Description
Key intermediates and impurities of the synthesis of Apixaban: Apixaban glycol esters
Technical Field
[0001] The present invention refers to a process for the preparation the active
pharmaceutical ingredient named Apixaban through new key
intermediates.
Background Art
[0002] Apixaban is an active pharmaceutical ingredient used as anticoagulant for
the treatment of venous thromboembolic events.
[0003] Apixaban has chemical name, 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-
oxopiperidin-1-yl)phenyl]-4,5-dihydropyrazolo[5,4-c]pyridine-3-
carboxamide and has the following chemical formula (I):
(I)
[0004] Some solvates of Apixaban are known, for example, are known the
solvates of Apixaban with formamide or with dimethylformamide, both
having stoichiometry 1: 1 .
[0005] Apixaban dihydrate, i.e. the hydrate form of Apixaban having two
molecules of water per one of Apixaban is also known.
[0006] In literature are disclosed some routes of synthesis of Apixaban, in
particular, in WO2007/0001385 is described in detail the first industrial
synthesis of Apixaban on multi-Kilos scale.
[0007] The PCT application WO2007/0001385 discloses in example 6 a process
for the preparation of Apixaban by amidation reaction on 10 Kg scale of
the Apixaban ethyl ester according to the following reaction scheme:
Apixaban ethyl ester Apixaban
[0008] According said procedure, using anhydrous ammonia in propylene glycol
and performing the reaction for at least 12 hours at 90°C , Apixaban was
obtained with 94.6% of isolated molar yield.
[0009] The biggest advantage of the method disclosed in example 6, also in
comparison with examples 7 and 9 of WO2007/0001385, is that such a
method provides Apixaban having the polymorphic form named N-1 , a
solid form which is well characterized in example 9 of the same application
and which is the thermodynamically stable form of Apixaban.
[0010] According to the regulatory information provided by the originator,
Apixaban form N-1 is the form currently on the market, so that, with the
aim of providing an active pharmaceutical ingredients which provides
exactly the same physical-chemical and therapeutical properties of that the
originator for the generic market, it is important to find a method for the
preparation of Apixaban which provides the polymorphic form N-1 .
[001 1] In the publication J. Med. Chem., 2007, vol. 50, 22, pag. 5339-5356,
Apixaban is prepared from Apixaban ethyl ester with aqueous ammonia at
5% in ethylene glycol heating to 120°C for 4 hours with a molar yield of
76%, according to the following reaction scheme:
[0012] Unfortunately, nothing is said relating to the solid form of Apixaban thus
prepared.
[0013] The publication Synthetic Communication, 43, pag. 72-79, (2013)
discloses a method for the preparation of Apixaban from the intermediate
Apixaban ethyl ester using 25% aqueous ammonia in methanol at 65°C for
5 hours with 9 1% molar yield.
[0014] Nevertheless such a method, probably because is carried out in Methanol
instead of in a glycol solvent, does not provide Apixaban form N-1 , indeed
the m.p. of the product is 171-173°C which is different from that of the
form N-1 being 235-237°C. Moreover no data relating to the purity of the
product are provided.
[0015] In the patent publication WO201 3/1 19328, example 2, the synthesis of
Apixaban was carried out from Apixaban ethyl ester using 5% aqueous
ammonia in propylene glycol at 100°C overnight. The reaction mixture was
not seeded with the form N-1 so that at the end of the work-up a different
solid form, named Form I, has been isolated. Apixaban Form I thus
prepared is Apixaban 1,2-propylen glycol hemisolvate.
[0016] Considering the above prior art, and our preliminary experimental results,
the presence of a glycol solvent, such as in example 6 of
WO2007/0001385, seems to promote the preparation of the polymorphic
form N-1 , while it appears that the presence of an alcohol solvent as in
example 7 and 9 WO2007/0001385 tends to provide the solid form H2-2.
[0017] Therefore, to prepare Apixaban form N-1 it appears convenient to isolate
Apixaban from a glycol solvent.
[0018] Nevertheless, although the industrial method for the preparation of
Apixaban form N-1 disclosed in WO2007/0001385 already uses a glycol
solvent, such a method has the drawback that it requires long reaction
times at high temperature, i.e. at least 12 hours at 90°C or, according to
WO20 13/1 19328, 100°C overnight, or 4 hours at 120°C (see above J.
Med. Chem. (2007)).
Description of the Figures
[0019] Figure 1 shows the kinetic study of the conversion of Apixaban glycol ester
to Apixaban in comparison to the conversion of Apixaban ethyl ester to
Apixaban, both conversion carried out under the same amidation
conditions.
Summary of invention
[0020] The problenn addressed by the present invention is therefore that of
providing an improved process for the preparation of Apixaban and
solvates or hydrates thereof which avoids long reaction times and/or high
temperatures.
[0021] This problem is solved by a process for the preparation of a Apixaban and
salts thereof as outlined in the annexed claims, whose definitions are
integral part of the present description.
[0022] Further features and advantages of the process according to the invention
will result from the description hereafter reported of examples of realization
of the invention, provided as an indication and not as a limitation of the
invention.
Description of embodiments
[0023] Object of the present invention is a process for the preparation of
Apixaban of formula (I) and solvates or hydrates thereof:
(I)
[0024] by amidation reaction of the compound of formula (II):
(II)
[0025] wherein R is chosen from the group comprising a linear or branched C2-C6
alkyl, -CH2-CH(OH)-CH 2- and -(R O)nR1- wherein R is a linear or
branched C2-C4 alkyl and n is an integer from 1 to 6.
[0026] It has been indeed surprisingly found that starting from the compound of
formula (II), i.e. from a glycol ester for Apixaban, the amidation reaction to
convert it to Apixaban proceed much more faster than using the
conventional Apixaban C1-C2 alkyl esters.
[0027] The effect provided by the compound of formula (II) of the present
invention is maybe due to the free oxydryl group that in any way favourites
the substitution of the alkoxy group operated by the ammonia, maybe
providing a sort of anchimeric assistance. Alternatively, such effect is
maybe due to the presence of another oxygen whose electronegativity
provide an ester which is easily substituted by the ammonia.
[0028] In other words, the glycol esters of Apixaban of formula (II) are converted
to Apixaban by means of an amidation reaction much more easily or
quickly than the conventional Apixaban esters.
[0029] Clear evidences of the effect provided by the process of the invention are
provided in the comparative Table 1 and Figure 1.
[0030] Although in some of the following examples the amidation reactions are
carried out for 6 hours (just to accomplish to an experimental standard
protocol), they were completed well before.
[0031] Indeed the amidation reaction of the compound of formula (II) to provide
Apixaban lasts typically only 3 hours at a temperature comprised between
80°C and 90°C, achieving 99.0% of conversion.
[0032] As shown in Table I, under exactly the same conditions the conversion of
Apixaban ethyl ester to Apixaban takes at least 6 hours.
[0033] According to the industrial process disclosed in WO2007/0001385, said
conversion lasts at least 12 hours at 90°C.
[0034] By comparison with the known amidation reaction of Apixaban esters, the
process of the present invention thus requires shorter reaction times.
[0035] As a further advantage of the method of the present invention is that the
by-product of reaction is a glycol, which can be the solvent medium of the
reaction, thus avoiding the presence of further residual solvents and
avoiding the presence of alcohol, e.g. ethanol, as by-product that,
according to example 7 and 9 of WO2007/0001385, seems to promote
the solid form H2-2.
[0036] The amidation reaction can be performed using anhydrous ammonia,
aqueous ammonia, ammonium salts such as, for example, ammonium
hydroxide, ammonium chloride, ammonium bromide, ammonium sulphate,
etc..
[0037] The amidation reaction of the process of the present invention can be
carried out in an aqueous medium and/or in an organic solvent.
[0038] The organic solvent can be an alcohol, glycol, ether, ester, nitrile,
hydrocarbon, chlorinated hydrocarbon, etc. and mixtures thereof.
[0039] The organic solvent can be an alcohol such as for example, methanol,
ethanol, isopropanol, butanol, etc.
[0040] The organic solvent can be an ether such as methyl-t-buthyl ether, an
ester such as ethylacetate or isopropylacetate, a nitrile such as
acetonitrile, an hydrocarbon such a toluene or xylene, a chlorinated
hydrocarbon such as chloroform, dichloromethane, chlorobenzene, etc..
[0041] The glycol solvents are preferred because they provides Apixaban in the
solid form N-1 and because, using a glycol solvent of formula HO-R-OH
wherein R is the same of that of the compound of formula (II), the by
product of the reaction is the same compound of the solvent, thus avoiding
an additional residual solvent to be monitored.
[0042] The organic solvent can be an a glycol chosen among ethylenglicol, 1,2-
propilenglycol, 1,3-propilenglycol, diethylenglycol, PEG200,
Polypropylenglycol, glycerol.
[0043] According to a preferred embodiment of the process of the present
invention, the preferred solvents are ethylenglicol, 1,2-propylenglycol, 1,3-
propylenglycol, diethylenglycol.
[0044] The glycol solvent of formula HO-R-OH used in the process of the
invention can have the R group with the same meaning of the R group of
the compound of formula (II), or, alternatively, R can have a different
meaning. According to a referred embodiment, the glycol solvent of
formula HO-R-OH has a R group with the same meaning of the R group of
the compound of formula (II).
[0045] In the compound of formula (II), the R group is chosen from the group
comprising a linear or branched C2-C6 alkyl, -CH2-CH(OH)-CH2-, -
(R O)nR1- wherein R is a linear or branched C2-C4 alkyl an n is an integer
from 1 to 6.
[0046] The linear or branched C2-C6 alkyl is a group chosen in the group
comprising -CH2CH2-, -CH2CH2CH2-, -CH2(CH3)-CH2- , -(CH2 )4-, -
CH(CH3)CH2CH2- , -CH2CH(CH3)CH2, -CH2CH2CH(CH3)-, -(CH2)5- , -
[0047] The group -(R O)nR1- , wherein n is an integer from 1 to 6 and wherein R
is a linear or branched C2-C4 alkyl, is a group chosen in the group
comprising -(C C O nC C -, -(C C C O nC C C -,
(CH(CH3)CH2O)nCH(CH3)CH2- , -(CH2CH(CH3)O)nCH2CH(CH3)-,
(CH2CH2CH2CH2O)nCH2CH2CH2CH2-, etc..
[0048] According to a preferred embodiment, the group -(R O)nR1- is
(CH2CH2O)nCH2CH2-.
[0049] According to a preferred embodiment of the present invention, the
compound of formula (II) is a compound isolated, i.e. a compound isolated
from the reaction mixture from which it is prepared. Therefore, the
compound of formula (II) is typically in form of a solid or of an isolated oil.
[0050] According to a preferred embodiment of the present invention, the
compound of formula (II) is a compound having a purity higher than 80%,
measured in HPLC A/A%, for example by using the analytical method of
example 13.
[0051] The process of the present invention is carried out at a temperature
comprised between 60°C and 140°C, preferably between 80°C and 120°C,
more preferable between 80°C and 90°C.
[0052] When the amidation reaction is carried out between 80°C and 90°C, the
reaction is completed (i.e. conversion higher than 99%) in about 3 hours.
[0053] According to a preferred embodiment, the process of the present invention
further comprises the step of preparation of the compound of formula (II):
(II)
wherein R is chosen from the group comprising a linear or branched C2-C6
alkyl, -CH2-CH(OH)-CH 2- and -(R O)nR1- wherein R is a linear or
branched C2-C4 alkyl and n is an integer from 1 to 6,
by means of a transesterification reaction of the compound of formula (III):
(Ml)
wherein R2 is a linear or branched C1-C6 alkyl.
[0055] The transesterification of the compound of formula (III) to provide the
compound of formula (II) is thus carried out by reaction of the compound
of formula (III) with a glycol, with a polyglycol or with glycerol.
[0056] The transesterification reaction is carried out by reaction of a glycol of
formula OH-R-OH where R is chosen from the group comprising a linear
or branched C2-C6 alkyl, -CH2-CH(OH)-CH 2- and -(R O)nR1- wherein R is
a linear or branched C2-C4 alkyl and n is an integer from 1 to 6.
[0057] Preferred glycols are ethylenglycol, 1,2-propylenglycol, 1,3-propylenglycol
and diethylenglycol.
[0058] Preferred polyglycols are Polyethylenglycol (200) (abbreviated PEG200)
and Propylenglicol 200 (PPG200). PEG has the following chemical
structure HO-(CH2CH2O)nCH2CH2-OH.
[0059] The transesterification reaction can be carried out at a pH comprised
between 7.5 and 10.0, preferably at pH comprised between 8.0 and 9.5.
[0060] The transesterification reaction can be carried out in presence of bases,
preferably inorganic bases such as, preferably, NaHCO3 or KH2PO 4 , i.e.
bibasic potassium phosphate.
[0061] The transesterification reaction is preferably carried out in presence of
bibasic potassium phosphate since it provides the higher and faster
conversions.
[0062] The transesterification reaction is carried out at a temperature comprised
between 60°C and 120°C, preferably between 70°C and 80°C, more
preferably at about 75°C.
[0063] The transesterification reaction is carried out using an excess of the
reactant glycol as reaction solvent.
[0064] According to an alternative route of synthesis, the compound of formula (II)
can be prepared by inner cyclization of the compound of formula (IV):
(IV)
[0065] wherein R is chosen from the group comprising a linear or branched C2-C6
alkyl, -CH2-CH(OH)-CH 2- and -(R O)nR1- wherein R is a linear or
branched C2-C4 alkyl and n is an integer from 1 to 6.
[0066] The compound of formula (IV) can be prepared adapting the known prior
art methods used for the preparation of the correspondent esters to the
preparation of said Apixaban glycol ester. Using this synthetic approach,
the compound of formula (II) can be prepared avoiding the preparation of
the previous Apixaban esters of formula (III). See reaction scheme below.
[0067] The cyclization of the compound of formula (IV) to provide the compound
of formula (II) can be carried out in presence of a base such, for example,
t-BuOK.
[0068] Moreover, the compound of formula (II) can be prepared according to
other synthetic approach which do not necessarily involve the preparation
of the compound of formula (III) or, however, esters intermediates.
[0069] In the following reaction scheme is described an example of direct
synthesis of the compound of formula (II) by reaction of the glycol ester of
the hydrazone starting material.
( II )
[0070] wherein R is chosen from the group comprising a linear or branched C2-C6
alkyl, -CH2-CH(OH)-CH 2- and -(R O)nR1- wherein R is a linear or
branched C2-C4 alkyl and n is an integer from 1 to 6.
[0071] According to a preferred embodiment, in the process of the present
invention for the preparation of Apixaban and/or in the further step of
preparation of the compound of formula (II) starting from the compound of
formula (III), the R group in the compound of formula (II) is chosen from
the group comprising -CH2CH2-, -CH2CH(CH3)-, CH(CH3)CH2- , -(CH2)3-
and CH2CH2OCH2CH2- , or according to another embodiment, chosen from
the group comprising -CH CH - , -(CH )3- and CH CH OCH CH - .
[0072] Said compounds of formula (II) can be conveniently prepared by
transesterification of the compound of formula (III), wherein R is for
example ethyl, respectively with ethylenglycol, 1,2-propylenglycol, 1,3-
propylenglycol and diethylenglycol or, according to another embodiment,
respectively with ethylenglycol, 1,3-propylenglycol and diethylenglycol.
[0073] The compound of formula (II) prepared from the compound of formula (III)
by transesterification with 1,2-propanediol, is a mixture of the two isomers
with ratio 1:2 wherein R is -CH(CH3)CH2, named Isomer A, or R is -
CH2CH(CH3)-, named Isomer B, i.e. having respectively the following
structures:
Isomer A Isomer B
[0074] It has been observed that the Isomer A (compound (II) with R= -
CH(CH3)CH2-) has RRT=1 ,18 according to the analytical method
described in example 13 while the Isomer B (compound (II) with R= -
CH2CH(CH3)-) has RRT=1 ,16 according to the same analytical method.
[0075] The compound of formula (II) wherein R is -C C CH )-, i.e. the isomer
B, is preferred since this is the main isomer compound prepared by
transesterification reaction of the compound of formula (III) with 1,2-
propylen glycol.
[0076] The compound of formula (II) prepared from the compound of formula (III)
by transesterification with ethylenglycol, has instead the only one following
structure:
[0077] According to a preferred embodiment of the process of the present
invention, the R substituent of the compound of formula (II) is chosen from
the group comprising -CH2CH2- , -CH2CH(CH3)-, -CH(CH3)CH2- , -(CH2)3-
and -CH2CH2OCH2CH2- .
[0078] According to a another preferred embodiment of the of the process of the
present invention, the R substituent of the compound of formula (II) is
chosen from the group comprising -CH2CH2- , -(CH2 )3- and -
CH2CH2OCH2CH2- .
[0079] Object of the present invention is thus also the compound of formula (II):
(II)
[0080] wherein R is chosen from the group comprising a linear or branched C2-C6
alkyl, -(R O)nR1- wherein R is a linear or branched C2-C4 alkyl an n is an
integer from 1 to 6, and the group -CH -CH(OH)-CH - .
[0081] According to a preferred embodiment of the compound of formula (II) of
the present invention, R is chosen from the group comprising -CH2CH2- , -
CH2CH(CH3)-, CH(CH3)CH2- , -(CH2)3- and -CH2CH2OCH2CH2- .
[0082] According to a another preferred embodiment of the compound of formula
(II) of the present invention, R is chosen from the group comprising -
CH2CH2- , -(CH2)3- and -CH2CH2OCH2CH2- .
[0083] The compound of formula (II) of the present invention can be thus used for
the preparation of Apixaban of formula (I) and solvates or hydrates thereof.
According to an embodiment of the present invention, the process for the
preparation of Apixaban of formula (I) and solvates or hydrates thereof:
(I)
comprises the following steps:
a) transesterification reaction of the compound of formula (III):
(III)
wherein R2 is a linear or branched C1-C6 alkyl,
to provide the compound of formula (II):
(II)
wherein R is chosen from the group comprising a linear or branched C2-C6
alkyl, -CH2-CH(OH)-CH 2- and -(R O)nR1- wherein R is a linear or
branched C2-C4 alkyl and n is an integer from 1 to 6,
b) isolating the compound of formula (II),
c) amidation reaction of the compound of formula (II):
(II)
to provide Apixaban of formula (I).
[0085] According to this above embodiment of the process of the present
invention, the compound of formula (II) is a useful intermediate for the
preparation of Apixaban which allows to insert a further step in the known
synthesis of Apixaban by direct amidation reaction of Apixaban esters.
[0086] Indeed, starting from Apixaban esters of formula (III) and preparing and
isolating of the compound of formula (II), allows to increase the purity the
said intermediate of formula (II), thus increasing the purity of the final
Apixaban.
[0087] In other words, performing the process of the invention according said
preferred embodiment, wherein the intermediate of formula (II) is isolated,
by comparison with the known process of direct conversion of Apixaban
esters of formula (III) to Apixaban, said process allows the improve the
purity of the final product Apixaban.
[0088] The isolation of the compound of formula (II) in the step (b) can be carried
out by the known techniques of organic synthesis, including precipitation
and filtration or centrifugation, or, alternatively, phases separation.
[0089] At the end of the step (b) the compound of formula (II) can be optionally
dried, in oven or within the filter drier.
[0090] Moreover, isolating the compound of formula (II) can be useful to satisfy
the requirements of Regulatory authorities, which require the process
being composed by at least three synthetic steps.
[0091] According to the just mentioned preferred embodiment of the process of
the present invention, it is a preferred process that wherein R2 in the
compound of formula (III) is ethyl.
[0092] According to the just mentioned preferred embodiment of the process of
the present invention, it is a preferred process that wherein R in the
compound of formula (II) is chosen from the group comprising -CH2CH2-, -
CH2CH(CH3)-, CH(CH3)CH2- , -(CH2)3- and -CH2CH2OCH2CH2-.
[0093] According to the just mentioned preferred embodiment of the process of
the present invention, it is another preferred process that wherein R in the
compound of formula (II) is chosen from the group comprising -CH2CH2-, -
(CH2 )3- and -CH2CH2OCH2CH2-.
[0094] According to the just mentioned preferred embodiment of the process of
the present invention, it is a preferred process wherein R2 in the
compound of formula (III) is ethyl and wherein R in the compound of
formula (II) is chosen from the group comprising -CH2CH2-, -C C CH )-,
CH(CH3)CH2- , -(CH2 )3- and -CH2CH2OCH2CH2-.
[0095] According to the just mentioned preferred embodiment of the process of
the present invention, it is another preferred process wherein R2 in the
compound of formula (III) is ethyl and wherein R in the compound of
formula (II) is chosen from the group comprising -CH2CH2-, -(CH2)3- and -
CH2CH2OCH2CH2-.
[0096] The conditions to performs the steps (a) and (c) of the preferred
embodiment of the invention just mentioned are the same of that already
described above for the process of the invention, including the preferred
embodiments thereof.
[0097] Just like any compound obtained by means of chemical synthesis,
Apixaban, and solvates or hydrates thereof, may contain small amounts of
foreign compounds referred to as impurities. These impurities may be the
raw materials, synthetic intermediaries, reaction by-products, degradation
products etc.
[0098] The impurities of Apixaban just like those of any other pharmaceutical
active ingredient or relative drug, referred to as "pharmaceutical
impurities", may affect both the efficiency and the safety of a drug which,
in extreme cases, could even be harmful for the patient. The purity of an
active ingredient like the Apixaban produced through a production process
based on subsequent chemical reactions represents a critical factor as
regards commercialization. The US Food and Drug Administration (FDA)
and the European Medicinal Agency (EMA) as well as the relative
pharmacopoea require that the impurities be maintained below given limit
values.
[0099] The product of a chemical reaction is rarely a single compound having
purity sufficient to meet the regulatory standards. By-products due to
secondary reactions of the reagents used in the reaction can also be
present in the isolated product. In some steps of the production process of
an active ingredient, such as Apixaban, the purity is analysed, generally by
means of high performance liquid chromatography (HPLC), gas
chromatography (GC) or thin layer chromatography (TLC), for defining if it
is suitable for the subsequent treatment and lastly for use in the
pharmaceutical product.
[00100] Generally, the impurities are identified spectroscopically, thus a
chromatographic peak position, such as that of a chromatogram or a spot
on a TLC panel, is associated thereto.
[00101] Once a peak position has been associated to a particular impurity, the
impurity can be identified in a sample for the relative position thereof in the
chromatogram, where the position in the chromatogram is measured in
minutes between the injection of the sample in a column and elution of the
impurity through the detector. The position in the chromatogram is known
as the retention time and the ratio between the retention times is known as
the relative retention time.
[00102] A man skilled in pharmaceutical art knows that a relatively pure compound
may be used as a reference standard. A reference standard is similar to a
reference marker, except for the fact that the latter can be used not only
for detecting the impurities, but also for quantifying the amount of
impurities present in the sample of active ingredient.
[00103] As known to those skilled in the art, the management of process impurities
is considerably improved by understanding the chemical structures
thereof, the synthetic process and identifying the parameters that affect
the amount of impurities in the final product for example by means of DOE.
The impurities of Apixaban, including the intermediaries not entirely
reacted, the impurities of the raw materials, the reaction by-products, the
degradation products, as well as other products, may affect the quality and
efficiency of the pharmaceutical form containing Apixaban. Thus, there
arises the need for a method for defining the level of impurities in samples
of Apixaban and methods for removing the impurities or limiting the
content thereof or preventing the formation thereof.
[00104] As one other aspect of the present invention, during the development of
the process for the preparation of Apixaban, it has been found that the
compound of formula (II) tends to remain into the product Apixaban, in
other words, the compound of formula (II) is both a starting material or
intermediate for the synthesis of Apixaban according to the method of the
present invention and is also an impurity of Apixaban.
[00105] In order to reduce the amount of an impurity in an active ingredient it is
necessary to detect the presence thereof using appropriate analytical
methods, it is convenient to identify it, quantify it and only afterwards one
can provide a method of synthesis capable of preventing the formation
and/or provide for the removal thereof. However, this essentially requires
providing the reference standard or reference marker of this impurity. For
such purpose the compound of formula (II) can be conveniently prepared
by means of the method described above.
[00106] The compound of formula (II) of the present invention can be thus used as
reference marker or reference standard for the identification and/or
quantification of said compound of formula (II) in Apixaban and solvates or
hydrates thereof.
[00107] The compound of formula (II) can be indeed used according to the
following analytical methods the identification and/or quantification of said
compound of formula (II) in Apixaban and solvates or hydrates thereof.
[00108] A method for detecting or identifying the compound of formula (II) in
Apixaban or a solvate or hydrate thereof comprises:
a) adding a known amount of compound of formula (II) to the Apixaban
sample or a solvate or hydrate thereof,
b) carrying out HPLC analysis of the Apixaban sample or a solvate or
hydrate thereof of step a),
c) detecting the HPLC peak of the compound of formula (I);
or,
a1) analysing the compound of formula (II) by means of HPLC,
b1) analysing the Apixaban sample or a solvate or hydrate thereof by
means of HPLC,
c1) detecting the HPLC peak of the compound of formula (II) by comparing
the retention times or relative retention times.
[00109] Substantially using the method above, it allowed identifying the peak in the
chromatogram of Apixaban sample or a solvate or hydrate thereof
regarding the impurity compound of formula (II). The analysis may be of
the HPLC type.
[001 10] Besides the identification of the impurity peak in Apixaban or a solvate or
hydrate, a method for the quantification of the compound of formula (II) in
Apixaban or a solvate or hydrate thereof comprises:
i) measuring the peak area corresponding to the compound of formula (II)
in a Apixaban sample or a solvate or hydrate thereof having an unknown
amount of compound of formula (II) by means of HPLC;
ii) measuring the peak area corresponding to a reference standard
containing a known amount of compound of formula (II) by means of
HPLC;
iii) defining the amount of compound of formula (II) in Apixaban or a
solvate or hydrate thereof comparing the area measured in step a) with
that measured in step ii).
[001 11] It is thus clear that the compound of formula (II) may be used as a
reference marker or reference standard respectively for the identification
and/or the quantification of the same in Apixaban or a solvate or hydrate
thereof.
[001 12] In particular, it has been observed that the impurity of Apixaban produced
according to the process of the present invention and having RRT=1 .16
with the analytical method reported in example 13 is the Isomer B of the
following structures, while the impurity having RRT=1 .18 has the is the
Isomer A of the following structures:
Isomer A Isomer B
[001 13] RRT=1 .18 RRT=1 .16
[001 14] As an aspect related to the solid form of Apixaban produced by the
process of the present invention, a study directed to the preparation of
Apixaban solid form N-1 was carried out.
[001 15] Repeating the experiment of example 6 of WO2007/0001385 but without
adding the seed of form N-1 , it has been obtained, prepared and
characterized Apixaban 1,2-Proprylen glycol hemisolvate of formula (V):
( V )
[001 16] Apixaban 1,2-propylene glycol solvate having stoichiometry (2:1) of
formula (V) is a white solid.
[001 17] After that, a study directed to find a method to obtain N-1 form from
Apixaban 1,2-proprylen glycol hemisolvate of formula (V) was carried out.
[001 18] A screening using Apixaban 1,2-propylene glycol hemisolvate
(abbreviated APX PG) as starting material was performed using form N-1
as a seed in all the cases. The experiments were performed with ICH
guideline class 3 solvents (except MeOH, ICH guideline class 2 with a
residual solvent permitted of 3000 ppm). The low solubility of APX PG with
common industrial solvents limited the use of the crystallization.
[001 19] The successful experiments that provided Apixaban form N-1 from
APX PG are collected in Table 1.
[00120] Table 1: Methods of preparation of Apixaban form N-1 from APX-PG.
[00121] Slurrying seems to be the best procedure to prepare form N-1 because a
high amount of solvent was required in crystallizations (36-60 volumes).
[00122] Due to their lower boiling points and the good industry acceptance, EtOH
and IPA were selected as solvents to perform a scale-up of the slurrying
experiments to 1 g. The transformation was monitored by XRPD:
[00123] - In EtOH the transformation was finished after 1 hour at room
temperature.
[00124] - In IPA the transformation is much slower: after 5 h at 50 °C, the
conversion was not complete, but finished after one night.
[00125] Propylene glycol was not detected in Apixaban form N-1 by H-NMR.
Unfortunately, residual solvent was detected by H-NMR in both
experiments (approx. 0.8 wt% of EtOH and 0.7 wt% of IPA). The NMR
analysis indicated also 0.7 wt% of residual solvent when EtOAc was used.
[00126] H-NMR analyses of form N-1 obtained in EtOH by crystallization and
slurrying indicated that the amount of residual solvent is lower in the case
of the crystallization (0,4 wt% instead of 0.8 wt%). The method of
preparation seems to have some effect on the final amount of residual
solvent (perhaps due to the different particle sizes or kind of aggregates).
[00127] Typically, the Apixaban form N-1 prepared according to the process of the
invention has a water content comprised between 0.05% and 0.1 %.
[001 28] EXPERIMENTAL SECTION
[00129] The starting material Apixaban ethyl ester can be prepared according to
Example 5 of WO2007/001385.
[00130] Example 1: Preparation of Apixaban 1,2-propylen glycol hemisolvate of
formula (V) from Apixaban ethyl ester.
Apixaban ethyl ester ( V )
[00131] In an autoclave inerted by nitrogen Apixaban ethyl ester (15g, 1.0eq) and
propylene glycol ( 1,2-propan diol, 135 ml_) were charged and the vessel
was pressurized with ammonia at p=4bar and T=80/85°C for 6h. The
mixture was then transferred in a round bottom flask, cooled to 45/50°C
and diluted with water (85 ml_). After stirring at T=45/50°C for additional
2h, the suspension was cooled to 20/25°C for 10h and filtered. The wet
cake was washed with water (2x30ml_). The solid was dried under vacuum
at T=75°C for 8h affording Apixaban 1,2-propylenglycol hemisolvate of
formula (V) (13.3g, 0.86eq). m.p. 195°C. H-NMR (400MHz, CDCIs, ppm),
d : 7.49 (d, J=9Hz, 2H), 7.35 (d, J=8Hz, 2H), 7.28 (d, J=8Hz, 2H), 6.95 (d,
J=9Hz, 2H), 6.91 (s, 1H), 5.91 (s, 1H), 4.13 (t, J=8Hz, 2H), 3.84 (bs, 3H +
0.5H CH propylene glycol), 3.62 (bm, 2H + 0.5H OH propylen glycol), 3.39
(bm, J=8Hz, 2H + 0.5H OH propylen glycol), 2,57 (bs, 2H + 1H CH2
propylen glycol), 1.95 (bs, 4H), 1.14 (d, J=6.4Hz, 1.5H CH3 propylen
glycol). 13C NMR and DEPT 135 NMR (100 MHz, CDC , ppm), d : 170.3
(C), 163.8 (C), 159.9 (C), 157.4 (C), 141 .4 (C), 140.7 (C), 140.0 (C), 133.4
(C), 132.5 (C), 126.8 (CH), 126.2 (CH), 126.5 (CH), 125.9 (CH), 113.8
(CH), 68.3 (CH), 68.1 (CH2) , 55.6 (CH3) , 5 1.6 (CH2) , 5 1.2 (CH2) , 32.8
(CH2) , 23.5 (CH2) , 2 1.4 (CH2) , 2 1.3 (CH2) , 18.8 (CH3) . ESI-MS m/z=460
([M+H] +) . IR (ATR, cm-1) : 3447, 3145, 2940, 2860, 1687, 1631 , 1543,
1512, 1465, 1441 , 1401 , 1380, 1350, 1326, 1297, 1243, 1170, 1144,
1111, 1027, 1016, 982, 945, 831 , 812, 761 , 705. X-RPD (2Q°): 6.6°, 7.6°,
8.1 ° , 9.9°, 11.7°, 12.7°, 13.7°, 14.5°, 15.1 ° , 15.6°, 16.3°, 16.9°, 17.2°,
17.9°, 18.2°, 19.5°, 20.0°, 20.5°, 20.8°, 2 1.4°, 22.8°, 23.8°, 24.8°, 25.5°,
29.0°, 3 1.2°, 33.0°.
[00132] The reworking of example 6 of WO2007/001385, carried out many times
but without adding the seeds of Apixaban form N-1 , always provided
Apixaban 1,2-propylen glycol hemisolvate of formula (V).
[00133] This is in agreement with the teachings of the patent publication
WO20 13/1 19328 wherein Apixaban form I, i.e. Apixaban 1,2-propylen
glycol hemisolvate was obtained without the seeding with form N-1 .
[00134] Example 2: Preparation of Apixaban form N-1 from Apixaban 1,2-propylen
glycol hemisolvate - without seeding of form N-1 .
[00135] In a round bottonn flask were charged Apixaban 1,2-propylene glycol
hemisolvate (10g, 1.0eq) and ethanol (400 ml_) and the mixture was
heated to reflux for 4 hours. The suspension was slowly cooled to 20/25°C
and stirred at this temperature for 8h, then filtered washing with ethanol (2
x 20 ml_). The wet solid was dried under vacuum at 75°C for 8h affording
8.1g of Apixaban N-1 form (0.87eq). mp 237°C. H-NMR (400MHz,
DMSO- , ppm), d : 7.74 (s, 1H), 7.53 (d, J=12Hz, 2H), 7.47 (s, 1H), 7.37
(d, J=8Hz, 2H), 7.30 (d, J=12Hz, 2H), 7.02 (d, J=8Hz, 2H), 4.07 (t, J=8Hz,
2H), 3.82 (s, 3H), 3.61 (t, J=4Hz, 2H), 3.23 (t, J=8Hz, 2H), 2.41 (t, J=4Hz,
2H), 1.87 (m, 4H). 3C-NMR and DEPT 135 NMR (100 MHz, DMSOppm),
d : 169.3 (C), 163.7 (C), 159.6 (C), 157.1 (C), 142.0 (C), 141 .9 (C),
140.3 (C), 133.5 (C), 133.1 (C), 127.3 (C), 126.8 (CH), 126.5 (CH), 125.7
(CH), 113.9 (CH), 56.0 (CH3), 5 1.3 (CH2) , 33.1 (CH2) , 23.5 (CH2) , 2 1.5
(CH2) , 2 1.4 (CH2) . ESI-MS m/z=460 ([M+H] +) .
[00136] Example 3: Characterization of Apixaban form N-1 .
Apixaban form N-1 obtained by crystallization in EtOH was characterized
by several techniques.
[00137] FT-IR
FTIR spectrum was recorded using a Thermo Nicolet Nexus 870 FT-IR,
equipped with a beamsplitter KBr system, a 35 mW He-Ne laser as the
excitation source and a DTGS KBr detector. The spectrum was acquired in
32 scans at a resolution of 4 cm-1.
IR (KBr): v = 3483 (m), 331 1 (m), 2909 (m), 2866 (W), 1683 (s), 1630 (s),
1595 (s), 1519 (m), 1295 (m), 1256 (m), 975 (m), 848 (s), 813 (m), 668
(m), 467 (m) cm-1 (see Figure 2).
[00138] DSC
DSC analysis was recorded with a Mettler DSC822 . A sample of 1.6770
mg was weighed into a 40 m I_ aluminium crucible with a pinhole lid and
was heated, under nitrogen (50 mL/min), at 10 °C/min from 30 to 300 °C.
[00139] Form N-1 is characterized by an endothermic sharp peak corresponding to
the melting point with an onset at 235.68 °C (fusion enthalpy -106.66 J/g),
measured by DSC analysis (10 °C/min).
[00140] TGA
Thermogravimetric analysis was recorded in a thermogravimetric analyzer
Mettler TGA/SDTA851 e. A sample of 4.2206 mg was weighed into a 70 m I_
alumina crucible with a pinhole lid and was heated at 10 °C/min from 30 to
400 °C, under nitrogen (50 mL/min).
The TG analysis of Form N 1 shows a 0.23% weight loss before the
melting point (between 130°C and 230°C). This loss of weight could come
from the elimination of EtOH traces.
X-RPD
XRPD analysis was performed using a PANalytical X'Pert diffractonneter
with Cu K radiation in Bragg-Brentano geometry. The system is equipped
with a monodimensional, real time multiple strip detector. The
diffractogrann was recorded from 3° to 40° (2Q) at a scan rate of 17.6° per
minute (see Figure 5). List of selected peaks (only peaks with relative
intensity greater than or equal to 1% are indicated):
Pos. Rel. Int.
[°2Th.] [%]
8,4 9
10.0 4
10,5 5
11. 1 5
12,3 8
12.8 4 1
13.9 58
15.1 2
16.2 14
16,9 100
18,4 30
18.8 14
19,6 8
2 1. 1 11
2 1.5 12
22,0 16
22.2 29
23.6 2
24,0 4
24.7 8
25.3 4
26,2 1
26.9 8
27,7 5
28.0 3
28,6 4
29,2 6
29,9 5
30,6 3
3 1,9 1
32,6 5
35.1 3
In the patent US7396932B2, form N-1 was described by SCXR and 3C
SSNMR. Using the data of SCXR (unit cell, symmetry and atom positions),
XRPD was simulated using the Mercury program. Comparison of this
simulated XRPD with the experimental XRPD obtained in example 2
confirmed the formation Apixaban N-1 form.
[00143] Karl Fischer
Karl Fischer analyses were recorded with a Metrohm 787 KF Trinito. The
product was dissolved in MeOH. Two samples were analyzed using the
following reactants: Hydranal-Composite 5 (Riedel de Haen Ref. 34805),
Hydranal Methanol Rapid (Riedel de Haen Ref. 37817) and Hydranal
Water Standard 1.0 (Riedel de Haen Ref. 34828 used to calculate the
factor).
[00144] The water content of form N-1 prepared in example 2 is 0.9%.
[00145] Example 4 : Preparation of Apixaban form N-1 from Apixaban 1,2-
Proprylen glycol hemisolvate on larger scale - with seeding of form N-1 .
[00146] To a three-necked round-bottomed flask equipped with a thermometer and
mechanical stirrer was added Apixaban 1,2-Propylene glycol hemisolvate
of formula (V) (85.1 g; 171 mmol), as prepared in Example 1, and a
mixture of EtOH/water (2:1) (850 ml_, 10 vol.). the resulting suspension
was seeded with form N-1 (as prepared in example 2) and it was heated at
50°C. The mixture was maintained at 50°c for 2.5 hours and then it was
cooled down to room temperature. The slurry was stirred at room
temperature for 2-3 hours. The solid was filtered with a sintered funnel
(porosity 2 - very good filtration), washed with EtOH: water (2:1) (170 ml_,
2 vol.), with water (170 ml_, 2 vol.) and dried under vacuum at 50°C
overnight. Apixaban form N-1 was obtained as off-white powder (65.4 g,
83% yield). H-NMR analysis shows that the product contains 0.13% of
residual Ethanol. K.F. 0.1 %. The chemical purity was determinate by
HPLC: 99.4%. The starting Apixaban 1,2-Propylene glycol hemisolvate
had a purity of 98.3%.
[00147] Repeating the above procedure starting from Apixaban 1,2-Propylene
glycol hemisolvate with a purity of 95.2%, Apixaban form N-1 was
prepared having a purity of 99.5%.
[00148] Example 5: Synthesis of the compound of formula (II) in which R is -
CH2CH2-.
[00149] A mixture of Apixaban ethyl ester (10.0 g, 1.0 eq), bibasic potassium
phosphate (K2HPO , 17.8 g, 5.0eq) and ethylenglycol ( 1,2-ethan diol, 70
ml_) was heated to T=75°C for 10h and then cooled to room temperature.
Water (70 ml_) and dichloromethane (70 ml_) were charged and the
resulting biphasic solution was stirred at room temperature for 10min.
Once cut the phases, the organic layer was treated with molecular sieves
to remove residual water and then concentrated to residue at reduced
pressure. The resulting solid was employed without further purification
(9.0g, 0.87eq). H-NMR (400MHz, DMSO- ppm), d: 7.52 (d, J=12 Hz,
2H), 7.37 (d, J=8 Hz, 2H), 7.30 (d, J=8 Hz, 2H), 7.03 (d, J=12 Hz, 2H),
4.98 (t, J=4 Hz, 1H), 4.35 (t, J=4 Hz, 2H), 4.09 (t, J=4 Hz, 2H), 3.82 (s,
3H), 3.74 (dd, J i =4 Hz, J 2=4 Hz, 2H), 3.60 (t, J =4 Hz, 2H), 3.24 (t, J =4 Hz,
2H), 2.40 (t, J=4 Hz, 2H), 1.85 (m, 4H). ^C-NMR and DEPT 135 NMR
(100 MHz, DMSO- ppm), d : 169.4(C), 162.0(C), 159.8(C), 156.9(C),
141 .9(C), 140.2(C), 139.0(C), 133.5(C), 132.9(C), 127.4 (CH), 127.2(CH),
126.8(CH), 126.5(CH), 114.0(CH), 66.8(CH 2) , 59.5(CH 2) , 56.0(CH 3) ,
5 1.3(CH2) , 5 1.2(CH2) , 33.1 (CH2) , 23.5(CH 2) , 2 1.6(CH2) , 2 1.4(CH2) . ESIMS
m/z=505 ([M+H] +) .
Example 6: Synthesis of the compound of formula (II) in which R is -
CH(CH 3)2CH2- and -CH2CH(CH 3)- .
Isomer A Isomer B
[00150] A mixture of Apixaban ethyl ester (30.0 g, 1.0 eq.), bibasic potassium
phosphate (K2HPO4, 53.4 g, 5.0 eq.) and propylenglycol ( 1,2-propan diol,
210 ml_) was heated to T=75°C for 10h and then cooled to room
temperature. Water (210 ml_) and dichloromethane (21 0 ml_) were
charged and the resulting biphasic solution was stirred at room
temperature for 10min. Once cut the phases, the organic layer was treated
with molecular sieves to remove residual water and then concentrated to
residue at reduced pressure. The resulting solid is a 1:2 mixture of the two
isomers (called isomer A and isomer B respectively in the H-NMR
characterization) was employed without further purification (25. 2g,
0.79eq). H-NMR (400 MHz, DMSO- ppm), d: 7.52 (m, 3H (2H isomer A
and 2H isomer B)), 7.37 (m, 3H (2H isomer A and 2H isomer B)), 7.30 (m,
3H (2H isomer A and 2H isomer B)), 7.03 (m, 3H (2H isomer A and 2H
isomer B)), 5.12 (m, 0.5H ( 1H isomer A), 4.96 (m, 1.5H ( 1H isomer A and
1H isomer B)), 4.20 (m, 2H (2H isomer B)), 4.1 1 (m, 3H (2H isomer A and
2H isomer B)), 3.98 (m, 1H ( 1H isomer B)), 3.83 (m, 4.5H (3H isomer A
and 3H isomer B)), 3.61 (m, 3H (2H isomer A and 2H isomer B)), 3.26 (m,
3H (2H isomer A and 2H isomer B)), 1.88 (m, 6H (4H isomer A and 4H
isomer B)), 1.29 (d, J=4 Hz, 1.5H (3H isomer A)), 1.17 (d, J=4 Hz, 3H (3H
isomer B)). ^C-NMR (100 MHz, DMSO- ppm), d: 169.4, 161 .8, 161 .6,
159.8, 156.9, 141 .9, 140.2, 139.3, 139.0, 133.5, 133.0, 127.3, 127.2,
126.8, 126.5, 114.0, 72.7, 69.9, 64.5, 64.1 , 56.0, 5 1.3, 5 1.2, 33.1 , 23.5,
2 1.7, 2 1.4, 20.5, 16.9 (overlap of some of the two isomers signals was
observed). ESI-MS m/z=519 ([M+H] +) .
[00151] IR (ATR, cm-1) : 3329, 2934, 2839, 1708, 1673, 1627, 1592, 151 1, 1438,
1403, 1372, 1325, 1301 , 1252, 1172, 1144, 1054, 1021 , 988, 949, 832,
788, 699. X-RPD (2Q°): 6.7°, 8.2°, 8.5°, 8.9°, 10.5°, 11. 1 ° , 11.6°, 12.1°,
13.0°, 15.3°, 15.9°, 16.8°, 17.2°, 17.9°, 19.3°, 20.1°, 20.4°, 2 1.3°, 22.8°,
23.2°, 23.8°, 24.5°, 25.4°, 27.9°, 30.3°.
[00152] Example 7: Synthesis of the compound of formula (II) in which R is -
CH2CH2OCH2CH2-.
[00153] A mixture of Apixaban ethyl ester (10.0 g, 1.0eq), bibasic potassium
phosphate (K2HPO 4, 17.8 g, 5.0eq) and diethylenglycol (70 ml_) was
heated to T=75°C for 10 h and then cooled to room temperature. Water
(70 ml_) and dichloromethane (70 ml_) were charged and the resulting
biphasic solution was stirred at room temperature for 10min. Once cut the
phases, the organic layer was treated with molecular sieves to remove
residual water and then concentrated to residue at reduced pressure. The
resulting solid was employed without further purification (10.5 g, 0.93 eq).
1H-NMR (400MHz, DMSO- , ppm), d : 7.53 (d, J=8Hz, 2H), 7.37 (d,
J=8Hz, 2H), 7.30 (d, J=8Hz, 2H), 7.03 (d, J=8Hz, 2H), 4.65 (t, J=4Hz, 1H),
4.45 (m, 2H), 4.10 (d, J=4Hz, 2H), 3.83 (s, 3H), 3.77 (m, 2H), 3.53 (m,
4H), 7.53 (d, J=8Hz, 2H), 3.24 (d, J=8Hz, 2H), 2.41 (m, 2H), 1.86 (m, 4H).
3C-NMR and DEPT 135 NMR (100 MHz, DMSO- , ppm), d : 169.4 (C),
161 .8 (C), 159.8 (C), 156.9 (C), 141 .9 (C), 140.2 (C), 138.8 (C), 133.5 (C),
132.9 (C), 127.5 (C), 127.2 (CH), 126.8 (CH), 126.5 (CH), 114.0 (CH),
72.8 (CH2) , 68.7 (CH2) , 64.3 (CH2) , 60.7 (CH2) , 56.0 (CH3) , 5 1.3 (CH2) ,
5 1.2 (CH2) , 33.1 (CH2) , 23.5 (CH2) , 2 1.6 (CH2) , 2 1.4 (CH2) . ESI-MS
m/z=549 ([M+H] +) . KF=0.06%.
[00154] Example 8: Synthesis of Apixaban form N-1 from the compound of formula
(II) in which R is -CH2CH2-.
[00155] In an autoclave inerted by nitrogen compound of Example 5 (compound of
formula (II) in which R is -CH2CH2-, 8.0g, 1.0eq) and propylene glycol
( 1 ,2-propandiol, 80 ml_) were charged and the vessel was pressurized with
ammonia at p=4bar and T=80/85°C for 6h. The mixture was then
transferred in a round bottom flask, heated to dissolution and diluted with
water (16 ml_). After stirring at T = 95/1 00°C for additional 2h, more water
was added (48ml_) and the solution was seeded with Apixaban N-1 form
(as prepared in Example 2 or 4). The sus-pension was stirred for 2h at
T=95/100°C, cooled to room temperature and diluted with ethanol (16 ml_).
After 3h stirring at T=20/25°C the slurry was filtered and the wet cake was
washed with water (2x8ml_). The solid was dried under vacuum at T=65°C
for 8h affording Apixaban N-1 form (6.7g, 0.92 eq). m.p. 237°C. H-NMR
(400MHz, DMSO- , ppm), d : 7.74 (s, 1H), 7.53 (d, J=12Hz, 2H), 7.47 (s,
1H), 7.37 (d, J=8Hz, 2H), 7.30 (d, J=12Hz, 2H), 7.02 (d, J=8Hz, 2H), 4.07
(t, J=8Hz, 2H), 3.82 (s, 3H), 3.61 (t, J=4Hz, 2H), 3.23 (t, J=8Hz, 2H), 2.41
(t, J=4Hz, 2H), 1.87 (m, 4H). 3C-NMR and DEPT 135 NMR (100 MHz,
DMSO- , ppm), d: 169.3 (C), 163.7 (C), 159.6 (C), 157.1 (C), 142.0 (C),
141 .9 (C), 140.3 (C), 133.5 (C), 133.1 (C), 127.3 (C), 126.8 (CH), 126.5
(CH), 125.7 (CH), 113.9 (CH), 56.0 (CH3) , 5 1.3 (CH2) , 33.1 (CH2) , 23.5
(CH2) , 2 1.5 (CH2) , 2 1.4 (CH2) . ESI-MS m/z=460 ([M+H] +) . KF=0.08%.
[00156] Example 9: Synthesis of Apixaban form N-1 from the compound of formula
(II) in which R is -CH(CH 3)2CH2- and -CH2CH(CH 3)-.
In an autoclave inerted by nitrogen compound of Example 6 (compound of
formula (II) in which R is -CH(CH 3)2CH2- and -CH 2CH(CH 3)-, 11g, 1.0eq)
and propylene glycol ( 1 ,2-propandiol, 100 ml_) were charged and the
vessel was pressurized with ammonia at p=4bar and T=80/85°C for 6h.
The mixture was then transferred in a round bottom flask, heated to
dissolution and diluted with water (20 ml_). After stirring at T=95/100°C for
additional 2h, more water was added (60 ml_) and the solution was seeded
with Apixaban N-1 form. The suspension was stirred for 2h at T=95/100°C,
cooled to room temperature and diluted with ethanol (20 ml_). After 3h
stirring at T=20/25°C the slurry was filtered and the wet cake was washed
with water (2x1 OmL). The solid was dried under vacuum at T=65°C for 8h
affording Apixaban N-1 form (8.6 g, 0.88 eq). m.p. 237°C. H-NMR
(400MHz, DMSO- , ppm), d: 7.74 (s, 1H), 7.53 (d, J=12Hz, 2H), 7.47 (s,
1H), 7.37 (d, J=8Hz, 2H), 7.30 (d, J=12Hz, 2H), 7.02 (d, J=8Hz, 2H), 4.07
(t, J=8Hz, 2H), 3.82 (s, 3H), 3.61 (t, J=4Hz, 2H), 3.23 (t, J=8Hz, 2H), 2.41
(t, J=4Hz, 2H), 1.87 (m, 4H). ^C-NMR and DEPT 135 NMR (100 MHz,
DMSO- ppm), d : 169.3 (C), 163.7 (C), 159.6 (C), 157.1 (C), 142.0 (C),
141 .9 (C), 140.3 (C), 133.5 (C), 133.1 (C), 127.3 (C), 126.8 (CH), 126.5
(CH), 125.7 (CH), 113.9 (CH), 56.0 (CH3) , 5 1.3 (CH2) , 33.1 (CH2) , 23.5
(CH2) , 2 1.5 (CH2) , 2 1.4 (CH2) . ESI-MS m/z=460 ([M+H] +) . KF=0.05%.
[00158] Example 10: Synthesis of Apixaban form N-1 from the compound of
formula (II) in which R is -CH2CH2OCH2CH2-.
[00159] In an autoclave inerted by nitrogen compound of Example 7 (compound of
formula (II) in which R is -CH2CH2OCH2CH2-, 9.0g, 1.0 eq) and propylene
glycol ( 1 ,2-propandiol, 105 ml_) were charged and the vessel was
pressurized with ammonia at p=4bar and T=80/85°C for 6h. The mixture
was then transferred in a round bottom flask, heated to dissolution and
diluted with water (20 ml_). After stirring at T=95/100°C for additional 2h,
more water was added (60ml_) and the solution was seeded with Apixa
ban N-1 form. The suspension was stirred for 2h at T=95/100°C, cooled to
room temperature and diluted with ethanol (20 ml_). After 3h stirring at
T=20/25°C the slurry was filtered and the wet cake was washed with water
(2x1 OmL). The solid was dried under vacuum at T=65°C for 8h affording
Apixaban N-1 form (6.5g, 0.86 eq). mp 237°C. H-NMR (400MHz, DMSOd6,
ppm), d : 7.74 (s, 1H), 7.53 (d, J=12Hz, 2H), 7.47 (s, 1H), 7.37 (d,
J=8Hz, 2H), 7.30 (d, J=12Hz, 2H), 7.02 (d, J=8Hz, 2H), 4.07 (t, J=8Hz,
2H), 3.82 (s, 3H), 3.61 (t, J=4Hz, 2H), 3.23 (t, J=8Hz, 2H), 2.41 (t, J=4Hz,
2H), 1.87 (m, 4H). NMR and DEPT 135 NMR (100 MHz, DMSOppm),
d : 169.3 (C), 163.7 (C), 159.6 (C), 157.1 (C), 142.0 (C), 141 .9 (C),
140.3 (C), 133.5 (C), 133.1 (C), 127.3 (C), 126.8 (CH), 126.5 (CH), 125.7
(CH), 113.9 (CH), 56.0 (CH3) , 5 1.3 (CH2) , 33.1 (CH2) , 23.5 (CH2) , 2 1.5
(CH2) , 2 1.4 (CH2) . ESI-MS m/z=460 ([M+H] +) . KF=0.06%.
[00160] Example 11: Synthesis of Apixaban form N-1- from the compound of
formula (III) in which R is -CH2CH3 (Example 6 of WO2007/0001385) -
Comparative example
[00161] In an autoclave inerted by nitrogen Apixaban ethyl ester (65g, 1.0eq) and
propylene glycol ( 1,2-propan diol, 455 ml_) were charged and the vessel
was pressurized with ammonia at p=4bar and T=80/85°C for 6h. The
mixture was then transferred in a round bottom flask washing the
autoclave with propylene glycol (65 ml_), heated to dissolution and diluted
with water (130 ml_). After stirring at T=95/100°C for additional 2h, more
water was added (390 ml_) and the solution was seeded with Apixaban N-
1 form. The suspension was stirred for 2h at T=95/100°C, cooled to room
temperature and diluted with ethanol (130 ml_). After 3h stirring at
T=20/25°C the slurry was filtered and the wet cake was washed with water
(2x130ml_). The solid was dried under vacuum at T=65°C for 8h affording
Apixaban N-1 form (56.0g, 0.917eq). m.p. 237°C. H-NMR (400MHz,
DMSO- , ppm), d: 7.74 (s, 1H), 7.53 (d, J=12Hz, 2H), 7.47 (s, 1H), 7.37
(d, J=8Hz, 2H), 7.30 (d, J=12Hz, 2H), 7.02 (d, J=8Hz, 2H), 4.07 (t, J=8Hz,
2H), 3.82 (s, 3H), 3.61 (t, J=4Hz, 2H), 3.23 (t, J=8Hz, 2H), 2.41 (t, J=4Hz,
2H), 1.87 (m, 4H). 3C-NMR and DEPT 135 NMR (100 MHz, DMSO- ,
ppm), d : 169.3 (C), 163.7 (C), 159.6 (C), 157.1 (C), 142.0 (C), 141 .9 (C),
140.3 (C), 133.5 (C), 133.1 (C), 127.3 (C), 126.8 (CH), 126.5 (CH), 125.7
(CH), 113.9 (CH), 56.0 (CH3) , 5 1.3 (CH2) , 33.1 (CH2) , 23.5 (CH2) , 2 1.5
(CH2) , 2 1.4 (CH2) . ESI-MS m/z=460 ([M+H] +) . KF=0.08%.
[00162] Example 12: Reaction rate of the synthesis of Apixaban from the
compound of formula (II) compared with the synthesis of Apixaban from
Apixaban ethyl ester. An effect of the invention.
[00163] Apixaban can be obtained from compound of formula (II) (as described in
Example 9, Example 10 or Example 11) or, according to prior art method,
from Apixaban ethyl ester (as described in comparative Example 11), (see
also example 6 of WO2007/0001385).
However, the reaction rate is considerably faster when starting from a
compound of formula (II), for example, the compound (II) in which R is -
CH2CH(CH 3)- and CH(CH 3)2CH2- (called Propylen glycol ester in Figure 1
and Table 1).
[00164] As depicted in Figure 1, reaction completion (conversion >99%) is reached
within 3h employing the Propylen glycol ester as starting material
(triangles in Figure 1) while it takes at least 6h, under exactly the same
reaction conditions, from the Apixaban ethyl ester to reach the same
conversion value (circles in Figure 1).
[00165] Moreover, comparing the data collected after 6 hours, the amount of
Apixaban is higher when it is prepared from the Propylene glycol ester
(99.25% versus 98.95%).
[00166] The detailed data are collected in Table 1 for the kinetic study of the
conversion of Propylen glycol ester to Apixaban and for the conversion of
Apixaban ethyl ester to Apixaban.
Table 1: Comparative kinetic study
Data are expressed as HPLC conversions.
[00167] Example 13: HPLC method for the identification and quantification of the
compound of formula (II) in which R is -CH(CH3)2CH2- (Isomer A) and -
CH2CH(CH3)- (Isomer B) which are typical process intermediates and
impurities.
[00168] As mentioned in Example 10, compound of formula (II), in particular the
compound of formula (II) in which R is -CH 2CH(CH 3)- and -CH(CH 3)2CH2- ,
is also a typical impurity found in the isolated Apixaban product obtained
by the process of the invention described in the Examples above.
[00169] This species could be identified and monitored via the following HPLC
method:
[00170] Chromatographic conditions:
Column: XBridge C 18 150x4.6 mm 3.5 mhh
Temp. Column: 40°C
Mobile Phase A: H2O MilliQ/Methanol 90/10
Mobile Phase B: Acetonitrile/Methanol 90/10
Gradient: Time (min) %A %B
0 83.5 16.5
20 5.5 94.5
25 5.5 94.5
Post run: 7 min.
Flow: 1.0 mL/min
Detector UV a 252 nm
Injection Volume
Run Time: 25 min
Sample diluent: CH2CI2/EtOH/H 2O 1:5:4
Applying the conditions described above the expected retention times are
as indicated below:
[00171] The amount of the compound of formula (II) into Apixaban is determ
percent area.

Claims
1. Process for the preparation of Apixaban of formula (I) and solvates or hydrates
thereof:
(I)
amidation reaction of the compound of formula (II)
(II)
wherein R is chosen from the group comprising a linear or branched C2-C6
alkyl, -CH2-CH(OH)-CH 2- and -(R O)nR1- wherein R is a linear or branched
C2-C4 alkyl and n is an integer from 1 to 6.
2. Process according to the claim 1 wherein the amidation reaction is carried out
by means of anhydrous ammonia, aqueous ammonia or an ammonium salt.
3. Process according to anyone of the claims from 1 to 2 wherein the amidation
reaction is carried out in an glycol solvent.
4. Process according to anyone of the claims from 1 to 3 wherein the amidation
reaction is carried out at a temperature comprised between 80°C and 90°C in
about 3 hours.
5. Process according to anyone of the claims from 1 to 4, further comprising the
step of preparation of the compound of formula (II):
(II)
wherein R is chosen from the group comprising a linear or branched C2-C6
alkyl, -CH2-CH(OH)-CH 2- and -(R O)nR1- wherein R is a linear or branched
C2-C4 alkyl and n is an integer from 1 to 6,
by means of a transesterification reaction of the compound of formula (III):
(Ml)
wherein R2 is a linear or branched C1-C6 alkyl.
6. Process according to the claim 5 wherein the transesterification reaction is
carried out in presence of bibasic potassium phosphate or NaHCO3.
7. Process according to anyone of the claims from 1 to 6, comprises the following
steps:
a) transesterification reaction of the compound of formula (III):
(Ml)
wherein R2 is a linear or branched C1-C6 alkyl,
to provide the compound of formula (II):
OMe
(II)
wherein R is chosen from the group comprising a linear or branched C2-C6
alkyl, -CH2-CH(OH)-CH 2- and -(R O)nR1- wherein R is a linear or branched
C2-C4 alkyl and n is an integer from 1 to 6,
b) isolating the compound of formula (II),
c) amidation reaction of the compound of formula (II):
(II)
to provide Apixaban of formula (I).
8. Process according to anyone of the claims from 1 to 7 wherein the R
substituent of the compound of formula (II) is chosen from the group
comprising -CH2CH2- , -CH2CH(CH3)-, -CH(CH3)CH2- , -(CH2)3- and -
CH2CH2OCH2CH2- .
9. Compound of formula (II):
(II)
wherein R is chosen from the group comprising a linear or branched C2-C6
alkyl, -CH2-CH(OH)-CH 2- and -(R O)nR1- wherein R is a linear or branched
C2-C4 alkyl an n is an integer from 1 to 6.
10. Compound of formula (II) according to the claim 9 wherein R is chosen from
the group comprising -CH2CH2-, -CH2CH(CH3)-, -CH(CH3)CH2- , -(CH2)3- and -
CH2CH2OCH2CH2- .
11. Process for the preparation of the compound of formula (II) according to
anyone of the claims from 9 to 10, by means of a transesterification reaction of
the compound of formula (III):
(IN)
wherein R2 is a linear or branched C2-C6 alkyl.
12. Use of the compound of formula (II) according to anyone of the claims from 9
to 10 for the preparation of Apixaban of formula (I) and solvates or hydrates
thereof.
13. Use of the compound of formula (II) according to anyone of the claims from 9
to 10 as reference marker or reference standard for the identification and/or
quantification of said compound of formula (II) in Apixaban and solvates or
hydrates thereof.
14. A method for detecting the compound of formula (II) according to anyone of
the claims from 9 to 10 in Apixaban or a solvate or hydrate thereof comprising:
a) adding a known amount of compound of formula (II) to the Apixaban sample
or a solvate or hydrate thereof,
b) carrying out HPLC analysis of the Apixaban sample or a solvate or hydrate
thereof of step a),
c) detecting the HPLC peak of the compound of formula (I);
or,
a1) analysing the compound of formula (II) by means of HPLC,
b1) analysing the Apixaban sample or a solvate or hydrate thereof by means
of HPLC,
c1) detecting the HPLC peak of the compound of formula (II) by comparing the
retention times or relative retention times.
15. Method for the quantification of the compound of formula (II) according to
anyone of the claims from 9 to 10 in Apixaban or a solvate or hydrate thereof
comprising:
i) measuring the peak area corresponding to the compound of formula (II) in a
Apixaban sample or a solvate or hydrate thereof having an unknown amount
of compound of formula (II) by means of HPLC;
ii) measuring the peak area corresponding to a reference standard containing
a known amount of compound of formula (II) by means of HPLC;
iii) defining the amount of compound of formula (II) in Apixaban or a solvate or
hydrate thereof comparing the area measured in step a) with that measured in
step ii).