2-{4-[(methylamino)carbonyl]- 1H-pyrazol-1-yl}adenosine

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

US FDA:link

313348-27-5  875148-45-1

CV Therapeutics (Originator), Fujisawa (Licensee)

Regadenoson (INN, code named CVT-3146) is an A_2A adenosine receptor agonist that is a coronary vasodilator. It produces hyperemia quickly and maintains it for a duration that is useful for radionuclide myocardial perfusion imaging.^[1]

It was approved by the United States Food and Drug Administration on April 10, 2008 and is marketed by Astellas Pharma under the tradename Lexiscan.^[2] It is approved for use in the European Union and under the name of Rapiscan. It is currently being marketed by GE Healthcare and is being sold in both the United Kingdom and Germany.

Regadenoson has a 2- to 3-minute biological half-life, as compared with adenosine‘s 30-second half-life. Regadenoson stress protocols using a single bolus have been developed, obviating the need for an intravenous line. Regadenoson stress tests are not affected by the presence of beta blockers, as regadenoson vasodilates but does not stimulate beta adrenergic receptors.

Regadenoson is an A_2A adenosine receptor agonist that is a coronary vasodilator [see CLINICAL PHARMACOLOGY]. Regadenoson is chemically described as adenosine, 2-[4-[(methylamino)carbonyl]-1H-pyrazol-1-yl]-, monohydrate. Its structural formula is:

The molecular formula for regadenoson is C₁₅H₁₈N₈O₅ • H₂O and its molecular weight is 408.37. Lexiscan is a sterile, nonpyrogenic solution for intravenous injection. The solution is clear and colorless. Each 1 mL in the 5 mL pre-filled syringe contains 0.084 mg of regadenoson monohydrate, corresponding to 0.08 mg regadenoson on an anhydrous basis, 10.9 mg dibasic sodium phosphate dihydrate or 8.7 mg dibasic sodium phosphate anhydrous, 5.4 mg monobasic sodium phosphate monohydrate, 150 mg propylene glycol, 1 mg edetate disodium dihydrate, and Water for Injection, with pH between 6.3 and 7.7.

Regadenoson is also referred to in the literature as CVT- 3146 or (1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6- aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide and has the formula:

Methods for synthesizing regadenoson and related compounds are set forth in U.S. Patent No. 6,403,567, the specification of which is incorporated herein by reference in its entirety.

Regadenoson may be administered by pharmaceutical administration methods that are known in the art. It is preferred that regadenoson is dosed i.v. It is more preferred that regadenoson is administered in a single dose i.v. The term “single dose” refers generally to a single quickly administered dose of a therapeutic amount of regadenoson. The term “single dose” does not encompass a dose or doses administered over an extended period of time by, for example continuous i.v. infusion.

Regadenoson will typically be incorporated into a pharmaceutical composition prior to use. The term “pharmaceutical composition” refers to the combination of regadenoson with at least one liquid carrier that together form a solution or a suspension. Lyophilized powders including compositions of this invention fall within the scope of “pharmaceutical compositions” so long as the powders are intended to be reconstituted by the addition of a suitable liquid carrier prior to use. Examples of suitable liquid carriers include, but are not limited to water, distilled water, de-ionized water, saline, buffer solutions, normal isotonic saline solution, dextrose in water, and combinations thereof.

Regadenoson [(l-{9-[(4S, 2R, 3R, 5R)-3,4-dihydroxy-5-(hydroxymethyl)oxalan-2-yl]-6- aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamine] is a selective A₂A-adenosine receptor agonist that is a coronary vasodilator. It is currently marketed in the form of a monohydrate as a pharmacologic stress agent indicated for radionuclide myocardial perfusion imaging (MPI) in patients unable to undergo adequate exercise stress.

U.S. Patent No. 8,106,183 describes amorphous regadenoson, and three forms of regadenoson, referred to as Form A (a monohydrate), Form B and Form C.

The synthesis of regadenoson is described, for example, in U.S. Patent Nos. 6,403,567 and 7,183,264. The syntheses disclosed are multi-step processes that proceed via 2- hydrazinoadenosine, which is prepared from the corresponding iodo-derivative (2- iodoadenosine).

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http://www.google.com/patents/WO2012149196A1?cl=en

EXAMPLE 1

Synthesis of N-Methyl-4-carboxamide

20 g (143 mmol, 1 equiv) of ethyl pyrazole-4-carboxylate and 200 mL (2310 mmol, 16.2 equiv) of a 40 % aqueous solution of methylamine were added to a three-necked flask equipped with a condenser and a heating mantle. The mixture was stirred to aid dissolution, and heated to 65 °C for 2 hours. The reaction was monitored using HPLC at 220 nm with a C18 column. The reaction mixture was then concentrated in vacuo to obtain a syrup / solid. The crude product was co-evaporated with acetonitrile (3 x 200 mL). 100 mL of acetonitrile was then added to the solids and the mixture was stirred for several hours until the solids were well suspended. The solids were then isolated by filtration, washed with 100 mL acetonitrile, and dried in an oven at 40°C to afford 14.4 g (80 % yield) of N-methyl-4-carboxamide with a purity of 93.5% by HPLC.

EXAMPLE 2

Synthesis of IDAAR-Cu⁺²

This preparation has reported in the literature. See, e.g., Chinese Chemical Letters, (21(1), 51-54, 2010.

An Erlenmeyer flask was charged with 350 mL of water and 75 g of Chelex 100 resin. With stirring, an aqueous solution of copper sulfate pentahydrate (59 g in 350 mL of water) was slowly added over a period of 15 minutes. The resulting slurry was stirred for 2 hours, then filtered. The resulting solids were washed with 100 – 200 mL of water and dried in a vacuum oven at 50 °C for 16 hours to afford 18 g of IDAAR-Cu⁺². The copper content of the product was determined to be 11 wt % using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES).

EXAMPLE 3

Synthesis of Regadenoson Monohydrate

5 g (17.5 mmol, 1 equiv) of 2-fluoroadenosine, 3.07g (24.5 mmol, 1.4 equiv) of N- methylpyrazole-4-carboxamide, and 32 mL of dimethylsulfoxide were added under a nitrogen atmosphere to a dry 3-necked reaction flask equipped with a condenser and a heating mantle.. The mixture was stirred to afford a solution. 100 mL of acetonitrile was then added followed by the addition of 2.2 g of IDAAR-Cu²⁺ and 5.34 g (5.24 mL, 35.1 mmol, 2 equiv) of

diazabicycloundecene (DBU). The reaction mixture was heated to 70 – 80 °C overnight and monitored by HPLC at 260 nm with a C18 column until the reaction was complete. Then, the reaction mixture was evaporated in vacuo to remove most of the acetonitrile. The remaining dimethylsufoxide solution was purified by reverse phase chromatography using methanol and water. The product was dried in vacuo at a temperature that did not exceed 40° C to afford 3 g (44% yield) of regadenoson monohydrate.

EXAMPLE 4

Synthesis of 2-Hydazineadenosine

2-fluoroadenosine (4g, 14 mmol) was dissolved in 100 mL ethanol in a 300 mL three- necked flask. Hydrazine hydrate (4.1 mL, 6 equivalents, 84 mmol) was added and the mixture was heated to reflux for 1 hour. The reaction mixture was allowed to cool to room temperature and stirred overnight (16 hours). The resulting white precipitate was isolated by filtration and dried in oven at 40°C overnight to afford 2-hydrazinoadenosine (yield: 94%, 3.5g, 96% purity).

EXAMPLE 5

Synthesis of Regadenoson Form D

2-Fluoroadenosine (45 g, 0.158 moL, 1 eq.), 4-(N-methylcarboxamido)pyrazole (27.64 g, 0.221 moL, 1.4 eq.), dimethylsulfoxide (DMSO) (320 mL) and acetonitrile (960 mL) were added to a dry 3000 ml 3-neck reaction flask equipped with a condenser and heating mantle. After stirring for 10 minutes, IDAAR-Cu (20.07 g, 0.032 moL, 0.2 eq.) and DBU (48.0 g, 0.316 moL, 2 eq.) were added. The resulting mixture was then heated to 65°C overnight (18 hours).

The reaction mixture was then filtered and the filtrate was evaporated followed by 2 x 500 mL co-evaporation with xylene. The residue was diluted with 5 L acetonitrile, transferred to a 10 L flask and kept in a cold room (4°C) overnight. The resulting white precipitate was isolated by filtration and stirred in 1.8 L of water. The mixture was heated to 80° C for 2 hours, then allowed to cool in a cold room (4°C) overnight.

The white precipitate was isolated by filtration, then dissolved in 200 ml of 1 : 1 mixture of DMSO and methanol. The clear and slightly yellow solution was loaded to a reverse phase column (10 L) and eluted with water/methanol (gradient with a 5% increase of MeOH every 10 L).

The fractions with HPLC purity of more than 99.9% were combined and concentrated to a paste. The supernatant liquid was decanted and the flask heated in an oil-bath at 150° C under reduced pressure of 20mmHg for 6 hours to afford 6.2 g of Regadenoson Form D as white solid (99.94% HPLC, KF analysis 0.8%).

The fractions with HPLC purity between 50 and 99.8% (~ 23g of product as indicated by HPLC) were combined and subjected to a second purification stage.

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WO 0078779

https://www.google.com/patents/WO2000078779A2?cl=en

Example 5

(l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2- yl}pyrazol-4-yl)-N-methylcarboxamide (16)

Compound 12 (0.05 mg, 0.12 mmol) was added to 4 mL methylamine (40% sol. In water). The mixture heated at 65 °C in for 24 h. After concentration in vacuo, the residue was purified using prep. TLC (10% MeOH:DCM). ‘HNMR (CD₃OD) 62.90 (s, 3 H), 3.78 (m, 1

H), 3.91 (m, 1 H), 4.13 (d, 1 H), 4.34 (d, 1 H), 4.64 (m, 1 H), 6.06 (d, 1 H), 8.11 (s, 1 H), 8.38

(s, 1 H), 9.05 (s, 1 H).

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https://www.google.com/patents/US6403567

U.S. Patent Nos. 6,403,567

Scheme 1.

Compound I can be prepared by reacting compound 1 with appropriately substituted 1,3 -dicarbonyl in a mixture of AcOH and MeOH at 80° C. (Holzer et al., J. Heterocycl. Chem. (1993) 30, 865). Compound II, which can be obtained by reacting compound I with 2,2-dimethoxypropane in the presence of an acid, can be oxidized to the carboxylic acid III, based on structurally similar compounds using potassium permanganate or pyridinium chlorochromate (M. Hudlicky, (1990) Oxidations in Organic Chemistry, ACS Monographs, American Chemical Society, Washington D.C.). Reaction of a primary or secondary amine having the formula HNR⁶R⁷, and compound III using DCC (M. Fujino et al., Chem. Pharm. Bull. (1974), 22, 1857), PyBOP (J. Martinez et al., J. Med. Chem. (1988) 28, 1874) or PyBrop (J. Caste et al. Tetrahedron, (1991), 32, 1967) coupling conditions can afford compound IV.

Compound V can be prepared as shown in Scheme 2. The Tri TBDMS derivative 4 can be obtained by treating compound 2 with TBDMSCl and imidazole in DMF followed by hydrolysis of the ethyl ester using NaOH. Reaction of a primary or secondary amine with the formula HNR⁶R⁷, and compound 4 using DCC (M. Fujino et al., Chem. Pharm. Bull. (1974), 22, 1857), PyBOP (J. Martinez et al., J. Med. Chem. (1988) 28, 1874) or PyBrop (J. Caste et al. Tetrahedron, (1991), 32, 1967) coupling conditions can afford compound V.

A specific synthesis of compound 11 is illustrated in Scheme 3. Commercially available guanosine 5 was converted to the triacetate 6 as previously described (M. J. Robins and B. Uznanski, Can. J. Chem. (1981), 59, 2601-2607). Compound 7, prepared by following the literature procedure of Cerster et al. (J. F. Cerster, A. F. Lewis, and R. K. Robins, Org. Synthesis, 242-243), was converted to compound 9 in two steps as previously described (V. Nair et al., J. Org. Chem., (1988), 53, 3051-3057). Compound 1 was obtained by reacting hydrazine hydrate with compound 9 in ethanol at 80° C. Condensation of compound 1 with ethoxycarbonylmalondialdehyde in a mixture of AcOH and MeOH at 80° C. produced compound 10. Heating compound 10 in excess methylamine afforded compound 11.

The synthesis of 1,3-dialdehyde VII is described in Scheme 4. Reaction of 3,3-diethoxypropionate or 3,3-diethoxypropionitrile or 1,1-diethoxy-2-nitroethane VI (R₃=CO₂R, CN or NO₂) with ethyl or methyl formate in the presence of NaH can afford the dialdehyde VII (Y. Yamamoto et al., J. Org. Chem. (1989) 54, 4734).

EXAMPLE 5

(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2 -yl}pyrazol-4N-methylcarboxamide which can also be identified as 2-(4-methylaminocarbonylpyrazol-1-yl)adenosine (16)

The mixture heated at 65° C. in for 24 h. After concentration in vacuo, the residue was purified using prep. TLC (10% MeOH:DCM). ¹HNMR (CD₃OD) δ2.90 (s, 3 H), 3.78 (m, 1 H), 3.91 (m, 1 H), 4.13 (d, 1 H), 4.34 (d, 1 H), 4.64 (m, 1 H), 6.06 (d, 1 H), 8.11 (s, 1 H), 8.38 (s, 1 H), 9.05 (s, 1 H).

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US 7,183,264

http://www.google.com/patents/US7183264

EXAMPLE 5

(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide (16)

Compound 12 (0.05 mg, 0.12 mmol) was added to 4 mL methylamine (40% sol. In water). The mixture heated at 65° C. in for 24 h. After concentration in vacuo, the residue was purified using prep. TLC (10% MeOH:DCM). ¹HNMR (CD₃OD) δ2.90 (s, 3 H), 3.78 (m, 1 H), 3.91 (m, 1 H), 4.13 (d, 1 H), 4.34 (d, 1 H), 4.64 (m, 1 H), 6.06 (d, 1 H), 8.11 (s, 1 H), 8.38 (s, 1 H), 9.05 (s, 1 H).

References

NEW PATENT

Novel process for the preparation of (1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl)-6-aminopurin-2-yl}pyrazole-4-yl)-N-methylcarboxamide

WO-2014068589

Biophore India Pharmaceuticals Pvt Ltd