM) solution in which diosgenin (200 mg, 0.48 mmol, 1 equiv) and p-toluenesulfonylchloride (230 mg, 1.21 mmol, 2.5 equiv) was dissolved in pyridine (97 ul, 1.2 mmol, 2.5 equiv) was added slowly to the solution for 15 min. After addition to pyridine, the reaction mixture was raised to room temperature and stirred for 3 hours under shading. The solvent was removed under reduced pressure and the remaining mixture was precipitated by addition of 5% HCl solution, filtered and washed with cold 5% HCl solution (3 × 25 ml) and distilled water (2 × 50 ml). The resulting solid was dissolved in DCM, dried over MgSO ₄ , filtered, the solvent was evaporated and then Compound 1 was obtained, which was used directly in the next step.

Step 2: Preparation of Compound 2

Compound 1 (200 mg, 0.48 mmol) obtained in Step 1 was added to 1,4-dioxane (2 ml), and the stirred solution was treated with anhydrous ethylene glycol (0.65 ml, 12 mmol) and 3 hours under a nitrogen atmosphere. Reflux for a while. The reaction solution was cooled and the solvent was removed by evaporation. A light yellow solid was obtained, which was dissolved in DCM and subsequently washed with NaHCO ₃ solution (2 × 10 ml), water (10 ml) and brine (20 ml), dried over MgSO 4, filtered and under reduced pressure. The crude product obtained by concentration was purified by flash chromatography to give compound 2 in 89% yield.

Step 3: Preparation of Compound 3

Compound 3 was prepared in the same manner as in Step 1, except that Compound 2, obtained in Step 2, was used as a starting material instead of the starting material, dioxenin, in Step 1.

Step 4: Preparation of the target compound 4a.

Dimethyl formamide (DMF) solution in which cesium hydroxide monohydrate is dissolved is added to activated powdered dry 4 molecular sieves and ethylenediamine (EDA), and then the compound obtained in step 3 DMF solution in which 3 was dissolved was added slowly and vigorously stirred at room temperature overnight. The reaction was terminated by addition of 1N NaOH solution (10 ml) to the reaction mixture and filtered to remove solids. The reaction solution was concentrated under reduced pressure to give a solid, which was dissolved in a small amount of methanol again, triturated with ethyl ether to remove inorganic salts, and then dried under vacuum. The crude product was purified by flash chromatography to give the title compound 4a.

¹ H NMR (300 MHz, CDCl ₃ ) δ 5.33 (1H, d, J = 5.03 Hz), 4.42 (1H, dd, J = 7.56, 14.52 Hz), 3.61 (1H, t, J = 5.59 Hz, 10.88 Hz ), 3.49 (2H, dd, J = 4.71, 11.04 Hz), 3.37 (1H, t, J = 11.04 Hz,), 3.16 (1H, m), 2.84 (4H, m), 2.72 (2H, t, J = 6.05, 12.06 Hz), 1.02 (3H, s), 0.97 (3H, d, J = 6.86 Hz), 0.78 (3H, s), 0.78 (3H, d, J = 6.28 Hz);

¹³ C NMR (300 MHz, CDCl ₃ ) δ 140.7, 121.5, 109.3, 80.8, 79.5, 77.4, 76.6, 69.6, 66.8, 65.4, 62.0, 56.5, 50.0, 41.6, 40.2, 39.7, 38.8, 37.0, 40.0, 32.0 , 31.8, 31.4, 30.3, 28.8, 28.3, 20.8, 19.3, 17.1, 16.3, 14.5;

Calcd for formula: C ₃₁ H ₅₂ N ₂ O ₃ , 500.76.

<Example 2> (3α, 25R) - Preparation of diethylene tri-amine (4b) - spy roast-5-en-3-oxy-ethane - N1, N3, N6

Example 4 was prepared in the same manner as in Example 1, except that diethylenetriamine (DETA) was used instead of ethylenediamine (EDA) in Step 4 of Example 1 to prepare a target compound 4b.

¹ H NMR (300 MHz, CDCl ₃ ) δ 5.34 (1H, d, J = 4.71 Hz), 4.67 (4H, s) 4.42 (1H, dd, J = 7.33, 14.42 Hz), 3.76 (2H, t), 3.48 (1H, dd, J = 4.71, 11.04 Hz), 3.37 (1H, t, J = 11.04 Hz,), 3.10 (1H, m), 2.95 (11H, m), 2.32 (1H, dd, J = 5.14 , 13.03 Hz), 2.16 (1H, t), 1.02 (3H, s), 0.97 (3H, d, J = 6.86 Hz), 0.78 (3H, s), 0.78 (3H, d, J = 6.28 Hz);

¹³ C NMR (300 MHz, CDCl ₃ ) δ 144.7, 140.5, 133.1, 129.7, 128, 121.5, 109.3, 80.8, 79.5, 77.4, 76.6, 69.6, 66.8, 65.4, 62.0, 56.5, 50.0, 41.6, 40.2, 39.7 , 38.8, 37.0, 36.9, 32.0, 31.8, 31.4, 30.3, 28.8, 28.1, 21.6, 20.8, 19.3, 17.1, 16.2, 14.5;

Calcd for formula: C ₃₃ H ₅₇ N ₃ O ₃ , 543.82.

< Example  3> ( 3α, 25R )- Spy roast -5-Yen-3- Oxyethanamine  Preparation of (4c)

Anhydrous DMF solution in which compound 3 (200 mg, 0.33 mmol) obtained in step 3 of Example 1 was dissolved was added to sodium azide (31.82 mg, 0.49 mmol), and the reaction solution was stirred at 50 ° C. for 14 hours. . After dilution with acetylacetate (20 ml), the reaction mixture was washed with distilled water (25 ml) and saturated brine (2 x 20 ml). The organic layer was dried over anhydrous MgSO ₄ , filtered and concentrated to give a (3α, 25R) -spirost-5-ene-3-oxyethanenitrile compound.

The azide intermediate (100 mg, 0.21 mmol) was then dissolved in THF (10 ml) and treated with triphenylphosphine (135.8 mg, 0.52 mmol). The reaction mixture was stirred at room temperature for 10 hours, treated with distilled water (0.1 ml), after 48 hours the solvent was removed under reduced pressure and the crude product was purified by flash chromatography to give the desired compound 4c as a white powder in 94% yield. Got it.

¹ H NMR (300 MHz, CDCl ₃ ) δ 5.34 (1H, d, J = 5.14 Hz), 4.40 (1H, dd, J = 7.41, 14.75 Hz), 3.64 (1H, t, J = 5.44 Hz), 3.59 (1H, dt, J = 1.14, 4.66 Hz), 3.43 (2H, dd, J = 4.71, 11.04 Hz), 3.37 (3H, t, J = 11.04 Hz,), 3.15 (1H, m), 2.88 (1H , t, J = 6.08, 11.82 Hz), 2.79 (1H, m), 2.39 (1H, dd, J = 5.14, 13.03 Hz), 2.20 (1H, dt), 1.02 (3H, s), 0.97 (3H, d, J = 6.86 Hz), 0.78 (3H, s), 0.78 (3H, d, J = 6.28 Hz);

¹³ C NMR (300 MHz, CDCl ₃ ) δ 140.8, 121.3,109.2, 80.8, 79.2, 77.4, 76.8, 69.2, 66.8, 62.0, 56.5, 50.0, 41.6, 40.2, 39.7, 39.0, 37.1, 37.0, 32.1, 32.0 , 31.4, 31.3, 30.3, 28.8, 28.3, 20.8, 19.4, 17.0, 16.3, 14.5;

Calcd for formula: C ₂₉ H ₄₇ NO ₃ , 457.69.

The compound of Example 4-9 was prepared using the following scheme.

<Example 4> (3α, 25R) - Preparation of roast spy-5-en-3-yl-oxy-carbonyl-1,2-diamino ethane (6a)

Step 1: Preparation of Compound 5

(5 mL) Triphosgene (213.9 mg, 0.72 mmol) was added to anhydrous THF, and 8 mL of diosgenin (600 mg, 1.44 mmol) was added to an ice bath, followed by reaction at room temperature (1-30 ° C.). Next, 0.1 mL of anhydrous pyridine diluted with anhydrous THF (2 mL) was added dropwise to the reaction solution over 1 hour. The precipitate was removed by filtration, and the supernatant was concentrated under vacuum to give a white solid. Foreign substance compound 5 was used in the next step of the reaction without purification.

Step 2: Preparation of the target compound 6a

Dioxenyl chloroformate (Compound 5) (200 mg, 0.419 mmol, 1 equivalent) obtained in Step 1 was added to anhydrous DCM (5 ml) under a nitrogen atmosphere in an ice bath, and the resulting solution was stirred. The reaction mixture was slowly added to anhydrous DCM (5 ml) solution in which aminoethane (EDA) (4.19 mmol, 20 equiv) was dissolved for 10 hours and kept at room temperature for 3 hours to warm to room temperature. The resulting mixture was diluted with DCM (15 m), washed sequentially with water, saturated aqueous NaCl solution (2 × 25 ml) and dried over anhydrous MgSO ₄ . The resulting mixture was filtered and dried to afford the crude product as a white solid, which was purified by flash chromatography to afford the desired compound 6a.

¹ H NMR (300 MHz, CDCl ₃ ) δ 5.82 (1H, s), 5.35 (1H, d, J = 5.14 Hz, H-6), 4.42 (2H, m), 3.49-3.31 (4H, t, J = 4.66 Hz,), 3.12 (2H, dt), 2.31 (1H, dd, J = 5.14, 13.03 Hz), 2.20 (1H, dt), 1.02 (3H, s), 0.97 (3H, d, J = 6.86 Hz,), 0.78 (3H, s), 0.78 (3H, d, J = 6.28 Hz);

¹³ C NMR (300 MHz, CDCl ₃ ) δ 156.5, 153.7, 140.8, 139.7, 139.5, 122.2, 121.3, 109.7, 109.2, 80.7, 77.4, 77.2, 76.6, 74.3, 71.5, 66.8, 62.0, 56.4, 49.8, 45.8 , 42.2, 41.6, 42.2, 40.2, 39.7, 38.5, 36.9, 36.6, 32.0, 31.8, 31.3, 30.2, 30.0, 29.6, 28.8, 28.1, 22.4, 20.8, 19.3, 17.1, 16.2, 14.5, 14.5;

Calcd for formula: C ₃₀ H _{4 8} N ₂ O ₄ , 500.71.

<Example 5> (3α, 25R) - Preparation of roast spy-5-en-3-yl-oxy-carbonyl-2,2'-diamino-diethyl amine (6b)

Example 2 was carried out in the same manner as in Example 4, except that 2,2'-diaminodiethylamine (DETA) was used instead of 1,2-diaminoethane (EDA) in Step 2 of Example 4 to obtain the target compound 6b. Was prepared.

¹ H NMR (300 MHz, CDCl ₃ ) δ 5.35 (1H, d, J = 5.14 Hz), 5.13 (1H, s) 4.49 (1H, dd, J = 7.41, 14.75 Hz), 3.71 (2H, t, J = 4.66 Hz), 3.59 (2H, dt, J = 1.14, 4.66 Hz), 3.46 (1H, dd, J = 4.71, 11.04 Hz), 3.37 (1H, t, J = 11.04 Hz), 3.20 (1H, m ), 2.39 (1H, dd, J = 5.14, 13.03 Hz), 2.20 (1H, dt, 2.62, 12.14 Hz), 1.02 (3H, s), 0.97 (3H, d, J = 6.86 Hz), 0.78 (3H , s), 0.78 (3H, d, J = 6.28 Hz);

¹³ C NMR (300 MHz, CDCl ₃ ) δ 156.3, 139.8, 122.3, 109.2, 80.6, 77.4, 77.2, 76.5, 74.1, 66.8, 62.0, 56.4, 49.9, 48.8, 41.5, 40.2, 39.7, 36.7, 32.0, 31.8 , 31.4, 30.3, 20.8, 19.3, 17.1, 16.3, 14.5;

Calcd for formula: C ₃₂ H ₅₃ N ₃ O ₄ , 543.78.

<Example 6> (3α, 25R) - Preparation of roast spy-5-en-3-yl-oxy-carbonyl-2,2'-thio-bis die ethanamine (6c)

Example 2, except that 2,2'-dithiobis (ethylamine) (synonymous 'cystamine') instead of 1,2-diaminoethane (EDA) in step 2 of Example 4 The target compound 6c was prepared.

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.35 (1H, s), 5.38 (1H, d, J = 3.91 Hz), 4.44-4.39 (2H, dd, J = 7.41, 14.75 Hz), 3.41-3.31 ( 6H, m), 3.12 (1H, bs), 2.87-2.46 (3H, m), 2.39 (1H, dd, J = 5.14, 13.03 Hz), 2.20 (1H, dt, 2.62, 12.14 Hz), 1.02 (3H , s), 0.97 (3H, d, J = 6.86 Hz), 0.78 (3H, s), 0.78 (3H, d, J = 6.28 Hz);

¹³ C NMR (300 MHz, CDCl ₃ ) δ 155.9, 139.7, 122.2, 109.2, 80.7, 77.4, 76.6, 74.6, 66.8, 62.0, 60.3, 56.4, 49.8, 42.4, 41.5, 40.5, 40.2, 39.7, 39.5, 38.6 , 38.1, 36.9, 36.6, 32.0, 31.8, 31.3, 28.7, 28.1, 21.0, 20.8, 19.3, 17.1, 16.2, 14.5, 14.2;

Calcd for formula: C ₃₂ H ₅₂ N ₂ O ₄ S ₂ 592.90.

<Example 7> (3α, 25R) - Preparation of roast spy-5-en-3-yl-oxy-carbonyl-1,2-diamino butane (6d)

Example 2 was prepared in the same manner as in Example 4, except that tetramethylenediamine (synonymous 'putrescine') was used instead of 1,2-diaminoethane (EDA) in Step 2 of Example 4, to thereby prepare 6d. .

¹ H NMR (300 MHz, CDCl ₃ ) δ 5.38 (1H, d, J = 6.21 Hz), 4.79 (1H, s), 4.48 (1H, m), 4.48-4.45 (1H, m, J = 6.79, 14.71 Hz), 4.3-4.34 (1H, m), 3.19-3.17 (2H, m) 2.75-2.71 (2H, t), 2.38 (1H, dd, J = 5.14, 13.03 Hz), 2.20 (1H, dt), 1.02 (3H, s), 0.97 (3H, d, J = 6.86 Hz), 0.78 (3H, s), 0.78 (3H, d, J = 6.28 Hz);

¹³ C NMR (300 MHz, CDCl ₃ ) δ 156.3, 140.1, 122.4, 109.5, 80.1, 77.7, 77.5, 76.6, 74.3, 71.5, 67.1, 62.3, 56.7, 50.2, 41.9, 40.5, 38.8, 37.2, 36.9, 32.3 , 32.1, 31.6, 30.5, 30.0, 21.1, 19.6, 17.4, 16.5, 14.8;

Calcd for formula: C ₃₂ H ₅₂ N ₂ O ₄ , 528.77.

<Example 8> (3α, 25R) - spy roast-5-en-3-yl-oxy-carbonyl - (N1, N2, N3 - Tris (tert- butoxy-carbonyl) - triethylene tetraamine (6e Manufacturing

Step 1: Preparation of Compound 5

Compound 5 was prepared in the same manner as Step 1 of Example 4 above.

Step 2: Preparation of the target compound 6e

Dry the dioszenyl chloroformate (Compound 5) (200 mg, 0.419 mmol, 1 equiv) and triethyleneamine (TEA) (87.7 ml, 0.629 mmol, 1.5 equiv) obtained in step 1 under nitrogen atmosphere in an ice bath (5 ml) was added to the mixture and stirred to treat the mixed solution with N1, N2, N3-tris (tert-butoxycarbonyl) -triethylenetetraamine (196.47 mg, 0.44 mmol, 1.05 equiv) and then 3 hours The reaction mixture obtained at room temperature was diluted with DCM (20 ml), washed with saturated aqueous NaCl solution (3 × 25 ml) and dried over anhydrous MgSO ₄ . The resulting mixture was filtered and dried to dissolve the desired product (300 mg, 0.338 mmol, 1 equiv) in dry DCM (5 ml) and treated with excess trifluoroacetic acid in an ice bath under nitrogen atmosphere. The reaction mixture was left at room temperature for 4 hours, the solvent was removed under reduced pressure, and then the residue was washed twice with dry ether to afford the desired compound 6e as a pale yellow amorphous powder.

¹ H NMR (300 MHz, CDCl ₃ ) δ 5.38 (1H, d, J = 5.21 Hz), 4.44-4.37 (2H, dd, J = 7.41, 14.75 Hz), 3.49-3.46 (2H, dd, J = 11.48 , 4.59 Hz), 3.41-3.31 (14H, bs), 2.31 (1H, dt), 1.02 (3H, s), 0.97 (3H, d, J = 6.86 Hz), 0.78 (3H, s), 0.78 (3H , d, J = 6.28 Hz);

¹³ C NMR (300 MHz, CDCl ₃ ) δ 155.9, 133.7, 109.3, 80.8, 77.4, 76.6, 71.5, 67.1, 66.8, 62.1, 56.9, 56.4, 49.9, 45.5, 41.6, 40.2, 39.7, 38.5, 36.7, 32.0 , 31.4, 30.3, 28.4, 27.6, 19.3, 17.1, 16.3, 14.5;

Calcd for formula: C ₄₉ H ₈₂ N ₄ O ₁₀ , 887.20.

<Example 9> (3α, 25R) - Preparation of triethylene Te trad amine (6f) - spy roast-5-en-3-yl-butyloxycarbonyl

Example 2 was carried out in the same manner as in Example 8 except that triethylenetetraamine (TETA) was used instead of N1, N2, N3-tris (tert-butoxycarbonyl) -triethylenetetraamine in Step 2 of Example 8. The desired compound 6f was prepared.

¹ H NMR (300 MHz, CDCl ₃ ) δ 5.38 (1H, d, J = 5.21 Hz), 4.89 (1H, bs), 3.65 (2H, m), 3.45-3.21 (12H m), 2.31 (1H, dt , 2.62, 12.14 Hz), 1.02 (3H, s, H-19), 0.97 (3H, d, J = 6.86 Hz), 0.78 (3H, s), 0.78 (3H, d, J = 6.28 Hz);

¹³ C NMR (300 MHz, CDCl ₃ ) δ 156.6, 139.8, 122.2, 109.2, 80.8, 77.4, 77.5, 76.6, 74.3, 71.5, 66.8, 62.0, 56.4, 50.4, 49.9, 41.6, 40.2, 39.7, 36.7, 32.0 , 31.8, 31.3, 30.2, 28.8, 20.8, 19.3, 17.1, 16.3, 14.5;

Calcd for formula: C ₃₄ H ₅₈ N ₄ O ₄ , 586.85.

Experimental Example 1 Cytotoxicity Evaluation

In order to determine the anticancer activity of the compound prepared in Example 1-9, it was tested as follows.

Specifically, HepG2 (human hepatocellular carcinoma), Hela (human cervical carcinoma), HT-29 (human colon carcinoma) and PC-3 (prostate carcinoma) cell lines were cultured in DMEM (10% FBS, 0.1 g / L penicillin) culture medium. In a humid environment, 37 ℃, 5% CO ₂ incubated in the atmosphere was used. MCF-7 (human breast carcinoma) and K562 (T-lymphoblastic leukemia) cell lines were used in culture in RPMI-1640 culture medium. The cell lines were aliquoted into 96-well plates (1.0 × 10 ⁵ cells / well), and 48 hours after treatment of the compounds of Examples 1-9, 10 μl of MTT solution (5 mg / ml) dissolved in PBS buffer ), Incubated for 4 hours, then the medium was removed and replaced with 100 μl of isopropanol at room temperature. Absorbance was quantified in each well using a microplate reader (570 nm wavelength), and the IC ₅₀ values of the compounds of Examples 1-9 were obtained. The results are shown in Table 1 below.

compound	IC 50 (μM) for cell line
	PC-3 (prostate carcinoma)	Hela (Human Cervical Carcinoma)	MCF-7 (Human Breast Carcinoma)	HT-29 (human colon carcinoma)	K562 (T-lymphoblastic leukemia)	HepG2 (Human Hepatocellular Carcinoma)
Example 1 (4a)	6.43	3.21	11.79	17.86	8.07	8.51
Example 2 (4b)	14.63	13.48	13.17	15.52	8.76	7.29
Example 3 (4c)	> 50	> 50	> 50	> 50	> 50	> 50
Example 4 (6a)	> 50	> 50	> 50	> 50	> 50	> 50
Example 5 (6b)	12.74	15.74	32.42	16.11	11.97	12.38
Example 6 (6c)	> 50	20.4	> 50	> 50	11.38	20.79
Example 7 (6d)	20.89	7.4	> 50	7.04	11.05	24.79
Example 8 (6e)	> 50	> 50	> 50	> 50	> 50	> 50
Example 9 (6f)	26.07	12.76	31.65	30.58	11.39	19.07
Diosgenin (DG)	> 50	> 50	> 50	> 50	> 50	> 50
Doxorubicin (Dox) positive control group	0.032	0.54	0.72	ND	0.28	0.43

As shown in Table 1, the diosgenin (DG) itself has a very low anticancer activity, the compounds of Example 1-9 primary aminated diosgenin according to the present invention was shown to significantly increase the anticancer activity.

Therefore, the compounds according to the present invention may be useful as an anticancer composition because the anti-cancer activity is remarkably improved compared to the diosgenin itself by introducing a hydrophilic linear polyamine moiety to the 3-OH group of the diosgenin.

Preparation Example 1 Preparation of Pharmaceutical Formulation

<1-1> Preparation of Powder

2 g of conjugate of formula 4 or 6

1 g lactose

After mixing the above components, the airtight cloth was filled to prepare a powder.

<1-2> Preparation of Tablet

100 mg conjugate of Formula 4 or 6

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

<1-3> Preparation of Capsule

100 mg conjugate of Formula 4 or 6

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

<1-4> Preparation of Injection Solution

10 μg / ml conjugate of Formula 4 or 6

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

The compound according to the invention was dissolved in an appropriate volume of sodium chloride BP for injection, the pH of the resulting solution was adjusted to pH 3.5 with dilute hydrochloric acid BP, and the volume was adjusted with sodium chloride BP for injection and thoroughly mixed. The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized with an autoclave at 120 ° C. for at least 15 minutes to prepare an injection solution.

Conjugates in which the hydrophilic linear primary amine and the diosgenin are linked with a linker according to the present invention, as the hydrophilic linear primary amine is introduced at the 3-OH position of the diosgenin, the cancer cell membrane permeability is improved and anticancer activity is increased. Since there is an effect that is significantly improved compared to the diosgenin itself, it may be useful as a pharmaceutical composition for preventing or treating cancer.

Claims (12)

1. Conjugates in which a linear primary amine and Diosgenin are linked with a linker, or a pharmaceutically acceptable salt thereof.
2. The method of claim 1,

   The linear primary amines are ethylenediamine (EDA), diethylenetriamine (DETA), amine, 2,2'-dithio (ethylamine), tetramethylenediamine, N1, N2, N3-tris ( A tert-butoxycarbonyl) -triethylenetetraamine or triethylenetetraamine (TETA), or a pharmaceutically acceptable salt thereof.
3. The method of claim 1,

   The linker is

   

   or

   

   Wherein X is a linear primary amine and DG is diosgenin, or a pharmaceutically acceptable salt thereof.
4. The method of claim 1,

   Conjugates in which the linear primary amine and diosgenin are linked with a linker are represented by the following formula (4) or (6), or a pharmaceutically acceptable salt thereof:

   [Formula 4]

   

   (In Formula 4,

   n is an integer from 0-2);

   [Formula 6]

   

   (In Chemical Formula 6,

   R is

   

   ,

   

   ,

   

   ,

   

   ,

   

   or

   

   to be).
5. As shown in Scheme 1 below,

   Reacting diosgenin (DG) with p -4-toluenesulfonyl chloride in an organic solvent to obtain compound 1 (step 1);

   Reacting compound 1 with ethylene glycol in an organic solvent to obtain compound 2 (step 2);

   Reacting compound 2 with p -4-toluenesulfonyl chloride in an organic solvent to obtain compound 3 (step 3); And

   Method of preparing a compound represented by the formula (4) comprising the step of reacting compound 3 with a primary amine in an organic solvent to obtain compound 4 (step 4):

   Scheme 1

   

   (In Scheme 1,

   n is an integer of 0-2).
6. The method of claim 5,

   The organic solvent is at least one selected from the group consisting of pyridine, dichloromethane (DCM), 1,4-dioxane, dimethylformamide (DMF) and tetrahydrofuran (THF).
7. As shown in Scheme 2 below,

   Reacting diosgenin with triphosgene in an organic solvent to obtain compound 5 (step 1); And

   Method of preparing a compound represented by the formula (6) comprising the step of preparing a compound 6 by reacting the compound 5 with a primary amine in an organic solvent (step 2):

   Scheme 2

   

   (In Scheme 2,

   R is

   

   ,

   

   ,

   

   ,

   

   ,

   

   or

   

   to be).
8. The method of claim 7, wherein

   The organic solvent is at least one selected from the group consisting of pyridine, dichloromethane (DCM), 1,4-dioxane, dimethylformamide (DMF), tetrahydrofuran (THF) and triethyleneamine (TEA). Characterized in the manufacturing method.
9. A pharmaceutical composition for preventing or treating cancer, comprising the conjugate of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
10. The method of claim 9,

    The cancer is a prostate cancer, cervical cancer, breast cancer, colon cancer, hematological cancer or liver cancer, characterized in that the pharmaceutical composition.
11. A method of claim 1 comprising administering a linear primary amine and a diosgenin conjugated linker conjugate, or a pharmaceutically acceptable salt thereof, to a patient in need of cancer treatment in a therapeutically effective amount. How to treat cancer.
12. Use of a conjugate wherein the linear primary amine and Diosgenin of claim 1 are bound with a linker, or a pharmaceutically acceptable salt thereof, in the manufacture of a cancer treatment.