KR0142110B1 - Production of 2:3:-dideoxy-2:3:-dihydronucleosides - Google Patents

Abstract

The present invention relates to an improved process for preparing 2 ', 3'-dideoxy-2', 3'-didehydronucleosides with ribonucleosides as starting material.

Description

Process for preparing 2 ', 3'-dideoxy-2', 3'-didehydronucleoside

The present invention relates to an improved process for preparing 2 ', 3'-dideoxy-2', 3'-didehydronucleosides.

AIDS is the result of infection with human immunodeficiency viruses (HIV).^(One)These retroviruses are helper / inducing factor T cells that consume them⁽²⁾Specific tropism for. The resulting immunosuppression leads to life-threatening opportunistic infections in HIV patients.

Although there is currently no cure for AIDS, 3'-azido-3'-deoxythymidine (AZT, Retrovir^TM) Has already been shown to be an effective drug for treating AIDS in clinical trials and has been recognized as a suitable modulator useful for AIDS patients.⁽³⁾Many other chemical and biological agents have been reported to be biologically active against HIV. 2 ', 3'-dideoxycytidine (ddC), 2', 3'-dideoxyadenosine (ddA)⁽⁴⁾, 2 ', 3'-dideoxy-2', 3'-didehydrocytidine (d4C)⁽⁵⁾, Suramin and the like⁽⁶⁾, Rivabarin⁽⁷⁾, Poscanet⁽⁸⁾, HPA-23⁽⁹⁾, d-penicillamine¹⁰, Castanospermine⁽¹¹⁾, Fusidic acid⁽¹²⁾, 3'-azidoguanosine (AZG)⁽¹³⁾And 3'-fluoro-3'-deoxythymidine (FDDT)⁽¹⁴⁾Are all reported to be active against HIV.

2 ', 3'-dideoxy-2', 3'-didehydrothymidine (d4T) has been reported in the literature as having in vitro activity against HIV in several cell lines ⁽¹⁵⁾ .

Horwitz et al. Produced 2 ', 3'-dideoxy-2', 3'-didehydrothymidine (d4T) via two different routes ^{(16) (17)} . The first synthetic route is to add 3 ', 5'-anhydro derivatives of thymidine to the removal reaction conditions. The second synthetic route is to subject the 5'-O-protected 2,3'-anhydro nucleoside derivative of thymidine to the ring opening elimination reaction conditions.

The use of anhydronucleosides as intermediates for synthesizing conventional nucleosides is well known in the art ⁽¹⁸⁾ .

As the efficacy of 2 ', 3'-dideoxy-2', 3'-didehydrothymidine (d4T) as an anti-HIV agent has recently been discovered, 2 ', 3'-dideoxy-2', including d4T, There is a growing importance for methods to produce 3'-didehydro-nucleosides in bulk inexpensively.

The Horzwitz route for preparing d4T from 3 ', 5'-anhydrous compound ⁽¹⁶⁾ is not feasible on a large scale because it is very difficult to completely remove the large amount of DMSO used in the Horbitz method and it is highly This is because a vacuum state of (0.01 mmHg and heating to a temperature range of about 40 ° C to 50 ° C) is required. Under these conditions, glycosidic bonds cleave to produce thymine, an unnecessary byproduct. In addition, prolonged exposure to the basic conditions required when using solvents other than DMSO (eg, THF, DMF) results in the degradation of d4T again to produce thymine, an unnecessary byproduct.

In a separate Horbitz method, the 5'-OH position must be protected before the 2,5'-anhydro nucleoside is formed. Such 2,5'-anhydro nucleosides can be ring-opened to form 5'-0-protected nucleosides.

Preferred 2,3'-anhydro nucleosides can be prepared directly by reacting thymidine with diethyl- (2-chloro-1,1-2-trifluoroethyl) amine ⁽¹⁹⁾ .

The present invention relates to a process for producing 2 ', 3'-dideoxy-2', 3'-didehydronucleoside represented by the following formula (1) in high yield and on a relatively large scale.

According to one feature of the present invention, the method of the present invention comprises (a) reacting a 2'-deoxynucleoside represented by the following formula (3) with a reactive 3 ', 5'-anhydro-2' Converting to a deoxynucleoside intermediate and (b) reacting the reactive 3 ′, 5′-anhydro-2′-deoxynucleoside obtained from step (a) in the presence of a strong base with the 2 ′, 3 Preparation of 2 ', 3'-dideoxy-2', 3'-didehydronucleoside represented by the following Chemical Formula 1, comprising converting to '-dideoxy-2', 3'-didehydronucleoside In the method,

(i) the 3 ', 5'-anhydro-2'-deoxynucleoside is selected from KOtBu, nBuLi, NaH, and LDA in the presence of a polar solvent selected from DMSO, THF, DMF, DME and mixtures thereof Reacting with a strong base;

(ii) triturating the resulting salt in the presence of an organic solvent;

(iii) collecting the crude crude salt intermediate obtained in step (ii) above;

(iv) dissolving the salt obtained in step (iii) in water;

(v) neutralizing the salt obtained in step (iv); And

(vi) A process for preparing 2 ', 3'-dideoxy-2', 3'-didehydronucleoside represented by the following formula (1), characterized by performing a step of obtaining a solid nucleoside free base product. It is about.

Formula 1

In each said formula,

The base moiety is selected from the group of unsubstituted and unsubstituted bases consisting of pyrimidine, aza-pyrimidine and deaza-pyrimidine;

X is selected from N and C-H;

Y is selected from CR ⁵ and N;

Z is selected from C-H and N;

R ⁴ is selected from OH and NH ₂ ;

R ⁵ is H, unsubstituted and halo substituted alkyl of formula C _n H _2n A and formula-(CH ₂ ) _m -CH = CHA wherein m is an integer selected from 0, 1, 2 and 3, n is Is an integer selected from 1, 2 and 3, and A is selected from H, F, Cl, Br and I.) and unsubstituted and halo substituted alkenyl.

According to another feature, the present invention provides a reactive 2,3'- anhydro-2'- deoxynucleoside intermediate represented by the following formula (a) 2'-deoxynucleoside represented by the following formula (3) Switching to;

(b) reacting the reactive 2,3'-anhydro-2'-deoxynucleoside obtained in step (a) in the presence of a base selected from non-nucleophilic and nucleophilic bases with the 2 ', 3'-dideoxy- Method for preparing 2 ', 3'-dideoxy-2', 3'-didehydronucleoside represented by the following Chemical Formula 1, comprising converting to 2,3'-didehydronucleoside It is about.

Formula 1

Formula 3

In each said formula,

The base moiety is selected from the group of substituted and unsubstituted bases consisting of pyrimidine, aza-pyrimidine and deaza-pyrimidine;

X is selected from N and C-H;

Y is selected from CR ⁵ and N;

Z is selected from C-H and N;

R ⁴ is selected from OH and NH ₂ ;

R ⁵ is H, unsubstituted and halo substituted alkyl represented by the formula C _n H _2n A and formula C _n H _n A (wherein n is an integer selected from 1, 2, and 3, and A is H, F, Cl , Br, and I), and unsubstituted and halo substituted alkenyl.

According to another feature, the present invention provides a method for preparing a reactive intermediate of formula (7), which comprises: (a) reacting a starting material ribonucleoside of formula (6) with trimethyl orthoformate in anhydrous conditions in the presence of a polar solvent;

(b) removing the intermediate obtained in step (a) by treating with p-TsOH at a high temperature of about 120 to 160 ° C. for about 4 to 8 hours; And

(c) treating the resulting 5'-OAC group under mild base hydrolysis conditions to deprotect it, wherein the 2 ', 3'-dideoxy-2', 3'- It relates to a process for preparing didehydronucleosides.

In each said formula,

B is selected from the group of bases consisting of purine, aza-purine, deaza-purine, pyrimidine, aza-pyrimidine, deaza-pyrimidine and triazole ring bases.

According to another feature, the present invention provides a process for reacting (a) a ribonucleoside of formula (6), which is a starting material, with a hydroxy protecting group reactant that is effective to selectively protect a 5′-hydroxyl (primary hydroxyl) group. step,

(b) reacting the 5′-OH-protected ribonucleoside obtained from step (a) with 1,1-thiocarbodiimidazole and thiophosgene under anhydrous conditions to obtain a reactive intermediate of formula ;

(c) removing and reacting the intermediate obtained in step (b) with P (OEt) ₃ in a polar solvent at about 140 to 175 ° C. for 30 minutes to 4 hours; And

(d) treating the resulting 5′-O-protecting group under mild acid hydrolysis conditions to deprotect the 2 ′, 3′-dideoxy-2 ′, 3 It relates to a process for the preparation of '-didehydronucleosides.

Formula 2

Formula 6

In each said formula,

B is selected from the group of bases consisting of purine, aza-purine, deaza-purine, pyrimidine, aza-pyrimidine, deaza-pyrimidine and triazole ring bases.

According to another feature, the present invention provides a process for preparing acyloxyisobutyryl bromide in a polar solvent for (a) a ribonucleoside of formula (6) as starting material for about 1 to 3 hours at an elevated temperature of about 75 to 100 ° C. under anhydrous conditions. Preferably, reacting with 2-acetoxy isobutyryl bromide to obtain a reactive intermediate of formula (9);

(b) treating the intermediate obtained in step (a) with a Zn / Cu reactant in an aprotic polar solvent to remove and react to obtain an intermediate of Formula 10; And

(c) treating the intermediate obtained in step (b) with a mild base, preferably methanolic ammonia, to deprotect the 5'-0-protecting group of the intermediate to obtain 2 ', 3'-dideoxy-2', 3 It relates to a method for preparing 2 ', 3'-dideoxy-2', 3'-didehydronucleoside of the formula (2), comprising the step of obtaining a '-didehydro nucleoside derivative.

Formula 2

Formula 6

In each said formula,

B is selected from the group of bases consisting of purine, aza-purine, deaza-purine, pyrimidine, aza-pyrimidine, deaza-pyrimidine and triazole ring bases;

R is an acyloxyisobutyryl group,

R 'is an acyl group of acyloxyisobutyryl bromide.

As described above, the present invention provides that the starting material is 2'-deoxynucleoside and the base component B is substituted or unsubstituted pyrimidine, or aza-pyrimidine or deaza-pyrimidine, preferably substituted or unsubstituted. Two specific examples of methods for preparing 2 ', 3'-dideoxy-2', 3'-didehydronucleosides derived from bases selected from the group of bases consisting of substituted pyrimidines are described. In these two specific examples, the base moiety is more preferably a substituted or unsubstituted pyrimidine corresponding to the descriptions and formulas described below in connection with appropriate substituted and unsubstituted pyrimidine bases. In these two specific examples, the base moiety is thymine (5-methyl-2,6-dihydroxy-pyrimidine), cytosine (2-hydroxy-6-aminopyrimidine), uracil (2,6-di Hydroxy-pyrimidine), 5-ethyl-2,6-dihydroxypyrimidin-3-yl, 5-vinyl-2,6, -dihydroxypyrimidin-3-yl, 5-halovinyl-2 , 6-dihydroxypyrimidin-3-yl, 5-halomethyl-2,6-dihydroxypyrimidin-3-yl and 5-haloethyl-2,6-dihydroxypyrimidin-3-yl It is more preferable to select from among. In these two specific examples it is most preferred that the base moiety is thymine.

As mentioned above, the starting materials of the invention are ribonucleosides and the base component B is substituted and unsubstituted purine, aza-purine, deaza-purine, pyrimidine, aza-pyrimidine, deaza-pyrimidine and tria Three embodiments of a method for preparing 2 ', 3'-dideoxy-2', 3'-didehydronucleosides derived from a base selected from the group of bases consisting of sol ring bases. The base is preferably selected from purine and pyrimidine bases. More preferably, the base is a pyrimidine base comprising one of uracil and thymine groups.

Suitable substituted and unsubstituted purine bases include purine bases represented by the formula:

In the above formula,

R ¹ and R ² are the same or different and are hydrogen, hydroxy, halo (F, Cl, Br), amino, monoalkylamino, dialkylamino, alkoxy and cyano groups, wherein the alkyl moiety is C ₁ to C ₃ It is selected from an alkyl group.

Suitable substituted and unsubstituted pyrimidine bases include pyrimidine bases represented by the formula:

In the above formula,

R ³ is selected from hydroxy, amino and sulfhydryl groups;

R ⁴ is selected from hydroxy, amino and sulfhydryl groups, or

R ⁴ is hydrogen;

R ⁵ is selected from hydroxy and amino;

R ⁶ is hydrogen, C ₁ -C ₁ alkyl, C ₂ -C ₃ alkenyl, C ₂ -C ₃ haloalkenyl, C ₂ -C ₃ alkynyl, alkyl having 1 to 5 halo groups as defined above The moiety is selected from alkoxy, cyano and halo (F, Cl, Br and I) with C ₁ -C ₃ .

Representative examples of B, when derived from a purine base, are 6-aminopurin-9-yl, 2-aminopurin-9-yl, 2,6-diaminopurin-9-yl, 2-amino-6-hydroxy Purin-9-yl (guanine-9-yl) and 6-hydroxypurin-9-yl.

In addition to the above examples, component B includes 2-halopurin-9-yl, 6-halopurin-9-yl, or 2,6-dihalopurin-9-yl, in which case step (e) It is not necessary to activate the base component, for example complete silylation, in order to cause a condensation or binding reaction in

When derived from pyrimidine bases, representative examples of B include 2,4-dihydroxypyrimidin-1-yl, 5-methyl-2,4-dihydroxypyrimidin-1-yl, 5-ethyl-2 , 4-aminopyrimidin-1-yl, 2-hydroxy-4-aminopyrimidin-1-yl, 5-vinyl-2,4-dihydroxypyrimidin-1-yl, 5-halovinyl-2 , 4-dihydroxypyrimidin-1-yl, 5-haloethyl-2,4-dihydroxypyrimidin-1-yl and 5-haloethyl-2,4-dihydroxypyrimidin-1-yl Can be mentioned.

Representative examples of the foregoing 5-methyl and 5-ethyl substituents are 5-alkyl substituents, and representative examples of the 5-vinyl substituents are 5-alkenyl substituents. Examples of halo groups on the 5-halovinyl (or 5-haloalkenyl) groups include 1-4 F, Cl, and Br groups.

In a first embodiment of the process of the invention, in its first step an intermediate 3 ', 5'-anhydro-2'-deoxynucleoside is prepared from the starting material 2'-deoxynucleoside. do. Such known intermediates are reacted with a corresponding amount of conventional activating hydroxy protecting group reactant, e.g., MsCl or TsCl, under conventional conditions to the first intermediate 3 ', 5'-0. After obtaining protecting group-2′-deoxynucleoside, the first intermediate is prepared by reacting a strong base selected from KOH and NaOH in a solvent selected from water and ethanol. Although known activated hydroxy protecting group reactants useful in the art related to the present invention may be used, mesyl chloride is most preferred according to the method of Horwitz et al.

In the second step of this specific example, the H atom removal reaction takes place in the presence of a strong base and in the presence of a polar solvent as is known, whereby the 3 ', 5'-anhydro ring is ring-opened, and the 2'-ene Double bonds are formed to form the preferred 2 ', 3'-dideoxy-2', 3'-didehydronucleosides.

In a first specific example of the process according to the invention described above, the invention is characterized by an improvement in the selection, handling and treatment of reactants, intermediates and products. This feature includes the following steps.

(i) the 3 ', 5'-anhydro-2'-deoxynucleoside is prepared in the presence of a polar solvent selected from DMSO, THF, DMF, DME, and mixtures thereof, preferably about 18-80 ° C. Reacting with a strong base selected from KOtBu, nBuLi, NaH, and LDA at a temperature in the range, more preferably about 18-22 ° C .;

(ii) milling the resulting salt in the presence of an organic solvent;

(iii) collecting the crude solid salt intermediate of step (ii);

(iv) dissolving the salt of step (iii) in water;

(v) neutralizing the salt of step (iv); And

(vi) obtaining a solid nucleoside free base product.

In addition to the features in step (b) as described above, in step (a), the preferred intermediate precipitate is subjected to filtration by reducing and neutralizing the amount of solvent upon KOH addition and then concentrating the slurry to about 20% of its original volume. While the byproduct KCl salt remained in the solvent. This eliminated the need for recovering the reactive intermediates by treating the fully evaporated reaction mixture of step (a) with hot acetone.

Another alternative for preparing preferred 2 ', 3'-dideoxy-2', 3'-didehydronucleosides from the corresponding 2'-deoxynucleosides is to use the corresponding ribonucleoside as starting material. . Thus, the inventors have found that treatment of uridine with trimethyl orthoformate can yield the corresponding ortho ester reactive intermediates.

In step (ii) of the first embodiment, as the solvent, any organic solvent compatible with the reactant and the intermediate salt formed in step (i) can be used. The solvent is preferably selected from toluene, acetone, and ethyl acetate, with the most preferred solvent being toluene. The temperature during the grinding process in step (ii) is preferably about 0 ° C. to 10 ° C., and most preferably 0 ° C. to 4 ° C.

In a second embodiment of the process of the invention, the first step is to form the corresponding starting material 2'-deoxynucleoside to form 2,3'-anhydro-2'-deoxynucleoside. Known reactive 2,3'-anhydro-2'-deoxynucleoside intermediates are prepared by reaction with an effective strong base. Suitable reactants for carrying out this ring forming step are the known reactants, diethyl (2-chloro-1,1,2-trifluoroethyl) amine. Such known intermediates were reacted with nucleophiles to obtain substituted nucleosides such as, for example, 3'-azido-2 ', 3'-dideoxythymidine (AZT). ²⁰

In the second step of this embodiment, the ring opening of the anhydrocyclic ring of the reactive intermediate is carried out, not the nucleophilic addition reaction. Suitable reactants for carrying out this ring opening reaction are non-nucleophilic bases such as tetrabutyl ammonium fluoride or nucleophilic bases selected from KOtBu, NaOH, KOH and the like.

In a third embodiment of the process of the invention, in the first step, the starting material ribonucleoside is reacted with trimethylortho formate in a polar solvent under anhydrous conditions ^(21,22) to represent an ortho ester reactivity represented by the following formula Obtain an intermediate.

Formula 7

In the next step, the reactive intermediate is removed and reacted by treatment with an organic acid such as p-TsOH in Ac ₂ O at a high temperature of about 120 to 160 ° C. for about 4 to 8 hours ⁽²³⁾ . Finally, the last step is deprotection by treating the 5'-OAc group of the ortho ester reactive intermediate under mild base hydrolysis conditions ^(15b) .

In a fourth embodiment of the process of the invention, the first step selectively protects the starting material ribonucleoside with a 5'-hydroxy group (i.e., a primary hydroxyl group that is distinct from a sugar-ring-binding secondary hydroxyl group). React with one of any conventional hydroxy protecting groups effective for In the second step, a reactive thiocarbonate intermediate represented by the following formula (8) is reacted by reacting 5′-O-protected ribonucleoside with one of 1,1-thiocarbonyldiimidazole and thiophosgene under anhydrous conditions. Get:

Formula 8

In the next step, the intermediate is removed by treating the reactive intermediate with P (OEt) ₃ in a polar solvent at a high temperature of about 140 to 175 ° C. for 30 minutes to 4 hours. Finally, in the fourth and final step, the 5'-O-protecting group of the reactive thiocarbonate intermediate is deprotected by treating it under mild acid hydrolysis conditions.

In a final fifth embodiment of the process of the invention, in the first step, the starting material ribonucleoside is reacted with 2-acetoxyisobutyryl bromide ⁽²⁴⁾ to obtain a reactive intermediate represented by the following formula (9 ⁾ .

Formula 9

Subsequently, the Zn / Cu reactant and the reactive intermediate mixture are reacted in an aprotic polar solvent to carry out the elimination reaction to obtain the desired 2 ', 3'-dideoxy-2', 3'-didehydronucleoside product. ²⁵ .

Scheme 1 below schematically illustrates a representative process according to the present invention. Route (A) illustrates the first embodiment, which proceeds via the reactive intermediate 3 ', 5'-anhydro, or "oxetane". Route (B) illustrates a second embodiment, which proceeds through the 2,3'-anhydro reactive intermediate. Route (C) describes the third and fourth embodiments, which proceed via ortho ester or thiocarbonate reactive intermediate, respectively, with ribonucleoside as starting material. Route (D) is a fifth sphere that proceeds via 3′-O-acetyl-2′-bromo-2′-deoxy ribonucleoside and / or 3′-bromo-2′-O-acetyl intermediate An example is explained.

Path (A):

Path (B):

Path (C):

Path (D):

The problems raised when trying to implement the so-called “oxetane” pathway by Horwitz et al., Which proceed through the 3 ', 5'-anhydro intermediates, are mainly related to large-scale final elimination reactions. When a large amount of the solvent is removed, a thymine removal reaction occurs because it is exposed to heat for a long time. The use of higher bases also results in unnecessary thymine.

A particular refinement of this “oxetane” route, which constitutes one specific example of the present invention, involves (1) the reaction under more concentrated conditions by using much less water to convert mesylate to oxetane. And (2) modifying the procedure of the final process to precipitate potassium salts from the final process by trituration, including KOtBu / DMSO treatment of the reactive intermediates. In addition, the procedure of step (a) was simplified by neutralizing the reaction solution, reducing the amount of water and collecting the product. This method is much easier than completely removing the water and suspending the formed salt in hot acetone, filtering and then washing the filtrate. This enables the collection, neutralization and recollection of the product. The advantage obtained by the present invention over the methods described in the prior art is that there is no need to remove large amounts of DMSO under vacuum, and that the product can be avoided from being destroyed by the long-term heating required when removing DMSO under vacuum. Is the point. As a result of these features, large quantities of relatively pure product are obtained.

The problem of the above-described method of preparing the desired 2 ', 3'-dideoxy-2', 3'-didehydronucleoside via the 2,3'-anhydroreactive intermediate is difficult to prepare It is to use diethyl (2-chloro-1,1,2-trifluoroethyl) amine, which is a kind of fluoroamine reactant requiring equipment. As an alternative, the document reports a long time four step process including 5'-0-tritylation, 3'-0-mesylation, detritylation and anhydrous formation. The document also reports on the production of various products and by-products other than the preferred 2 ', 3'-dideoxy-2', 3'-didehydronucleoside products.

Certain refinements performed in the above known process, which proceed through the 2,3'-anhydroreactive intermediate, utilize tetrabutyl ammonium fluoride (TBAF) under non-nucleophilic conditions to produce the desired product in high yield and high purity. It includes the discovery that it can be prepared by. As an alternative, use of NatN / DMF or DBU / DMF or KOtBu / DMSO and NaOH / DMF rather than NaOH / MeOH or KOtBu / BuOH in place of TBAF in THF or DMF yields the desired product, but yields are low, NaOH / DMF Some unwanted unwanted byproducts (3'-epi-thymidine) are produced.

Thus, the method according to the invention has a number of 2 ', 3'-dideoxy-2', 3'-didehydronus having antiviral, anti-metabolic and anti-tumor activity as well as activity against human immunodeficiency viruses. Useful for preparing cleosides, in particular pyrimidine and purine nucleosides.

The following examples illustrate some representative embodiments of the methods of the present invention and are presented to teach those skilled in the art how to practice the present invention, and the scope of protection of the present invention is not limited to these examples. Unless otherwise noted, all parts and percentages are by weight and temperatures are in degrees Celsius.

Biological data including anti-HIV data of d4T prepared by the method of the present invention are shown in Table I below. These data are identical to the published data.

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(21). Davidson, V. J., Davis, D. R., Dixit, V. M.; Mr. Poulter. D. J. Org. Chem. 1987, 52, 1794.

(22). Griffin, rain. this.; But, J .; Reese, mr. ratio.; Schulston, Jay. Tetrahedron, 1967, 23, 230.

(23). Ando, M .; O'Hara, H .; Takase, K. Chem. Lett., 1986, 879.

(24). Jane, T. Seed.; Jenkins, child. D.; Rush, F .; Burden, J. P. H .; Mopart, Jay. H., J. Org. Chem., 1974, 39, 80.

Experiment

Melting points were measured with an electronic heated capillary device and were not corrected. TLC has this. Column chromatography was performed on silica gel 60F-254 plates purchased from Merck and Company, and column chromatography was performed on flash silica gel (particle size 40 μΜ, Baker). Elemental analysis was performed in Bristol-Meyers' analysis at Wallingford. ¹ H and ¹³ C NMR spectra were recorded on an AM360 Bruker NMR spectrometer using tetramethylsilane as internal standard. Chemical shifts are reported in ppm. HPLC analysis was performed on a Waters C18 reversed phase column.

3 ', 5'-di-O- (methanesulfonyl) thymidine

A 3-liter three necked round bottom flask was equipped with a top stirrer and paddle, a 500 mL dropping funnel and a Klysene adapter with drying tube and thermometer. To the flask was added thymidine (200 g, 0.82 M) and pyridine (750 mL). The mixture was stirred and warmed in a water bath (20 minutes) to give a clear solution. Subsequently, the solution was cooled to 0 to 3 ° C. in an ice bath, and then methanesulfonyl chloride (206.5 g, 1.08 M) was added to the dropping funnel. Subsequently, methanesulfonyl chloride was added dropwise for 40 minutes, at which time no exothermic phenomenon was observed. The solution was stirred at 0 ° C. for 1 hour and then stored at 5 ° C. for 18 hours. Then the light brown mixture was added to rapid stirred water (3 L) containing ice (about 500 g). The desired product immediately crystallized. After stirring for 30 minutes, the product was collected by filtration and washed several times with water (3 times in 100 mL). The white solid was then dried under vacuum overnight (crude weight 322 g, yield 98%). The product was recrystallized from hot acetone to give 267 g of a white solid (yield 81%). mp: 169-171 degreeC (document description: 170-171 degreeC).

¹ H NMR (360 MHz, d ₆ -DMSO) 11.40 (s, 1H, NH), 7.50 (s, 1H, H-6), 6.21 (t, 1H, H-1 '), 5.29 (m, 1H, H-3 '), 4.45 (m, 2H, H-5'), 4.35 (m, 1H, H-4 '), 3.31 (s, 6H, SO ₂ CH ₃ ), 2.50 (m, 2H, H- 2 '), 1.78 (s, 3H, CH ₃ ).

Elemental Analysis (C ₁₂ H ₁₈ N ₂ O ₉ S ₂ ) C, H, N.

1- (3,5-Anhydro-2-deoxy-β-D-threo-pentofuranosyl) thymine

To the stirred solution was dissolved sodium hydroxide (74.7 g, 1.87 M) in water (1.6 L) and 3 ', 5'-di-O- (methanesulfonyl) thymidine (248 g, 0.62 M) was added in portions. . Upon addition, the reaction mixture turned into a yellow-orange solution. The stirred solution was then heated to reflux for 2 hours. Once the reaction mixture was cooled to room temperature, 6 N hydrochloric acid (100 mL) was added. 1.3 L of water was removed and the reaction mixture was concentrated in vacuo. The resulting slurry was cooled in an ice bath for 2 hours. The solid was then filtered, washed slightly with ice water and vacuum dried until constant weight (103.7 g, 74%). The product (3) having a melting point of 188 ° C to 190 ° C (document literature, 190 ° C to 193 ° C) was used without further purification.

¹ H NMR (360 NHz, d ₆ -DMSO) 11.35 (s, 1H, NH), 8.01 (s, 1H, H-6), 6.49 (q, 1H, H-1 '), 5.47 (m, 1H, H-3 '), 4.88 and 4.67 (m, 2H, H-5'), 4.22 (d, 1H, H-4 '), 2.47 (m, 2H, H-2'), 1.77 (s, 3H, CH ₃ ).

¹³ C NMR (75 MHz, d ₆ -DMSO) 163.64 (C2), 151.10 (C4), 136.57 (C6), 109.62 (C5), 88.29 (C4 '), 86.85 (C1'), 79.83 (C3 '), 75.14 (C5 ′), 37.17 (C2 ′), 12.33 (CH ₃ ).

Elemental Analysis (C ₁₀ H ₁₂ N ₂ O ₄ ) C, H, N.

1- (2,3-dideoxy-β-D-glycero-pent-2-enofuranosyl) thymine

Anhydrous DMSO (400 mL) and oxetane (90.0 g, 0.402M) were added to a 1 L three neck round bottom flask equipped with a mechanical stirrer, thermometer and nitrogen inlet. 97% KOtBu (74 g, 0.643 M) was added to the solution over 1.5 minutes at 1.5 g each. The temperature was maintained at 18 ° C. to 22 ° C. using an external ice bath. After the addition was complete, the reaction was stirred for an additional hour, with no further temperature rise observed. TLC analysis showed that the reaction was approximately 90% complete. The reaction solution was stirred at 21 ° C. for 16 hours, after which TLC analysis showed that the reaction was complete. The resulting viscous solution was injected into low temperature (4 ° C.) toluene (3 L), yielding a beige precipitate. The temperature of the mixture rose to 7 ° C. when DMSO solution was added. The compound was stirred intermittently over 20 minutes and then filtered through a 18.5 cm Buchner funnel. The collected yellow solid was washed twice with cold toluene and suction dried for 1 hour. Two layers formed as the solid was dissolved in 300 mL of water. The mixture was placed in a separating funnel to remove the upper layer (containing residual toluene). The aqueous layer was placed in a 1 L beaker equipped with a pH probe, magnetic stir bar and thermometer. The temperature was cooled to 10 ° C. using an external ice bath. Concentrated HCl was added dropwise to the stirred solution at a rate such that the temperature was maintained at 15 ° C. or lower. After addition of HCl (50.5 mL, 0.61 M), the pH was 7 ± 0.1 and a precipitate began to form. Potassium chloride (70 g) was added to this thick mixture and stirring continued at 5 ° C for 1 hour. The precipitate was collected, suction dried for 2 hours and then air dried for 16 hours. The resulting solid was completely triturated, slurried in hot acetone (500 mL) and filtered. The residue on the filter paper was washed with hot acetone (2 × 200 mL), then slurried using hot acetone (300 mL), filtered and washed again with hot acetone (100 mL each). Episode 2). The combined filtrates were concentrated to dryness to give 51.3 g (57%) of d4T as an off-white solid. mp 165-166 ° C. [α] ²⁰ _D -46.1 (c = O.7, water).

¹ H NMR (360 MHz, d ₆ -DMSO) 11.29 (s, 1H, NH), 7.63 (s, 1H, H-6), 6.80 (d, 1H, J = 1.2 Hz, H-1 '), 6.38 (d, 1H, J = 5.9 Hz, H-3 '), 5.90 (dd, 1H, J = 1.1, 4.7 Hz, H-3'), 5.01 (m, 1H, OH), 4.76 (s, 1H, H-4 '), 3.60 (dd, 2H, J = 4.8, 3.6 Hz, H-5'), 1.71 (d, 3H, J = 1.2 Hz, CH ₃ ).

¹³ C NMR (75 MHz, d ₆ -DMSO) 164.42 (C4), 151.30 (C2), 137.23 (C2 '), 135.36 (C3'), 126.35 (C6), 109.33 (C5), 89.15 (C1 '), 87.56 (C4 ′), 62.41 (C5 ′), 12.15 (C5CH ₃ ). MS m / e (methane DCI) (relative strength) 225 (M + H, 20), 207 (15), 193 (8), 155 (13), 127 (100), 90 (20).

IR (cm- ¹ ) 3463, 3159, 3033, 1691, 1469, 1116, 1093.

Elemental Analysis (C ₁₀ H ₁₂ N ₂ O ₄ ) C, H, N.

1- (2,3-dideoxy-β-D-glycero-pent-2-enofuranosyl) thymine

Tetrabutyl ammonium fluoride (0.22 mL, 0.22 mM, 1.0 M) was added to the suspension of anhydronucleoside (25 mg, 0.11 mM) in dry THF (3 mL). After stirring for 3 h at 22 ° C., TLC analysis showed only starting material. The mixture was heated to reflux for 18 hours at which time the reaction was found to be complete. After cooling, the solvent was removed in vacuo and the residue was dissolved in CH ₂ Cl ₂ / MeOH / NH ₄ OH (90: 10: 1). CH ₂ Cl ₂ / MeOH / NH ₄ OH (90: 10: 1) was purified on a 20 mm flash chromatography column using eluent. Fractions containing the product were concentrated to give 18 mg (72%) of d4T.

1- (5'-O-trityl-2 ', 3'-thiocarbonyllipofranosyl) uracil

5'-0-trityluridine (10.6 gm, 22 mM) was placed in a dry 250 mL round bottom flask under an argon atmosphere. Anhydrous tetrahydrofuran (110 mL) was added and stirred until the reaction mixture became homogeneous. When 1,1-thiocarbonyldiimidazole (4.3 gm, 27 mM) was added to the solution, the reaction turned yellow. Then stirred at room temperature for 72 hours. The solvent was removed in vacuo and the resulting syrup was purified by flash chromatography on silica using ethyl acetate / hexane (75:25) as eluent. The product was separated and then recrystallized from anhydrous ethanol to give an off-white powder (0.8 g, 77%).

¹ H NMR (360 MHz, CDCl ₃ ) 8.9 (br s, 1H, NH), 7.3 (m, 16H, 3xC ₆ H ₅ , H6), 5.7 (d, 1H, H5), 5.6 (m, 2H, H2 ', H3'), 5.4 (m, 1H, H1 '), 3.4 (q, 2H, H5').

5'-O-trityl-2 ', 3'-dideoxy-2', 3'-didehydrouridine

1- (5'-O-trityl-2 ', 3'-thiocarbonylibofuranosyl) uracil (6.0 g, 11.5 mM) was added to triethyl phosphite (30 mL). The triethyl phosphite was preheated to 160 ° C. The reaction mixture was heated at 160 ° C. for 1 hour. The solvent was then removed in vacuo and the resulting glassy solid was purified by flash chromatography on silica using ethyl acetate / hexanes (75:25) as eluent. The desired product was separated from the column and then recrystallized in ethyl acetate / hexanes to collect as a white solid. (2.0 g, 40%). m.p. 188 to 191 ° C.

¹ H NMR (360 MHz, CDCl ₃ ) 8.95 (br 2, 1 H, NH), 8.00 (d, 1 H, H ₆ ), 7.5 (m, ₁₅ H, 3 × C ₆ H ₅ ), 7.2 (m, 1H, H 1 ′), 6.7 (m, 1H, H2 '), 6.05 (m, 1H, H3'), 5.2 (dd, 1H, H5), 5.10 (br s, 1H, H4 '), 3.6 (m, 2H, H5').

2 ', 3'-dideoxy-2', 3'-didehydrouridine (d4U)

5'-O-trityl-2 ', 3'-dideoxy-2', 3'-didehydrouridine (0.5 g, 1.1 mM) in chloroform (2 mL) containing 2% p-toluenesulfonic acid ) And methanol (2 mL). The reaction mixture was stirred at room temperature for 45 minutes and then neutralized with 2N of NaOH (0.5 mL). The solvent was removed in vacuo and the residue was purified by chromatography on silica using chloroform / acetone (2: 1) as eluent. The desired product was isolated as a white crystalline solid with the same physical and spectroscopic properties as d4U produced by other methods. m.p. 155 ° C.

¹ H NMR (360 MHz, D ₂ O / DMSO), 7.8 (d, 1H, H6), 6.7 (m, 1H, H1 '), 6.37 (m, 1H, H2'), 5.8 (m, 1H, H3 '), 5.56 (d, 1H, H5), 4.7 (m, 1H, H4'), 3.6 (m, 2H, H5 '). ¹³ C NMR (70Mhz, D ₂ O / DMSO) 163 (C4), 151 (C2), 141 (C2 '), 135 (C3'), 126 (C6), 101 (C5), 89 (C1 '), 87 (C4 '), 62 (C5').

2 ', 3'-methoxymethylideneuridine

Uridine (50 g, 205 M) was placed in a 1 liter round bottom flask under a nitrogen atmosphere. Quickly distilled anhydrous tetrahydrofuran (500 mL) and pyridinium p-toluene sulfonate (5 g, 20 mM) were added to the reaction mixture. Trimethyl orthoformate (109 g, 1.03 M) was then added slowly via addition funnel. The reaction mixture was stirred at room temperature for 18 hours during which time the reaction was homogenized. Water (18 g, 1 M) was added and the reaction further stirred for 30 minutes before pyridine (20 mL) was added. The reaction was stirred further for 18 hours at room temperature, then the solvent was removed in vacuo. The resulting white solid was purified by flash chromatography on silica to give the desired product as a white solid (40 g, 68%). m.p. 188-190 degreeC. (Documentary description, 189-190 degreeC).

5-O-acetyl-2 ', 3'-dideoxy-2', 3'-didehydrouridine

The methoxymethylidene compound (11.8 g, 41 mM) was dissolved in acetic acid (110 mL), then p-toluene sulfonate (20 mg) was added and heated to 140 ° C. for 6 hours. After cooling the reaction, triethylamine (1 mL) was added. The solvent was removed in vacuo and purified by chromatography on silica using chloroform / acetone (4: 1) as eluent to afford the desired product as a clear oily material.

¹ H NMR (360 MHz, DMSO), 11.3 (br s, 1H, NH), 7.4 (d, 1H, H6), 6.8 (m, 1H, H1 '), 6.4 (m, 1H, H2'), 5.9 (m, 1H, H3 '), 5.6 (d, 1H, H5), 5.0 (m, 1H, H4'), 4.2 (m, 2H, H5 '). 2.0 (s, 3H, CH ₃ ).

5'-O- (2'-acetoxyisobutyryl) 3-O-acetyl-2'-bromo-2'-deoxyuridine

Uridine (5.0 g, 0.021 M) is suspended in acetonitrile (90 mL) and 2-acetoxyisobutyryl bromide (12.85 g, 0.063 M) is added over 15 minutes, and then the reaction is added at 80 ° C. in 3 Heated for hours. After cooling the homogeneous solution to room temperature, the solvent was removed under vacuum. The resulting syrup was dissolved in EtOAc (200 mL) and washed with NaHCO ₃ (3 × 100 mL). The organic layer was dried over MgSO ₄ and the solvent was removed in vacuo. Purification by chromatography on SiO ₂ (75% EtOAc / 25% Hex) gave 6.7 g of a white foam (67%). mp 68 ° C. to 70 ° C. (ms m ⁺ 477).

2 ', 3'-dihydro-2', 3'-dideoxyuridine (D4U)

Bromouridine (2 g, 4.2 mM) was dissolved in 3 mL of DMF and added dropwise to a slurry of Zn / Cu (0.70 g, 10.5 mM) in anhydrous DMF (25 mL). The reaction was stirred at room temperature for 2 hours 30 minutes until no starting material was observed by TLC analysis. The reaction was filtered through celite and the filtrate was concentrated at 20 ° C. under vacuum in a high vacuum system. The resulting white solid (1.1 g, 85%) was dissolved in MeOH and then cooled to 0 ° C. using an ice water bath. Anhydrous ammonia was bubbled through for 20 minutes and then the solution was warmed to 60 ° C. over 18 hours. TLC analysis revealed spots corresponding to d4u. The solvent was removed and the resulting white solid was purified by chromatography on SiO ₂ (10% MeOH / CH ₂ Cl ₂ ) to afford 0.5 g (55%) of the desired product. mp 155 ° C (document description: 154 to 155 ° C).

Comparison of in vivo anti-HIV efficiencya and cytotoxicity of AZT and d4T compound ID ₅₀ ^c (μM) TCID ₅₀ ^d (μM) AZT 0.45 54.0 d4T 0.33 39.0 (E.g. p. 28)

Antiviral tests were performed on CEM cells infected with a HIV (LAV strain).

b Cytotoxicity was measured in CEM cells.

c 50% inhibitory dose.

d 50% tissue culture inhibition dose.

According to the present invention, there is provided a method capable of producing a large amount and inexpensively 2 ', 3'-dideoxy-2', 3'-didehydronucleoside having utility in the medical art.

Claims (12)

(a) reacting the starting material ribonucleoside of formula (6) with trimethylorthoformate in the presence of a polar solvent under anhydrous conditions to obtain a reactive intermediate of formula (7); (b) subjecting the intermediate obtained in step (a) to a removal reaction by treating with an organic acid in Ac ₂ O at a high temperature of about 120 to 160 ° C. for about 4-8 hours, and (c) deprotecting by treating the formed 5′-OAc group under mild base hydrolysis conditions, To prepare a 2 ', 3'-dideoxy-2', 3'-didehydronucleoside of formula (2): Formula 2

Formula 6

Formula 7

In the above formula, B is selected from purine, aza-purine, deaza-purine, pyrimidine, aza-pyrimidine, deaza-pyrimidine, and triazole ring bases. The method of claim 1, Wherein base B is selected from purine and pyrimidine bases. The method of claim 2, Wherein said base B is a pyrimidine base. The method of claim 3, wherein Wherein said pyrimidine base is selected from uridine and 5-methyl-uridine. (a) reacting the starting material ribonucleoside of Formula 6 with a hydroxy protecting group reactant effective to selectively protect the 5'-hydroxy (primary hydroxy) group; (b) reacting the 5′-OH-protected ribonucleoside obtained in step (a) with a reactant selected from 1,1-thiocarbodiimidazole and thiophosgene under anhydrous conditions to obtain a reactive intermediate of formula (8) Generating; (c) subjecting the intermediate obtained in step (b) to a removal reaction by treating the intermediate obtained in step (b) with P (OEt) ₃ for about 30 minutes to 4 hours in a polar solvent at a high temperature of about 140 ° C to 175 ° C; And (d) deprotecting by treating the formed 5'-0-protecting group under mild acid hydrolysis conditions, wherein the 2 ', 3'-dideoxy-2', 3'-didehydronude of Formula 2 How to prepare cleoside: Formula 2

Formula 6

Formula 8

In the above formula, B is selected from purine, aza-purine, deaza-purine, pyrimidine, aza-pyrimidine, deaza-pyrimidine, and triazole ring bases. The method of claim 5, Wherein base B is selected from purine and pyrimidine bases. The method of claim 6, Wherein said base B is a pyrimidine base. The method of claim 7, wherein Wherein said pyrimidine base is selected from uridine and 5-methyluridine. (a) Reactivating the ribonucleoside of Formula 6, which is a starting material, with acyloxyisobutyryl bromide for about 1 to 3 hours in a polar solvent under anhydrous conditions at a high temperature of about 75 to 100 ℃ Creating an intermediate; (b) treating the intermediate obtained in step (a) with a Zn / Cu reactant in an aprotic polar solvent to remove and react to produce an intermediate represented by the following Chemical Formula 10; And (c) treating the intermediate obtained in step (b) with a mild base to remove the 5'-O-protecting group of the intermediate, thereby removing 2 ', 3'-dideoxy-2', 3'-didehydro- A method of preparing the 2 ', 3'-dideoxy-2', 3'-didehydro nucleoside, comprising generating a nucleoside: Formula 2

Formula 6

Formula 9

Formula 10

In the above formula, B is selected from a base crowd consisting of purine, aza-purine, deaza-purine, pyrimidine, aza-pyrimidine, dea-pyrimidine, and triazole ring bases; R represents an acyloxyisobutyryl group; R 'represents an acyl group of acyloxyisobutyryl bromide. The method of claim 9, Wherein base B is selected from purine and pyrimidine bases. The method of claim 10, Wherein said base B is a pyrimidine base. The method of claim 11, Wherein said pyrimidine base is selected from uridine and 5-methyluridine.