(12) United States Patent
`Ionescu et al.
`
`I 1111111111111111 11111 lllll 111111111111111 11111 1111111111 lll111111111111111
`US006887855B2
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 6,887,855 B2
`May 3, 2005
`
`(54) FORMS OF 5-AZACYTIDINE
`
`(75)
`
`Inventors: Dumitru Ionescu, Ann Arbor, MI (US);
`Peter Blumbergs, Royal Oak, MI (US);
`Gary L. Silvey, Overland Park, KS
`(US)
`
`(73) Assignees: Pharmion Corporation, Boulder, CO
`(US); Ash Stevens, Inc., Detroit, MI
`(US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 45 days.
`
`(21) Appl. No.: 10/390,578
`
`(22) Filed:
`
`Mar. 17, 2003
`
`(65)
`
`(51)
`(52)
`
`(58)
`
`(56)
`
`Prior Publication Data
`
`US 2004/0186065 Al Sep. 23, 2004
`
`Int. Cl.7 ................... A61K 31/4436; C07D 411/04
`U.S. Cl. ........................... 514/43; 514/50; 514/274;
`514/547; 514/575; 536/23.1; 536/24.5;
`536/24.31; 536/24.33; 536/28.1; 536/28.3;
`435/6; 424/93.7
`Field of Search ............................ 514/43, 274, 50,
`514/547, 575; 536/23.1, 24.5, 24.31, 24.33,
`28.1, 28.3; 435/6; 424/93.7
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`3,350,388 A
`3,817,980 A
`3,891,623 A
`4,082,911 A
`4,209,613 A
`6,723,728 B2 *
`
`10/1967
`6/1974
`6/1975
`4/1978
`6/1980
`4/2004
`
`Sorm et al.
`Vorbriiggen et al.
`Vorbriiggen et al.
`Vorbriiggen
`Vorbriiggen
`Hu et al.
`.................... 514/274
`
`FOREIGN PATENT DOCUMENTS
`
`DE
`GB
`
`2012888
`1227691
`
`9/1971
`4/1971
`
`OTHER PUBLICATIONS
`
`Wimkley et al. J. Org. Chem., 35 (2), 491-495, 1970.*
`Beisler, Journal of Medicinal Chemistry, 21(2):204 (1978).
`Niedballa & Vorbriiggen, Journal of Organic Chemistry,
`39(25):3672 (1974).
`
`(Continued)
`
`Primary Examiner-James 0. Wilson
`Assistant Examiner----Devesh Khare
`(74) Attorney, Agent, or Firm-Swanson & Bratschun,
`LLC.
`
`(57)
`
`ABSTRACT
`
`The invention provides novel polymorphic and pseudopoly(cid:173)
`morphic crystalline forms of 5-azacytidine, along with
`methods for preparing said forms, wherein 5-azacytidine is
`represented by the formula:
`
`NH2
`
`NAN
`
`lA
`
`HO~N
`
`O
`
`H
`
`H
`
`H
`OH
`
`H
`OH
`
`The invention also includes pharmaceutical compositions
`comprising said forms.
`
`DE
`
`1922702
`
`4/1971
`
`8 Claims, 8 Drawing Sheets
`
`X•ra7 Powder Difrnclion Patte,n .r Ar.adlldiDe, , _ I, Labeled with the
`more Promintnt 28 Anps (Ca Ka Radiation)
`
`5500
`
`5000
`
`4500
`
`4000
`
`3500
`
`3000 1 2500
`
`2000
`
`1500
`
`1000
`
`500
`
`~
`
`~~
`3-
`
`-•
`
`ts .i
`
`t
`Ii
`
`10
`
`15
`
`25
`20
`Tw1>-'neta
`
`30
`
`35
`
`40
`
`CELGENE 2035
`APOTEX v. CELGENE
`IPR2023-00512
`
`

`

`US 6,887,855 B2
`Page 2
`
`OIBER PUBLICATIONS
`
`Komblith et al., J. Clin Oneal. 20: 2441 (2002).
`Piskala & Sorm, Collect. Czech. Chem. Commun. 29:2060
`(1964).
`Piskala & Sorm, Nucleic Acid Chemistry 1: 435 (1978).
`Piskala & Sorm, Nucleic Acids Research, Special Publica(cid:173)
`tion No. 1: sl 7 (1975).
`Silverman et al., J. Clin Oneal. 20: 2429 (2002).
`Vorbriiggen et al, Chem. Ber. 114: 1234 (1981).
`
`Vorbriiggen & Bennua, Chem Ber. 114: 1279 (1981).
`Vorbriiggen & Ruh-Pohlenz in Organic Reactions, vol. 55,
`plO0 (L. A Paquette Ed., John Wiley & Sons, New York,
`2000).
`Winkley & Robins, Journal of Organic Chemistry, 35(2):491
`(1970).
`Wittenburg, Z. Chem. 4:303 (1964).
`
`* cited by examiner
`
`

`

`U.S. Patent
`
`May 3, 2005
`
`Sheet 1 of 8
`
`US 6,887,855 B2
`
`F1cure 1. X-ray Powder Diffnction Pattun of A:r.adtidine, Form I, Labeled with the
`moft Promintnt 28 Angles (Cu Ka Radiation)
`
`5500
`
`5000
`
`4500
`
`4000
`
`3500
`
`3000
`
`.e-
`.... 2500
`!
`~
`....
`~ 2000
`
`1500
`
`1000
`
`500
`
`0
`
`ij
`!:I
`
`a
`
`I:;
`
`i;:..,;
`
`. ~
`~-
`
`~ ~
`~ ..
`
`~
`
`~;
`
`!!: jl
`
`~
`:
`
`t
`Ji
`
`5
`
`10
`
`15
`
`20
`25
`Two-Theta
`
`30
`
`35
`
`40
`
`

`

`U.S. Patent
`
`May 3, 2005
`
`Sheet 2 of 8
`
`US 6,887,855 B2
`
`Figure 2 X-ray Powder Diffraction Pattern of Azadtidine1 Mixed F!Jase Forms I and II,
`Labeled with the more Prominent 29 Angl..s {Cu Ka Radiation)
`
`5000 ,...,...,._ ...... ..--.,""T""'r ......... .,...,__,,_...F"'T"" ......... ~ - - - - - - - - . - - - -..........
`• lndical• .,.. dilllnew• to Form II
`
`4500
`
`4000
`
`3500
`
`3000
`
`1500
`
`1000
`
`A1aciliwne
`500 Milled Fonm I & D
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`Two-Theta
`
`

`

`U.S. Patent
`
`May 3, 2005
`
`Sheet 3 of 8
`
`US 6,887,855 B2
`
`Figure 3 X-ray Powder Diffnction Patt.em of Azacitidine, Fonn III, Labeled with the
`more Prominent 28 Angles (CU Ka. Radiation)
`
`4000
`
`3500
`
`3000
`
`2500
`
`~ 2000
`
`-la
`~ 1500
`....
`Q
`
`1000
`
`500
`
`0
`
`.
`..
`
`~
`~
`
`:.
`i:::
`f;!
`
`~
`~
`
`~
`
`~
`~ !:;
`ti
`
`t
`~
`
`~
`....
`
`N
`
`~
`
`;!:
`
`;
`~
`~
`
`~
`
`~
`
`.., .
`~
`~
`..,
`"'
`
`~
`~ ~
`;
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`Two-Theta
`
`

`

`U.S. Patent
`
`May 3, 2005
`
`Sheet 4 of 8
`
`US 6,887,855 B2
`
`Figure 4 X-ray Powder Diffraction Pattern of ADcitidine, Form IV, Labeled with the
`more Prominent 28 An~ (Cu Ka R2d.iation)
`
`3000
`
`2500
`
`2000
`
`1~00
`
`-
`c
`la
`:J
`.s 1000
`
`500
`
`0
`
`~ t ;!
`
`i
`~
`~
`:!
`
`~
`:;;
`
`~
`=
`
`~
`
`~
`~ls
`i:i
`
`. ~
`~ ...
`!il~
`~
`
`N
`
`5
`
`~
`
`?:l
`. ~
`~
`
`t.
`
`j~.
`~Iii i
`
`§
`~
`
`5
`
`· 10
`
`1.5
`
`20
`
`'25
`
`30
`
`35
`
`40
`
`Two-Theta
`
`

`

`U.S. Patent
`
`May 3, 2005
`
`Sheet 5 of 8
`
`US 6,887,855 B2
`
`Figure S X-ray Powdrr Diffraction Pattern of A.zacitidine, Form V, Labeled with the
`morr Prominent l8 Anglf:s (Cu Ka RadJation)
`
`. .. :;j
`
`;::
`
`?.
`~
`::
`
`~
`~
`
`.
`- l;:
`..,
`. .
`~
`~
`. ~
`... ..,
`'" ,..
`~~
`' .
`"1
`...
`~ ::! •
`t
`:~ ,:, .
`~
`2!
`
`:l
`
`~ t.
`t; !:;
`;;i i:j
`
`~
`~
`
`~
`
`i:;
`
`...
`...
`f·
`i!!, ~
`;:illl ~~
`.. ...
`
`2000
`
`....
`~ 1.500
`l9
`- 1000
`i
`
`.500
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`Two-Theta
`
`

`

`U.S. Patent
`
`May 3, 2005
`
`Sheet 6 of 8
`
`US 6,887,855 B2
`
`Figure 6 X-ray Powder Diffraction Pattern of Azacltidine, Form VI, Labeled with the
`more Prominent 28 Angles (Cu Ka Radiation)
`·
`
`1500
`
`1000
`
`c
`·;
`~
`....
`C
`
`500
`
`~ =
`
`~
`~
`
`.,..,
`~ f.
`.;;
`I
`
`;,,
`~
`i;!
`
`~
`~
`J ~
`
`:.
`~
`
`5
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`

`

`U.S. Patent
`
`May 3, 2005
`
`Sheet 7 of 8
`
`US 6,887,855 B2
`
`Figure 7 X-ray Powder Diffraction Pattern of Azacltidtne, Mixed PbJR Fonm I and
`VII, Labeled with tM more Prominent 28 Angles (Cu Ka Radiation)
`
`3500 ~.--r-.,..,...,..,.....-r--r-T",-r"T""T"'T"'T""T""T~""T""T..,...,""T""l~rr-r-,-r-,-r-,--r-,--,-,
`
`3000
`
`2500
`
`2000
`
`~
`
`.$
`
`·-! 1500
`-
`=
`
`)(XX)
`
`500
`
`0
`
`~~
`~~
`~~-
`• .,
`;,
`I:
`);I -
`!::!
`~ ~
`
`~ ~ *
`~
`~ 2
`::I ;
`...
`
`,.(
`
`!
`
`~
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`
`~
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`l!!l
`~
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`
`..
`f;
`"'
`
`•
`ll'l ..
`
`:!.I
`
`5
`
`10
`
`15
`
`20
`
`2.5
`
`30
`
`35
`
`40
`
`Two-Theta
`
`

`

`U.S. Patent
`
`May 3, 2005
`
`Sheet 8 of 8
`
`US 6,887,855 B2
`
`Figure 8 X-ny Powder Diflraction Pattern or Azacitidlne, Form VIII, Labeled with the
`more Pnuninent 28 Angles (Cu Ka Radiation)
`
`3500
`
`3000
`
`2500
`
`2000
`
`1500
`
`1000
`
`500
`
`0
`
`....
`. t ;a
`~ .s
`
`~
`"' ~
`
`0
`~
`
`~
`!:!
`
`. !,. t
`..
`a
`~ -
`
`~ ,._
`
`. ~
`"''"
`;ga
`~i:l
`
`~
`
`i
`g
`
`~
`...,
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`
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`"'
`
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`....
`
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`
`5
`
`ID
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`Two-Theta
`
`

`

`US 6,887,855 B2
`
`1
`FORMS OF 5-AZACYTIDINE
`
`FIELD OF THE INVENTION
`
`The invention relates to the isolation of crystalline poly(cid:173)
`morphic and pseudopolymorphic forms of 5-azacytidine
`(also known as azacitidine and 4-amino-l-~-D(cid:173)
`ribofuranosyl-S-triazin-2(1H)-one). 5-azacytidine may be
`used in the treatment of disease, including the treatment of
`myelodysplastic syndromes (MDS).
`
`BACKGROUND OF THE INVENTION
`
`2
`phic crystalline forms (Forms I-VIII), in addition to an
`amorphous form. Form I is a polymorph found in prior art
`retained samples of 5-azacytidine drug substance. Form II is
`a polymorph found in some prior art retained samples of the
`5 5-azacytidine drug substance; in those samples, Form II is
`always found in mixed phase with Form I. Form III is a
`hydrate, and is formed when prior art retained and current
`samples of the drug product are reconstituted with water to
`form a "slurry" prior to administration to the patient. Form
`10 VI is found in prior art retained samples of the 5-azacytidine
`drug product, either substantially free of other polymorphs,
`or in mixed phase with Form I.
`The invention provides novel crystalline forms referred to
`as Form IV, Form V, Form VII and Form VIII. Forms I-VIII
`15 each have characteristic X-ray power diffraction (XRPD)
`patterns and are easily distinguished from one another using
`XRPD.
`Also included in the present invention are methods for
`robustly and reproducibly synthesizing 5-azacytidine drug
`substance substantially as Form IV, Form V, or Form VIII.
`Also provided are methods for robustly and reproducibly
`synthesizing a Form I/VII mixed phase. The invention also
`provides pharmaceutical compositions comprising the vari(cid:173)
`ous forms of 5-azacytidine together with one or more
`pharmaceutically acceptable excipients, diluents, or carriers.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`35
`
`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 result(cid:173)
`ing 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 20
`hydrate. The polymorphic and pseudopolymorphic solids
`display different physical properties, including those due to
`packing, and various thermodynamic, spectroscopic, inter(cid:173)
`facial and mechanical properties (See H. Brittain, Polymor(cid:173)
`phism in Pharmaceutical Solids, Marcel Dekker, New York, 25
`N.Y., 1999, pp. 1-2). Polymorphic and pseudopolymorphic
`forms of the drug substance (also known 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 known 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 Modem Drug Discoveries, March 2000:
`53).
`5-azacytidine (also known as azacitidine and 4-amino-l(cid:173)
`~-D-ribofuranosyl-1,3,5-triazin-2(1H)-one; Nation Service
`Center designation NSC-102816; CAS Registry Number
`320-67-2) has undergone NCI-sponsored clinical trials for
`the treatment of myelodysplastic syndromes (MDS). See
`Komblith et al., J. Clin. Oneal. 20(10): 2441-2452 (2002)
`and Silverman et al., J. Clin. Oneal. 20(10): 2429-2440
`(2002). 5-azacytidine may be defined as having a formula of
`C8 H 12N 4 0 5 , a molecular weight of 244.20 and a structure
`of:
`
`FIG. 1 presents the X-Ray Powder Diffraction (XRPD)
`30 pattern of 5-azacytidine, Form I, labeled with the most
`prominent 28 angles (Cu Ka radiation).
`FIG. 2 presents the XRPD pattern of 5-azacytidine, mixed
`phase Form I and Form II, labeled with the most prominent
`28 angles (Cu Ka radiation).
`FIG. 3 presents the XRPD pattern of 5-azacytidine, Form
`III, labeled with the most prominent 28 angles (Cu Ka
`radiation).
`FIG. 4 presents the XRPD pattern of 5-azacytidine, Form
`IV, labeled with the most prominent 28 angles (Cu Ka
`radiation).
`FIG. 5 presents the XRPD pattern of 5-azacytidine, Form
`V, labeled with the most prominent 28 angles (Cu Ka
`radiation).
`FIG. 6 presents the XRPD pattern of 5-azacytidine, Form
`VI, labeled with the most prominent 28 angles (Cu Ka
`radiation).
`FIG. 7 presents the XRPD pattern of 5-azacytidine, mixed
`phase Form I and Form VII, labeled with the most prominent
`50 28 angles (Cu Ka radiation).
`FIG. 8 presents the XRPD pattern of 5-azacytidine, Form
`VIII, labeled with the most prominent 28 angles (Cu Ka
`radiation).
`
`40
`
`45
`
`NH2
`
`NAN
`
`lA
`
`HO~N
`
`O
`
`H
`
`H
`
`H
`OH
`
`H
`OH
`
`The polymorphic form of 5-azacytidine drug substance and
`drug product has never been characterized. It is an object of
`the present invention to characterize the polymorphic forms
`of 5-azacytidine.
`
`SUMMARY OF THE INVENTION
`
`It has been unexpectedly found that 5-azacytidine exists
`in at least eight different polymorphic and pseudopolymor-
`
`55
`
`DETAILED DESCRIPTION OF THE
`PREFERRED EMBODIMENTS
`5-azacytidine Crystalline Forms I-VIII
`It has been discovered that 5-azacytidine exists in at least
`eight different polymorphic and pseudopolymorphic crys-
`60 talline forms, and also in an amorphous form.
`Form I
`A single sample of the 5-azacytidine drug substance was
`synthesized from 5-azacytosine and 1,2,3,5,-Tetra-O-acetyl(cid:173)
`~-D-ribofuranose according to the prior art method provided
`65 in Example 1. The last step of this method is a recrystalli(cid:173)
`zation of the crude synthesis product from a DMSO/
`methanol co-solvent system. Specifically, the crude synthe-
`
`

`

`US 6,887,855 B2
`
`3
`sis 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.
`The X-Ray Powder Diffraction (XRPD; see Example 5)
`pattern of the resulting 5-azacytidine is shown in FIG. 1
`along with some of the 28 values. Table 1 provides the most
`prominent 28 angles, d-spacing and relative intensities for
`this material, which is designated as Form I.
`
`5
`
`10
`
`4
`
`TABLE 2-continued
`
`5-azacytidine, Mixed Phase Forms I and II - the most prominent
`28 angles cl-spacing and relative intensities (Cu Ka radiation)
`
`20Angle (°)
`
`d-spacing (A)
`
`Relative Intensity
`
`27.158
`29.309
`29.609
`30.384
`32.074
`
`3.281
`3.045
`3.015
`2.939
`2.788
`
`46.0
`27.3
`12.7
`10.5
`12.0
`
`TABLE 1
`
`5-azacytidine Form I - the most prominent 20 angles,
`cl-spacing and relative intensities (Cu Ka radiation)
`
`20 Angle(°)
`
`d-spacing (A)
`
`Relative Intensity
`
`12.182
`13.024
`14.399
`16.470
`18.627
`19.049
`20.182
`21.329
`23.033
`23.872
`26.863
`27.135
`29.277
`29.591
`30.369
`32.072
`
`7.260
`6.792
`6.146
`5.378
`4.760
`4.655
`4.396
`4.162
`3.858
`3.724
`3.316
`3.284
`3.048
`3.016
`2.941
`2.788
`
`39.1
`44.1
`31.5
`27.1
`16.0
`35.9
`37.0
`12.4
`100.0
`28.0
`10.8
`51.5
`25.6
`11.5
`10.8
`13.4
`
`20
`
`25
`
`These results indicate that the prior art 5-azacytidine
`synthesis procedures for the drug substance produce either
`15 Form I substantially free of other forms, or a Form 1/11 mixed
`phase i.e. a solid material in which 5-azacytidine is present
`in a mixed phase of both Form I and Form II.
`Thermal analysis of mixed phase Form 1/11 is presented in
`Example 6.
`Form III
`An additional crystalline form of 5-azacytidine, desig(cid:173)
`nated Form III, is found in slurries of 5-azacytidine. See
`Example 8. Moreover, it has been found that all forms of
`5-azacytidine (including the 5-azacytidine in the prior art
`drug product) convert to Form III in water. See Example 8.
`Thus, reconstitution of the drug product used in the afore(cid:173)
`mentioned NCI trials would have led to the formation of a
`saturated solution ( or "slurry") in which the remaining solid
`5-azacytidine was Form III. The XRPD powder pattern of
`30 Form III is shown in FIG. 3 along with some of the 28
`values. Table 3 provides the most prominent 28 angles,
`d-spacing and relative intensities for this crystalline mate(cid:173)
`rial. The XRPD powder pattern for Form III is distinctly
`different from that of all of the other forms of 5-azacytidine.
`
`Thermal analysis of Form I indicates that this form of
`5-azacytidine is anhydrous. See Example 6.
`Form II
`Retained samples of the drug substance previously used to 35
`the formulate the drug product in the NCI-sponsored Cancer
`and Leukaemia Group B (CALGB) investigations (Phase 2
`trial 8291 and Phase 3 trial 9221) for the treatment of MDS
`(Investigational New Drug (IND) 7574) were also analyzed
`by XRPD. The retained drug substance samples comprised 40
`either Form I, or a mixed phase of Form I and another
`polymorph: Form II. See Example 5.
`The XRPD powder pattern of mixed phase Forms I and II
`is shown in FIG. 2 along with some of the 28 values. Peaks
`distinctive to Form II are observed at 13.5, 17.6 and 22.3° 45
`28. Table 2 provides the most prominent 28 angles,
`d-spacing and relative intensities for this mixed phase.
`
`TABLE 3
`
`5-azacytidine, Form III - the most prominent 20 angles,
`cl-spacing and relative intensities (Cu Ka radiation)
`
`20Angle (°)
`
`d-spacing (A)
`
`Relative Intensity
`
`6.566
`11.983
`13.089
`15.138
`17.446
`20.762
`21.049
`22.776
`24.363
`25.743
`26.305
`28.741
`31.393
`32.806
`33.043
`33.536
`36.371
`39.157
`41.643
`
`13.450
`7.380
`6.758
`5.848
`5.079
`4.275
`4.147
`3.901
`3.651
`3.458
`3.385
`3.104
`2.847
`2.728
`2.709
`2.670
`2.468
`2.299
`2.167
`
`32.9
`52.5
`71.0
`38.9
`48.2
`10.8
`34.8
`89.5
`13.7
`22.8
`39.9
`100.0
`22.5
`11.8
`10.1
`15.1
`11.0
`19.3
`12.1
`
`50
`
`55
`
`TABLE 2
`
`5-azacytidine, Mixed Phase Forms I and II - the most prominent
`28 angles cl-spacing and relative intensities (Cu Ka radiation)
`
`20 Angle(°)
`
`d-spacing (A)
`
`Relative Intensity
`
`12.244
`13.082
`13.458*
`14.452
`16.521
`17.648*
`18.677
`19.093
`20.231
`21.353
`22.309
`23.070
`23.909
`26.641
`26.813
`
`7.223
`6.762
`6.574
`6.124
`5.361
`5.022
`4.747
`4.645
`4.386
`4.158
`3.982
`3.852
`3.719
`3.343
`3.322
`
`34.8
`37.0
`29.2
`25.4
`19.0
`12.1
`12.7
`41.3
`42.1
`15.5
`35.1
`100.0
`18.9
`18.2
`12.6
`
`Thermal analysis and proton (1H) NMR spectroscopy
`indicate that Form III is a pseudopolymorphic form of
`60 5-azacytidine, specifically a monohydrate. See Examples
`6-7.
`Form IV
`Form IV is a novel crystalline form of 5-azacytidine.
`Form IV was recovered by slow recrystallization from a
`65 DMSO/toluene co-solvent system (see Example 2) or by fast
`recrystallization from the DMSO/chloroform co-solvent
`system (see Example 3). The XRPD powder pattern of Form
`
`

`

`US 6,887,855 B2
`
`5
`IV is shown in FIG. 4 along with some of the 28 values.
`Table 4 provides the most prominent 28 angles, d-spacing
`and relative intensities for this crystalline material. The
`XRPD powder pattern for Form IV is distinctly different
`from that of any other form.
`
`TABLE 4
`
`5-azacytidine Form IV - the most prominent 20 angles,
`d-s12acing and relative intensities (Cu Ka radiation)
`
`20 Angle(°)
`
`d-spacing (A)
`
`Relative Intensity
`
`5.704
`11.571
`12.563
`14.070
`15.943
`16.993
`18.066
`20.377
`20.729
`21.484
`21.803
`22.452
`22.709
`23.646
`24.068
`25.346
`25.346
`26.900
`27.991
`28.527
`28.723
`30.124
`30.673
`31.059
`35.059
`38.195
`38.403
`
`15.408
`7.642
`7.040
`6.289
`5.555
`5.213
`4.906
`4.355
`4.281
`4.132
`4.073
`3.957
`3.913
`3.760
`3.695
`3.526
`3.511
`3.312
`3.185
`3.126
`3.106
`2.964
`2.912
`2.877
`2.557
`2.354
`2.342
`
`24.9
`97.8
`22.2
`100.0
`67.4
`51.0
`20.1
`44.7
`49.0
`36.30
`11.2
`66.7
`64.0
`17.3
`19.4
`12.0
`12.5
`11.0
`11.4
`25.7
`34.1
`14.7
`53.6
`15.7
`18.1
`15.0
`12.6
`
`35
`
`Thermal analysis of Form IV is presented in Example 6.
`Form V
`Form Vis a novel crystalline form of 5-azacytidine. Form
`V was recovered by fast recrystallization of 5-azacytidine
`from a DMSO/toluene co-solvent system (see Example 3). 40
`The XRPD powder pattern of Form Vis shown in FIG. 5
`along with some of the 28 values. Table 5 provides the most
`prominent 28 angles, d-spacing and relative intensities for
`this crystalline material. The XRPD powder pattern for
`Form V is distinctly different from that of any other form. 45
`
`TABLE 5
`
`5-azacytidine Form V - the most prominent 20 angles,
`cl-spacing and relative intensities (Cu Ka radiation)
`
`20 Angle(°)
`
`d-spacing (A)
`
`Relative Intensity
`
`50
`
`6
`
`TABLE 5-continued
`
`5
`
`10
`
`5-azacytidine Form V - the most prominent 20 angles,
`cl-spacing and relative intensities (Cu Ka radiation)
`
`20Angle (°)
`
`d-spacing (A)
`
`Relative Intensity
`
`27.224
`28.469
`29.041
`29.429
`30.924
`31.133
`37.938
`
`3.273
`3.133
`3.072
`3.033
`2.889
`2.870
`2.370
`
`50.2
`24.2
`24.8
`15.0
`15.6
`22.6
`10.7
`
`15
`
`Thermal analysis indicates that Form V is a solvate. See
`Example 6.
`Form VI
`The drug product used in the aforementioned NCI inves(cid:173)
`tigation was typically prepared by lypohilizing a solution of
`20 ~;~~:~r~~:p~~~d ~~noni!! ~/s:~:~;t~:t::~tafciod::
`mannitol as a lyophilized cake in a vial and was adminis(cid:173)
`tered by subcutaneous injection as an aqueous suspension
`("slurry"). XRPD analysis of retained samples of the drug
`product used in the NCI investigation revealed the existence
`25 of another polymorph, Form VI. The retained drug product
`samples comprised either Form VI alone, or a Form I/VI
`mixed phase. Table 6 provides the most prominent 28
`angles, d-spacing and relative intensities for Form VI.
`
`30
`
`TABLE 6
`
`5-azacytidine Form VI - the most prominent 20 angles,
`cl-spacing and relative intensities (Cu Ka radiation)
`
`20Angle (°)
`
`d-spacing (A)
`
`Relative Intensity
`
`12.533
`12.963
`13.801
`18.929
`20.920
`21.108
`21.527
`22.623
`22.970
`24.054
`26.668
`27.210
`28.519
`29.548
`30.458
`33.810
`35.079
`37.528
`
`7.057
`6.824
`6.411
`4.6843
`4.243
`4.205
`4.125
`3.922
`3.869
`3.697
`3.340
`3.275
`3.127
`3.021
`2.932
`2.649
`2.556
`2.411
`
`10.1
`10.2
`100.0
`10.0
`34.2
`49.4
`47.0
`10.7
`13.8
`77.8
`23.0
`33.7
`12.9
`27.2
`50.3
`11.6
`12.6
`24.7
`
`11.018
`12.351
`13.176
`13.747
`14.548
`15.542
`16.556
`17.978
`18.549
`19.202
`19.819
`20.329
`21.518
`21.970
`22.521
`23.179
`24.018
`24.569
`
`8.024
`7.160
`6.714
`6.436
`6.084
`5.697
`5.350
`4.930
`4.780
`4.618
`4.476
`4.365
`4.126
`4.042
`3.948
`3.834
`3.702
`3.620
`
`40.0
`29.6
`28.3
`42.9
`18.3
`14.2
`47.8
`18.1
`83.9
`25.0
`12.1
`28.6
`100.0
`65.6
`11.5
`66.5
`13.0
`40.7
`
`55
`
`Thermal analysis and proton (1H) NMR spectroscopy of
`Form VI is presented in Examples 6-7.
`Form VII
`Form VII is a novel crystalline form of 5-azacytidine.
`Form VII was produced by fast recrystallization from a
`DMSO/methanol co-solvent system (see Example 3). Form
`VII was always isolated by this recrystallization method as
`a mixed phase with Form I. The XRPD powder pattern of
`60 mixed phase Forms I and VII is shown in FIG. 7 along with
`some of the 28 values and the Form VII distinctive peaks
`indicated with asterisks. Table 7 provides the most promi(cid:173)
`nent 28 angles, d-spacing and relative intensities for this
`mixed phase. Form VII exhibits distinctive peaks at 5.8,
`65 11.5, 12.8, 22.4 and 26.6° 28 in addition to peaks displayed
`in the Form I XRPD powder pattern. The XRPD pattern for
`mixed phase Forms I and VII is distinctly different from that
`
`

`

`US 6,887,855 B2
`
`of any other form.
`
`7
`
`TABLE 7
`
`5-azacytidine, mixed Forms I and VII - the most prominent
`28 angles cl-spacing and relative intensities (Cu Ka radiation)
`
`20 Angle(°)
`
`d-spacing (A)
`
`Relative Intensity
`
`5.779
`11.537
`12.208
`12.759
`13.048
`14.418
`16.489
`18.649
`19.101
`20.200
`20.769
`21.355
`22.365
`23.049
`23.884
`26.628
`27.145
`29.296
`29.582
`32.078
`
`15.281
`7.664
`7.244
`6.932
`6.780
`6.138
`5.372
`4.754
`4.643
`4.392
`4.273
`4.157
`3.972
`3.856
`3.723
`3.345
`3.282
`3.046
`3.017
`2.788
`
`14.7
`8.3
`28.0
`21.7
`34.4
`22.5
`21.6
`13.5
`34.7
`34.4
`10.5
`11.7
`29.9
`100.0
`23.1
`13.3
`52.9
`26.2
`11.3
`12.9
`
`Thermal analysis of Form VII is presented in Example 6
`Form VIII
`Form VIII is a novel crystalline form of 5-azacytidine.
`Form VIII was recovered by recrystallizing 5-azacytidine
`Form I from a N-methyl-2-pyrrolidone (NMP) single sol(cid:173)
`vent system (see Example 4). The XRPD powder pattern of
`Form VIII is shown in FIG. 8 along with some of the 28
`values. Table 8 provides the most prominent 28 angles,
`d-spacing and relative intensities for this material. The
`XRPD pattern for Form VIII is distinctly different from that
`of any other form.
`
`TABLE 8
`
`5-azacytidine, Form VIII - the most prominent 20 angles,
`cl-spacing and relative intensities (Cu Ka radiation)
`
`20 Angle(°)
`
`d-spacing (A)
`
`Relative Intensity
`
`6.599
`10.660
`12.600
`13.358
`15.849
`17.275
`20.243
`20.851
`21.770
`22.649
`25.554
`25.740
`29.293
`32.148
`35.074
`38.306
`
`13.384
`8.292
`7.020
`6.623
`5.587
`5.129
`4.383
`4.257
`4.079
`3.923
`3.483
`3.458
`3.046
`2.782
`2.556
`2.348
`
`2.9
`2.2
`23.4
`2.6
`2.0
`4.2
`5.8
`7.8
`74.4
`32.1
`100.0
`7.8
`3.8
`8.8
`7.4
`2.5
`
`Amorphous 5-azacytidine
`Amorphous 5-azacytidine may be recovered from equi(cid:173)
`librium saturated solutions of 5-azacytidine in propylene
`glycol, polyethylene glycol and DMSO. See Example 8.
`Pharmaceutical Formulations
`For the most effective administration of drug substance of
`the present invention, it is preferred to prepare a pharma(cid:173)
`ceutical formulation (also known as the "drug product")
`preferably in unit dose form, comprising one or more of the
`5-azacytidine forms of the present invention and one or 65
`more pharmaceutically acceptable carrier, diluent, or excipi(cid:173)
`ent.
`
`15
`
`8
`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 excipi-
`5 ent 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).
`10 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, microc(cid:173)
`rystalline cellulose, polyvinylpyrrolidone, cellulose, water,
`syrup, and methyl cellulose. The formulations can addition(cid:173)
`ally include lubricating agents such as, for example, talc,
`20 magnesium stearate and mineral oil; wetting agents; emul(cid:173)
`sifying 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
`25 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
`30 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,
`35 or delayed release of the drug substance after administration
`to the patient by employing procedures well known in the
`art.
`In certain embodiments of the invention, the
`5-azacytidine forms(s) may be made into the form of dosage
`40 units for oral administration. The 5-azacytidine forms(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
`45 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
`50 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 com(cid:173)
`pounds or with different amounts of the active compound
`55 present.
`Soft gelatin capsules may be prepared in which capsules
`contain a mixture of the 5-azacytidine form(s) and vegetable
`oil or non-aqueous, water miscible materials such as, for
`example, polyethylene glycol and the like. Hard gelatin
`60 capsules may contain granules or powder of the
`5-azacytidine polymorph in combination with a solid, pul(cid:173)
`verulent 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
`
`

`

`US 6,887,855 B2
`
`9
`particle size, and a binding agent is homogenized and
`suspended in a suitable solvent. The 5-azacytidine form(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 5
`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 10
`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 15
`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 com(cid:173)
`prises sterile, aqueous and non-aqueous injection solutions
`comprising one or more 5-azacytidine forms for which
`preparations are preferably isotonic with the blood of the
`intended recipient. These preparations may contain anti(cid:173)
`oxidants, buffers, bacteriostats, and solute; which render the
`formulation isotonic with the blood of the intended recipi(cid:173)
`ent. Aqueous and non-aqueous suspensions may include 25
`suspending agents and thickening agents. The formulations
`may be present in unit-dose or multi-dose containers, for
`example, sealed ampules and vials. Extemporaneous injec(cid:173)
`tion solutions and suspensions may be prepared from sterile
`powders, granules, and tablets of the kind previously 30
`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, 35
`glycerol, and propylene glycol. If desired, such liquid prepa(cid:173)
`rations contain coloring agents, flavoring agents, and sac(cid:173)
`charin. Thickening agents such as carboxymethylcellulose
`may also be used.
`As such, the pharmaceutical formulations of the present 40
`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 form(s). In
`liquid form, dosage unit contains from about 5 to about 200
`mg, more usually about 100 mg of the 5-azacytidine form(s). 45
`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 pro(cid:173)
`duce the desired therapeutic effect, in association with 50
`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.
`
`55
`
`10
`
`HN(Si(CH3)3)2
`
`(NH2hS04, Heat
`
`(1)
`
`+
`
`AcOfiOAc
`
`H
`
`H
`
`H
`
`H
`
`OAc OAc
`
`(2)
`
`NH2
`
`NAN lA
`HOAN
`
`O
`
`H
`
`H
`
`H
`
`H
`
`OH
`
`OH
`
`(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
`
`Slow Recrystallization from DMSO/Toluene
`
`EXAMPLES
`
`Example 1
`
`Prior Art Procedure for Synthesis of 5-azacytidine
`Drug Substance
`
`Using commercially available 5-azacytosine (1) and 1,2, 65
`3,5-Tetra-O-~-acetyl-ribofuranose (2) (RTA), 5-azacytidine
`(3) may be synthesized according to the pathway below.
`
`Dimethyl sulf