WO2018066947A1 - Entecavir derivative compound bound with fatty acid and pharmaceutical use thereof - Google Patents

Abstract

The present invention relates to an entecavir derivative compound bound with a fatty acid and a pharmaceutical use thereof. The entecavir derivative covalently bound with a fatty acid according to the present invention exhibits a significantly lower solubility and dissolution rate than entecavir. The fatty acid polymer of entecavir according to the present invention can exert a sustained effect for several weeks or more after a single administration when a preparation is made for parenteral administration such as an injection, and can drastically improve patient medication compliance .

Description

Entercavir derivative compounds bound to fatty acids and pharmaceutical uses thereof

The present invention relates to an enticavir derivative compound to which a fatty acid is bound and its pharmaceutical use. It is to prepare a drug for sustained release parenteral administration by pro-drug entecavir has been used only for conventional oral use.

Entecavir, ie, compound 2-amino-9-[(1S, 3R, 4S) -4-hydroxy-3- (hydroxymethyl) -2-methylenecyclopentyl] -1H as shown in formula (1) -Purine-6 (9H) -one is an antiviral agent of the nucleoside type.

[Formula 1]

Entercavir is the drug that has the highest anti-HBV activity among commercial anti-HBV (Hepatitis B virus) drugs. The anti-HBV activity of entecavir is known to be 100 and 30 times higher than lamivudine and adefovir difficile, respectively. It also has fewer side effects and therapeutic effects in patients who are resistant to lamivudine. Therefore, entecavir is used as a useful treatment for hepatitis B.

Treatment of HBV infection requires long-term administration of entecavir preparations, but entecavir must be administered orally every day, avoiding mealtimes, resulting in poor patient compliance. Therefore, there is a need for the development of a drug that can exhibit long-term anti-HBV activity through a single drug administration. Accordingly, subcutaneous injection formulations have been designed by designing microsphere formulations based on biodegradable polymers, or sustained-release injection formulations (depots) based on lipid materials have been attempted. However, due to the low affinity for the hydrophobic polymer and lipid material of entecavir, and the high solubility in the water-soluble medium, there is a problem that the drug loaded in these formulations is not released slowly and is rapidly released in the medium. In addition, the loss of the drug is inevitable in the process of mounting the drug on the carrier of the polymer or lipid material, there is a problem that the drug mounted therein can be quickly escaped if the carrier is partially destroyed or modified.

On the other hand, prodrugization is a method of improving the undesired drug dynamics that occur during the use of the drug, that is, during the administration, absorption, distribution, excretion, and metabolism of the drug by changing the structure of the drug. In particular, prodrugation techniques that covalently bind, ie, acylate, fatty acids can be utilized to design sustained release or sustained release formulations by reducing the water solubility of the drug. When the acylated prodrug is administered as an intramuscular or subcutaneous injection, it is present as a solid storage form in vivo and then slowly dissolved and hydrolyzed to break down into active drugs and fatty acids. In most cases, the rate of absorption of the drug is determined by the rate of dissolution of the drug.

Acids generated as byproducts through hydrolysis are removed through metabolic processes in vivo, which can cause side effects of inflammation, acute and chronic toxicity. The method of acylating fatty acids such that non-toxic or minimized bio-derived substances are generated as by-products is one of the good methods of prodrug-induced sustained-release drug development and prevention of toxicity by by-products.

Examples of prodrug-formed drugs that exhibit a sustained release by acylating fatty acids include paripridone and iripyrazole, which are mental nervous system drugs. Considering the fact that the patient is reluctant to take the drug, such a mental nervous system drug has been required in the clinical field for a certain period of time, which has been developed as a sustained release injection. . However, since the process of prodrugation is different depending on the structure of the individual drug, it is difficult to synthesize acylated entecavir by conventional techniques.

It is an object of the present invention to provide an enticavir derivative compound having a fatty acid bound thereto and a pharmaceutical use thereof.

In order to achieve the above object, the present invention provides an enticavir derivative compound, a pharmaceutically acceptable salt thereof or a hydrate thereof covalently bonded to a fatty acid represented by the following formula (2).

[Formula 2]

Wherein R ¹ is ^one of C4 to C20 straight or branched alkyl, alkene or alkane.

The present invention also provides a pharmaceutical composition for preventing or treating type B infection, which comprises an enticavir derivative compound covalently bonded to a fatty acid represented by Formula 2, a pharmaceutically acceptable salt thereof, or a hydrate thereof as an active ingredient.

[Formula 2]

Wherein R ¹ is ^one of C4 to C20 straight or branched alkyl, alkene or alkane.

The present invention relates to an enticavir derivative compound having a fatty acid bound thereto and a pharmaceutical use thereof, wherein the entecavir derivative covalently bonded to a fatty acid according to the present invention shows a significantly lower solubility and dissolution rate than entecavir. When the fatty acid polymer of entecavir according to the present invention is prepared by parenteral administration preparations such as injections, it can express a sustained effect of several weeks or more after a single administration, and can dramatically improve the patient's compliance with the medication.

1 is a view showing the dissolution test results according to the present invention.

Accordingly, the present inventors attempted to reduce the water solubility of entecavir, which has difficulty in formulating sustained release due to high water solubility, and for this purpose, established a method of acylating various fatty acids in entecavir, and thereby synthesized a derivative of entecavir to complete the present invention. Came to.

The present invention provides an enticavir derivative compound, a pharmaceutically acceptable salt thereof, or a hydrate thereof in which a fatty acid represented by Formula 2 is covalently bonded.

[Formula 2]

Wherein R ¹ may be ^one of C4 to C20 straight or branched alkyl, alkene or alkane.

In addition, when specifically exemplified the enticavir derivative compound covalently bonded to the fatty acid of Formula 2 as follows, but is not limited thereto.

(1) [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl hexanoate

(2) [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl octanoate

(3) [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl decanoate

(4) [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl dodecanoate

(5) [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl tetradecano Eight

(6) [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl hexadecano Eight

(7) [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl octadecano Eight

The structure of the enticavir derivative compound to which the fatty acid of Formula 2 is covalently bonded according to the present invention exemplified above is shown in Table 1 below.

Example	rescue
One
2
3
4
5
6
7

In addition, the present invention may include not only enticavir derivative compounds in which the fatty acid of Formula 2 is covalently bonded, but also all possible solvates, hydrates, isomers, and the like, which can be prepared therefrom.

On the other hand, the present invention, as shown in the following Scheme 1, it is possible to prepare an enticavir derivative compound is a covalently bonded fatty acid of formula (2).

Scheme 1

Wherein R ¹ may be ^one of C4 to C20 straight or branched alkyl, alkene or alkane, and R ² may be hydroxyl, halogen, —OR ³ or —O ₂ R ³ .

In the above, the halogen may be fluorine, chlorine, bromine or iodine.

In the above, R ³ may be an alkyl group of 1 to 20 carbon atoms.

The term "alkyl" as used herein, whether used alone or in combination with other groups, refers to a straight or branched monovalent hydrocarbon group consisting of carbon atoms and hydrogen atoms.

In detail, the present invention may be obtained by reacting an enticavir of Formula 1 or a hydrate thereof with a fatty acid of Formula 3 to obtain an entecavir derivative compound of Formula 2.

Scheme 2

R <^1> in the said reaction formula means a C4-20 alkyl group.

In Scheme 2, the entecavir derivative compound to which the fatty acid of Formula 2 is bound may be obtained by performing an acylation reaction using a coupling reagent.

Coupling reagents mentioned above include dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide (N, N'-diisopropylcarbodiimide; DIC), 1-ethyl-3- (3- Reagents of carbodiimide structure, such as dimethylaminopropyl) carbodiimide (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC)).

In addition, the coupling reagent is benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate (benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP), benzotriazol-1-yljade Phosphonium structure reagents such as benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate (PyBOP) and the like.

In addition, the coupling reagent is 2- (1H-benzotriazol-1-yl) -N, N, N ', N'-tetramethylammonium tetrafluoroborate (2- (1H-benzotriazol-1-yl)- N, N, N'N'-tetramethylammonium tetrafluoroborate (TBTU), 2- (7-aza-1H-benzotriazol-1-yl) -N, N, N ', N'-tetramethylammonium hexaflofuophosphate Ammonium structure reagents such as (2- (7-aza-1H-benzotriazol-1-yl) -N, N, N ', N'-tetramethylammonium hexafluorophosphate (HATU)).

On the other hand, the pharmaceutically acceptable salts are hydrochloride, bromate, sulfate, phosphate, nitrate, citrate, acetate, lactate, tartarate, maleate, gluconate, succinate, formate, trifluoroacetate, oxalate , Fumarate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, camphorsulfonate, sodium salt, potassium salt, lithium salt, calcium salt and magnesium salt.

The present invention also provides a pharmaceutical composition for preventing or treating type B infection, which comprises an enticavir derivative compound covalently bonded to a fatty acid represented by Formula 2, a pharmaceutically acceptable salt thereof, or a hydrate thereof as an active ingredient.

[Formula 2]

Wherein R ¹ may be ^one of C4 to C20 straight or branched alkyl, alkene or alkane.

The entecavir derivative compound to which the fatty acid represented by Formula 2 is covalently bonded is [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydride Roxy-2-methylenecyclopentyl] methyl hexanoate, [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy- 2-methylenecyclopentyl] methyl octanoate, [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2- Methylenecyclopentyl] methyl decanoate, [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclo Pentyl] methyl dodecanoate, [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] Methyl tetradecanoate, [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl Hexadecanoate or [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-meth Alkylene cyclopentyl] no means may be a methyl octadecanoate, limited.

The enticavir derivative compounds to which the fatty acid is covalently bonded according to the present invention may be provided in the form of pharmaceutically acceptable salts thereof, and the pharmaceutically acceptable salts thereof include hydrochloride, bromate, sulfate, phosphate, nitrate and citrate. Acetate, lactate, tartarate, maleate, gluconate, succinate, formate, trifluoroacetate, oxalate, fumarate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, camphorsulfonate, It may be any one selected from the group consisting of sodium salt, potassium salt, lithium salt, calcium salt and magnesium salt.

The pharmaceutical composition may further include a suitable carrier, excipient or diluent commonly used in the manufacture of the pharmaceutical composition.

Carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

The pharmaceutical compositions according to the invention can be used in the form of injections, granules, tablets, pills, capsules, gels, syrups, suspensions, emulsions or solutions, respectively, according to conventional methods.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient, for example, starch, calcium carbonate, sucrose ( sucrose, lactose, gelatin and the like can be mixed and prepared.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, 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, glycerogelatin and the like can be used.

The amount of the compound which is an active ingredient of the pharmaceutical composition according to the present invention may vary depending on the age, sex, weight, and disease of the patient, but is 0.001 to 100 mg / kg, preferably 0.01 to 10 mg / kg once or several times a day. May be administered.

In addition, the dosage of the compound according to the present invention may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The pharmaceutical composition may be administered to various mammals such as mice, mice, livestock, humans, and the like. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrabronchial inhalation, intrauterine dural or intracerebroventricular injection.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

1. Analyzer

The instrument used to confirm the structure of the product obtained in the present invention is as follows. Nuclear magnetic resonance spectra ( ¹ H NMR) were used as JEOL JNM-LA 300, Bruker Analytik ADVANCE digital 500 or ADVANCE digital 600, and the solvent was DMSO-d ₆ . Mass spectra were used and expressed in m / z form.

2. TLC and tube chromatography

Thin layer chromatography (TLC) was used as a silica gel (Merck F254) manufactured by Merk, and silica (Merck EM9385, 230-400 mesh) was used for column chromatography. In addition, using a UV lamp (254 nm) or soaked in anisealdehyde coloring reagent to identify the material separated on the TLC, the plate was confirmed by heating.

3. Used reagent

Reagents and solvents used in the present invention were purchased from Sigma-aldrich and Tsiyi (TCI) products. The entecavir monohydrate used for the derivative synthesis was purchased from J & H Chem.

4. HPLC  Analytical Method

Example 1-7 was analyzed by HPLC using the following conditions.

analysis Sample	Example 1	Example 2	Example 3	Example 4	Example 5	Example 6	Example 7
column	C18, 150 x 4.6mm	C18, 150 x 4.6mm	C18, 150 x 4.6mm	C18, 150 x 4.6mm	C18, 150 x 4.6mm	C18, 150 x 4.6mm	C18, 150 x 4.6mm
column Temperature	20 ℃	20 ℃	20 ℃	20 ℃	20 ℃	20 ℃	20 ℃
Mobile phase	Isopropyl alcohol (IPA): Distilled water (DW) = 6: 4	IPA: DW = 6: 4	IPA: DW = 5: 5	IPA: DW = 5: 5	IPA: DW = 4: 6	IPA: DW = 4: 6	IPA: DW = 3: 7
Flow rate	1ml / min	1ml / min	1ml / min	1ml / min	1ml / min	1ml / min	1ml / min
Analysis wavelength	253nm	253 nm	253nm	253 nm	253 nm	253nm	253nm
Injection volume	40 uL	40 uL	40 uL	40 uL	40 uL	40 uL	40 uL

5. General Synthetic Process

(1) Preparation method 1: general manufacturing process of enticavir with covalently bonded fatty acid using coupling reagent

Scheme 2

R <^1> in the said reaction formula means a C4-20 alkyl group.

Pyridine (20-30 mL) in enticavir hydrate (500 mg-2000 mg, 1 equivalent), fatty acid (1 equivalent), 1-ethyl-3- (3-dimethylaminopropyl) Bodyimide (EDC) (2 equiv) and N, N-dimethylaminopyridine (DMAP) (0.05 equiv) were added and stirred. Diisopropylethylamine (3 equivalents) was slowly added dropwise to the reaction solution at room temperature. After stirring for 6 hours at the same temperature, water (3 ~ 5 mL) was added to the reaction solution to terminate the reaction. The reaction solution was concentrated under vacuum at 50 ° C. to evaporate the solvent, and the concentrate was dissolved by adding a solvent containing 10% methanol in dichloromethane. The mixed solution was washed with 1N aqueous hydrochloric acid solution, the organic layer was washed with brine and dried over magnesium sulfate. After removing the solvents in vacuo, the reaction mixture was purified by column chromatography (silica gel) using 10% methanol solvent in dichloromethane to afford the desired compound.

(2) Preparation method 2: general manufacturing process of enticavir with covalently bonded fatty acid using acid anhydride

Scheme 3

R <^1> in the said reaction formula means a C4-20 alkyl group.

To pyridine (20-30 mL) was added enticavir hydrate (500 mg, 1 equiv.) Of chemical 1 and then stirred. To the reaction solution was slowly added dropwise a fatty acid anhydride (1 equivalent) of Formula 5 at 0 degrees. After stirring for 6 hours at room temperature, the reaction was terminated by adding water (3 ~ 5 mL) to the reaction solution. The reaction solution was concentrated under vacuum at 50 ° C. to evaporate the solvent, and the concentrate was dissolved by adding a solvent containing 10% methanol in dichloromethane. The mixed solution was washed with 1N aqueous hydrochloric acid solution, the organic layer was washed with brine and dried over magnesium sulfate. After removing the solvents in vacuo, the reaction mixture was purified by column chromatography (silica gel) using 10% methanol solvent in dichloromethane to afford the desired compound.

< Example  1> [( 1R, 3S, 5S ) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5- Hydroxy Preparation of 2-methylenecyclopentyl] methyl hexanoate

According to the general preparation method of Preparation 2, hexanoic anhydride (0.39 mL, 1.69 mmol) was added to entecavir hydrate (500 mg, 1 equivalent, 1.69 mmol) to obtain the desired [(1R, 3S, 5S). 260 mg (41%) of) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl hexanoate were obtained.

MS m / z 376 (M + H ⁺ )

1 H NMR (500 MHz, DMSO-d6) δ 10.60 (s, 1H), 7.64 (s, 1H), 6.42 (s, 2H), 5.38-5.34 (m, 1H), 5.14-5.06 (m, 2H), 4.60 (s, 1H), 4.17-4.14 (m, 3H), 2.72-2.71 (m, 1H), 2.31 (t, 2H, J = 7.4 Hz), 2.29-2.27 (m, 1H), 2.09-2.06 ( m, 1H), 1.53 (t, 2H, J = 7.3 Hz), 1.25-1.23 (m, 4H), 0.84 (t, 3H, J = 6.9 Hz).

< Example  2> [( 1R, 3S, 5S ) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5- Hydrolock Cy-2-methylenecyclopentyl] methyl Octanoate  Produce

According to the general preparation of Preparation 1, the desired [(1R, 3S, 5S) -3- (2-amino from entecavir hydrate (500 mg, 1 equiv, 1.69 mmol) and octanoic acid (243 mg, 1.69 mmol) -6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl octanoate (347 mg, 51%) was obtained.

MS m / z 404 (M + H ⁺ )

1 H NMR (500 MHz, DMSO-d6) δ 10.71 (s, 1H), 7.65 (s, 1H), 6.45 (s, 2H), 5.39-5.35 (m, 1H), 5.14 (m, 1H), 5.08 ( s, 1H), 4.62-4.61 (m, 1H), 4.19-4.15 (m, 3H), 2.74-2.72 (m, 1H), 2.34-2.27 (m, 3H), 2.09-2.05 (m, 1H), 1.53 (t, 2H, J = 7.2 Hz), 1.25-1.22 (m, 8H), 0.84 (t, 3H, J = 6.9 Hz).

< Example  3> [( 1R, 3S, 5S ) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5- Hydrolock Preparation of Cy-2-methylenecyclopentyl] methyl decanoate

According to the general preparation of Preparation 1, the desired [(1R, 3S, 5S) -3- (2-amino from entecavir hydrate (1 g, 1 equiv, 3.39 mmol) and decanoic acid (582 mg, 3.39 mmol) -6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl decanoate (724 mg, 49%) was obtained.

MS m / z 432 (M + H ⁺ )

1 H NMR (500 MHz, DMSO-d6) δ 10.58 (s, 1H), 7.64 (s, 1H), 6.39 (s, 2H), 5.38-5.34 (m, 1H), 5.13 (s, 1H), 5.06 ( s, 1H), 4.60 (s, 1H), 4.17-4.13 (m, 3H), 2.72-2.71 (m, 1H), 2.32-2.29 (m, 3H), 2.08-2.06 (m, 1H), 1.51 ( t, 2H, J = 7.1 Hz), 1.23-1.21 (m, 12H), 0.83 (t, 3H, J = 6.9 Hz).

< Example  4> [( 1R, 3S, 5S ) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5- Hydroxy Preparation of 2-methylenecyclopentyl] methyl dodecanoate

According to the general preparation of Preparation 1, the desired [(1R, 3S, 5S) -3- (2-amino from entecavir hydrate (500 mg, 1 equiv, 1.69 mmol) and dodecanoic acid (339 mg, 1.69 mmol) -6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl dodecanoate (367 mg, 47%) was obtained.

MS m / z 460 (M + H ⁺ )

1 H NMR (500 MHz, DMSO-d6) δ 10.59 (s, 1H), 7.65 (s, 1H), 6.43 (s, 2H), 5.39-5.34 (m, 1H), 5.14 (m, 1H), 5.08 ( s, 1H), 4.61 (s, 1H), 4.19-4.15 (m, 3H), 2.72-2.71 (m, 1H), 2.34-2.28 (m, 3H), 2.09-2.05 (m, 1H), 1.53 ( t, 2H, J = 7.3 Hz), 1.24-1.22 (m, 16H), 0.84 (t, 3H, J = 6.9 Hz).

< Example  5> [( 1R, 3S, 5S ) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5- Hydrolock Preparation of Cy-2-methylenecyclopentyl] methyl tetradecanoate

According to the general preparation of Preparation 1, the desired [(1R, 3S, 5S) -3- (2-) from entecavir hydrate (500 mg, 1 equivalent, 1.69 mmol) and tetradecanoic acid (386 mg, 1.69 mmol) Amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl tetradecanoate (405 mg, 49%) was obtained.

MS m / z 510 (M + Na +)

1 H NMR (500 MHz, DMSO-d6) δ 10.60 (s, 1H), 7.64 (s, 1H), 6.37 (s, 2H), 5.38-5.34 (m, 1H), 5.13 (s, 1H), 5.06 ( m, 1H), 4.60 (s, 1H), 4.18-4.14 (m, 3H), 2.72-2.71 (m, 1H), 2.32-2.27 (m, 3H), 2.09-2.07 (m, 1H), 1.52 ( t, 2H, J = 7.3 Hz), 1.23-1.21 (m, 20H), 0.83 (t, 3H, J = 6.9 Hz).

< Example  6> [( 1R, 3S, 5S ) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5- Hydroxy 2-Methylenecyclopentyl] methyl hexadecanoate

According to the general preparation of Preparation 1, the desired [(1R, 3S, 5S) -3- (2- from entecavir hydrate (2 g, 1 equiv, 6.77 mmol) and hexadecanoic acid (1.74 g, 6.77 mmol) Amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl hexadecanoate (2.06 g, 59%) was obtained.

MS m / z 516 (M + H ⁺ )

1 H NMR (600 MHz, DMSO-d6) δ 10.61 (s, 1H), 7.65 (s, 1H), 6.42 (s, 2H), 5.40-5.36 (m, 1H), 5.14 (s, 1H), 5.07 ( d, 1H, J = 3.2 Hz), 4.62 (s, 1H), 4.21-4.15 (m, 3H), 2.75-2.71 (m, 1H), 2.32 (t, 2H, J = 7.4 Hz), 2.30-2.27 (m, 1H), 2.09-2.06 (m, 1H), 1.27-1.22 (m, 26H), 0.84 (t, 3H, J = 6.9 Hz).

< Example  7> [( 1R, 3S, 5S ) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5- Hydroxy Preparation of 2-methylenecyclopentyl] methyl octadecanoate

According to the general preparation of Preparation 2, the desired [(1R, 3S, 5S) -3- (2) from entecavir hydrate (500 mg, 1 equiv, 1.69 mmol) and octadecanoic acid chloride (0.57 mL, 1.69 mmol) -Amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl octadecanoate (432 mg, 47%) was obtained.

MS m / z 566 (M + Na ⁺ )

1 H NMR (300 MHz, DMSO-d6) δ 10.64 (s, 1H), 7.70 (s, 1H), 6.46 (s, 2H), 5.41-5.35 (m, 1H), 5.14-5.06 (m, 2H), 4.61 (s, 1H), 4.18-4.16 (m, 3H), 2.77-2.73 (m, 1H), 2.34-2.25 (m, 3H), 2.10-2.04 (m, 1H), 1.28-1.22 (m, 30H ), 0.84 (t, 3H, J = 6.6 Hz).

< Experimental Example  1> Solubility Evaluation

1 ml of 10 mM phosphate buffered saline and 10 mg of entecavir or fatty acid polymers (Examples 1 to 7) were added to an Eppendorf tube and stirred using a shaking incubator at room temperature for 24 hours. After centrifugation at 13,000 rpm for 10 minutes, the supernatant was taken, diluted with a suitable mobile phase, and analyzed by HPLC.

The experimental results are shown in Table 3 below. As shown in Table 3, entecavir had a solubility in phosphate buffer solution of 1700 μg / ml, whereas Examples 1 to 7 all showed low water solubility of 100 μg / ml or less. Low water solubility enables the preparation of suspension injections and ensures a sustained release pattern after parenteral administration in the body.

matter	Solubility (μg / ml, average value) (n = 3)
Entecavir	1718.1
Example 1	4.9
Example 2	4.2
Example 3	5.2
Example 4	5.3
Example 5	3.3
Example 6	1.1
Example 7	13.3

< Experimental Example 2> elution  evaluation

Drug dissolution evaluation was performed using the USP paddle method. The enticavir fatty acid polymers of Examples 6 and 7 corresponding to 40 mg as entecavir and entecavir were added to 900 ml of medium, respectively, and stirred at 50 rpm to evaluate the dissolution rate of the drug. The medium used was 0.001 N hydrochloric acid solution containing 0.5% Polysorbate 20 to secure the sink condition. The temperature of the medium was 37.5 degrees.

After taking the medium at regular time, the insoluble material was removed by centrifugation and the concentration of drug in the medium was analyzed by HPLC. The experimental results are shown in FIG.

Entecavir had high water solubility and dissolved more than 80% of the drug at 45 minutes, while Examples 6 and 7 showed low dissolution rates of 13% and 36%, respectively. Such slowly dissolving characteristics can be expected to ensure a sustained release pattern after injection.

< Experimental Example 3> Hydrolysis  evaluation

5 mg of each of the entecavir fatty acid polymers of Examples 6 and 7 were completely dissolved in 100 ml of 0.001 N hydrochloric acid solution containing 0.5% Polysorbate 20 and stirred at 37 ° C. for 48 hours. After centrifugation at 13,000 rpm for 10 minutes, the supernatant was taken, and the concentration of entecavir in the supernatant was analyzed by HPLC. As shown in Table 4, the amount of hydrolysis was less than 1% compared to the amount added. Such good water stability can provide high chemical stability in the preparation of aqueous suspensions in the future.

matter	Hydrolyzed fraction (μg / ml, mean) (n = 3)
Example 6	0.5
Example 7	0.4

< Experimental Example  4> internal Mother drug  conversion to parent drug

Example 6 was added to human liver microsomes (0.5 mg / ml) and 0.1 M phosphate buffer (pH 7.4) at a concentration of 1 μM, pre-incubated at 37 ° C. for 5 minutes, and then NADPH Regeneration system solution was added at 37 ° C. Incubate for 30 minutes. Then, to terminate the reaction by adding an acetonitrile solution containing a chlorpropamide, centrifuged for 5 minutes (14,000 rpm, 4 ℃) and the supernatant was injected into the LC-MS / MS system 6 and entercavir were analyzed to evaluate the conversion to the parent drug of Example 6. The amount of entecavir, the parent drug of Example 6 and Example 6 remaining through the reaction, was used using an Agilent 1290 infinity series pump system (Agilent, USA) and Triple Quad 5500 LC-MS / MS system (Applied Biosystems, USA). And analyzed. As a result of the test, it was confirmed that after 6 minutes, Example 6 was converted into entecavir, which is about 76.8% of the parent drug. Through this, it was confirmed that the fatty acid-bound entecavir derivative compound (Example) can be converted into enticavir which is an active substance quickly and completely after administration in the body and exhibit its activity.

As mentioned above, although this invention was demonstrated by the limited embodiment and drawing, this invention is not limited by this, The person of ordinary skill in the art to which this invention belongs, Of course, various modifications and variations are possible within the scope of equivalents of the claims to be described.

Claims (7)

1. An enticavir derivative compound to which a fatty acid represented by Formula 2 is covalently bonded, a pharmaceutically acceptable salt thereof, or a hydrate thereof:

   [Formula 2]

   

   Wherein R ¹ is ^one of C4 to C20 straight or branched alkyl, alkene or alkane.
2. The entecavir derivative compound to which the fatty acid represented by Formula 2 is covalently bonded is [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purine-9 (6H)- Yl) -5-hydroxy-2-methylenecyclopentyl] methyl hexanoate, [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl octanoate, [(1R, 3S, 5S) -3- (2-amino-6-oxo-1 H-purin-9 (6H) -yl) -5 -Hydroxy-2-methylenecyclopentyl] methyl decanoate, [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydr Roxy-2-methylenecyclopentyl] methyl dodecanoate, [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy- 2-methylenecyclopentyl] methyl tetradecanoate, [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2 Methylenecyclopentyl] methyl hexadecanoate and [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-ha Entekavir derivative compounds covalently bonded to a fatty acid represented by the formula (2), characterized in that any one selected from the group consisting of hydroxy-2-methylenecyclopentyl] methyl octadecanoate, pharmaceutically acceptable salts thereof Carb.
3. The method of claim 1, wherein the pharmaceutically acceptable salt is hydrochloride, bromate, sulfate, phosphate, nitrate, citrate, acetate, lactate, tartarate, maleate, gluconate, succinate, formate, trifluoroacetic acid Salt, oxalate, fumarate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, camphorsulfonate, sodium salt, potassium salt, lithium salt, calcium salt and magnesium salt Entekavir derivative compound, a pharmaceutically acceptable salt thereof, or a hydrate thereof wherein the fatty acid represented by Formula 2 is covalently bonded.
4. A pharmaceutical composition for hepatitis B prophylaxis or treatment containing an enticavir derivative compound covalently bonded to a fatty acid represented by Formula 2, a pharmaceutically acceptable salt thereof, or a hydrate thereof as an active ingredient:

   [Formula 2]

   

   Wherein R ¹ is ^one of C4 to C20 straight or branched alkyl, alkene or alkane.
5. The entecavir derivative compound to which the fatty acid represented by Formula 2 is covalently bonded is [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purine-9 (6H)-. Yl) -5-hydroxy-2-methylenecyclopentyl] methyl hexanoate, [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylenecyclopentyl] methyl octanoate, [(1R, 3S, 5S) -3- (2-amino-6-oxo-1 H-purin-9 (6H) -yl) -5 -Hydroxy-2-methylenecyclopentyl] methyl decanoate, [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydr Roxy-2-methylenecyclopentyl] methyl dodecanoate, [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy- 2-methylenecyclopentyl] methyl tetradecanoate, [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2 Methylenecyclopentyl] methyl hexadecanoate and [(1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-ha Hepatitis B prophylaxis or treatment pharmaceutical composition, characterized in that any one selected from the group consisting of hydroxy-2-methylenecyclopentyl] methyl octadecanoate.
6. The method of claim 4, wherein the pharmaceutically acceptable salt is hydrochloride, bromate, sulfate, phosphate, nitrate, citrate, acetate, lactate, tartarate, maleate, gluconate, succinate, formate, trifluoroacetic acid Salt, oxalate, fumarate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, camphorsulfonate, sodium salt, potassium salt, lithium salt, calcium salt and magnesium salt Pharmaceutical composition for the prevention or treatment of hepatitis B.
7. The method of claim 4, wherein the pharmaceutical composition is any one formulation selected from the group consisting of injections, granules, tablets, pills, capsules, gels, syrups, suspensions, emulsions and solutions. Therapeutic pharmaceutical composition.

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