WO2004082822A2 - Methods for isolating crystalline form i of 5-azacytidine - Google Patents

Methods for isolating crystalline form i of 5-azacytidine Download PDF

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Publication number
WO2004082822A2
WO2004082822A2 PCT/US2004/007896 US2004007896W WO2004082822A2 WO 2004082822 A2 WO2004082822 A2 WO 2004082822A2 US 2004007896 W US2004007896 W US 2004007896W WO 2004082822 A2 WO2004082822 A2 WO 2004082822A2
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Prior art keywords
solvent
azacytidine
propanol
isolating
dimethylsulfoxide
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PCT/US2004/007896
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French (fr)
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WO2004082822A3 (en
Inventor
Dumitru Ionescu
Peter Blumbergs
Gary L. Silvey
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Pharmion Corporation
Ash Stevens Inc
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Priority to EP04721078.6A priority Critical patent/EP1608634B1/en
Priority to ES04721078T priority patent/ES2425474T3/en
Priority to SI200432065T priority patent/SI1608634T1/en
Priority to BRPI0408506-0A priority patent/BRPI0408506A/en
Priority to MXPA05009969A priority patent/MXPA05009969A/en
Application filed by Pharmion Corporation, Ash Stevens Inc filed Critical Pharmion Corporation
Priority to AU2004222274A priority patent/AU2004222274B2/en
Priority to DK04721078.6T priority patent/DK1608634T3/en
Priority to CA002518973A priority patent/CA2518973A1/en
Priority to PL04721078T priority patent/PL1608634T3/en
Priority to JP2006507211A priority patent/JP4792550B2/en
Publication of WO2004082822A2 publication Critical patent/WO2004082822A2/en
Publication of WO2004082822A3 publication Critical patent/WO2004082822A3/en
Priority to AU2010241198A priority patent/AU2010241198B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/12—Triazine radicals

Definitions

  • the invention relates to the isolation of crystalline polymorphic Form I of 5-azacytidine (also known as azacitidine and 4-amino-l- ⁇ -D-ribofuranosyl-S-triazin-2(lH)-one).
  • 5- azacytidine may be used in the treatment of disease, including the treatment of myelodysplastic syndromes (MDS).
  • Polymorphs exist as two or more crystalline phases that have different arrangements and/or different conformations of the molecule in a crystal lattice.
  • a solvent molecule(s) is contained within the crystal lattice the resulting crystal is called a pseudopolymorph, or solvate.
  • the solvent molecule(s) within the crystal structure is a water molecule, then the pseudopolymorph/solvate is called a hydrate.
  • the polymorphic and pseudopolymorphic solids display different physical properties, including those due to packing, and various thermodynamic, spectroscopic, interfacial and mechanical properties (See ⁇ . Brittain,
  • API active pharmaceutical ingredient
  • 5-Azacytidine also known as azacitidine and 4-amino-l- ⁇ -D-ribofu ⁇ -anosyl-S-triazin- 2(lH)-one; National Service Center designation NSC-102816; CAS Registry Number 320-67-2) has undergone NCI-sponsored trials for the treatment of myelodysplastic syndromes (MDS).
  • MDS myelodysplastic syndromes
  • 5-azacytidine may be defined as having a formula of
  • Forms I-NIII polymorphic and pseudopolymorphic forms of 5-azacytidine
  • XRPD X-Ray Powder Diffraction
  • 5-azacytidine drug substance used in the previous clinical trials has typically been synthesized from 5-azacytosine and 1,2,3,5,-tetra-O-acetyl- ⁇ -D-ribofuranose by the method presented in Example 1.
  • the last step of this method is a recrystallization of the crude synthesis product from a methanol/DMSO co-solvent system. Specifically, the crude synthesis product is dissolved in DMSO (preheated to about 90°C), and then methanol is added to the DMSO solution. The product is collected by vacuum filtration and allowed to air dry.
  • the present invention provides methods for robustly and reproducibly isolating 5- azacytidine as polymorphic Form I substantially free of other forms.
  • the methods involve recrystallizing dissolved 5-azacytidine from a primary solvent/co-solvent mixture and then collecting the resultant crystals.
  • the invention also provides pharmaceutical compositions comprising Form I of 5-azacytidine together with a pharmaceutically acceptable excipient, diluent, or carrier.
  • Form I of 5-azacytidine may be reproducibly isolated substantially free of other forms by recrystallizing dissolved 5-azacytidine and collecting the resultant crystals.
  • 5- azacytidine is first dissolved completely in at least one suitable primary solvent, preferably a polar solvent, more preferably a polar aprotic solvent.
  • suitable polar aprotic solvents include, but are not limited to, dimethylformamide (DMF), dimethylacetamide (DMA), dimethylsulfoxide (DMSO), and N-methylpyrrolidinone (NMP).
  • the most preferred polar aprotic solvent is DMSO.
  • Mixtures of two or more primary solvents are also contemplated for dissolving the 5-azacytidine, for example a mixture of DMSO and DMF.
  • the 5-azacytidine used to form the solution may be synthesized by any procedure known in the art; an exemplary prior art synthesis scheme is provided in Example 1. Any polymorphic or pseudopolymorphic form(s) of 5-azacytidine, including mixed phases, may be used to form the solution. Amorphous 5-azacytidine may also be used to form the solution. It is preferred, but not required, that the primary solvent is preheated to an elevated temperature in order to ensure that the 5-azacytidine is dissolved completely.
  • An especially preferred primary solvent is dimethyl sulfoxide, (DMSO), most preferably preheated to a temperature in the range of about 40°C to about 90°C.
  • co-solvents include C 2 -C 5 alcohols (which term hereinafter refers to C 2 -C 5 alcohols that are independently: branched or unbranched, substituted or unsubstituted), aliphatic ketones (which term hereinafter refers to aliphatic ketones that are indepedently: branched or unbranched, substituted or unsubstituted), and alkyl cyanides (which term hereinafter refers to alkyl cyanides that are independently: branched or unbranched, substituted or unsubstituted).
  • Class 2 aliphatic ketones, and alkyl cyanides, along with other suitable solvents, are listed below as Class 2 (solvents to be limited) and Class 3 (solvents of low toxic potential) per the International Conference on Harmonization's (ICH) Guideline for Residual Solvents, July 1997).
  • ICH International Conference on Harmonization's
  • co-solvents are preheated before mixing with the primary solvent, preferably to a temperature below the temperature at which a substantial portion of the co-solvent would boil, most preferably to about 50°C. It is also preferred, but not required, that the co-solvent(s) is added gradually to the primary solvent(s).
  • the primary solvent(s)/co-solvent(s) mixture is then equilibrated at different temperatures in order to promote either a slow recrystallization or a fast recrystallization of Form I of 5-azacytidine, as described below.
  • slow recrystallization is meant that the co-solvent/DMSO solution is allowed to equilibrate at a temperature in the range from about 0°C to about 40°C, preferably in the range of about 15°C to about 30°C, and most preferably at about ambient temperature.
  • Slow recrystallization of Form I of 5-azacytidine is preferably performed using C 2 -C 5 alcohols, aliphatic ketones, or alkyl cyanides as the co-solvent. More preferably, slow recrystallization is performed with Class 3 C 2 -C 5 alcohols, Class 3 aliphatic ketones, or acetonitrile (Class 2).
  • the most preferred Class 3 C 2 -C 5 alchohols are ethanol, isopropyl alcohol, and 1 -propanol, and the most preferred Class 3 aliphatic ketone is methylethyl ketone.
  • fast recrystallization is meant that the co-solvent solution is allowed to equilibrate at a temperature of below 0°C, preferably below about -10°C, and most preferably at about -20°C.
  • Fast recrystallization of Form I of 5-azacytidine is preferably performed with a C 3 - C 5 alcohol (which term hereinafter refers to C -C 5 alcohols which are independently: branched or unbranched, substituted or unsubstituted) or an alkyl cyanide as the co-solvent. More preferably the C 3 - C 5 alcohol is a Class 3 solvent, and the alkyl cyanide is acetonitrile. The most preferred Class 3 C 3 -C 5 alcohols are isopropyl alcohol (2 -propanol) and 1 -propanol.
  • Non-limiting examples of protocols for the recrystallization of Form I according to the methods described herein are provided in Examples 2 (slow recrystallization with DMSO as the primary solvent and ethanol, isopropyl alcohol, acetonitrile, or methylethyl ketone as the co-solvent) and 3 (fast recrystallization with DMSO as the primary solvent, and isopropyl alcohol or acetonitrile as the co-solvent) below.
  • the Form I of 5-azacytidine crystals may be isolated from the co-solvent mixture by any suitable method known in the art.
  • the Form I crystals are isolated using vacuum filtration through a suitable filter medium or by centrifugation.
  • Form I of 5-azacytidine as the drug substance reproducibly and robustly.
  • isopropyl alcohol and acetonitrile reliably produce Form I independent of cooling rate (either slow recrystallization or fast recrystallization) and are preferred as the recrystallization co-solvents to recover Form I.
  • Form I is isolated using isopropyl alcohol as the co- solvent since isopropyl alcohol carries a Class 3 risk classification (solvent of low toxic potential), whereas acetonitrile carries a Class 2 risk classification (solvent to be limited).
  • Form I of 5-azacytidine may be reliably recovered for the first time from solvents of low toxic potential without requiring control over the rate of recrystallation.
  • Form I of 5-azacytidine may be recovered simply by dissolving 5-azacytidine in DMSO (preferably heated to a temperature in the range of about 40°C to about 90°C prior to the addition of 5-azacytidine), adding isopropyl alcohol, and allowing the resulting solvent mixture to equilibrate at about ambient temperature.
  • Form I of 5-azacytidine may be recovered from a primary solvent(s)/co-solvent(s) mixture by "seeding" with a small amount of Form I of 5- azacytidine either prior to, or during, the addition of the co-solvent(s).
  • seeding with Form I, it is possible to expand the list of suitable co-solvents and co-solvent classes beyond those listed above. For example, it is known that recrystallization from the DMSO/ ethanol system produces either Form I, or a Form I/II mixed phase (see Example 1).
  • Form I of 5- azacytidine may be reliably isolated.
  • the present invention allows for the first time the production of 5-azacytidine drug substance with uniform and consistent properties from batch to batch, which properties include but are not limited to solubility and dissolution rate. In turn, this allows one to provide 5-azacytidine drug product (see below) which also has uniform and consistent properties from batch to batch.
  • compositions for the most effective administration of drug substance of the present invention, it is preferred to prepare a pharmaceutical formulation (also known as the "drug product” or “pharmaceutical composition") preferably in unit dose form, comprising one or more of the 5- azacytidine polymorphs of the present invention and one or more pharmaceutically acceptable carrier, diluent, or excipient. Most preferably, Form I 5-azacytidine prepared according to the methods provided herein is used to prepare the pharmaceutical formulation.
  • Such pharmaceutical formulation may, without being limited by the teachings set forth herein, include a solid form of the present invention which is blended with at least one pharmaceutically acceptable excipient, diluted by an excipient or enclosed within such a carrier that can be in the form of a capsule, sachet, tablet, buccal, lozenge, paper, or other container.
  • a pharmaceutically acceptable excipient diluted by an excipient or enclosed within such a carrier that can be in the form of a capsule, sachet, tablet, buccal, lozenge, paper, or other container.
  • the excipient serves as a diluent, it may be a solid, semi-solid, or liquid material which acts as a vehicle, carrier, or medium for the 5-azacytidine polymorph(s).
  • the formulations can be in the form of tablets, pills, powders, elixirs, suspensions, emulsions, solutions, syrups, capsules (such as, for example, soft and hard gelatin capsules), suppositories, sterile injectable solutions, and sterile packaged powders.
  • excipients include, but are not limited to, starches, gum arabic, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose.
  • the formulations can additionally include lubricating agents such as, for example, talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propyl- hydroxybenzoates; sweetening agents; or flavoring agents.
  • lubricating agents such as, for example, talc, magnesium stearate and mineral oil
  • wetting agents such as talc, magnesium stearate and mineral oil
  • emulsifying and suspending agents such as methyl- and propyl- hydroxybenzoates
  • sweetening agents or flavoring agents.
  • Polyols, buffers, and inert fillers may also be used.
  • polyols examples include, but are not limited to: mannitol, sorbitol, xylitol, sucrose, maltose, glucose, lactose, dextrose, and the like.
  • Suitable buffers encompass, but are not limited to, phosphate, citrate, tartrate, succinate, and the like.
  • Other inert fillers which may be used encompass those which are known in the art and are useful in the manufacture of various dosage forms.
  • the solid pharmaceutical compositions may include other components such as bulling agents and/or granulating agents, and the like.
  • the compositions of the invention can be formulated so as to provide quick, sustained, controlled, or delayed release of the drug substance after administration to the patient by employing procedures well l ⁇ iown in the art.
  • the 5-azacytidine polymorph(s) may made into the form of dosage units for oral administration.
  • the 5-azacytidine polymorph(s) may be mixed with a solid, pulverant carrier such as, for example, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives or gelatin, as well as with an antifriction agent such as for example, magnesium stearate, calcium stearate, and polyethylene glycol waxes.
  • a solid, pulverant carrier such as, for example, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives or gelatin
  • an antifriction agent such as for example, magnesium stearate, calcium stearate, and polyethylene glycol waxes.
  • coated tablets, capsules, or pulvules may be coated with a concentrated solution of sugar, which may contain gum arabic, gelatin, talc, titanium dioxide, or with a lacquer dissolved in the volatile organic solvent or mixture of solvents.
  • a concentrated solution of sugar which may contain gum arabic, gelatin, talc, titanium dioxide, or with a lacquer dissolved in the volatile organic solvent or mixture of solvents.
  • various dyes may be added in order to distinguish among tablets with different active compounds or with different amounts of the active compound present.
  • Soft gelatin capsules may be prepared in which capsules contain a mixture of the 5- azacytidine polymorph(s) and vegetable oil or non-aqueous, water miscible materials such as, for example, polyethylene glycol and the like.
  • Hard gelatin capsules may contain granules or powder of the 5-azacytidine polymorph in combination with a solid, pulverulent carrier, such as, for example, lactose, saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives, or gelatin. Tablets for oral use are typically prepared in the following manner, although other techniques may be employed.
  • the solid substances are gently ground or sieved to a desired particle size, and a binding agent is homogenized and suspended in a suitable solvent.
  • the 5- azacytidine polymorph(s) and auxiliary agents are mixed with the binding agent solution.
  • the resulting mixture is moistened to form a uniform suspension.
  • the moistening typically causes the particles to aggregate slightly, and the resulting mass is gently pressed through a stainless steel sieve having a desired size.
  • the layers of the mixture are then dried in controlled drying units for a pre-determined length of time to achieve a desired particle size and consistency.
  • the granules of the dried mixture are gently sieved to remove any powder.
  • disintegrating, anti-friction, and anti-adhesive agents are added.
  • the mixture is pressed into tablets using a machine with the appropriate punches and dies to obtain the desired tablet size.
  • such a formulation typically comprises sterile, aqueous and non-aqueous injection solutions comprising one or more 5-azacytidine polymorphs for which preparations are preferably isotonic with the blood of the intended recipient.
  • preparations may contain anti- oxidants, buffers, bacteriostats, and solute; which render the formulation isotonic with the blood of the intended recipient.
  • Aqueous and non-aqueous suspensions may include suspending agents and thickening agents.
  • the formulations may be present in unit-dose or multi-dose containers, for example, sealed ampules and vials.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Liquid preparations for oral administration are prepared in the form of solutions, syrups, or suspensions with the latter two forms containing, for example, 5-azacytidine polymorph(s), sugar, and a mixture of ethanol, water, glycerol, and propylene glycol. If desired, such liquid preparations contain coloring agents, flavoring agents, and saccharin. Thickening agents such as carboxymethylcellulose may also be used.
  • the pharmaceutical formulations of the present invention are preferably prepared in a unit dosage form, each dosage unit containing from about 5 mg to about 200 mg, more usually about 100 mg of the 5-azacytidine polymorph(s).
  • dosage unit contains from about 5 to about 200 mg, more usually about 100 mg of the 5-azacytidine polymorph(s).
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human subjects/patients or other mammals, each unit containing a predetermined quantity of the 5-azacytidine polymorph calculated to produce the desired therapeutic effect, in association with preferably, at least one pharmaceutically acceptable carrier, diluent, or excipient.
  • 5-azacytidine may be synthesized using commercially available 5-azacytosine and 1,2,3,5- tetra-O-acetyl- ⁇ -D-ribofuranose (RTA) according to the following pathway:
  • the crude synthesis product is dissolved in DMSO (preheated to about 90°C), and then methanol is added to the DMSO solution.
  • the co-solvent mixture is equilibrated at approximately -20°C to allow 5-azacytidine crystal formation.
  • the product is collected by vacuum filtration and allowed to air dry.
  • Form I of 5-azacvtidine Slow Recrystallization of 5-azacytidine from Co-Solvent Systems Approximately 250 mg of 5-azacytidine was dissolved with approximately 5 ml of dimethyl sulfoxide (DMSO), preheated to approximately 90 °C, in separate 100-mL beakers.
  • DMSO dimethyl sulfoxide
  • 5-azacytidine was dissolved with approximately 5 mL of DMSO, preheated to approximately 90 °C, in separate 100-ml beakers. The solids were allowed to dissolve to a clear solution. Approximately 45 mL of isopropyl alcohol or acetonitrile co-solvent, preheated to approximately 50 °C, was added to the solution and the resultant solution was mixed. The solution was covered and placed in a freezer to equilibrate at approximately -20°C to allow crystal formation. Solutions were removed from the freezer after crystal formation. The product was collected by vacuum filtration using a Buchner funnel.
  • X-ray powder diffraction (XRPD) patterns for each sample were obtained on a Scintag XDS 2000 or a Scintag X 2 ⁇ / ⁇ diffractometer operating with copper radiation at 45 kN and 40 mA using a Kevex Psi Peltier-cooled silicon detector or a Thermo ARL Peltier-cooled solid state detector. Source slits of 2 or 4 mm and detector slits of 0.5 or 0.3 mm were used for data collection. Recrystallized material was gently milled for approximately one minute using an agate mortar and pestle. Samples were placed in a stainless steel or silicon sample holder and leveled using a glass microscope slide. Powder diffraction patterns of the samples were obtained from 2 to 42° 2 ⁇ at l°/minute. Calibration of the X 2 diffractometer is verified annually using a silicon powder standard.
  • XRPD performed according to this method revealed that the Form I of 5-azacytidine was isolated in Example 2 by slow recrystallization using either ethanol, isopropyl alcohol, acetonitrile, or methyl ethyl ketone as the co-solvent, and in Example 3 by fast recrystallization using isopropyl alcohol or acetonitrile as the co-solvent.
  • the results indicate that Form I of 5-azacytidine may be reliably recovered from the DMSO/isopropyl alcohol and DMSO/acetonitrile solvent systems without control of the rate of recrystallization.

Abstract

The invention includes methods for isolating crystalline Form I of 5-azacytidine substantially free of other forms, wherein 5-azacytidine is represented by the formula: The invention also includes pharmaceutical compositions comprising Form I of 5-azacytidine.

Description

METHODS FOR ISOLATING CRYSTALLINE FORM I OF
5-AZACYTIDINE
Field of the Invention
The invention relates to the isolation of crystalline polymorphic Form I of 5-azacytidine (also known as azacitidine and 4-amino-l-β-D-ribofuranosyl-S-triazin-2(lH)-one). 5- azacytidine may be used in the treatment of disease, including the treatment of myelodysplastic syndromes (MDS).
Background of the Invention
Polymorphs exist as two or more crystalline phases that have different arrangements and/or different conformations of the molecule in a crystal lattice. When a solvent molecule(s) is contained within the crystal lattice the resulting crystal is called a pseudopolymorph, or solvate. If the solvent molecule(s) within the crystal structure is a water molecule, then the pseudopolymorph/solvate is called a hydrate. The polymorphic and pseudopolymorphic solids display different physical properties, including those due to packing, and various thermodynamic, spectroscopic, interfacial and mechanical properties (See Η. Brittain,
Polymorphism in Pharmaceutical Solids, Marcel Dekker, New York, NY, 1999, pp. 1-2). Polymorphic and pseudopolymorphic forms of the drug substance (also lαiown as the "active pharmaceutical ingredient" (API)), as administered by itself or formulated as a drug product (also known as the final or finished dosage form, or as the pharmaceutical composition) are well lαiown and may affect, for example, the solubility, stability, flowability, fractability, and compressibility of drug substances and the safety and efficacy of drug products, (see, e.g., Knapman, K Modern Drug Discoveries, March 2000: 53).
5-Azacytidine (also known as azacitidine and 4-amino-l-β-D-ribofuι-anosyl-S-triazin- 2(lH)-one; Nation Service Center designation NSC-102816; CAS Registry Number 320-67-2) has undergone NCI-sponsored trials for the treatment of myelodysplastic syndromes (MDS).
See Kornblith et al., J. Clin. Oncol. 20(10): 2441-2452 (2002) and Silverman et al, J. Clin.
Oncol. 20(10): 2429-2440 (2002). 5-azacytidine may be defined as having a formula of
C8Ηι N O5, a molecular weight of 244.20 and a structure of:
Figure imgf000003_0001
In the United States Patent Application Serial No. 10/390,578 entitled "Forms of 5- azacytidine," filed March 17, 2003 and incorporated herein by reference in its entirety, eight different polymorphic and pseudopolymorphic forms of 5-azacytidine (Forms I-NIII), in addition to an amoiphous form, are described. Forms I-NIII each have characteristic X-Ray Powder Diffraction (XRPD) patterns and are easily distinguished from one another using XRPD.
5-azacytidine drug substance used in the previous clinical trials has typically been synthesized from 5-azacytosine and 1,2,3,5,-tetra-O-acetyl-β-D-ribofuranose by the method presented in Example 1. The last step of this method is a recrystallization of the crude synthesis product from a methanol/DMSO co-solvent system. Specifically, the crude synthesis product is dissolved in DMSO (preheated to about 90°C), and then methanol is added to the DMSO solution. The product is collected by vacuum filtration and allowed to air dry.
In In the United States Patent Application Serial No. 10/390,578 entitled "Forms of 5- azacytidine," filed March 17, 2003 and incorporated herein by reference in its entirety, it is demonstrated that this prior art method for the recrystallization of the crude synthesis product does not control for the polymorphic forms of 5-azacytidine. Specifically, the prior art recrystallization procedure produces either Form I substantially free of other forms, or a Form I/II mixed phase i.e. a solid material in which 5-azacytidine is present in a mixed phase of both polymorphic Form I and polymorphic Form II. Thus, the prior art procedures do not allow one to reliably target Form I as the single polymorphic form in the drug substance. The present invention provides methods that allow one to recrystallize 5-azacytidine as polymorphic Form I robustly and reproducibly. Summary of the Invention
The present invention provides methods for robustly and reproducibly isolating 5- azacytidine as polymorphic Form I substantially free of other forms. The methods involve recrystallizing dissolved 5-azacytidine from a primary solvent/co-solvent mixture and then collecting the resultant crystals. The invention also provides pharmaceutical compositions comprising Form I of 5-azacytidine together with a pharmaceutically acceptable excipient, diluent, or carrier.
Detailed Description of the Preferred Embodiments
Polymorphic Form I of 5-azacytidine
Form I of 5-azacytidine is described in United States Patent Application Serial No. 10/390,578 entitled "Forms of 5-azacytidine," filed March 17, 2003 and incorporated herein by reference in its entirety. Table 1 provides the most prominent 2Θ angles, d-spacing and relative intensities for Form I observed using X-Ray Powder Diffraction (XRPD) performed according the method of Example 4:
20 Angle (°) d-spacing (A) Relative Intensity
12.182 7.260 39.1
13.024 6.792 44.1
14.399 6.146 31.5
16.470 5.378 27.1
18.627 4.760 16.0
19.049 4.655 35.9
20.182 4.396 37.0
21.329 4.162 12.4
23.033 3.858 100.0
23.872 3.724 28.0
26.863 3.316 10.8
27.135 3.284 51.5
29.277 3.048 25.6
29.591 3.016 11.5
30.369 2.941 10.8
32.072 2.788 13.4
Table 1 : 5-azacytidine Form I - the most prominent 2Θ angles, d-spacing and relative intensities (Cu Kα radiation) Isolation of Polymorphic Form I of 5-azacytidine by Recrystallization
Form I of 5-azacytidine may be reproducibly isolated substantially free of other forms by recrystallizing dissolved 5-azacytidine and collecting the resultant crystals. Specifically, 5- azacytidine is first dissolved completely in at least one suitable primary solvent, preferably a polar solvent, more preferably a polar aprotic solvent. Suitable polar aprotic solvents include, but are not limited to, dimethylformamide (DMF), dimethylacetamide (DMA), dimethylsulfoxide (DMSO), and N-methylpyrrolidinone (NMP). The most preferred polar aprotic solvent is DMSO. Mixtures of two or more primary solvents are also contemplated for dissolving the 5-azacytidine, for example a mixture of DMSO and DMF.
The 5-azacytidine used to form the solution may be synthesized by any procedure known in the art; an exemplary prior art synthesis scheme is provided in Example 1. Any polymorphic or pseudopolymorphic form(s) of 5-azacytidine, including mixed phases, may be used to form the solution. Amorphous 5-azacytidine may also be used to form the solution. It is preferred, but not required, that the primary solvent is preheated to an elevated temperature in order to ensure that the 5-azacytidine is dissolved completely. An especially preferred primary solvent is dimethyl sulfoxide, (DMSO), most preferably preheated to a temperature in the range of about 40°C to about 90°C.
Following solvation of the 5-azacytidine in the primary solvent, at least one co-solvent is added to the solution of 5-azacytidine. Suitable co-solvents include C2-C5 alcohols (which term hereinafter refers to C2-C5 alcohols that are independently: branched or unbranched, substituted or unsubstituted), aliphatic ketones (which term hereinafter refers to aliphatic ketones that are indepedently: branched or unbranched, substituted or unsubstituted), and alkyl cyanides (which term hereinafter refers to alkyl cyanides that are independently: branched or unbranched, substituted or unsubstituted). Preferred C2-C5 alcohols, aliphatic ketones, and alkyl cyanides, along with other suitable solvents, are listed below as Class 2 (solvents to be limited) and Class 3 (solvents of low toxic potential) per the International Conference on Harmonization's (ICH) Guideline for Residual Solvents, July 1997). The use of mixtures of two or more of any of the aforementioned co-solvents is also included within the scope of the invention. Class 2
Acetonitrile
Chlorobenzene
Cyclohexane
1 ,2-Dichloroethene Dichloromethane
1 ,2-Dimethoxyethane
N,N-Dimethylformamide
N,N-Dimethylacetamide
1,4-Dioxane 2-Ethoxyethanol
Ethyleneglycol
Formamide
2-Methoxyethanol
Methylbutyl ketone Methylcyclohexane
Nitromethane
Pyridine
Sulfolane
Tetralin 1,1,2-Trichloroethene
Class 3
1-Butanol
1-Pentanol 1-Propanol
2-Butanol
2-Methyl- 1 -propanol
2-Propanol (isopropyl alcohol)
3-Methyl- 1 -butanol Acetone
Anisole
Butyl acetate
Cumene Ethanol
Ethyl acetate
Ethyl ether
Ethyl formate
Isobutyl acetate Isopropyl acetate
Methyl acetate
Methylethyl ketone
Methylisobutyl ketone
Propyl acetate tert-Butylmethyl ether
Tetrahydrofuran
It is preferred, but not required, that the co-solvents are preheated before mixing with the primary solvent, preferably to a temperature below the temperature at which a substantial portion of the co-solvent would boil, most preferably to about 50°C. It is also preferred, but not required, that the co-solvent(s) is added gradually to the primary solvent(s).
Following mixing, the primary solvent(s)/co-solvent(s) mixture is then equilibrated at different temperatures in order to promote either a slow recrystallization or a fast recrystallization of Form I of 5-azacytidine, as described below.
By slow recrystallization is meant that the co-solvent/DMSO solution is allowed to equilibrate at a temperature in the range from about 0°C to about 40°C, preferably in the range of about 15°C to about 30°C, and most preferably at about ambient temperature. Slow recrystallization of Form I of 5-azacytidine is preferably performed using C2-C5 alcohols, aliphatic ketones, or alkyl cyanides as the co-solvent. More preferably, slow recrystallization is performed with Class 3 C2-C5 alcohols, Class 3 aliphatic ketones, or acetonitrile (Class 2). The most preferred Class 3 C2-C5 alchohols are ethanol, isopropyl alcohol, and 1 -propanol, and the most preferred Class 3 aliphatic ketone is methylethyl ketone.
By fast recrystallization is meant that the co-solvent solution is allowed to equilibrate at a temperature of below 0°C, preferably below about -10°C, and most preferably at about -20°C. Fast recrystallization of Form I of 5-azacytidine is preferably performed with a C3 - C5 alcohol (which term hereinafter refers to C -C5 alcohols which are independently: branched or unbranched, substituted or unsubstituted) or an alkyl cyanide as the co-solvent. More preferably the C3 - C5 alcohol is a Class 3 solvent, and the alkyl cyanide is acetonitrile. The most preferred Class 3 C3-C5 alcohols are isopropyl alcohol (2 -propanol) and 1 -propanol.
Non-limiting examples of protocols for the recrystallization of Form I according to the methods described herein are provided in Examples 2 (slow recrystallization with DMSO as the primary solvent and ethanol, isopropyl alcohol, acetonitrile, or methylethyl ketone as the co-solvent) and 3 (fast recrystallization with DMSO as the primary solvent, and isopropyl alcohol or acetonitrile as the co-solvent) below.
Following recrystallization, the Form I of 5-azacytidine crystals may be isolated from the co-solvent mixture by any suitable method known in the art. Preferably, the Form I crystals are isolated using vacuum filtration through a suitable filter medium or by centrifugation.
Using the novel methods provided herein, it is possible for the first time to target Form I of 5-azacytidine as the drug substance reproducibly and robustly. In particular, isopropyl alcohol and acetonitrile reliably produce Form I independent of cooling rate (either slow recrystallization or fast recrystallization) and are preferred as the recrystallization co-solvents to recover Form I. Most preferably, Form I is isolated using isopropyl alcohol as the co- solvent since isopropyl alcohol carries a Class 3 risk classification (solvent of low toxic potential), whereas acetonitrile carries a Class 2 risk classification (solvent to be limited). The use of the DMSO/isopropyl alcohol system allows Form I of 5-azacytidine to be reliably recovered for the first time from solvents of low toxic potential without requiring control over the rate of recrystallation. In the most preferred embodiment, Form I of 5-azacytidine may be recovered simply by dissolving 5-azacytidine in DMSO (preferably heated to a temperature in the range of about 40°C to about 90°C prior to the addition of 5-azacytidine), adding isopropyl alcohol, and allowing the resulting solvent mixture to equilibrate at about ambient temperature.
In some embodiments of the invention, Form I of 5-azacytidine may be recovered from a primary solvent(s)/co-solvent(s) mixture by "seeding" with a small amount of Form I of 5- azacytidine either prior to, or during, the addition of the co-solvent(s). By seeding with Form I, it is possible to expand the list of suitable co-solvents and co-solvent classes beyond those listed above. For example, it is known that recrystallization from the DMSO/ ethanol system produces either Form I, or a Form I/II mixed phase (see Example 1). If a small amount of Form I is added to the solution of 5-azacytidine in DMSO prior to addition of the methanol co- solvent, or is added during the addition of the methanol co-solvent, then Form I of 5- azacytidine may be reliably isolated.
By allowing the isolation of a single polymorphic form, one skilled in the art will appreciate that the present invention allows for the first time the production of 5-azacytidine drug substance with uniform and consistent properties from batch to batch, which properties include but are not limited to solubility and dissolution rate. In turn, this allows one to provide 5-azacytidine drug product (see below) which also has uniform and consistent properties from batch to batch.
Pharmaceutical Formulations For the most effective administration of drug substance of the present invention, it is preferred to prepare a pharmaceutical formulation (also known as the "drug product" or "pharmaceutical composition") preferably in unit dose form, comprising one or more of the 5- azacytidine polymorphs of the present invention and one or more pharmaceutically acceptable carrier, diluent, or excipient. Most preferably, Form I 5-azacytidine prepared according to the methods provided herein is used to prepare the pharmaceutical formulation.
Such pharmaceutical formulation may, without being limited by the teachings set forth herein, include a solid form of the present invention which is blended with at least one pharmaceutically acceptable excipient, diluted by an excipient or enclosed within such a carrier that can be in the form of a capsule, sachet, tablet, buccal, lozenge, paper, or other container. When the excipient serves as a diluent, it may be a solid, semi-solid, or liquid material which acts as a vehicle, carrier, or medium for the 5-azacytidine polymorph(s). Thus, the formulations can be in the form of tablets, pills, powders, elixirs, suspensions, emulsions, solutions, syrups, capsules (such as, for example, soft and hard gelatin capsules), suppositories, sterile injectable solutions, and sterile packaged powders.
Examples of suitable excipients include, but are not limited to, starches, gum arabic, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include lubricating agents such as, for example, talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propyl- hydroxybenzoates; sweetening agents; or flavoring agents. Polyols, buffers, and inert fillers may also be used. Examples of polyols include, but are not limited to: mannitol, sorbitol, xylitol, sucrose, maltose, glucose, lactose, dextrose, and the like. Suitable buffers encompass, but are not limited to, phosphate, citrate, tartrate, succinate, and the like. Other inert fillers which may be used encompass those which are known in the art and are useful in the manufacture of various dosage forms. If desired, the solid pharmaceutical compositions may include other components such as bulling agents and/or granulating agents, and the like. The compositions of the invention can be formulated so as to provide quick, sustained, controlled, or delayed release of the drug substance after administration to the patient by employing procedures well lαiown in the art.
In certain embodiments of the invention, the 5-azacytidine polymorph(s) may made into the form of dosage units for oral administration. The 5-azacytidine polymorph(s) may be mixed with a solid, pulverant carrier such as, for example, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives or gelatin, as well as with an antifriction agent such as for example, magnesium stearate, calcium stearate, and polyethylene glycol waxes. The mixture is then pressed into tablets or filled into capsules. If coated tablets, capsules, or pulvules are desired, such tablets, capsules, or pulvules may be coated with a concentrated solution of sugar, which may contain gum arabic, gelatin, talc, titanium dioxide, or with a lacquer dissolved in the volatile organic solvent or mixture of solvents. To this coating, various dyes may be added in order to distinguish among tablets with different active compounds or with different amounts of the active compound present.
Soft gelatin capsules may be prepared in which capsules contain a mixture of the 5- azacytidine polymorph(s) and vegetable oil or non-aqueous, water miscible materials such as, for example, polyethylene glycol and the like. Hard gelatin capsules may contain granules or powder of the 5-azacytidine polymorph in combination with a solid, pulverulent carrier, such as, for example, lactose, saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives, or gelatin. Tablets for oral use are typically prepared in the following manner, although other techniques may be employed. The solid substances are gently ground or sieved to a desired particle size, and a binding agent is homogenized and suspended in a suitable solvent. The 5- azacytidine polymorph(s) and auxiliary agents are mixed with the binding agent solution. The resulting mixture is moistened to form a uniform suspension. The moistening typically causes the particles to aggregate slightly, and the resulting mass is gently pressed through a stainless steel sieve having a desired size. The layers of the mixture are then dried in controlled drying units for a pre-determined length of time to achieve a desired particle size and consistency. The granules of the dried mixture are gently sieved to remove any powder. To this mixture, disintegrating, anti-friction, and anti-adhesive agents are added. Finally, the mixture is pressed into tablets using a machine with the appropriate punches and dies to obtain the desired tablet size.
In the event that the above formulations are to be used for parenteral administration, such a formulation typically comprises sterile, aqueous and non-aqueous injection solutions comprising one or more 5-azacytidine polymorphs for which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain anti- oxidants, buffers, bacteriostats, and solute; which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous suspensions may include suspending agents and thickening agents. The formulations may be present in unit-dose or multi-dose containers, for example, sealed ampules and vials. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.
Liquid preparations for oral administration are prepared in the form of solutions, syrups, or suspensions with the latter two forms containing, for example, 5-azacytidine polymorph(s), sugar, and a mixture of ethanol, water, glycerol, and propylene glycol. If desired, such liquid preparations contain coloring agents, flavoring agents, and saccharin. Thickening agents such as carboxymethylcellulose may also be used.
As such, the pharmaceutical formulations of the present invention are preferably prepared in a unit dosage form, each dosage unit containing from about 5 mg to about 200 mg, more usually about 100 mg of the 5-azacytidine polymorph(s). In liquid form, dosage unit contains from about 5 to about 200 mg, more usually about 100 mg of the 5-azacytidine polymorph(s). The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects/patients or other mammals, each unit containing a predetermined quantity of the 5-azacytidine polymorph calculated to produce the desired therapeutic effect, in association with preferably, at least one pharmaceutically acceptable carrier, diluent, or excipient.
The following examples are provided for illustrative purposes only, and are not to be construed as limiting the scope of the claims in any way.
Examples
Example 1
Prior Art Procedure for Synthesis and Recrystallization of 5-azacvtidine Drug Substance
5-azacytidine may be synthesized using commercially available 5-azacytosine and 1,2,3,5- tetra-O-acetyl-β-D-ribofuranose (RTA) according to the following pathway:
Figure imgf000013_0001
(2) (3)
The crude synthesis product is dissolved in DMSO (preheated to about 90°C), and then methanol is added to the DMSO solution. The co-solvent mixture is equilibrated at approximately -20°C to allow 5-azacytidine crystal formation. The product is collected by vacuum filtration and allowed to air dry.
Example 2
Form I of 5-azacvtidine: Slow Recrystallization of 5-azacytidine from Co-Solvent Systems Approximately 250 mg of 5-azacytidine was dissolved with approximately 5 ml of dimethyl sulfoxide (DMSO), preheated to approximately 90 °C, in separate 100-mL beakers.
The solids were allowed to dissolve to a clear solution. Approximately 45 mL of ethanol, isopropyl alcohol, acetonitrile, or methyl ethyl ketone co-solvent, preheated to approximately
50 °C, was added to the solution and the resultant solution was mixed. The solution was covered and allowed to equilibrate at ambient conditions. The product was collected by vacuum filtration using a Buchner funnel. Example 3
Form I of 5-azacvtidine: Fast Recrystallization of 5-azacvtidine from Co- Solvent Systems
Approximately 250 mg of 5-azacytidine was dissolved with approximately 5 mL of DMSO, preheated to approximately 90 °C, in separate 100-ml beakers. The solids were allowed to dissolve to a clear solution. Approximately 45 mL of isopropyl alcohol or acetonitrile co-solvent, preheated to approximately 50 °C, was added to the solution and the resultant solution was mixed. The solution was covered and placed in a freezer to equilibrate at approximately -20°C to allow crystal formation. Solutions were removed from the freezer after crystal formation. The product was collected by vacuum filtration using a Buchner funnel.
Example 4
X-Ray Powder Diffraction of Recrystallized 5-azacvtidine
X-ray powder diffraction (XRPD) patterns for each sample were obtained on a Scintag XDS 2000 or a Scintag X2 θ/θ diffractometer operating with copper radiation at 45 kN and 40 mA using a Kevex Psi Peltier-cooled silicon detector or a Thermo ARL Peltier-cooled solid state detector. Source slits of 2 or 4 mm and detector slits of 0.5 or 0.3 mm were used for data collection. Recrystallized material was gently milled for approximately one minute using an agate mortar and pestle. Samples were placed in a stainless steel or silicon sample holder and leveled using a glass microscope slide. Powder diffraction patterns of the samples were obtained from 2 to 42° 2Θ at l°/minute. Calibration of the X2 diffractometer is verified annually using a silicon powder standard.
XRPD performed according to this method revealed that the Form I of 5-azacytidine was isolated in Example 2 by slow recrystallization using either ethanol, isopropyl alcohol, acetonitrile, or methyl ethyl ketone as the co-solvent, and in Example 3 by fast recrystallization using isopropyl alcohol or acetonitrile as the co-solvent. The results indicate that Form I of 5-azacytidine may be reliably recovered from the DMSO/isopropyl alcohol and DMSO/acetonitrile solvent systems without control of the rate of recrystallization.

Claims

What is claimed is:
1. A method for isolating crystalline Form I of 5-azacytidine substantially free of other forms, the method comprising: recrystallizing 5-azacytidine from a solvent mixture comprising at least one primary solvent and at least one co-solvent selected from the group consisting of:
1 ,1 ,2-Trichloroethene
1 ,2-Dichloroethene
152-Dimethoxyethane
1,4-Dioxane 1-Butanol
1-Pentanol
1 -Propanol
2-Butanol
2-Ethoxyethanol 2-Methoxyethanol
2-Methyl- 1 -propanol
2-Propanol (isopropyl alcohol)
3 -Methyl- 1-butanol
Acetone Acetonitrile
Anisole
Butyl acetate
Chlorobenzene
Cumene Cyclohexane
Dichloromethane
Ethanol
Ethyl acetate
Ethyl ether Ethyl formate
Ethyleneglycol
Formamide
Isobutyl acetate Isopropyl acetate
Methyl acetate Methylbutyl ketone Methylcyclohexane Methylethyl ketone Methylisobutyl ketone
N.N-Dimethylacetamide N,N-Dimethylformamide Nitromethane Propyl acetate Pyridine
Sulfolane tert-Butylmethyl ether Tetrahydrofuran, and Tetralin
by cooling said solvent mixture from a temperature selected to allow said 5-azacytidine to dissolve completely to about ambient temperature; and isolating the recrystallized 5-azacytidine.
2. The method of claim 1 wherein said primary solvent is a polar solvent.
3. The method of claim 2 wherein said polar solvent is a polar aprotic solvent.
4. The method of claim 3 wherein said polar aprotic solvent is selected from the group consisting of dimethylsulfoxide, dimethylfomiamide, dimethylacetamide, and - methylpyrrolidinone.
5. The method of claim 4 wherein said polar aprotic solvent is dimethylsulfoxide.
6. The method of claim 1 wherein said co-solvent is acetonitrile.
7. The method of claim 1 wherein said co-solvent is ethanol.
8. The method of claim 1 wherein said co-solvent is methylethyl ketone.
9. The method of claim 1 wherein said co-solvent is 2-propanol (isopropyl alcohol).
10. The method of claim 1 wherein said co-solvent is 1 -propanol.
11. A method for isolating crystalline Form I of 5-azacytidine substantially free of other forms, the method comprising: recrystallizing 5-azacytidine from a solvent mixture comprising at least one primary solvent and at least one co-solvent selected from the group consisting of C -C5 alcohols, aliphatic ketones, and alkyl cyanides, by cooling said solvent mixture from a temperature selected to allow said 5-azacytidine to dissolve completely to about ambient temperature; and isolating the recrystallized 5-azacytidine.
12. The method of claim 11 wherein said primary solvent is a polar solvent.
13. The method of claim 12 wherein said polar solvent is a polar aprotic solvent.
14. The method of claim 13 wherein said polar aprotic solvent is selected from the group consisting of dimethylsulfoxide, dimethylfomiamide, dimethylacetamide, and N- methylpyrrolidinone.
15. The method of claim 14 wherein said polar aprotic solvent is dimethylsulfoxide.
16. The method of claim 11 wherein said co-solvent is acetonitrile.
17. The method of claim 11 wherein said co-solvent is ethanol.
18. The method of claim 11 wherein said co-solvent is methylethyl ketone.
19. The method of claim 11 wherein said co-solvent is 2-propanol (isopropyl alcohol).
20. The method of claim 11 wherein said co-solvent is 1 -propanol.
21. A method for isolating crystalline Form I 5-azacytidine substantially free of other foπns, the method comprising: recrystallizing 5-azacytidine from a solvent mixture comprising at least one primary solvent and at least one co-solvent selected from the group consisting of:
1 , 1 ,2-Trichloroethene
1 ,2-Dichloroethene
1 ,2-Dimethoxyethane
1 ,4-Dioxane 1-Butanol
1-Pentanol
1 -Propanol
2-Butanol
2-Ethoxyethanol 2-Methoxyethanol
2-Methyl- 1 -propanol
2-Propanol
3 -Methyl- 1-butanol
Acetone Acetonitrile
Anisole
Butyl acetate
Chlorobenzene
Cumene Cyclohexane
Dichloromethane
Ethyl acetate
Ethyl ether
Ethyl formate Ethyleneglycol
Formamide
Isobutyl acetate
Isopropyl acetate Methyl acetate
Methylbutyl ketone
Methylcyclohexane
Methylisobutyl ketone
N,N-Dimethylacetamide N,N-Dimethylforman ide
Nitromethane >
Propyl acetate
Pyridine
Sulfolane tert-Butylmethyl ether
Tetrahydrofuran, and
Tetralin
by cooling said solution from a temperature selected to allow said 5-azacytidine to dissolve completely to about -20°C ; and isolating the recrystallized 5-azacytidine.
22. The method of claim 21 wherein said primary solvent is a polar solvent.
23. The method of claim 22 wherein said polar solvent is a polar aprotic solvent.
24. The method of claim 23 wherein said polar aprotic solvent is selected from the group consisting of dimethylsulfoxide, dimethylformamide, dimethylacetamide, andN- methylpyrrolidinone.
25. The method of claim 24 wherein said polar aprotic solvent is dimethylsulfoxide.
26. The method of claim 21 wherein said co-solvent is acetonitrile.
27. The method of claim 21 wherein said co-solvent is 2-propanol (isopropyl alcohol).
28. The method of claim 21 wherein said co-solvent is 1-propanol.
29. A method for isolating crystalline Form I of 5-azacytidine substantially free of other forms, the method comprising: recrystallizing 5-azacytidine from a solvent mixture comprising at least one primary solvent and at least one co-solvent selected from the group consisting of C3 - C5 alcohols and alkyl cyanides by cooling said solution from a temperature selected to allow said 5- azacytidine to dissolve completely to about -20°C ; and isolating the recrystallized 5-azacytidine.
30. The method of claim 29 wherein said primary solvent is a polar solvent.
31. The method of claim 30 wherein said polar solvent is a polar aprotic solvent.
32. The method of claim 31 wherein said polar aprotic solvent is selected from the group consisting of dimethylsulfoxide, dimethylformamide, dimethylacetamide, andN- methylpyrrolidinone.
33. The method of claim 32 wherein said polar aprotic solvent is dimethylsulfoxide.
34. The method of claim 29 wherein said co-solvent is acetonitrile.
35. The method of claim 29 wherein said co-solvent is 2-propanol (isopropyl alcohol).
36. The method of claim 29 wherein said co-solvent is 1 -propanol.
37. A method for isolating crystalline Form I of 5-azacytidine substantially free of other forms, the method comprising: recrystallizing 5-azacytidine from a solvent mixture comprising dimethylsulfoxide and isopropyl alcohol; and isolating the recrystallized 5-azacytidine.
38. A method for isolating crystalline Form I of 5-azacytidine substantially free of other forms, the method comprising: recrystallizing 5-azacytidine from a solvent mixture comprising dimethylsulfoxide and 1- propanol; and isolating the recrystallized 5-azacytidine.
39. A method for isolating crystalline Form I of 5-azacytidine substantially free of other forms, the method comprising: recrystallizing 5-azacytidine from a solvent mixture comprising dimethylsulfoxide and methanol by seeding said solvent mixture with Form I of 5-azacytidine; and isolating the recrystallized 5-azacytidine.
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US7038038B2 (en) * 2003-03-17 2006-05-02 Pharmion Corporation Synthesis of 5-azacytidine
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US7192781B2 (en) * 2004-04-13 2007-03-20 Pharmion Corporation Methods for stabilizing 5-azacytidine in plasma
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US20110288042A1 (en) * 2007-08-02 2011-11-24 Chemagis Ltd. Stable highly pure azacitidine and preparation methods therefor
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WO2010014883A2 (en) 2008-08-01 2010-02-04 Dr. Reddy's Laboratories Ltd. Azacitidine process and polymorphs
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US20110042247A1 (en) * 2009-06-25 2011-02-24 Chandrasekhar Kocherlakota Formulations of azacitidine and its derivatives
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US9968627B2 (en) 2013-03-26 2018-05-15 Celgene Corporation Solid forms comprising 4-amino-1-β-D-ribofuranosyl-1,3,5-triazin-2(1H)-one and a coformer, compositions and methods of use thereof
CN103450303A (en) * 2013-09-04 2013-12-18 重庆泰濠制药有限公司 Azacitidine crystal form A and azacitidine crystal form B as well as preparation method thereof
US10034872B2 (en) 2014-08-22 2018-07-31 Celgene Corporation Methods of treating multiple myeloma with immunomodulatory compounds in combination with antibodies
US20160095925A1 (en) * 2014-10-01 2016-04-07 Cadila Healthcare Limited Stable formulation of azacitidine or salts thereof and their process for preparation
JP7033061B2 (en) 2015-10-15 2022-03-09 アジオス ファーマシューティカルズ， インコーポレイテッド Combination therapy to treat malignant tumors
KR20180064529A (en) 2015-10-15 2018-06-14 셀진 코포레이션 Combination Therapy for the Treatment of Malignant Tumors
TW201818934A (en) * 2016-10-17 2018-06-01 日商第一三共股份有限公司 Treatment method by combined use of MDM2 inhibitor and DNA methyltransferase inhibitor
WO2022251663A2 (en) * 2021-05-27 2022-12-01 Emory University Novel universal anti-rna virus agents

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1227691A (en) 1968-05-08 1971-04-07
US3817980A (en) 1970-03-14 1974-06-18 Schering Ag 5-azapyrimidine nucleosides
US3891623A (en) 1971-05-04 1975-06-24 Schering Ag Process for preparing cytidines
US6723728B2 (en) 2001-03-01 2004-04-20 Gilead Sciences, Inc. Polymorphic and other crystalline forms cis-FTC
US6877855B2 (en) 2003-07-08 2005-04-12 Dave Ng Spectacles with peripheral lens support
US6887855B2 (en) 2003-03-17 2005-05-03 Pharmion Corporation Forms of 5-azacytidine

Family Cites Families (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1050899A (en) 1963-12-22
CH507969A (en) 1968-11-12 1971-05-31 Ceskoslovenska Akademie Ved Process for the preparation of 1-glycosyl-5-azacytosines
FR2123632A6 (en) 1971-01-26 1972-09-15 Ceskoslovenska Akademie Ved Prepn of 5-azacytosines and 5-azacytidines - from bis trimethylsilyl-5-azacytosine
DE2508312A1 (en) 1975-02-24 1976-09-02 Schering Ag NEW PROCESS FOR THE PRODUCTION OF NUCLEOSIDES
DE2757365A1 (en) 1977-12-20 1979-06-21 Schering Ag NEW PROCESS FOR THE PRODUCTION OF NUCLEOSIDES
US5700640A (en) 1994-09-16 1997-12-23 Basf Aktiengesellschaft Inducers of gamma globin gene expression and screening assays therefor
US5539209A (en) 1994-10-17 1996-07-23 Trojan Technologies Inc. Method of cleaning fouling materials from a radiation module
TW513409B (en) 1996-06-07 2002-12-11 Eisai Co Ltd Polymorphs of donepezil hydrochloride
JPH1053576A (en) * 1996-06-07 1998-02-24 Eisai Co Ltd Polymorphic crystal of donepezil hydrochloride and its production
SK284054B6 (en) 1996-10-16 2004-08-03 Icn Pharmaceuticals, Inc. Monocyclic L-nucleosides, analogs and uses thereof
WO2000075143A1 (en) * 1999-06-03 2000-12-14 Eisai Co., Ltd. Crystals of carbapenem derivatives and pharmaceutical preparations for injection
ATE312823T1 (en) * 1999-07-09 2005-12-15 Boehringer Ingelheim Pharma METHOD FOR PRODUCING HETEROARYL-SUBSTITUTED UREA COMPOUNDS
IL150524A0 (en) 2000-01-07 2003-02-12 Transform Pharmaceuticals Inc High-throughput formation, identification, and analysis of diverse solid-forms
PL205567B1 (en) * 2000-09-07 2010-05-31 Kaneka Corp Methods for crystallization of hydroxycarboxylic acids
AU2003278904A1 (en) 2002-09-24 2004-04-19 Kornis Pharmaceuticals, Incorporated 1, 3, 5-triazines for treatment of viral diseases
US7189740B2 (en) 2002-10-15 2007-03-13 Celgene Corporation Methods of using 3-(4-amino-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione for the treatment and management of myelodysplastic syndromes
EP1556010A4 (en) 2002-10-31 2007-12-05 Supergen Inc Pharmaceutical formulations targeting specific regions of the gastrointestinal tract
WO2004069796A2 (en) * 2003-02-03 2004-08-19 Teva Pharmaceutical Industries Ltd. Process for producing levetiracetam
US7038038B2 (en) 2003-03-17 2006-05-02 Pharmion Corporation Synthesis of 5-azacytidine
US6943249B2 (en) 2003-03-17 2005-09-13 Ash Stevens, Inc. Methods for isolating crystalline Form I of 5-azacytidine
AU2004270150C1 (en) 2003-08-29 2011-07-14 Merck Hdac Research, Llc Combination methods of treating cancer
US7192781B2 (en) 2004-04-13 2007-03-20 Pharmion Corporation Methods for stabilizing 5-azacytidine in plasma
US20060063735A1 (en) 2004-09-17 2006-03-23 Supergen, Inc. Salts of 5-azacytidine
US20060069060A1 (en) 2004-09-27 2006-03-30 Sanjeev Redkar Salts of decitabine
US20060128654A1 (en) 2004-12-10 2006-06-15 Chunlin Tang Pharmaceutical formulation of cytidine analogs and derivatives
WO2008028193A2 (en) 2006-09-01 2008-03-06 Pharmion Corporation Colon-targeted oral formulations of cytidine analogs
US7759481B2 (en) 2007-01-11 2010-07-20 Ivax Corporation Solid state forms of 5-azacytidine and processes for preparation thereof
WO2008092127A1 (en) 2007-01-25 2008-07-31 Nevada Cancer Institute Use of acetylated or esterificated azacytidine, decitabine, or other nucleoside analogs as oral agents for the treatment of tumors or other dysplastic syndromes sensitive to hypomethylating agents
US20110288042A1 (en) 2007-08-02 2011-11-24 Chemagis Ltd. Stable highly pure azacitidine and preparation methods therefor
EP2048151A1 (en) 2007-10-10 2009-04-15 Cilag AG Method for producing nucleosides by direct glycosylation of the nucleoside base
EP2211870A1 (en) 2007-11-01 2010-08-04 Celgene Corporation Cytidine analogs for treatment of myelodysplastic syndromes
DK2695609T3 (en) 2008-05-15 2020-02-10 Celgene Corp Oral formulations of cytidine analogues and methods for their use
WO2010014883A2 (en) 2008-08-01 2010-02-04 Dr. Reddy's Laboratories Ltd. Azacitidine process and polymorphs
EP2324042B1 (en) 2008-08-06 2012-11-28 Sicor, Inc. Process for preparing azacytidine intermediate
JP5650643B2 (en) 2008-08-08 2015-01-07 サイノファーム タイワン リミテッド Method for producing 5-azacytosine nucleoside and derivatives thereof
US20110042247A1 (en) 2009-06-25 2011-02-24 Chandrasekhar Kocherlakota Formulations of azacitidine and its derivatives
WO2011014541A1 (en) 2009-07-30 2011-02-03 Eagle Pharmaceuticals, Inc. Stable formulations of azacitidine
IT1399195B1 (en) 2010-03-30 2013-04-11 Chemi Spa PROCESS FOR THE SYNTHESIS OF AZACITIDINE AND DECITABINE

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1227691A (en) 1968-05-08 1971-04-07
US3817980A (en) 1970-03-14 1974-06-18 Schering Ag 5-azapyrimidine nucleosides
US3891623A (en) 1971-05-04 1975-06-24 Schering Ag Process for preparing cytidines
US6723728B2 (en) 2001-03-01 2004-04-20 Gilead Sciences, Inc. Polymorphic and other crystalline forms cis-FTC
US6887855B2 (en) 2003-03-17 2005-05-03 Pharmion Corporation Forms of 5-azacytidine
US6877855B2 (en) 2003-07-08 2005-04-12 Dave Ng Spectacles with peripheral lens support

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
A. PISCALA ET AL., COLLECTION OF CZECHOSLOVAK CHEMICAL COMMUNICATIONS, 1964, pages 2060 - 2069
A. PISCALA ET AL., NUCLEID ACID CHEMISTRY, 1978, pages 435 - 441
BEISLER J.A. ET AL., JOURNAL OF CARBOHYDRATES, 1977, pages 281 - 299
BEISLER J.A. ET AL., JOURNAL OF MEDICINAL CHEMISTRY, 1978, pages 204 - 208
H. BRITTAIN: "Polymorphism in Pharmaceutical Solids", 1999, MARCEL DEKKER, pages: 1 - 2
KNAPMAN, K, MODEM DRUG DISCOVERIES, vol. 53, March 2000 (2000-03-01)
KORNBLITH ET AL., J. CLIN. ONCOL., vol. 20, no. 10, 2002, pages 2441 - 2452
M.W.WINKLEY; R.K. ROBINS, THE JOURNAL OF ORGANIC CHEMISTRY, 1970, pages 491 - 495
See also references of EP1608634A4
SILVERMAN ET AL., J. CLIN. ONCOL., vol. 20, no. 10, 2002, pages 2429 - 2440

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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WO2008044041A1 (en) 2006-10-12 2008-04-17 Astex Therapeutics Limited Pharmaceutical combinations
WO2008044045A1 (en) 2006-10-12 2008-04-17 Astex Therapeutics Limited Pharmaceutical combinations
US10220050B2 (en) 2008-05-15 2019-03-05 Celgene Corporation Isotopologues of 5-azacytidine
US10463683B2 (en) 2008-05-15 2019-11-05 Celgene Corporation Isotopologues of 5-azacytidine
US11571436B2 (en) 2008-05-15 2023-02-07 Celgene Corporation Oral formulations of cytidine analogs and methods of use thereof
US8846628B2 (en) 2008-05-15 2014-09-30 Celgene Corporation Oral formulations of cytidine analogs and methods of use thereof
US10646503B2 (en) 2008-05-15 2020-05-12 Celgene Corporation Isotopologues of 5-azacytidine
US9393255B2 (en) 2011-01-31 2016-07-19 Celgene Corporation Pharmaceutical compositions of cytidine analogs and methods of use thereof
US8980850B2 (en) 2011-11-03 2015-03-17 Millennium Pharmaceuticals, Inc. Administration of a NEDD8-activating enzyme inhibitor and hypomethylating agent
WO2014076616A3 (en) * 2012-11-19 2014-07-10 Shilpa Medicare Limited Formulations of 5-azacytidine
WO2014076616A2 (en) * 2012-11-19 2014-05-22 Shilpa Medicare Limited Formulations of 5-azacytidine
CN109988207A (en) * 2017-12-29 2019-07-09 江苏豪森药业集团有限公司 The preparation method of azacitidine crystal form
CN110225918A (en) * 2017-12-29 2019-09-10 江苏豪森药业集团有限公司 The preparation method of azacitidine crystal form
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CN110225918B (en) * 2017-12-29 2022-10-11 江苏豪森药业集团有限公司 Preparation method of azacitidine crystal form
WO2019129260A1 (en) * 2017-12-29 2019-07-04 江苏豪森药业集团有限公司 Method for preparing crystal form of azacitidine
CN110642901A (en) * 2019-11-11 2020-01-03 扬子江药业集团有限公司 Azacitidine methanolate, preparation method thereof, pharmaceutical composition and application

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PT2181988E (en) 2016-06-15
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US7700770B2 (en) 2010-04-20
US8211862B2 (en) 2012-07-03
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