WO2018131950A1

WO2018131950A1 - Carbocyclic nucleoside derivative and antiviral agent comprising same

Info

Publication number: WO2018131950A1
Application number: PCT/KR2018/000644
Authority: WO
Inventors: 정낙신; 신영섭; 김규동; 윤지성; 김홍래; 김혜옥; 쿠마 사후프라모드; 리테오간도 에스. 나타샤 리마; 코바시코바크리스티나; 클라라 포스츄마씨.; 에릭 스나이더제이.; 허머트제이. 마틴 반
Original assignee: 퓨쳐메디신 주식회사; 서울대학교산학협력단
Priority date: 2017-01-12
Filing date: 2018-01-12
Publication date: 2018-07-19

Abstract

The present invention relates to a carbocyclic nucleoside derivative represented by chemical formula A or a pharmaceutically acceptable salt thereof, and an antiviral agent comprising the same.

Description

Carbocyclic nucleoside derivatives and antiviral agents comprising the same

The present invention relates to carbocyclic nucleoside derivatives and antiviral agents comprising the same.

Virus is a representative cause of numerous intractable diseases that threaten human health, and invests enormous capital to prevent or treat them worldwide. Recently, intractable RNA viruses such as Chikungunya Virus, SARS virus, MERS virus, and Zika virus have caused serious risks to humans, thus inhibiting their proliferation. Although there is an urgent need to develop a virus treatment that can be done, there are currently no treatments or preventive agents that can effectively suppress the viruses.

On the other hand, antiviral agents can inhibit the virus through various mechanisms (mechanism), two of which are known to be effectively used to suppress the virus.

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

SAH hydrolase is a tetramer type enzyme that uses NAD + as a coenzyme. It reversibly hydrolyzes SAH into adenosine (Ado) and homocysteine (Hcy). In addition to nucleosides, it is an important enzyme for the methylation of substances like histamine and norepinephrine.

The inhibition of SAH hydrolase causes the accumulation of SAH, and the excess SAH in turn inhibits S-adenosylmethionine (AdoMet) -dependent transmethylase and capping of viral mRNAs, thereby reducing the protein required for virus replication. It can't be made properly, resulting in an antiviral effect.

SAH hydrolase is considered an essential component in the development of a broad spectrum antiviral agent, as most animal DNA viruses, as well as RNA viruses, require methylation enzymes (viral mRNA guanosine N7-methytransferases, O-2′-methytransferase) for mRNA capping. do. In other words, the development of RNA virus therapeutics and the development of SAH hydrolase inhibitors seem to have a high correlation.

The second is to inhibit viral RNA polymerase.

RNA viruses replicate by inserting Nucleoside-5'-triphosphate (NTP), a substrate, into an RNA chain by RNA polymerase. Thus, substances that inhibit RNA polymerase may also act as antiviral agents, and are converted to triphosphate in the body to selectively inhibit viral RNA polymerase or directly inserted into the viral RNA chain, thereby resulting in chain termination. ), It is thought that the development of effective antiviral agents is possible.

When an antiviral agent functions as an inhibitor of RNA polymerase against normal cells as well as viruses, it is difficult to increase its selectivity because it may be toxic to normal cells.

Accordingly, the problem to be solved by the present invention is that it has a function of inhibiting SAH hydrolase, and can effectively suppress the virus, but has little toxicity in the body, and is very suitable for the inhibition of the virus and the prevention and treatment of viral diseases. It is to provide a novel carbocyclic nucleoside derivative that can be utilized.

The objects of the present invention are not limited to the above-mentioned technical problem, and other technical problems not mentioned will be clearly understood by those skilled in the art from the following description.

Carbocyclic nucleoside derivative or a pharmaceutically acceptable salt thereof according to an embodiment of the present invention for solving the above problems is represented by the following formula (A).

(In Formula A, n is 1 or 2, R1 and R2 are each independently hydrogen or fluorine, B is the following formula B-1 or B-2, P is hydrogen or phosphoamidate group to be)

(In the above formula B-1, X1 is a chlorine, a hydroxyl group (OH group), an amino group or an alkylamino group, Y1 is hydrogen or an amino group.

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

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

Formula A may be the following Formula a.

R 2 in Formula A may be fluorine.

Formula a may be the following Formula 5a, 5b or 5c.

Formula A may be the following Formula b.

In Formula b, R 1 may be fluorine.

Formula b may be the following Formula 15a or Formula 15b.

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 problems is represented by the following formula (A).

Formula A may be the following Formula a.

R 2 in Formula A may be fluorine.

Formula a may be the following Formula 5a, 5b or 5c.

Formula A may be the following Formula b.

In Formula b, R 1 may be fluorine.

Formula b may be the following Formula 15a or Formula 15b.

The present invention is expected to be developed as a new antiviral agent with little toxicity in the body, which can contribute to the development of the new drug development field, a representative national knowledge-based business in the future, can be expected to improve the public health It is, of course, expected to contribute to economic development.

1 to 8 summarize the experimental results.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the present embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the art to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

Hereinafter, the present invention will be described in detail.

Carbocyclic nucleoside derivatives according to one embodiment of the present invention may be represented by the following formula (A).

In Formula A, n is 1 or 2, R ₁ and R ₂ are each independently hydrogen or fluorine, B is the following formula B-1 or B-2, P is hydrogen or phosphoamidate It may be a flag.

In Formula B-1, X1 may be a chlorine, a hydroxyl group, an amino group, or an alkylamino group, and Y1 may be 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.

In Formula B-2, X2 may be a hydroxyl group or an amino group, and Y2 may be hydrogen, a methyl group, or a halogen.

In an embodiment, n may be 1 in Formula A, and P may be hydrogen. That is, the carbocyclic nucleoside derivative represented by Formula A may be a carbocyclic nucleoside derivative represented by Formula A below.

Specifically, in Chemical Formula a, n is 1, P is hydrogen or a phosphoramidate group, and B may be Chemical Formula B-1. More specifically, the carbocyclic nucleoside derivative represented by Chemical Formula a may be a compound represented by Chemical Formula 5a, a compound represented by 5b, or a compound represented by Chemical Formula 5c.

In another embodiment, in Formula A, n is 2, and P may be hydrogen. That is, the carbocyclic nucleoside derivative represented by Formula A may be a carbocyclic nucleoside derivative represented by Formula b below.

Specifically, in Formula b, R1 may be fluorine, and B may be Formula B-1. More specifically, the carbocyclic nucleoside derivative represented by Formula b may be a compound represented by Formula 15a or a compound represented by Formula 15b.

Carbocyclic nucleoside derivatives represented by Formula A may be provided in the form of a pharmaceutically acceptable salt. As salts are acid addition salts formed with various pharmaceutically acceptable organic or inorganic acids. Suitable organic acids include, for example, tetracarboxylic acid, phosphonic acid, sulfonic 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, aspartic acid, Maleic acid, benzoic acid, salicylic acid, phthalic acid, phenylacetic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, methyl sulfuric acid, ethyl sulfuric acid, dodecyl sulfuric acid, and the like can be used. Or phosphoric acid may be used.

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

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

An antiviral agent refers to a pharmaceutical composition that can be used not only to inhibit the activity, replication, etc. of a virus, but also to prevent and / or treat all diseases caused by a virus.

The virus is, for example, Chikungunya Virus (CHIKV), Samryki Forrest Virus (SFV), MERS virus (Middle East Respiratory Syndrome Coronavirus (MERS-CoV), SARS Virus (Severe Acute) RNA viruses such as Respiratory Syndrome Coronavirus (SARS-CoV), Equine Arteritis Virus (EAV), Mouse Hepatitis Virus (MHV), Sindbis Virus (SINV), and the like.

The antiviral agent including the carbocyclic nucleoside derivative described above may be a pharmaceutical composition that effectively inhibits a virus by using the main mechanism of inhibiting SAH hydrolase.

The antiviral agent may be administered systemically or locally, and formulated with excipients (or diluents) such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like, which are generally used for oral or parenteral administration. Can be. Hereinafter, although an excipient and a formulation method are illustrated concretely, it is not limited to these examples.

Solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ( sucrose, lactose, gelatin and the like can be mixed and prepared. In addition to the simple excipients, lubricants such as magnesium stearate, talc and the like can also be used. Liquid formulations for oral administration include, for example, suspensions, solutions, emulsions, syrups, and the like, and liquid formulations include various excipients, for example, wetting agents, in addition to water, liquid paraffin, which are commonly used simple diluents, Sweeteners, fragrances, preservatives and the like.

Formulations for parenteral administration may include injections, emulsions, inhalants, suppositories, and the like. Injectables may include sterile aqueous solvents, non-aqueous solvents and suspending agents, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, esters such as ethyl oleate, and the like, suppositories being based on witepsol, Macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like. The antiviral agents of the invention may also be formulated in ointments or creams for topical application.

The preferred dosage of the antiviral agent depends on a number of factors, including the condition and weight of the patient, the extent of the disease, the form of the drug, the route and duration of administration, and can be appropriately selected by those skilled in the art. The route of administration may also vary depending on the condition of the patient and its severity.

Because the carbocyclic nucleoside derivatives of the present invention have antiviral efficacy against RNA viruses and at the same time have biocompatible properties with little or no toxicity in the body (see Experimental Examples), inhibition of viruses and prevention of viral diseases and / Or as a pharmaceutical composition well suited for treatment.

Hereinafter, a method for preparing the carbocyclic nucleoside derivative will be described in detail.

Preparation Example 1

References [1] Tetrahedron: Asymmetry, 2002, 13, 1189-1193 and [2] J. Med. Chem. With reference to 2001, 44, 3985-3993, the compound of Formula 1 may be synthesized.

Preparation Example 2-1

The compound of formula 1 (6 g, 24.8 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 was slowly added dropwise 1.0 M lithium bis (trimethylsilyl) amide / THF solution (LiHMDS 1.0 M solution in THF) (50 mL, 50 mmol), followed by stirring the reaction well under the same conditions for 30 minutes to obtain silyl enol ether. It makes me angry. After confirming that the reaction was terminated by TLC, the reaction mixture was terminated with an aqueous saturated ammonium chloride (NH 4 Cl) solution, the aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was sufficiently washed with water and brine, dried over magnesium sulfate (MgSO 4), and the residual solid was removed by filtration under reduced pressure, and then concentrated under reduced pressure. This concentrated residue is taken up in anhydrous DMF and then cooled to 0 ° C. To the cooled mixture was added 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo [2.2.2] octane bis (tetrafluoroborate) (trade name Selectfluor®) (10.15 g, 28.65 mmol) to 1.5. When the equivalent was added and stirred for 15 hours under the same conditions, fluorinated compounds 3a and 3b were produced in a ratio of 3: 1 (identified by NMR). When TLC confirms the completion of the reaction, the reaction mixture is terminated with saturated aqueous ammonium chloride (NH 4 Cl) solution, the aqueous layer is extracted with ethyl acetate, and the organic layer is separated. The separated organic layer was washed with water and brine sufficiently, dried over magnesium sulfate (MgSO4), removed under reduced pressure, and concentrated under reduced pressure. The concentrated residue was separated and purified through silica gel chromatography to obtain the compound of Formula 2a (6.591 g, 58%) and the compound of Formula 2b (3.078 g, 27%) having the following 1H-NMR spectrum. Respectively obtained.

2a: 1H NMR (400 MHz, CDCl3) 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, 1 H), 3.61 (dd, J = 1.6, 8.6 Hz, 1 H) 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: 1H NMR (600 MHz, CDCl3) 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 (s, 3H), 1.33 (s, 3H), 1.13 (s, 9 H)

Preparation Example 2-2

The same procedure as in Preparation Example 2-1 was carried out except that the compound of Formula 2a (3.29 g, 12.6 mmol) synthesized in Preparation Example 2-1 was used as the starting material. The compound of formula 2c (2.95 g, 84%) having 1 H-NMR spectrum was obtained.

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 peaks presumed to be equilibrated in diol form.)

Preparation Example 3-1

The compound of formula 2a (3.33 g, 12.8 mmol) obtained in Preparation Example 2-1 was dissolved in methanol, and then cooled sufficiently to 0 ° C or less. To the cooled solution is added sodium borohydride (NaBH 4) (1.45 g, 38.4 mmol) slowly. After stirring the reaction under the same conditions and confirming the reaction termination by TLC, the reaction mixture was terminated with a saturated aqueous solution of 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 with water and brine sufficiently, dried over magnesium sulfate (MgSO4), removed under reduced pressure, and concentrated under reduced pressure. The intermediate (2.54 g, 76%) obtained by separating the produced alcohol compound by silica gel chromatography was dissolved in anhydrous pyridine and cooled to 0 ° C. Anhydrous trifluoromethanesulfonic anhydride (3.26 ml, 19.36 mmol) was added dropwise to the cooled mixture, followed by stirring for 30 minutes under the same conditions to trifluoromethylsulfonated. After confirming that the reaction was terminated by TLC, the reaction mixture was terminated with a saturated aqueous solution of 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 with water and brine sufficiently, dried over magnesium sulfate (MgSO4), removed under reduced pressure, and concentrated under reduced pressure. The resulting trifluoromethylsulfonated compound and sodium azide (NaN 3) (1.89 g, 29.04 mmol) are mixed with anhydrous DMF, and then agitated by stirring at 60 ° C. under heating. After stirring for about 4 hours, the reaction was terminated by TLC, and the reaction was terminated with water. The aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was washed with water and brine sufficiently, dried over magnesium sulfate (MgSO4), removed under reduced pressure, and concentrated under reduced pressure. The obtained azide compound is separated and purified through silica gel chromatography and the like, and the obtained compound (4.07 g, 42%) is dissolved in methanol. An appropriate amount of palladium / carbon is added to the solution, and the reaction vessel is hydrogen-substituted to carry out a hydrogenation reaction to reduce the azide group to an amine group. After confirming that the reaction was terminated by TLC, and when the reaction was terminated by removing the residual solid by vacuum filtration and concentrated under reduced pressure to obtain a compound of formula 3a having the following 1H-NMR spectrum. The obtained compound of Formula 3a proceeds to the next step without separate 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, 2H), 3.48-3.54 (m, 2H), 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)

Preparation Example 3-2

Except for using the compound of formula 2b (5.2 g, 19.97 mmol) as a starting material, except that the compound of Formula 3b having the following 1H NMR spectrum Compound (3.24 g, 62%) was synthesized.

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, 1 H ), 3.50 (m, 2 H), 2.40 (m, 1 H), 1.48 (s, 3 H), 1.27 (s, 3 H), 1.16 (s, 9 H)

Preparation Example 3-3

Except for using the compound of formula 2c (4.95 g, 17.79 mmoL) as a starting material, the compound of formula 3c having the following 1H NMR spectrum 2.98 g, 60%). Lithium borohydride (LiBH4) is used instead of sodium borohydride (NaBH4) in the alcoholation of ketones, and 10 equivalents of sodium azide is used in the azide reaction and the temperature is raised to 100 ° C. In addition, the stirring time should be increased to about 15 hours.

1 H NMR (800 MHz, CDCl 3) d 4.45-4.46 (m, 1 H), 4.25-4.27 (m, 1 H), 3.58 (dd, J = 4.5, 9.2 Hz, 1 H), 3.54 (dd, 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)

Preparation Example 4-1

Compound of formula 3a (982 mg, 3.757 mmol) and 5-amino-4,6-dichloropyrimidine (1.85 g, 11.27 mmol) synthesized in Preparation Example 3-1, Diisopropylethylamine (DIPEA) (6.54 mL, 37.57 mmol) was mixed with n-butanol, heated to 170 ° C. using microwave or the like and stirred for 4 hours. After confirming that the reaction was completed by TLC, the reaction mixture was mixed with methanol, concentrated under reduced pressure, and the residue was purified by silica gel chromatography to obtain an intermediate (964 mg, 66%). This intermediate was dissolved in diethoxymethyl acetate, and then stirred at 140 ° C. for about 3 hours using a microwave or the like. After confirming that the reaction was terminated by TLC, the reaction mixture was mixed with methanol, concentrated under reduced pressure, and purified by silica gel chromatography. The compound of Chemical Formula 4a having the following 1H NMR spectrum (643 mg, 65%). Got.

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, 2H), 4.61 (t, J = 5.0 Hz, 1H), 3.65-3.69 (m, 1H), 3.61 (t, J = 9.2 Hz, 1H), 2.56-2.71 (m, 1 H), 1.56 (s, 3 H), 1.32 (s, 3 H), 1.17 (s, 9 H)

Preparation Example 4-2

Except for using the compound of Formula 3b (1.592 g, 6.09 mmol) synthesized in Preparation Example 3-2 other than the compound of Formula 3a as a starting material was the same as in Preparation Example 4-1, The compound of formula 4b (943 mg, 39%) having 1 H NMR spectrum was synthesized.

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 H), 4.91 (m, 1 H), 4.69 (m, 1 H), 3.63 (m, 1 H), 2.56 (m, 1 H), 1.59 (s, 3 H), 1,30 (s, 3 H), 1.22 (s, 9 H)

Preparation Example 4-3

Except for using the compound of Formula 3c (182 mg, 0.625 mmol) synthesized in Preparation Example 3-3 other than the compound of Formula 3a as a starting material was the same as in Preparation Example 4-1, The compound of Chemical Formula 4c (223 mg, 50%) having a 1 H NMR spectrum was synthesized. In the first reaction step, however, the temperature should be raised to 200 ° C and the stirring 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, 1H), 4.59-4.64 (m, 1H), 3.64-3.74 (m, 2H), 2.81-2.96 (m, 1H), 1.58 (s, 3H), 1.33 (s, 3 H), 1.20 (s, 9 H)

Preparation Example 5-1

The compound of formula 4a (220 mg, 0.54 mmol) synthesized in Preparation Example 4-1 was dissolved in tert-butanol, and then transferred to a stainless steel bomb reactor. After saturated ammonia / tert-butanol (third butanol) was added dropwise to the mixture, the reaction vessel was sealed and stirred at 120 ° C. for about 15 hours. After confirming that the reaction was terminated by TLC, the reaction mixture was diluted with methanol and concentrated under reduced pressure. The concentrated residue is dissolved in THF, and then dropwise added a solution of trifluoroacetic acid (TFA) and water in a 2: 1 mixture. After stirring the reaction mixture for about 60 to 15 hours, the reaction was terminated by TLC and concentrated under reduced pressure. The concentrated residue was separated and purified by silica gel chromatography to obtain a compound of formula 5a (66 mg, 43%) having a 1H NMR spectrum as follows.

1 H NMR (400 MHz, CD3OD) 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, 1 H), 5.05-5.16 (m, 1 H), 4.66-4.69 (m, 1 H), 3.64-3.71 (m, 2 H), 2.51-2.66 (m, 1 H), 1.55 (s , 3 H), 1.35 (s, 3 H), 1.22 (s, 9 H)

Preparation Example 5-2

The same procedure as in Preparation Example 5-1 was carried out except for using the compound of Formula 4b (1.1 g, 2.758 mmol) synthesized in Preparation Example 4-2 as a starting material, and not the compound of Formula 4a. A compound of formula 5b (556 mg, 71%) was obtained having an NMR spectrum.

1 H NMR (400 MHz, CD3OD) 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, 1 H), 4.07 (m, 1 H), 3.80 (d, J = 5.9 Hz, 2 H), 2.35 (m, 1 H)

Preparation Example 5-3

The same procedure as in Preparation Example 5-1 was carried out except for using the compound of Formula 4c (223 mg, 0.534 mmol) synthesized in Preparation Example 3-3 instead of the compound of Formula 4a. A compound of formula 5c (99 mg, 61%) with 1 H NMR spectrum was synthesized.

1 H NMR (400 MHz, CD3OD) δ 8.29 (s, 1 H), 8.21 (s, 1 H), 5.29-5.36 (m, 2 H), 4.66 (bs, 1 H), 3.75-3.79 (m, 1 H), 3.66-3.70 (m, 1H), 2.80-2.89 (m, 1H), 1.57 (s, 3H), 1.34 (s, 3H), 1.22 (s, 9H)

Preparation Example 5-4

The compound of formula 4b (21 mg, 0.0527 mmol) synthesized in Preparation Example 4-2 was dissolved in methanol, and heated to 80 ° C. under acidic conditions such as a mixed solution of trifluoroacetic acid and water (1: 1). After stirring for about 15 hours, the reaction was terminated by TLC or the like and concentrated under reduced pressure. The concentrated residue was separated and purified by silica gel chromatography to obtain the compound of Chemical Formula 5d (10 mg, 67%) having the following 1H NMR spectrum.

1 H NMR (300 MHz, DMSO-d 6) δ 12.3 (s, 1 H, D 2 O exchanged), 8.22 (s, 1 H), 8.03 (d, J = 3.66, 1 H), 4.92-5.24 (m, 2 H ), 4.30 (m, 1 H), 3.84 (m, 1 H), 3.57 (d, J = 5.67, 2 H), 2.07-2.19 (m, 1 H)

Preparation Example 6-1

The compound of formula 4a (113 mg, 0.283 mmol) synthesized in Preparation Example 4-1 was dissolved in ethanol and then transferred to a glass seal bomb. 40% methylamine aqueous solution was added dropwise to the mixture, and the reaction vessel was sealed and stirred at room temperature for 2 hours. After confirming that the reaction was terminated by TLC, the reaction mixture was concentrated under reduced pressure. The concentrated residue is dissolved in THF, and then dropwise added a solution of trifluoroacetic acid (TFA) and water in a 2: 1 mixture. After stirring the reaction mixture for 50 to 30 hours, the reaction was terminated by TLC and concentrated under reduced pressure. The concentrated residue was separated and purified by silica gel chromatography to obtain the compound of Chemical Formula 6a (66 mg, 67%) having the following 1H NMR spectrum.

1 H NMR (500 MHz, CD3OD) δ 8.28 (s, 1 H), 8.24 (s, 1 H), 5.15-528 (dt, J = 54.7, 3.85 Hz, 1 H), 4.96-5.04 (ddd, J = 29.55, 9.95, 3.25 Hz, 1 H), 4.75 (dd, J = 9.55, 6.80 Hz, 1 H), 4.28 (dd, J = 6.65, 4.90 Hz, 1 H), 3.83 (m, 1 H), 3.12 (bs, 3H), 2.44-2.53 (m, 1H)

Preparation Example 6-2

Except for using the compound of Formula 4c (223 mg, 0.535 mmol) obtained in Preparation Example 3-3 other than the compound of Formula 4a as a starting material, the same procedure as in Preparation Example 6-1 was performed. The compound of Chemical Formula 6b (128 mg, 76%) having a spectrum was synthesized.

1 H NMR (500 MHz, CD3OD) δ 8.24 (s, 1 H), 8.20 (s, 1 H), 5.33 (m, 1H), 4.79 (dd, J = 10.3, 10.2 Hz, 1 H), 4.17 (s , 1 H), 3.81-3.90 (m, 2 H), 3.10 (bs, 3 H), 2.67 (m, 1 H)

Preparation Example 7-1

After dissolving the compound of Formula 5c (20 mg, 0.066 mmol) synthesized in Preparation Example 5-3 in acetone, a catalytic amount of sulfuric acid was added dropwise at 0 ° C., followed by stirring at room temperature for about 8 hours. After confirming by TLC that the reaction is complete, the reaction mixture is neutralized with sodium hydrogen carbonate (NaHCO 3) and the reaction mixture is concentrated under reduced pressure. The concentrated residue was separated and purified by silica gel chromatography to obtain a 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 prepared as a suspension using hexamethyldisilazane (HMDS) as a solvent. Subsequently, trimethylsilyl methanesulfonate (TMSOTf) (5 μL, cat.) Was added dropwise at room temperature, followed by heating at 75 ° C. for 2 hours. Thereafter, the reaction mixture was concentrated under reduced pressure, and then the concentrated residue was dissolved in anhydrous THF, and di-tert-butyl dicarbonate (Boc 2 O) (63 mg, 0.29 mmol) was added dropwise to the mixture. The reaction mixture is stirred at room temperature for about 4 hours and then concentrated under reduced pressure. The concentrated residue is dissolved in methanol: triethylamine = 5: 1 mixed solvent and then heated to 55 ° C. and stirred for about 16 hours. After confirming that the reaction was terminated by TLC or the like, and concentrated under reduced pressure. The concentrated residue is diluted with water and then extracted with ethyl acetate to separate the organic layer. The separated organic layer was washed with water and brine sufficiently, dried over magnesium sulfate (MgSO4), removed under reduced pressure, and concentrated under reduced pressure. The concentrated residue was separated and purified through silica gel chromatography to obtain N, N-di (tertbutyl carboxylic acid) (N, N-diBoc) -ized intermediate (8 mg, 0.015 mmol) and N-tertbutyl carboxylic acid ( N-Boc) intermediate (13 mg, 0.029 mmol) can be obtained. After dissolving these two intermediates (21 mg, 0.043 mmol) in dry THF, 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-phenoxyphosphenyl) phenoxyphosphinyl] -L-alanine (N-[(S)-(2 , 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, and the reaction mixture was allowed to stand at room temperature for 36 hours. Stir about. After confirming that the reaction was terminated by TLC, the reaction mixture was terminated with a saturated aqueous solution of 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 with water and brine sufficiently, dried over magnesium sulfate (MgSO4), removed under reduced pressure, and concentrated under reduced pressure. The concentrated residue can be separated and purified through silica gel chromatography or the like to obtain a precursor (10 mg, 0.0215 mmol) that has not reacted with the intermediate (15 mg, 0.0215 mmol). Among them, the reacted intermediate (15 mg, 0.0215 mmol) is suspended in an aqueous formic acid solution (50 v / v%) and stirred at room temperature for about 8 hours to proceed with the protecting group removal reaction. After confirming that the reaction was terminated by TLC, the reaction mixture was concentrated under reduced pressure, and the residue was separated and purified through silica gel chromatography, obtaining a compound of Formula 7b (12 mg, 100%) having the following 1H NMR spectrum. .

1 H NMR (CD3OD, 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, 1 H ), 5.00 (ddd, J = 20.75, 8.05, 8.05 Hz, 1 H), 4.92 (ddd, J = 12.45, 6.25, 6.25 Hz, 1 H), 4.53 (dd, J = 9.3, 5.35 Hz, 1 H) , 4.36 (t, J = 5.45 Hz, 2H), 4.12 (dd, J = 4.5, 4.0 Hz, 1H), 3.99-3.93 (m, 1H), 2.59-2.49 (m, 1H), 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)

Preparation Example 7-2

The same procedure as in Preparation Example 7-1 was carried out except for using the compound of Formula 5b (100 mg, 0.35 mmol) synthesized in Preparation Example 5-2 instead of the compound of Formula 5c. The compound of Formula 7a (27 mg, 27%) having 1 H NMR spectrum was synthesized.

1 H NMR (CD3OD, 500 MHz): 8.20 (s, 1 H), 8.18 (bs, 1 H), 7.38-7.18 (m, 5 H), 5.33 (ddd, J = 18.5, 9.7, 9.7 Hz, 1 H ), 4.95 (ddd, J = 12.3, 6.15, 6.15 Hz, 1 H), 4.76 (dd, J = 9.95, 5.05 Hz, 1 H), 4.42-4.32 (m, 2H), 4.19 (bs, 1 H ), 3.92-3.86 (m, 1H), 2.92-2.83 (m, 1H), 1.33 (d, J = 7 Hz, 3H), 1.20 (d, J = 6.2 Hz, 3H), 1.17 ( d, J = 6.2 Hz, 3 H)

Preparation Example 8-1

Methyl 3-methoxyacrylate (20 mL, 186 mmol) was added to a 2 M aqueous sodium hydroxide solution (103 ml) and stirred at room temperature for 2 hours to completely dissolve. The reaction mixture is completely acidified with 2M aqueous hydrochloric acid solution and filtered to collect the precipitated solid. 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 sodium salt. This is also dried in a reduced pressure desiccator with phosphorus pentoxide (phosphorus (V) oxide) 3 days. The dried organic sodium salt (6.20 g, 50 mmol) was made into a suspension with anhydrous diethyl ether, and then thionyl chloride (5.45 mL, 75 mmol) was added dropwise and heated under reflux for 4 hours. Let it be. Thereafter, the reaction mixture is stirred at 30 ° C. for about 15 hours to acid chloride. The reaction mixture is cooled to room temperature and filtered under reduced pressure while washing with anhydrous diethyl ether. The filtrate was concentrated under reduced pressure under nitrogen gas, and the concentrated residue was distilled under reduced pressure to obtain an organic acid chloride (3.08 g, 51%). The dried silver cyanate (8.44 g, 56.32 mmol) was made into a suspension with benzene and heated to reflux for 30 minutes. The obtained organic acid chloride (1.45 g, 12.03 mmol) was added dropwise to the prepared suspension and heated to reflux for 30 minutes to obtain isocyanate, which precipitated the reaction suspension without any other procedure, and then the supernatant was added to the next reaction. Use it. The compound of formula 3a (2.572 g, 9.84 mmol) synthesized in Preparation Example 3-1 was dissolved in DMF and cooled sufficiently to below -20 ° C. The isocyanic acid (45 mL, 19.68 mmol) synthesized above was added dropwise to the reaction mixture and stirred for about 15 hours while being slowly heated to room temperature. The reaction was terminated by TLC, washed with methylene chloride, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure. At this time, an azeotrope mixed with toluene, ethanol, etc. is concentrated sufficiently under reduced pressure to solidify the residue, and the solidified residue is separated and purified by silica gel chromatography to obtain urea derivative (2.903 g, 76%) as an intermediate. have. After dissolving the obtained urea derivative in 1,4-dioxane, a small amount of 2M sulfuric acid aqueous solution (˜1: 10 = 2M sulfuric acid: 1,4-dioxane) was added dropwise, followed by heating to reflux for about 1 hour to urea derivative. The cyclization reaction and the protecting group removal reaction are carried out simultaneously. After confirming that the reaction was terminated by TLC, the reaction mixture was neutralized using a basic resin such as DOWEX 66 ion-exchange resin, and the residue obtained through filtration under reduced pressure and concentrated under reduced pressure was purified by silica gel. When separated and purified through chromatography, the compound of Formula 8a (1.097 g, 56%) having the following 1H NMR spectrum may be obtained.

1 H NMR (400 MHz, CD3OD) 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, 1 H ), 4.81-4.93 (m, 1 H), 4.46 (m, 1 H), 3.93 (t, J = 5.00 Hz, 1 H), 3.76 (m, 2 H), 2.30-2.41 (m, 1 H)

Preparation Example 8-2

Except for using the compound of Formula 3b (1.3 g, 4.97 mmol) instead of the compound of Formula 3a and proceeding in the same manner as in Preparation Example 8-1, the compound of Formula 8b having the following 1H NMR spectrum (597 mg , 53%).

1 H NMR (500 MHz, CD3OD) 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, 5.10 Hz, 1 H), 4.61-4.69 (ddd, J = 22.6, 8.75, 7.35 Hz, 1 H), 4.32 (dd, J = 9.00, 5.25 Hz, 1 H), 3.98 (t, J = 3.75 Hz, 1 H) , 3.70 (m, 2 H), 2.24 (m, 1 H)

Preparation Example 8-3

Except for using the compound of Formula 3c (1.942 g 6.952 mmol) instead of the compound of Formula 3a and proceeding in the same manner as in Preparation Example 8-1, the compound of Formula 8b having the following 1H NMR spectrum (1 g, 52%).

1 H NMR (500 MHz, CD3OD) 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 Hz, 1 H ), 4.41 (dd, J = 10.7, 5.15 Hz, 1 H), 4.07 (m, 1 H), 3.73-3.82 (m, 2 H), 2.53 (m, 1 H)

Preparation Example 9-1

The compound of Formula 8a (100 mg, 0.384 mmol) synthesized in Preparation Example 8-1 was dissolved in methylene chloride, and then pyridine (0.21 mL, 2.57 mmol) was added dropwise and cooled to 0 ° C. Benzoyl chloride (0.27 mL, 2.31 mmol) was added dropwise to the cooled reaction mixture, which was then slowly heated to room temperature and stirred for about 15 hours. After confirming that the reaction was terminated by TLC, the reaction was terminated with water, the aqueous layer was extracted with methylene chloride, and the organic layer was separated. The separated organic layer was washed with water and brine sufficiently, dried over magnesium sulfate (MgSO4), removed under reduced pressure, and concentrated under reduced pressure. The concentrated residue can be separated and purified through silica gel chromatography to obtain benzoylated intermediate (164 mg, 75%). 1,2,4-triazole (1,2,4-triazole) (198 mg, 2.87 mmol) is made a suspension in anhydrous acetonitrile, cooled to 0 ° C., and then phosphoryl chloride (0.27 mL , 2.87 mmol). The reaction mixture was stirred at 0 ° C. for 30 minutes, after which the intermediate (164 mg, 0.286 mmol) obtained above was dissolved in anhydrous acetonitrile and added dropwise, followed by trimethylamine (0.4 mL, 2.87 mmol) at 0 ° C. Add it down. The reaction mixture is slowly heated to room temperature and stirred for about 15 hours. After confirming that the reaction was terminated by TLC, the reaction mixture was concentrated under reduced pressure. The concentrated residue is diluted with methylene chloride and aliquoted twice with a small amount of water to remove residual 1,2,4-triazole. The residue obtained by concentrating the washed organic layer under reduced pressure is dissolved in 1,4-dioxane and transferred to a sealable reaction vessel, and saturated ammonia water is then added at a rate of 20 v / v% of the solution. After the reaction mixture was stirred at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure. The concentrated residue can be separated and purified by silica gel chromatography to obtain an intermediate (42 mg, 26%) converted to cytosine. The cytosine intermediate (42 mg, 0.0735 mmol) was dissolved in methanol, transferred to a sealable reaction vessel, saturated aqueous ammonia / methanol solution was added, stirred at room temperature for 2 days, and the reaction mixture was concentrated under reduced pressure. The concentrated residue was diluted with a small amount of water and extracted about 10 times with methylene chloride to remove residual benzoyl adduct. Thus, the compound of formula 9a having the following 1H NMR spectrum (17 mg, 85%) was obtained.

1 H NMR (500 MHz, CD3OD) 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, 2 H), 2.36 (m, 1 H)

Preparation Example 9-2

Except for using the compound of Formula 8c (100 mg, 0.359 mmol) in place of the compound of Formula 8a and proceeding in the same manner as in Preparation Example 9-1, the compound of Formula 9b having the following 1H NMR spectrum ( 20 mg, 20%). However, the residue concentrated in the final step was purified by acidic resin (acidic resin) and the like.

1 H NMR (500 MHz, CD3OD) 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, 1 H ), 4.37 (dd, J = 10.6, 5.25 Hz, 1 H), 4.06 (m, 1 H), 3.73-3.83 (m, 2 H), 2.54 (m, 1 H)

Preparation Example 10-1

After dissolving the compound of Formula 8a (40 mg, 0.15 mmol) synthesized in Preparation Example 8-1 in acetone, a catalytic amount of sulfuric acid was added dropwise at 0 ° C., followed by stirring at room temperature for 2 hours. After confirming that the reaction was terminated by TLC, neutralized with sodium hydrogen carbonate (NaHCO 3) and the reaction mixture was concentrated under reduced pressure. The concentrated residue is separated and purified by silica gel chromatography 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 then tert-butylmagnesium chloride 1.0 M solution in THF (0.75 ml, 0.75 mmol) was dissolved. Add dropwise at 0 ° C. To this reaction mixture was added 1-methylethyl N-[(S)-(2,3,4,5,6-phenoxyphosphenyl) phenoxyphosphinyl] -L-alanine (N-[(S)-(2 A solution of 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, and the reaction mixture was allowed to stand at room temperature for 36 hours. Stir about. After confirming that the reaction was terminated by TLC, the reaction mixture was terminated with a saturated aqueous solution of 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 with water and brine sufficiently, dried over magnesium sulfate (MgSO4), removed under reduced pressure, and concentrated under reduced pressure. The concentrated residue was separated and purified through silica gel chromatography to obtain a precursor (25 mg, 0.083 mmol) that did not react with the intermediate (28 mg, 0.0495 mmol). Among them, the reacted intermediate (28 mg, 0.0495 mmol) is suspended in an aqueous formic acid solution (50 v / v%) and stirred at room temperature for about 8 hours to proceed with the protecting group removal reaction. After confirming that the reaction was terminated by TLC, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography to obtain the compound of Chemical Formula 10a having the following 1H NMR spectrum (24 mg, 90%). .

1 H NMR (CD3OD, 500 MHz): 7.64 (d, J = 8.05 Hz, 1 H), 7.34-7.37 (m, 2 H), 7.17-7.24 (m, 3H), 5.68 (d, J = 8.05 Hz , 1H), 5.04 (ddd, J = 55, 3.9, 3.9 Hz, 1H), 4.93-4.98 (m, 1H), 4.89-4.92 (m, 1H), 4.44 (dd, J = 9.7, 6.6 Hz, 1 H), 4.26 (t, J = 7 Hz, 2 H), 3.99 (t, J = 5.4 Hz, 1 H), 3.91-3.85 (m, 1 H), 2.51-2.57 (m, 1 H), 1.33 (d, J = 7 Hz, 3 H), 1.21 (d, J = 6.15 Hz, 6 H)

Preparation Example 10-2

Except for using the compound of Formula 8c (30 mg, 0.10 mmol) synthesized in Preparation Example 8-3 other than the compound of Formula 8a, and proceeding in the same manner as in Preparation Example 10-1, 1H NMR spectrum as follows Compound of Formula 10b (31 mg, 32%) was obtained.

1 H NMR (CD3OD, 500 MHz): 7.53 (dd, J = 8.05, 2.1 Hz, 1 H), 7.38-7.35 (m, 2H), 7.25-7.18 (m, 3H), 5.69 (d, J = 8.1 Hz, 1 H), 5.34 (ddd, J = 18.85, 9.7, 9.7 Hz, 1 H), 4.97 (ddd, J = 12.5, 6.2, 6.2 Hz, 1 H), 4.24-4.36 (m, 3 H) , 4.08 (bs, 1H), 3.87-3.90 (m, 1H), 2.70-2.80 (m, 1H), 1.34 (d, J = 7.15 Hz, 3H), 1.22 (d, J = 6.25 Hz , 6 H) Preparation Example 11

Copper bromide dimethylsulfide complex (933mg, 4.54mmol) was dissolved in anhydrous THF (300ml) and 1.0M vinylmagnesium bromide 1.0M solution in THF (69.3ml) at -78 ° C. , 69.3 mmol) and hexamethylphosphoramide (HMPA) (33.4 ml, 192 mmol) were added. Di-cyclopentenone (5 g, 32.4 mmol) and chlorotrimethylsilane (TMSCl) (20.6 ml, 162 mmol) were dissolved together in anhydrous THF (180 ml) and then slowly added dropwise to the reaction flask at -78 ° C. Stirred for 2 h. After confirming that the reaction was completed by TLC, a saturated aqueous ammonium chloride (NH 4 Cl) solution was added, the aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was sufficiently washed with water and brine, dried over magnesium sulfate (MgSO 4), and the residual solid was removed by filtration under reduced pressure, and then concentrated under reduced pressure. The concentrated residue was separated and purified through 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, 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 sufficiently washed with water and brine, dried over magnesium sulfate (MgSO 4), and the residual solid was removed by filtration under reduced pressure, and then concentrated under reduced pressure. The concentrated residue was separated and purified through silica gel chromatography to obtain a second intermediate (4.6 g, 97%).

The second intermediate (4.6 g, 25.2 mmol) was dissolved in DMF (30 mL), followed by imidazole (8.6 g, 126.2 mmol) and chlorotertbutyldiphenylsilane (TBDPSCl) (10.4 g, 37.8 mmol). Add at 0 ° C. The reaction mixture was stirred at room temperature for 1 hour, water was added, the mixture was extracted with diethyl ether, and the organic layer was separated. The separated organic layer was sufficiently washed with water and brine, dried over magnesium sulfate (MgSO 4), and the residual solid was removed by filtration under reduced pressure, and then concentrated under reduced pressure. The concentrated residue was separated and purified through silica gel chromatography to obtain a third intermediate (10.2 g, 92%).

After dissolving the third intermediate (10.0 g, 23.6 mmol) in THF (125 mL), the 2.0 M borane dimethyl sulfide complex 2.0 M solution in THF (35.5 mL, 71.0 mmol) was 0. Add at 캜. After the reaction mixture was stirred at room temperature for 4 hours, sodium perborate (7.2 g, 71.0 mmol) was added thereto at 0 ° C., and water (30 mL) was slowly added thereto. After stirring the reaction mixture 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 sufficiently washed with water and brine, dried over magnesium sulfate (MgSO 4), and the residual solid was removed by filtration under reduced pressure, and then concentrated under reduced pressure. The concentrated residue was separated and purified through silica gel chromatography to obtain a fourth intermediate (7.5 g, 72%).

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

The fifth intermediate (3.3 g, 16.3 mmol) was dissolved in methylene chloride (40 mL), and then 4-dimethylaminopyridine (DMAP) (0.2 g, 1.6 mmol) and triethylamine (6.8 mL, 48.9) at 0 ° C. mmol) and chlorotertbutyldiphenylsilane (TBDPSCl) (4.9 g, 17.9 mmol) were added. The reaction mixture was stirred at rt for 12 h. After the reaction was completed by adding water, the aqueous layer was extracted with methylene chloride and the organic layer was separated. The organic layer was sufficiently washed with water and brine, dried over magnesium sulfate (MgSO 4), and the residue was concentrated under reduced pressure by filtration under reduced pressure. The concentrated residue was separated and purified through 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 Å molecular sieves at room temperature (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 and then filtered under reduced pressure through a pad of Celite and silica gel to remove residual solids and then concentrated under reduced pressure. The concentrated residue was separated and purified through silica gel chromatography to obtain a compound of formula 11 (4.8 g, 98%) having a 1 H-NMR spectrum as follows.

1 H NMR (400 MHz, CDCl 3) δ 7.64 (m, 4 H), 7.41 (m, 6 H), 4.57 (d, J = 5.6 Hz, 1 H), 4.17 (m, 1 H), 3.72 (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)

Preparation Example 12-1

The compound of formula 11 (4.2 g, 9.5 mmol) synthesized in Preparation Example 11 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 was slowly added dropwise 1.0M lithium bis (trimethylsilyl) amide / THF solution (LiHMDS 1.0M solution in THF) (50mL, 50mmol), and the reaction was stirred under the same conditions for 30 minutes to carry out silyl enol etherification. After confirming by TLC that the reaction was terminated, the reaction mixture was added with saturated aqueous ammonium chloride (NH 4 Cl) solution, the aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was sufficiently washed with water and brine, dried over magnesium sulfate (MgSO 4), and the residual solid was removed by filtration under reduced pressure, and then concentrated under reduced pressure. This concentrated residue was taken up in anhydrous acetonitrile and then cooled to 0 ° C. 1.5 equivalents of 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo [2.2.2] octane bis (tetrafluoroborate) (Selectfluor®) (10.15 g, 28.65 mmol) to the cooled mixture After dropwise addition, the mixture was stirred for 15 hours under the same conditions to obtain a mixture of fluorinated compounds.

After confirming the completion of the reaction of the mixture by TLC, a saturated ammonium chloride (NH 4 Cl) aqueous solution 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 with water and brine sufficiently, dried over magnesium sulfate (MgSO 4), removed from the residual solid by filtration under reduced pressure, and concentrated under reduced pressure. The concentrated residue was separated and purified through silica gel chromatography to obtain a compound of formula 12a (3.7 g, 84%) having a 1H NMR spectrum as follows.

1 H NMR (300 MHz, CDCl 3) δ 7.65 (m, 4 H), 7.39 (m, 6 H), 5.43 (dd, J = 50.6, 7.14 Hz, 1 H), 4.77 (m, 1 H), 4.46 ( m, 1 H), 3.74 (m, 2 H), 2.51 (m, 1 H), 1.79 (m, 2 H), 1.45 (s, 3 H), 1.29 (s, 3 H), 1.03 (s, 9 H) (mixed with peaks presumed to be equilibrated in diol form.)

Preparation Example 12-2

Except for using the compound of Formula 12a (2.5g, 5.4mmol) synthesized in Preparation Example 12-1 other than the compound of Formula 11 as a starting material was the same as in Preparation Example 12-1, The compound of formula 12b (1.6g, 61%) having 1H NMR spectrum was obtained.

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, 1 H), 4.30 (m, 1 H), 4.21 (br , s, 1 H), 3.78 (ddd, J = 6.4, 10.4, 3.2 Hz, 2 H), 2.88 (br, s, 1 H), 2.65 (m, 1 H), 1.96 (m, 1 H), 1.71 (m, 1H), 1.54 (s, 3H), 1.33 (s, 3H), 1.04 (s, 9H)

Preparation Example 13-1

The compound of formula 12a (2.5 g, 5.4 mmol) synthesized in Preparation Example 12-1 was dissolved in methanol, and then cooled to -40 ° C. Sodium borohydride (NaBH 4) (1.45 g, 38.4 mmol) was slowly added to the cooled solution. After the reaction was stirred under the same conditions, the reaction was terminated by TLC, and then saturated ammonium chloride (NH 4 Cl) aqueous solution 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 with water and brine sufficiently, dried over magnesium sulfate (MgSO 4), removed from the residual solid by filtration under reduced pressure, and concentrated under reduced pressure. The intermediate (2.54 g, 76%) obtained by separating and purifying the alcohol compound produced through silica gel chromatography was dissolved in anhydrous pyridine and cooled to 0 ° C. Anhydrous trifluoromethanesulfonic anhydride (3.26ml, 19.36mmol) was added dropwise to the cooled mixture, followed by stirring for 30 minutes under the same conditions to trifluoromethylsulfonated. After confirming that the reaction was terminated by TLC, an aqueous saturated ammonium chloride (NH 4 Cl) solution 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 with water and brine sufficiently, dried over magnesium sulfate (MgSO 4), and the residual solid was removed by filtration under reduced pressure, and then concentrated under reduced pressure. The trifluoromethylsulfonated compound and 10 equivalents of sodium azide (NaN3) thus obtained were mixed with anhydrous DMF, and then agitated by stirring at 100 ° C. After stirring for about 4 hours, it was confirmed that the reaction was terminated by TLC, water was added, the aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was washed with water and brine sufficiently, dried over magnesium sulfate (MgSO 4), removed from the residual solid by filtration under reduced pressure, and concentrated under reduced pressure. The resulting azide compound was separated and purified through silica gel chromatography to dissolve the compound obtained in methanol. An appropriate amount of palladium / carbon was added to the solution, and the reaction vessel was hydrogen-substituted to proceed with hydrogenation to reduce the azide group to the amine group. After confirming that the reaction was terminated by TLC, the residual solid was removed by filtration under reduced pressure and concentrated under reduced pressure to obtain a compound of formula 13a (1.6 g, 61%) having a 1H NMR spectrum as follows.

1 H NMR (400 MHz, CDCl 3) 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 (m, 2 H), 3.26 (m, 1 H), 2.30 (m, 1 H), 1.88 (m, 2 H), 1.51 (s, 3 H), 1.30 (s, 3 H), 1.06 (s, 9 H )

Preparation Example 13-2

Using the compound of formula 12b (2.117g, 4.636mmoL) synthesized in Preparation Example 12-2 other than the compound of formula 12a as starting materials, the temperature conditions of sodium borohydride reduction reaction were adjusted to 0 ° C or lower, and Except for using sodium azide as 1.89g, 29.04mmol in the azide reaction and heating the temperature to 60 ℃ the same process as in Preparation Example 13-1, having the following 1H NMR spectrum Compound 13b (1.28 g, 60%) was obtained.

Preparation Example 14-1

Compound (300 mg, 0.65 mmol) synthesized in Preparation Example 13-1 (5-amino-4,6-dichloropyrimidine) (1.85 g, 11.27 mmol) and di Isopropylethylamine (DIPEA) (6.54 mL, 37.57 mmol) and n-butanol were mixed and heated to 170 ° C. using microwave and then stirred for 12 hours. After confirming that the reaction was terminated by TLC, the reaction mixture was concentrated under reduced pressure by mixing with methanol, and 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 then stirred at 140 ° C. for 3 hours using microwave. After confirming that the reaction was terminated by TLC, the reaction mixture was concentrated under reduced pressure by mixing with methanol, and then purified by silica gel chromatography to obtain a compound of Chemical Formula 14a (240 mg, 61.6%) having the following 1H NMR spectrum as follows. Got it.

1 H NMR (400 MHz, CDCl 3) 8.78 (s, 1 H), 8.32 (d, J = 2.4 Hz, 1 H), 7.68 (m, 4 H), 7.4 (m, 6 H), 5.18 (m, 1 H), 5.08 (m, 2H), 4.62 (m, 1H), 3.81 (m, 2H), 2.64 (m, 1H), 1.95 (m, 2H), 1.59 (s, 3H) , 1.34 (s, 3 H), 1.06 (s, 9 H)

Preparation Example 14-2

The compound of formula 13b (250 mg, 0.52 mmol) synthesized in Preparation Example 13-2 was dissolved in 1,4-dioxane (50 mL), and then 4,6-dichloro-5-formamidopi Limidine (120 mg, 0.65 mmol) and trimethylamine (0.55 ml, 3.86 mmol) were added. After the reaction mixture was heated to reflux 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 confirming that the reaction was terminated by TLC, the reaction mixture was mixed with methanol, concentrated under reduced pressure, and purified by silica gel chromatography to obtain a compound of Chemical Formula 14b (100 mg, 31%) having the following 1H NMR spectrum as follows. Got it.

1 H NMR (400 MHz, CDCl 3) 8.80 (s, 1 H), 8.25 (d, J = 2.4 Hz, 1 H), 7.70 (m, 4 H), 7.40 (m, 6 H), 5.35 (m, 1 H), 5.16 (superimposed dd, J = 6.8 Hz, 1 H), 4.54 (superimposed dd, J = 6.4 Hz, 1 H), 3.80 (m, 2 H), 2.96 (m, 1 H), 2.08 (m , 1 H), 1.84 (m, 1 H), 1.61 (s, 3H), 1.33 (s, 3 H), 1.07 (s, 9 H)

Preparation Example 15-1

After dissolving the compound of Formula 14a (200 mg, 0.336 mmol) synthesized in Preparation Example 14-1 in methanol, a solution of 1: 1 mixed trifluoroacetic acid (TFA) (5 mL) and water at 0 ° C. was added dropwise. . After the reaction mixture was stirred at room temperature for 2 hours, the reaction was terminated by TLC and concentrated under reduced pressure. The concentrated residue was separated and purified by silica gel chromatography to obtain an intermediate (80 mg, 75.4%).

The intermediate (70 mg, 0.22 mmol) was dissolved in tert-butanol, and then transferred to a stainless steel bomb reactor. Saturated ammonia / terbutanol was added dropwise to this mixture, and then the reaction vessel was sealed and stirred at 120 ° C. for 15 hours. After confirming that the reaction was terminated 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 a compound of formula 15a (55 mg, 84.6%) having a 1H NMR spectrum as follows.

1 H NMR (400 MHz, CD3OD) 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)

Preparation Example 15-2

A compound of Formula 14b (100 mg, 0.3 mmol) synthesized in Preparation Example 14-2 other than the compound of Formula 14a was used as a starting material, except that the mixture was stirred at 90 ° C. for about 3 hours during the amination reaction. The same procedure as in Preparation Example 15-1 was carried out to obtain a compound of Formula 15b (30 mg, 32%) having the following 1H NMR spectrum.

1 H NMR (300 MHz, CD3OD) 8.27 (d, J = 2.1 Hz, 1 H), 8.20 (s, 1 H), 5.28 (m, 1 H), 4.76 (m, 1 H), 4.06 (m, 1 H), 3.71 (m, 2 H), 2.64 (m, 1 H), 1.98 (m, 1 H), 1.79 (m, 1 H)

Preparation Example 16-1

After dissolving the compound of Formula 14a (570 mg, 0.966 mmol) synthesized in Preparation Example 14-1 in methanol, a solution of 1: 1 mixture of trifluoroacetic acid (TFA) (5 mL) and water was added dropwise at 0 ° C. . After the reaction mixture was stirred at room temperature for 2 hours, the reaction was terminated by TLC and concentrated under reduced pressure. The concentrated residue was separated and purified by silica gel chromatography to give an intermediate (230 mg, 75%).

The purified intermediate (55 mg, 0.17 mmol) was dissolved in methanol and then transferred to a stainless steel bomb reactor. Methylamine aqueous solution (40w / v%) was added dropwise to this mixed solution, and then the reaction vessel was sealed and stirred at 80 to 5 hours. After confirming that the reaction was terminated by TLC, the reaction mixture was concentrated under reduced pressure. The concentrated residue was separated and purified by silica gel chromatography to obtain a compound of formula 16a (37 mg, 68%) having a 1H NMR spectrum as follows.

1 H NMR (400 MHz, CD3OD) 8.25 (s, 1 H), 8.2 (d, J = 1.4 Hz, 1 H), 5.09 (dt, J = 3.6, 54.8 Hz, 1 H), 4.96 (ddd, J = 3.0 , 9.4, 26.7 Hz, 1 H), 4.74 (t, J = 7.4 Hz, 1 H), 4.02 (t, J = 6.0 Hz, 1 H), 3.74-3.67 (m, 2H), 3.20-3.00 ( brs, 3H), 2.42-2.31 (m, 1H), 1.94-1.84 (m, 2H)

Preparation Example 16-2

Except for using the compound of Formula 14b (100mg, 0.163mmol) synthesized in Preparation Example 14-2 other than the compound of Formula 14a as starting material, the same procedure as in Preparation Example 16-1 was performed. The compound of formula 16b having a spectrum (35 mg, 66%) was obtained.

1 H NMR (500 MHz, CD3OD) 8.25 (s, 1 H), 8.21 (s, 1 H), 5.27 (ddd, J1 = J2 = 8.4, 17.8 Hz, 1 H), 4.76 (dd, J = 6.6, 8.7 Hz, 1H), 4.06 (t, J = 4.7 Hz, 1H), 3.74-3.68 (m, 2H), 3.11 (brs, 3H), 2.66-2.61 (m, 1H), 2.00-1.94 (m, 1 H), 1.83-1.77 (m, 1 H)

Preparation Example 17

After dissolving the compound of Formula 14a (570 mg, 0.966 mmol) synthesized in Preparation Example 14-1 in methanol, a solution of 1: 1 mixture of trifluoroacetic acid (TFA) (5 mL) and water was added dropwise at 0 ° C. . After the reaction mixture was stirred at room temperature for 2 hours, the reaction was terminated by TLC and concentrated under reduced pressure. The concentrated residue was separated and purified by silica gel chromatography to obtain an intermediate (230 mg, 75%).

The purified intermediate (50 mg, 0.158 mmol) was dissolved in 1,4-dioxane and 1 N HCl (3 mL) was added dropwise at room temperature. The reaction mixture was heated to reflux and stirred for about 14 hours, after which the reaction was terminated by TLC and concentrated under reduced pressure. The concentrated residue was separated and purified by silica gel chromatography to obtain the compound of formula 17 having the following 1H NMR spectrum (33 mg, 70%).

1 H NMR (800 MHz, D2O) 8.20 (s, 1H), 8.08 (s, 1H), 5.07 (dt, J = 3.7, 54.2 Hz, 1H), 4.91 (ddd, J = 3.2, 9.8, 30.2 Hz, 1H ), 4.74-4.72 (m, 1H), 4.05 (t, J = 5.9 Hz, 1H), 3.65 (t, J = 6.6 Hz, 2H), 2.33-2.25 (m, 1H), 1.87-1.77 (m, 2H)

Preparation Example 18

Methyl 3-methoxyacrylate (20 mL, 186 mmol) was added to a 2 M aqueous sodium hydroxide solution (103 ml), and stirred at room temperature for 2 hours to completely dissolve. The reaction mixture was completely acidified with 2M aqueous hydrochloric acid solution and filtered to collect the precipitated solid. The solid organic acid obtained above was dissolved in water and neutralized with a 2 M aqueous sodium hydroxide solution to obtain an organic acid in the form of sodium salt. This is also dried in a reduced pressure desiccator with phosphorus pentoxide (phosphorus (V) oxide) 3 days. The dried organic sodium salt (6.20 g, 50 mmol) was made into a suspension with anhydrous diethyl ether, and then thionyl chloride (5.45 mL, 75 mmol) was added dropwise and heated under reflux for 4 hours. I was. Thereafter, the reaction mixture is stirred at 30 ° C. for about 15 hours to acid chloride. The reaction mixture is cooled to room temperature and filtered under reduced pressure while washing with anhydrous diethyl ether. The filtrate was concentrated under reduced pressure under nitrogen gas, and the concentrated residue was purified by distillation under reduced pressure to obtain an organic acid chloride (3.08 g, 51%). The dried silver cyanate (8.44g, 56.32mmol) is made into a suspension with benzene and heated under reflux for 30 minutes. The obtained organic acid chloride (1.45 g, 12.03 mmol) was added dropwise to the prepared suspension and heated to reflux for 30 minutes to obtain isocyanate, which precipitated the reaction suspension without any other procedure, and then the supernatant was used for the next reaction. It was. The compound of formula 13a (0.2 g, 0.41 mmol) synthesized in Preparation Example 13-1 was dissolved in DMF and cooled sufficiently to below -20 ° C. The isocyanic acid (45 mL, 19.68 mmol) synthesized above was added dropwise to the reaction mixture and stirred for about 15 hours while being slowly heated to room temperature. The reaction was terminated by TLC, washed with methylene chloride, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure. At this time, an azeotrope is made with toluene, ethanol, etc., and concentrated under reduced pressure to make the residue solid. Then, the solidified residue is separated and purified by silica gel chromatography to obtain urea derivative (2.903 g, 76%) as an intermediate. . After dissolving the obtained urea derivative in 1,4-dioxane, a small amount of 2M sulfuric acid aqueous solution (˜1: 10 = 2M sulfuric acid: 1,4-dioxane) was added dropwise, followed by heating to reflux for about 1 hour to urea derivative. The cyclization reaction and the protecting group removal reaction are carried out simultaneously. After confirming that the reaction was terminated by TLC, the reaction mixture was neutralized using a basic resin such as DOWEX 66 ion-exchange resin, and the residue obtained through filtration under reduced pressure and concentrated under reduced pressure was purified by silica gel. Separation and purification through chromatography or the like gave a compound of the following Chemical Formula 18 (0.535 g, 48%) having the following 1H NMR spectrum.

1 H NMR (500 MHz, CD3OD) 7.68 (d, J = 8.0 Hz, 1H), 5.69 (d, J = 8.1 Hz, 1H), 4.99 (dt, J = 3.6, 55.4 Hz, 1H), 4.88 (ddd, J = 3.3, 9.8, 30.7, 1H), 4.45 (merged dd, J1 = J2 = 7.1, 1H), 3.91 (t, J = 5.9 Hz, 1H), 3.71-3.64 (m, 2H), 2.29-2.20 ( m, 1H), 1.88-1.75 (m, 2H)

Experimental Example 1 Antiviral Activity and Cytotoxicity Experiment

In order to determine the antiviral activity and cytotoxicity of the carbocyclic nucleoside derivatives of the present invention, the following experiment was performed.

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

Compound of Formula 15a synthesized in Preparation Example 15-1 was diluted to 200 μM in Iscove's modified Dulbecco's medium (IMDM) containing 1% FCS (fetal calf serum) and antibiotics. Vero E6 cells were cultured in 96-well plates to contain 2 × 10 4 cells per well. After incubating the cells overnight at 37 ° C., 50 μl of the compound dilution was injected into each well, and mixed with 100 μl of Eagle's minimal essential medium (EMEM) containing 2% FCS and 50 μl of the CHIKV inoculum. Subsequently, the survival difference between cells caused by virus-induced cytopathic effect (CPE) and side effects of the compound at 3 dpi (day postinfection) was analyzed using CellTiter 96 Aqueous nonradioactive cell proliferation assay (Promega). Cytotoxicity by compound treatment was confirmed by culturing the cells in the same manner as above except that the normal medium was injected instead of the virus inoculum.

Using Graphpad Prism 5 software, 50% effective concentration (EC50) and 50% cytotoxic concentration (CC50) were calculated based on the results of cell viability for CPE and cell viability for compound side effects, respectively. EC50 represents the minimum concentration of compound for which 50% of the pathogens (viruses) are to be inhibited, and CC50 represents the compound concentration when 50% of host cells are killed. The degree of relative potency of a compound against a particular virus was defined as selectivity (SI; CC50 / EC50).

In addition, the experiment was carried out in the same manner as above except that the mixture with the SFV inoculum was calculated EC50, CC50 and SI values for the SFV of the compound of Formula 15a, the compound of Formula 15b synthesized in Preparation Example 15-2 Except for the use of compounds, the same experiments for CHIKV above were performed to calculate EC50, CC50 and SI values of the compound of Formula 15b.

Table 1 shows the EC50, CC50 and SI values calculated by the above method.

Virus 종류Virus type	화합물compound	EC50(μM)EC50 (μM)	CC50(μM)CC50 (μM)	SI(CC50/EC50)SI (CC50 / EC50)
CHIKV LS3CHIKV LS3	화학식 15aFormula 15a	0.20.2	>250> 250	>1,000> 1,000
CHIKV LS3CHIKV LS3	화학식 15bFormula 15b	4.24.2	>250> 250	6060
SFVSFV	화학식 15aFormula 15a	6.46.4	>250> 250	>30> 30

As shown in Table 1, the carbocyclic nucleoside derivatives of the present invention have a low EC50 for the RNA viruses CHIKV and SFV and a high CC50 for the host cell, thus preventing and treating viral inhibition and viral diseases. It can be seen that it can be utilized as a pharmaceutical composition very suitable for.

Experimental Example 2-SAH hydrolase inhibition efficacy test

In order to determine the SAH hydrolase inhibition effect of the carbocyclic nucleoside derivative of the present invention, the following experiment was performed.

A reaction mixture (250 μl) of 50 mM sodium phosphate (pH 8.0) and AdoHcy hydrolase (2 μM as monomer; 5 μM as tetramer) was synthesized in Preparation Example 15-1 and Preparation Example 15-2, respectively. One of the compounds of Formula 15b was preincubated at 37 ° C. for 10 minutes. After preincubation, 100 μM of AdoHcy was added to initiate the reaction and allowed to proceed for 20 minutes. The reaction mixture was then further incubated in 200 μM DTNB, and the maximum absorbance at 412 nm of the product TNB (5-thio-2-nitrobenzoic acid) was measured with a spectrophotometer. IC50 values of the compound of Formula 15a and the compound of Formula 15b were calculated using the measured maximum absorbance and the molar extinction coefficient of the TNB (ε412 = 13700 M-1 cm-1).

화합물compound	IC50(μM)IC50 (μM)
화학식 5aFormula 5a	0.10.1
화학식 5bFormula 5b	0.090.09

As shown in Table 2, since the carbocyclic nucleoside derivative of the present invention has a low IC50 value for SAH hydrolase, it is suitable as a pharmaceutical composition having an antiviral effect through the SAH hydrolase inhibition mechanism. It can be seen that it can be used as.

Preparation of cells, viruses, substances

Vero E6 and Vero CCL81 cell lines in Dulbecco's modified Eagle's Medium (DMEM; Lonza), 8% foetal calf serum (FCS; PAA), 2 mM L-glutamine, penicillin 100 IU / ml, streptomycin 100 g / ml was used to incubate at 37 ° C, 5% carbon dioxide conditions in a thermo-hygrostat.

Vero cell lines were described above using a combination of Eagle's Minimum Essential Medium (EMEM; Lonza) with 8% FCS, 2 mM L-glutamine, penicillin 100 IU / ml and streptomycin 100 g / ml Cultured under the same conditions.

The HuH7 cell line contains a medium containing 8% FCS, 2 mM L-glutamine, penicillin 100 IU / ml, streptomycin 100 μg / ml and 1x non-essential amino acids (NEAA; Lonza) in MDEM. Cultured under the same conditions as described above.

MRC-5 cell line was subjected to the same conditions described above using a medium containing 8% FCS, 2 mM L-glutamine, penicillin 100 IU / ml, streptomycin 100 μg / ml, 1 × NEAA in EMEM Incubated at.

In case of virus infection, EMEM and 25 mM N-2-Hydroxyethylpiperazine-N ''-2-ethanesulfonic acid (HEPES; Lonza) in 2% FCS, mM L-glutamine, penicillin 100 IU / ml, streptomycin (streptomycin) ) 100 μg / ml combined medium was used.

Clonal-derived chikungunya virus (CHIKV LS3) infectious agents were produced by the method described in PLOS ONE 8 (2013): e71047 published by the Scholte group.

Zika virus (ZIKV) strain SL1602 was developed by Sci. Rep. (2017) isolated from infected travelers returning from Suriname by the method described in in press.

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

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

Each test substance was dissolved in DMSO and used at 20 mM and 10 μM.

Experiments with infectious agents CHIKV, MERS-CoV, SARS-CoV, and ZIKV were all conducted in a biological safety cabinet at LUMC's biological safety level 3 facility.

CPE-reduction assays

VeroE6 cell lines were inoculated at 100 μl volume into each well to reach 5,000 cells / well (CHIKV) in 96-well clusters.

Vero (CCL81) cell lines were inoculated at 96-well clusters to 5,000 cells / well (ZIKV).

For MERS-CoV CPE reduction assays, 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 SARS-CoV CPE reduction assays, VeroE6 cell lines were seeded at 96 cells / well in 96-well clusters.

The next day inoculations of each cell line were prepared in the above-described virus infection medium which was serially diluted twice at a starting concentration of 100 μM and then exchanged with the medium in the existing 96-well clusters.

Each cell is identical to 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 (MOI 0.01) Infected with quadruplicate.

In the case of uninfected cells, the evaluation material was treated in the same manner with diluted media, and the cytotoxicity test (CC50 evaluation) was performed simultaneously.

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

Assay was terminated by treatment with 30 μl 37% formaldehyde after 2 ~ 2.5 hours.

Absorbance was measured using a Berthold Mithras LB 940 plate reader at 495 nm and normalized to cells that were not treated with infectious agents and analytes.

CC50 measurements were normalized to cells not treated with infectious agents and test substances.

The results were expressed as the mean of the values obtained under the same four replicates and expressed as mean ± standard deviation.

EC50 values were determined using a non-linear regression method using Graphpad Prism.

The experimental results are summarized in FIGS. 1 to 8.

While the embodiments of the present invention have been described above, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims

Carbocyclic nucleoside derivative represented by the following formula (A) or a pharmaceutically acceptable salt thereof.

<Formula A>

(In Formula A, n is 1 or 2, R1 and R2 are each independently hydrogen or fluorine, B is the following formula B-1 or B-2, P is hydrogen or phosphoamidate group to be)

<Formula B-1>

(In the above formula B-1, X1 is a chlorine, a hydroxyl group (OH group), an amino group or an alkylamino group, Y1 is hydrogen or an amino group.

The alkylamino group is 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 formula B-2, X2 is a hydroxyl group or an amino group, Y2 is hydrogen, methyl group or halogen)
The method of claim 1,

Formula A is a carbocyclic nucleoside derivative represented by Formula a or a pharmaceutically acceptable salt thereof.

<Formula a>
The method of claim 2,

R 2 is a fluorine carbocyclic nucleoside derivative or a pharmaceutically acceptable salt thereof
The method of claim 2,

Formula a is a carbocyclic nucleoside derivative of Formula 5a, Formula 5b or Formula 5c or a pharmaceutically acceptable salt thereof.

<Formula 5a>

<Formula 5b>

<Formula 5c>
The method of claim 1,

Formula A is a carbocyclic nucleoside derivative represented by Formula b or a pharmaceutically acceptable salt thereof.

<Formula b>
The method of claim 5,

R 1 is fluorine carbocyclic nucleoside derivative or a pharmaceutically acceptable salt thereof.
The method of claim 5,

Formula b is a carbocyclic nucleoside derivative of Formula 15a or Formula 15b or a pharmaceutically acceptable salt thereof.

<Formula 15a>

<Formula 15b>
An antiviral agent comprising a carbocyclic nucleoside derivative represented by Formula A or a pharmaceutically acceptable salt thereof.

<Formula A>

(In Formula A, n is 1 or 2, R1 and R2 are each independently hydrogen or fluorine, B is the following formula B-1 or B-2, P is hydrogen or phosphoamidate group to be)

<Formula B-1>

(In the above formula B-1, X1 is a chlorine, a hydroxyl group (OH group), an amino group or an alkylamino group, Y1 is hydrogen or an amino group.

The alkylamino group is 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 formula B-2, X2 is a hydroxyl group or an amino group, Y2 is hydrogen, methyl group or halogen)
The method of claim 8,

Formula A is an antiviral agent of Formula a.

<Formula a>
The method of claim 9,

R 2 is fluorine.
The method of claim 9,

Formula (a) is an antiviral agent of Formula 5a, Formula 5b or Formula 5c.

<Formula 5a>

<Formula 5b>

<Formula 5c>
The method of claim 8,

Formula A is an antiviral agent of formula b.

<Formula b>
The method of claim 12,

R 1 is fluorine.
The method of claim 12,

Formula b is an antiviral agent of Formula 15a or Formula 15b.

<Formula 15a>

<Formula 15b>