KR100385281B1 - Novel inhibitors of cell cycle-related kinases - Google Patents

Abstract

The present invention relates to an L-arabinose derivative represented by the following Chemical Formula 1, an L-xylose derivative represented by the following Chemical Formula 2, a preparation method thereof, and a pharmaceutical composition comprising the same. It is characterized by the fact that the part is in the L-form rather than the D-form, and because it has an excellent ability to inhibit cell cycle regulators of cancer cells, it can be usefully used as an anticancer agent.

In Formula 1 and Formula 2, X, Y, R and Q are as described in the specification.

Description

Novel inhibitors of cell cycle-related kinases

The present invention relates to a L- arabinose derivative represented by the formula (1), L- xylose derivative represented by the formula (2), a preparation method thereof and a pharmaceutical composition comprising the same, specifically, the compound according to the present invention It is characterized by the L-form rather than the D-form.

Anti-cancer agents currently clinically applied have a mechanism of action that inhibits the growth of cancer cells by directly or indirectly inhibiting DNA synthesis of cancer cells. However, cancer cells have a mechanism of defense against the action of inhibiting DNA synthesis, and thus drug resistance to these anticancer drugs, resulting in poor drug efficacy and eventually cancer recurrence. In addition, due to the side effects of the anticancer agent itself, it has already been found that there is a limit in the treatment of cancer using an anticancer agent having a mechanism of action that inhibits DNA synthesis. Thus, recently, as an alternative, the development of anticancer drugs having a mechanism of action that regulates a signal transduction pathway that regulates the cell cycle has attracted attention.

Eukaryotic cells proliferate along the cell cycle when they receive growth signals from the outside through a signaling system. Eukaryotic cells DNA synthesis (Synthesis phase; S group) cleavage procedure (Mitosis phase; M group) of the cells in groups to proliferate by, between the S group and M groups include, but are not limited to G ₁ group G ₂ G ₁ → S → G ₂ → Cells proliferate in cycles of phase M. G ₁ phase is the most active period of intracellular metabolism, and most cells are in G ₁ phase for the longest time, where the cells are in diploid, ie 2n state. In the S phase, DNA replication occurs so that 2n becomes 4n, or tetraploid. Cells passing through S phase enter the M phase via G ₂ phase, where 4n divides into 2n in this short M phase, and the cell returns to G ₁ phase. Normal cells enter the S phase when there is an external signal in the G ₁ stage, divide and multiply the cell cycle, and when there is no external signal, the cell cycle is interrupted and is present in the G ₀ phase which is stopped at the G ₁ stage _.

Recently, it has been known that some points in this cell cycle are regulated by protein kinase complexes. Protein kinase complexes consist of a catalytically active subunit (CDK), a cyclin dependent kinase, and a regulatory activity, a regulatory subunit (cyclin), from yeast to humans. CDK is very similar.

In the somatic cell cycle, no other event begins until the preceding event is completed in time. However, if a mutation occurs that inhibits the progression of a particular cell cycle, the cell cycle is interrupted, and in most cases cell proliferation is stopped at a time that requires the gene product of the mutation. In the case of yeast, Saccharomyces as MY process three Levy jiae cdc28 (Saccharomyces cerevisiae cdc28) gene and the ski irradiation Caro My process pombe cdc2 (Schizosaccharomyces pombe cdc2) occurs, a mutation in gene G of each cell cycle ₁ → S, G ₂ → M I This acts to stop the cycle. The two genes are so similar that they can be functionally replaced, and both express a 34 kDa serine / threonine kinase, called p34. p34 maintains a constant amount during the cell cycle, and forms a heterodimer by combining with cyclin, which is synthesized and accumulated at the transition stage of the cell cycle and then degraded after cell division.

Higher organisms, on the other hand, differ from yeast in that the G ₁ → S, G ₂ → M transition groups are regulated by two different CDKs, namely the CDK2-cyclin A complex and the CDK2-cyclin B complex, respectively. As with cdc2, it binds to cyclin.

These cell cycle studies have shown that cancer is caused by abnormal differentiation of cells and abnormal proliferation during development. Based on these results, the development of new anticancer agents worldwide is currently under development of cell differentiation promoters to normalize the differentiation of cancer cells, and within signal transduction systems that regulate the growth and division of cells to inhibit the continuous growth and division of cancer cells. There are two directions for developing cell cycle regulator inhibitors that inhibit the activity of various protein kinases (eg, protein kinase C, protein kinase A, cdc2 kinase, CDK2 kinase).

In particular, anti-cancer drugs have been actively developed in the United States and Japan based on a mechanism of action that inhibits cell cycle regulators. Human cancer cells differ greatly from normal cells in the expression and regulation of cell cycle regulators, CDK and other protein kinases (eg, casein kinase II). Therefore, by inhibiting the activity of these cell cycle regulators and blocking signal transduction systems that regulate DNA synthesis, DNA synthesis and cell division can be suppressed in an indirect manner, thereby selectively inhibiting the growth of cancer cells. Experimental evidence has also been reported that apoptosis can be induced to selectively kill cancer cells. Based on this, recently, cell cycle regulators such as UCN-01 butyrolactone, olomoucine and roscovitine have been reported in the US and Japan. It is entering the application stage (Meijer, L., Trends Cell Biol. 1996, 6,390).

On the other hand, the present inventors have isolated and purified a substance having a relatively selective and high inhibitory activity against the cell cycle control factors casein kinase II and CDK from soil microorganisms and natural medicinal plant extracts. In addition, it was revealed that the inhibitory components of cdc2 kinase activity isolated from about 200 soil bacteria were D-toyocamycin and D-sangivamycin, which showed high inhibitory activity and selectivity against cdc2. Have been confirmed (Park, SG et al ., Mol. Cells , 1996, 6 (6), 679-683).

Therefore, the present inventors have tried to develop a new compound related to the cell cycle control of cancer cells using the D-toyokamycin as a leading substance, the sugar portion of the sugar and base, characterized in that the L- form sugar portion, not D- form It is useful as a new anticancer agent because it synthesizes a condensate (a compound named 'L-arabinose derivative' or 'L-xylose derivative' in the present specification) and has an excellent effect of inhibiting cell cycle regulation factors of cancer cells. The present invention has been completed by revealing that it can be used.

It is an object of the present invention to provide L-arabinose derivatives and L-xylyl derivatives, pharmaceutically acceptable salts thereof, and methods for preparing them, which are excellent in inhibiting cell cycle control factors of cancer cells.

It is another object of the present invention to provide a cell cycle control factor inhibitor comprising the compound as an active ingredient.

It is also an object of the present invention to provide a pharmaceutical composition for an anticancer agent comprising the compound as an active ingredient.

1 shows the effect of L-xylose derivative ( 12 ) inhibiting the activity of cdc2, PKA, casein kinase II and PKC,

2 shows the effect of L-xylose derivative ( 16 ) inhibiting the activity of cdc2, PKA, casein kinase II and PKC,

3 is an MTT assay showing the effect of L-xylose derivative ( 12 ) inhibiting the growth of SK-Hep-1 cells,

4 is an MTT assay showing the effect of L-xylose derivative ( 16 ) inhibiting the growth of SK-Hep-1 cells,

Figure 5 shows the change in the amount of ³ H thymidine introduced into the DNA of SK-Hep-1 cells by treatment concentration of L- xylose derivative ( 12 ),

Figure 6 shows the change in the amount of ³ H thymidine introduced into the DNA of SK-Hep-1 cells by treatment concentration of L- xylose derivative ( 16 ),

Figure 7 shows the micrographs with time when the L-xylose derivative ( 12 ) 60 μM administered to SK-Hep-1 cells and the control group,

Figure 8 shows the micrographs with time when the L-xylose derivative ( 16 ) 60 μM administered to SK-Hep-1 cells and the control group,

9 shows the results of electrophoresis of fragmented DNA by proteinase K after treatment of SK-Hep-1 cells with L-xylose derivatives 12 at different concentrations.

M: DNA marker

1: control group

2: L-Xylose derivative (12) 0.03 μM

3: L-xyl derivative (12) 0.3 μM

4: L-xyl derivative (12) 3 μM

5: L-Xylose derivative (12) 30 μM

6: 100 μM of L-Xylose derivative (12)

7: L-xyl derivative (12) 300 μM

10 shows the results of electrophoresis of DNA fragmented by proteinase K after treatment of SK-Hep-1 cells with L-xylose derivatives ( 16 ) at different concentrations.

M: DNA marker

1: control group

2: L-xyl derivative (16) 0.03 μM

3: L-Xyl Derivative (16) 0.3 μM

4: L-xyl derivative (16) 3 μM

5: L-Xylose Derivative (16) 30 μM

6: 100 μM of L-Xylose Derivative (16)

7: L-Xylose Derivative (16) 300 μM

FIG. 11 shows Western blot using PARP, p21, p27 and caspase-3 after treating L-xylose derivatives ( 16 ) with SK-Hep-1 cells at different concentrations. Shows the results

FIG. 12 shows the results of Western blot using CDK2 and PCNA after treatment of SK-Hep-1 cells with L-xylose derivatives ( 16 ) for each concentration.

In order to achieve the above object, the present invention provides a L-arabinose derivative represented by the following formula (1) and salts thereof.

Formula 1

In Chemical Formula 1,

R is H or a halogen element,

X is CN, , , , , or ego,

Y is NH ₂ or ═O,

Q is N or NH,

The bond between the carbon atom to which Y is bonded and Q is a single bond or a double bond.

In addition, the present invention provides an L-xylose derivative represented by the following Chemical Formula 2 and a salt thereof.

Formula 2

In Chemical Formula 2,

R is H or a halogen element,

X is CN, , , , , or ego,

Y is NH ₂ or ═O,

Q is N or NH,

The bond between the carbon atom to which Y is bonded and Q is a single bond or a double bond.

More preferably, in the present invention, in Chemical Formula 1, R is H or Br; X is CN, , , or ego; Y is NH ₂ ; Q is N; The bond between the carbon atom to which Y is bonded and Q is a double bond provides an L-arabinose derivative and salt thereof.

More preferably, in the present invention, in Chemical Formula 2, R is H or Br; X is CN, , , or ego; Y is NH ₂ ; Q is N; The bond between the carbon atom to which Y is bonded and Q provides a L-xyl derivative and salt thereof, wherein the bond is a double bond.

Particularly preferred compounds among the compounds of Formula 1 or Formula 2 are specifically as follows.

1) 4-amino-5-cyano-7- (2 ', 3', 5'-trihydroxy-β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine ( 12 ) ;

2) 4-amino-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamidine ( 14 );

3) 4-amino-6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamide ( 16 );

4) ethyl 4-amino-6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamide oxime.HCl ( 18 );

5) 4-amino-6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-thiocarboxamide ( 22 );

6) 4-amino-5-cyano-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine ( 28 );

7) 4-amino-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamide ( 30 );

8) ethyl 4-amino-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamideoxime.HCl ( 32 ); And

9) 4-amino-7- (β-L-xylfuranosyl) pyrrolo [2,3-d] pyrimidine-5-thiocarboxamide ( 36 ).

The compound of Formula 1 or Formula 2 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Compounds of Formula 1 or Formula 2 may form pharmaceutically acceptable acid addition salts according to methods conventional in the art. Organic acids and inorganic acids may be used as the free acid, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, and fumaric acid may be used as the organic acid. (fumaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Can be used.

In another aspect, the present invention provides a method for preparing the L- arabinose derivative of Formula 1 and the L- xylose derivative of Formula 2.

Specifically, the preparation method of the compounds according to the present invention

1) preparing a base ( 3 ), represented by Scheme 1 below (step 1);

2) preparing an L-type sugar derivative ( 6 or 8 ) represented by Scheme 2 or Scheme 3 below (step 2);

3) L-arabinose derivative ( 10 ) or L-xyl by reacting the base ( 3 ) of step 1 with the L-type sugar derivative ( 6 or 8 ) of step 2, represented by Scheme 4 or Scheme 5 below Preparing a loose derivative ( 12 ) (step 3); And

4) optionally, by modifying the base moiety through an appropriate reaction using the L-arabinose derivative ( 10 ) or L-xylose derivative ( 12 ) of step 3 above as a starting material, the L-arabinose derivative of Formula 1 or Formula 2 Preparing L-Xylose Derivatives of (Step 4)

Is made of.

Hereinafter, the manufacturing method according to the present invention will be described in more detail step by step.

(1) step 1

The method for preparing the base ( 3 ) according to the present invention

1) preparing tetracyanoethane by reacting tetracyanoethylene and HI in a solvent under anhydrous conditions;

2) preparing 2-amino-5-bromo-3,4-dicyanopyrrole by adding HBr gas to tetracyanoethane under anhydrous solvent; And

3) preparing a base ( 3 ) by reacting 2-amino-5-bromo-3,4-dicyanopyrrole with formamidine

Is made of.

(2) step 2

The preparation method of the L-type sugar derivative ( 6 or 8 ) according to the present invention

1) reacting L-arabinose or L-xyl with HCl / methanol solution, reacting with pyridine, and adding benzene chloride (BzCl) to react to prepare a sugar derivative ( 5 or 7 ); And

2) preparing an L-type sugar derivative ( 6 or 8 ) by adding acetic acid, acetic anhydride and concentrated sulfuric acid to the sugar derivative ( 5 or 7 ).

Is made of.

(3) step 3

The reaction of the base ( 3 ) of step 1 with the L-type sugar derivative ( 6 or 8 ) of step 2 to prepare an L-arabinose derivative ( 10 ) or L-xylose derivative ( 12 )

1) N, O-bis (trimethylsilyl) acetamide (BSA) is added to the base ( 3 ) of step 1 under anhydrous solvent and reacted with the L-type sugar derivative ( 6 or 8 ) and trimethylsilyl of step 2 Adding trifluoromethanesulfonate (TMSOTf) to react to prepare L-type sugar derivatives ( 9 or 11 ); And

2) preparing the L- hyeongdang derivatives (10 or 11) according to the present invention by hydrolyzing the derivative (9 or 11) per the type L-

Is made of.

(4) step 4

Substituents of the base moiety can be modified through appropriate reactions according to conventional methods known in the art, and those skilled in the art will readily be able to determine the reaction reagents and reaction conditions.

Hereinafter, a method of modifying the base moiety will be described in detail, but it is only an example and does not limit the content of the present invention.

① X Substitute with

L-arabinose derivatives ( 10 , 27 , 25 , 39 ) or L-xylose derivatives ( 12 , 28 , 26 , 40 ), wherein X is CN, with hydrogen peroxide (H ₂ O ₂ ) and ammonium hydroxide (NH ₄ OH) React.

② X Substitute with

L-arabinose derivative ( 10 , 27 , 25 , 39 ) or L-xylose derivative ( 12 , 28 , 26 , 40 ) wherein X is CN is reacted with ethanol and HCl gas

③ X Substitute with

L-arabinose derivatives ( 10 , 27 , 25 , 39 ) or L-xylose derivatives ( 12 , 28 , 26 , 40 ), wherein X is CN, can be prepared by anhydrous H ₂ S and React with metal salts of alcohols (eg NaOMe).

④ X Substitute with

L-arabinose derivatives ( 10 , 27 , 25 , 39 ) or L-xylyl derivatives ( 12 , 28 , 26 , 40 ), wherein X is CN, are reacted with hydroxylamine (HONH ₂ ).

⑤ X R is replaced by H

X The phosphorus L-arabinose derivative ( 19 , 47 ) or L-xylose derivative ( 20 , 48 ) is reacted with hydrogen gas under Pd / C catalyst.

⑥ X Substitute with

L-arabinose derivatives ( 10 , 27 , 25 , 39 ) or L-xylyl derivatives ( 12 , 28 , 26 , 40 ), wherein X is CN, are reacted with hydrazine hydrate.

⑦ Replace R with H

L-arabinose derivatives ( 10 , 25 ) or L-xylose derivatives ( 12 , 26 ), wherein X is CN, are reacted with zinc in acetic acid.

⑧ Replace Y with = O and Q with NH

L-arabinose derivative ( 10 , 27 ) or L-xylose derivative ( 12 , 28 ), wherein X is CN, is reacted with water, acetic acid, NaNO ₂ or higher at 50 ° C or higher.

The present invention provides a cell cycle control factor inhibitor comprising the L-arabinose derivative of Formula 1 or the L-xylose derivative of Formula 2 as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for an anticancer agent comprising the L- arabinose derivative of Formula 1 or the L- xylose derivative of Formula 2 as an active ingredient.

Compounds of Formula 1 or Formula 2 can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical formulations.

That is, the compound according to the present invention may be administered in various oral and parenteral dosage forms in actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used, or Formulated using excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations comprise at least one excipient such as starch, calcium carbonate, water It is prepared by mixing cross or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.

Dosage units may contain, for example, one, two, three or four times, or 1/2, 1/3 or 1/4 times the individual dosage. Individual dosages preferably contain an amount in which the active compound is administered at one time, which usually corresponds to a total, 1/2, 1/3 or 1/4 of the daily dose.

The effective dose of the compound according to the invention is 10-500 mg / kg, preferably 50-200 mg / kg, and can be administered 1-3 times a day.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are illustrative of the present invention, and the contents of the present invention are not limited to Examples or Experimental Examples.

Preparation Example 1 4-Amino-6-bromo-5-cyanopyrrole [2,3-d] pyrimidine ( 3 Manufacturing

(Step 1) Preparation of Tetracyanoethane

12.80 g (100 mmol) of tetracyanoethylene was dissolved in 50 ml of anhydrous acetone, and then 50 ml of concentrated aqueous HI solution (57 w / v%) was added dropwise while maintaining the temperature at 0 ° C. After the reaction solution was stirred at 0 ° C. for 1 hour, the resulting solid was filtered and washed sufficiently with cold water. The obtained solid was recrystallized and refine | purified with the mixed solvent of n -hexane: ethyl acetate = 8: 1.

(Step 2) Preparation of 2-amino-5-bromo-3,4-dicyanopyrrole

The tetracyanoethane of step 1 was dissolved in anhydrous acetone, and then saturated with HBr gas to the solution while maintaining the temperature at -10 to 0 ℃. The resulting solid was recrystallized from ethanol to give 13.72 g (yield: 65%) of purple needle-like long needles.

¹ H-NMR (300 MHz, DMSO- d ₆ ) δ 11.75 (bs, 1H, NH), 6.37 (bs, 2H, NH ₂ )

(Step 3) 4-amino-6-bromo-5-cyanopyrrole [2,3-d] pyrimidine ( 3 Manufacturing

16.3 g (77.24 mmol) of 2-amino-5-bromo-3,4-dicyanopyrrole and 16.10 g (155 mmol) of formamidine of step ₂ were dissolved in 200 ml of EtOCH ₂ CH ₂ OH. Reflux for 36 hours. The reaction solution which turned dark brown was decolorized and filtered with charcoal. The obtained residue was recrystallized with a mixed solvent of DMF (dimethylformamide): methanol = 1: 1 to obtain 9.74 g (yield: 53%) of a pale yellow solid.

¹ H-NMR (300 MHz, DMSO- d ₆ ) δ 8.20 (bs, 1H, H-2), 7.94 (bs, 1H, NH), 7.15 (bs, 2H, NH ₂ )

Preparation Example 2 1-O-acetyl-2,3,5-tri-O-benzoyl-L-arabinofuranose ( 6 Manufacturing

(Step 1) 1-methoxy-2,3,5-tri-O-benzoyl-L-arabinofuranose ( 5 Manufacturing

200 ml of 1% HCl-methanol solution was added to 9.01 g (60.01 mmol) of L-arabinose, followed by stirring at room temperature for 24 hours. Anhydrous pyridine was added to the transparently dissolved reaction solution to neutralize it, and the solution was concentrated under reduced pressure. 10 ml of pyridine was added twice to the obtained syrup, and it concentrated under reduced pressure again. 100 ml of anhydrous pyridine was added to the obtained residue at 0 ° C, and 36 ml of benzene chloride (BzCl) was slowly added dropwise while maintaining the temperature at 0 ° C, followed by stirring at room temperature for 17 hours. After confirming that the reaction was completed by TLC (Thin Layer Chromatography), the reaction solution was concentrated under reduced pressure and extracted with ethyl acetate and water. The organic layer was washed with HCl, saturated NaHCO ₃ solution and brine in that order, dried over anhydrous MgSO ₄ and filtered.

(Step 2) 1-O-acetyl-2,3,5-tri-O-benzoyl-L-arabinofuranose ( 6 Manufacturing

To compound ( 5 ) of step 1 was added 100 ml of acetic acid and 25 ml of acetic anhydride at 0 ° C., and then 3 ml of concentrated sulfuric acid was added dropwise. The reaction solution was stirred at room temperature for 20 hours, and the reaction was terminated by TLC, and extracted with ice water and CHCl ₃ . The organic layer was washed with saturated aqueous NaHCO ₃ and brine, dried over anhydrous MgSO ₄ , filtered through a Celite pad, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane: ethyl acetate = 5: 1) to obtain 25.55 g (yield: 84%) of a colorless oily product in the form of an anomeric mixture.

¹ H-NMR (300 MHz, DMSO- d ₆ ) δ 8.06, 7.46 (m, 15H, Ar-H), 6.49 (s, 1H, H-1), 5.64 (m, 2H, H-2 and H- 3)), 4.69 (m, 3H, H-4 and H-5), 2.19 (s, 3H, OAc)

Preparation Example 3 1-O-Acetyl-2,3,5-tri-O-benzoyl-L-xylfuranose ( 8 Manufacturing

A colorless oily product (yield: 82%) was obtained in the form of an anomeric mixture by the same method as Preparation Example 2, except that L-xylose was used as a starting material.

¹ H-NMR (300 MHz, DMSO- d ₆ ) δ 8.08, 7.47 (m, 15H, Ar-H), 6.48 (s, 1H, H-1), 5.62 (m, 2H, H-2 and H- 3), 4.71 (m, 3H, H-4 and H-5), 2.18 (s, 3H, OAc)

I. Preparation of 7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidine derivatives (compounds of Formula 1)

Example 1 4-amino-5-cyano-7- (2 ', 3', 5'-trihydroxy-α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidine ( 10 Manufacturing

(Step 1) 4-amino-5-cyano-7- (2 ', 3', 5'-tri-O-benzoyl-α-L-arabinofuranosyl) pyrrolo [2,3-d] pyri Midine ( 9 Manufacturing

4.59 g of 4-amino-6-bromo-5-cyanopyrrolo [2,3-d] pyrimidine ( 3 ) of Preparation Example 1 was added to the flask, and 300 ml of anhydrous CH ₃ CN was added under argon gas. Stirred for about 10 minutes. 7.84 g (2 equivalents to the base ( 3 )) of N, O-bis (trimethylsilyl) acetamide was added dropwise to the reaction solution, followed by stirring at room temperature for 30 minutes. Compound ( 6 ) of Preparation Example 2 (about 1 equivalent to the base ( 3 )) dissolved in anhydrous CH ₃ CN was added to the reaction solution, and 11.17 ml of trimethylsilyltrifluoromethanesulfonate as Lewis acid. (3 equivalents to the base ( 3 )) was added slowly. The reaction solution was stirred at room temperature for 30 minutes, and the reaction temperature was raised to 80 ° C. and further stirred for 3 hours. After confirming that the reaction was terminated by TLC, cold ethyl acetate and sodium bicarbonate water were added and stirred for 30 minutes. The resulting solid was filtered through a pad of celite and the filtrate was concentrated under reduced pressure. Ethyl acetate and water were added to the residue, and the organic layer was washed with brine, dried over anhydrous MgSO ₄ , and filtered. The filtrate was concentrated under reduced pressure, and the residue was recrystallized from ethanol to obtain 9.98 g (yield: 76%) of the target compound ( 9 ).

¹ H-NMR (300 MHz, DMSO- d ₆ ) δ 8.25 (s, 1H, H-2), 8.06, 7.59 (m, 15H, Ar-H), 6.52 (d, 1H, H'-1), 6.41 (m, 1H, H-2 '), 5.97 (dd, 1H, H-3'), 4.74 (m, 3H, H-4 'and H-5')

(Step 2) 4-amino-5-cyano-7- (2 ', 3', 5'-trihydroxy-α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidine ( 10 Manufacturing

4.01 g of Compound ( 9 ) of Step 1 was dissolved in 400 ml of anhydrous methanol, and 2 ml of 28% sodium methoxide solution was added thereto, followed by stirring at room temperature for 30 minutes. After the reaction was completed, the reaction solution was neutralized with acetic acid and then concentrated under reduced pressure. The residue was separated by silica gel column chromatography (methylene chloride: methanol = 10: 1) to obtain 2.06 g (yield: 95%) of the target compound ( 10 ).

Example 2 4-Amino-6-bromo-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamide ( 15 Manufacturing

To 441 mg (1.19 mmol) of Compound ( 10 ) in Example 1, 24 ml of a 2: 1 (volume ratio) mixed solvent of water and metalol was added, followed by 1.5 ml of 25% NH ₄ OH aqueous solution. 0.1 ml of 30% aqueous hydrogen peroxide solution was added to the reaction solution three times at 30 minute intervals, followed by stirring at room temperature for 1 hour. After confirming that the starting material disappeared completely by TLC, the reaction solution was concentrated under reduced pressure and recrystallized with ethanol to obtain 420 mg (yield: 91%) of the target compound ( 15 ).

Example 3 Ethyl 4-amino-6-bromo-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamide oxime · HCl (17) Manufacture

Example 1 of the compound (10) 375 mg was then stirred at room temperature for 6 hours, was added a saturated HCl gas for 40 minutes was added to the ethanol to 40 ㎖ (1.01 mmol) it was refluxed for 30 minutes after. The residue was concentrated under reduced pressure to remove ethanol, and the residue was added to ethanol, crushed finely, and then concentrated under reduced pressure again and again to remove excess HCl. The obtained residue was recrystallized from a mixed solvent of ethanol: water = 8: 1 to give 246 mg (yield: 54%) of the target compound ( 17 ).

Example 4 4-Amino-6-bromo-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamide oxime ( 19 Manufacturing

To 500 mg (1.35 mmol) of Compound ( 10 ) of Example 1 was added 50 ml of ethanol and 500 mg of hydroxylamine (hydroxylamine), and the reaction solution was refluxed for 2 hours. The reaction solution was cooled to room temperature, filtered and recrystallized with water to obtain 473 mg (yield: 87%) of the target compound ( 19 ).

Example 5 4-amino-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamidine ( 13 Manufacturing

200 mg (0.50 mmol) of Compound ( 19 ) in Example 4 were dissolved in 25 ml of boiling ethanol and cooled to 50 ° C. 10% Pd / C 200 mg was added to the reaction solution, cooled to room temperature, and reduced at 45 psi for 5 hours. The residue was filtered through Celite Par and washed with boiling water. Concentrated HCl was added to the filtrate, the mixture was adjusted to pH 5, concentrated under reduced pressure, and recrystallized with ethanol to obtain 44 mg of the target compound ( 13 ) (yield: 29%).

Example 6 4-Amino-6-bromo-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidine-5-thiocarboxamide ( 21 Manufacturing

Anhydrous H ₂ S gas was added to 50 mL of anhydrous methanol solution containing 108 mg (2.00 mmol) of NaOMe for 30 minutes to saturate. To this solution was added 370 mg (1.00 mmol) of the compound of Example 1 ( 10 ), followed by stirring in a sealed pressure tube for 24 hours. 1N-HCl was added to the reaction solution, neutralized to pH 7, and concentrated under reduced pressure. The obtained solid was recrystallized with a mixed solvent of water: methanol = 5: 1 to obtain 355 mg (yield: 88%) of the target compound ( 21 ).

Example 7 4-amino-6-bromo-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamidelazone ( 23 Manufacturing

Example 1 of the compound (10) 300 mg (0.81 mmol ) in ethanol 10 ㎖ and hydrazine hydrate (hydrazine hydrate) (85%) was added to 0.4 ㎖ After refluxing for 2 hours keeping the temperature at 5 ℃ for 12 hours. The resulting solid was filtered and recrystallized with water to give 247 mg (yield: 76%) of the target compound ( 23 ).

Example 8 6-Bromo-5-cyano-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] -4-pyrimidone ( 25 Manufacturing

To 1.50 g (4.05 mmol) of Compound ( 10 ) in Example 1, a mixed solution of 100 ml of water warmed to 55 ° C. and 7.5 ml of glacial acetic acid was added, followed by 1.96 g (28.41 mmol) of NaNO ₂ for 1 hour. Divided in two. The temperature of the reaction solution was maintained at 70 ° C. for 10 hours, and the water was evaporated under reduced pressure, and 2-propanol was added thereto to evaporate under reduced pressure to remove excess acetic acid. The remaining solid was recrystallized from water to give 1.32 g (yield: 88%) of the target compound ( 25 ).

Example 9 4-amino-5-cyano-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidine ( 27 Manufacturing

220 ml of acetic acid (99.7%) was added to 1.10 g (2.97 mmol) of the compound ( 10 ) of Example 1, and 2.1 g of Zn dust was added at a time. The reaction mixture was stirred at room temperature for 2 hours, and the filtrate obtained by filtration was concentrated under reduced pressure. Toluene was added to the resulting solid and crushed. Then, hot ethyl acetate was added to dissolve the solid and filtered through a pad of celite. The filtrate was concentrated under reduced pressure, and then purified by silica gel column chromatography (using solvent-methylene chloride: methanol = 9: 1) to obtain 778 mg (yield: 90%) of the target compound ( 27 ).

Example 10 4-amino-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamide ( 29 Manufacturing

To 147 mg (0.50 mmol) of the compound ( 27 ) in Example 9 was added 8 ml of a 2: 1 (volume ratio) mixed solvent of water and methanol, and 0.5 ml of 25% NH ₄ OH aqueous solution was added thereto. 0.1 ml of hydrogen peroxide (30%) was added to the reaction solution three times at 30 minute intervals, and then stirred at room temperature for 1 hour. After confirming that the starting material disappeared completely by TLC, the reaction solution was concentrated under reduced pressure and recrystallized with ethanol to obtain 142 mg (yield: 92%) of the target compound ( 29 ).

Example 11 ethyl 4-amino-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamide oxime · HCl ( 31 Manufacturing

To 125 mg (0.43 mmol) of Compound ( 27 ) in Example 9 was added 13 ml of ethanol, followed by saturation with HCl gas for 30 minutes, followed by stirring at room temperature for 6 hours and refluxing for 30 minutes. The reaction solution was concentrated under reduced pressure to remove ethanol, and the residue was added to ethanol again, crushed finely, and then concentrated under reduced pressure again and again to remove excess HCl. The finally obtained residue was recrystallized with a mixed solvent of ethanol: water = 10: 1 to obtain 90 mg of the target compound ( 31 ) (yield: 56%).

Example 12 4-Amino-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamide oxime ( 33 Manufacturing

Example 9 Compound (27) 100 mg (0.34 mmol ) in ethanol was added to 10 ㎖ and hydroxylamine 100 mg and refluxed the reaction solution for 2 hours. The reaction solution was cooled to room temperature, filtered, and recrystallized with water to obtain 98 mg (yield: 88%) of the title compound ( 33 ).

Example 13 4-Amino-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidine-5-thiocarboxamide ( 35 Manufacturing

Anhydrous H ₂ S gas was added to 50 mL of anhydrous methanol solution containing 109 mg (2.02 mmol) of NaOMe for 30 minutes to saturate. To this solution was added 294 mg (1.01 mmol) of the compound of Example 9 ( 27 ), followed by stirring in a pressure tube for 24 hours. 1N-HCl was added to the reaction solution, neutralized to pH 7, and concentrated under reduced pressure. The resulting solid was recrystallized with a mixed solvent of water: methanol = 4: 1 to give 292 mg (yield: 89%) of the target compound ( 35 ).

Example 14 4-Amino-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamidelazone ( 37 Manufacturing

To 300 mg (1.03 mmol) of Compound ( 27 ) was added 10 ml of ethanol and 0.4 ml of hydrazine hydrate (85%), refluxed for 2 hours, and the temperature was maintained at 5 ° C. for 12 hours. The resulting solid was filtered and recrystallized with water to give 259 mg (yield: 78%) of the target compound ( 37 ).

Example 15 5-Cyano-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] -4-pyrimidone ( 39 Manufacturing

(Method A)

To 3.01 g (10.33 mmol) of Compound ( 27 ) in Example 9, a mixed solution of 200 ml of warmed water at 55 ° C. and 15 ml of glacial acetic acid was added, followed by dividing 5.0 g (72.46 mmol) of NaNO ₂ twice at an hourly interval. Was added. The temperature of the reaction solution was maintained at 70 ° C. for 1 hour, and then the water was evaporated under reduced pressure, and 2-propanol was added thereto under reduced pressure to remove excess acetic acid. The remaining solid was recrystallized from water to give 2.72 g (yield: 90%) of the target compound ( 39 ).

(Method B)

2.02 g (5.44 mmol) of Compound ( 25 ) in Example 8 was added to dissolve 440 ml of acetic acid (99.7%), and then 4.2 g of Zn dust was added at a time. The reaction mixture was stirred at room temperature for 2 hours, and the filtrate obtained by filtration was concentrated under reduced pressure. Toluene was added to the resulting solid and crushed. Then, hot ethyl acetate was added to dissolve the solid and filtered through a pad of celite. The filtrate was concentrated under reduced pressure, and then purified by silica gel column chromatography (using solvent-methylene chloride: methanol = 12: 1) to obtain 1.40 g (yield: 88%) of the target compound ( 39 ).

Example 16 6-Bromo-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidone-5-carboxamide ( 43 Manufacturing

To 441 mg (1.19 mmol) of Compound ( 25 ) in Example 8 was added 24 ml of a 2: 1 (volume ratio) mixed solvent of water and methanol, followed by 1.5 ml of 25% NH ₄ OH aqueous solution. 0.3 ml of hydrogen peroxide (30%) was added to the reaction solution three times at 30 minute intervals, followed by stirring at room temperature for 1 hour. After confirming that the starting material disappeared completely by TLC, the reaction solution was concentrated under reduced pressure and recrystallized with ethanol to obtain 412 mg of the target compound ( 43 ) (yield: 89%).

Example 17 Ethyl 6-Bromo-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-carboxamide oxime HCl ( 45 Manufacturing

To 250 mg (0.67 mmol) of Compound ( 25 ) in Example 8 was added 26 ml of ethanol, followed by saturation with HCl gas for 30 minutes, followed by stirring at room temperature for 3 hours, followed by reflux for 30 minutes. The reaction solution was concentrated under reduced pressure to remove ethanol, and the residue was added to ethanol again, crushed finely, and then concentrated under reduced pressure again and again to remove excess HCl. The obtained residue was recrystallized from a mixed solvent of ethanol: water = 7: 1 to give 176 mg (yield: 58%) of the target compound ( 45 ).

Example 18 6-Bromo-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-carboxamide oxime ( 47 Manufacturing

To 650 mg (1.75 mmol) of Compound ( 25 ) in Example 8 was added 10 ml of ethanol and 650 mg of hydroxylamine, and the reaction solution was refluxed for 4 hours. The reaction solution was cooled to room temperature, filtered, and recrystallized with water to obtain 516 mg (yield: 73%) of the target compound ( 47 ).

Example 19 7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidone-5-carboxamidine ( 41 Manufacturing

Example 18 Compound 47 was dissolved in boiling ethanol for 20 ㎖ 551 mg (1.36 mmol) cooled to 50 ℃ of. 550 mg of 10% Pd / C was added to the reaction solution, which was cooled to room temperature and reduced for 5 hours at 45 psi. The residue was filtered through a pad of celite and washed with boiling water. Concentrated HCl was added to the filtrate, the mixture was adjusted to pH 5, concentrated under reduced pressure, and recrystallized with ethanol to obtain 117 mg (yield: 28%) of the title compound ( 41 ).

Example 20 6-Bromo-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-thiocarboxamide ( 49 Manufacturing

Anhydrous H ₂ S gas was added to 40 mL of anhydrous methanol solution containing 111 mg (1.60 mmol) of NaOMe for 30 minutes to saturate. To this solution was added 302 mg (0.81 mmol) of Compound ( 25 ) of Example 8 and then stirred in a pressure tube for 24 hours. 1N-HCl was added to the reaction solution, neutralized to pH 7, and concentrated under reduced pressure. The resulting solid was recrystallized from a mixed solvent of water: methanol = 6: 1 to give 262 mg (yield: 80%) of the target compound ( 49 ).

Example 21 6-Bromo-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidone-5-carboxamidelazone ( 51 Manufacturing

To 420 mg (1.13 mmol) of Compound ( 25 ) in Example 8 was added 14 ml of ethanol and 0.56 ml of hydrazine hydrate (85%), refluxed for 2 hours, and the temperature was maintained at 5 ° C. for 12 hours. The resulting solid was filtered and recrystallized with water to give 342 mg (yield: 75%) of the target compound ( 51 ).

Example 22 6-Bromo-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidone-5-carboxamide ( 53 Manufacturing

To 220 mg (0.75 mmol) of Compound ( 39 ) in Example 15 was added 12 ml of a 2: 1 (volume ratio) mixed solvent of water and methanol, and 0.75 ml of 25% NH ₄ OH aqueous solution was added. 0.15 mL of hydrogen peroxide (30%) was added to the reaction solution three times at 30 minute intervals, and then stirred at room temperature for 1 hour. After confirming that the starting material disappeared completely by TLC, the reaction solution was concentrated under reduced pressure and recrystallized with ethanol to obtain 209 mg (yield: 90%) of the target compound ( 53 ).

Example 23 Ethyl 7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-carboxamide oxime · HCl ( 55 Manufacturing

To 250 mg (0.86 mmol) of Compound ( 39 ) in Example 15 was added 26 ml of ethanol, followed by saturation with HCl gas for 30 minutes, followed by stirring at room temperature for 3 hours, followed by reflux for 30 minutes. The reaction solution was concentrated under reduced pressure to remove ethanol, and ethanol was added to the residue, followed by crushing. The residue was concentrated under reduced pressure again and again to remove excess HCl. The obtained residue was recrystallized with a mixed solvent of ethanol: water = 8: 1 to give 193 mg (yield: 60%) of the target compound ( 55 ).

Example 24 7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-carboxamide oxime ( 57 Manufacturing

Example 15 of the compound (39) 401 mg (1.37 mmol ) in ethanol was added to 40 ㎖ and hydroxylamine 400 mg and refluxed the reaction solution for 4 hours. The reaction solution was cooled to room temperature, filtered, and recrystallized with water to obtain 334 mg (yield: 75%) of the target compound ( 57 ).

Example 25 6-Bromo-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-thiocarboxamide ( 59 Manufacturing

Anhydrous H ₂ S gas was added to 50 ml of anhydrous methanol solution containing 135 mg (1.96 mmol) of NaOMe for 30 minutes to saturate. To this solution was added 286 mg (0.98 mmol) of Compound ( 39 ) of Example 15, followed by stirring in a pressure tube for 24 hours. 1N-HCl was added to the reaction solution, neutralized to pH 7, and concentrated under reduced pressure. The obtained solid was recrystallized with a mixed solvent of water: methanol = 5: 1 to give 250 mg (yield: 87%) of the target compound ( 59 ).

Example 26 6-Bromo-7- (α-L-arabinofuranosyl) pyrrolo [2,3-d] pyrimidone-5-carboxamidelazone ( 61 Manufacturing

To 451 mg (1.54 mmol) of Compound ( 39 ) in Example 15 was added 15 ml of ethanol and 0.6 ml of hydrazine hydrate (85%), refluxed for 2 hours, and the temperature was maintained at 5 ° C. for 12 hours. The resulting solid was filtered and recrystallized with water to give 385 mg (yield: 77%) of the target compound (61).

¹ H NMR (300 MHz, solvent DMSO- d ₆ ) of the compound of Chemical Formula 1 prepared through Examples 1 to 26 are shown in Table 1 below.

¹ H NMR Results of the Compound of Formula 1 Example number (compound number) H-2H-1 ' H-2'H-3 ' H-4'H-5 ' _{a, b} Guitar peak 1 (10) 8.41 (s) 6.29 (d) 4.62 (dd) 4.33 (m) 4.05 (m) 3.51-3.61 (m) 7.05 (bs, 2H, C ₄ -NH ₂ ), 5.47 (d, 1H, 2'-OH), 5.13 (d, 1H, 3'-OH), 4.85 (t, 1H, 5'-OH) 2 (15) 8.36 (s) 6.14 (d) 4.57 (dd) 4.45 (m) 4.06 (m) 3.50-3.59 (m) 8.00, 7.98 (2bs, 2H, CONH ₂ ), 7.52 (bs, 2H, C ₄ -NH ₂ ) 5.45 (d, 1H, 2'-OH), 5.12 (d, 1H, 3'-OH), 4.83 ( t, 1H, 5'-OH) 3 (17) 8.30 (s) 6.15 (d) 4.58 (dd) 4.46 (m) 4.06 (m) 3.51-3.60 (m) 8.40 (bs, 1H, C = NH), 7.43 (bs, 2H, C ₄ -NH ₂ ), 5.46 (d, 1H, 2'-OH), 5.14 (d, 1H, 3'-OH), 4.84 ( t, 1H, 5'-OH), 3.79 (q, 2H, OCH ₂ ), 1.46 (t, 3H, CH ₃ ) 4 (19) 8.33 (s) 6.92 (d) 4.54 (dd) 4.42 (m) 4.04 (m) 3.47-3.57 (m) 9.93 (s, 1H, NOH), 7.48 (bs, 2H, C ₄ -NH ₂ ), 6.20 (bs, 2H, C (NOH) NH ₂ ), 5.42 (d, 1H, 2'-OH), 5.10 ( d, 1H, 3'-OH), 4.81 (t, 1H, 5'-OH) 5 (13) 8.39 (s) 6.05 (d) 4.52 (dd) 4.11 (m) 3.96 (m) 3.42-3.51 (m) 8.30 (s, 1H, H-6), 8.17 (bs, 1H, C = NH), 7.46 (bs, 2H, C ₄ -NH ₂ ), 5.93 (bs, 2H, C (NH) NH ₂ ), 5.38 (d, 1H, 2'-OH), 5.06 (d, 1H, 3'-OH, 4.85 (t, 1H, 5'-OH) 6 (21) 8.40 (s) 6.15 (d) 4.65 (dd) 4.53 (m) 4.14 (m) 3.58-3.68 (m) 9.54, 9.69 (2bs, 2H, CSNH ₂ ), 812 (bs, 2H, C ₄ -NH ₂ ) 5.43 (d, 1H, 2'-OH), 5.09 (d, 1H, 3'-OH), 4.80 ( t, 1H, 5'-OH) 7 (23) 8.35 (s) 6.30 (d) 4.59 (dd) 4.44 (m) 4.07 (m) 3.51-3.60 (m) 9.58 (bs, 2H, C = N NH ₂ ), 7.50 (bs, 2H, C ₄ -NH ₂ ), 5.91 (bs, 2H, C (NNH ₂ ) NH ₂ ), 5.44 (d, 1H, 2'- OH), 5.11 (d, 1H, 3'-OH), 4.83 (t, 1H, 5'-OH) 8 (25) 8.32 (d) 6.16 (d) 4.60 (dd) 4.32 (m) 4.04 (m) 3.50-3.60 (m) 12.11 (d, 1H, NH), 5.46 (d, 1H, 2'-OH), 5.04 (d, 1H, 3'-OH), 4.84 (t, 1H, 5'-OH) 9 (27) 8.50 (s) 6.09 (d) 4.32 (dd) 4.04 (m) 3.85 (m) 3.41-3.51 (m) 8.41 (s, 1H, H-6), 6.92 (bs, 2H, C ₄ -NH ₂ ), 5.40 (d, 1H, 2'-OH), 5.08 (d, 1H, 3'-OH), 4.87 ( t, 1H, 5'-OH) 10 (29) 8.45 (s) 6.04 (d) 4.27 (dd) 4.09 (m) 3.86 (m) 3.39-3.48 (m) 8.36 (s, 1H, H-6), 7.98 (bs, 2H, CONH ₂ ), 7.39 (bs, 2H, C ₄ -NH ₂ ), 5.38 (d, 1H, 2'-OH), 5.07 (d, 1H, 3'-OH), 4.85 (t, 1H, 5'-OH) 11 (31) 8.39 (s) 6.05 (d) 4.28 (dd) 4.17 (m) 3.86 (m) 3.41-3.51 (m) 8.42 (bs, 1H, C = NH), 8.30 (s, 1H, H-6), 7.30 (bs, 2H, C ₄ -NH ₂ ), 5.39 (d, 1H, 2'-OH), 5.09 (d , 1H, 2'-OH), 4.86 (t, 1H, 5'-OH) 3.79 (q, 2H, OCH ₂ ), 1.46 (t, 3H, CH ₃ ) 12 (33) 8.42 (s) 6.03 (d) 4.24 (dd) 4.13 (m) 3.84 (m) 3.37-3.47 (m) 9.80 (s, 1H, NOH), 8.33 (s, 1H, H-6), 7.35 (bs, 2H, C ₄ -NH ₂ ), 6.07 (bs, 2H, C (NOH) NH ₂ ), 5.35 (d , 1H, 2'-OH), 5.05 (d, 1H, 3'-OH), 4.83 (t, 1H, 5'-OH)

¹ H NMR Results of the Compound of Formula 1 Example number (compound number) H-2H-1 ' H-2'H-3 ' H-4'H-5 ' _{a, b} Guitar peak 13 (35) 8.49 (s) 6.04 (d) 4.35 (dd) 4.24 (m) 3.94 (m) 3.47-3.57 (m) 9.40, 9.59 (2bs, 2H, CSNH ₂ ), 8.40 (s, 1H, 6-H), 8.09 (bs, 2H, C ₄ -NH ₂ ), 5.36 (d, 1H, 2'-OH), 5.04 ( d, 1H, 3'-OH), 4.82 (t, 1H, 5'-OH) 14 (37) 8.45 (s) 6.10 (d) 4.29 (dd) 4.15 (m) 3.87 (m) 3.41-3.51 (m) 9.45 (bs, 2H, C = N NH ₂ ), 8.36 (s, 1H, 6-H), 7.37 (bs, 2H, C ₄ -NH ₂ ), 5.77 (bs, 2H, C (NNH ₂ ) NH ₂ ), 5.37 (d, 1H, 2'-OH), 5.06 (d, 1H, 3'-OH), 4.85 (t, 1H, 5'-OH) 15 (39) 8.41 (d) 6.06 (d) 4.31 (dd) 4.03 (m) 3.84 (m) 3.41-3.50 (m) 11.98 (d, 1H, NH), 8.32 (s, 1H, 6-H), 5.39 (d, 1H, 2'-OH), 4.99 (d, 1H, 3'-OH), 4.86 (t, 1H, 5'-OH) 16 (43) 8.27 (d) 6.22 (d) 4.55 (dd) 4.44 (m) 4.05 (m) 3.49-3.59 (m) 12.13 (d, 1H, NH), 7.97, 7.94 (2bs, 2H, CONH ₂ ), 5.44 (d, 1H, 2'-OH), 5.11 (d, 1H, 3'-OH), 4.82 (t, 1H , 5'-OH) 17 (45) 8.21 (d) 6.22 (d) 4.56 (dd) 4.45 (m) 4.05 (m) 3.50-3.60 (m) 12.10 (d, 1H, NH), 8.37 (bs, 1H, C = NH), 5.45 (d, 1H, 2'-OH), 5.13 (d, 1H, 3'-OH), 4.83 (t, 1H, 5'-OH), 3.78 (q, 2H, OCH ₂ ), 1.44 (t, 3H, CH ₃ ) 18 (47) 8.24 (d) 6.09 (d) 4.52 (dd) 4.41 (m) 4.03 (m) 3.46-3.59 (m) 12.13 (d, 1H, NH), 9.90 (s, 1H, NOH), 6.18 (bs, 2H, C (NOH) NH ₂ ), 5.40 (d, 1H, 2'-OH), 5.08 (d, 1H, 3'-OH), 4.79 (t, 1H, 5'-OH) 19 (41) 8.30 (d) 6.02 (d) 4.50 (dd) 4.01 (m) 3.95 (m) 3.41-3.50 (m) 12.02 (d, 1H, NH), 8.24 (bs, 1H, C = NH), 8.21 (s, 1H, H-6), 5.90 (bs, 2H, C (NH) NH ₂ ), 5.37 (d, 1H , 2'-OH), 5.05 (d, 1H, 3'-OH), 4.84 (t, 1H, 5'-OH) 20 (49) 8.31 (d) 6.22 (d) 4.62 (dd) 4.52 (m) 4.13 (m) 3.57-3.67 (m) 12.16 (d, 1H, NH), 9.52, 9.68 (2bs, 2H, CSNH ₂ ), 5.42 (d, 1H, 2'-OH), 5.08 (d, 1H, 3'-OH), 4.78 (t, 1H , 5'-OH) 21 (51) 8.26 (d) 6.16 (d) 4.57 (dd) 4.43 (m) 4.05 (m) 3.50-3.61 (m) 12.12 (d, 1H, NH), 9.56 (bs, 2H, C = N NH ₂ ), 5.88 (bs, 2H, C (NNH ₂ ) NH ₂ ), 5.42 (d, 1H, 2'-OH), 5.10 (d, 1H, 3'-OH), 4.82 (t, 1H, 5'-OH) 22 (53) 8.36 (d) 6.02 (d) 4.26 (dd) 4.15 (m) 3.85 (m) 3.39-3.49 (m) 12.04 (d, 1H, NH), 8.27 (s, 1H, 6-H), 7.89, 7.87 (2bs, 2H, CONH ₂ ), 5.37 (d, 1H, 2'-OH), 5.06 (d, 1H, 3'-OH), 4.84 (t, 1H, 5'-OH) 23 (55) 8.30 (d) 6.03 (d) 4.27 (dd) 4.15 (m) 3.84 (m) 3.41-3.46 (m) 12.01 (d, 1H, NH), 8.28 (bs, 1H, C = NH), 8.21 (s, 1H, 6-H), 5.38 (d, 1H, 2'-OH), 5.08 (d, 1H, 3 '-OH), 4.85 (t, 1H, 5'-OH), 3.76 (q, 2H, OCH ₂ ), 1.42 (t, 3H, CH ₃ )

¹ H NMR Results of the Compound of Formula 1 Example number (compound number) H-2H-1 ' H-2'H-3 ' H-4'H-5 ' _{a, b} Guitar peak 24 (57) 8.33 (d) 6.00 (d) 4.32 (dd) 4.12 (m) 3.76 (m) 3.36-3.45 (m) 12.04 (d, 1H, NH), 9.81 (s, 1H, NOH), 8.25 (s, 1H, 6-H), 6.09 (bs, 2H, C (NOH) NH ₂ ), 5.33 (d, 1H, 2 '-OH), 5.03 (d, 1H, 3'-OH), 4.81 (t, 1H, 5'-OH) 25 (59) 8.40 (d) 6.02 (d) 4.31 (dd) 4.22 (m) 3.93 (m) 3.47-3.57 (m) 12.07 (d, 1H, NH), 9.43, 9.58 (2bs, 2H, CSNH ₂ ), 8.31 (s, 1H, 6-H), 5.35 (d, 1H, 2'-OH), 5.03 (d, 1H, 3'-OH), 4.79 (t, 1H, 5'-OH) 26 (61) 8.35 (d) 6.06 (d) 4.27 (dd) 4.14 (m) 3.85 (m) 3.41-3.50 (m) 12.03 (d, 1H, NH), 9.47 (bs, 2H, C = N NH ₂ ), 8.26 (s, 1H, 6-H), 5.79 (bs, 2H, C (NNH ₂ ) NH ₂ ), 5.37 ( d, 1H, 2'-OH), 5.05 (d, 1H, 3'-OH), 4.84 (t, 1H, 5'-OH)

II. Preparation of 7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine derivative (compound of formula 2)

Example 27 4-Amino-5-cyano-7- (2 ', 3', 5'-trihydroxy-β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine ( 12 Manufacturing

(Step 1) 4-amino-5-cyano-7- (2 ', 3', 5'-tri-O-benzoyl-β-L-xylofuranosyl) pyrrolo [2,3-d] pyri Midine ( 11 Manufacturing

A target compound ( 11 ) (yield: 71%) was obtained by the same method as Step 1 of Example 1, except that Compound ( 8 ) of Preparation Example 3 was used instead of the compound of Preparation Example 2 as the reactant.

¹ H-NMR (300 MHz, DMSO- d ₆ ) δ 8.24 (s, 1H, H-2), 8.08, 7.60 (m, 15H, Ar-H), 6.51 (d, 1H, H'-1), 6.42 (m, 1H, H-2 '), 5.93 (dd, 1H, H-3'), 4.76 (m, 3H, H-4 'and H-5')

(Step 2) 4-amino-5-cyano-7- (2 ', 3', 5'-trihydroxy-β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine ( 12 Manufacturing

A target compound ( 12 ) (yield: 95%) was obtained by the same method as Step 2 of Example 1, except that the compound of Step 1 was used as a starting material.

The compounds of Examples 28 to 52 were prepared by the method of Examples 2 to 26, and the starting materials and preparation methods used at this time are shown in Table 2 below.

Preparation Conditions of Compound (2) Example Number Starting material (compound number) Manufacturing method Compound Name (Compound Number) 28 Example 27 (12) Same as Example 2 4-amino-6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamide (16) 29 Example 27 (12) Same as Example 3 Ethyl 4-amino-6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamideoximeHCl (18) 30 Example 27 (12) Same as Example 4 4-amino-6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamideoxime (20) 31 Example 30 (20) Same as Example 5 4-amino-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamidine (14) 32 Example 27 (12) Same as Example 6 4-amino-6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-thiocarboxamide (22)

Preparation Conditions of Compound (2) Example Number Starting material (compound number) Manufacturing method Compound Name (Compound Number) 33 Example 27 (12) Same as Example 7 4-amino-6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamidelazone (24) 34 Example 27 (12) Same as Example 8 6-bromo-5-cyano-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] -4-pyrimidone (26) 35 Example 27 (12) Same as Example 9 4-amino-5-cyano-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine (28) 36 Example 35 (28) Same as Example 10 4-amino-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamide (30) 37 Example 35 (28) Same as Example 11 Ethyl 4-amino-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamideoximeHCl (32) 38 Example 35 (28) Same as Example 12 4-amino-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamideoxime (34) 39 Example 35 (28) Same as Example 13 4-amino-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-thiocarboxamide (36) 40 Example 35 (28) Same as Example 14 4-amino-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamidelazone (38) 41 Example 35 (28) or Example 34 (26) Same as Example 15 5-cyano-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] -4-pyrimidone (40) 42 Example 34 (26) Same as Example 16 6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidone-5-carboxamide (44) 43 Example 34 (26) Same as Example 17 Ethyl 6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-carboxamideoximeHCl (46) 44 Example 34 (26) Same as Example 18 6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-carboxamideoxime (48) 45 Example 44 (48) Same as Example 19 7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidone-5-carboxamidine (42) 46 Example 34 (26) Same as Example 20 6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-thiocarboxamide (50) 47 Example 34 (26) Same as Example 21 6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidone-5-carboxamidelazone (52) 48 Example 41 (40) Same as Example 22 6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidone-5-carboxamide (54) 49 Example 41 (40) Same as Example 23 Ethyl 7- (β-L-xylofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-carboxamideoximeHCl (56)

Preparation Conditions of Compound (2) Example Number Starting material (compound number) Manufacturing method Compound Name (Compound Number) 50 Example 41 (40) Same as Example 24 7- (β-L-xylofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-carboxamideoxime (58) 51 Example 41 (40) Same as Example 25 6-Bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-thiocarboxamide (60) 52 Example 41 (40) Same as Example 26 6-Bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidone-5-carboxamidelazone (62)

¹ H NMR (300 MHz, solvent DMSO- d ₆ ) of the compound of Chemical Formula 2 prepared through Examples 27 to 52 are shown in Table 3 below.

¹ H NMR Results of the Compound of Formula 2 Example number (compound number) H-2H-1 ' H-2'H-3 ' H-4'H-5 ' _{a, b} Guitar peak 27 (12) 8.45 (s) 6.06 (d) 4.31 (m) 4.10 (dd) 3.96 (m) 3.75-3.79 (m) 7.05 (bs, 2H, C ₄ -NH ₂ ), 5.45 (d, 1H, 2'-OH), 5.16 (d, 1H, 3'-OH), 4.88 (t, 1H, 5'-OH) 28 (16) 8.40 (s) 6.01 (d) 4.26 (dd) 4.22 (m) 3.97 (m) 3.73-3.78 (m) 8.26, 8.24 (2bs, 2H, CONH ₂ ), 7.62 (bs, 2H, C ₄ -NH ₂ ) 5.43 (d, 1H, 2'-OH), 5.15 (d, 1H, 3'-OH), 4.86 ( t, 1H, 5'-OH) 29 (18) 8.34 (s) 6.02 (d) 4.27 (dd) 4.22 (m) 3.97 (m) 3.74-3.80 (m) 8.44 (bs, 1H, C = NH), 7.53 (bs, 2H, C ₄ -NH ₂ ), 5.44 (d, 1H, 2'-OH), 5.17 (d, 1H, 3'-OH), 4.87 ( t, 1H, 5'-OH), 3.81 (q, 2H, OCH ₂ ), 1.48 (t, 3H, CH ₃ ) 30 (20) 8.38 (s) 5.99 (d) 4.24 (dd) 4.19 (m) 3.95 (m) 3.69-3.75 (m) 9.97 (s, 1H, NOH), 7.58 (bs, 2H, C ₄ -NH ₂ ), 6.27 (bs, 2H, C (NOH) NH ₂ ), 5.40 (d, 1H, 2'-OH), 5.13 ( d, 1H, 3'-OH), 4.84 (t, 1H, 5'-OH) 31 (14) 8.43 (s) 5.92 (d) 4.21 (dd) 4.16 (m) 3.91 (m) 3.68-3.73 (m) 8.34 (s, 1H, H-6), 8.30 (bs, 1H, C = NH), 7.56 (bs, 2H, C ₄ -NH ₂ ), 5.99 (bs, 2H, C (NH) NH ₂ ), 5.36 (d, 1H, 2'-OH), 5.10 (d, 1H, 3'-OH), 4.89 (t, 1H, 5'-OH)

¹ H NMR Results of the Compound of Formula 2 Example number (compound number) H-2H-1 ' H-2'H-3 ' H-4'H-5 ' _{a, b} Guitar peak 32 (22) 8.44 (s) 6.02 (d) 4.34 (dd) 4.30 (m) 4.05 (m) 3.81-3.86 (m) 9.76, 9.91 (2bs, 2H, CSNH ₂ ), 8.23 (bs, 2H, C ₄ -NH ₂ ) 5.41 (d, 1H, 2'-OH), 5.12 (d, 1H, 3'-OH), 4.83 ( t, 1H, 5'-OH) 33 (24) 8.39 (s) 6.07 (d) 4.28 (dd) 4.23 (m) 3.98 (m) 3.74-3.79 (m) 9.80 (bs, 2H, C = N NH ₂ ), 7.61 (bs, 2H, C ₄ -NH ₂ ), 6.12 (bs, 2H, C (NNH ₂ ) NH ₂ ), 5.42 (d, 1H, 2'- OH), 5.14 (d, 1H, 3'-OH), 4.86 (t, 1H, 5'-OH) 34 (26) 8.36 (d) 6.03 (d) 4.29 (m) 4.09 (dd) 3.95 (m) 3.74-3.79 (m) 12.21 (d, 1H, NH), 5.44 (d, 1H, 2'-OH), 5.15 (d, 1H, 3'-OH), 4.87 (t, 1H, 5'-OH) 35 (28) 8.54 (s) 5.96 (d) 4.01 (dd) 3.81 (m) 3.76 (dd) 3.65-3.69 (m) 8.45 (s, 1H, H-6), 7.02 (bs, 2H, C ₄ -NH ₂ ) 5.38 (d, 1H, 2'-OH), 5.11 (d, 1H, 3'-OH), 4.90 (t , 1H, 5'-OH) 36 (30) 8.49 (s) 5.91 (d) 3.96 (dd) 3.86 (m) 3.77 (m) 3.63-3.68 (m) 8.39 (s, 1H, H-6), 8.13 (bs, 2H, CONH ₂ ), 7.49 (bs, 2H, C ₄ -NH ₂ ) 5.36 (d, 1H, 2'-OH), 5.09 (d, 1H , 3'-OH), 4.88 (t, 1H, 5'-OH) 37 (32) 8.43 (s) 5.92 (d) 3.97 (dd) 3.93 (m) 3.77 (m) 3.64-3.69 (m) 8.46 (bs, 1H, C = NH), 8.34 (s, 1H, H-6), 7.40 (bs, 2H, C ₄ -NH ₂ ), 5.37 (d, 1H, 2'-OH), 5.12 (d , 1H, 3'-OH), 4.89 (t, 1H, 5'-OH) 3.81 (q, 2H, OCH ₂ ), 1.48 (t, 3H, CH ₃ ) 38 (34) 8.47 (s) 5.90 (d) 3.94 (dd) 3.90 (m) 3.75 (m) 3.59-3.64 (m) 9.84 (s, 1H, NOH), 8.38 (s, 1H, H-6), 7.46 (bs, 2H, C ₄ -NH ₂ ), 6.11 (bs, 2H, C (NOH) NH ₂ ), 5.33 (d , 1H, 2'-OH), 5.08 (d, 1H, 3'-OH), 4.86 (t, 1H, 5'-OH) 39 (36) 8.52 (s) 5.53 (d) 4.35 (dd) 4.00 (m) 3.85 (m) 3.71-3.75 (m) 9.62, 9.82 (2bs, 2H, CSNH ₂ ), 8.43 (s, 1H, 6-H), 8.20 (bs, 2H, C ₄ -NH ₂ ), 5.34 (d, 1H, 2'-OH), 5.07 ( d, 1H, 3'-OH), 4.85 (t, 1H, 5'-OH) 40 (38) 8.48 (s) 5.97 (d) 3.98 (dd) 3.94 (m) 3.78 (m) 3.64-3.68 (m) 9.67 (bs, 2H, C = N NH ₂ ), 8.39 (s, 1H, 6-H), 7.48 (bs, 2H, C ₄ -NH ₂ ), 5.99 (bs, 2H, C (NNH ₂ ) NH ₂ ), 5.35 (d, 1H, 2'-OH), 5.09 (d, 1H, 3'-OH), 4.88 (t, 1H, 5'-OH) 41 (40) 8.45 (d) 5.53 (d) 4.00 (dd) 3.81 (m) 3.75 (m) 3.64-3.70 (m) 12.06 (d, 1H, NH), 8.36 (s, 1H, 6-H), 5.37 (d, 1H, 2'-OH), 5.10 (d, 1H, 3'-OH), 4.89 (t, 1H, 5'-OH) 42 (44) 8.32 (d) 5.98 (d) 4.24 (dd) 4.20 (m) 3.95 (m) 3.71-3.76 (m) 12.21 (d, 1H, NH), 8.21, 8.19 (2bs, 2H, CONH ₂ ), 5.42 (d, 1H, 2'-OH), 5.14 (d, 1H, 3'-OH), 4.85 (t, 1H , 5'-OH) 43 (46) 8.25 (d) 5.97 (d) 4.25 (dd) 4.21 (m) 3.96 (m) 3.73-3.78 (m) 12.17 (d, 1H, NH), 8.41 (bs, 1H, C = NH), 5.43 (d, 1H, 2'-OH), 5.16 (d, 1H, 3'-OH), 4.85 (t, 1H, 5'-OH), 3.80 (q, 2H, OCH ₂ ), 1.46 (t, 3H, CH ₃ )

¹ H NMR Results of the Compound of Formula 2 Example number (compound number) H-2H-1 ' H-2'H-3 ' H-4'H-5 ' _{a, b} Guitar peak 44 (48) 8.28 (d) 5.96 (d) 4.22 (dd) 4.18 (m) 3.94 (m) 3.68-3.73 (m) 12.21 (d, 1H, NH), 9.94 (s, 1H, NOH), 6.23 (bs, 2H, C (NOH) NH ₂ ), 5.39 (d, 1H, 2'-OH), 5.12 (d, 1H, 3'-OH), 4.83 (t, 1H, 5'-OH) 45 (42) 8.34 (d) 5.89 (d) 4.19 (dd) 4.15 (m) 3.90 (m) 3.67-3.72 (m) 12.10 (d, 1H, NH), 8.27 (bs, 1H, C = NH), 8.25 (s, 1H, H-6), 5.96 (bs, 2H, C (NH) NH ₂ ), 5.35 (d, 1H , 2'-OH), 5.09 (d, 1H, 3'-OH), 4.88 (t, 1H, 5'-OH) 46 (50) 8.35 (d) 5.99 (d) 4.32 (dd) 4.29 (m) 4.04 (m) 3.80-3.85 (m) 12.24 (d, 1H, NH), 9.74, 9.91 (2bs, 2H, CSNH ₂ ), 5.39 (d, 1H, 2'-OH), 5.11 (d, 1H, 3'-OH), 4.82 (t, 1H , 5'-OH) 47 (52) 8.31 (d) 6.04 (d) 4.27 (dd) 4.22 (m) 3.97 (m) 3.73-3.78 (m) 12.20 (d, 1H, NH), 9.77 (bs, 2H, C = N NH ₂ ), 6.10 (bs, 2H, C (NNH ₂ ) NH ₂ ), 5.41 (d, 1H, 2'-OH), 5.13 (d, 1H, 3'-OH), 4.85 (t, 1H, 5'-OH) 48 (54) 8.41 (d) 5.89 (d) 3.94 (dd) 3.90 (m) 3.75 (m) 3.61-3.67 (m) 12.12 (d, 1H, NH), 8.31 (s, 1H, 6-H), 8.12, 8.10 (2bs, 2H, CONH ₂ ), 5.35 (d, 1H, 2'-OH), 5.09 (d, 1H, 3'-OH), 4.87 (t, 1H, 5'-OH) 49 (56) 8.34 (d) 5.89 (d) 3.96 (dd) 3.92 (m) 3.85 (m) 3.41-3.51 (m) 12.08 (d, 1H, NH), 8.32 (bs, 1H, C = NH), 8.25 (s, 1H, 6-H), 5.36 (d, 1H, 2'-OH), 5.11 (d, 1H, 3 '-OH), 4.87 (t, 1H, 5'-OH), 3.78 (q, 2H, OCH ₂ ), 1.44 (t, 3H, CH ₃ ) 50 (58) 8.38 (d) 5.87 (d) 3.92 (dd) 3.88 (m) 3.76 (m) 3.58-3.63 (m) 12.12 (d, 1H, NH), 9.85 (s, 1H, NOH), 8.29 (s, 1H, 6-H), 6.14 (bs, 2H, C (NOH) NH ₂ ), 5.32 (d, 1H, 2 '-OH), 5.07 (d, 1H, 3'-OH), 4.85 (t, 1H, 5'-OH) 51 (60) 8.45 (d) 5.90 (d) 4.02 (dd) 3.99 (m) 3.84 (m) 3.71-3.76 (m) 12.15 (d, 1H, NH), 9.65, 9.80 (2bs, 2H, CSNH ₂ ), 8.36 (s, 1H, 6-H), 5.32 (d, 1H, 2'-OH), 5.06 (d, 1H, 3'-OH), 4.84 (t, 1H, 5'-OH) 52 (62) 8.39 (d) 5.95 (d) 3.98 (dd) 3.94 (m) 3.77 (m) 3.63-3.69 (m) 12.11 (d, 1H, NH), 9.88 (bs, 2H, C = N NH ₂ ), 8.31 (s, 1H, 6-H), 6.01 (bs, 2H, C (NNH ₂ ) NH ₂ ), 5.34 ( d, 1H, 2'-OH), 5.08 (d, 1H, 3'-OH), 4.87 (t, 1H, 5'-OH)

Production Example 4 Culture of Cell Line

SK-HEp-1 human hematoma cells were purchased from the Cancer Research Center of Seoul National University and 37 ° C in DMEM (Dulbecco's Modified Eagle's Medium) containing 50 mg / L of 5% bovine serum and gentamycin. The cells were incubated in culture conditions supplied with 5% CO ₂ .

Experimental Example 1 Inhibitory Effect on Serine-Threonine Protein Kinases

The following experiment was conducted to determine whether the compounds of the present invention have a selective inhibitory effect on various serine-threonine protein kinases involved in the signal transduction system in cancer cells.

1) Inhibitory effect on cdc2 kinase

SK-HEp-1 cell extract of Preparation Example 4 was treated with nocadazole for 17 hours to fix the cell cycle in M phase (M phase), which was used as a raw material for cdc2 kinase. Reaction solution [50 mM Tris, pH 7.4, 2 mM DTT, 10 mM MgCl ₂ , 1 mM EGTA, 40 mM β-glycerophosphate, 0.1 mM sodium vanadate, 1 μCi [γ- ³² P] ATP (50 μM APT), 50 μM Hl peptide] and the SK-HEp-1 cell extract and the compound of Formula 1 or Formula 2 according to the present invention were added at different concentrations, and distilled water was added to adjust the final volume to 25 μl and reacted for 15 minutes at 30 ° C. . Thereafter, 6 μl of 50% trichloroacetic acid solution was added to the reaction solution, and the mixture was left on ice for 40 minutes, centrifuged at 10,000 × g for 15 minutes, and 15 μl of the supernatant was dipped onto P-81 paper. The paper was washed three times with 75 mM phosphoric acid three times for 15 minutes and dried, and then radioactivity was measured with a liquid scintillation counter.

2) Inhibitory Effect on Protein Kinase A

Reaction solution [50 mM Tris, pH 7.4, 2 mM DTT, 10 mM MgCl ₂ , 1 mM EGTA, 40 mM β-glycerophosphate, 0.1 mM sodium vanadate, 1 μCi [γ- ³² P] ATP (50 μM ATP), 30 μM cAMP, 1 mM kemptide] and 2 units of purified protein kinase A (PKA, Promega) and the compound of formula 1 or formula 2 according to the present invention were added at different concentrations, and distilled water was added thereto. The final volume was adjusted to 25 μl and reacted at 30 ° C. for 15 minutes. Thereafter, 6 µl of 50% trichloroacetic acid was added to the reaction solution, which was allowed to stand on ice for 40 minutes, centrifuged at 10,000 × g for 15 minutes, and 15 µl of the supernatant was dipped onto p-81 paper. The paper was washed three times for 15 minutes with 75 mM phosphoric acid solution and dried, and then radioactivity was measured with a liquid scintillation counter.

3) Inhibitory Effect on Casein Kinase II

Reaction solution [50 mM Tris, pH 7.4, 2 mM DTT, 10 mM MgCl ₂ , 1 mM EGTA, 40 mM β-glycerophosphate, 0.1 mM sodium vanadate, 1 μCi [γ- ³² P] ATP (50 μM ATP), 0.6 mM peptide substrate ( SEQ ID NO: 1 )] and 3 units of purified casein kinase II (CKII, Promega) and the compound of formula 1 or formula 2 according to the present invention were added at different concentrations, and distilled water was added to the final volume. The reaction was adjusted to 25 μl for 15 minutes at 30 ° C. Thereafter, 6 µl of 50% trichloroacetic acid was added to the reaction solution, left for 40 minutes on ice, centrifuged at 10,000 x g for 15 minutes, and 15 µl of the supernatant was dipped onto p-81 paper. The paper was washed three times for 15 minutes with 75 mM phosphoric acid solution and dried, and then radioactivity was measured with a liquid scintillation counter.

4) Inhibitory Effect on Protein Kinase C

Reaction solution [20 mM Tris, pH 7.4, 10 mM MgCl ₂ , 0.25 mM EGTA, 0.4 CaCl ₂ , 40.1 mg / ml BSA, 0.3 mg / ml phosphatidylserine, 0.03 mg / ml diacylglycerol, 2.5 μCi [γ- ³² P] ATP (100 μM ATP), 50 μM neurogranin] and 0.3 μl of purified protein kinase C (PKC, Promega) and the compound of formula 1 or formula 2 according to the present invention were added according to the concentration, and distilled water was added to the final volume. The reaction was adjusted to 25 µl for 15 minutes at 30 ° C. Thereafter, 6 µl of 100% trichloroacetic acid was added to the reaction solution, left for 40 minutes on ice, centrifuged at 10,000 x g for 15 minutes, and 10 µl of the supernatant was dipped onto p-81 paper. The paper was washed three times for 15 minutes with 75 mM phosphoric acid solution and dried, and then radioactivity was measured with a liquid scintillation counter.

The inhibitory effect of various seryl-threonine protein kinases through the experiments 1) to 4) is shown in Table 4 and FIGS. 1 and 2 .

Inhibitory Effect on Serine-Threonine Protein Kinase Kinase IC ₅₀ (μM) L-Xylose Derivatives (12) L-Xylose Derivatives (16) cdc2 2.2 2.4 PKA 70.0 21.0 Casein Kinase II 319.6 55.5 PKC 3728.2 86.0

As can be seen in Table 4 , the IC ₅₀ for the cdc2 kinase of the L-xylose derivative ( 12 ) and L-xylose derivative ( 16 ), which are the compounds according to the present invention, is 2.2 μM and 2.4 μM, respectively. In comparison, it was up to 1700 times lower. As described above, the compound according to the present invention selectively inhibits activity against cdc2 kinase related to the cell cycle, and thus may be usefully used as an anticancer agent by a selective cell cycle regulator inhibitor mechanism as well as a cell cycle regulation mechanism.

Experimental Example 2 Cancer Cell Growth Inhibition Effect

In order to determine whether the compounds according to the present invention inhibit the growth of cancer cells, in vitro experiments were carried out by MTT assay method which is generally well known using SK-HEP-1 cells of Preparation Example 4.

The cells were diluted to 5 × 10 ⁴ cells / 200 μl per well in a 96-well plate and incubated overnight in 5% bovine serum / DMEM. The next day, the medium was removed and DMEM added with the compound of Formula 1 or Formula 2 by concentration, and the control drug was added without the test drug. 50 μl of MTT (3- [4,5-Dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide) reagent was added to the culture solution, followed by further incubation for 4 hours. After 4 hours, the supernatant was completely removed, 100 μl of DMSO (dimethylsulfoxide) was added to dissolve formazan formed in the culture solution, and the absorbance at 595 nm was measured with an ELISA reader. The results are shown in FIGS . 3 and 4 .

As a result of the above experiment, the compound according to the present invention showed an effect of inhibiting the proliferation of cancer cells. In particular, as shown in FIGS. 3 and 4, the IC 50 of the L-xylose derivative ( 12 ) and the L-xylose derivative ( 16 ) is The cancer cell proliferation effect was particularly excellent at 60 μM and 3 μM, respectively.

Experimental Example 3 DNA Synthesis Inhibitory Effect of Cancer Cells

In order to determine whether the compounds of the present invention inhibit the DNA synthesis of cancer cells, the following experiment was conducted.

SK-HEP-1 cells of Preparation Example 4 were diluted with 1 × 10 ⁵ cells / ml in a 24-well plate with [ ³ H] -thymidine and cultured overnight in 5% bovine serum / DMEM. ³ H was introduced into the DNA. The next day, the medium was removed and DMEM added with the compound of Formula 1 or Formula 2 by concentration, and the control drug was added without the test drug. After incubation for 24 hours again, the supernatant was completely removed by micro pipette, fixed with 0.5 ml of methanol and washed with phosphate buffer solution (PBS). 500 µl of cold 10% trichloroacetic acid was added to the reaction solution, and the mixture was allowed to stand on ice for 30 minutes, and 170 µl of 0.2 N NaOH-0.5% SDS solution was added thereto, and dissolved at 37 ° C for 30 minutes. After adding 34 μl of 1N HCl to the reaction solution, 102 μl of the dissolved solution was taken into a 1.5 mL tube, and 1 ml of a cocktail solution was added to the solution, followed by vigorous shaking. After the solution was put a 10 ㎕ on P-81 paper buried dried to wash the paper with a 75 mM phosphoric acid solution for 15 minutes three times were measured for ³ H radioactivity introduced in the DNA in a liquid scintillation counter, 5, and the results 6 is shown.

As a result of the experiment, when the compound according to the present invention was added, the thymidine introduction rate was significantly reduced compared to the control group, and it was confirmed that there was an effect of inhibiting DNA synthesis of cancer cells. In particular, as can be seen in Figures 5 and 6 , the IC50 of L-xylose derivative ( 12 ) and L-xylyl derivative ( 16 ) was 60 μM and 30 μM, respectively, which was particularly excellent in inhibiting DNA synthesis of cancer cells.

Experimental Example 4 Growth Inhibition Effect of Cancer Cells by Morphological Analysis

In order to determine the growth inhibition effect of cancer cells by the compound of the present invention, the following morphological analysis was performed.

SK-HEP-1 cells of Preparation Example 4 were treated with 60 μM of the compound of Formula 1 or Formula 2 according to the present invention, followed by a microscope with a Kodak iso400 film after 8, 16 and 24 hours in a 60 φ dish. I took a picture. As a control, a test drug was not added. Results of the L-xylose derivative ( 12 ) and L-xylose derivative ( 16 ) among the compounds of the present invention are shown in FIGS . 7 and 8 , respectively.

As can be seen in Figures 7 and 8, in the case of the control group, many new blood vessels were generated over time, but when the compound of the present invention was added, there was a marked difference in the cell morphology from the control group and the apoptosis effect (apoptosis) It was confirmed that there is.

Experimental Example 5 DNA Fragmentation

When apoptosis occurs and cancer cells die, DNA fragmentation of cancer cells occurs. Thus, DNA fragmentation experiments were conducted to determine whether the compounds of the present invention have an apoptosis effect on cancer cells.

SK-HEP-1 cells of Preparation Example 4 were treated with a compound of Formula 1 or Formula 2 according to the present invention at each concentration, incubated for 24 hours, harvested and centrifuged at 1000 rpm for 5 minutes. The supernatant was removed and the remainder washed twice with PBS, followed by DNA separation buffer [50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.5% SDS, 0.5 mg / ml proteinase K]. Cells were lysed. Thereafter, the mixture was extracted once with a phenol / chloroform (1: 1) mixed solution and twice with chloroform / isopentanol (24: 1). An aqueous sodium chloride solution was added to bring the final concentration to 0.5 L, and two volumes of ethanol were added to precipitate. Electrophoresis was performed on 1.8% agarose gel to isolate quantitated DNA, stained with EtBr, and photographed on a UV camera. Results of L-xylose derivative (12) and L-xylose derivative (16) among the compounds of the present invention are shown in FIGS . 9 and 10 .

As a result of the experiment it was confirmed that the DNA ladder appears when the compound of the present invention is added. In particular, as shown in FIGS. 9 and 10, in the case of L-xylose derivative (12) and L-xylose derivative (16), DNA ladders appeared at 100 μM and 30 μM, respectively.

Experimental Example 6 Expression Analysis of Cell Cycle Regulatory Protein by Immunoblot

Immunoblot was performed using antibodies against proteins known to play an important role in animal cell cycle regulation to investigate how the expression of each protein was altered by the compounds of the present invention.

SK-HEP-1 cells of Preparation Example 4 were treated with a compound of Formula 1 or Formula 2 according to the present invention at each concentration, and the IP buffer [50 mM Tris-Cl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 1 % Triton X-100, 1 mM PMSF), and then quantified by extracting the protein by BCA method.

30-50 μg of quantified protein was isolated on 10-15% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF). The membranes were washed with TBS-T buffer (50 mM Tris-HCl, PH7.5, 150 mM NaCl, 0.05% Tween 20) containing 5% non-fat dried milk to produce a non-specific binding site. Was inhibited and washed again with TBS-T buffer. CDK2, p21, p27, proliferating cell nuclear antigen (PCNA), poly (ADP-ribose) polymerase (PARP), caspase-3 and the like are known as antibodies of proteins involved in apoptosis. Incubated overnight in TBS-T buffer containing% skim milk powder and the secondary antibody (polyclonal antibody or monoclonal antibody) was incubated for 1 hour. After incubation, the membrane was reacted with ECL-plus reagent (Amersham) and exposed to X-ray film to observe the expression pattern of proteins attached to the membrane. The results for the L- xylose derivative (16) of the compounds of the invention are shown in FIGS.

As a result of this experiment, the protein amount of various enzymes involved in apoptosis was reduced by the addition of the compound according to the present invention. In particular, as shown in FIGS . 11 and 12 , PARP showed a truncated form at a concentration of 3 μM of L-xylose derivative ( 16 ), and the protein amounts of p21 and p27 gradually decreased as the concentration of the test drug increased. , Caspase-3 also decreased gradually. Therefore, it was confirmed that the compounds of the present invention induce apoptosis in cancer cells and also participate in the cell cycle by reducing the amount of protein of PCNA.

Experimental Example 7 Oral Acute Toxicity in Rats

In order to determine the acute toxicity of the compound according to the present invention was carried out the following experiment.

Using a 6-week-old SPF SD rat, the compound of Examples 1-52, suspended in 0.5% methylcellulose solution in 2 animals per group, was orally administered at a dose of 1 g / kg / 15 ml. Administered. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, no significant clinical symptoms were observed in all animals administered the test substance, no animals died, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. These results the test compound does not represent a change in toxicity both to 1 g / ㎏ in rats orally minimum lethal dose (LD ₅₀₎ was determined to be a safe substance less than 1 g / ㎏.

Experimental Example 8 Anticancer Effect in Experimental Animal Model

Six mice (C3H / He mice, four-week old males) were allocated to one group and divided into five groups. MH134 hepatoma cells were injected into mice at 1 × 10 ⁵ cell counts / mouse into the abdominal cavity of mice to make a solid animal experimental animal model, and the test substance was administered from the following day.

L-Xylose derivatives ( 12 ) were suspended in 0.5% CMC-saline (carboxymethyl cellulose sodium-saline) and 50 mg / kg and 100 mg / kg, respectively, in the first and second test groups, respectively. 16 ) was orally administered to the third and fourth test groups daily for 4 weeks as described above. The fifth test group was orally administered 0.5% CMC- saline daily for 4 weeks as a control.

On the 35th day after the test substance administration, the solid cancer was extracted from the mouse and its weight was measured. The results are shown in Table 5 below.

Solid cancer proliferation inhibitory effect Test medication Dosage Tumor Weight (g) ^a Control - - 4.77 ± 0.67 Test group 1 L-Xylose Derivatives ( 12 ) 50 mg / kg 3.75 ± 0.21 ^b Test group 2 L-Xylose Derivatives ( 12 ) 100 mg / kg 2.85 ± 0.67 Test group 3 L-Xylose Derivatives ( 16 ) 50 mg / kg 3.81 ± 0.14 Test group 4 L-Xylose Derivatives ( 16 ) 100 mg / kg 2.80 ± 0.68 a: mean ± standard deviation b: p <0.05

As can be seen in Table 5, in the test group to which the L-xylose derivative (12) and the L-xylose derivative (16) were administered, solid cancers were significantly reduced compared to the control group, especially when administered at a concentration of 100 mg / kg. Statistically reliable results were obtained.

As such, the compounds according to the present invention have an effect of inhibiting the proliferation of cancer, and thus may be usefully used as anticancer agents.

As described above, the L-arabinose derivative represented by the formula (1), the L-xylose derivative represented by the formula (2), and salts thereof according to the present invention have excellent ability to inhibit cell cycle regulators of cancer cells and thus, the cell cycle. It can be usefully used as an anticancer agent as well as a regulator.

Claims (9)

L-arabinose derivatives represented by the following formula (1) and salts thereof. Formula 1 In Chemical Formula 1, R is H or a halogen element, X is CN, , , , , or ego, Y is NH ₂ or ═O, Q is N or NH, The bond between the carbon atom to which Y is bonded and Q is a single bond or a double bond. The compound of claim 1, wherein R is H or Br; X is CN, , , or ego; Y is NH ₂ ; Q is N; L-arabinose derivatives and salts thereof, wherein the bond between Y-bonded carbon atom and Q is a double bond. L-xylose derivatives represented by the following formula (2) and salts thereof. Formula 2 In Chemical Formula 2, R is H or a halogen element, X is CN, , , , , or ego, Y is NH ₂ or ═O, Q is N or NH, The bond between the carbon atom to which Y is bonded and Q is a single bond or a double bond. The compound of claim 3, wherein R is H or Br; X is CN, , , or ego; Y is NH ₂ ; Q is N; L-xyl derivatives and salts thereof, wherein the bond between Y-bonded carbon atom and Q is a double bond. The compound of claim 3 wherein 1) 4-amino-5-cyano-7- (2 ', 3', 5'-trihydroxy-β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine ( 12 ) ; 2) 4-amino-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamidine ( 14 ); 3) 4-amino-6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamide ( 16 ); 4) ethyl 4-amino-6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamide oxime.HCl ( 18 ); 5) 4-amino-6-bromo-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-thiocarboxamide ( 22 ); 6) 4-amino-5-cyano-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine ( 28 ); 7) 4-amino-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamide ( 30 ); 8) ethyl 4-amino-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-carboxamideoxime.HCl ( 32 ); And 9) 4-amino-7- (β-L-xylofuranosyl) pyrrolo [2,3-d] pyrimidine-5-thiocarboxamide ( 36 ) L-xyl derivative and its salt characterized by the above-mentioned. 1) tetracyanoethylene is reacted with HI in anhydrous solvent to produce tetracyanoethane; HBr gas was added to tetracyanoethane under anhydrous solvent to prepare 2-amino-5-bromo-3,4-dicyanopyrrole; Reacting 2-amino-5-bromo-3,4-dicyanopyrrole with formamidine to prepare base ( 3 ) (step 1, scheme 1); 2) reacting L-arabinose or L-xyl with HCl / methanol solution by adding thereto, reacting with pyridine, and then adding benzene chloride (BzCl) to react sugar derivatives ( 5 or 7 ); Reacting the sugar derivative ( 5 or 7 ) with acetic acid, acetic anhydride and concentrated sulfuric acid to prepare an L-type sugar derivative ( 6 or 8 ) (step 2, scheme 2 or scheme 3); 3) N, O-bis (trimethylsilyl) acetamide (BSA) is added to the base ( 3 ) of step 1 under anhydrous solvent and reacted with the L-type sugar derivative ( 6 or 8 ) and trimethylsilyl of step 2 Trifluoromethanesulfonate (TMSOTf) is added to react to prepare L-type sugar derivatives ( 9 or 11 ); Hydrolyzing the L-type sugar derivative ( 9 or 11 ) to prepare an L-arabinose derivative ( 10 ) or L-xylose derivative ( 12 ) (step 3, scheme 4 or scheme 5); And 4) optionally, by modifying the base moiety through an appropriate reaction using the L-arabinose derivative ( 10 ) or L-xylose derivative ( 12 ) of step 3 above as a starting material, the L-arabinose derivative of Formula 1 or Formula 2 Preparing L-Xylose Derivatives of (Step 4) A method of producing the L-arabinose derivative of claim 1 or the L-xylose derivative of claim 2. Scheme 1 Scheme 2 Scheme 3 Scheme 4 Scheme 5 The method of claim 6, wherein step 4 is performed by reacting the L-arabinose derivative ( 10 ) or L-xylose derivative ( 12 ) of step 3 with hydrogen peroxide (H ₂ O ₂ ) and ammonium hydroxide (NH ₄ OH). Substituent X of the base moiety A process for producing the L-arabinose derivative of claim 1 or the L-xylose derivative of claim 3. A pharmaceutical composition for an anticancer agent comprising the compound of claim 1 or 3 as an active ingredient. delete