FIELD OF THE INVENTION
The present invention relates to salts of Pralatrexate and their polymorphic forms, process for
the preparation thereof, pharmaceutical compositions thereof and the use of pharmaceutical
composition for the treatment of conditions related to human tumors.
BACKGROUND OF THE INVENTION
Pralatrexate chemically known as "(25)-2-[[4-[(li?S)-l-[(2,4-diaminopteridin-6-
yl)methyl]but-3-ynyl]benzoyl]- aminojpentanedioic acid" also known as "10-Propargyl-lOdeazaminopterin"
or "PDX" is a compound which has been tested and foimd useful in the
treatment of cancer. In its racemic form, 25)-2-[[4-[(li?5)-l-[(2,4-diaminopteridin-6-
yl)methyl]but-3-ynyl]benzoyl]amino]- pentanedioic acid has been approved by the U.S. Food
and Drug Administration (FDA) as a treatment for relapsed and refractory peripheral T-cell
lymphoma.
Pralatrexate molecule (I) was first disclosed in Journal of Medicinal Chemistry. 36.- 2228-
2231 (1993) by DeGraw et al., and subsequently in US 5374726, US 5354741 and US
6028071.
o V'
NH, r TT NH ^ - ^ >f
XXJ (I)
Tiseni et al., in International Application Publication No. WO 2012/061469 discloses
crystalline forms A, B, and C of Pralatrexate. The publication also discloses amorphous form
of Pralatrexate.
Polymorphism, the occurrence of different crystal forms, is a property of some molecules and
molecular complexes. A single molecule, like Pralatrexate, may give rise to a variety of
polymorphic forms having distinct crystal structures and physical properties like melting
point. X-ray diffraction pattern, infrared absorption fingerprint, Raman spectnmi, density,
crystal group, and solid state NMR spectrum.
2
• One polymorphic form may give rise to thermal behavior different from that of another
polymorphic form. Thermal behavior can be measured in the laboratory by such techniques
as capillary melting point, thermogravimetric analysis ("TGA"), and differential scanning
calorimetry ("DSC"), which have been used to distinguish polymorphic forms.
The difference in the physical properties of different polymorphic forms results from the
orientation and intermolecular interactions of adjacent molecules or complexes in the bulk
solid. Accordingly, polymorphs are distinct solids sharing the same molecular formula yet
having distinct advantageous physical properties compared to other polymorphic forms of the
same compound or complex.
The discovery of new polymorphic forms and solvates of a pharmaceutically useful
compound provides a new opportunity to improve the performance characteristics of a
pharmaceutical product. It enlarges the repertoire of materials that a formulation scientist has
available for designing, for example, a pharmaceutical dosage form of a drug with a targeted
release profile or other desired characteristic. Therefore, there is a need for additional
polymorphic forms of Pralatrexate.
It is also important that the processes for the preparation of the salts in different polymorphic
forms be robust and reproducible, so that the processes are easily scaled up in the plant.
The present invention addresses a need in the art by providing salts of Pralatrexate in
different polymorphic forms, which are believed to provide significant advantages for making
pharmaceutical compositions, as compared to the polymorphic form of Pralatrexate free acid
disclosed in the prior art.
As is well known to the skilled artisan, variations in the salt form and/or crystal structure of a
pharmaceutical drug substance often affect the dissolution rate (which may affect
bioavailability, etc.), manufacturability (e.g., ease of handling, ability to consistently prepare
doses of known strength) and stability (e.g., thermal stability, shelf life, etc.) of a
pharmaceutical drug product. Salts of Pralatrexate and their Polymorphic forms, such as the
crystalline forms and amorphous form of the present invention are believed to provide
advantages over Pralatrexate free acid disclosed in the prior art.
Pralatrexate has been described as the free acid of formula (I) in US 6028071, as a treatment
for relapsed and refractory peripheral T-cell lymphoma.
The present invention provides salts of Pralatrexate. The present invention also provides
processes for their preparation, which is robust and reproducible. The present invention
3
' further provides pharmaceutical compositions comprising said salts of Pralatrexate and at
least one pharmaceutically acceptable excipient. The present invention still further provides a
method for treatment of tumors using pharmaceutical composition comprising salts of
Pralatrexate.
SUMMARY OF THE INVENTION
First aspect of the present invention provides a salt of Pralatrexate of formula (II), wherein M
is an alkali or alkaline earth metal.
o V
xXJ I
HjN"^ N N ^^::;v
(II)
In one variation, the salt is represented by Formula (IF),
I ll
XXJ I
(II')
In another variation, the salt of Formula (IF) is Crystalline.
In yet another variation, the salt of Formula (IF) is Amorphous.
In a further variation, the salt is represented by formula (Ila)
Ox. ,OLi
O ^V^
NH, r iT NH ^ ^ >r
I II
XXJ I
H2N'^ N N'^ ^ ^^
(Ila)
In a yet further variation, the salt is represented by the formula (lib)
4
0 V
^^;\ ^^ ' ^ ^^'"v^ /OK
NH2 r II ^ " ^ ^ ^ [f^
XXJ I
(lib)
Second aspect of the present invention provides a process for producing a salt of formula (II)
comprising the steps of
a) reacting Pralatrexate of formula (I) with a source of alkali or alkaline earth metal in
presence of a suitable solvent to obtain a reaction mixture;.
b) stirring the reaction mixture of step (a) and;
c) isolating the salt from the reaction mixture.
In one variation, step (a) is followed by addition of a second suitable solvent.
In another variation, step (b) is followed by cooling.
In yet another variation, salt of step (c) is isolated by freeze drying or filtration.
In a still fiirther variation, the compound of Formula (II) is converted to a compoimd of
Formula (I).
Third aspect of the present invention provides pharmaceutical composition comprising salt of
formula (II) and at least one pharmaceutically acceptable excipient.
Fourth aspect of the present invention provides a method for treatment of tumors using
pharmaceutical composition comprising salt of formula (II).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the X-ray Powder diffractogram of Pralatrexate.
Figure 2 depicts the X-ray Powder diffractogram of crystalline Pralatrexate disodium of
formula (IF).
Figure 3 depicts the X-ray Powder diffractogram of amorphous Pralatrexate disodium of
formula (IF).
Figure 4 depicts the X-ray Powder diffractogram of amorphous Pralatrexate dilithium of
formula (Ila).
Figure 5 depicts the X-ray Powder diffractogram of amorphous Pralatrexate dipotassium of
formula (lib).
Figure 6 depicts the DSC thermogram of amorphous Pralatrexate dilithium of formula (Ila).
5
* Figure 7 depicts the TGA profile of amorphous Pralatrexate dilithium of formula (Ila).
Figure 8 depicts the DSC thermogram of amorphous Pralatrexate dipotassium of formula
(lib).
Figure 9 depicts the TGA profile of amorphous Pralatrexate dipotassium of formula (lib).
DETAILED DESCRIPTION OF THE INVENTION
The alkali metal in the compound of formula II may be selected fi-om the group comprising of
sodium, lithium, potassium and the like.
The alkaline-earth metal in the compound of formula II may be selected fi-om the group
comprising of calcium, magnesium and the like.
The salt of Formula (II) may be characterized using various techniques, which are well
known to those of ordinary skill in the art. Examples of characterization methods include, but
are not limited to, single crystal X-ray diffraction, powder X-ray diffraction (PXRD),
simulated powder X-ray patterns, differential scanning calorimetry (DSC), solid-state ^'^CNMR,
Raman spectroscopy, infrared spectroscopy, moisture sorption isotherms, thermal
gravimetric analysis (TGA), and hot stage techniques.
The crystalline salt of Formula (IF) may be characterized as having powder X-ray diffraction
pattern peaks of 29 at 4.32, 6.08, 6.81, 9.93, 17.4, 23.13, 25.48, 27.82 ± 0.2°. The crystalline
salt of formula (IF) may be characterized by having a powder X-ray diffraction pattern
substantially as shown in Figure 2. The crystalline polymorphic form of formula (IF) may be
fiirther characterized by having water content between about 1% and about 20 %.
The amorphous salt of formula (IF) may be characterized by powder X-ray diffraction of one
broad halo at 26 of about 26°. The amorphous polymorphic form of salt of formula (IF) may
be characterized by having a powder X-ray diffraction pattern substantially as shown in
Figure 3. The amorphous form may be fiirther characterized by having water content between
about 1 % and about 20 %.
The amorphous salt of formula (Ila) may be characterized by having a powder X-ray
diffraction pattern substantially as shown in Figure 4. The amorphous form may be fiirther
characterized by having water content between about 1% and about 20 %. The amorphous
salt of formula (Ila) may be further characterized by having a DSC thermogram substantially
as shown in Figure 6. The amorphous salt of formula (Ila) may be still fiirther characterized
by having a TGA profile substantially as shown in Figure 7. The amorphous form may be
fiirther characterized by having water content between about 1% and about 20 %.
6
• The amoqjhous salt of formula (lib) may be characterized by having a powder X-ray
diffraction pattern substantially as shown in Figure 5. The amorphous form may be further
characterized by having water content between about 1% and about 20 %. The amorphous
salt of formula (lib) may be further characterized by having a DSC thermogram substantially
as shown in Figure 8. The amorphous salt of formula (lib) may be still further characterized
by having a TGA Profile substantially as shown in Figure 9. The amorphous form may be
further characterized by having water content between about 1% and about 20 %.
Furthermore, the present salt forms may be anhydrous or contain solvent(s), such as water. In
some embodiments, the present salt forms comprise solvate, such as hydrate.
Pralatrexate used as starting material may be obtained by any of the methods known in the art
including as described in Example-1.
The source of alkali or alkaline earth metal, used in the process for producing salt of formula
(II) may be selected from inorganic bases like metallic hydroxides such as but not limited to
those of sodium hydroxide, potassium hydroxide, lithium hydroxide, barium hydroxide,
calcium hydroxide and magnesium hydroxide; metal carbonate such as lithium carbonate,
sodium carbonate, potassium carbonate, barium carbonate, calcium carbonate and magnesium
carbonate; metal bicarbonate such as sodium bicarbonate, potassium bicarbonate, barium
bicarbonate, calcium bicarbonate.
The suitable solvent, used in the process for producing salt of formula (II) may be selected
from the group comprising of water; alcohols, such as methanol, ethanol and isopropanol;
nitriles, such as acetonitrile; chlorinated hydrocarbons, such as methylene chloride,
ethylenedichloride; dipolar aprotic solvents, such as dimethylsulfoxide, dimethyacetamide
and dimethylformamide; esters, such as ethyl acetate and isopropyl acetate; cyclic ethers,
such as dioxane and tetrahydrofiiran; ketone such as acetone, diisobutyl ketone,
cyclohexanone, methylcyclohexanone, methyl ethyl ketone, methyl isobutyl ketone,
acetylacetone or mixtures thereof
The process for producing a salt of formula (II) may be carried out by reacting Pralatrexate of
formula (I) at a temperature range of 0°C-60°C. Preferably the temperature is between 20-
30°C.The process for producing a salt of formula (II) may be carried out by reacting
Pralatrexate of formula (I) for about 1 minute to 24 hours. Preferably reaction is carried out
for 10 minutes to 8 Hours. A second suitable solvent may be used in the process for
producing salt of formula (II) as defined herein above. The reaction mixture may be further
cooled in the process for producing salt of formula (II) at a temperature range of -80° to
20°C. The salt of formula (II) may be isolated from the reaction mixture by freeze drying or
7
' filtration. The salt of formula (II) may be isolated from the reaction mixture at a temperature
rangeof-200°Cto30°C.
The Salt of formula (II) may be converted to compound of formula (I).
The process for producing crystalline of salt of formula (IT) may be carried out in an aqueous
medium. The crystalline salt of formula (11') may be obtained by adding a suitable second
solvent to the reaction mixture. The second solvent may be selected from methanol, ethanol,
2-propanol, Tetrahydrofuran, acetic acid, acetonitrile and acetone.
The process for producing a crystalline salt of formula (IF) may be carried out at a
temperature range of 0°C - 60°C. Preferably the temperature is between 20-30°C.
The process for producing a crystalline salt of formula (IF) may be carried out for about 1
minute to 24 hours. Preferably reaction is carried out for 10 minutes to 15 minutes.
The reaction mixture may be isolated by filtration to obtain a crystalline salt of formula (IF).
The process for producing an amorphous salt of formula (IF) may be carried out in an
aqueous medium.
The process for producing an amorphous salt of formula (IF) may be carried out at a
temperature range of 0°C - 60°C. Preferably the temperature is between 20-30°C.
The process for producing an amorphous sah of formula (IF) may be carried out for about 1
minute to 24 hours. Preferably reaction is carried out for 10 minutes to 20 minutes.
The amorphous salt of formula (IF) may be obtained by isolating the reaction mixture by
freeze drying.
The process for producing an amorphous salt of formula (Ila) may be carried out in a suitable
solvent.
The reaction mixture may be cooled in the process for producing salt of formula (Ila) in the
temperature range of-80° to 20°C. Preferably the temperature is between -20 to 15°C.
The process for producing an amorphous salt of formula (Ila) may be carried out for about 1
minute to 24 hours. Preferably reaction is carried out for 1 hour to 8 hours.
The reaction mixture may be isolated by filtration to obtain amorphous salt of formula (Ila).
The process for producing an amorphous salt of formula (lib) may be carried out in an
aqueous medium.
The amorphous salt of formula (lib) may be obtained by adding a suitable second solvent to
the reaction mixture. The second solvent may be selected from methanol, ethanol, 2-
propanol, Tetrahydrofuran, acetic acid, acetonitrile and acetone.
The process for producing an amorphous salt of formula (lib) may be obtained by cooling the
reaction mixture in the temperature range of-25°C to 25°C.
8
' The reaction mixture may be isolated by filtration to obtain a salt of formula (lib).
As used herein the term "isolation" includes filtration, filtration under vacuum, freeze drying,
and decantation.
The term "freeze drying" as used herein describes a process in which a solution or slurry
containing the sah of formula (IT) is placed within a container and cooled or frozen under
vacuum. The container is connected to one or more vacuum sources, and a vacuum (pressure
less than 760 torr) is applied. The container is then cooled to a temperature less than 0°C,
preferably between -200° C and 0° C.
In further aspect is provided a pharmaceutical composition comprising salts of formula (II)
and at least one pharmaceutically acceptable excipient.
In still ftirther aspect frirther is provided a method for treatment of tumors using
pharmaceutical composition comprising salt of formula (II), including both solid tumors and
leukemias. The composition may be used in the treatment of human mammary tumors and
human lung cancer, particularly in the treatment of T-cell lymphomas.
The following examples are provided to illustrate the invention and do not limit the scope
thereof One skilled in the art will appreciate that although specific reagents and conditions
are outlined in the following examples, modifications can be made which are meant to be
encompassed by the spirit and scope of the invention.
Example-l
Pralatrexate
To aqueous sodium hydroxide (11.6 g NaOH in 472 mL water) and Methanol (944 mL), 10-
Propargyl-lO-deazaminopterin dimethyl ester (59.0 g) was added at 20-25°C and stirred the
reaction mass for 8 hours. After completion of reaction which was monitored by HPLC, pH
of the reaction mass was adjusted to 6.6 with acetic acid. Excess methanol was evaporated
under reduced pressure below 40° C and water (1298mL) was added to the residual solution.
The pH of the residual solution was adjusted to 4.5 with dilute acetic acid. The reaction mass
was stirred for 30 minutes at 20-25° C and precipitated solid was filtered. The solid was
furthered purified with water (590 mL) by stirring at 20-25°C for 30-35 minutes. The solid
was filtered and dried under vacuum at 35-40° C to give 39 g (70 %) of the title compound.
Purity: 99.56 %
Water content = 4.8 % (w/w)
9
*H NMR (DMSO-d6; 400MHz): 6 1.91 (m, IH), 2.05 (m, IH), 2.33 (t, J=7.2 Hz, 2H), 2.59
(bm, 2H), 2.78 (s, IH), 3.14-3.20 (bm, IH), 3.28 (dd, J=14.4 Hz & 6.4 Hz, IH), 3.64 (quintet,
J=7.2 Hz), 4.35 (bm, IH), 6.30 (bs, 2H, NHj), 7.39 (d, J=8.0 Hz, 2H), 7.61 & 7.63 (2xbs, 2H,
NH2), 7.73 (d, J=8.0Hz, 2H), 8.39 (bs, IH), 8.50 (d, J=7.6 Hz, IH, NH), 12.20 (bs, 2H,
2XCO2H).
*^C NMR (DMSO-d6; lOOMHz): 5 24.84 (CH2), 25.94 (CH2), 30.46 (CH2), 39.08 (CH2),
43.05 (CH), 51.93 (CH), 72.90 (CH), 82.57 (C), 121.51 (C), 127.35 (2xCH), 127.35 (2xCH),
132.22 (C), 146.69 (C), 147.20 (C), 150.56 (CH), 154.17 (C), 162.41 (C), 162.77 (C), 166.42
& 166.46 (CONH), 173.54 (CO2H), 173.94 (CO2H).
MS (ES+) m/z: 478 [M+H]^.
IR(KBr, cm-^): 1540,1557, 1639, 1704, 3300, 3420.
X R D (<'2Theta; Cu): 8.47, 10.85, 12.28, 14.34, 15.00, 15.78, 18.90, 21.79, 24.20, 27.5,
28.92, 34.28.
Example-2
Crystalline Pralatrexate disodium salt
To sodium hydroxide (0.10 g) dissolved in water (12 mi) was added Pralatrexate (0.6 g) at
20-25°C and stirred for 10 min at 20-25°C. Isopropanol (85 ml) was added to the resulting
solution and stirred for further 4 hours at 20-25°C. The solid was filtered and dried the solid
under vacuum at 35-40°C to give 0.50 g (76.3 %) of the title compound.
Weight : 0.50 g
Yield : 76.3%
Water content : 10.3% w/w
XRD (°2e;Cu) : 4.32, 6.08, 6.81, 9.93, 17.4, 23.13, 25.48, 27.82
Example-3
Amorphous Pralatrexate disodium salt
To sodium hydroxide (0.086 g) dissolved in water (5 ml) was added Pralatrexate (0.50 g) at
20-25°C and stirred for 15-20 min at 20-25°C. The resulting reaction mixture was freeze
dried under vacuum for 24 hours to give 0.5 g (91.5 %) of the title compound.
Weight : 0.50 g
Yield 91.5%
Water content 8.2% w/w
XRD (°2e; Cu) : Halo at 26°
10
Example-4
Pralatrexate dilithium salt
To Pralatrexate (2.0 g) in Methanol (20 mL), Lithium hydroxide (0.501 g) was added at 20-
25 °C and stirred for 5 hours. The reaction mixture was cooled to 0-5 °C and the product was
filtered. The cooling and filtration was carried out in presence of nitrogen. The filtered
product was washed with cold (0-5°C) Methanol (4 mL), suck-dried for 2-3 hours and fiirther
dried under vacuum at 50-55°C for 14-18 hours. The product was characterized by XRD,
TGA and DSC.
Weight : 1.1 g
Yield : 44 %
Purity by HPLC (%) : 99.8
Water content : 1.7% w/w
Example-5
Pralatrexate dipotassium salt
To Pralatrexate (2.0 g) in Demineralized water (16 mL), aqueous Potassium hydroxide (0.5 g
in 4mL Demineralized water) was added at 20-25°C and stirred for 25-30 min. To this
agitating reaction mixture, Isopropanol (110 mL) was added. The reaction mixture was stored
at -20 to -15°C for 12 hours and then was allowed to warm to 0-5°C with stirring. The
product was filtered, washed with Isopropanol (10 mL), suck-dried for 1-2 hours followed by
drying at 50-55°C for 8 hours. The product was characterized by XRD, TGA and DSC.
Weight : 1.7 g
Yield : 73.9%
Purity by HPLC : 99.8 %
Water content : 1.7% w/w
11

WE CLAIM:
1. A salt of Pralatrexate of fonnula (II)
o ^
NH2 r^ Y ^ NH ^ ^ jr^
xXJ I
H2N'^ N N-^ ^ ^^
(II)
wherein M is an alkali or alkaline earth metal selected from the group comprising of
sodium, potassium, lithium, calcium and magnesium.
2. The salt of claim 1, wherein M is sodium.
3. The salt of claim 2, wherein the salt is crystalline characterized by X-ray diffraction
pattern with peaks at about 4.32, 6.08, 6.81, 9.93, 17.4, 23.13, 25.48, 27.82 ± 0.2°twotheta.
4. The salt of claim 2, wherein the salt is in amorphous form.
5. The salt of claim 1, wherein M is lithium.
6. The salt of claim 5 wherein the salt is in amorphous form.
7. The salt of claim 1 wherein M is potassium.
8. The salt of claim 7 wherein the salt is in amorphous form.
9. A process for preparing a salt of Pralatrexate of formula (II) comprising the steps of
a) reacting Pralatrexate of formula (I)
Ov. .OH
o "V
NH, r n"^ NH ^ ^ >f
(I)
with a source of alkali or alkaline earth metal in a suitable solvent to obtain a reaction
mixture;
b) stirring the reaction mixture of step (a) and;
c) isolating the salt from the reaction mixture.
12
10. A process of claim 9 wherein the source of alkali metal of step (a) is selected from the
group comprising of sodium hydroxide, potassium hydroxide, lithium hydroxide, lithium
carbonate, sodiimi carbonate, potassium carbonate, sodium bicarbonate, potassium
bicarbonate.
11. A process of claim 9 wherein step (a) is optionally followed by addition of a second
suitable solvent.
12. A process of claim 9 or claim 11 wherein the suitable solvent is selected from the group
comprising of water, methanol, ethanol, isopropanol, acetonitrile, methylene chloride,
ethylenedichloride, dimethylsulfoxide, dimethyacetamide and dimethylformamide, ethyl
acetate, isopropyl acetate, dioxane, tetrahydrofuran, acetone, diisobutyl ketone,
cyclohexanone, methylcyclohexanone, methyl ethyl ketone, methyl isobutyl ketone,
acetylacetone.
13. A process of claim 9 wherein step (b) is optionally followed by cooling.
14. A process of claim 13 wherein the said cooling is from - 80°C to 20°C.
15. A process of claim 9 wherein salt of step (c) is isolated by freeze drying or filtration.
16. A pharmaceutical composition comprising a salt of claim 1 and at least one
pharmaceutically acceptable excipient.
17. A method of treating tumors using pharmaceutical composition comprising a salt of
Pralatrexate of claim 1.
Dated this 31'' day of July 2013
Dr. Sanchita Ganguli
ofS.MAJUMDAR«&CO.
Applicant's Agent
13