RU2443708C2 - Method of producing a2a-adenosine receptor agonist and polymorphic modifications thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing a monohydrate of (1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide through contact of a compound of formula

with aqueous methylamine at temperature equal to approximately 2.5-7.5°C. The invention also relates to a method of producing an intermediate compound of formula (4): involving reaction of a compound of formula (1) with 14.3-16.7-fold molar excess hydrazine hydrate at temperature equal to approximately 60-65°C to obtain the corresponding hydrazine of formula (2), followed by contact between the compound of formula (2) and excess ethyl-2-formyl-3-oxopropionate, optionally in the presence of an acid.

EFFECT: method enables to obtain, in a single step, a crystalline compound in form of a monohydrate and also exclude undesirable impurities of the compound of formula 2 in the end product owing to use of intermediate product 4.

15 cl, 7 ex, 5 dwg

Description

This application is a partial continuation of US patent application Serial No. 11/701699, filed February 2, 2007, which claimed priority on the basis of provisional patent application US Serial No. 60/801857, filed May 18, 2006, and provisional patent application US Serial No. 60/765114, filed February 3, 2006, the full disclosure of which is incorporated into this description by reference.

FIELD OF THE INVENTION

The present invention relates to a method for large-scale production of an A _2A- adenosine receptor agonist, and also relates to polymorphic modifications of this compound and to methods for isolating a specific polymorphic modification.

State of the art

Adenosine is a naturally occurring nucleoside that exerts its biological properties through interaction with a family of adenosine receptors known as A ₁ , A _2A , A _2B and A ₃ , each of which modulates important physiological processes. One of the biological properties of adenosine is its action as a coronary vasodilator, and this property is the result of interaction with adenosine A _2A receptor. It was found that this effect of adenosine is useful in cardiac imaging when it is necessary to expand the coronary arteries before using a radiopharmaceutical (for example, thallium 201) and as a result, by observing the images thus obtained, determine the presence or absence of coronary artery disease. The advantage of this technology is that they avoid the more traditional method of inducing coronary vasodilation by means of an exercise on a treadmill, which is undoubtedly undesirable for a patient with coronary disease.

However, the use of adenosine has several disadvantages. Adenosine has a very short half-life in humans (less than 10 seconds), and has all the properties associated with agonists of A _1, A _2A, A _2B and A ₃ receptors. Thus, the use of a selective adenosine A _2A receptor agonist would provide an excellent way to accomplish coronary vasodilation, especially an agonist with longer half-life and some or no side effects.

A class of compounds possessing these desirable properties is disclosed in US Pat. No. 6,035,367, the entire disclosure of which is hereby incorporated by reference. In particular, it is shown that one compound disclosed in this patent is (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6 -aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide is a highly selective agonist of the A _2A- adenosine receptor and is currently undergoing clinical trials as a coronary vasodilator suitable for cardiac imaging.

Given the increased interest in this and similar compounds, it became desirable to discover new synthesis methods that provide a convenient way to obtain large quantities of the substance with good yield and high purity. A patent that discloses a compound of interest (US Patent No. 6403567) provides several methods for preparing the compound. However, although these methods are suitable for synthesis on a small scale, all synthesis methods disclosed in the patent use protecting groups, which is undesirable for synthesis on a large scale.

In addition, it was found that the desired product (i.e. (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurine- 2-yl} pyrazol-4-yl) -N-methylcarboxamide) can exist in at least three different crystalline forms, the most stable of which is monohydrate. This polymorphic modification is stable in stress states of relative humidity, up to its melting point. Accordingly, it is desirable that the final product produced by a new synthesis be obtained as a stable monohydrate.

SUMMARY OF THE INVENTION

Thus, it is an object of the present invention to provide convenient synthesis for large scale production of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6 -aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide and its polymorphic modifications, preferably in the form of its monohydrate. Accordingly, in a first aspect, the invention relates to a method for producing a compound of formula I:

Formula I

including

contacting a compound of formula (3):

with methylamine.

In one embodiment, the reaction is carried out in an aqueous solution of methylamine, initially at a temperature of about 0-5 ° C, followed by heating to about 50-70 ° C. Alternatively, the reaction is carried out as described above, but in a sealed reactor under pressure.

In a second embodiment, the product is isolated as pure monohydrate by dissolving the product in a solvent, for example dimethyl sulfoxide, adding purified water, filtering the suspension thus formed, washing the contents of the filter with water and then ethanol, and drying the solid, which is under vacuum at a temperature that does not exceed 40 ° C.

In a second aspect, the invention relates to a method for producing a compound of formula (3):

including

contacting a compound of formula (2):

with ethyl 2-formyl-3-oxopropionate.

In one embodiment, the reaction is carried out in ethanol at a temperature of about 80 ° C, with about 1.1 molar equivalents of ethyl 2-formyl-3-hydroxypropionate.

In a third aspect, the invention relates to a method for producing a compound of formula (2):

including

contacting a compound of formula (1):

with hydrazine.

A 14.3-16.7-fold molar excess of hydrazine can be used and the reaction can be carried out at a temperature of approximately 60-65 ° C. In one embodiment, hydrazine is first heated to about 60-65 ° C., followed by the addition of a compound of formula (1). The compound of formula (2) can be isolated by (a) cooling the reaction mixture to approximately 40 ° C, (b) adding 4.2-4.9 mass equivalent amounts of water, while maintaining the temperature at approximately 40 ° C, (s ) cooling the mixture to about 10 ° C and maintaining at this temperature for at least about 1 hour, (d) filtering, (e) washing the contents of the filter with water and then ethanol, and (f) drying the solid, which is under vacuum at temperature, which does not exceed 30 ° С, during at least 12 hours.

The method described above is suitable for large-scale synthesis of the desired product, which is achieved in good yield, despite the presence of one minor impurity in the final product. This impurity has been shown to be an unchanged intermediate of formula (2); that is, a compound of the formula:

Although this impurity can be removed from the final product by crystallization, it was decided to look for an alternative synthesis that has all the advantages of the above synthesis, but does not give the compound of formula (2) as an impurity in the final product.

Thus, in a fourth aspect, the invention relates to a method for the synthesis of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurine- 2-yl} pyrazol-4-yl) -N-methylcarboxamide by contacting a compound of formula (4):

with methylamine.

In one embodiment, the reaction is carried out in an aqueous solution of methylamine, initially at a temperature of about 0-5 ° C, followed by heating to about 50-70 ° C. Preferably, the reaction is carried out in a sealed reactor under pressure.

In another embodiment, the reaction is carried out at a temperature of about 2.5-7.5 ° C.

In yet another embodiment, the product is isolated as pure monohydrate by dissolving the product in a solvent, for example dimethyl sulfoxide, adding purified water, filtering the suspension thus obtained, washing the contents of the filter with water and then ethanol, and drying the solid, which remains under vacuum at a temperature that does not exceeds 40 ° C. In some other embodiments, the product can be isolated as pure monohydrate by (a) degassing the reaction mixture under vacuum at no more than 35 ° C to remove excess methylamine, (b) venting and cooling to 0-5 ° C for about 15 minutes to one hour, (c) filtering the suspension thus formed, (d) washing the contents of the filter with water and then ethanol, and (e) drying the solid, which remains under vacuum at a temperature that does not exceed 40 ° C.

In some embodiments, (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4 -yl) -N-methylcarboxamide is further purified to provide a monohydrate form by (i) dissolving the compound in a solvent such as dimethyl sulfoxide heated to about 78-88 °, (ii) filtering any solid impurities from the solution, (iii) washing with an additional solvent , (iv) adding a solution to the purified water, which is maintained at approximately 78-88 ° C, thereby forming a suspension, (v) curing the suspension, (vi) cooling the suspension, (vii) filtering, (viii) washing the contents of the filter with water and then ethanol, and (ix) drying the solid, which is kept under vacuum at a temperature that does not exceed 40 ° C.

In a fifth aspect, the invention relates to a method for synthesizing a compound of formula (4):

comprising contacting a compound of formula (2):

with an excess of ethyl 2-formyl-2-oxopropionate, preferably about 2-10-fold molar excess, more preferably about 5-10-fold excess. In one embodiment, the reaction is carried out in ethanol at a temperature of about 80 ° C. Ethyl 2-formyl-3-oxopropionate is present in a 5-10-fold excess.

In an additional embodiment, an additional method for synthesizing a compound of formula (4) is also provided, comprising contacting a compound of formula (2)

with excess ethyl 2-formyl-3-oxopropionate in the presence of acid. The reaction takes place at the boil under reflux, and it is usually carried out in ethanol. The reaction can proceed both in the presence and in the absence of HCl as a catalyst. Up to 0.1 molar equivalent, preferably about 0.05 molar excess of HCl and about 5-10 times molar excess, preferably about 6.8-7.5 times molar excess of ethyl 2-formyl-3-oxopropionate are used. The product of this reaction can be isolated by (a) cooling the reaction mixture after completion of the reaction to about 10 ° C, (b) filtering, (c) washing the contents of the filter with ethanol, and (d) drying the solid, which remains under vacuum at a temperature that does not exceeds 40 ° C.

Definitions and main parameters

Figure 1 presents the ¹ H NMR spectrum of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurine monohydrate 2-yl} pyrazol-4-yl) -N-methylcarboxamide (Form A).

Figure 2 shows the thermal analysis of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl monohydrate } pyrazol-4-yl) -N-methylcarboxamide.

Figure 3 shows the x-ray diffraction pattern of the monohydrate (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl } pyrazol-4-yl) -N-methylcarboxamide.

Figure 4 shows an x-ray diffraction pattern of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide Form B.

5 shows an X-ray diffraction pattern of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide form C compared to form A.

In the context of the present description, the following terms and expressions are generally intended to define the meanings set forth below, unless otherwise indicated.

“Optional” or “optional” means that the event or circumstance described sequentially may or may not occur and that the description includes cases where the specified event or circumstance occurs and cases in which it does not occur.

The term “therapeutically effective amount” refers to an amount of a compound of formula 1 that is sufficient for effective treatment, as defined below, when administered to a mammal, if necessary, such treatment. A therapeutically effective amount will vary depending on the subject and the conditions of the disease to be treated, the weight and age of the subject, the severity of the condition of the disease, route of administration and the like, which can be easily determined by an ordinary specialist.

The term “treatment” or “treating” means any treatment for a disease in a mammal, including:

(i) prevention of the disease, that is, an obstacle to the development of clinical symptoms of the disease;

(ii) inhibition of the disease, i.e., suspension of clinical symptoms; and / or

(iii) alleviation of the disease, i.e., regression of clinical symptoms.

As used herein, a “pharmaceutically acceptable carrier” includes any and all solvents, dispersible media, coatings, antimicrobial and antifungal agents, isotonic and absorption preventing agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except where any conventional medium or agent is incompatible with the active ingredient, their use in therapeutic compositions is contemplated. You can also enter in the composition of additional active ingredients.

The term “polymorphic modification” is intended to include amorphous modifications and solvates of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurine -2-yl} pyrazol-4-yl) -N-methylcarboxamide.

This compound has been found to exist in at least three different crystalline forms, referred to herein as Form A, Form B, Form C, and an amorphous product.

Form A : This polymorphic modification can be obtained by crystallization of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2 -yl} pyrazol-4-yl) -N-methylcarboxamide from protic solvents, for example ethanol or mixtures of ethanol and water, or from a polar solvent, for example water and dimethyl sulfoxide. It was shown that Form A is a monohydrate and is the most stable of various polymorphic modifications at ambient temperatures. It is stable in relatively wet stresses up to its melting point.

Form B : This polymorphic modification is obtained by vacuum evaporation of a solution of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurine -2-yl} pyrazol-4-yl) -N-methylcarboxamide in trifluoroethanol at ambient temperatures. The X-ray analysis of the crystals was noticeably different from any other polymorphic modification (see Fig. 4), but its composition was difficult to determine, since the X-ray analysis yielded disordered wide peaks and the polymorphic modification contained various amounts of water. It was found that it is difficult to reproduce the receipt of this polymorphic modification with high probability.

Form C : This polymorphic modification is obtained by suspending (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2- il} pyrazol-4-yl) -N-methylcarboxamide in acetonitrile for a long period of time at 60 ° C. X-ray crystal analysis was noticeably different from any other polymorphic modification (see figure 5). It was shown that polymorphic modification C is an unstable hydrate, which at elevated temperatures desolvates into an unstable form.

Amorphous material : This polymorphic modification is obtained by heating the polymorphic modification of Form A at temperatures up to 200 ° C. This polymorphic modification is unstable in the presence of atmospheric moisture, forming various hydrates.

Methods of analysis of forms A, B, C and amorphous material

X-ray powder diffraction

X-ray powder diffraction (XRPD) analysis was performed on a Shimadzu XRD-6000 X-ray powder diffractometer using Cu Kα radiation. The device was equipped with a fine-focus X-ray tube and the voltage and current in the tube were set to 40 kV and 40 mA, respectively. Divergence and scattering slots were set to 1 ”and the receiving slit was set to 0.15 mm. The diffracted radiation was determined by a NaI scintillation detector. A theta-two theta continuous scan was used at 3 ° / min (step 0.4 sec / 0.02 °) from 2.5-40 ° 2θ. To check the settings, a silicon standard was used. Data was collected and analyzed using XRD-6000 v. 4.1 software.

X-ray powder diffraction (XRPD) analyzes are also performed using an Inel XRG-3000 diffractometer equipped with a CPS (sensitive with bent position) detector with 28 intervals of 120 °. Calibration of the device was performed using a silicon reference standard. The voltage and current in the tube were set to 40 kV and 30 mA, respectively. A monochrome gap was set at 5 mm near 80 μm. Samples were placed on an aluminum sample holder with a silicon insert or in XRPD-quality glass capillaries. Each capillary was installed in the head of the goniometer, which was actuated to provide torsion of the capillary during data collection. Real-time data was obtained using Cu-Kα radiation at a resolution of 0.03 ° 2θ. Typically, data was collected over a period of 300 seconds. XRPD drawings show only data points within the interval 2.5-40 ° 2θ.

Thermal analysis

Thermogravimetric (TG) analysis was performed on a TA Instruments 2050 or 2950 thermogravimetric analyzer. Calibration standards were nickel and Alumel ^TM . Samples were placed in an aluminum sample tray, inserted into a TG furnace, and accurately weighed. Samples were heated in nitrogen at a rate of 10 ° C / min or up to 300 or up to 350 ° C. Unless otherwise stated otherwise, the masses of the samples were balanced before analysis at 25 ° C. in a TGA furnace.

Differential Scanning Calorimetry (DSC) analysis was performed on a TA Instruments 2920 Differential Scanning Calorimeter. Accurately weighed samples were placed in either crimped trays or hermetically sealed trays that contain a puncture to allow pressure relief. Each sample was heated in nitrogen at a rate of 10 ° C / min or up to 300 or 350 ° C. Indium metal was used as the calibration standard. Temperatures were held at the transition maximum.

Infrared spectroscopy

Infrared spectra were obtained on a Magna 860 ^® Fourier transformed infrared (FT-IR) spectrophotometer (Nicolet Instrument Corp.) equipped with an Ever-Glo mid / far infrared source, an extended range potassium bromide beam splitter and a deuterated triglycine sulfate detector (DTGS). Unless otherwise stated, Spectra-Tech, Inc. was used for the sample study. auxiliary device for the coefficient of diffuse reflection. Each spectrum represents 256 co-added scans at a spectral resolution of 4 cm ^-1 . Obtaining samples for the connection consisted of placing the sample in a microwall and leveling the material with a frosted glass slide. The installation of the underlying data was obtained using a centered reflector in place. Spectra represent the ratio of a data set with a single beam of a sample to a data set with a background single beam. The wavelength calibration of the device was carried out using polystyrene.

NMR spectroscopy

¹ H NMR spectra in the solution phase were obtained at ambient temperature on a Bruker AM-250 spectrometer model, operating at 5.87 T (Larmor frequency: ¹ N = 250 MHz). Time lapse data was obtained using a ripple width of 7.5 ps and a collection time of 1.6834 seconds on a spectral window of 5000 Hz. A total of 16384 data points was collected. Between transition states, a delay time of 5 seconds of relaxation was used. Each data set typically consisted of 128 co-averaged transition states. The spectra were processed using GRAMS132 A1 software, version 6.00. Attenuation of free induction (FID) was zeroed to four times the number of data points and exponentially multiplied by the line broadening factor of 0.61 Hz before the Fourier transform. In the ¹ H spectra, signals were assigned internally to tetramethylsilane (0 ppm), which was added as an internal standard.

Alternative NMR analysis was performed as described in example 4.

Moisture sorption / desorption analysis

Moisture sorption / desorption data were collected on a VTI SCA-100 Steam Sorption Analyzer. Sorption and desorption data were collected in the range from 5% to 95% relative humidity (RK) with 10% relative humidity intervals with nitrogen purging. Sodium chloride (NaCl) and polyvinylpyrrolidone (PVP) were used as calibration standards. The equilibrium criteria used for the analysis were less than 0.0100% of the mass change in 5 minutes with a maximum equilibrium time of 180 minutes if the mass criterion was not reached. The plotted data has not been adjusted for the initial moisture content.

Nomenclature

Structure of the compound (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl ) -N-methylcarboxamide is the following:

Synthesis of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide

One method for large-scale synthesis of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol- 4-yl) -N-methylcarboxamide is shown in Reaction Scheme I. All reactions are usually carried out under a nitrogen atmosphere.

SCHEME OF REACTION I

Stage 1 - Obtaining the formula (2)

A compound of formula (2) is prepared from a compound of formula (1) by reaction with hydrazine monohydrate in the absence of a solvent. The reaction is carried out at a temperature of approximately 45-55 ° C. When the reaction is complete, the product of formula (2) is isolated by mixing with a protic solvent, in which the compound of formula (2) has limited solubility, for example ethanol or isopropanol. The mixture is stirred for about 1-5 hours and then filtered. The solid is purified by stirring with water, filtration and washing with water and then isopropanol and drying under vacuum and taken for the next step without purification.

Stage 2 - Obtaining the formula (3)

The compound of formula (2) is then converted to the compound of formula (3) by reaction with approximately 1-1.2 molar equivalents of ethyl 2-formyl-3-oxopropionate. The reaction is carried out in a protic solvent, preferably ethanol, at about reflux temperature for about 2-4 hours. After cooling to approximately 0 ° C., the solid was filtered off, washed with cold ethanol and dried under reduced pressure. The product of formula (3) is taken for the next step without purification.

Stage 3 - Getting the final product

The final product is obtained from a compound of formula (3) by reaction with methylamine, preferably aqueous methylamine. The reaction is carried out at approximately room temperature for approximately 4 hours. The product of formula 1 is isolated in a conventional manner, for example by filtration, washing the solid with cold ethanol and drying under reduced pressure.

Preparation of starting materials

The starting material used in step 1 is (4S, 2R, 3R, 5R) -2- (6-amino-2-chloropurin-9-yl) -5- (hydroxymethyl) oxolan-3,4-diol. This compound is available in the market.

Ethyl 2-formyl-3-oxopropionate is used as the starting material in step 2. It is commercially available or can be obtained as shown in Reaction Scheme II.

SCHEME OF REACTION II

Ethyl 3,3-diethoxypropionate reacts with ethyl formate in the presence of a strong base, preferably sodium hydride. The reaction is carried out at approximately 0-5 ° C for approximately 24 hours. The product is isolated in a conventional manner, for example, by adding water and extracting impurities with a traditional solvent, for example tert-butyl methyl ether, acidifying the aqueous phase, for example, hydrochloric acid, followed by extraction with a solvent, such as dichloromethane, and removing the solvent from the dried extract under reduced pressure. Ethyl 2-formyl-3-oxopropionate is purified by distillation under reduced pressure.

The preferred method for large-scale synthesis of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol- 4-yl) -N-methylcarboxamide is shown in Reaction Scheme III.

RESPONSE DIAGRAM III

Stage 1 - Obtaining the formula (2)

As before, a compound of formula (2) is prepared from a compound of formula (1) by reaction with hydrazine monohydrate in the absence of a solvent. However, in this method, a 14.3-16.7-fold excess of hydrazine monohydrate is first heated to about 60-65 ° C., followed by the addition of a compound of formula (1). The temperature is maintained at about 60-65 ° C. during the reaction, which takes about 1 to 3 hours to complete. When the level of the residual compound of formula (1) in the mixture is not more than about 0.10%, the reaction mixture is cooled to about 40 ° C. The temperature is maintained while water is slowly added. As soon as approximately 4.2-4.9 mass equivalence is added, the mixture is cooled to approximately 10 ° C. and held at this temperature for at least 1 hour.

Then the product is isolated by filtration, washed with water and then washed with absolute ethanol. The solid is dried under vacuum at up to 30 ° C for at least 12 hours and then taken to the next step without further purification.

Stage 2 - Obtaining the formula (4)

The compound of formula (2) is then converted to the compound of formula (4) by reaction with an excess of ethyl 2-formyl-3-oxopropionate, for example, a 2-10-fold excess, preferably about 5-10-fold, ideally about 6.8-7 A 5-fold excess. The reaction can be carried out under the same conditions as the conditions described for obtaining the compounds of formula (3) in the reaction scheme (I).

Alternatively, approximately 0.05 molar equivalents of an acid such as HCl can also be added to the reaction mixture. The reactions make it possible to continue at the boiling point under reflux for approximately 2-4 hours, until the level of the residual compound of formula (2) is not more than 0.50% and the amount of any compound of formula (3) that can be formed is not will be no more than 2.5%. After cooling to approximately 10 ° C, the solid is filtered off, washed with absolute ethanol and dried under vacuum at up to 40 ° C to remove residual ethanol. The product of formula (4) is taken for the next step without purification.

The compound of formula (4) is depicted as a derivative of (2E) alkene, since it is the main isomer formed in this reaction. However, it should be noted that a significant amount of a (2Z) alkene derivative can also be formed in this reaction; i.e

named as ethyl (2Z) -3 - ({9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -2- [4- (ethoxycarbonyl) pyrazolyl] purin-6-yl} amino) -2-formylprop-2-enoate.

Accordingly, although the compound of formula (4) is presented only as a derivative of (2E) alkene, the term “compound of formula (4)” is intended to include both the case where it is the only (2E) isomer and the case where the bulk of the product is ( 2E) isomer and a minor portion (2Z) of the isomer is also present. The conversion of the compound of formula (4) to the final product by reaction with methylamine as described in step 3 proceeds in the same way, whether the compound of formula (4) is present as a (2E) isomer or as a mixture of the (2E) isomer and (2Z) isomer.

Stage 3 - Getting the final product

The final product is obtained from a compound of formula (4) by reaction with methylamine, preferably aqueous methylamine. The reaction is initially carried out at about 0-5 ° C for about 8 hours, preferably in a pressure reactor, followed by raising the temperature to 50-60 ° C after about 1 hour and maintaining the temperature for 15-30 minutes.

Alternatively, methylamine is first placed in a pressure vessel and cooled to about 2.5-7.5 ° C., and then the compound of formula (4) is added while the temperature is maintained. The reaction proceeds until the level of the residual compound of the formula (3) is less than about 0.10%.

After completion, the product is isolated in a conventional manner, for example, by degassing under vacuum at no more than about 35 ° C. to remove excess methylamine. Then the vacuum is lowered and the mixture is cooled to approximately 0-5 ° C and maintained at a temperature of 15 minutes to one hour, followed by filtration. The solid thus obtained is washed with water and then ethanol and dried under reduced pressure at a temperature of not more than about 40 ° C.

This method provides (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4- il) -N-methylcarboxamide as its monohydrate. This polymorphic modification can be further purified by dissolving in dimethyl sulfoxide (DMSO), filtering any solid impurities from the solution, washing with additional DMSO, and precipitating the monohydrate from the solution by adding to water. This method is particularly effective when the DMSO solution and water are heated to about 78-88 ° C and kept at this temperature with stirring for about 1 hour. After stirring, the suspension is slowly cooled to approximately 20 ° C. Then the final product is isolated by filtration, washed with purified water and then ethanol and dried, as described previously.

EXAMPLE 1

Obtaining ethyl 2-formyl-3-oxopropionate

A three- or four-necked round-bottom flask equipped with a magnetic stir bar, thermocouple, thermometer with digital indication, gas inlet and outlet, and additional funnel were purged with argon. Ethyl 3,3-diethoxypropionate (64.5 g) in tetrahydrofuran was charged to an additional funnel. Sodium hydride (21.2 g of a 60% dispersion) was charged into the reaction flask following tetrahydrofuran. The contents of the flask were cooled to 0-5 ° C in an ice bath and ethyl formate (257 g) was added. The mixture was cooled to 0-5 ° C and the contents of the additional funnel was added dropwise, keeping the internal temperature less than 5 ° C. The ice bath was removed and the contents were allowed to warm to ambient temperature. The consumption of ethyl 3,3-diethoxypropionate was monitored by TLC analysis. The reaction was quenched by addition of ice water (10.6 vol.), Extracted three times with methyl tert-butyl ether (5.4 vol. Each) and the organic layers were discarded. The aqueous phase was acidified with concentrated hydrochloric acid to a pH of from 1 to 1.5. The acidified aqueous layer was extracted three times with dichloromethane and the combined organic layers were dried over sodium sulfate. The solvent was removed under reduced pressure and the residue was vacuum distilled to provide ethyl 2-formyl-3-oxopropionate, 27.92 g, 70% yield.

EXAMPLE 2

A. Preparation of 2-hydrazinoadenosine (2)

A flask equipped with a mechanical stirrer, gas inlet and outlet, and a thermocouple was purged with argon. 2-Chloradenosine hemihydrate (53.1 g) was added followed by hydrazine monohydrate (134 g). The mixture was stirred under heating at 40-45 ° C for 2 hours. The progress of the reaction was monitored by TLC analysis. Upon completion of the reaction, the heat source was removed and ethanol (800 ml) was added. The mixture was stirred for 2 hours at ambient temperature, then the precipitate was collected by filtration. The filter cake was washed with ethanol and dried under reduced pressure for 30 minutes. The solids were transferred to a clean flask equipped with a mechanical stirrer and water (300 ml) was added. The suspension was stirred at room temperature for 18 hours and the solids were isolated by filtration. The filter cake was washed with ice water (300 ml), followed by washing with ice ethanol (300 ml). The solid was dried under reduced pressure to give 2-hydrazinoadenosine (41.38 g, 81.4% yield, 99.3% purity).

B. Alternative Preparation of 2-Hydrazinoadenosine (2)

The reaction vessel containing hydrazine hydrate (258 g, 250 ml) was heated to 40-50 ° C. To the warm mixture, 2-chloradenosine hemihydrate (100 g) was added in portions, maintaining the temperature between 45-55 ° C. The temperature was maintained at this value for two hours, and then deionized water (500 ml) was added over a period of 30 minutes, maintaining the temperature at 45-55 ° C. The mixture was then gradually cooled to 0-5 ° C over a period of 3 hours, then stirred at this temperature for an additional 30 minutes. The solid was further filtered and washed with cold (2-5 ° C) deionized water (200 ml), followed by washing with ethanol (400 ml). The solid was dried under vacuum for 12 hours to give 2-hydrazinoadenosine.

C. Alternative Preparation of 2-Hydrazinoadenosine (2)

Hydrazine hydrate (1285 g) was charged into the reaction vessel. The solution was heated to approximately 62 ° C and 2-chloradenosine (500 g) was added while maintaining a temperature of approximately 62 ° C. The mixture is maintained at 62 ° C. for at least 2 hours, until the level of residual 2-chloradenosine in the mixture is not more than 0.10%. The mixture was cooled to approximately 40 ° C and the mixture was checked to confirm the presence of solids. Slowly add water (2275 g), while maintaining a temperature of approximately 40 ° C. The mixture is cooled to approximately 10 ° C and incubated for at least 1 hour. The product was isolated by filtration, washed with water (1195 g) and then with absolute ethanol (1885 g). The product is dried under vacuum at temperatures up to 30 ° C for at least 12 hours to obtain 2-hydrazinoadenosine.

EXAMPLE 3

Preparation of Ethyl 1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazole-4-carboxylate (3)

Ethyl 2-formyl-3-oxopropionate (23.93 g, 0.17 mol) was placed in a flask equipped with a mechanical stirrer, gas inlet and outlet and a reflux condenser. 2-propanol was added to the flask, followed by 2-hydrazinoadenosine (44.45 g, 0.15 mol). The mixture was heated at the boil under reflux with stirring for 2-4 hours, monitoring the progress of the reaction using TLC analysis. When the reaction was deemed complete, the heat source was removed and the mixture was cooled to room temperature. The suspension was cooled with stirring in an ice bath for 1.5-2 hours. The solids were suction filtered and washed with ice-cold 2-propanol. Product, ethyl-1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4- carboxylate, dried under reduced pressure to constant weight.

EXAMPLE 4

Preparation of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide

A mixture of ethyl 1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazole-4-carboxylate (46.4 g) and methylamine (40% in water, 600 ml) were stirred at ambient temperature for approximately 4 hours, monitoring the progress of the reaction by HPLC analysis. The main amount of excess methylamine was removed under reduced pressure and the remaining mixture was cooled to 0 ° C for 2 hours. The solid was filtered off, washed with 200 ml ice-cold ethanol and dried under reduced pressure to obtain (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl ] -6-aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide as its monohydrate.

The structure of the substance was confirmed using ¹ H NMR (see figure 1 and below). Thermal analysis (see figure 2) presented results that coincide with the presence of one water molecule. Received x-ray diffraction bands (figure 3).

Spectra ¹ H - and ¹³ C-NMR were obtained as follows. Two samples of the material obtained above were weighed and dissolved in d ₆ -DMSO - 5.3 mg was used for ¹ H-spectra and 20.8 mg was used for ¹³ C-spectra. All spectra were recorded at ambient temperature on a JEOL Eclipse ⁺ 400 spectrometer with an operating frequency of 400 MHz for ¹ H and 100 MHz for ¹³ C.

Designation ¹³ C shift (parts per million) ¹ N shift (ppm) Multiplicity, splitting (Hz) 2 150.5 or 150.3 - four 156.4 - 4a 117.9 - 6 140.0 8.41 from 7a 150.5 or 150.3 - one' 86.9 5.94 d, 6.2 2 ' 73.7 4.62 m 2'-OH - 5.50 d, 6.2 3 ' 70.5 4.17 m 3'-OH - 5.23 d, 4.7 four' 85.7 3.96 m 5' 61.5 3.67, 3.57 m 5'-OH - 5.02 d, 5.7 A 140.9 8.07 d, 0.8 B 120,2 - C 129.6 8.95 d, 0.8 D 161.7 - E 25.6 2.76 d, 4.6 NH ₂ - 7.77 broad s NH - 8.35 sq., 4.6

EXAMPLE 5

A. Preparation of Ethyl (2E) -3 - ({9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -2- [4- ( ethoxycarbonyl) pyrazolyl] purin-6-yl} amino) -2-formylprop-2-enoate (4)

A mixture of 2-hydrazinoadenosine (100 g, 0.34 mol), ethyl 2-formyl-3-oxopropionate (242 g, 1.7 mol) and absolute ethanol was charged into the reactor and the mixture was heated under reflux for 2 hours . When the reaction was deemed complete, the heat source was removed and the mixture was gradually cooled to 5-10 ° C. over a period of 3 hours. The suspension was stirred for 30 minutes at this temperature and the mixture was filtered. The solid was washed with cold (5-10 ° C) absolute ethanol and then dried under vacuum at a temperature that did not exceed 40 ° C to obtain ethyl (2E) -3 - ({9 - [(4S, 2R, 3R, 5R ) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -2- [4- (ethoxycarbonyl) pyrazolyl] purin-6-yl} amino) -2-formylprop-2-enoate.

B. Alternative Preparation of Ethyl (2E) -3 - ({9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -2- [4- (ethoxycarbonyl) pyrazolyl] purin-6-yl} amino) -2-formylprop-2-enoate (4)

2-hydrazinoadenosine (450 g) was loaded into the reaction vessel and absolute ethanol (11376 g), HCl (7.47 g) and ethyl 2-formyl-3-oxopropionate (1557 g) were added. The mixture is heated at the boil under reflux and samples are taken from it until the level of residual 2-hydrazinoadenosine in the mixture is more than 0.50% and the level of the compound of formula (3) is more than 2.5%. The mixture is slowly cooled to approximately 10 ° C. The product, a compound of formula (4), was isolated by filtration and washed with absolute ethanol (5121 g). The product is dried under vacuum at a temperature of up to 40 ° C until the residual ethanol is more than 5000 ppm to obtain ethyl- (2E) -3 - ({9 - [(4S, 2R, 3R, 5R) -3, 4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -2- [4- (ethoxycarbonyl) pyrazolyl] purin-6-yl} amino) -2-formylprop-2-enoate.

Elemental analysis of the product of example 5A gave the following results: C, 48.75%; H, 4.86%; N, 18.05%; Oh, 27.57. Theoretical values: C, 49.72%; H, 4.74%; N, 18.45%; Oh, 27.09. The analysis results correspond to the experimental error limits for the hemihydrate of the desired product (C, 48.89%; H, 4.81%; N, 18.1%; O, 28.12).

Spectra ¹ H - and ¹³ C-NMR were obtained as follows. 20.2 mg of the compound of formula (4) was dissolved in ~ 0.75 ml of DMSO-d ₆ and the spectra were obtained at ambient temperature on a JEOL ECX-400 spectrometer with an operating frequency of 400 MHz for ¹ H and 100 MHz for ¹³ C. Chemical shifts are treated with DMSO solvent, 2.50 ppm for ¹ H and 39,5 ppm to ¹³ C.

RESULTS

Chemical shifts for ¹ H and ¹³ C are shown in Table 1. Two isomers in a ratio of ~ 60/30 was observed in the spectra of ¹ H and ¹³ C, designated as the primary and minor in the table.

Atom ^{a 13} C chemical shift (ppm) ¹ N chemical shift (ppm) Multiplicity ^b , splitting (Hz) 21 (main) 192.4 9.96 d, 3.6 21 (minor) 187.6 9.83 from 22 (minor) 167.1 - - 22 (main) 165.2 - - 15 (minor) 161.8 - - 15 (main) 161.7 - - 6 (main) 153.1 - - 6 (minor) 152.9 - - 2 (minor) 149.4 - - 2 (main) 149.3 - - 19 (minor) 148.0 9.22 d, 13.0 4 (minor) 147.9 - - 4 (main) 147.8 - - 19 (main) 147.5 9.26 d, 12.4, d, 3.6 8 (main) 144.9 8.87 from 8 (minor) 144.7 8.85 from 12 143.1 8.20-8.23 m 14 (minor) 132.8 9.20 d, ~ 0.7 14 (main) 132.6 9.12 d, ~ 0.7 5 (main) 120.7 - - 5 (minor) 120.6 - - 13 116.7 - - 20 (minor) 107,2 - - 20 (main) 106.1 - - 1 '(main) 87.9 6.07 d, 5.3 1 '(minor) 87.9 6.06 d, 5.3 four' 85.8 4.02 sq. 3.9 2 '(minor) 74.1 4.62 sq, ~ 5.4 2 '(main) 74.1 4.61 sq, ~ 5.4 3 ' 70.1 4.22 sq. 4.2 5' 61.0 3.62, 3.73 m 23, 16 60.3-60.8 4.25-4.39 m 17, 24 14.1-14.2 1.28-1.38 m 18 (main) - 12.51 d, 12.4 18 (minor) - 11.47 d, 13.0 2'-OH (basic) - 5.63 d, 6.1 2'-OH (minor) - 5.62 d, 6.1 3'-OH - 5.30 d, 5.1 5'-OH - 5.08 t, 5.5

It was confirmed that the compound of formula (4) is a mixture of the following two isomers:

EXAMPLE 6

A. Preparation of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4- il) -N-methylcarboxamide from compound (4)

An aqueous 40% methylamine solution (1300 ml) was placed in a reactor under pressure, cooled to 0-5 ° C, and the product of Example 5A, (ethyl- (2E) -3 - ({9 - [(4S, 2R, 3R, 5R) was added ) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -2- [4- (ethoxycarbonyl) pyrazolyl] purin-6-yl} amino) -2-formylprop-2-enoate (100 g) The mixture was stirred at 0-5 ° C for at least 8 hours, monitoring the completion of the reaction.After completion, the mixture was heated, maintaining the temperature between 50 and 60 ° C for 1 hour, and then cooled to less than 30 ° C for 1 hour. When the temperature dropped below 30 ° C, the mixture was degassed using pressure equal to 100-150 mm Hg, allowing the temperature to drop to 0-5 ° C. The mixture was stirred at 0-5 ° C for at least 1 hour, maintaining the pressure at 100-150 mm Hg. The vacuum was then interrupted and replaced nitrogen, maintaining the temperature at 0-5 ° C for at least 30 minutes.The solid product was then filtered, washed with water (3 × 500 ml), then with absolute ethanol (625 ml). The product was dried under vacuum, not allowing the temperature to exceed 40 ° C, to give (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol- 4-yl) -N-methylcar oxamide in the form of its monohydrate.

B. Alternative Preparation of (1- {9 - [(4S, 2R, 3R , 5R) -3,4- dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurine-2-yl} pyrazol-4 -yl) -N-methylcarboxamide from Compound (4)

The pressure vessel was charged with a solution of 47% methylamine (10080 g). The solution was cooled and the compound of formula (4) was added as was obtained in Example 5B above (600 g) while maintaining a temperature of 5 ° C. The mixture is stirred at a temperature of 5 ° C until the level of the residual compound of formula (3) in the mixture drops below 0.10%. The reaction mixture is degassed under vacuum at no more than 35 ° C to remove excess methylamine. The vacuum is discharged and the mixture is cooled to 2.5 ° C and held for at least 30 minutes. The product is further isolated by filtration, washed with water (not less than 9000 g) and then absolute ethanol (not less than 2964 g). The product is dried under vacuum at a temperature of up to 40 ° C until the residual ethanol is not more than 5000 ppm to obtain crude (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide as its monohydrate.

Spectra ¹ H - and ¹³ C-NMR were obtained as follows. Two samples of the substance obtained in Example 6A were weighed and dissolved in d ₆ -DMSO — 5.3 mg was used for ¹ H-spectra and 20.8 mg was used for ¹³ C-spectra. All spectra were recorded at ambient temperature on a JEOL Eclipse ⁺ 400 spectrometer with an operating frequency of 400 MHz for ¹ H and 100 MHz for ¹³ C.

Designation ¹³ C shift (parts per million) ¹ N shift (ppm) Multiplicity, splitting (Hz) 2 150.5 or 150.3 - four 156.4 - 4a 117.9 - 6 140.0 8.41 from 7a 150.5 or 150.3 - one' 86.9 5.94 d, 6.2 2 ' 73.7 4.62 m 2'-OH - 5.50 d, 6.2 3 ' 70.5 4.17 m 3'-OH - 5.23 d, 4.7 four' 85.7 3.96 m 5' 61.5 3.67, 3.57 m 5'-OH - 5.02 d, 5.7 A 140.9 8.07 d, 0.8 B 120,2 - C 129.6 8.95 d, 0.8 D 161.7 - E 25.6 2.76 d, 4.6 NH ₂ - 7.77 broad s NH - 8.35 sq., 4.6

Elemental analysis of the product gave the following results: C, 43.96%; H, 4.94%; N, 27.94%; Oh, 27.57%. Theoretical values: C, 44.12%; H, 4.94%; N, 27.44%; Oh, 27.09%. The results of the analysis correspond to the experimental error limits for the monohydrate.

EXAMPLE 7

A. Purification of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4 monohydrate -yl) -N-methylcarboxamide

Solution (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl) The -N-methylcarboxamide obtained as in Example 4 (100 g) in dimethyl sulfoxide (300 ml) was filtered through a 0.6-0.8 micron prefilter and a 0.2 micron filter to remove any solid impurities. The filtrate was then slowly added over 1 hour to deionized water (1 liter) with stirring, and the suspension thus obtained was stirred for at least 1 hour. The solid was filtered off, washed with deionized water (2 × 1 liter) and dried under vacuum for at least 1 hour.

The dried product was then re-suspended in deionized water (1.5 liters) for at least 2 hours, filtered, washed with deionized water (1 liter), followed by washing with absolute ethanol (750 ml). The purified product was dried under vacuum at a temperature of not more than 40 ° C for at least 12 hours to obtain the monohydrate (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide free of any impurity of 2-hydrazinadenosine.

B. Alternative Purification of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol- monohydrate 4-yl) -N-methylcarboxamide

Solution of (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl monohydrate ) -N-methylcarboxamide (400 g) was dissolved in DMSO (1320 g) and the solution was filtered through a series of successive filters of 0.6-0.8-micron and 0.2-micron. An additional DMSO (880 g) is used to flush the filter system. The solution is slowly added to purified water (5000 g) while maintaining a temperature mark of 83 ° C. The product begins to crystallize during the addition, and the suspension is stirred at 83 ° C. for approximately 1 hour. The mixture is slowly cooled to 20 ° C. The product was isolated by filtration and washed with purified water (8000 g).

Solids are loaded into a vessel and purified water (6000 g) is added. The suspension is stirred for approximately 1 hour. The product was isolated by filtration and washed with purified water (4000 g) and then with absolute ethanol (3160 g). The product is dried under vacuum at a temperature of up to 40 ° С until the content of residual water becomes no more than 5.5% and the residual ethanol no more than 2000 ppm to produce monohydrate (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide.

Claims (15)

1. The method of obtaining monohydrate (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol- 4-yl) -N-methylcarboxamide

by contacting a compound of formula (4):

with aqueous methylamine at a temperature of approximately 2.5-7.5 ° C. 2. The method according to claim 1, where the reaction is carried out in a sealed reactor under pressure. 3. The method according to claim 2, where the product is isolated by
(a) degassing under vacuum at a temperature not exceeding 35 ° C to remove excess methylamine,
(b) venting and cooling to 0-5 ° C. for about 15 minutes to an hour,
(c) filtering the suspension thus formed,
(d) washing the contents of the filter with water and then ethanol; and
(e) drying the remaining solid under vacuum at a temperature that does not exceed 40 ° C. 4. The method according to claim 3, where the final product is (1- {9 - [(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurine- 2-yl} pyrazol-4-yl) -N-methylcarboxamide is further purified by
(i) dissolving the dried solid obtained in step (e) according to claim 3 in a solvent such as dimethyl sulfoxide,
(ii) filtering any solid impurities from the solution,
(iii) washing with an additional solvent such as dimethyl sulfoxide,
(iv) adding a solution to the purified water, which is maintained at a temperature of about 78-88 ° C, thereby forming a suspension,
(v) mixing the suspension,
(vi) cooling the suspension,
(vii) filtering,
(viii) washing the contents of the filter with water and then ethanol; and
(ix) drying the remaining solid under vacuum at a temperature that does not exceed 40 ° C. 5. The method according to claim 4, where the residual water content in the final product is not more than 5.5%, and the residual ethanol is not more than 2000 ppm. 6. A method of obtaining a compound of formula (4):

comprising the interaction of the compounds of formula (1)

with a 14.3-16.7-fold molar excess of hydrazine hydrate at a temperature of about 60-65 ° C. to give a compound of formula (2)

followed by contacting the resulting compound of formula (2) with an excess of ethyl 2-formyl-3-oxopropionate, optionally in the presence of an acid. 7. The method according to claim 6, where the step of contacting the compounds of formula (2) is carried out in ethanol. 8. The method according to claim 6, where an acid is used, which is HCl in an amount of up to 0.1 molar equivalent. 9. The method of claim 8, where the reaction is carried out at the boil under reflux. 10. The method according to claim 6, where approximately 5-10-fold molar excess of ethyl 2-formyl-3-oxopropionate is used. 11. The method of claim 10, wherein an approximately 6.8-7.5-fold molar excess of ethyl 2-formyl-3-oxopropionate is used. 12. The method according to claim 11, where the final product is isolated by
(a) cooling the mixture obtained after completion of the reaction to about 10 ° C,
(b) filtering,
(c) washing the contents of the filter with ethanol; and
(d) drying the remaining solid under vacuum at a temperature that does not exceed 40 ° C. 13. The method of obtaining the compounds of formula (2):

including
the interaction of the compounds of formula (1)

with a 14.3-16.7-fold molar excess of hydrazine hydrate at a temperature of approximately 60-65 ° C. 14. The method according to item 13, where the hydrazine hydrate is initially heated to a temperature of approximately 60-65 ° C, followed by the addition of a compound of formula (1). 15. The method according to item 13, where the compound of formula (2) is isolated by
(a) cooling the reaction mixture to about 40 ° C,
(b) adding 4.2-4.9 weight equivalents of water while maintaining a temperature of approximately 40 ° C,
(c) cooling the mixture to a temperature of approximately 10 ° C and maintaining this temperature for at least 1 hour,
(d) filtering,
(e) washing the contents of the filter with water and then ethanol; and
(f) drying the remaining solid in vacuo at a temperature that does not exceed 30 ° C. for at least 12 hours

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