KR20180128252A - Carbocyclic nucleosides derivatives and the antivirus compositions containing the same - Google Patents

Abstract

The purpose of the present invention is to provide new carbocyclic nucleoside derivatives which not only can effectively suppress virus by having a function of suppressing an SAH hydrolase, but also can be very appropriately used in suppression of virus and prevention and treatment of viral diseases since the carbocyclic nucleoside derivatives rarely have toxicity in body. According to the present invention, provided are Carbocyclic nucleoside derivatives or pharmaceutically acceptable salts thereof. The carbocyclic nucleoside derivatives or pharmaceutically acceptable salts thereof are resented by chemical formula A. In chemical formula A, R_1 and R_2 are each independently hydrogen or fluorine, and B is chemical formula B-1 or B-2. In chemical formula B-1, X_1 is chlorine, a hydroxyl group, an amino group or an alkylamino group, and Y_1 is hydrogen or an amino group. In chemical formula B-2, X_2 is a hydroxyl group or an amino group, and Y_2 is hydrogen, a methyl group or a halogen.

Description

[0001] CARBOCYCLIC NUCLEOSIDES DERIVATIVES AND THE ANTIVIRUS COMPOSITIONS CONTAINING THE SAME [0002] The present invention relates to carbocyclic nucleoside derivatives,

The present invention relates to a carbocyclic nucleoside derivative and an antiviral agent comprising the same.

Viruses are one of the leading causes of many intractable diseases that pose a threat to the health of human beings, and they are investing heavily in capital to prevent or treat them worldwide. In recent years, intractable RNA viruses such as Chickungunya virus, SARS virus, MERS virus and Zika virus have posed a serious risk to mankind and inhibited their proliferation. Although there is an urgent need for the development of antiviral drugs, currently there are no antiviral drugs or prophylactics that can effectively inhibit these viruses.

Antiviral agents, on the other hand, can inhibit the virus through a variety of mechanisms, including the following two mechanisms that can be used to effectively suppress the virus.

The first is to inhibit S-adenosylhomocysteine (hereinafter, SAH) hydrolase.

SAH hydrolytic enzyme is a tetramer type enzyme that uses NAD ⁺ as a coenzyme. It plays a role of reversible hydrolysis of SAH to adenosine (Ado) and homocysteine (Hcy) Lipid and nucleic acids as well as methylation of substances in the body such as histamine and norepinephrine.

Inhibition of SAH hydrolytic enzymes induces accumulation of SAH. Excess SAH sequentially inhibits the inhibition of S-adenosylmethionine (AdoMet) -dependent transmethylase and capping of virus mRNA, It prevents the virus from being properly produced, resulting in an antiviral effect.

Since viral mRNA guanosine N7-methytransferases (O-2'-methytransferases) are essential for mRNA encapsulation as well as most animal DNA viruses, SAH hydrolase is considered to be an essential element in the development of broad- spectrum antiviral agents do. That is, the development of a therapeutic agent for RNA viruses and the development of a SAH hydrolase inhibitor are considered to have a high correlation.

The second is to inhibit the viral RNA polymerase.

RNA viruses are replicated by inserting the substrate Nucleoside-5'-triphosphate (NTP) into the RNA chain by RNA polymerase. Therefore, a substance that inhibits RNA polymerase can also act as an antiviral agent and can be converted into triphosphate in the body to selectively inhibit viral RNA polymerase or directly inserted into a viral RNA chain to perform chain termination ) Can be used to develop an effective antiviral agent.

However, since it is difficult to increase selectivity when an antiviral agent also has an inhibitory function of an RNA polymerase, it may exhibit toxicity to normal cells as well as viruses.

Accordingly, a problem to be solved by the present invention is to provide a pharmaceutical composition which is capable of effectively inhibiting a virus because it has a function of inhibiting SAH hydrolase, has little toxicity in the body, is well suited for the inhibition of viruses and for the prevention and treatment of viral diseases And to provide novel carbocyclic nucleoside derivatives that can be utilized.

The present invention has been made in view of the above problems, and it is an object of the present invention to provide a method of manufacturing the same.

In order to solve the above-mentioned problems, a carbocyclic nucleoside derivative or a pharmaceutically acceptable salt thereof according to an embodiment of the present invention is represented by the following formula (A).

≪ Formula (A)

In the formula (A), R ₁ and R ₂ are each independently hydrogen or fluorine, and B is B-1 or B-2.

<Formula B-1>

In the above formula (B-1), X ₁ is chlorine, a hydroxyl group, an amino group or an alkylamino group, and Y ₁ is a hydrogen atom or an amino group.

<Formula B-2>

In the above formula (B-2), X ₂ is a hydroxyl group or an amino group, and Y ₂ is hydrogen, a methyl group or a halogen.

The R &lt; ₁ &gt; may be fluorine.

The formula (A) may be represented by the following formula (5a) or (5b).

&Lt; Formula 5a &

&Lt; Formula 5b >

An antiviral agent comprising a carbocyclic nucleoside derivative or a pharmaceutically acceptable salt thereof according to another embodiment of the present invention for solving the above-mentioned problems is represented by the following formula (A).

&Lt; Formula (A)

In the formula (A), R ₁ and R ₂ are each independently hydrogen or fluorine, and B is B-1 or B-2.

<Formula B-1>

In the above formula (B-1), X ₁ is chlorine, a hydroxyl group, an amino group or an alkylamino group, and Y ₁ is a hydrogen atom or an amino group.

<Formula B-2>

In the above formula (B-2), X ₂ is a hydroxyl group or an amino group, and Y ₂ is hydrogen, a methyl group or a halogen.

The R &lt; ₁ &gt; may be fluorine.

The formula (A) may be represented by the following formula (5a) or (5b).

&Lt; Formula 5a &

&Lt; Formula 5b >

The present invention can be considered as a new antiviral agent having little toxicity in the body. This can contribute to the development of the new drug development field, which is a representative national knowledge base, and can improve the public health. But also contribute to economic development.

1 to 3 show the results of experiments.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

Hereinafter, the present invention will be described in detail.

A carbocyclic nucleoside derivative according to an embodiment of the present invention may be represented by the following formula (A).

&Lt; Formula (A)

In the formula (A), R ₁ and R ₂ are each independently hydrogen or fluorine, and B may be represented by the following formula (B-1) or (B-2).

<Formula B-1>

In the above formula (B-1), X ₁ is chlorine, a hydroxy group, an amino group or an alkylamino group, and Y ₁ may be hydrogen or an amino group.

The alkylamino group may be a monoalkyl amino group (-NHR), or -NMe _2, -NMeEt, -NEt _2, such as a dialkyl amino group (-NR _2), such as -NHMe, -NHEt.

<Formula B-2>

In the above formula (B-2), X ₂ is a hydroxyl group or an amino group, and Y ₂ may be hydrogen, a methyl group or a halogen.

Specifically, in the above formula (A), R ₁ may be fluorine, and B may be the above formula (B-1).

More specifically, the carbocyclic nucleoside derivative represented by the above formula (A) may be a compound represented by the following formula (5a) or a compound represented by the following formula (5b).

&Lt; Formula 5a &

&Lt; Formula 5b >

The carbocyclic nucleoside derivative represented by the above formula (A) may be provided in the form of a pharmaceutically acceptable salt. Salts are useful as acid addition salts formed by various pharmaceutically acceptable organic or inorganic acids. Suitable organic acids include, for example, organic acids such as formic acid, phosphonic acid, acetic acid, propionic acid, octanoic acid, decanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, malic acid, tartaric acid, citric acid, glutamic acid, There may be used maleic acid, benzoic acid, salicylic acid, phthalic acid, phenylacetic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, methylsulfuric acid, ethylsulfuric acid and dodecylsulfuric acid. Suitable inorganic acids include, for example, Or phosphoric acid can be used.

However, the present invention is not limited thereto. The carbocyclic nucleoside derivative of the present invention may be provided in the form of all salts, hydrates and solvates which can be prepared by conventional methods.

The antiviral agent according to one embodiment of the present invention may include the above-mentioned carbocyclic nucleoside derivative as an active ingredient.

An antiviral agent means a pharmaceutical composition which can be used not only for inhibiting the activity and replication of a virus but also for preventing and / or treating all diseases caused by viruses.

The virus may be, for example, Chickungunya Virus (CHIKV), Semliki Forrest Virus (SFV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Viruses such as Respiratory Syndrome Coronavirus (SARS-CoV), Equine Arteritis Virus (EAV), Mouse Hepatitis Virus (MHV), and Sindbis Virus (SINV).

The antiviral agent comprising the above-described carbocyclic nucleoside derivative may be a pharmaceutical composition that effectively inhibits the virus by inhibiting the SAH hydrolase.

The antiviral agent may be administered systemically or locally and may be formulated using excipients (or diluents) such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that can be generally used for oral or parenteral administration . Hereinafter, the excipient and the formulation method will be specifically exemplified, but are not limited thereto.

Solid formulations for oral administration may include tablets, pills, powders, granules, capsules and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose sucrose), lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Examples of liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, and the like. The liquid formulations may contain various excipients such as water, liquid paraffin, which are simple diluents commonly used, Flavoring agents, fragrances, preservatives, and the like.

Formulations for parenteral administration may include injections, emulsions, inhalants, suppositories, and the like. The injectable preparation may contain sterilized aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, esters such as ethyl oleate, non-aqueous solvents and suspensions, and the suppositories may contain witepsol, Macrogol, tween 61, cacao paper, laurin, glycerogelatin, and the like. In addition, the antiviral agent of the present invention may be formulated into ointments or creams for topical application.

The preferred dosage of the antiviral agent varies depending on a number of factors such as the condition and weight of the patient, the severity of the disease, the type of the drug, the route of administration and the period of time, but may be suitably selected by those skilled in the art. The route of administration may also vary depending on the condition of the patient and its severity.

Since the carbocyclic nucleoside derivative of the present invention has antiviral efficacy against RNA viruses and has biocompatible properties with little toxicity in the body (see Experimental Example), it is possible to inhibit viruses and prevent and / Or as a pharmaceutical composition that is well suited for treatment.

Manufacturing example  One

(CuBr / Me ₂ S complex) (933 mg, 4.54 mmol) was dissolved in anhydrous THF (300 ml) at -78 ° C. and then a 1.0 M solution of vinylmagnesium bromide in THF 69.3 ml, 69.3 mmol) and hexamethylphosphoramide (HMPA) (33.4 ml, 192 mmol). D-cyclopentenone (5 g, 32.4 mmol) and chlorotrimethylsilane (TMSCl) (20.6 ml, 162 mmol) were dissolved together in anhydrous THF (180 ml), and the mixture was slowly added dropwise to the reaction flask. Lt; / RTI &gt; for 2 h. After completion of the reaction was confirmed by TLC, an aqueous solution of saturated ammonium chloride (NH ₄ Cl) was added thereto. The aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), and filtered under reduced pressure to remove the remaining solid and then concentrated under reduced pressure. The concentrated residue was purified by silica gel chromatography to obtain a first intermediate (4.48 g, 76%).

The first intermediate (4.7 g, 26.0 mmol) was dissolved in methanol (100 mL) and sodium borohydride (NaBH ₄ ) (1.1 g, 28.6 mmol) was added at 0 ° C. The reaction mixture was stirred at the same temperature for 30 minutes, and neutralized with water and a small amount of acetic acid. The aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), and filtered under reduced pressure to remove the remaining solid and then concentrated under reduced pressure. The concentrated residue was purified by silica gel chromatography to obtain a second intermediate (4.6 g, 97%).

The imidazole (8.6 g, 126.2 mmol) and chlorotetrabutyldiphenylsilane (TBDPSCl) (10.4 g, 37.8 mmol) were dissolved in DMF (30 mL), and the second intermediate (4.6 g, 25.2 mmol) At 0 &lt; 0 &gt; C. The reaction mixture was stirred at room temperature for 1 hour, water was added, and the mixture was extracted with diethyl ether and the organic layer was separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), and filtered under reduced pressure to remove the remaining solid and then concentrated under reduced pressure. The concentrated residue was purified by silica gel chromatography to obtain a third intermediate (10.2 g, 92%).

The tertiary intermediate (10.0 g, 23.6 mmol) was dissolved in THF (125 mL), and a borane-dimethyl sulfide complex 2.0 M solution in THF (35.5 mL, 71.0 mmol) Lt; 0 &gt; C. The reaction mixture was stirred at room temperature for 4 hours, then sodium perborate (7.2 g, 71.0 mmol) was added at 0 ° C and water (30 mL) was slowly added thereto. After the reaction mixture was stirred at room temperature for 12 hours, the aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), and filtered under reduced pressure to remove the remaining solid and then concentrated under reduced pressure. The concentrated residue was purified by silica gel chromatography to obtain a fourth intermediate (7.5 g, 72%).

The fourth intermediate (7.5g, 17.0mmol) of was dissolved in THF (200mL) at 0 ℃ 1.0M tetra - n - butylammonium fluoride / THF solution (tetra- n -butylammonium fluoride 1.0M solution in THF) (51.0 mL, 51.1 mmol). The reaction mixture was stirred at room temperature for 12 hours, and the solvent was removed under reduced pressure. The concentrated residue was separated and purified by silica gel chromatography to obtain a fifth intermediate (3.3 g, 93%).

4-Dimethylaminopyridine (DMAP) (0.2 g, 1.6 mmol) and triethylamine (6.8 mL, 48.9 mmol) were dissolved in 40 mL of methylene chloride. mmol) and chlorotetrabutyldiphenylsilane (TBDPSCl) (4.9 g, 17.9 mmol). The reaction mixture was stirred at room temperature for 12 hours. After water was added to terminate the reaction, the aqueous layer was extracted with methylene chloride, and the organic layer was separated. The organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), and filtered under reduced pressure to remove the remaining solid, which was then concentrated under reduced pressure. The concentrated residue was purified by silica gel chromatography to obtain a sixth intermediate (5.7 g, 80%).

After dissolving the sixth intermediate (5.0 g, 11.3 mmol) in methylene chloride (25 mL), N-methylmorpholine N-oxide (2.0 g, 17.0 mmol) and 4 molecular sieves (1.0 g) and tetrapropylammonium perruthenate (0.2 g, 0.5 mmol) were added. The reaction mixture was stirred at room temperature for 30 minutes, then filtered through Celite and silica gel pad under reduced pressure to remove the remaining solid and then concentrated under reduced pressure. The concentrated residue was separated and purified by silica gel chromatography to obtain 4.8 g (98%) of a compound represented by the following formula (1) having a ¹ H-NMR spectrum as described below.

¹ H NMR (400 MHz, CDCl ₃ )? 7.64 (m, 4 H), 7.41 (m, 6 H), 4.57 (d, J = 5.6 Hz, m, 3 H), 2.73 ( dd, J = 8.8, 18.4 Hz, 1 H), 2.61 (m, 1 H), 2.03 (dt, J = 1.6, 17.6 Hz, 1 H), 1.68 (m, 1 H ), 1.43 (s, 3 H), 1.33 (s, 3 H), 1.05 (s, 9 H)

&Lt; Formula 1 >

Manufacturing example  2-1

Compound (1) (4.2 g, 9.5 mmol) synthesized in Preparation Example 1 was dissolved in anhydrous THF, and chlorotriethylsilane (TESCl) (16.7 mL, 99.2 mmol) was added dropwise and cooled to -78 ° C. To the cooled mixture, a 1.0 M lithium bis (trimethylsilyl) amide / THF solution (LiHMDS 1.0M solution in THF) (50 mL, 50 mmol) was slowly added dropwise and the reaction was stirred for 30 minutes under the same conditions for silylenol etherification. After confirming that the reaction was completed by TLC, the reaction mixture was added with an aqueous solution of saturated ammonium chloride (NH ₄ Cl), the aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), and filtered under reduced pressure to remove the remaining solid and then concentrated under reduced pressure. The concentrated residue was dissolved in anhydrous acetonitrile and then cooled to 0 &lt; 0 &gt; C. To the cooled mixture was added a solution of 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo [2.2.2] octane bis (tetrafluoroborate) (Selectfluor ^® ) (10.15 g, 28.65 mmol) The mixture was stirred for 15 hours under the same conditions to obtain a mixture of fluorinated compounds.

After confirming that the reaction for producing the mixture was completed by TLC, an aqueous solution of saturated ammonium chloride (NH ₄ Cl) was added to the reaction mixture, the aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), and filtered under reduced pressure to remove the remaining solid, which was then concentrated under reduced pressure. The concentrated residue was separated and purified by silica gel chromatography to obtain the compound of the following formula (2a) (3.7 g, 84%) having the ¹ H NMR spectrum as described below.

¹ H NMR (300 MHz, CDCl ₃ )? 7.65 (m, 4 H), 7.39 (m, 6 H), 5.43 (dd, J = 50.6, 7.14 Hz, 3H), 1.29 (s, 3H), 1.03 (m, 2H), 1.46 s, 9 H) (mixed with a peak estimated to be equilibrated in diol form).

&Lt; EMI ID =

Manufacturing example  2-2

The same procedure was followed as in Production Example 2-1, except that the compound of Formula 2a (2.5 g, 5.4 mmol) synthesized in Production Example 2-1, which was not the compound of Formula 1, was used as the starting material, To obtain the compound of the following formula (2b) (1.6 g, 61%) having ¹ H NMR spectrum.

¹ H NMR (400 MHz, CDCl ₃ )? 7.69 (m, 4 H), 7.40 (m, 6 H), 4.35 (dd, J = 3.6, 7.6 Hz, (Br s, 1H), 2.65 (m, 1H), 1.96 (m, 1 H), 3.78 (ddd, J = 6.4, 10.4, 3.2 Hz, 2H) H), 1.71 (m, 1H), 1.54 (s, 3H)

(2b)

Manufacturing example  3-1

Compound (2a) (2.5 g, 5.4 mmol) synthesized in Preparation Example 2-1 was dissolved in methanol and cooled to -40 ° C. Sodium borohydride (NaBH ₄ ) (1.45 g, 38.4 mmol) was slowly added to the cooled solution. Thereafter, the reaction mixture was stirred under the same conditions, and after completion of the reaction was confirmed by TLC, an aqueous solution of saturated ammonium chloride (NH ₄ Cl) was added to the reaction mixture, the aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), and filtered under reduced pressure to remove the remaining solid, which was then concentrated under reduced pressure. The resulting alcohol compound was separated and purified by silica gel chromatography to obtain an intermediate (2.54 g, 76%) which was dissolved in pyridine anhydride and cooled to 0 ° C. Trifluoromethanesulfonic anhydride (3.26 ml, 19.36 mmol) was added dropwise to the cooled mixture, and the mixture was stirred for 30 minutes under the same conditions to perform trifluoromethylsulfone oxidation. After completion of the reaction was confirmed by TLC, an aqueous solution of saturated ammonium chloride (NH ₄ Cl) was added to the reaction mixture, the aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), and filtered under reduced pressure to remove the remaining solid and then concentrated under reduced pressure. The resulting trifluoromethylsulfone compound and 10 equivalents of sodium azide (NaN ₃ ) were mixed in anhydrous DMF, and the mixture was stirred under heating at 100 ° C. After stirring for about 4 hours, it was confirmed that the reaction was terminated by TLC. After adding water, the aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), and filtered under reduced pressure to remove the remaining solid, which was then concentrated under reduced pressure. The azide compound thus obtained was isolated and purified by silica gel chromatography, and the resulting compound was dissolved in methanol. An appropriate amount of palladium / carbon was added to the solution, and the hydrogenation reaction was carried out by hydrogen substitution in the reaction vessel to reduce the azide group to an amine group. After the completion of the reaction was confirmed by TLC, the remaining solid was removed by filtration under reduced pressure, and the filtrate was concentrated under reduced pressure to obtain 1.6 g (61%) of a compound of the following formula (3a) having the following ¹ H NMR spectrum.

^{1 H NMR (400 MHz, CDCl} 3) δ 7.69 (m, 4 H), 7.40 (m, 6 H) 4.75 (dt, J = 2.8, 53.2 Hz, 1 H), 4.36 (m, 2 H), 3.78 (s, 3 H), 1.30 (s, 3 H), 1.06 (s, 3 H) , 9 H)

&Lt; EMI ID =

Manufacturing example  3-2

(2.117 g, 4.636 mmol) synthesized in Preparation Example 2-2, which was not the compound of Formula 2a, was used as the starting material, the temperature condition of the sodium borohydride reduction was adjusted to 0 ° C or lower, The procedure of Preparation Example 3-1 was repeated except that 1.89 g of sodium azide was used as the azide and 29.04 mmol of the azide was used and the temperature was heated to 60 ° C to obtain the following ¹ H NMR spectrum To obtain the compound of Formula 6b (1.28 g, 60%).

¹ H NMR (400 MHz, CDCl ₃ )? 7.69 (m, 4 H), 7.40 (m, 6 H), 4.35 (dd, J = 3.6, 7.6 Hz, (Br s, 1H), 2.65 (m, 1H), 1.96 (m, 1 H), 3.78 (ddd, J = 6.4, 10.4, 3.2 Hz, 2H) H), 1.71 (m, 1H), 1.54 (s, 3H)

&Lt; EMI ID =

Manufacturing example  4-1

(300 mg, 0.65 mmol) synthesized in Production Example 3-1 was reacted with 5-amino-4,6-dichloropyrimidine (1.85 g, 11.27 mmol) and di diisopropylethylamine (DIPEA) (6.54mL, 37.57mmol) and n - butanol was heated to 170 ℃ using that later, microwave mixing the (n -butanol) was stirred for 12 hours. After confirming that the reaction was completed by TLC, the reaction mixture was mixed with methanol and concentrated under reduced pressure. The residue was separated and purified by silica gel chromatography to obtain an intermediate (964 mg, 66%). The intermediate was dissolved in diethoxymethyl acetate and stirred at 140 ° C for 3 hours using a microwave. After that the checking by TLC the reaction was completed, the reaction mixture was mixed with methanol to having a ¹ H NMR spectrum as described below, to obtain separation in the back was concentrated under reduced pressure, silica gel chromatography, compounds of formula 8a (240mg, 61.6%) &Lt; / RTI &gt;

¹ H NMR (400 MHz, CDCl ₃ )? 8.78 (s, 1H), 8.32 (d, J = 2.4 Hz, 1H), 7.68 1 H), 1.95 (m, 2H), 1.59 (s, 1H), 5.08 (m, 2H), 4.62 3 H), 1.34 (s, 3 H), 1.06 (s, 9 H)

&Lt; Formula 4a &

Manufacturing example  4-2

The compound of Formula 3b (250 mg, 0.52 mmol) synthesized in Preparation Example 3-2 was dissolved in 1,4-dioxane (50 mL), and 4,6-dichloro-5- (120 mg, 0.65 mmol) and trimethylamine (0.55 ml, 3.86 mmol) were added. After the reaction mixture was refluxed for 2 days, the reaction was terminated by TLC, and the reaction mixture was concentrated under reduced pressure. The residue was separated and purified by silica gel chromatography to obtain an intermediate. Diethoxymethyl acetate (5 mL) was added to the intermediate, followed by stirring at 140 ° C for about 12 hours. After that the checking by TLC the reaction was completed, a mixture as the reaction mixture with methanol to having a ¹ H NMR spectrum as described below, to obtain separated then concentrated under reduced pressure, silica gel chromatography, compounds of formula 4b (100mg, 31%) &Lt; / RTI &gt;

¹ H NMR (400 MHz, CDCl ₃ )? 8.80 (s, 1H), 8.25 (d, J = 2.4 Hz, 1H), 7.70 m, 1 H), 5.16 (superimposed dd, J = 6.8 Hz, 1H), 4.54 (superimposed dd, J = 6.4 Hz, 1H), 3.80 (S, 3 H), 1.33 (s, 3 H), 1.07 (s, 9 H)

<Formula 4b>

Manufacturing example  5-1

The compound of Formula 4a (200 mg, 0.336 mmol) synthesized in Production Example 4-1 was dissolved in methanol, and a solution of trifluoroacetic acid (TFA) (5 mL) and water at 1: 1 was added dropwise at 0 ° C . After the reaction mixture was stirred at room temperature for 2 hours, it was confirmed by TLC that the reaction was terminated and concentrated under reduced pressure. The concentrated residue was separated and purified by silica gel chromatography to obtain an intermediate (80 mg, 75.4%).

Tert after the intermediate (70mg, 0.22mmol) was dissolved in butanol (tert -butanol), was transferred to a high-temperature closed container (stainless steel bomb reactor). Saturated ammonia / butanol was added dropwise to the mixed solution, and the reaction vessel was sealed and stirred at 120 ° C for 15 hours. After confirming that the reaction was completed by TLC, the reaction mixture was diluted with methanol and concentrated under reduced pressure. The concentrated residue was separated and purified by silica gel chromatography to obtain the following compound of formula (5a) (55 mg, 84.6%) having ¹ H NMR spectrum as described below.

^{1 H NMR (400 MHz, CD} 3 OD) δ 8.26 (d, J = 2.0 Hz, 1 H), 8.20 (s, 1 H), 5.11 (dt, J = 3.6, 55.2 Hz, 1 H), 4.99 ( ddd, J = 3.2, 9.6, 26.4 Hz, 1 H), 4.75 (m, 1 H), 4.04 (m, 1 H), 3.71 (m, 2 H), 2.38 (m, 1 H), 1.89 (m , 2 H)

&Lt; Formula 5a &

Manufacturing example  5-2

Except that the compound of the formula 4b (100 mg, 0.3 mmol) synthesized in Production Example 4-2, which was not the compound of the formula 4a, was used as a starting material and the mixture was stirred at 90 ° C for about 3 hours during the amination reaction The procedure of Preparation Example 5-1 was repeated to give the compound of Formula 5b (30 mg, 32%) having the ¹ H NMR spectrum as described below.

^{1 H NMR (300 MHz, CD} 3 OD) δ 8.27 (d, J = 2.1 Hz, 1H), 8.20 (s, 1H), 5.28 (m, 1H), 4.76 (m, 1H), 4.06 , 1H), 1.98 (m, 1H), 1.79 (m, 1H)

&Lt; Formula 5b >

Experimental Example  1 - Antiviral activity and cytotoxicity experiments

In order to examine the antiviral activity and cytotoxicity of the carbocyclic nucleoside derivative of the present invention, the following experiment was conducted.

Vero E6 cells were cultured and infected with CHIKV (Chikungunya Virus) LS3 and SFV (Semliki Forrest Virus) and titrated to obtain CHIKV inoculum and SFV inoculum, respectively.

The compound of Chemical Formula 5a synthesized in Preparation Example 5-1 was diluted with 200 μM in IMD (Iscove's modified Dulbecco's medium) containing 1% fetal calf serum (FCS) and an antibiotic. Vero E6 cells were cultured in 96-well plates containing 2 x 10 ⁴ cells per well. The cells were incubated overnight at 37 ° C, then 50 μl of the compound dilution was injected into each well, mixed with 100 μl of EMEM (Eagle's minimal essential medium) containing 2% FCS and 50 μl of the CHIKV inoculum. Subsequently, the cell-to-cell survival difference caused by virus-induced CPP (cytopathic effect) and compound side effects at 3 dpi (day postinfection) was analyzed using the CellTiter 96 Aqueous nonradioactive cell proliferation assay (Promega). The cytotoxicity by the compound treatment was confirmed by culturing the cells in the same manner as above except that a general medium was injected instead of the inoculated virus.

Using Graphpad Prism 5 software, cell viability for CPE and cell viability for compound adverse events were calculated for 50% effective concentration (EC ₅₀ ) and 50% cytotoxic concentration (CC ₅₀ ), respectively, based on the respective results . EC ₅₀ represents the minimum concentration of the compound for which 50% of the pathogen (virus) is inhibited, and CC ₅₀ represents the concentration of the compound when 50 pro of the host cell is killed. The degree of relative efficacy of the compounds against a particular virus was defined as selective (SI; CC ₅₀ / EC ₅₀ ).

Further, experiments were carried out in the same manner as above except that the compounds were mixed with the inoculum of SFV to calculate the EC ₅₀ , CC ₅₀ and SI values for the SFV of the compound of the formula 5a. The experiment for CHIKV above proceeded in the same way except that the compound of 5b was used to calculate the EC ₅₀ , CC ₅₀ and SI values of the compound of formula 5b.

Table 1 shows the EC ₅₀ , CC ₅₀ and SI values calculated by the above method.

Virus Type compound EC ₅₀ ( μM ) CC ₅₀ ( μM ) SI (CC ₅₀ / EC ₅₀ ) CHIKV  LS3 5a 0.2 > 250 > 1,000 5b 4.2 > 250 60 SFV 5a 6.4 > 250 > 30

As shown in Table 1, since the carbocyclic nucleoside derivative of the present invention has a low EC ₅₀ for the RNA viruses CHIKV and SFV and a high CC ₅₀ for the host cells, the inhibition of the virus and the prevention of viral diseases And as a pharmaceutical composition that is well suited for treatment.

Experimental Example  2 - SAH  Hydrolytic enzyme inhibitory effect experiment

In order to examine the SAH hydrolase inhibitory effect of the carbocyclic nucleoside derivative of the present invention, the following experiment was conducted.

A reaction mixture (250 μl) of 50 mM sodium phosphate (pH 8.0) and an AdoHcy hydrolase (2 μM as a monomer; 5 μM as a tetramer) was mixed with the compound of Formula 5a synthesized in Preparation Example 5-1 and the compound Preincubated with a compound of formula 5b for 10 minutes at 37 &lt; 0 &gt; C. After the pre-incubation, 100 μM AdoHcy was added to initiate the reaction and the reaction was allowed to proceed for 20 minutes. Thereafter, the reaction mixture was further cultured in 200 [mu] M DTNB and the maximum absorbance of the resulting product, TNB (5-thio-2-nitrobenzoic acid) at 412 nm, was measured with a spectrophotometer. The IC ₅₀ values of the compounds of formulas (5a) and (5b) were calculated using the maximum absorbance measured and the molar extinction coefficient of TNB (? 412 = 13700 M ^{- 1} cm ^{- 1} ).

compound IC ₅₀ ( μM ) 5a 0.1 5b 0.09

As shown in Table 2, since the carbocyclic nucleoside derivative of the present invention has a low IC ₅₀ value against the SAH hydrolase, it is suitable as a pharmaceutical composition having an antiviral effect through the SAH hydrolase inhibiting mechanism It can be seen that it can be used as a material.

A carbocyclic nucleoside derivative according to another embodiment of the present invention can be represented by the following formula (a).

<Formula a>

In Formula (a), n is 2, R 1 and R 2 are each independently hydrogen or fluorine, B is the following formula b-1 or b-2, and P is hydrogen or a phosphoamidate group.

<Formula b-1>

In the above formula (b-1), X1 may be chlorine, an OH group, an amino group or an alkylamino group, and Y1 may be a hydrogen atom or an amino group.

The alkylamino group may be a monoalkylamino group (-NHR) such as -NHMe or -NHEt or a dialkylamino group (-NR2) such as -NMe2, -NMeEt or -NEt2.

<Formula b-2>

In the above formula (b-2), X2 is a hydroxyl group or an amino group, and Y2 may be hydrogen, a methyl group or a halogen.

Specifically, in the above formula (a), R1 is fluorine, B is the above formula (b-1), and P may be hydrogen.

More specifically, the carbocyclic nucleoside derivative represented by Formula (a) may be a compound represented by Formula (5a) or a compound represented by Formula (5b) described in the following production method.

Hereinafter, the production method of the carbocyclic nucleoside derivative will be described in detail.

Method for producing substance 1

References [1] Tetrahedron: Asymmetry, 2002, 13, 1189-1193 and [2] J. Med. Chem . 2001, 44, 3985-3993.

Method for producing substances 2a and 2b

The previously synthesized substance 1 (6 g, 24.8 mmol) was dissolved in anhydrous THF, and then chloro-ethyl triethylsilane (TESCl) (16.7 mL, 99.2 mmol) was added dropwise and cooled to -78 ^° C. To the cooled mixture was slowly added dropwise a 1.0 M lithium bis (trimethylsilyl) amide / THF solution (LiHMDS 1.0 M solution in THF) (50 mL, 50 mmol) and the reaction was stirred well under the same conditions for 30 minutes to obtain silylenol ether It makes me angry. After confirming the completion of the reaction by TLC, the reaction mixture is quenched with an aqueous solution of saturated ammonium chloride (NH ₄ Cl), the aqueous layer is extracted with ethyl acetate, and the organic layer is separated. The separated organic layer was washed with water and brine, dried over magnesium sulfate (MgSO ₄ ), and filtered under reduced pressure to remove the remaining solid, which was then concentrated under reduced pressure. The concentrated residue is dissolved in anhydrous DMF and then cooled to 0 ^° C. To a cooled mixture was added 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo [2.2.2] octane bis (tetrafluoroborate) (Selectfluor ^® ) (10.15 g, 28.65 mmol) And the mixture is stirred under the same conditions for 15 hours to produce fluorinated compounds 3a and 3b at a ratio of 3: 1, respectively (confirmed by NMR). When the reaction is confirmed by TLC, the reaction mixture is quenched with an aqueous solution of saturated ammonium chloride (NH ₄ Cl), the aqueous layer is extracted with ethyl acetate, and the organic layer is separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), filtered off under reduced pressure, and the residue was removed under reduced pressure. The concentrated residue was purified by silica gel chromatography or the like to obtain 2a (6.591 g, 58%) and 2b (3.078 g, 27%), respectively.

^{2a: 1 H NMR (400 MHz} , CDCl 3) 5.29 (dd, J = 8.2, 49.5 Hz, 1 H), 4.70 (t, J = 5.7 Hz, 1 H), 4.20 (dd, J = 2.4, 6.1 Hz , 1H), 3.61 (dd, J = 1.6, 8.6 Hz, 1H) 3.38-3.41 (m, 1 H), 2.75 (d, J = 8.2 Hz, 1 H), 1.41 (s, 3 H), 1.30 (s, 3 H), 1.06 (s, 9 H)

^{2b: 1 H NMR (600 MHz} , CDCl 3) 5.21-5.36 (ddd, J = 1.3, 4.5, 50.8 Hz, 1 H,), 4.55 (d, J = 5.9 Hz, 1 H), 4.50 (d, J = 5.9 Hz, 1H), 3.63 (d, J = 2.2 Hz, 2H), 2.52-2.58 (m, 1H), 1.41 , 9 H)

Method for producing substance 2c

2c (2.95 g, 84%) can be synthesized by proceeding in a similar manner to the synthesis of the above-synthesized substance 2a (3.29 g, 12.6 mmol) from the above-mentioned substance 1 to the substances 2a and 2b.

^{1 H NMR (500 MHz, CDCl} 3) 4.72 (t, J = 6.1 Hz, 1 H), 4.35-4.38 (m, 1 H), 3.68 (d, J = 8.6 Hz, 1 H), 3.68 (d, J = 8.6 Hz, 1 H) 3.46 (d, J = 8.5 Hz, 1 H), 2.67 (d, J = 17.4 Hz, 1 H), 1.42 (s, 3 H), 1.33 (s, 3 H), 1.05 (s, 9 H). (Mixed with a peak estimated to be equilibrated in diol form).

Method for producing material 3a

The material 2a (3.33 g, 12.8 mmol) is dissolved in methanol and cooled to below 0 ^° C sufficiently. Sodium borohydride (NaBH ₄ ) (1.45 g, 38.4 mmol) is slowly added to the cooled solution. Thereafter, the reaction is stirred under the same conditions, and the reaction is terminated by TLC. The reaction mixture is then quenched with an aqueous solution of saturated ammonium chloride (NH ₄ Cl), the aqueous layer is extracted with ethyl acetate, and the organic layer is separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), filtered off under reduced pressure, and the residue was removed under reduced pressure. The resulting alcohol compound was separated and purified by silica gel chromatography or the like (2.54 g, 76%) was dissolved in anhydrous pyridine and cooled to 0 ^° C. Trifluoromethanesulfonic anhydride (3.26 ml, 19.36 mmol) was added dropwise to the cooled mixture, and the mixture was stirred under the same conditions for 30 minutes to give trifluoromethylsulfone oxidation. After confirming that the reaction is terminated by TLC, the reaction mixture is quenched with an aqueous solution of saturated ammonium chloride (NH ₄ Cl), the aqueous layer is extracted with ethyl acetate, and the organic layer is separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), filtered off under reduced pressure, and the residue was removed under reduced pressure. The resulting trifluoromethylsulfone oxide compound and sodium azide (NaN ₃ ) (1.89 g, 29.04 mmol) were mixed in anhydrous DMF and stirred at 60 ^° C under heating to azide. After stirring for about 4 hours, the reaction was terminated by TLC. After completion of the reaction with water, the aqueous layer was extracted with ethyl acetate and the organic layer was separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), filtered off under reduced pressure, and the residue was removed under reduced pressure. The obtained azide compound was isolated and purified by silica gel chromatography or the like, and the obtained compound (4.07 g, 42%) was dissolved in methanol. An appropriate amount of palladium / carbon is added to the solution, and the reaction vessel is subjected to hydrogen substitution to proceed the hydrogenation reaction, thereby reducing the azide group to an amine group. After completion of the reaction, the reaction mixture is filtered to remove the remaining solid, and the filtrate is concentrated under reduced pressure to obtain the desired substance 3a. The resulting material 3a proceeds to the next step without separation and purification.

^{1 H NMR (600 MHz, CDCl} 3) d 4.96 (dt, J = 52.5, 3.3 Hz, 1 H), 4.31-4.36 (m, 2 H), 3.48-3.54 (m, 2 H), 3.27 (td, J = 4.1, 28.4 Hz, 1 H), 2.24-2.34 (m, 1 H), 1.80 (s, 2 H), 1.45 (s, 3 H), 1.26 (s, 3 H), 1.16 (s, 9 H)

Method for producing substance 3b

3b (3.24 g, 62%) can be synthesized by proceeding in the same manner as in the synthesis of the above-synthesized substance 2b (5.2 g, 19.97 mmol) from the above-mentioned substance 2a to substance 3a.

^{1 H NMR (300 MHz, CDCl} 3) d 4.59 (dt, J = 52.7, 6.0 Hz, 1 H), 4.48, (t, J = 4.02 Hz, 1 H), 4.15 (dd J = 6.42, 4.23 Hz, 3 H), 1.16 (s, 3 H), 1.16 (s, 3 H), 3.50 (m,

Method for producing substance 3c

3c (2.98 g, 60%) can be synthesized by proceeding in the same manner as in the synthesis of the above-synthesized substance 2c (4.95 g, 17.79 mmol) from the above-mentioned substance 2a to substance 3a. However, sodium beam hydride in the reaction of alcohol screen a ketone (NaBH ₄₎ instead of lithium to view it hydride (LiBH ₄₎ the uses, Oh the azide reaction of sodium azide to 10 times the amount used and the temperature at 100 ^o C And the stirring time should be increased to about 15 hours.

¹ H NMR (800 MHz, CDCl ₃ ) d 4.45-4.46 (m, 1H), 4.25-4.27 (m, 1H), 3.58 (dd, J = 4.5, 9.2 Hz, 1H) J = 5.1, 9.2 Hz, 1 H), 3.35-3.38 (m, 1 H), 2.56-2.59 (m, 1 H), 1.94 (bs, 3 H), 1.47 (s, 3 H), 1.28 (s , 3 H), 1.18 (s, 9 H)

Method for producing substance 4a

(982 mg, 3.757 mmol), 5-amino-4,6-dichloropyrimidine (1.85 g, 11.27 mmol) and diisopropylethylamine (DIPEA ) (6.54 mL, 37.57 mmol) of n - using the back were mixed in butanol (n -butanol), microwave, etc., heated to 170 ^o C and the mixture was stirred for 4 hours. After confirming that the reaction was completed by TLC, the reaction mixture was mixed with methanol and concentrated under reduced pressure. The residue was purified by silica gel chromatography or the like to obtain an intermediate (964 mg, 66%). The intermediate is dissolved in diethoxymethyl acetate, and the mixture is stirred at 140 ^° C for 3 hours using a microwave or the like. After confirming that the reaction was completed by TLC, the reaction mixture was mixed with methanol, concentrated under reduced pressure, and then purified by silica gel chromatography or the like to obtain substance 4a (643 mg, 65%).

^{1 H NMR (400 MHz, CDCl} 3) δ 8.74 (s, 1 H), 8.34 (d, J = 2.4 Hz, 1 H), 5.28-5.43 (dt, J = 52.8, 2.8 Hz, 1 H), 5.12 -5.23 (m, 2 H), 4.61 (t, J = 5.0 Hz, 1 H), 3.65-3.69 (m, 1 H), 3.61 (t, J = 9.2 Hz, 1 H), 2.56-2.71 (m , 1 H), 1.56 (s, 3 H), 1.32 (s, 3 H), 1.17 (s, 9 H)

Method for producing substance 4b

4b (943 mg, 39%) can be synthesized by proceeding in the same manner as in the synthesis of the above-synthesized substance 3b (1.592 g, 6.09 mmol) from the above-described substance 3a to the compound 4a.

^{1 H NMR (300 MHz, CDCl} 3) δ 8.72 (s, 1 H), 8.15 (s, 1 H), 5.59 (dt, J = 53.6, 8.42 Hz, 1 H), 5.05 (t, J = 5.52 Hz , 1.91 (m, 1H), 4.69 (m, 1H), 3.63 (m, 1H), 2.56 , 3 H), 1.22 (s, 9 H)

Method for producing substance 4c

7c (223 mg, 50%) can be synthesized by proceeding in the same manner as in the synthesis of the above synthesized substance 3c (182 mg, 0.625 mmol) from the above-mentioned substance 3a to the substance 4a. However, in the first reaction step, the temperature should be increased to 200 ^° C and the agitation time should be increased to about 7 hours.

^{1 H NMR (300 MHz, CDCl} 3) δ 8.76 (s, 1 H), 8.76 (s, 1 H), 8.29 (d, J = 2.4 Hz, 1 H), 5.26-5.37 (m, 1 H), 5.11 (t, J = 6.9 Hz , 1 H), 4.59-4.64 (m, 1 H), 3.64-3.74 (m, 2 H), 2.81-2.96 (m, 1 H), 1.58 (s, 3 H) , 1.33 (s, 3 H), 1.20 (s, 9 H)

Method for producing substance 5a

The material 4a (220 mg, 0.54 mmol) synthesized above was dissolved in tert- butanol and transferred to a stainless steel bomb reactor. Saturated ammonia / tert-butanol (tert-butanol) is added dropwise to the mixture, the reaction vessel is sealed and stirred at 120 ^° C for about 15 hours. After confirming that the reaction is completed by TLC, the reaction mixture is diluted with methanol and then concentrated under reduced pressure. The concentrated residue is dissolved in THF, and a solution of trifluoroacetic acid (TFA) and water (2: 1) is added dropwise to the mixture. The reaction mixture was stirred at 60 ^° C for about 15 hours, and then the reaction was terminated by TLC and concentrated under reduced pressure. The concentrated residue is purified by silica gel chromatography or the like to obtain the final substance 5a (66 mg, 43%).

^{1 H NMR (400 MHz, CD} 3 OD) δ 8.31 (d, J = 2.4 Hz, 1 H), 8.23 (s, 1 H), 5.36-5.39 (m, 1 H), 5.20-5.35 (td, J = 2.8, 53.6 Hz, 1H), 5.05-5.16 (m, 1H), 4.66-4.69 (m, 1H), 3.64-3.71 (m, 2H), 2.51-2.66 1.55 (s, 3 H), 1.35 (s, 3 H), 1.22 (s, 9 H)

Method for producing substance 5b

5b (556 mg, 71%) can be synthesized by proceeding in the same manner as in the synthesis of the above-synthesized substance 4b (1.1 g, 2.758 mmol) from the above-mentioned substance 4a to the substance 5a.

^{1 H NMR (400 MHz, CD} 3 OD) δ 8.19 (s, 1 H), 8.18 (s, 1 H), 5.40 (dt, J = 54.2, 7.32 Hz, 1 H), 5.03 (m, 1 H) , 4.59 (dd, J = 9.9,5.3 Hz, 1H), 4.07 (m, 1H), 3.80 (d, J = 5.9 Hz, 2H)

Method for producing substance 5c

5c (99 mg, 61%) can be synthesized by proceeding in the same manner as in the synthesis of the above synthesized substance 4c (223 mg, 0.534 mmol) from the above-mentioned substance 4a to the substance 5a.

¹ H NMR (400 MHz, CD ₃ OD)? 8.29 (s, 1H), 8.21 (s, 1H), 5.29-5.36 (m, 2H), 4.66 (bs, 1H), 3.75-3.79 3 H), 1.22 (s, 3 H), 1.22 (s, 3 H), 1.22

Method for producing substance 5d

The material 4b (21 mg, 0.0527 mmol) synthesized above was dissolved in methanol, heated at 80 ^° C under acidic conditions such as a mixed solution of trifluoroacetic acid and water (1: 1) and stirred for about 15 hours, The completion of the reaction is confirmed by TLC or the like, and the reaction is concentrated under reduced pressure. The concentrated residue is separated and purified by silica gel chromatography or the like to obtain the final substance 5d (10 mg, 67%).

^{1 H NMR (300 MHz, DMSO} -d 6) δ 12.3 (s, 1 H, D2O exchanged), 8.22 (s, 1 H), 8.03 (d, J = 3.66, 1 H), 4.92-5.24 (m, J = 5.67, 2H), 2.07-2.19 (m, 1H), 4.30 (m, 1H)

Method for producing substance 6a

The material 4a (113 mg, 0.283 mmol) synthesized above was dissolved in ethanol and transferred to a glass seal bomb. To the mixed solution, 40% methylamine aqueous solution was added dropwise, the reaction vessel was sealed, and stirred at room temperature for about 2 hours. After confirming that the reaction is terminated by TLC, the reaction mixture is concentrated under reduced pressure. The concentrated residue is dissolved in THF, and a solution of trifluoroacetic acid (TFA) and water (2: 1) is added dropwise to the mixture. The reaction mixture was stirred at 50 ^° C for about 30 hours, and the reaction was terminated by TLC and concentrated under reduced pressure. The concentrated residue is separated and purified by silica gel chromatography or the like to obtain the final substance 6a (66 mg, 67%).

¹ H NMR (500 MHz, CD ₃ OD)? 8.28 (s, 1H), 8.24 (s, 1H), 5.15-528 (dt, J = 54.7, 3.85 Hz, 1H), 4.96-5.04 J = 29.55, 9.95, 3.25 Hz, 1H), 4.75 (dd, J = 9.55,6.80 Hz, 1H), 4.28 (dd, J = 6.65, 4.90 Hz, 1H) ), 3.12 (bs, 3 H), 2.44-2.53 (m, 1 H)

Method for producing substance 6b

6b (128 mg, 76%) can be synthesized by proceeding in the same manner as in the synthesis of substance 4c (223 mg, 0.535 mmol) synthesized above in the same manner as in the synthesis of substance 6a from the above-mentioned substance 4a.

¹ H NMR (500 MHz, CD ₃ OD)? 8.24 (s, 1H), 8.20 (s, 1H), 5.33 (m, 1H), 4.79 (dd, J = 10.3, 10.2 Hz, 1H) 3.17 (s, 1H), 3.81-3.90 (m, 2H), 3.10 (bs,

Method for producing substance 7b

The material 5c (20 mg, 0.066 mmol) synthesized above was dissolved in acetone, and a catalytic amount of sulfuric acid was added dropwise at 0 ^° C., followed by stirring at room temperature for about 8 hours. After confirming that the reaction is completed by TLC, it is neutralized with sodium bicarbonate (NaHCO ₃ ) and the reaction mixture is concentrated under reduced pressure. The concentrated residue is purified by silica gel chromatography or the like to obtain 2 ', 3'-isopropylidene compound (21 mg, 0.063 mmol). The obtained 2 ', 3'-isopropylidene compound (21 mg, 0.063 mmol) and dimethylaminopyridine (DMAP) (0.7 mg, 0.058 mmol) were dissolved in hexamethyldisilazane (HMDS) Then, trimethylsilyl methanesulfonate (TMSOTf) (5 μL, cat.) Was added dropwise at room temperature, and the mixture was heated to about 75 ^° C. and stirred for about 2 hours. Then, after which the reaction mixture was concentrated under reduced pressure, the concentrated residue was added dropwise to the mixture solution was dissolved in anhydrous THF dicarboxylic acid di-tert-butyl _{(di- tert -butyl dicarbonate, Boc 2} O) (63 mg, 0.29 mmol). The reaction mixture is stirred at room temperature for about 4 hours and then concentrated under reduced pressure. The concentrated residue is dissolved in a mixed solvent of methanol: trimethylamine = 5: 1, heated to 55 ^° C, and stirred for about 16 hours. After completion of the reaction is confirmed by TLC or the like, it is concentrated under reduced pressure. The concentrated residue is diluted with water, extracted with ethyl acetate, and the organic layer is separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), filtered off under reduced pressure, and the residue was removed under reduced pressure. The concentrated residue was separated by silica gel chromatography or the like to give the N, N - di (tert-butyl carbonic acid) (N, N -diBoc) an intermediate screen (8 mg, 0.015 mmol) and N - tert-butyl carboxylic acid ( N- Boc) intermediate (13 mg, 0.029 mmol). After dissolving the two intermediates (21 mg, 0.043 mmol) in anhydrous THF, 1.0 M THF solution of tert- butylmagnesium chloride 1.0 M solution in THF (0.215 ml, 0.215 mmol) was added dropwise at 0 ^° C . To this reaction mixture was added 1-methylethyl N - [(S) - (2,3,4,5,6-octylphenoxy) phenoxyphosphinyl] -L-alanine (N - [(S) - , 3,4,5,6-pentafluorophenoxy) phenoxyphosphinyl] -L-alanine 1-Methylethyl ester (0.21 mg, 0.45 mmol) in anhydrous THF was added dropwise at 0 ^° C., The mixture is stirred for about an hour. After confirming that the reaction is terminated by TLC, the reaction mixture is quenched with an aqueous solution of saturated ammonium chloride (NH ₄ Cl), the aqueous layer is extracted with ethyl acetate, and the organic layer is separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), filtered off under reduced pressure, and the residue was removed under reduced pressure. The concentrated residue was purified by silica gel chromatography or the like to obtain a precursor (10 mg, 0.0215 mmol) which was not reacted with the intermediate (15 mg, 0.0215 mmol). The reaction intermediate (15 mg, 0.0215 mmol) is suspended in formic acid aqueous solution (50 v / v%) and stirred at room temperature for about 8 hours to carry out the protecting group elimination reaction. After confirming that the reaction was completed by TLC, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography or the like to obtain Compound 7b (12 mg, 100%).

_{^{1 H NMR (CD 3 OD,}} 500 MHz): 8.17 (s, 1 H), 8.14 (s, 1 H), 7.33-7.14 (m, 5 H), 5.42 (ddd, J = 54.1, 6.35, 6.35 Hz J = 9.3, 5.35 Hz, 1 H), 5.00 (ddd, J = 20.75, 8.05, 8.05 Hz, 1H), 4.92 (ddd, J = 12.45, 6.25, 6.25 Hz, 1H) 1 H), 3.99-3.93 (m, 1H), 2.59-2.49 (m, 1H), 4.36 (t, J = 5.45 Hz, 2H), 4.12 (dd, J = ), 1.34 (d, J = 7 Hz, 3 H), 1.20 (d, J = 6.25 Hz, 3 H), 1.16 (d, J = 6.25 Hz, 3 H)

Method for producing substance 7a

7a (27 mg, 27%) can be synthesized by proceeding in the same manner as in the synthesis of the above synthesized substance 5b (100 mg, 0.35 mmol) from the above-mentioned substance 5c in the same manner as in the synthesis of substance 7b.

¹ H NMR (CD ₃ OD, 500 MHz): 8.20 (s, 1H), 8.18 (bs, 1H), 7.38-7.18 (m, 5H), 5.33 (ddd, J = 18.5, 9.7, 9.7 Hz , 4.95 (ddd, J = 12.3, 6.15, 6.15 Hz, 1H), 4.76 (dd, J = 9.95, 5.05 Hz, 1H), 4.42-4.32 (m, 2H), 4.19 , 1 H), 3.92-3.86 (m , 1 H), 2.92-2.83 (m, 1 H), 1.33 (d, J = 7 Hz, 3 H), 1.20 (d, J = 6.2 Hz, 3 H) , 1.17 (d, J = 6.2 Hz, 3 H)

Method for producing substance 8a

3-methoxyacrylate (20 mL, 186 mmol) was added to a 2 M sodium hydroxide aqueous solution (103 mL) and stirred at room temperature for about 2 hours to dissolve completely. The reaction mixture is completely acidified with 2 M aqueous hydrochloric acid solution and collected by filtration. The solid organic acid obtained above is dissolved in water and neutralized with a 2 M aqueous sodium hydroxide solution to obtain an organic acid in the form of a sodium salt. This is dried in a vacuum desiccator for 3 days with phosphorus (V) oxide. The dried organic acid sodium salt (6.20 g, 50 mmol) was suspended in anhydrous diethyl ether and then thionyl chloride (5.45 mL, 75 mmol) was added dropwise. The mixture was heated to reflux for about 4 hours . Thereafter, the reaction mixture is stirred at 30 ^° C for about 15 hours to form an organic acid chloride. The reaction mixture is cooled to room temperature and filtered under reduced pressure while washing with anhydrous diethyl ether. The obtained filtrate was concentrated under reduced pressure in a nitrogen gas, and the concentrated residue was distilled under reduced pressure to obtain an organic acid chloride (3.08 g, 51%). Dry silver cyanate (8.44 g, 56.32 mmol) is suspended in benzene and heated to reflux for 30 minutes. The organic acid chloride (1.45 g, 12.03 mmol) obtained above was added dropwise to the prepared suspension, and heated to reflux for 30 minutes to obtain isocyanate, which was precipitated without any further procedure. The supernatant was added to the next reaction Use. The material 3a (2.572 g, 9.84 mmol) synthesized above is dissolved in DMF and sufficiently cooled to below -20 ^° C. To this reaction mixture was added isocyanic acid (45 mL, 19.68 mmol) synthesized previously, and the mixture was stirred for about 15 hours while being gradually heated to room temperature. The reaction is terminated by TLC, washed with methylene chloride, filtered under reduced pressure, and the filtrate is concentrated under reduced pressure. At this time, an azeotropic mixture with toluene, ethanol and the like was made and sufficiently concentrated under reduced pressure to solidify the residue. The solidified residue was purified by silica gel chromatography or the like to obtain an urea derivative (2.903 g, 76%) as an intermediate have. After the obtained urea derivative was dissolved in 1,4-dioxane, a small amount of a 2 M aqueous sulfuric acid solution (~1: 10 = 2 M sulfuric acid: 1,4-dioxane) was added dropwise, And the elimination reaction of the protecting group are simultaneously carried out. After confirming that the reaction was completed by TLC, the reaction mixture was neutralized using a basic resin such as DOWEX 66 ion-exchange resin, and the residue obtained through vacuum filtration and concentration under reduced pressure was purified by silica gel Separation and purification through chromatography or the like affords substance 8a (1.097 g, 56%).

^{1 H NMR (400 MHz, CD} 3 OD) δ 7.69 (d, J = 8.08 Hz, 1 H), 5.68 (d, J = 8.04 Hz, 1 H), 5.02-5.18 (td, J = 55.28, 3.9 Hz (M, 1H), 4.76 (m, 1H), 3.93 (t, J = 5.00 Hz, 1H), 3.76 (m, 2H), 2.30-2.41 1 H)

Method for producing substances 8b and 8c

(597 mg, 53%), 8c (1 g (53%)), respectively, by proceeding in the same manner as in the synthesis of the precursor substance 3b (1.3 g, 4.97 mmol) and 3c , 52%).

^{8b 1 H NMR (500 MHz,} CD 3 OD) δ 7.60 (d, J = 7.95 Hz, 1 H), 5.69 (d, J = 7.90 Hz, 1 H), 5.07-5.21 (ddd, J = 55.2, 6.85 (Dd, J = 22.6, 8.75, 7.35 Hz, 1H), 4.32 (dd, J = 9.00, 5.25 Hz, 1H), 3.98 (t, J = 3.75 Hz, 1H), 4.61-4.69 , 1H), 3.70 (m, 2H), 2.24 (m, 1H)

^{8c 1 H NMR (500 MHz,} CD 3 OD) δ 7.67 (dd, J = 8.15, 2.35 Hz, 1 H) 5.71 (d, J = 8.05 Hz, 1 H), 5.36, (dt, J = 17.7, 10.3 (M, 1H), 3.73-3.82 (m, 2H), 2.53 (m, 1H), 4.41 (dd, J = 10.7,5.15 Hz, 1H)

Method for producing substance 9a

After dissolving material 8a (100 mg, 0.384 mmol) in methylene chloride, pyridine (0.21 mL, 2.57 mmol) was added dropwise and cooled to 0 ^° C. Benzyl chloride (0.27 mL, 2.31 mmol) was added dropwise to the cooled reaction mixture, and the mixture was slowly heated to room temperature and stirred for about 15 hours. After confirming that the reaction is completed by TLC, the reaction is terminated by water, the aqueous layer is extracted with methylene chloride, and the organic layer is separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), filtered off under reduced pressure, and the residue was removed under reduced pressure. The concentrated residue can be isolated and purified by silica gel chromatography or the like to obtain a benzoylated intermediate (164 mg, 75%). 1,2,4-triazole (198 mg, 2.87 mmol) was made a suspension in anhydrous acetonitrile, cooled to 0 ^° C and phosphoryl chloride (0.27 mL, 2.87 mmol). Intermediate obtained above The reaction mixture was stirred for about 30 minutes at ^{0 o C (164 mg, 0.286} mmol) was added dropwise then followed by triethylamine (trimethylamine) dissolved in anhydrous acetonitrile (0.4 mL, 2.87 mmol) to 0 ^o C in the fall. The reaction mixture is slowly heated to room temperature and stirred for about 15 hours. After confirming that the reaction is terminated by TLC, the reaction mixture is concentrated under reduced pressure. The concentrated residue is diluted with methylene chloride and separated twice with a small amount of water to remove residual 1,2,4-triazole. The washed organic layer is concentrated under reduced pressure, dissolved in 1,4-dioxane, transferred to a sealable reaction vessel, and saturated aqueous ammonia is added at a rate of 20 v / v% of the solution. The reaction mixture is stirred at room temperature for about 2 hours, and then the reaction mixture is concentrated under reduced pressure. The concentrated residue is separated and purified by silica gel chromatography or the like to obtain an intermediate (42 mg, 26%) which is converted into cytosine. After dissolving the cytosine intermediate (42 mg, 0.0735 mmol) in methanol, the reaction mixture was transferred to a hermetically sealed reaction vessel, saturated ammonia / methanol solution was added thereto, and the mixture was stirred at room temperature for about 2 days. The concentrated residue is diluted with a small amount of water and extracted with methylene chloride approximately 10 times to remove the remaining benzoyl adduct to obtain substance 9a (17 mg, 85%).

^{1 H NMR (500 MHz, CD} 3 OD) δ 7.67 (d, J = 7.45, 1 H), 5.88 (d, J = 7.45, 1 H) 5.05-5.17 (dt, J = 55.4, 3.65 Hz, 1 H ), 4.89-4.97 (ddd, J = 30.6, 10.2, 3.2 Hz, 1 H), 4.44 (dd, J = 10.4, 6.7 Hz, 1 H), 3.92 (t, J = 4.75 Hz, 1 H), 3.75 (m, 2H), 2.36 (m, 1H)

Method for producing substance 9b

Substance 8c (100 mg, 0.359 mmol) is treated in the same manner as in the synthesis of substance 9a from the above-described substance 8a to obtain substance 9b (20 mg, 20%). However, the residue concentrated in the final step should be separated and purified with an acidic resin or the like.

^{1 H NMR (500 MHz, CD} 3 OD) δ 7.62 (dd, J = 7.45, 2.35 Hz, 1 H), 5.90 (d, J = 7.40 Hz, 1 H), 5.51 (dt, J = 18.2, 10.0 Hz , 3.17 (m, 1H), 3.73-3.83 (m, 2H), 2.54 (m, 1H), 4.37 (dd, J = 10.6,5.25 Hz, 1H)

Method for producing substance 10a

Substance 8a (40 mg, 0.15 mmol) was dissolved in acetone, and a catalytic amount of sulfuric acid was added dropwise at 0 ^° C., followed by stirring at room temperature for about 2 hours. After confirming that the reaction is completed by TLC, it is neutralized with sodium bicarbonate (NaHCO ₃ ) and the reaction mixture is concentrated under reduced pressure. The concentrated residue is separated and purified by silica gel chromatography or the like to obtain 2 ', 3'-isopropylidene compound (45 mg, 0.15 mmol). Intermediate 2 ', 3'-isopropylidene compound (45 mg, 0.15 mmol) was dissolved in anhydrous THF and tert- butylmagnesium chloride 1.0 M solution in THF (0.75 ml, 0.75 mmol) It drops at 0 ^o C. To this reaction mixture was added 1-methylethyl N - [(S) - (2,3,4,5,6-octylphenoxy) phenoxyphosphinyl] -L-alanine (N - [(S) - , 3,4,5,6-pentafluorophenoxy) phenoxyphosphinyl] -L-alanine 1-Methylethyl ester (68 mg, 0.15 mmol) in anhydrous THF was added dropwise at 0 ^° C., The mixture is stirred for about an hour. After confirming that the reaction is terminated by TLC, the reaction mixture is quenched with an aqueous solution of saturated ammonium chloride (NH ₄ Cl), the aqueous layer is extracted with ethyl acetate, and the organic layer is separated. The separated organic layer was washed thoroughly with water and brine, dried over magnesium sulfate (MgSO ₄ ), filtered off under reduced pressure, and the residue was removed under reduced pressure. The concentrated residue was purified by silica gel chromatography or the like to obtain a precursor (25 mg, 0.083 mmol) which was not reacted with the intermediate (28 mg, 0.0495 mmol). (28 mg, 0.0495 mmol) is suspended in an aqueous formic acid solution (50 v / v%) and stirred for about 8 hours at room temperature to carry out the protecting group elimination reaction. After confirming that the reaction is completed by TLC, the reaction mixture is concentrated under reduced pressure, and the residue is purified by silica gel chromatography or the like to obtain Compound 10a (24 mg, 90%).

_{^{1 H NMR (CD 3 OD,}} 500 MHz): 7.64 (d, J = 8.05 Hz, 1 H), 7.34-7.37 (m, 2 H), 7.17-7.24 (m, 3 H), 5.68 (d, J = 8.05 Hz, 1 H), 5.04 (ddd, J = 55, 3.9, 3.9 Hz, 1 H), 4.93-4.98 (m, 1 H), 4.89-4.92 (m, 1 H), 4.44 (dd, J = 9.7,6.6 Hz, 1H), 4.26 (t, J = 7 Hz, 2H), 3.99 (t, J = 5.4 Hz, 1H), 3.91-3.85 (m, 1H), 2.51-2.57 m, 1 H), 1.33 (d, J = 7 Hz, 3 H), 1.21 (d, J = 6.15 Hz,

Method for producing substance 10b

Substance 8c (30 mg, 0.10 mmol) was subjected to the same procedure for synthesis of substance 10a from the above-mentioned substance 8a to obtain substance 10b (31 mg, 32%).

_{^{1 H NMR (CD 3 OD,}} 500 MHz): 7.53 (dd, J = 8.05, 2.1 Hz, 1 H), 7.38-7.35 (m, 2 H), 7.25-7.18 (m, 3 H), 5.69 (d J = 8.1 Hz, 1H), 5.34 (ddd, J = 18.85, 9.7, 9.7 Hz, 1H), 4.97 (ddd, J = 12.5, 6.2, 6.2 Hz, 1H), 4.24-4.36 3 H), 4.08 (bs, 1 H), 3.87-3.90 (m, 1 H), 2.70-2.80 (m, 1 H), 1.34 (d, J = 7.15 Hz, 3 H), 1.22 (d, J = 6.25 Hz, 6 H)

Preparation of cells, viruses and substances

Vero E6 and Vero CCL81 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Lonza) supplemented with 8% fetal calf serum (FCS), 2 mM L-glutamine, 100 IU / ml penicillin, 100 g streptomycin / ml in the presence of 5% carbon dioxide at 37 ° C in a constant-temperature and constant-humidity incubator.

Vero cell lines were grown in medium supplemented with 8% FCS, 2 mM L-glutamine, penicillin 100 IU / ml and streptomycin 100 g / ml in Eagle's Minimum Essential Medium (EMEM; Lonza) And cultured under the same conditions described above.

HuH7 cell lines were grown in MDEM supplemented with 8% FCS, 2 mM L-glutamine, 100 IU / ml penicillin, streptomycin 100 μg / ml, and 1x non-essential amino acids (NEAA; Lonza) Were cultured under the same conditions as described above.

The MRC-5 cell line was cultured in EMEM under the same conditions described above using 8% FCS, 2 mM L-glutamine, 100 IU / ml of penicillin, 100 μg / ml of streptomycin and 1 × NEAA Lt; / RTI &gt;

For viral infection, EMEM and 2% FCS, mM L-glutamine, penicillin 100 IU / ml, streptomycin (1 mg / ml) were added to 25 mM N-2-Hydroxyethylpiperazine-N "-2-ethanesulfonic acid ) 100 [mu] g / ml were used.

The clone-derived chikungunya virus (CHIKV LS3) infectious agent was produced by the method described in Scholte Group's PLOS ONE 8 (2013): e71047.

The Zika virus (ZIKV) strain SL1602 was produced by the Van Boheemen group Sci . Rep. (2017) in press from an infected traveler returning from Suriname.

Middle east respiratory syndrome coronavirus (MERS-CoV; strain EMC / 2012) was developed by Fakeeh Hospital of Jeddah, Saudi Arabia. Soliman was isolated from patient tissue (2012, Van Boheemen group) and received at the Erasmus Medical Center in Rotterdam, The Netherlands.

Severe acute respiratory syndrome coronavirus (SARS-CoV strain Frankfurt 1) was obtained from H. F. Rabenau and H. W. Doerr of Johann Wolfgang Goethe University in Frankfurt am Main, Germany. (2006, Snijder Group)

Each of the evaluation materials was dissolved in DMSO and used at 20 mM and 10 μM.

Experiments using infectious agents CHIKV, MERS-CoV, SARS-CoV, and ZIKV were performed within the biological safety cabinet in the LUMC Biosafety Level 3 facility.

CPE -reduction assays

The VeroE6 cell line was inoculated in 96-well clusters at a concentration of 5,000 cells / well (CHIKV) in each well in a volume of 100 μl.

The Vero (CCL81) cell line was inoculated into 96-well clusters at 5,000 cells / well (ZIKV).

Vero, HuH7 and MRC-5 cell lines were inoculated in 96-well clusters at 20,000, 10,000 and 15,000 cells / well, respectively, for MERS-CoV CPE reduction assays.

To perform the SARS-CoV CPE reduction assays, the VeroE6 cell line was inoculated into 96-well clusters at 10,000 cells / well.

On the next day after inoculation in each cell line, the evaluation materials were prepared in the above-described virus infection medium by serial dilution at a starting concentration of 100 [mu] M in duplicate, and then exchanged with the medium in the existing 96-well clusters.

The cells were incubated in the same incubation medium for CHIKV (MOI 0.005), ZIKV (MOI 0.050), MERS-CoV (MOI 0.01 in HuH7, MOI 0.005 in Vero and MOI 0.03 in MRC-5 cells) or SARS-CoV (Quadruplicate).

In the case of uninfected cells, the test material was treated with diluted media in the same manner, and a cytotoxicity test (CC ₅₀ evaluation) was performed simultaneously.

After 96 hours, the virus was subjected to colorimetric viability assays using CellTiter 96 ^® AQ _ueous One Solution Cell Proliferation Assay (MTS) reagent (Promega).

Assay was terminated by treatment with 30 μl 37% formaldehyde for 2 to 2.5 hours.

Absorbance was measured using a Berthold Mithras LB 940 plate reader at 495 nm and normalized to cells not treated with the infectious agent and the assessed material.

For the CC ₅₀ measurement, normalization was performed on the infected cells and the untreated cells.

The results were expressed as the mean value of the values obtained under the same 4-iteration conditions and expressed as mean ± standard deviation.

EC ₅₀ values were determined using a non-linear regression method using Graphpad Prism.

The experimental results are summarized in Figs.

While the present invention has been described in connection with certain exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (1)

A carbocyclic nucleoside derivative represented by the following formula (a) or a pharmaceutically acceptable salt thereof.
<Formula a>

(Wherein, in the above formula (a), n is 2, R 1 and R 2 are each independently hydrogen or fluorine, B is the following formula b-1 or b-2 and P is hydrogen or a phosphoamidate group.
<Formula b-1>

(In the above formula (b-1), X1 is chlorine, an OH group, an amino group or an alkylamino group, and Y1 is hydrogen or an amino group.
The alkylamino group may be a monoalkylamino group (-NHR) such as -NHMe or -NHEt or a dialkylamino group (-NR2) such as -NMe2, -NMeEt or -NEt2,
<Formula b-2>

(In the above formula (b-2), X2 is a hydroxyl group or an amino group, and Y2 is hydrogen, a methyl group or a halogen)

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