JP2013067662A - Process for preparing a2a-adenosine receptor agonist and polymorph of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a synthesis suitable for the large-scale preparation of a process of an A-adenosine receptor agonist.SOLUTION: The present invention relates to a process for the large-scale preparation of the A-adenosine receptor agonist, and to polymorphs of the compound and a method of isolating a specific polymorph. The present invention provides a method of synthesizing (1-{9-[(4S, 2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide. Adenosine is a naturally occurring nucleoside, which exerts its biological effects by interacting with a family of adenosine receptors. The family of adenosine receptors is known as A, A, Aand A, all of which modulate important physiological processes.

Description

  This application gives priority to US Provisional Patent Application No. 60 / 801,857 filed May 18, 2006 and US Provisional Patent Application No. 60 / 765,114 filed February 3, 2006. This is a continuation-in-part of US patent application Ser. No. 11 / 701,699 filed Feb. 2, 2007, the entire disclosure of which is incorporated herein by reference.

The present invention relates to a method for large-scale preparation of A _2A -adenosine receptor agonists. It also relates to polymorphs of the compound and to methods for isolating specific polymorphs.

Adenosine is a naturally occurring nucleoside that exerts its biological effects by interacting with a family of adenosine receptors. The family of adenosine receptors is known as A ₁ , A _2A , A _2B and A ₃ , all of which regulate important physiological processes. One of the biological effects of adenosine is to act as a coronary vasodilator, and this result is produced by interaction with the _A2A adenosine receptor. This effect of adenosine has been found to be useful as an aid in cardiac imaging, dilating the coronary artery prior to administration of contrast agent (eg, thallium 201), and thus viewing the image thus projected The presence or absence of coronary artery disease can be determined. The advantage of such a technique is that it avoids the more traditional method of promoting coronary vasodilation by exercise on a treadmill, which is clearly undesirable for patients with coronary disease.

However, administration of adenosine has several disadvantages. Adenosine has a very short half-life in humans (less than 10 seconds) and has all of the effects associated with A ₁ , A _2A , A _2B and A ₃ receptor agonism. Thus, the use of selective A _2A adenosine receptor agonists, particularly those with longer half-lives and fewer or no side effects, provides an excellent method of producing coronary vasodilation.

A class of compounds having these desirable properties is disclosed in US Pat. No. 6,403,567, the entire disclosure of which is hereby incorporated by reference. In particular, one compound disclosed in this patent, (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6 - amino-purin-2-yl} pyrazol-4-yl) -N- methyl-carboxamide, highly selective a _{2A -} adenosine receptors has been shown to be an agonist, useful for imaging of the heart coronary A clinical trial is currently underway as a vasodilator.

US Pat. No. 6,403,567

  In view of the growing interest in this and similar compounds, it has become desirable to find new synthetic methods that provide a convenient way to make large quantities of material in good yield and purity. . A patent disclosing the compound of interest (US Pat. No. 6,403,567) provides several methods for preparing the compound. However, although these methods are suitable for small scale synthesis, all synthetic methods disclosed in this patent utilize protecting groups, which is undesirable for large scale synthesis.

  In addition, the desired product (ie (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 crystal forms, of which the most stable one has been found to be a monohydrate. This polymorph is stable to its melting point under relative humidity stress conditions. Therefore, it is desirable that the final product produced by the new synthesis be obtained as a stable monohydrate.

  Accordingly, the object of the present invention is to provide (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurine-2. -Yl} pyrazol-4-yl) -N-methylcarboxamide and polymorphs thereof, preferably providing a convenient synthesis for large scale preparation as the monohydrate. Therefore, in a first aspect, the present invention provides a compound of formula I

の化合物の調製であって、
式（３）

A compound comprising:
Formula (3)

の化合物をメチルアミンと接触させることを含む調製に関する。

The preparation comprising contacting the compound with methylamine.

  In one embodiment, the reaction is first carried out in an aqueous solution of methylamine at a temperature of about 0-5 ° C. and subsequently warmed to about 50-70 ° C. Alternatively, the reaction is carried out as described above, but in a closed pressure reactor.

  In a second embodiment, the product is dissolved in a solvent, such as dimethyl sulfoxide, purified water is added, the slurry thus formed is filtered, the filter contents are washed with water, followed by The product is isolated as pure monohydrate by washing with ethanol and drying the remaining solid under vacuum at a temperature not exceeding 40 ° C.

  In a second aspect, the present invention provides a compound of formula (3)

の化合物の調製であって、
式（２）

A compound comprising:
Formula (2)

の化合物をエチル２−ホルミル−３−オキソプロピオネートと接触させることを含む調製に関する。

In contact with ethyl 2-formyl-3-oxopropionate.

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

  In a third aspect, the present invention provides a compound of formula (2)

の化合物の調製であって、
式（１）

A compound comprising:
Formula (1)

の化合物をヒドラジンと接触させることを含む調製に関する。

The preparation comprising contacting said compound with hydrazine.

  A 14.3-16.7-fold molar excess of hydrazine may be used and the reaction can be carried out at a temperature of about 60-65 ° C. In one embodiment, the hydrazine is first heated to about 60-65 ° C. followed by the addition of the compound of formula (1). The compound of formula (2) is obtained by (a) cooling the reaction mixture to about 40 ° C. and (b) adding 4.2 to 4.9 mass equivalents of water while maintaining the temperature at about 40 ° C. (C) cooling the mixture to about 10 ° C. and maintaining at that temperature for at least about 1 hour; (d) filtering; (e) washing the contents of the filter with water followed by ethanol And (f) drying the remaining solid under vacuum at a temperature not exceeding 30 ° C. for at least 12 hours.

  The above synthesis is suitable for large scale synthesis of the desired product and is produced in good yield, but one small amount of impurity is found in the final product. This impurity is represented by the formula (2), that is, the formula

の化合物の未変化中間体であることが示されている。この不純物は、最終生成物から結晶化によって除去することができるとはいえ、上述の合成の利点をすべて有するが、式（２）の化合物を最終生成物中の不純物として生じさせない代替合成を探索することに決めた。

It is shown to be an unchanged intermediate of the compound Although this impurity can be removed from the final product by crystallization, it seeks an alternative synthesis that has all the advantages of the synthesis described above but does not result in the compound of formula (2) as an impurity in the final product. Decided to do.

  Accordingly, in a fourth aspect, the present invention provides (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6- Aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide having the formula (4)

の化合物をメチルアミンと接触させることによる方法に関する。

In contact with methylamine.

  In one embodiment, the reaction is first carried out in an aqueous solution of methylamine at a temperature of about 0-5 ° C. and subsequently warmed to about 50-70 ° C. Preferably the reaction is carried out in a closed pressure reactor.

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

  In yet another embodiment, the product is dissolved in a solvent such as dimethyl sulfoxide, purified water is added, the slurry thus formed is filtered, the filter contents are washed with water, followed by The product is isolated as pure monohydrate by washing with ethanol and drying the remaining solid under vacuum at a temperature not exceeding 40 ° C. In some other embodiments, (a) the reaction mixture is degassed under vacuum at 35 ° C. or lower to remove excess methylamine, and (b) the vacuum is released to about 0-5 ° C. Cooling from 15 minutes to 1 hour; (c) filtering the slurry thus formed; (d) washing the contents of the filter with water followed by ethanol; and (e) remaining. By drying the solid under vacuum at a temperature not exceeding 40 ° C., the product can be isolated as pure monohydrate.

  In some embodiments, (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurine-2- Yl} pyrazol-4-yl) -N-methylcarboxamide is obtained by (i) dissolving the compound in a solvent such as dimethyl sulfoxide and heating to about 78-88 °; and (ii) any solid impurities from the solution. (Iii) rinsing with additional solvent; (iv) forming a slurry by adding the solution to purified water maintained at about 78-88 °; and (v) the slurry. (Vi) cooling the slurry; (vii) filtering; (viii) washing the contents of the filter with water followed by ethanol; ix) Further purification by drying the remaining solid under vacuum at a temperature not exceeding 40 ° C. to produce the monohydrate form.

  In a fifth aspect, the present invention provides formula (4)

の化合物を合成する方法であって、式（２）

A method of synthesizing a compound of formula (2)

の化合物を過剰の、好ましくは約２〜１０倍モル過剰の、より好ましくは約５〜１０倍過剰のエチル２−ホルミル−３−オキソプロピオネートと接触させることを含む方法に関する。一実施形態では、反応は、エタノール中、約８０℃の温度で行われる。エチル２−ホルミル−３−オキソプロピオネートは、５〜１０倍過剰で存在する。

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

  In a further embodiment, the formula (2)

の化合物を、酸の存在下で、過剰のエチル２−ホルミル−３−オキソプロピオネートと接触させることを含む、式（４）の化合物を合成する追加の方法も提示される。反応は、還流下で行い、一般にエタノール中で行われる。反応は、触媒としてＨＣｌを使用して、または使用せずに行われる。最大０．１モル当量まで、好ましくは約０．０５モル過剰のＨＣｌおよび約５〜１０倍モル過剰、好ましくは約６．８〜７．５倍モル過剰のエチル２−ホルミル−３−オキソプロピオネートが使用される。この反応の生成物は、（ａ）反応が終了した混合物を約１０℃に冷却することと、（ｂ）ろ過することと、（ｃ）フィルターの内容物をエタノールで洗浄することと、（ｄ）残った固体を真空下にて４０℃を超えない温度で乾燥することとによって単離することができる。

An additional method for synthesizing a compound of formula (4) is also presented, comprising contacting a compound of formula (I) with an excess of ethyl 2-formyl-3-oxopropionate in the presence of an acid. The reaction is carried out under reflux and is generally carried out in ethanol. The reaction is carried out with or without HCl as a catalyst. Up to 0.1 molar equivalents, preferably about 0.05 molar excess of HCl and about 5-10 fold molar excess, preferably about 6.8-7.5 fold molar excess of ethyl 2-formyl-3-oxopro Pionate is used. The product of this reaction consists of (a) cooling the finished reaction mixture to about 10 ° C., (b) filtering, (c) washing the contents of the filter with ethanol, (d ) The remaining solid can be isolated by drying under vacuum at a temperature not exceeding 40 ° C.

(1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl ) -N- is a ¹ H NMR spectrum of methyl carboxamide monohydrate (a type). (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl ) -N-methylcarboxamide monohydrate thermal analysis. (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl FIG. 3 is an X-ray diffraction pattern of —N-methylcarboxamide monohydrate. (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl FIG. 3 is an X-ray diffraction pattern of —N-methylcarboxamide Form B; (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl FIG. 4 is a diagram showing an X-ray diffraction pattern of Form -N-methylcarboxamide C in comparison with Form A.

Definitions and General Parameters As used herein, the following words and phrases are generally intended to have the meanings set forth below. However, it excludes to the extent that the situation where they are used is indicated elsewhere.

  “Optional” or “optionally” means that an event or situation described later may or may not occur, and an explanation is given when the event or situation occurs and the event or It means that the situation does not happen.

  The term “therapeutically effective amount” when administered to a mammal in need of treatment, refers to an amount of a compound of formula I sufficient to effect such treatment, as defined below. The therapeutically effective amount will vary depending on the subject and disease state being treated, the weight and age of the subject, the severity of the disease state, the manner of administration, etc., and can be readily determined by one skilled in the art.

The term “treatment” or “treating” means any treatment of a disease in a mammal, including:
(I) preventing the disease, ie preventing the development of clinical symptoms of the disease, (ii) preventing the disease, ie suppressing the onset of clinical symptoms, and / or (iii) It includes relieving, i.e., regressing clinical symptoms.

  As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. Is included. The use of such media and agents for pharmaceutically active substances is well known in the art. It is contemplated to use it in therapeutic compositions unless any conventional vehicle or agent is incompatible with the active ingredient. Supplementary active ingredients can also be incorporated into the compositions.

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

  It has been discovered that this compound can exist in at least three different crystalline forms, which are referred to herein as A-form, B-form, C-form and amorphous product.

  Form A: This polymorph is 1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurine-2- Yl} pyrazol-4-yl) -N-methylcarboxamide can be produced by crystallization from a protic solvent such as ethanol or an ethanol / water mixture or from a polar solvent such as dimethyl sulfoxide / water. Form A has been shown to be a monohydrate and is the most stable of the various polymorphs at ambient temperature. Form A is stable up to its melting point under relative humidity stress conditions.

  Form B: This polymorph is represented by 1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurine-2- It is produced by evaporating a solution of (Il} pyrazol-4-yl) -N-methylcarboxamide in trifluoroethanol at ambient temperature under vacuum. X-ray analysis of this crystal was clearly different from any of the other polymorphs (see Figure 4), but X-ray analysis produced a broad, turbulent peak, and the polymorphs contained varying amounts of water. It was difficult to determine its structure. It has proven difficult to reliably reproduce the preparation of this polymorph.

  Form C: This polymorph 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 produced by slurrying in acetonitrile at 60 ° C. for an extended period of time. X-ray analysis of this crystal was clearly different from any of the other polymorphs (see FIG. 5). Polymorph C is a highly variable hydrate and has been shown to desolvate into an unstable form at high temperatures.

Amorphous: This polymorph is produced by heating the A polymorph at temperatures up to 200 ° C. This polymorph is unstable in the presence of atmospheric moisture and forms a hydrate that is susceptible to change.
Analytical techniques for Form A, Form B, Form C and Amorphous X-ray powder diffraction X-ray powder diffraction (XRPD) analysis was performed using a Shimadzu XRD-6000 X-ray powder diffractometer with CuKα radiation. This instrument was equipped with a high-precision focal X-ray tube, and the number of bolts and amperage of the tube were set to 40 kV and 40 mA, respectively. The divergence and scattering slits were set at 1 ° and the light receiving slit was set at 0.15 mm. Diffracted radiation was detected with a NaI scintillation detector. A θ-2θ continuous scan from 2.5 to 40 ° 2θ at 3 ° / min (0.4 sec / 0.02 ° step) was used. The instrument alignment was checked using a silicon standard. XRD-6000 v. Data was collected and analyzed using 4.1 software.

X-ray powder diffraction (XRPD) analysis was also carried out using a Inel XRG-3000 diffractometer equipped with a CPS (curved position sensitive) detector and a 28 ° range of 120 °. The instrument was calibrated using a silicon standard. The tube bolt and amperage were set to 40 kV and 30 mA, respectively. The monochromator slit was set to 5 mm long and 80 μm wide. Samples were loaded into aluminum sample holders with silicon inserts or XRPD quality glass capillaries. Each capillary was mounted on the tip of a goniometer. The goniometer is equipped with a motor that allows the capillary to rotate during data acquisition. Real-time data was collected with a resolution of 0.03 ° 2θ using Cu-Kα radiation. Typically, data was collected over 300 seconds. Only data points within the range of 2.5-40 ° 2θ are displayed in the plotted XRPD pattern.
Thermal Analysis Thermogravimetric (TG) analysis was performed using a TA Instruments 2050 or 2950 thermogravimetric analyzer. The calibration standards were nickel and Alumel ™. The sample was placed in an aluminum sample pan, inserted into a TG furnace, and accurately weighed. Samples were heated to 300 ° C. or 350 ° C. at a rate of 10 ° C./min in nitrogen. Unless otherwise stated, sample weights were equilibrated at 25 ° C. in a TGA furnace prior to analysis.

Differential scanning calorimetry (DSC) analysis was performed using a TA Instruments differential scanning calorimeter 2920. An accurately weighed sample was placed in a crimped or sealed pan. These pans contained pinholes that allowed pressure relief. Each sample was heated to 300 ° C. or 350 ° C. at a rate of 10 ° C./min under nitrogen. Indium metal was used as a calibration standard. The temperature was recorded at the transition maximum.
Infrared Spectroscopy Magna860® Fourier Transform Infrared (FT-) with Ever-Glo mid-infrared / far-infrared light source, extended range potassium bromide beam splitter and deuterated triglycine sulfate (DTGS) detector An IR spectrum was obtained using an IR) spectrophotometer (Nicolet Instrument Corp.). Unless otherwise stated, Spectra-Tech, Inc. A diffuse reflection accessory (Collector ™) was used for sampling. Each spectrum represents a scan accumulated 256 times with a spectral resolution of 4 cm ⁻¹ . Compound sample preparation consisted of placing the sample in a microcup and flattening this material with a glass slide. A background data set was obtained using an alignment mirror mounted in the appropriate position. The spectrum represents the ratio of the sample single beam data set to the background single beam data set. The instrument wavelength calibration was performed using polystyrene.
NMR spectroscopy A ¹ H NMR spectrum of the solution phase was obtained at ambient temperature using a Bruker model AM-250 spectrometer operating at 5.87 T (Larmor frequency: ¹ H = 250 MHz). Time domain data was acquired over a spectral window of 5000 Hz using a pulse width of 7.5 ps and an acquisition time of 1.6834 seconds. A total of 16,384 data points were collected. A relaxation delay time of 5 seconds was used during the transition. Each data set typically consisted of 128 transients averaged together. The spectra were processed using GRAMS132 A1 software, version 6.00. The free induction decay (FID) was zero-filled to quadruple the number of data points and increased exponentially using a 0.61 Hz linewidth expansion factor, followed by Fourier transform. ^{The 1} H spectrum was based on tetramethylsilane (0 ppm) added as an internal standard.

Alternatively, NMR analysis was performed as described in Example 4.
Moisture Adsorption / Desorption Analysis Moisture adsorption / desorption data was collected using a VTI SGA-100 Vapor Sorption Analyzer. Adsorption / desorption data was collected at 10% RH intervals over a relative humidity (RK) range of 5% to 95% under a nitrogen purge. Sodium chloride (NaCl) and polyvinylpyrrolidone (PVP) were used as calibration standards. The equilibrium criterion used for the analysis was a change in weight of less than 0.0100% in 5 minutes and a maximum equilibrium time of 180 minutes if the weight criterion was not met. The plotted data is not corrected for the initial moisture content.
Nomenclature Compound (1- {9-[(4S, 2R, 3R, 5R) -3,4-Dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazole- The structure of 4-yl) -N-methylcarboxamide is as follows:

（１−｛９−［（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−３，４−ジヒドロキシ−５−（ヒドロキシメチル）オキソラン−２−イル］−６−アミノプリン−２−イル｝ピラゾール−４−イル）−Ｎ−メチルカルボキサミドの合成
（１−｛９−［（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−３，４−ジヒドロキシ−５−（ヒドロキシメチル）オキソラン−２−イル］−６−アミノプリン−２−イル｝ピラゾール−４−イル）−Ｎ−メチルカルボキサミドの大規模合成のための１つの方法を反応スキームＩに示す。すべての反応は通常、窒素雰囲気下で行われる。

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

工程１−式（２）の調製
式（２）の化合物は、式（１）の化合物から、溶媒の存在下でヒドラジン一水和物との反応によって調製される。反応は、約４５〜５５℃の温度で行われる。反応が終了すると、式（２）の生成物は、式（２）の化合物の溶解度が制限されているプロトン性溶媒、例えばエタノールまたはイソプロパノールを用いて撹拌することによって単離される。混合物を約１〜５時間撹拌し、次いでろ過する。固体は、水を用いて撹拌することと、ろ過することと、水で洗浄し、続いてイソプロパノールで洗浄することと、真空下で乾燥することとによって精製され、これは精製することなく次工程に用いられる。
工程２−式（３）の調製
式（２）の化合物は、次いで、約１〜１．２モル当量のエチル２−ホルミル−３−オキソプロピオネートと反応することによって、式（３）の化合物に変換される。反応は、プロトン性溶媒中、好ましくはエタノール中、約還流温度で約２〜４時間行われる。約０℃に冷却した後、固体をろ過分離し、冷エタノールで洗浄し、減圧下で乾燥させる。式（３）の生成物は、精製することなく次工程に用いられる。
工程３−最終生成物の調製
最終生成物は、式（３）の化合物から、メチルアミン、好ましくはメチルアミン水溶液と反応することによって調製される。反応は、約室温で、約４時間行われる。式Ｉの生成物は、従来の手段によって、例えば、ろ過することと、固体を冷エタノールで洗浄することと、減圧下で乾燥することとによって単離される。
出発物質の調製
（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−２−（６−アミノ−２−クロロプリン−９−イル）−５−（ヒドロキシメチル）オキソラン−３，４−ジオールが、工程１における出発物質として使用される。この化合物は市販されている。

Step 1—Preparation of Formula (2) The compound of formula (2) is prepared from the compound of formula (1) by reaction with hydrazine monohydrate in the presence of a solvent. The reaction is carried out at a temperature of about 45-55 ° C. When the reaction is complete, the product of formula (2) is isolated by stirring with a protic solvent in which the solubility of the compound of formula (2) is limited, 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, filtering, washing with water followed by washing with isopropanol and drying under vacuum, which is the next step without purification. Used for.
Step 2—Preparation of Formula (3) The compound of formula (2) is then reacted with about 1-1.2 molar equivalents of ethyl 2-formyl-3-oxopropionate to give a compound of formula (3) Converted to a compound. The reaction is carried out in a protic solvent, preferably in ethanol, at about reflux temperature for about 2-4 hours. After cooling to about 0 ° C., the solid is filtered off, washed with cold ethanol and dried under reduced pressure. The product of formula (3) is used in the next step without purification.
Step 3-Preparation of the final product The final product is prepared from the compound of formula (3) by reacting with methylamine, preferably with an aqueous methylamine solution. The reaction is carried out at about room temperature for about 4 hours. The product of formula I is isolated by conventional means, for example by filtration, washing the solid with cold ethanol and drying under reduced pressure.
Preparation of starting material (4S, 2R, 3R, 5R) -2- (6-Amino-2-chloropurin-9-yl) -5- (hydroxymethyl) oxolane-3,4-diol Used as a substance. This compound is commercially available.

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

エチル３，３−ジエトキシプロピオネートを、強塩基、好ましくは水素化ナトリウムの存在下でギ酸エチルと反応させる。反応は、約０〜５℃で、約２４時間行われる。生成物は、従来の手段によって、例えば、水を加え、従来の溶媒、例えばｔ−ブチルメチルエーテルで不純物を抽出することと、水相を例えば塩酸で酸性化することと、続いてジクロロメタンなどの溶媒で抽出することと、乾燥させた抽出物から溶媒を減圧下で除去することとによって単離される。エチル２−ホルミル−３−オキソプロパノエートは、減圧下で蒸留することによって精製される。

Ethyl 3,3-diethoxypropionate is reacted with ethyl formate in the presence of a strong base, preferably sodium hydride. The reaction is carried out at about 0-5 ° C. for about 24 hours. The product can be obtained by conventional means, for example by adding water and extracting impurities with a conventional solvent such as t-butyl methyl ether, acidifying the aqueous phase with eg hydrochloric acid, followed by dichloromethane and the like. Isolated by extracting with a solvent and removing the solvent from the dried extract under reduced pressure. Ethyl 2-formyl-3-oxopropanoate is purified by distillation under reduced pressure.

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

工程１−式（２）の調製
前述のように、式（２）の化合物は、式（１）の化合物から、溶媒の存在下でヒドラジン一水和物との反応によって調製される。しかしこの方法では、１４．３〜１６．７倍モル過剰のヒドラジン一水和物を、最初に約６０〜６５℃に加熱し、続いて式（１）の化合物を加える。温度は反応中、約６０〜６５℃に維持され、この反応は終了するまでに約１から３時間を要する。混合物中に残存する式（１）の化合物のレベルが約０．１０％以下である場合、次いで反応混合物は、約４０℃に冷却される。水がゆっくり加えられている間、温度は維持される。約４．２〜４．９質量当量が加えられると、混合物は約１０℃に冷却され、その温度で１時間以上維持される。

Step 1—Preparation of Formula (2) As described above, a compound of formula (2) is prepared from a compound of formula (1) by reaction with hydrazine monohydrate in the presence of a solvent. However, in this method, a 14.3-16.7-fold molar excess of hydrazine monohydrate is first heated to about 60-65 ° C. followed by the addition of the compound of formula (1). The temperature is maintained at about 60-65 ° C. during the reaction and the reaction takes about 1 to 3 hours to complete. If the level of compound of formula (1) remaining in the mixture is about 0.10% or less, then the reaction mixture is cooled to about 40 ° C. The temperature is maintained while water is slowly added. When about 4.2 to 4.9 mass equivalents are added, the mixture is cooled to about 10 ° C. and maintained at that temperature for over 1 hour.

The product is then isolated by filtration, washed with water and then with absolute ethanol. The solid is dried under vacuum at up to 30 ° C. for 12 hours or more and then used in the next step without further purification.
Step 2—Preparation of Formula (4) The compound of formula (2) is then in excess, eg, 2-10 fold excess, preferably about 5-10 fold excess, ideally about 6.8-7.5. Conversion to the compound of formula (4) by reaction with a fold excess of ethyl 2-formyl-3-oxopropionate. The reaction may be carried out under the same conditions as described for the preparation of the compound of formula (3) in Reaction Scheme I.

  Alternatively, about 0.05 molar equivalents of an acid such as HCl may be added to the reaction mixture. The reaction is carried out at reflux temperature for about 2-4 hours, the level of remaining compound of formula (2) is 0.50% or less, and the amount of any compound of formula (3) that can be formed is 2.5. Up to a state of less than or equal to%. After cooling to about 10 ° C., the solid is filtered off, washed with absolute ethanol and dried under vacuum at a maximum of 40 ° C. to remove residual ethanol. The product of formula (4) is used in the next step without purification.

  Compounds of formula (4) are depicted as (2E) alkene derivatives. This is because it is the major isomer formed in this reaction. However, it should be noted that significant amounts of (2Z) alkene derivatives can also be formed in this reaction. That is,

であり、エチル（２Ｚ）−３−（｛９−［（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−３，４−ジヒドロキシ−５−（ヒドロキシメチル）−オキソラン−２−イル］−２−［４−（エトキシカルボニル）ピラゾリル］プリン−６−イル｝アミノ）−２−ホルミルプロプ−２−エノエートと名付けられる。

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.

Hence, the compound of formula (4) is represented only as the (2E) alkene derivative, but the term “compound of formula (4)” is used when the compound is solely the (2E) isomer, and It is intended to include both where the major part of the product is the (2E) isomer and the minor part (2Z) isomer is also present. Conversion of the compound of formula (4) to the final product by reaction with methylamine, as described in Step 3, may occur if the compound of formula (4) exists as the (2E) isomer, or ( It may be present as a mixture of 2E) and (2Z) isomers, but in the same way.
Step 3—Preparation of the Final Product The final product is prepared from the compound of formula (4) by reaction with methylamine, preferably an aqueous methylamine solution. The reaction is first carried out at about 0-5 ° C. for about 8 hours, preferably in a pressurized reactor, followed by raising the temperature to 50-60 ° C. over about 1 hour and maintaining that temperature for 15-30 minutes. To do.

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

  At the end, the product is isolated by conventional means, for example by degassing under vacuum at about 35 ° C. or less to remove excess methylamine. The vacuum is then released and the mixture is cooled to about 0-5 ° C. and maintained at that temperature for 15 minutes to 1 hour, followed by filtration. The solid thus obtained is washed with water followed by ethanol and dried under reduced pressure at a temperature below about 40 ° C.

  This step comprises (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazole -4-yl) -N-methylcarboxamide is provided as its monohydrate. This polymorph is dissolved in dimethyl sulfoxide (DMSO), filtering any solid impurities from this solution, rinsing with additional DMSO, and adding to water to precipitate the monohydrate from the solution. Can be further purified. This method is particularly effective when the DMSO solution and water are heated to about 78-88 ° C. and maintained at that temperature for about 1 hour with stirring. After stirring, the slurry is slowly cooled to about 20 ° C. The final product is then isolated by filtration as described previously, washed with purified water followed by ethanol and dried.

Example 1
Preparation of ethyl-2-formyl-3-oxopropionate

磁気撹拌子、熱電対、デジタル温度計、ガス注入口および排出口ならびに添加用漏斗を備え付けた三つ口または四つ口丸底フラスコをアルゴンで洗い流した。エチル３，３−ジエトキシプロピオネート（６４．５ｇ）のテトラヒドロフラン中溶液を添加用漏斗に投入した。水素化ナトリウム（２１．２ｇの６０％分散物）を反応フラスコに投入し、続いてテトラヒドロフランを加えた。フラスコの内容物を氷浴中で０〜５℃に冷却し、ギ酸エチル（２５７ｇ）を加えた。混合物を０〜５℃に冷却し、５℃未満の内部温度を維持しながら、添加用漏斗の内容物を滴下した。氷浴を取り外し、内容物を周囲温度に温めた。エチル３，３−ジエトキシプロピオネートの消費を、ＴＬＣ分析によってモニターした。氷水（１０．６ｖｏｌ）を加えることによって反応物をクエンチし、メチルｔ−ブチルエーテルで３回抽出し（各５．４ｖｏｌ）、有機層を廃棄した。水相を濃塩酸でｐＨ１から１．５に酸性化した。酸性化した水層をジクロロメタンで３回抽出し、合わせた有機層を硫酸ナトリウムで乾燥させた。溶媒を減圧下で除去し、残渣を真空下で蒸留して、エチル−２−ホルミル−３−オキソプロピオネート２７．９２ｇを７０％収率で得た。

A three-necked or four-necked round bottom flask equipped with a magnetic stir bar, thermocouple, digital thermometer, gas inlet and outlet, and addition funnel was flushed with argon. A solution of ethyl 3,3-diethoxypropionate (64.5 g) in tetrahydrofuran was charged to the addition funnel. Sodium hydride (21.2 g of a 60% dispersion) was charged to the reaction flask followed by the addition of 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 addition funnel were added dropwise while maintaining an internal temperature below 5 ° C. The ice bath was removed and the contents were warmed to ambient temperature. The consumption of ethyl 3,3-diethoxypropionate was monitored by TLC analysis. The reaction was quenched by adding ice water (10.6 vol), extracted 3 times with methyl t-butyl ether (5.4 vol each), and the organic layer was discarded. The aqueous phase was acidified with concentrated hydrochloric acid to pH 1 to 1.5. The acidified aqueous layer was extracted 3 times with dichloromethane and the combined organic layers were dried over sodium sulfate. The solvent was removed under reduced pressure and the residue was distilled under vacuum to give 27.92 g of ethyl-2-formyl-3-oxopropionate in 70% yield.

(Example 2)
A. Preparation of 2-hydrazinoadenosine (2)

撹拌機、ガス注入口、ガス排出口および熱電対を備え付けたフラスコをアルゴンで洗い流した。２−クロロアデノシン半水和物（５３．１ｇ）を加え、続いてヒドラジン一水和物（１３４ｇ）を加えた。混合物を撹拌すると同時に、４０〜４５℃に２時間加熱した。反応の進行を、ＴＬＣ分析によって追跡した。反応が終了すると、熱源を取り外し、エタノール（８００ｍｌ）を加えた。混合物を周囲温度で２時間撹拌し、次いで沈殿物をろ過によって収集した。ろ過ケーキをエタノールで洗浄し、減圧下で３０分間乾燥させた。撹拌機を備え付けたきれいなフラスコに固体を移し、水（３００ｍｌ）を加えた。懸濁液を室温で１８時間撹拌し、固体をろ過によって単離した。ろ過ケーキを氷水（３００ｍｌ）で洗浄し、続いて氷冷エタノール（３００ｍｌ）で洗浄した。固体を減圧下で乾燥させて、２−ヒドラジノアデノシン（４１．３８ｇ、収率８１．４％、純度９９．３％）を得た。
Ｂ．２−ヒドラジノアデノシン（２）の代替調製
ヒドラジン水和物（２５８ｇ、２５０ｍｌ）を含む反応容器を４０〜５０℃に加熱した。温めた混合物に、４５〜５５℃の間の温度を維持しながら、２−クロロアデノシン半水和物（１００ｇ）を少しずつ加えた。温度をこの温度で２時間保ち、次いで、温度を４５〜５５℃で維持しながら、脱イオン水（５００ｍｌ）を３０分間かけて加えた。次いで混合物を３時間かけて０〜５℃に徐々に冷却し、次いでこの温度でさらに３０分間撹拌した。次いで固体をろ過分離し、冷（２〜５℃）脱イオン水（２００ｍｌ）で洗浄し、続いてエタノール（４００ｍｌ）で洗浄した。固体を真空下で１２時間乾燥させて、２−ヒドラジノアデノシンを得た。
Ｃ．２−ヒドラジノアデノシン（２）の代替調製
反応容器をヒドラジン水和物（１２８５ｇ）で満たす。溶液を約６２℃に加熱し、約６２℃の温度を維持しながら、２−クロロアデノシン（５００ｇ）を加える。混合物中に残存する２−クロロアデノシンのレベルが０．１０％以下である状態まで、混合物を６２℃を目標に、少なくとも２時間維持する。混合物を約４０℃に冷却し、混合物をチェックして、固体が存在していることを確認する。温度を約４０℃に維持しながら、水（２２７５ｇ）をゆっくり加える。混合物を約１０℃に冷却し、１時間以上維持する。生成物を、ろ過によって単離し、水（１１９５ｇ）で洗浄し、次いで無水エタノール（１８８５ｇ）で洗浄する。生成物を、真空下にて最大３０℃で１２時間以上乾燥させ、２−ヒドラジノアデノシンを得る。

The flask equipped with a stirrer, gas inlet, gas outlet and thermocouple was flushed with argon. 2-Chloroadenosine hemihydrate (53.1 g) was added, followed by hydrazine monohydrate (134 g). The mixture was stirred and heated to 40-45 ° C. for 2 hours. The progress of the reaction was followed by TLC analysis. When the reaction was completed, the heat source was removed and ethanol (800 ml) was added. The mixture was stirred at ambient temperature for 2 hours and then the precipitate was collected by filtration. The filter cake was washed with ethanol and dried for 30 minutes under reduced pressure. The solid was transferred to a clean flask equipped with a stirrer and water (300 ml) was added. The suspension was stirred at room temperature for 18 hours and the solid was isolated by filtration. The filter cake was washed with ice water (300 ml) followed by ice cold ethanol (300 ml). The solid was dried under reduced pressure to give 2-hydrazinoadenosine (41.38 g, yield 81.4%, purity 99.3%).
B. Alternative preparation of 2-hydrazinoadenosine (2) A reaction vessel containing hydrazine hydrate (258 g, 250 ml) was heated to 40-50 ° C. To the warmed mixture, 2-chloroadenosine hemihydrate (100 g) was added in portions while maintaining a temperature between 45-55 ° C. The temperature was maintained at this temperature for 2 hours and then deionized water (500 ml) was added over 30 minutes while maintaining the temperature at 45-55 ° C. The mixture was then gradually cooled to 0-5 ° C. over 3 hours and then stirred at this temperature for an additional 30 minutes. The solid was then filtered off and washed with cold (2-5 ° C.) deionized water (200 ml) followed by ethanol (400 ml). The solid was dried under vacuum for 12 hours to give 2-hydrazinoadenosine.
C. Alternative Preparation of 2-Hydrazinoadenosine (2) Fill the reaction vessel with hydrazine hydrate (1285 g). The solution is heated to about 62 ° C. and 2-chloroadenosine (500 g) is added while maintaining a temperature of about 62 ° C. The mixture is maintained at 62 ° C. for at least 2 hours until the level of 2-chloroadenosine remaining in the mixture is below 0.10%. Cool the mixture to about 40 ° C. and check the mixture to ensure that solids are present. Water (2275 g) is slowly added while maintaining the temperature at about 40 ° C. The mixture is cooled to about 10 ° C. and maintained for over 1 hour. The product is isolated by filtration, washed with water (1195 g) and then with absolute ethanol (1885 g). The product is dried under vacuum at a maximum of 30 ° C. for over 12 hours to give 2-hydrazinoadenosine.

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

撹拌機、ガス注入口、ガス排出口および還流冷却器を備え付けたフラスコに、エチル２−ホルミル−３−オキソプロピオネート（２３．９３ｇ、０．１７ｍｏｌ）を入れた。２−プロパノールをフラスコに加え、続いて２−ヒドラジノアデノシン（４４．４５ｇ、０．１５ｍｏｌ）を加えた。混合物を加熱し、撹拌下で２〜４時間還流させ、反応の進行をＴＬＣ分析によって追跡した。反応が終了したと判断されると、熱源を取り外し、混合物を室温に冷却した。懸濁液を撹拌下にて氷浴中で１．５から２時間冷却した。固体を真空ろ過によって単離し、氷冷２−プロパノールで洗浄した。生成物であるエチル１−｛９−［（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−３，４−ジヒドロキシ−５−（ヒドロキシメチル）オキソラン−２−イル］−６−アミノプリン−２−イル｝ピラゾール−４−カルボキシレートを、恒量に至るまで減圧下で乾燥させた。

Ethyl 2-formyl-3-oxopropionate (23.93 g, 0.17 mol) was placed in a flask equipped with a stirrer, gas inlet, gas outlet and reflux condenser. 2-Propanol was added to the flask followed by 2-hydrazinoadenosine (44.45 g, 0.15 mol). The mixture was heated and refluxed with stirring for 2-4 hours and the progress of the reaction was followed by TLC analysis. When the reaction was judged complete, the heat source was removed and the mixture was cooled to room temperature. The suspension was cooled in an ice bath with stirring for 1.5 to 2 hours. The solid was isolated by vacuum filtration and washed with ice cold 2-propanol. The product ethyl 1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazole The -4-carboxylate was dried under reduced pressure until a constant weight was reached.

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

エチル１−｛９−［（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−３，４−ジヒドロキシ−５−（ヒドロキシメチル）オキソラン−２−イル］−６−アミノプリン−２−イル｝ピラゾール−４−カルボキシレート（４６．４ｇ）およびメチルアミン（水中４０％、６００ｍｌ）の混合物を周囲温度で約４時間撹拌し、反応の進行をＨＰＬＣ分析によって追跡した。過剰なメチルアミンの大部分を減圧下で除去し、残りの混合物を０℃で２時間冷却した。固体物質をろ過分離し、氷冷した２００プルーフエタノールで洗浄し、減圧下で乾燥させて、（１−｛９−［（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−３，４−ジヒドロキシ−５−（ヒドロキシメチル）オキソラン−２−イル］−６−アミノプリン−２−イル｝ピラゾール−４−イル）−Ｎ−メチルカルボキサミドをその一水和物として得た。

Ethyl 1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazole-4-carboxy A mixture of the rate (46.4 g) and methylamine (40% in water, 600 ml) was stirred at ambient temperature for about 4 hours and the progress of the reaction was followed by HPLC analysis. Most of the excess methylamine was removed under reduced pressure and the remaining mixture was cooled at 0 ° C. for 2 hours. The solid material was separated by filtration, washed with ice-cold 200 proof ethanol and dried under reduced pressure to give (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- ( Hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide was obtained as its monohydrate.

The structure of this material was confirmed by ¹ H NMR (see FIG. 1 and below). The result of the thermal analysis obtained (see FIG. 2) was consistent with the presence of one molecule of water. An X-ray powder diffraction pattern was obtained (FIG. 3).

^１Ｈおよび^１３Ｃ ＮＭＲスペクトルを、以下のようにして得た。上記で得られた物質の２つのサンプルを量り分け、ｄ_６−ＤＭＳＯに溶かした。５．３ｍｇは^１Ｈスペクトル用に使用し、２０．８ｍｇは^１３Ｃスペクトル用に使用した。^１Ｈについては４００ＭＨｚで、および^１３Ｃについては１００ＭＨｚで稼働するＪＥＯＬ Ｅｃｌｉｐｓｅ^＋４００分光計を使用して、すべてのスペクトルを周囲温度で得た。

¹ H and ¹³ C NMR spectra 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 the ¹ H spectrum and 20.8 mg was used for the ¹³ C spectrum. All spectra were acquired at ambient temperature using a JEOL Eclipse ⁺ 400 spectrometer operating at 400 MHz for ¹ H and 100 MHz for ¹³ C.

（実施例５）
Ａ．エチル（２Ｅ）−３−（｛９−［（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−３，４−ジヒドロキシ−５−（ヒドロキシメチル）−オキソラン−２−イル］−２−［４−（エトキシカルボニル）ピラゾリル］プリン−６−イル｝アミノ）−２−ホルミルプロプ−２−エノエート（４）の調製

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

２−ヒドラジノアデノシン（１００ｇ、０．３４ｍｏｌ）、エチル２−ホルミル−３−オキソプロピオネート（２４２ｇ、１．７ｍｏｌ）および無水エタノールの混合物を反応器に投入し、この混合物を加熱して２時間還流させた。反応が終了したと判断されると、熱源を取り外し、混合物を３時間かけて５〜１０℃に徐々に冷却した。スラリーをこの温度で３０分間撹拌し、混合物をろ過した。固体物質を冷（５〜１０℃）無水エタノールで洗浄し、次いで真空下にて４０℃を超えない温度で乾燥させて、エチル（２Ｅ）−３−（｛９−［（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−３，４−ジヒドロキシ−５−（ヒドロキシメチル）オキソラン−２−イル］−２−［４−（エトキシカルボニル）−ピラゾリル］プリン−６−イル｝アミノ）−２−ホルミルプロプ−２−エノエートを得た。
Ｂ．エチル（２Ｅ）−３−（｛９−［（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−３，４−ジヒドロキシ−５−（ヒドロキシメチル）−オキソラン−２−イル］−２−［４−（エトキシカルボニル）ピラゾリル］プリン−６−イル｝アミノ）−２−ホルミルプロプ−２−エノエート（４）の代替調製
反応容器を２−ヒドラジノアデノシン（４５０ｇ）および無水エタノール（１１３７６ｇ）で満たす。ＨＣｌ（７．４７ｇ）およびエチル２−ホルミル−３−オキソプロピオネート（１５５７ｇ）を加える。混合物を加熱して還流させ、サンプル採取し、混合物中に残存する２−ヒドラジノアデノシンのレベルが０．５０％以下であり、式（３）の化合物のレベルが２．５％以下である状態まで行う。混合物を約１０℃にゆっくり冷却する。生成物である式（４）の化合物をろ過によって単離し、無水エタノール（５１２１ｇ）で洗浄する。生成物を真空下にて最大４０℃で乾燥させ、残存するエタノールが５０００ｐｐｍ以下である状態まで行い、エチル（２Ｅ）−３−（｛９−［（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−３，４−ジヒドロキシ−５−（ヒドロキシメチル）オキソラン−２−イル］−２−［４−（エトキシカルボニル）−ピラゾリル］プリン−６−イル｝アミノ）−２−ホルミルプロプ−２−エノエートを得る。

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 to the reactor and the mixture was heated to 2 Reflux for hours. When the reaction was judged complete, the heat source was removed and the mixture was gradually cooled to 5-10 ° C. over 3 hours. The slurry was stirred at this temperature for 30 minutes and the mixture was filtered. The solid material is washed with cold (5-10 ° C.) absolute ethanol and then dried under vacuum at a temperature not exceeding 40 ° C. to give 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- I got enoate.
B. Ethyl (2E) -3-({9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) -oxolan-2-yl] -2- [4- (ethoxycarbonyl Alternative preparation of)) pyrazolyl] purin-6-yl} amino) -2-formylprop-2-enoate (4) The reaction vessel is filled with 2-hydrazinoadenosine (450 g) and absolute ethanol (11376 g). Add HCl (7.47 g) and ethyl 2-formyl-3-oxopropionate (1557 g). The mixture is heated to reflux, sampled, and the level of 2-hydrazinoadenosine remaining in the mixture is below 0.50% and the level of the compound of formula (3) is below 2.5% Do until. Cool the mixture slowly to about 10 ° C. The product, compound of formula (4), is isolated by filtration and washed with absolute ethanol (5121 g). The product is dried under vacuum at a maximum of 40 ° C. until the residual ethanol is 5000 ppm or less, and 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 is obtained.

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

¹ H and ¹³ C NMR spectra were obtained as follows. Dissolve 20.2 mg of the compound of formula (4) in about 0.75 ml DMSO-d6 and use a JEOL ECX-400 NMR spectrometer operating at 400 MHz for ¹ H and 100 MHz for ¹³ C, and the spectra Obtained at ambient temperature. Chemical shifts were referenced to DMSO solvent, 2.50 ppm for ¹ H and 39.5 ppm for ¹³ C.
result
The chemical shifts for ¹ H and ¹³ C are listed in Table 1. Two isomers present in a ratio of about 60/30 were found in both ¹ H and ¹³ C spectra and were classified as major and minor in the table.

式（４）の化合物は、以下の２つの異性体

The compound of formula (4) has the following two isomers:

の混合物であることが確認された。

It was confirmed to be a mixture of

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

４０％メチルアミン水溶液（１３００ｍｌ）を加圧反応器に入れ、０〜５℃に冷却し、実施例５Ａの生成物（エチル（２Ｅ）−３−（｛９−［（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−３，４−ジヒドロキシ−５−（ヒドロキシメチル）オキソラン−２−イル］−２−［４−（エトキシカルボニル）ピラゾリル］プリン−６−イル｝アミノ）−２−ホルミルプロプ−２−エノエート（１００ｇ）を加えた。混合物を０〜５℃で少なくとも８時間撹拌し、終了を得るために反応をモニターした。終了すると、混合物を温め、５０〜６０℃の間の温度を１時間維持し、次いで１時間かけて３０℃未満に冷却した。温度が３０℃未満になると、１００〜１５０ｍｍＨｇの圧力を使用して混合物を脱気し、温度を０〜５℃に下げた。１００〜１５０ｍｍＨｇの圧力を維持しながら、混合物を０〜５℃で少なくとも１時間撹拌した。次いで、真空を中止して窒素で置換し、温度を０〜５℃で３０分間以上維持した。次いで、固体生成物をろ過分離し、水（５００ｍｌ×３回）で洗浄し、次いで無水エタノール（６２５ｍｌ）で洗浄した。生成物を、真空下で温度が４０℃を超えないようにしながら乾燥させて、（１−｛９−［（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−３，４−ジヒドロキシ−５−（ヒドロキシメチル）オキソラン−２−イル］−６−アミノプリン−２−イル｝ピラゾール−４−イル）−Ｎ−メチルカルボキサミドをその一水和物として得た。
Ｂ．化合物（４）からの（１−｛９−［（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−３，４−ジヒドロキシ−５−（ヒドロキシメチル）オキソラン−２−イル］−６−アミノプリン−２−イル｝ピラゾール−４−イル）−Ｎ−メチルカルボキサミドの代替調製
加圧容器を４７％メチルアミン水溶液（１００８０ｇ）で満たす。溶液を冷却し、５℃の目標温度を維持しながら、上述の実施例５Ｂにおいて調製された式（４）の化合物（６００ｇ）を加える。混合物を５℃の目標温度で撹拌し、混合物中に残存する式（３）の化合物のレベルが０．１０％未満である状態まで行う。反応混合物を真空下にて３５℃以下で脱気して過剰なメチルアミンを除去する。真空を解き、混合物を２．５℃に冷却し、少なくとも３０分間維持する。次いで、生成物をろ過によって単離し、水（９０００ｇ以上）で洗浄し、次いで無水エタノール（２９６４ｇ以上）で洗浄する。生成物を真空下にて最大４０℃で乾燥させ、残存するエタノールが５０００ｐｐｍ以下である状態まで行い、粗（１−｛９−［（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−３，４−ジヒドロキシ−５−（ヒドロキシメチル）オキソラン−２−イル］−６−アミノプリン−２−イル｝ピラゾール−４−イル）−Ｎ−メチルカルボキサミドをその一水和物として得る。

A 40% aqueous methylamine solution (1300 ml) was placed in a pressure reactor, cooled to 0-5 ° C., and the product of Example 5A (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 ( 100 g) was added, the mixture was stirred at 0-5 ° C. for at least 8 hours, and the reaction was monitored to obtain completion, upon completion the mixture was warmed and maintained at a temperature between 50-60 ° C. for 1 hour, It was then cooled over 1 hour to below 30 ° C. When the temperature was below 30 ° C., the mixture was degassed using a pressure of 100-150 mmHg and the temperature was reduced to 0-5 ° C. The pressure of 100-150 mmHg The mixture was stirred for at least 1 hour at 0-5 ° C. while maintaining the vacuum, then the vacuum was turned off and replaced with nitrogen, and the temperature was maintained over 30 minutes at 0-5 ° C. The solid product was then filtered Separated and washed with water (3 x 500 ml) and then with absolute ethanol (625 ml) The product was dried under vacuum with the temperature not exceeding 40 ° C. (1- {9 -[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl) -N-methyl Carboxamide was obtained as its monohydrate.
B. (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl] from compound (4) } Alternative Preparation of Pyrazol-4-yl) -N-methylcarboxamide Fill a pressurized vessel with 47% aqueous methylamine (10080 g). Cool the solution and add the compound of formula (4) (600 g) prepared in Example 5B above while maintaining the target temperature of 5 ° C. The mixture is stirred at a target temperature of 5 ° C. until the level of the compound of formula (3) remaining in the mixture is less than 0.10%. The reaction mixture is degassed under 35 ° C. under vacuum to remove excess methylamine. The vacuum is released and the mixture is cooled to 2.5 ° C. and maintained for at least 30 minutes. The product is then isolated by filtration, washed with water (9000 g or more) and then with absolute ethanol (2964 g or more). The product is dried under vacuum at a maximum of 40 ° C. until the residual ethanol is less than or equal to 5000 ppm, and crude (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy- 5- (Hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide is obtained as its monohydrate.

¹ H and ¹³ C NMR spectra were obtained as follows. Two samples of the material obtained in Example 6A were weighed out and dissolved in d ₆ -DMSO. 5.3 mg was used for the ¹ H spectrum and 20.8 mg was used for the ¹³ C spectrum. All spectra were acquired at ambient temperature using a JEOL Eclipse ⁺ 400 spectrometer operating at 400 MHz for ¹ H and 100 MHz for ¹³ C.

元素分析によって、以下の結果を得た：Ｃ、４３．９６％；Ｈ、４．９４％；Ｎ、２７．９４。理論上：Ｃ、４４．１２％；Ｈ、４．９４％；Ｎ、２７．４４％；Ｏ、２７．０９。分析は、一水和物に対する実験誤差限界内に相当する。

Elemental analysis gave the following results: C, 43.96%; H, 4.94%; N, 27.94. Theoretical: C, 44.12%; H, 4.94%; N, 27.44%; O, 27.09. The analysis corresponds to within experimental error limits for monohydrate.

(Example 7)
A. (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl ) Purification of -N-methylcarboxamide monohydrate (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolane- prepared in Example 4) A solution of 2-yl] -6-aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide monohydrate (100 g) in dimethyl sulfoxide (300 ml) was added to 0.6 to 0.8 microns. And filtered through a 0.2 micron filter to remove any solid impurities. The filtrate was then slowly added to deionized water (1 liter) over 1 hour with stirring, and the slurry thus produced was stirred for over 1 hour. The solid was separated by filtration, washed with deionized water (1 liter x 2), and dried under vacuum for 1 hour or more.

The dried product is then re-slurried with deionized water (1.5 liters) for more than 2 hours, filtered off and washed with deionized water (1 liter) followed by absolute ethanol (750 ml). Washed with. The purified product is dried under vacuum at a temperature of 40 ° C. or lower for 12 hours or more, and is free of 2-hydrazinoadenosine impurity (1- {9-[(4S, 2R, 3R, 5R) -3,4-Dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide monohydrate was obtained.
B. (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl ) Alternative purification of -N-methylcarboxamide monohydrate (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6 A solution of -aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide monohydrate (400 g) was dissolved in DMSO (1320 g), and the solution was 0.6 to 0.8 microns and 0. Filter through a 2 micron in-line filter in sequence. Rinse the filter system using additional DMSO (880 g). Slowly add the solution to purified water (5000 g) while maintaining a target temperature of 83 ° C. The product begins to crystallize during the addition and the slurry is stirred for about 1 hour at a target temperature of 83 ° C. Cool the mixture slowly to 20 ° C. The product is isolated by filtration and washed with purified water (8000 g).

を合成する方法であって、式（４）

の化合物をメチルアミン水溶液と、約２．５〜７．５℃の温度で接触させることによる方法。
（項２）
反応を密閉加圧反応器中で行う、上記項１に記載の方法。
（項３）
生成物を、
（ａ）真空下にて３５℃以下で脱気して過剰なメチルアミンを除去することと、
（ｂ）前記真空を解き、０〜５℃に約１５分から１時間冷却することと、
（ｃ）こうして形成されたスラリーをろ過することと、
（ｄ）フィルターの内容物を水で洗浄し、続いてエタノールで洗浄することと、
（ｅ）残った固体を真空下にて４０℃を超えない温度で乾燥することと
によって単離する、上記項２に記載の方法。
（項４）
前記（１−｛９−［（４Ｓ，２Ｒ，３Ｒ，５Ｒ）−３，４−ジヒドロキシ−５−（ヒドロキシメチル）オキソラン−２−イル］−６−アミノプリン−２−イル｝ピラゾール−４−イル）−Ｎ−メチルカルボキサミドの最終生成物を、
（ｉ）上記項３に記載の工程（ｅ）由来の前記乾燥固体を溶媒に溶かすことと、
（ｉｉ）前記溶液からいずれの固体不純物もろ過することと、
（ｉｉｉ）追加の溶媒ですすぐことと、
（ｉｖ）約７８〜８８°に維持されている精製水に溶液を加えることによって、スラリーを形成することと、
（ｖ）前記スラリーを撹拌することと、
（ｖｉ）前記スラリーを冷却することと、
（ｖｉｉ）ろ過することと、
（ｖｉｉｉ）フィルターの内容物を水で洗浄し、続いてエタノールで洗浄することと、
（ｉｘ）残った固体を真空下にて４０℃を超えない温度で乾燥することと
によってさらに精製する、上記項３に記載の方法。
（項５）
工程（ｉ）および（ｉｉｉ）において使用される前記溶媒がジメチルスルホキシドである、上記項４に記載の方法。
（項６）
最終生成物中に残存する水分含量が５．５％以下であり、残存するエタノールが２０００ｐｐｍ以下である、上記項５に記載の方法。
（項７）
式（４）

の化合物を合成する方法であって、
式（２）

の化合物を、任意選択で酸の存在下で、過剰のエチル２−ホルミル−３−オキソプロピオネートと接触させることを含む方法。
（項８）
反応をエタノール中で行う、上記項６に記載の方法。
（項９）
前記酸はＨＣｌであり、最大０．１モル当量までを使用する、上記項６に記載の方法。
（項１０）
反応を還流下で行う、上記項９に記載の方法。
（項１１）
約５〜１０倍モル過剰のエチル２−ホルミル−３−オキソプロピオネートを使用する、上記項６に記載の方法。
（項１２）
約６．８〜７．５倍モル過剰のエチル２−ホルミル−３−オキソプロピオネートを使用する、上記項１１に記載の方法。
（項１３）
生成物を、
（ａ）反応が終了した混合物を約１０℃に冷却することと、
（ｂ）ろ過することと、
（ｃ）フィルターの内容物をエタノールで洗浄することと、
（ｄ）残った固体を真空下にて４０℃を超えない温度で乾燥することと
によって単離する、上記項１２に記載の方法。
（項１４）
式（２）

の化合物の調製方法であって、
式（１）

の化合物を１４．３〜１６．７倍モル過剰のヒドラジンと、約６０〜６５℃の温度で反応させることを含む方法。
（項１５）
前記ヒドラジンを、最初に約６０〜６５℃に加熱し、続いて式（１）の前記化合物を加える、上記項１４に記載の方法。
（項１６）
式（２）の前記化合物を、
（ａ）前記反応混合物を約４０℃に冷却することと、
（ｂ）温度を約４０℃で維持しながら、４．２〜４．９質量当量の水を加えることと、
（ｃ）前記混合物を約１０℃に冷却し、その温度で少なくとも約１時間維持することと、（ｄ）ろ過することと、
（ｅ）フィルターの内容物を水で洗浄し、続いてエタノールで洗浄することと、
（ｆ）残った固体を真空下にて３０℃を超えない温度で少なくとも１２時間乾燥することと
によって単離する、上記項１４に記載の方法。 The solid is put into a container and purified water (6000 g) is added. The slurry is mixed for about 1 hour. The product is isolated by filtration, washed with purified water (4000 g) and then with absolute ethanol (3160 g). The product is dried under vacuum at a maximum of 40 ° C. until the residual water content is 5.5% or less and the residual ethanol is 2000 ppm or less, (1- {9-[(4S, 2R , 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl) -N-methylcarboxamide monohydrate. obtain.
In a preferred embodiment of the present invention, the following is provided.
(Claim 1)
(1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-yl ) -N-methylcarboxamide

Wherein the formula (4)

By contacting the compound of claim 1 with an aqueous methylamine solution at a temperature of about 2.5-7.5 ° C.
(Section 2)
Item 2. The method according to Item 1, wherein the reaction is carried out in a closed pressure reactor.
(Section 3)
Product
(A) degassing under vacuum at 35 ° C. or less to remove excess methylamine;
(B) breaking the vacuum and cooling to 0-5 ° C. for about 15 minutes to 1 hour;
(C) filtering the slurry thus formed;
(D) washing the contents of the filter with water followed by ethanol;
(E) The method according to Item 2, wherein the remaining solid is isolated by drying under vacuum at a temperature not exceeding 40 ° C.
(Claim 4)
Said (1- {9-[(4S, 2R, 3R, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-aminopurin-2-yl} pyrazole-4- Yl) -N-methylcarboxamide final product
(I) dissolving the dry solid derived from step (e) according to item 3 in a solvent;
(Ii) filtering any solid impurities from the solution;
(Iii) rinsing with additional solvent;
(Iv) forming a slurry by adding the solution to purified water maintained at about 78-88 °;
(V) stirring the slurry;
(Vi) cooling the slurry;
(Vii) filtering;
(Viii) washing the contents of the filter with water followed by ethanol;
(Ix) The method according to Item 3, wherein the remaining solid is further purified by drying under vacuum at a temperature not exceeding 40 ° C.
(Section 5)
Item 5. The method according to Item 4, wherein the solvent used in steps (i) and (iii) is dimethyl sulfoxide.
(Claim 6)
Item 6. The method according to Item 5, wherein the water content remaining in the final product is 5.5% or less and the remaining ethanol is 2000 ppm or less.
(Claim 7)
Formula (4)

A method of synthesizing a compound of
Formula (2)

Contacting with an excess of ethyl 2-formyl-3-oxopropionate, optionally in the presence of an acid.
(Section 8)
Item 7. The method according to Item 6, wherein the reaction is carried out in ethanol.
(Claim 9)
Item 7. The method of Item 6, wherein the acid is HCl and a maximum of 0.1 molar equivalent is used.
(Section 10)
Item 10. The method according to Item 9, wherein the reaction is carried out under reflux.
(Item 11)
Item 7. The method according to Item 6, wherein an about 5- to 10-fold molar excess of ethyl 2-formyl-3-oxopropionate is used.
(Clause 12)
Item 12. The method according to Item 11, wherein an about 6.8 to 7.5-fold molar excess of ethyl 2-formyl-3-oxopropionate is used.
(Section 13)
Product
(A) cooling the finished reaction mixture to about 10 ° C .;
(B) filtering,
(C) washing the contents of the filter with ethanol;
(D) The method according to Item 12, wherein the remaining solid is isolated by drying under vacuum at a temperature not exceeding 40 ° C.
(Item 14)
Formula (2)

A method for preparing a compound of
Formula (1)

And a 14.3 to 16.7-fold molar excess of hydrazine at a temperature of about 60-65 ° C.
(Section 15)
Item 15. The method of Item 14, wherein the hydrazine is first heated to about 60-65 ° C, followed by addition of the compound of formula (1).
(Section 16)
Said compound of formula (2)
(A) cooling the reaction mixture to about 40 ° C .;
(B) adding 4.2 to 4.9 mass equivalents of water while maintaining the temperature at about 40 ° C;
(C) cooling the mixture to about 10 ° C. and maintaining at that temperature for at least about 1 hour; (d) filtering;
(E) washing the contents of the filter with water followed by ethanol;
(F) The process of paragraph 14, wherein the remaining solid is isolated by drying under vacuum at a temperature not exceeding 30 ° C. for at least 12 hours.

Claims (1)

Invention described in this specification or drawings .

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